CN120035444A - Affinity binding entities for PSMA and methods of use thereof - Google Patents

Abstract

Aspects of the disclosure include affinity binding entities that target Prostate Specific Membrane Antigen (PSMA), chimeric Antigen Receptors (CARs) comprising the same, modified immune cells comprising the CARs, and compositions and methods comprising the same for treating disorders associated with PSMA expression. In embodiments, the modified immune cell is an engineered γδ T cell.

Description

Affinity binding entities for PSMA and methods of use thereof

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application serial No. 63/397,296, filed 8-11 of 2022, the disclosure of which is expressly incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates generally to affinity binding entities directed to Prostate Specific Membrane Antigen (PSMA). Provided herein are Chimeric Antigen Receptors (CARs) capable of binding PSMA, polynucleotides, host cells comprising the polynucleotides and/or CARs, and methods of treating PSMA-related disorders in a patient.

Background

Adoptive cell therapy has undergone nearly constant iterations for over thirty years (30) from early focus on basic lymphokine activation and/or tumor infiltration to recent strategies of engineering immune cells to express genetically engineered antigen receptors, such as Chimeric Antigen Receptors (CARs). Although there have been some implications and indications in this process that indicate the healing potential of these methods, there is still much work to do. In particular, whether CAR-T lymphocytes successfully eradicate a tumor depends on the persistence and effector function of the CAR-T cells, but either excess triggers the graft versus host (GvH) effect in the patient. Furthermore, while adoptive transfer of CAR-expressing T cells has shown some success in treating hematological malignancies, only limited efficacy has been shown in other cancer types, particularly solid tumors. Solid tumors face unique challenges compared to hematological diseases, including but not limited to tumor microenvironments with high immunosuppressive and metabolic challenges.

Prostate Specific Membrane Antigen (PSMA), also known as glutamate carboxypeptidase II, or N-acetylated α -linked acidic dipeptidase 1 or folate hydrolase 1 (FOLH 1), is a dimeric type 2 transmembrane glycoprotein. PSMA is a prostate cancer associated cell membrane antigen that is often overexpressed in neovasculature of Prostate Intraepithelial Neoplasia (PIN), a condition in which some prostate cells begin to appear abnormal in appearance and behavior, primary and metastatic prostate cancer, and other solid tumors (e.g., breast, lung, bladder, kidney). PSMA expression correlates with disease progression and Gleason score (Gleason score). PSMA expression is increased in metastatic disease, hormone refractory cases, and high grade lesions, and is further up-regulated in androgen insensitive tumors.

Adoptive cell therapy methods for PSMA have heretofore had toxicity issues, limiting the practical transformation of such methods. Phase I clinical study performed in month 8 2020, poseida Therapeutics was suspended by death of a patient treated with P-PSMA-101, an autologous CAR-T cell therapy with αβ T cells designed to target PSMA-expressing prostate cancer cells. The patient in this example presented with symptoms consistent with Macrophage Activation Syndrome (MAS), a severe and sometimes fatal immune system overactivation associated with CAR-T therapy. The results of a phase I clinical trial conducted at the university of pennsylvania, published at month 3 of 2021, which tested the efficacy of using autologous CAR-T cells designed to target the alpha beta T cells of PSMA and equipped with dominant negative Transforming Growth Factor (TGF) -beta (Narayan et al, (2022) Nature medicine. Doi: 10.1038). The results revealed that of 13 patients receiving all four dose level therapies, 5 patients developed grade 2 Cytokine Release Syndrome (CRS), including one patient dying from grade 4 CRS with concomitant sepsis. Thus, PSMA-targeted CAR-T has been examined to date in the context of αβ T cells, where potential efficacy appears to be compromised by generally higher alloreactive potential and its propensity to trigger complications such as CRS and MAS.

Gamma delta (γδ) T cells are thymus-derived lymphocytes that differ from αβt cells in activation and functional mechanisms and anatomical distribution. Specifically, while αβ T cells play a role exclusively in adaptive immunity, γδ T cells are congenital-like immune cells that recognize malignant cells in an MHC-independent manner through a range of activating receptors, similar to NK cells (Welsh et al, immunol Rev.1997; 159:79-93). Thus, and in contrast to αβt cells, γδ T cells can potentially be used in an allogeneic setting without risk of causing graft versus host disease (GvHD). Furthermore, recent studies have shown that engineered γδ -T cells can produce fewer pro-inflammatory cytokines than αβ -T cells, thereby reducing the risk of patients developing CRS (Harrer et al, BMC Cancer 2017;17 (1): 551).

Although CAR-T therapies develop rapidly and robustly, the optimal parameters of CAR-T cell-target interactions that will lead to in vivo and human efficacy are not yet clear. Given the mechanism of action and the difference in function of αβt cells compared to γδ T cells, the function and effectiveness of CARs displayed in the αβt cell context cannot be predicted for CARs in the γδ T cell context. Thus, the PSMA-targeted CAR T treatment approach is at best uncertain for the actual transformation of γδ T cells.

Thus, it is apparent that there remains a need for improved strategies to increase the activity, survival and/or expansion of cells following administration, while increasing the safety of adoptive cell therapies targeting PSMA.

Disclosure of Invention

The present disclosure provides methods, cells, compositions, and kits for improving the safety and effectiveness of adoptive cell therapy methods for targeting PSMA. In one aspect, an affinity binding entity is provided that comprises an antigen binding domain that specifically binds to a Prostate Specific Membrane Antigen (PSMA). In embodiments, the antigen binding domain comprises a heavy chain variable region/light chain variable region (HCVR/LCVR) sequence pair ：SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24、25/26、27/28、29/30、31/32、33/34 and 35/36 selected from the group consisting of, or six CDR：SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24、25/26、27/28、29/30、31/32、33/34 and 35/36 selected from the group consisting of HCVR/LCVR sequence pairs. In embodiments, the numbering system used is Kabat et al.

In embodiments, the antigen binding domain comprises a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NO 1/2, 3/4, 5/6, 7/8, 9/10, 11/12 and 13/14, or six CDRs of a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NO 1/2, 3/4, 5/6, 7/8, 9/10, 11/12 and 13/14.

In embodiments, the antigen binding domain specifically binds to an epitope within residues 574-686 of human PSMA, the residues being numbered according to SEQ ID NO. 329 of FIG. 18B. In embodiments, the antigen binding domain specifically binds to an epitope consisting of residues 574-686 of human PSMA, the residues being numbered according to SEQ ID NO. 329 of FIG. 18B. In embodiments, the antigen binding domain specifically binds to an epitope comprising or consisting of residues 574-580, 644-649 and 674-686 of human PSMA, the residues being numbered according to SEQ ID NO:329 in FIG. 18B. In embodiments, the epitope is localized by phage panning using biotinylated recombinant human PSMA protein bound to streptavidin beads. In embodiments, the antigen binding domain comprises a HCVR/LCVR sequence pair of SEQ ID NO 1/2.

In embodiments, the antigen binding domain specifically binds to an epitope within residues 150-261 of human PSMA, residues numbered according to SEQ ID NO. 330 of FIG. 18B. In embodiments, the antigen binding domain specifically binds to an epitope consisting of residues 150-261 of human PSMA, the residues numbered according to SEQ ID NO. 330 of FIG. 18B. In embodiments, the antigen binding domain specifically binds to an epitope comprising or consisting of residues 150-161, 167-172, and 256-261 of human PSMA, the residues being numbered according to SEQ ID NO:330 in FIG. 18B. In embodiments, the epitope is localized by phage panning using biotinylated recombinant human PSMA protein bound to streptavidin beads. In embodiments, the antigen binding domain comprises a HCVR/LCVR sequence pair of SEQ ID NO 9/10.

In embodiments, the affinity binding entity is an antibody or antibody fragment. In embodiments, the antibody or antibody fragment is bispecific. In embodiments, the antibody or antibody fragment is chimeric, humanized or human. In embodiments, the antibody or antibody fragment is monoclonal. In embodiments, the affinity binding entity is selected from the group consisting of scFv, fab, fab ', fv, F (ab') ₂, dsFv, dAb, and any and combinations or more thereof.

According to one aspect of the present invention there is provided a Chimeric Antigen Receptor (CAR) comprising an affinity binding entity comprising an antigen binding domain that specifically binds to a Prostate Specific Membrane Antigen (PSMA), wherein the antigen binding domain comprises a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NO:1/2, 3/4, 5/6, 7/8, 9/10, 11/12 and 13/14, or six CDRs of a HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NO:1/2, 3/4, 5/6, 7/8, 9/10, 11/12 and 13/14.

In embodiments, the CAR further comprises a hinge domain. In embodiments, the hinge domain comprises a glycine polymer, a glycine-serine polymer, a glycine-alanine polymer, an alanine-serine polymer, an immunoglobulin heavy chain hinge, or a receptor-derived hinge. In embodiments, the receptor-derived hinge is a CD8 a hinge domain. In embodiments, the CD 8. Alpha. Hinge domain comprises the amino acid sequence shown as SEQ ID NO. 156.

In embodiments, the CAR further comprises a Transmembrane (TM) domain. In embodiments, the TM domain comprises the TM region of 4-1BB/CD137, activated NK cell receptor, immunoglobulin 、B7-H3、BAFFR、BLAME(SLAMF8)、BTLA、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137 or CD154、CD100(SEMA4D)、CD103、CD160(BY55)、CD18、CD19、CD19a、CD2、CD247、CD27、CD276(B7-H3)、CD28、CD29、CD3δ、CD3ε、CD3γ、CD3ζ、CD30、CD4、CD40、CD49a、CD49D、CD49f、CD69、CD7、CD84、CD8、CD8α、CD8β、CD96(Tactile)、CD11a、CD11b、CD11c、CD11d、CDS、CEACAM1、CRT AM、 cytokine receptor, DAP10, DNAM1 (CD 226), fc gammA receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ig alphA (CD 79A), IL-2 RbetA, IL-2 RgammA, IL-7 RalphA, induced T cell co-stimulatory factor (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, ligand that specifically binds CD83, LIGHT, LTBR, ly9 (CD 229), lymphocyte function-associated antigen-1 (LFA-1; CD1A/CD 18), MHC class 1 molecule, NKG2C, NKG2D, NKp, NKG 44, NKp46, NKp80 (KLRF 1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PD-62, CD 62, SLF-6 or A combination thereof, SLF-6, SLF-35, SLF-6 or A ligand thereof. In embodiments, the TM domain comprises the TM domain of CD8, preferably wherein the CD8 TM domain is the TM domain of CD8 a. In embodiments, the TM domain comprises the amino acid sequence shown as SEQ ID NO. 158.

In embodiments, the CAR further comprises a co-stimulatory domain. In embodiments, the co-stimulatory domain comprises co-stimulatory domain ：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、B7-H3、CEACAM1、CRTAM、CD2、CD3C、CD4、CD7、CD8α、CD8β、CD11a、CD11b、CD11c、CD11d、IL2Rβ、IL2γ、IL7Rα、IL4R、IL7R、IL15R、IL21R、CD18、CD19、CD19a、CD27、CD28、CD29、CD30、CD40、CDS、CD49a、CD49D、CD49f、CD54(ICAM)、CD69、CD70、CD80、CD83、CD84、CD86、CD96(Tactile)、CD100(SEMA4D)、CD103、CD134(OX40)、CD137(4-1BB)、CD152(CTLA-4)、CD160(BY55)、CD162(SELPLG)、CD244(2B4)、CD270(HVEM)、CD226(DNAM1)、CD229(Ly9)、CD278(ICOS)、ICAM-1、LFA-1(CD11a/CD18)、FcR、FcγRI、FcγRII、FcγRIII、LAT、NKG2C、SLP76、TRIM、ZAP70、GITR、BAFFR、LTBR、LAT、GADS、LIGHT、HVEM(LIGHTR)、KIRDS2、ITGA4、ITGA6、ITGAD、ITGAE、ITGAL、ITGAM、ITGAX、ITGB1、ITGB2、ITGB7、NKG2C、NKG2D、IA4、VLA-1、VLA-6、SLAM(SLAMF1、CD150、IPO-3)、SLAMF4、SLAMF6(NTB-A、Ly108)、SLAMF7、SLAMF8(BLAME)、SLP-76、PAG/Cbp、NKp80(KLRF1)、NKp44、NKp30、NKp46、BTLA、JAML、CD150、PSGL1、TSLP、TNFR2 or TRANCE/RANKL, or a portion or combination thereof. In embodiments, the costimulatory domain is a 4-1BB costimulatory domain. In embodiments, the 4-1BB co-stimulatory domain comprises the amino acid sequence set forth in SEQ ID NO. 162.

In embodiments, the CAR further comprises an intracellular signaling domain. In embodiments, the intracellular signaling domain is a cd3ζ intracellular signaling domain. In embodiments, the intracellular signaling domain of CD3 zeta comprises the amino acid sequence shown as SEQ ID NO. 164, 166 or 167.

In embodiments, the CAR further comprises a signal peptide. In an embodiment, the signal peptide comprises the amino acid sequence shown as SEQ ID NO. 152.

In one aspect of the invention, an isolated polynucleotide is provided comprising a nucleic acid sequence encoding any one of the foregoing affinity binding entities. In embodiments, the expression vector comprises the polynucleotide. In embodiments, the polynucleotide is operably linked to a cis-acting regulatory element.

According to an aspect of the invention there is provided a cell comprising any one or more of the aforementioned affinity binding entities, polynucleotides and/or expression vectors.

According to one aspect of the invention there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding any one of the foregoing CARs. In embodiments, the polynucleotide further comprises a nucleic acid sequence encoding at least one polycistronic linker region. In some embodiments, the polycistronic region encodes a cleavage sequence. In embodiments, the cleavage sequence is selected from T2A, F2A, P2A, E a, furin, and furin-P2A (FP 2A). In embodiments, the polycistronic linker region encodes an Internal Ribosome Entry Site (IRES).

In embodiments, the isolated polynucleotide further comprises a nucleic acid sequence encoding one or more additional polypeptides. In embodiments, the one or more additional polypeptides are selected from the group consisting of lymphotoxin beta receptor (LTBR), low affinity nerve growth factor receptor (LNGFR), dominant negative (dn) receptor of TGF-beta or Fas, truncated forms of human epidermal growth factor receptor (EGFRt), and membrane bound IL-12 (mbiL-12), or any combination thereof. In embodiments, the one or more additional polypeptides are selected from the group consisting of fluorescent proteins, gamma chain cytokines, CD19, CD20, LNGFR, EGFRt, LTBR, dnTGF βr2, and any combination thereof.

In embodiments, one or more additional polypeptides are dominant negative receptors for TGF-beta. In embodiments, the dominant negative receptor for TGF- β is dnTGF βR2. In an embodiment dnTGF βr2 comprises the amino acid sequence shown as SEQ ID No. 265. In embodiments, the one or more additional polypeptides is a lymphotoxin β receptor (LTBR). In an embodiment, the LTBR comprises the amino acid sequence shown as SEQ ID NO 267. In embodiments, the one or more additional polypeptides are truncated forms of the epidermal growth factor receptor (EGFRt). In an embodiment, EGFRt comprises the amino acid sequence shown as SEQ ID NO: 261. In embodiments, the one or more additional polypeptides are low affinity nerve growth factor receptors (LNGFR). In an embodiment, LNGFR comprises the amino acid sequence shown as SEQ ID NO. 273. In embodiments, the one or more additional polypeptides are dominant negative Fas (dnFas). In embodiments, one or more additional polypeptides is membrane-bound IL-12 (mbiL-12). In embodiments, the one or more additional polypeptides are CARs that bind to CD 70.

In embodiments, the one or more additional polypeptides are operably linked to the nucleic acid sequence encoding the signal peptide. In embodiments, the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:259, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:271, and SEQ ID NO: 248.

In embodiments, the isolated polynucleotide comprising a nucleic acid sequence encoding a CAR comprises the nucleic acid sequence of SEQ ID NO 205, 209, 213, 217, 221, 225, 229, or 233. In embodiments, the expression vector comprises an isolated polynucleotide comprising a nucleic acid sequence encoding a CAR. In embodiments, the polynucleotide is operably linked to a cis-acting regulatory element.

According to one aspect, provided herein is a γδ T cell comprising a) a nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an affinity binding domain that specifically binds to a Prostate Specific Membrane Antigen (PSMA), and/or (b) a polypeptide comprising a CAR comprising an amino acid sequence encoded by the nucleic acid sequence of (a), wherein the γδ T cell functionally expresses the polypeptide or the binding domain of the nucleic acid encoded CAR on the surface of the γδ T cell. In embodiments, the γδ T cell is a δ1, δ2, δ3 or δ4γδ T cell, preferably δ2 ^- γδ T cell, more preferably δ1γδ T cell.

According to one aspect, provided herein is a modified immune cell comprising a CAR, an isolated polynucleotide comprising a nucleic acid sequence encoding a CAR, and/or an expression vector comprising a polynucleotide comprising a nucleic acid sequence encoding a CAR, as described herein. In embodiments, the modified immune cell is a γδ T cell, γδ NKT cell, αβ T cell, NK cell, NKT cell or macrophage. In embodiments, the modified immune cell is a γδ T cell. In embodiments, the γδ T cell is a δ1, δ2, δ3 or δ4γδ T cell, preferably δ2 ^- γδ T cell, more preferably δ1γδ T cell.

In embodiments, the modified immune cell or the γδ T cell exhibits tumor cell killing activity in vitro and/or in vivo against tumor cells exhibiting PSMA cell surface expression. In embodiments, the cell killing activity is greater than the congenital level of in vitro and/or in vivo tumor cell killing activity in a control modified immune cell or control γδ T cell of the same type that does not comprise the CAR construct. In embodiments, the modified immune cells or γδ T cells proliferate in response to contact with tumor cells exhibiting PSMA cell surface expression. In embodiments, the modified immune cells or γδ T cells exhibit increased proliferation in response to contact with tumor cells that exhibit PSMA cell surface expression, as compared to control modified immune cells or γδ T cells of the same type that do not comprise the CAR construct.

In embodiments, the modified immune cells or γδ T cells are propagated in a host organism comprising tumor cells that exhibit PSMA cell surface expression.

In embodiments, the modified immune cell or γδ T cell expresses a pro-inflammatory cytokine upon contact with a tumor cell exhibiting PSMA cell surface expression.

In embodiments, the modified immune cell or γδ T cell comprises at least one disrupted gene. In embodiments, the at least one disrupted gene is a cytokine-induced SH 2-containing protein (CISH). In embodiments, the at least one disrupted endogenous gene is Cbl proto-oncogene B (Cbl-B). In embodiments, the at least one disrupted endogenous gene is zinc finger protein 91 (ZFP 91). In embodiments, the at least one disrupted endogenous gene is Roquin. In embodiments, the at least one disrupted endogenous gene is CD58 and/or ICAM-1.

According to one aspect, there is provided a plurality of modified immune cells as disclosed herein.

According to one aspect, there is provided a plurality of γδ T cells as disclosed herein, preferably wherein the γδ T cells comprise (a) a nucleic acid encoding a CAR as disclosed herein, the CAR comprising an affinity binding domain that specifically binds to PSMA, and/or (b) a polypeptide comprising a CAR comprising an amino acid sequence encoded by the nucleic acid of (a), wherein the γδ T cells functionally express the polypeptide or the binding domain of the nucleic acid encoded CAR on the surface of the γδ T cells.

In embodiments, the plurality of modified immune cells or the plurality of γδ T cells comprises a composition of δ1, δ2, δ3 or δ4γδ T cells, preferably δ1 or δ2γδ T cells, more preferably δ2 ^- γδ T cells, most preferably δ1γδ T cells of at least 60%, 80% or about 60% or 80% to about 90% or 95%.

In embodiments, the plurality of modified immune cells or the plurality of γδ T cells comprise at least about 10 ⁷ modified immune cells or γδ T cells, respectively, preferably from about 10 ⁸ modified immune cells or γδ T cells to about 10 ¹¹ modified immune cells or γδ T cells, respectively.

According to one aspect, there is provided a method of making a modified immune cell, γδ T cell, a plurality of modified immune cells, or a plurality of γδ T cells, wherein the method comprises transfecting an immune cell or γδ T cell with an expression vector comprising a nucleic acid encoding a CAR as disclosed herein, optionally wherein the cell has at least one disrupted gene. In embodiments, the method comprises retroviral transduction. In embodiments, the method comprises ex vivo expansion of the immune cells or γδ T cells, wherein ex vivo expansion is performed before and/or after transfection of the immune cells or γδ T cells.

According to an aspect of the invention there is provided an antibody-drug conjugate (ADC) comprising any one of the foregoing affinity binding entities.

According to an aspect of the invention there is provided a pharmaceutical composition comprising any of the foregoing affinity binding entities, modified immune cells, γδ T cells or ADCs, and a pharmaceutically acceptable carrier.

According to an aspect of the present invention, there is provided a method of inhibiting the growth of a cell exhibiting PSMA cell surface expression, comprising contacting the cell with any of the foregoing affinity binding entities, modified immune cells, γδ T cells, ADCs, or pharmaceutical compositions.

According to an aspect of the present invention, there is provided a method of killing a tumor cell exhibiting PSMA cell surface expression, the method comprising contacting the tumor cell with a therapeutically effective amount of any of the foregoing affinity binding entities, modified immune cells, γδ T cells, ADCs, or pharmaceutical compositions. In embodiments, the method comprises introducing a therapeutically effective amount of an affinity binding entity, a modified immune cell, a γδ T cell, an ADC, or a pharmaceutical composition into a host organism comprising a tumor cell.

In embodiments of the method of killing tumor cells, the method further comprises administering one or more methods of increasing the common gamma chain cytokine simultaneously or sequentially. In embodiments, the one or more methods of administering an elevated shared gamma chain cytokine comprise administering an amount of the shared gamma chain cytokine effective to increase proliferation, cytotoxic activity, persistence, or a combination thereof of the introduced modified immune cells or γδ T cells, either simultaneously and/or sequentially before and/or after the introduction of the modified immune cells or γδ T cells. In embodiments, one or more methods of increasing the common gamma chain cytokine comprises lymphocyte depletion (lymphodepletion) prior to introducing the modified immune cells or γδ T cells. In embodiments, one or more methods of increasing the common gamma chain cytokine comprise secreting one or more common gamma chain cytokines from the introduced modified immune cells or γδ T cells.

In embodiments of the methods of inhibiting growth of cells exhibiting PSMA cell surface expression or methods of killing tumor cells exhibiting PSMA cell surface expression herein, the methods reduce the in vivo tumor burden and/or increase the mean survival time of a host organism compared to a control organism, wherein the control organism is not treated with an affinity binding entity, modified immune cells, γδ T cells, ADC, or pharmaceutical composition. In embodiments, the host organism is a human. In embodiments, the method is a method of treating cancer in a subject in need thereof.

According to one aspect, there is provided the use of any of the aforementioned affinity binding entities, modified immune cells, γδ T cells, ADCs or pharmaceutical compositions for the manufacture of a medicament for the treatment of cancer.

In one aspect, there is provided a method of reducing or inhibiting graft versus host response of immune cells administered to a subject in need thereof, comprising administering a therapeutically effective amount of γδ T cells according to the present invention. In embodiments, γδ T cells can comprise a dual CAR that binds to CD70 and PSMA, or the method can further comprise co-administering γδ T cells according to the invention simultaneously or sequentially with immune cells (e.g., T cells or NK cells) comprising a CAR that binds to PSMA and a CAR that binds to CD 70.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Drawings

FIG. 1 illustrates the binding profile of anti-PSMA antibodies to both PSMA-expressing 22Rv1 cells and PSMA-expressing knocked-out 22Rv1 cells.

Fig. 2A depicts a list of EC50 of anti-PSMA antibodies against recombinant human PSMA protein.

Fig. 2B illustrates a differential binding profile of selected anti-PSMA antibodies to the monomeric or dimeric state of recombinant human PSMA protein.

Figures 3A-3H are graphs illustrating in vitro cytotoxicity of various PSMACAR constructs of the present disclosure against PSMA expressing cell lines (PSMA expressing 22Rv1 target cells, figures 3A, 3C; PC3 cells engineered to express PSMA, figures 3E, 3G) or corresponding negative controls (PSMA expressing knockdown 22Rv1, figures 3B, 3D; parent PC3 cell lines not expressing PSMA, figures 3F, 3H).

FIGS. 4A-4B are graphs illustrating the in vitro cytotoxicity profile of PSMACAR constructs modified to express dominant negative TGF-beta receptor II (dnTGF. Beta. RII). The cytotoxicity profile of the modified PSMA CAR construct was comparable to that of the similar PSMACAR construct lacking dnTGF βrii (fig. 4A). No cytotoxicity was observed against PSMA knockout cell lines (fig. 4B).

Fig. 5A illustrates that expression of the PSMACAR construct modified to express dnTGF βrii was unchanged relative to the expression of a similar PSMACAR lacking dnTGF βrii.

Fig. 5B illustrates the detection of dnTGF βrii expression in the PSMACAR construct modified to express dnTGF βrii, but not in the similar unmodified PSMACAR construct.

Figure 5C illustrates that γδ T cells containing the PSMACAR construct modified to express dnTGF βrii have reduced CD103 expression compared to control cells containing a similar PSMACAR construct lacking dnTGF βrii.

FIG. 5D illustrates the reduced expression of pSMAD2/3 in γδ T cells containing the PSMACAR construct modified to express dnTGF βRII compared to control cells containing a similar PSMACAR construct lacking dnTGF βRII.

FIG. 6 illustrates a 15 day cell expansion profile of anti PSMACAR transduced γδ T cells.

Fig. 7A-7B are graphs illustrating in vivo efficacy of anti-PSMACAR transduced γδ T cells in the NOD scid γ (NSG) mouse subcutaneous human xenograft 22Rv1 clone E7 model.

FIG. 8 is a graph illustrating the gene knockout efficiency of two different guide RNAs targeting cytokine-induced SH 2-containing proteins (CISH).

Fig. 9A is a graph illustrating that CISH knockdown vδ1t cells can be enriched after αβt cell depletion.

FIG. 9B is a graph illustrating the viability of CISH-knocked-out V.delta.1T cells.

Fig. 10A illustrates the binding profile of anti-PSMA antibodies to both PSMA expressing 22Rv1 cells and PSMA expressing knockdown 22Rv1 cells. Fig. 10B summarizes the binding profile of anti-PSMA antibodies to 3 different psma+ prostate cancer (PCa) cell lines (different PSMA expression levels).

Figure 11A illustrates the EC50 of anti-PSMA antibodies against recombinant human PSMA protein. Fig. 11B illustrates the differential binding profile of some anti-PSMA antibodies to the monomeric or dimeric state of recombinant human PSMA protein.

FIG. 12 illustrates the in vitro cytotoxicity of different PSMACAR constructs against PSMA expressing PCa cell lines 22Rv1 and PC3-PSMA and corresponding knockout or parent lines, respectively, lacking PSMA expression.

FIG. 13 illustrates cytotoxicity PSMACAR in vitro in the presence of TGF-beta 1.

Fig. 14A illustrates that the expression of CAR remains unchanged relative to a bare CAR in the "bolt-on" modified PSMACAR construct. Fig. 14B illustrates expression of dominant negative tgfβ receptor II (dnTGF βrii) in a "bolt-on" modified PSMACAR construct when compared to an unmodified naked CAR. Figure 14C illustrates the reduced CD103 expression in the "bolt-on" modified PSMACAR construct compared to the naked CAR. FIG. 14D illustrates the reduced expression of pSMAD2/3 in the "bolting" modified PSMACAR construct following addition of exogenous TGF-beta as compared to the unmodified naked CAR.

Fig. 15 illustrates cell expansion profiles of anti-PSMA CAR transduced γδ T cells with and without "bolting" in 3 donors and baseline (J591).

Figure 16 illustrates in vivo efficacy of anti PSMACAR transduced γδ T cells expanded in3 donors in the NOD scid γ (NSG) mouse subcutaneous human xenograft 22Rv1 clone E7 model compared to reference J591 transduced γδ T cells.

Figure 17 illustrates in vivo efficacy of anti-PSMA CARs with and without dnTGF βrii "bolting" (including sub-optimal doses) in the subcutaneous human xenograft PC3-PIP model of NSG mice.

Fig. 18A illustrates binding agent epitopes in two anti-PSMACAR transduced γδ T cells that map to the crystal structure of human PSMA. The predicted linear epitope, conformational epitopes of leader 1 and leader 2 for benchmark (J591) are indicated in the figure. FIG. 18B lists the sequences on human PSMA (SEQ ID NOS: 329-330) elucidated as binding agent epitopes in leads 1 and 2 using cross-linked mass spectrometry (XL-MS).

FIG. 19 illustrates the design of the CAR-mbiL-12 construct.

FIGS. 20A-20D illustrate the expansion and expression of CAR-mbiL-12 in V.delta.1T cells.

FIG. 21 illustrates the enhanced in vitro cytotoxicity of CAR-mbiL-12 in V.delta.1T cells.

Figure 22 illustrates the in vivo therapeutic efficacy of CAR-mbIL-12 in vδ1T cells in a subcutaneous human xenograft Raji cell NSG mouse model.

FIGS. 23A-23B illustrate that CISH KO enhances cytotoxicity of V.delta.1T cells in vitro.

FIGS. 24A-24B illustrate that CBL-BKO enhances cytotoxicity of V.delta.1T cells in vitro.

FIGS. 25A-25B illustrate Roquin KO enhance in vitro cytotoxicity of V.delta.1T cells.

FIGS. 26A-26B illustrate that CD58 or ICAM-1KO enhanced in vitro cell survival of V.delta.1T cells in an allogeneic MLR assay.

Detailed Description

I. Definition of the definition

For the purposes of explaining the present specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. If any of the definitions set forth conflict with any document incorporated herein by reference, the definitions set forth below shall control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used herein, "about" when referring to measurable values such as amount, duration, etc., is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% of the specified value, as such variations are suitable for performing the disclosed methods.

As used herein, "w/v" refers to the weight of a component in a given volume of solution.

"Scope" throughout this disclosure, various aspects of the disclosure may be presented in a scope format. It should be understood that the description in range format is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have explicitly disclosed all possible sub-ranges as well as individual values within that range. For example, a range description such as 1 to 6 should be considered to have the explicitly disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1,2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal that is amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

As used herein, the term "diagnosis" or "diagnosis (diagnosing)" refers to a process of identifying a disease, such as cancer, by its signs, symptoms, and/or the results of various tests. The conclusion drawn through this process is a diagnosis. Commonly performed test formats include blood testing, medical imaging, urine analysis, biopsy, and the like.

As used herein, the term "agent" refers to any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, other molecule or cell of interest (e.g., a cell engineered to express a chimeric antigen receptor). The agent may include a therapeutic agent, a diagnostic agent, or a pharmaceutical agent. A therapeutic agent or agent is one that, when administered to a subject (including treating a subject suffering from cancer or other disease/disorder), alone or in combination with additional agents, induces a desired response, such as inducing a therapeutic or prophylactic effect.

The term "therapeutically effective amount" (or simply "effective amount") refers to the amount of an agent or composition (e.g., an agent-containing composition) that will elicit the biological or medical response of a tissue, system or subject that is being sought by a researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes an amount of an agent or composition comprising an agent that, when administered, is sufficient to prevent the development of, or to some extent reduce, one or more signs or symptoms of the disorder or disease being treated (e.g., prostate cancer). The therapeutically effective amount will vary depending on the composition, the disease and its severity, the age, weight, etc., of the subject to be treated.

As used herein, the term "γδ T cells (gamma T cells)" refers to a subset of T cells expressing different T Cell Receptors (TCRs) consisting of one γ chain and one δ chain, namely γδ TCRs, on their surfaces. The term "γδ T cells" specifically includes all subsets of γδ T cells including, but not limited to, vδ1 and vδ2, vδ3γδ T cells, as well as initial, effector memory, central memory, and terminal differentiated γδ T cells. As another example, the term "γδ T cells" includes vδ4, vδ5, vδ7, and vδ8γδ T cells, as well as vγ2, vγ3, vγ5, vγ8, vγ9, vγ10, and vγ1γδ T cells. In embodiments, γδ T cells are vδ1 ^-、Vδ2^- or vδ1 ^- and vδ2 ^-. Compositions and methods for making and using engineered and non-engineered γδ T cells and/or subtypes thereof include, but are not limited to, those described in US2016/0175358, wo 2017/197347;US 9499788;US2018/0169147;US 9907820;US 2018/0125889, and US2017/0196910, the contents of each of which are incorporated by reference for all purposes, including the compositions and methods for making and using engineered and non-engineered γδ T cells and/or subtypes thereof. The application further contemplates T cells or other engineered leukocytes or lymphocytes expressing a gamma chain or a delta chain, optionally in combination with a second polypeptide to form a functional TCR. Such engineered leukocytes or lymphocytes expressing a gamma or delta chain can be used in the methods described herein or are present in the compositions described herein.

The γδ T cells described herein can be δ1, δ2, δ3, or δ4γδ T cells or combinations thereof. In some cases, γδ T cells are predominantly (> 50%), substantially (> 90%), substantially all or completely δ2γδ T cells. In some cases, γδ T cells are predominantly (> 50%), substantially (> 90%), substantially all or completely δ1γδ T cells. In some cases, γδ T cells are predominantly (> 50%), substantially (> 90%), substantially all or all δ3γδ T cells.

Γδ T cells for use as used herein can be obtained from allogeneic or autologous donors. γδ T cells may be partially or fully purified or not purified and may be expanded ex vivo. Methods and compositions for ex vivo amplification include, but are not limited to, those described in WO 2017/197347. Amplification may be performed before or after, or both before and after, the CAR polypeptides of the present disclosure are introduced into γδ T cells. Other additional or alternative methods of amplification include the use of, for example, artificial antigen presenting cells (aAPCs), aminobisphosphonates, cytokine mixtures and feeder cells (Cortes-Selva, D et al, (2021) Trends Pharmacol Sci.42 (1): 45-59).

As used herein, the term "αβ T cell" refers to a T cell that expresses the α and β chains of a TCR as part of a complex with a CD3 chain molecule. Each of the α and β chains contains a variable domain and a constant domain. αβ T cells predominantly recognize peptide antigens presented by Major Histocompatibility Complex (MHC) class I and II molecules, with most of the receptor diversity contained within the third complementarity determining region (CDR 3) of the TCR α and β chains.

As used herein, the term "Natural Killer (NK) cells" refers to CD56 ⁺CD3^- granule lymphocytes, which play an important role in immune surveillance against viruses and tumors, and constitute a critical subset of cells of the innate immune system (Godfrey J et al Leuk Lymphoma 2012 53:1666-1676). NK cells express a markedly diverse repertoire of inhibitory and activating receptors on their cell surface (repertoire) that regulate their immune response. NK cells can kill transformed or infected cells by releasing perforin and granzyme or by using effector molecules of the Tumor Necrosis Factor (TNF) family such as TNF, TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand, which induce apoptosis of target cells. In addition, NK cells produce chemokines and cytokines rapidly after activation, including Interferon (IFN) -gamma, GM-CSF, and IL-10, which recruit and affect the function of hematopoietic and non-hematopoietic cells of the host. Unlike cytotoxic CD8 ⁺ T lymphocytes, NK cells can be cytotoxic to tumor cells without prior sensitization, and can also destroy MHC-I negative cells (Narni-MANCINELLI E et al Int Immunol 2011 23:427-431). NK cells are considered quite safe effector cells because they can avoid cytokine storms (Morgan R A et al Mol Ther 201018:843-851), oncolytic syndrome (Porter D L et al N Engl J Med 20110265:725-733), and potentially fatal complications in target, tumor-free effects.

NK cells may be obtained from allogeneic or autologous donors. NK cells may be partially or fully purified, or not purified, and may be expanded ex vivo. Methods and compositions for ex vivo amplification include, but are not limited to, those described in Becker et al, (2016) Cancer immunol. Immunother.65 (4): 477-84). Amplification may be performed before or after the CAR is introduced into the NK cells or both. Briefly, but not limited to, NK cell expansion can include the use of engineered feeder cells, cytokine mixtures (e.g., IL-2, IL-15) and/or aAPC (Cortes-Selva, D et al, (2021) Trends Pharmacol Sci.42 (1): 45-59).

In some embodiments, placental hematopoietic stem cell-derived natural killer (PNK) cells or immortalized cell lines (e.g., NK-92) can be engineered to express chimeric adapter polypeptides of the disclosure. In other examples, NK cells useful for engineering CAR expression herein can be differentiated from human embryonic stem cells (hescs) and induced pluripotent stem cells (ipscs). As used herein, the term "Natural Killer T (NKT) cells" are T lineage cells that have the same morphological and functional characteristics as T cells and NK cells. NKT cells are fast responders to the innate immune system and mediate powerful immune regulation and effector functions in a variety of disease settings. Ligand recognition in NKT cells results in rapid secretion of pro-inflammatory cytokines (such as IFN- γ and TNF- α) and anti-inflammatory cytokines (such as IL-4, IL-10 and IL-13) that enhance immune responses to e.g. cancer by targeting tumor cells directly and by modulating anti-tumor responses indirectly (by releasing different cytokines) or by altering TME. Upon activation, NKT cells can begin cytokine secretion immediately without prior differentiation into effector cells. The rapidity of their response makes NKT cells play an important role in the first line of innate defense against infection by some types of bacteria and viruses. In addition, many cytokines secreted by NKT cells have a strong impact on the differentiation and function of αβ T cells, linking NKT cells with adaptive defenses. NKT cells bridge the adaptive immune system and the innate immune system. Unlike conventional T cells, which recognize peptide antigens presented by Major Histocompatibility Complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1 d. NKT cells may be obtained from allogeneic or autologous donors. NKT cells may be partially or fully purified, or not purified, and may be expanded ex vivo. Briefly, NKT cells can be expanded by using ex vivo IL-2 and/or monoclonal antibodies specific for the TCR alpha chain CDR3 loop (Cortes-Selva, D et al, (2021) Trends Pharmacol Sci.42 (1): 45-59).

As used herein, the term "γδ natural killer T cells" or "γδ NKT cells" refers to iPSC-derived cells expressing γδ TCR and NK receptors but lacking expression of a marker γδ T cell marker (cores-Selva, D et al, (2021) Trends Pharmacol sci.42 (1): 45-59). These cells have been shown to have anti-tumor activity against a number of cancer cell lines, but not normal cells, and to exhibit more potent killing than donor-derived γδ T cells or donor-derived NK cells (Zeng J et al, (2019) PLoS ONE 14 (5): e 0216815). In embodiments herein, the CAR can be expressed in γδ NKT cells for use according to the methods disclosed herein.

As used herein, the term "myeloid cells" refers to a subpopulation of leukocytes represented by granulocytes, monocytes, macrophages and Dendritic Cells (DCs). They circulate through the blood and lymphatic systems and are rapidly recruited to sites of tissue injury and infection via various chemokine receptors. Within tissues, they are activated to phagocytose and secrete inflammatory cytokines, playing an important role in protective immunity. Myeloid cells can also be found in tissues under steady state conditions, where they control development, homeostasis, and tissue repair.

As used herein, the term "macrophage" refers to an innate cell with high plasticity whose functional and phenotypic characteristics can develop in response to a variety of stimuli. Macrophage polarization is largely reduced to two distinct states, the M1 phenotype (classical activation) in response to factors such as Lipopolysaccharide (LPS) or IFN- γ, or the M2 phenotype in response to cytokines such as IL-4, IL-5 and IL-13. Examples of M1-like macrophages express iNOS and pro-inflammatory cytokines such as TNF- α, IL1- β, IL-6, IL-12 and IL-23. Examples of M2 macrophages show increased expression of CD209, CD200R, CD a and CD1b in humans and are associated with wound healing and anti-tumor responses. The ability of macrophages to infiltrate solid tumors and reprogram, as well as the anti-tumor effects associated with conversion to the M1 phenotype, make macrophages relevant to the present disclosure in terms of engineered macrophages expressing the CARs described herein. For example, it has been demonstrated that in a mouse ovarian cancer model, macrophages can be reprogrammed to an anti-tumor M1 phenotype cell that is capable of producing nitric oxide and inducing IL-12 dependent NK-mediated anti-tumor effects by inhibiting NK- κB signaling (Zhang F et al, (2019) Nat Commun 10:3974).

Macrophages may be obtained/derived from allogeneic or autologous donors. Macrophages may be partially or fully purified, or not purified, and may be cultured ex vivo (see, e.g., davies JQ and Gordon A (2005) Methods Mol Biol 290:105016). In embodiments, the disclosure encompasses macrophages derived from hESCs (Karlsson, KR et al, (2008) Exp Hematol 36:1167-1175) or iPSC-derived macrophages (Takata K. Et al, (2017) Immunity 47:183-198).

As used herein, the term "T lymphocyte" or "T cell" refers to an immune cell that expresses or has expressed CD3 (cd3+) and a T cell receptor (tcr+). T cells play a central role in cell-mediated immunity. T cells that "have expressed" CD3 and TCR are engineered to eliminate CD3 and/or TCR cell surface expression.

As used herein, the term "TCR" or "T cell receptor" refers to dimeric heterologous cell surface signaling proteins that form an alpha-beta or gamma-delta receptor, or a combination thereof. While αβ TCRs recognize antigens presented by MHC molecules, γδ TCRs can recognize antigens independent of MHC presentation.

The term "MHC" (major histocompatibility complex) refers to a subset of genes encoding cell surface antigen presenting proteins. In humans, these genes are called Human Leukocyte Antigen (HLA) genes. The abbreviation MHC or HLA is used interchangeably herein.

As used herein, "prostate specific membrane antigen" or "PSMA" refers to any natural PSMA from any vertebrate source, including mammals, such as primates (e.g., humans, non-human primates, and rodents), unless otherwise indicated. The term encompasses "full-length" untreated PSMA as well as any form of PSMA produced by intracellular processing. The term also encompasses naturally occurring variants of PSMA, e.g., splice variants, allelic variants, and isoforms. PSMA is a type II membrane protein, initially characterized by the murine monoclonal antibody (mAb) 7E 11-C5.3. PSMA proteins have a 3-part structure, an inner 19 amino acid portion, a 24 amino acid transmembrane portion, and an outer 707 amino acid portion (e.g., an extracellular domain). An exemplary amino acid sequence for human PSMA is set forth herein as SEQ ID NO. 150. An exemplary amino acid sequence for the extracellular domain of human PSMA is shown in SEQ ID NO. 151.

As used herein, "activation" refers to the state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cell" refers to, among other things, a T cell that is undergoing cell division.

The "co-stimulatory domain" (also referred to herein as a Chimeric Antigen Receptor (CAR)) in the context of the chimeric receptor of the present disclosure enhances cell proliferation, cell survival, and memory cell development of cytotoxic cells expressing the chimeric receptor. The chimeric receptors of the invention may comprise one or more co-stimulatory domains selected from the group consisting of co-stimulatory domains ：CD28、CD137(4-1BB)、CD134(OX40)、Dapl0、CD27、CD2、CD7、CD5、ICAM-1、LFA-1(CD1 la/CD18)、Lck、TNFR-I、PD-1、TNFR-II、Fas、CD30、CD40、ICOS LIGHT、NKG2C、B7-H3 of proteins in the TNFR superfamily or a combination thereof. If the chimeric receptor comprises more than one co-stimulatory domain, these domains may be arranged in tandem, optionally separated by a linker. The co-stimulatory domain is an intracellular domain that can be located between (truncated or full length) CD70 and the intracellular signaling domain within the chimeric receptor.

As used herein, the term "co-stimulatory domain" also encompasses any modification thereof, examples of which are described in U.S. patent application No. 20200129554, U.S. patent application No. 20200317777, wo2019010383, li, w., et al, (2020) Immunity 53:456-470, and Li, g., et al, (2017) J Immunity 198 (1 support): 198.4, the contents of each of which are incorporated herein in their entirety.

The "intracellular signaling domain" in the context of the chimeric receptors of the present disclosure converts effector function signals and directs cytotoxic cells to perform their specialized function, i.e., damage and/or destruction of the target cell. Examples of suitable intracellular signaling domains include, for example, zeta chains of the T cell receptor complex or any homologue thereof, e.g., eta, fcsRly and beta chains, MB 1 (Iga) chains, B29 (Ig) chains, etc., human CD3 zeta chains, CD3 polypeptides (delta, delta and epsilon), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, fyn, lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5, and CD28. In embodiments, the intracellular signaling domain of the chimeric receptor can be the human cd3ζ chain, the cytoplasmic tail of FcyRIII, fcsRI, fc receptors, cytoplasmic receptors carrying an immune receptor tyrosine activation motif (ITAM), and combinations thereof.

The intracellular signaling domain may include several types of intracellular signaling domains of various other immune signaling receptors, including but not limited to first, second and third generation T cell signaling proteins, including CD3, B7 family co-stimulatory receptors and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (Park et al, "ARE ALL CHIMERIC ANTIGEN receptors created equal. Other intracellular signaling domains include those used by NK and NKT cells (Hermanson et al, "Utilizing CHIMERIC ANTIGEN receptors to direct natural KILLER CELL ACTIVITY," Front immunol., volume 6, page 195, 2015), signaling domains such as NKp30 (B7-H6) (Zhang et al ,"An NKp30-based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo,"J Immunol.,, pages 2290-2299, 2012) and DAP12 (Topfer et al, "DAP12-based ACTIVATING CHIMERIC ANTIGEN receptor for NK cell tumor immunotherapy," J immunol., volume 194, pages 3201-3212, 2015), NKG2D, NKp, NKp46, DAP10 and CD3z. In addition, intracellular signaling domains also include those of human immunoglobulin receptors containing an immune receptor tyrosine activation motif (ITAM), such as FCGAMMARI, FCGAMMARIIA, FCGAMMARIIC, FCGAMMARIIIA, FCRL (Gillis et al, volume ,"Contribution of Human Fc.gamma.Rs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies,"Front Immunol.,, page 5, 254,2014).

In embodiments, the intracellular signaling domain comprises a cytoplasmic signaling domain of TCR ζ, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, or CD66 d. In exemplary embodiments, the intracellular signaling domain in the chimeric receptor comprises the cytoplasmic signaling domain of human cd3ζ. The term "intracellular signaling domain" as used herein also encompasses any modification thereof, examples of which are described in U.S. patent application Ser. No. 2020/0317777 and Roda-Navarro, P., and Reyburn, HT., (2009) J Biol Chem 284 (24): 16463-16472; giurisato, E., et al, (2007) Mol Cell Biol 27 (24): 8583-8599; and Wu, J., et al, (2000) J Exp Med 192 (7): 1059-1068, the respective contents of which are incorporated herein in their entirety.

The term "affinity binding entity" refers to a binding moiety that binds a specific antigen with a higher affinity than a non-specific antigen and is conferred an affinity of at least 10 ^-6 M, as determined by assays well known in the art, including Surface Plasmon Resonance (SPR). According to specific embodiments, the affinity is 500nM to 0.01nM, 100nM to 0.01nM, 50nM to 0.01nM, 10nM to 0.01nM, 5nM to 0.01nM.

According to an embodiment, the affinity binding entity is an antibody. The term "antibody" is used in the broadest sense and specifically covers, for example, single anti-PSMA monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full length or intact monoclonal antibodies), anti-PSMA antibody compositions having multi-epitope specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), single chain anti-PSMA antibodies, and fragments of anti-PSMA antibodies (see below) (including Fab, fab ', F (ab') 2, and Fv fragments), diabodies, single domain antibodies (sdabs), so long as they exhibit the desired biological or immunological activity. anti-PSMA antibodies, and particularly fragments, also include portions of the anti-PSMA antibodies (and combinations of portions of the anti-PSMA antibodies, e.g., scFv) that can be used as targeting arms against, e.g., PSMA epitopes in chimeric antigen receptors of the present disclosure. Such fragments are not necessarily proteolytic fragments, but rather portions of the polypeptide sequence that can confer affinity to the target. The term "immunoglobulin" (Ig) is used interchangeably herein with antibody. The antibody may be, for example, a human antibody, a humanized antibody, and/or an affinity matured antibody.

Methods for preparing antibodies and antibody fragments are known in the art. (see, e.g., harlow and Lane,Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory,New York,1988,incorporated herein by reference).

Antibodies can be produced by immunizing a variety of animals (including mice, rats, rabbits, goats, primates, humans, and chickens) with a target antigen (such as PSMA or PSMA peptide fragments containing an anti-PSMA epitope of the present disclosure). Antibodies can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al, nature,352:624-8 (1991) and Marks et al, J.mol.biol.,222:581-97 (1991). The antibodies or antigen binding fragments of the invention may be purified by methods known in the art, such as gel filtration, ion exchange, affinity chromatography, and the like. Affinity chromatography or any of a number of other techniques known in the art may be used to isolate polyclonal or monoclonal antibodies from, for example, serum, ascites fluid, or hybridoma supernatants.

The terms "anti-PSMA antibody", "PSMA antibody" and "antibody that binds PSMA" are used interchangeably. The anti-PSMA antibody is preferably capable of binding with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent, whether the antibody is isolated or as part of a fusion protein, cell, or cell composition.

An "isolated antibody" is an antibody that has been identified and isolated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that interfere with the therapeutic use of antibodies and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H chain and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the alpha and gamma chains, and four CH domains of the mu and epsilon isoforms. Each L chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end. VL is aligned with VH, and CL is aligned with the first constant domain of the heavy chain (CH 1). It is believed that the particular amino acid residues form an interface between the light chain and heavy chain variable domains. The VH and VL pair together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see for example Basic AND CLINICAL Immunology, 8 th edition, daniel p.Stites, abba I.terr and Tristram G.Parslow (ed.), appleton & Lange, norwalk, CT,1994, pages 71 and chapter 6.

L chains from any vertebrate species can be classified into one of two distinct types called kappa and lambda based on the amino acid sequences of their constant domains. Immunoglobulins are assigned to different classes or isotypes based on the amino acid sequence of the constant domain of their heavy Chain (CH). Immunoglobulins are of five classes IgA, igD, igE, igG and IgM, with heavy chains designated α, δ, ε, γ and μ, respectively. The gamma and alpha classes are further divided into subclasses based on relatively small differences in CH sequence and function, e.g., humans express subclasses IgG1, igG2, igG3, igG4, igA1, and IgA2.

"Variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH" or "V _H". The variable domain of the light chain may be referred to as "VL" or "V _L". These domains are typically the most variable parts of an antibody and contain antigen binding sites.

The term "variable" refers to the fact that certain segments of the variable domain vary greatly in sequence from antibody to antibody. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed over the 110 amino acids of the variable domains. Instead, the V region consists of relatively invariant segments, known as Framework Regions (FR), of 15-30 amino acids, separated by extremely variable shorter regions, known as "hypervariable regions", each about 9-12 amino acids long. The variable domains of the natural heavy and light chains each comprise four FR, principally in the β -sheet configuration, joined by three hypervariable regions, which form loops that connect the β -sheet structure and in some cases form part of the β -sheet structure. The hypervariable regions in each chain are held together by the FR and together with the hypervariable regions from the other chain contribute to the formation of the antigen binding site of the antibody (see Kabat et al Sequences of Proteins of Immunological Interest, 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD. (1991)).

An "intact" antibody is an antibody comprising an antigen binding site, CL and at least heavy chain constant domains CH1, CH2 and CH 3. The constant domain may be a natural sequence constant domain (e.g., a human natural sequence constant domain) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding region or one or more variable regions of an intact antibody. Examples of antibody fragments include Fab, fab ', F (ab') 2 and Fv fragments, diabodies, linear antibodies (see U.S. Pat. No. 5,641,870, example 2; zapata et al, protein Eng.8 (10): 1057-62 (1995)), single chain antibody molecules, and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment comprises the antigen binding site of an intact antibody, thus preserving the ability to bind antigen. Also included in the anti-PSMA antibody fragments are the portions of the anti-PSMA antibodies (and combinations of portions of the anti-PSMA antibodies, e.g., scFv) that can be used as targeting arms against, for example, PSMA epitopes in chimeric antigen receptors of the present disclosure. Such fragments are not necessarily proteolytic fragments, but rather portions of the polypeptide sequence that can confer affinity to the target.

Papain digestion of antibodies produces two identical antigen-binding fragments (referred to as "Fab" fragments) and a residual "Fc" fragment (the name reflecting the ability to crystallize readily). The Fab fragment consists of the complete L chain as well as the variable region domain of the H chain (VH) and the first constant domain of one heavy chain (CH 1). Each Fab fragment is monovalent in terms of antigen binding, i.e. it has a single antigen binding site. Pepsin treatment of the antibodies produced a single large F (ab') 2 fragment, which corresponds approximately to two disulfide-linked Fab fragments with bivalent antigen binding activity, and was still able to crosslink the antigen. Fab' fragments differ from Fab fragments in that there are additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is herein the name of Fab' in which the cysteine residue of the constant domain bears a free thiol group. F (ab ') 2 antibody fragments were initially produced as Fab' fragment pairs with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of two H chains held together by disulfide bonds. The effector function of antibodies is determined by sequences in the Fc region, which is also the part recognized by Fc receptors (fcrs) found on certain types of cells.

"Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in close non-covalent association. In a single chain Fv (scFv) material, one heavy chain variable domain and one light chain variable domain may be covalently linked by a flexible peptide linker such that the light and heavy chains may associate similar to the "dimeric" structure in a double chain Fv material. Six hypervariable loops (from 3 loops each of the H and L chains) are created by folding of these two domains, which contribute amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with less affinity than the entire binding site.

"Single chain Fv" also abbreviated "sFv" or "scFv" is an antibody fragment comprising VH and VL antibody domains linked into a single polypeptide chain. In embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For reviews of sFvs, see, for example, pluckaphun, the Pharmacology of Monoclonal Antibodies, vol.113, rosenburg and Moore, springer-Verlag, new York, pp.269-315 (1994); borrebaeck 1995, see below. In one embodiment, the anti-PSMA antibody-derived scFv is used as a targeting arm for a CAR modified immune cell as disclosed herein. For scFv antibody fragments in which the order of VH and VL regions in the binding domain is explicitly or implicitly described, the present disclosure also includes alternative embodiments in which the order of VH and VL regions is reversed (e.g., in scFv or CAR comprising scFv binding domains). Thus, for example, in a scFv or CAR comprising a scFv binding domain, the description of a VH-VL sequence also describes an alternative VL-VH sequence. Furthermore, the description of VL-VH sequences also describes alternative VH-VL sequences, for example in scFv or CARs comprising scFv binding domains. The VH region and VL region may be joined directly or by a peptide-encoding linker that links the N-terminus of VH to the C-terminus of VL, or links the C-terminus of VH to the N-terminus of VL.

ScFv linkers are typically rich in glycine to increase flexibility, and serine or threonine to increase solubility. The linker may connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al, anal. Chem.80 (6): 1910-1917 (2008) and WO 2014/087010, the contents of which are hereby incorporated by reference in their entirety. Various linker sequences are known in the art, including but not limited to Glycine Serine (GS) linkers such as (GS) n, (GSGGS) n (SEQ ID NO: 275), (GGGS) n (SEQ ID NO: 276) and (GGGGS) n (SEQ ID NO: 277), where n represents an integer of at least 1. Exemplary linker sequences can include amino acid sequences including, but not limited to GG SG(SEQ ID NO:278)、GGSGG(SEQ ID NO:279)、GSGSG(SE Q ID NO:280)、GSGGG(SEQ ID NO:281)、GGGSG(SEQ ID N O:282)、GSSSG(SEQ ID NO:283)、GGGGS(SEQ ID NO:284)、GGGGSGGGGSGGGGS(SEQ ID NO:154) and the like. One skilled in the art can select an appropriate linker sequence for use in the present invention. In one embodiment, the antigen binding domain of the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and V L are separated by a linker sequence having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 154), which may be encoded by the nucleic acid sequence GGAGGCGGAGGATCT GGTGGTGGTGGATCTGGCGGCGGAGGCTCT (SEQ ID NO: 155).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Monoclonal antibodies have the advantage in addition to their specificity that they can be synthesized without being contaminated with other antibodies. The modifier "monoclonal" is not to be construed as requiring antibody production by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma method described first by Kohler et al, nature,256:495 (1975), or may be prepared in bacterial, eukaryotic, or plant cells using recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al, nature,352:624-8 (1991) and Marks et al, J.mol.biol.,222:581-97 (1991).

The term "hypervariable region", "HVR" or "HV" as used herein refers to a region of an antibody variable domain that is hypervariable in sequence and/or forms a structurally defined loop. Typically, an antibody comprises six hypervariable regions, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Many descriptions of hypervariable regions are in use and are encompassed herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are most commonly used (Kabat et al, sequences of Proteins of Immunological Interest, 5 th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, MD. (1991)). While Chothia refers to the position of the structural loop (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop (since the Kabat numbering scheme will insert at H35A and H35B; the loop ends at 32 if both 35A and 35B are absent; the loop ends at 33 if only 35A is present; the loop ends at 34 if both 35A and 35B are present). The AbM hypervariable region represents a tradeoff between Kabat CDRs and Chothia structural loops and is used by Oxford Molecular AbM antibody modeling software. The "contact" hypervariable region is based on analysis of available complex crystal structures. Residues from each of these hypervariable regions are shown below.

The hypervariable regions may comprise "extended hypervariable regions" of 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in VL, and 26-35B (H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al, supra.

"Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues defined herein.

The term "variable domain residue number as in Kabat" or "amino acid position number as in Kabat" and variations thereof refers to the numbering system of the heavy chain variable domain or the light chain variable domain used in antibody assembly, as in Kabat et al. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to or inserted into the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion following residue 52 of H2 (residue 52a according to Kabat) and an insertion residue following heavy chain FR residue 82 (e.g., residues 82a, 82b, 82c, etc. according to Kabat). For a given antibody, the Kabat numbering of residues may be determined by sequence alignment in the homologous region of the antibody sequence with the "standard" Kabat numbering.

When referring to residues in the variable domain (about residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., kabat et al, supra). When referring to residues in the immunoglobulin heavy chain constant region, the "EU numbering system" or "EU index" is generally used (e.g., kabat et al, EU index as reported above). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise indicated herein, reference to residue numbering in the variable domains of antibodies means that the residue numbering is by the Kabat numbering system.

A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. In one embodiment, an anti-PSMA antibody is provided, which is an antagonist antibody.

An antibody that "binds" an antigen or epitope of interest is an antibody that binds the antigen or epitope with sufficient affinity to measurably differ from non-specific interactions. Specific binding can be measured, for example, by determining binding of a molecule as compared to binding of a control molecule, which is typically a similarly structured molecule that does not have binding activity.

As used herein, the term "antigen" or "Ag" is defined as a molecule that causes an immune response. Such an immune response may involve antibody production, or activation of specific immune competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including proteins or peptides, may be used as an antigen.

The term "epitope" includes any protein, lipid or carbohydrate determinant capable of specific binding to an immunoglobulin or T cell receptor. Epitope determinants generally consist of active surface groupings of molecules such as amino acids, lipids or sugar side chains and generally have specific three dimensional structural characteristics as well as specific charge characteristics. Exemplary epitopes of certain anti-PSMA antigen-binding domains according to the invention are shown in fig. 18B.

As used herein, the term "specifically binds" refers to a receptor (which may include, but is not limited to, an antibody or antibody fragment) that recognizes a particular molecule/ligand, but does not substantially recognize or bind other molecules in a sample. For example, a receptor that specifically binds to a molecule from one species may also bind to the molecule from one or more other species. But this cross-species reactivity does not itself alter the specific classification. In another example, a receptor that specifically binds to a molecule may also bind to a different allelic form of the molecule. However, this cross-reactivity does not itself change the specific classification. In some cases, the term "specific binding" or "specific binding (SPECIFICALLY BINDING)" may be used to refer to the interaction of a protein (or peptide) with a second chemical substance, meaning that the interaction is dependent on the presence of a particular structure (e.g., an epitope or epitope) on the chemical substance, e.g., the receptor recognizes and binds to a particular structure rather than the protein in general. If the receptor is specific for epitope "A", then the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A bound to the receptor in a reaction containing labeled "A" and the receptor.

In embodiments, specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10 ^-8 M or less (e.g., a smaller KD indicates a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancerous conditions. "anti-tumor effects" can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention to initially prevent tumorigenesis.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid system malignancy. Cancers may include, but are not limited to, prostate cancer, lung cancer, liver cancer, pancreatic cancer, colon cancer, gastric cancer, breast cancer, ovarian cancer, renal cancer, prostate cancer, bladder cancer, melanoma, and glioma.

As used herein, the term "autologous" refers to any material derived from an individual that is subsequently reintroduced into the same individual.

As used herein, the term "allogenic" refers to materials derived from animals that are subsequently introduced into different animals of the same species.

As used herein, "modification" of an amino acid residue/position refers to a change in the primary amino acid sequence as compared to the starting amino acid sequence, wherein the change is caused by a sequence change involving the amino acid residue/position. For example, typical modifications include substitution of a residue (or at the position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more (typically less than 5 or 3) amino acids adjacent to the residue/position, and deletion of the residue/position. "amino acid substitution" or variations thereof refers to the replacement of an existing amino acid residue in a predetermined (starting) amino acid sequence with a different amino acid residue. In general, the modification results in an alteration of at least one physical biochemical activity of the variant polypeptide as compared to a polypeptide comprising the starting (or "wild-type") amino acid sequence. For example, in the case of antibodies, the altered physical biochemical activity may be binding affinity, binding capacity and/or binding effect to the target molecule.

The term "treating or preventing" a disease as used herein means reducing the frequency or severity of at least one sign or symptom of the disease or disorder to which the subject is subjected. In one example, a therapy (e.g., administration of a therapeutic agent of the present disclosure) treats a disease or condition by reducing one or more signs or symptoms associated with the disease or condition, e.g., as compared to a response in the absence of the therapy. For example, administration of a therapeutic agent may provide an anti-tumor effect that reduces one or more signs or symptoms associated with cancer. Treatment or prevention may refer to delaying the appearance of symptoms, reducing the severity of episodes, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, improving symptoms, reducing secondary infections, prolonging patient survival, preventing disease recurrence, reducing the number or frequency of recurrent episodes, increasing latency between episodes of symptoms, increasing the time to progression, accelerating remission, inducing remission, enhancing remission, accelerating recovery, or increasing the efficacy of or decreasing resistance to an alternative therapy. In one embodiment, "treatment" refers to therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or reduce a target pathological condition or disorder as described herein.

As used herein, the term "administering" means providing or administering one or more agents to a subject, such as an agent that treats one or more signs or symptoms associated with a condition/disorder or disease by any effective route, including but not limited to cancer (e.g., lymphoma), viral infection, bacterial infection, and the like. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes. Administration "in combination with one or more additional therapeutic agents" includes simultaneous (concurrent) administration and sequential administration in any order.

As used herein, the term "pharmaceutically acceptable" means that the material (including but not limited to salts, carriers, or diluents) does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material can be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences, by e.w. martin, mack Publishing co., easton, pa., 19 th edition (1995), describes compositions and formulations suitable for drug delivery of one or more agents, such as one or more modulators. Generally, the nature of the carrier will depend on the particular mode of administration employed. For example, a parenteral formulation may include injectable fluids including pharmaceutically and physiologically acceptable fluids (such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like) as vehicles. In addition to the bio-neutral carrier, the medicament to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives and pH buffering agents and the like, for example sodium acetate or sorbitol monolaurate, sodium lactate, potassium chloride, calcium chloride and triethanolamine oleate. For example, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and, as described herein, e.g., γδ T cells, preferably γδ T cells engineered to express a CAR against PSMA.

"Coding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules having defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences in biological processes and the biological properties resulting therefrom. Thus, if transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes the protein. The nucleotide sequence is identical to the mRNA sequence and both the coding strand, which is typically provided in the sequence listing, and the non-coding strand, which serves as a transcription template for a gene or cDNA, may be referred to as a protein or other product encoding the gene or cDNA.

"Isolated" means altered or removed from a natural state. For example, a nucleic acid or peptide that is naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from coexisting materials in its natural state is "isolated. The isolated nucleic acid or protein may be present in a substantially pure form or may be present in a non-natural environment (such as, for example, a host cell).

Unless otherwise indicated, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The nucleotide sequences encoding proteins and RNAs may comprise introns.

The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal that is amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

An "expression cassette" refers to a nucleic acid comprising an expression control sequence operably linked to a nucleic acid encoding a transcript or polypeptide to be expressed. The expression cassette contains sufficient cis-acting expression elements and other expression elements may be provided by the host cell or in an in vitro expression system. The expression cassette may be a component of a vector such as a cosmid, a plasmid (e.g., naked or contained in a liposome) or a virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus). The expression cassette may be in a host cell (such as γδ T cells).

Compositions and methods of the invention

A. anti-PSMA antibodies

In one embodiment, the invention provides anti-PSMA antibodies that can be used herein as therapeutic agents. Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies.

1. Polyclonal antibodies

Polyclonal antibodies may be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to conjugate the relevant antigen (especially when using synthetic peptides) with a protein that is immunogenic in the species to be immunized. For example, antigens may be conjugated to Keyhole Limpet Hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, such as maleimide benzoyl sulfosuccinimide ester (conjugated via a cysteine residue), N-hydroxysuccinimide (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, SOCl2, or R' N ═ C ═ NR, where R and R1 are different alkyl groups.

Animals are immunized against antigen, immunogenic conjugate or derivative by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbit or mouse, respectively) with 3 volumes of freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals were boosted by subcutaneous injections at multiple sites with 1/5 to 1/10 of the original amount of peptide or conjugate in Freund's complete adjuvant. Seven to 14 days later, animals were bled and serum antibody titers were determined. Animals were boosted until the titer reached a stable level. Conjugates can also be prepared as protein fusions in recombinant cell cultures. In addition, aggregating agents such as alum are suitably used to enhance the immune response.

2. Monoclonal antibodies

Monoclonal antibodies (mabs) to the antigen of interest can be prepared by employing any technique known in the art. These techniques include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975,Nature 256,495-497), the human B cell hybridoma technique (Kozbor et al, 1983,Immunology Today 4:72), and the EBV-hybridoma technique (Cole et al, 1985,Monoclonal Antibodies and Cancer Therapy,Alan R.Liss,Inc., pages 77-96). Selective Lymphocyte Antibody Methods (SLAM) (Babcook, J.S. et al ,A novel strategy for generating monoclonal antibodies from single,isolated lymphocytes producing antibodies of defined specificities.Proc Natl Acad Sci U S A,1996.93(15): pages 7843-8.) and (Mclean G et al 2005,J Immunol.174 (8): 4768-78. Such antibodies can fall into any immunoglobulin class, including IgG, igM, igE, igA and IgD and any subclass thereof. Hybridomas producing mAbs useful in the invention can be cultured in vitro or in vivo.

Monoclonal antibodies can be prepared using the hybridoma method described for the first time by Kohler et al, nature,256:495 (1975), or can be prepared by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other suitable host animal such as hamster is immunized as described above to obtain lymphocytes producing or capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Following immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding, monoclonal Antibodies: PRINCIPLES AND PRACTICE, pages 59-103 (ACADEMIC PRESS, 1986)).

The hybridoma cells so prepared are inoculated into a suitable medium and grown, which may contain one or more substances that inhibit the growth or survival of the unfused parent myeloma cells (also referred to as fusion partners). For example, if the parent myeloma cell lacks the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective medium for the hybridoma will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high levels of antibody production by selected antibody-producing cells, and are sensitive to selective media selected for unfused parent cells. Preferred myeloma cell lines are murine myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors, which are available from Salk Institute Cell Distribution Center, san Diego, calif. USA, and SP-2 and derivatives, such as X63-Ag8-653 cells available from AMERICAN TYPE Culture Collection, manassas, va., USA. Human myeloma and mouse-human heterologous myeloma cell lines are also described for the production of human monoclonal antibodies (Kozbor, J.Immunol.,133:3001 (1984)), and Brodeur et al, monoclonal Antibody Production Techniques and Applications, pages 51-63 (MARCEL DEKKER, inc., new York, 1987)).

The production of monoclonal antibodies directed against the antigen in the medium in which the hybridoma cells are grown is determined. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as a Radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).

The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis as described in Munson et al, anal biochem.107:220 (1980).

Once hybridoma cells producing antibodies of the desired specificity, affinity and/or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, monoclonal Antibodies: PRINCIPLES AND PRACTICE, pages 59-103 (ACADEMIC PRESS, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. Furthermore, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by intraperitoneal injection of the cells into mice.

Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as affinity chromatography (e.g., using protein a or protein G-sepharose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or the like.

DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells are used as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell, such as an e.coli cell, simian COS cell, chinese Hamster Ovary (CHO) cell, or myeloma cell that does not otherwise produce antibody protein, to obtain synthesis of monoclonal antibodies in the recombinant host cell. A review article on recombinant expression of DNA encoding antibodies in bacteria includes Skerra et al, curr. Opinion in Immunol.5:256-62 (1993) and Pluckthun, immunol. Rev.130:151-88 (1992).

In another embodiment, monoclonal antibodies or antibody fragments can be isolated from an antibody phage library generated using the techniques described in McCafferty et al, nature,348:552-54 (1990). Clackson et al, nature,352:624-28 (1991) and Marks et al, J.mol.biol.,222:581-97 (1991), describe the use of phage libraries to isolate murine and human antibodies, respectively. The subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al, bio/Technology,10:779-83 (1992)) and combined infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al, nuc. Acids. Res.21:2265-6 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

The DNA encoding the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example by substituting human heavy and light chain constant domains (CH and CO sequences for homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison et al, proc. Natl. Acad. Sci. USA,81:6851 (1984)), or by fusing an immunoglobulin coding sequence to all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide).

3. Chimeric, humanized and human antibodies

In embodiments, the anti-PSMA antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567, and Morrison et al, proc.Natl. Acad.Sci.USA,81:6851-5 (1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another example, the chimeric antibody is a "class switch (CLASS SWITCHED)" antibody, in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Typically, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody will optionally further comprise at least a portion of a human constant region. In embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., an antibody from which CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

The anti-PSMA antibodies of the invention may comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., fv, fab, fab ', F (ab') 2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases, fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues found in neither the recipient antibody nor the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al Nature,321:522-5 (1986); riechmann et al Nature,332:323-9 (1988); and Presta, curr.Op. Struct. Biol.,2:593-6 (1992)).

The humanized antibodies of the invention may comprise one or more human and/or human consensus non-hypervariable region (e.g., framework) sequences in their heavy and/or light chain variable domains. In embodiments, one or more additional modifications are present within the human and/or human consensus non-hypervariable region sequence. In one embodiment, the heavy chain variable domain of an antibody of the invention comprises a human consensus framework sequence, which in one embodiment is a subgroup III consensus framework sequence. In one embodiment, the antibodies of the invention comprise variant subgroup III consensus framework sequence modified at least one amino acid position.

As known in the art, the amino acid positions/boundaries that define the antibody hypervariable regions may vary, depending on the context and various definitions known in the art. Some positions within the variable domain may be considered hybrid hypervariable positions, as these positions may be considered to be within the hypervariable region under one set of criteria and outside the hypervariable region under another set of criteria. One or more of these locations may also be found in extended hypervariable regions (as further defined below). The invention provides antibodies comprising modifications at these hybridization hypervariable positions. In one embodiment, these hypervariable positions include one or more positions 26-30, 33-35B, 47-49, 57-65, 93, 94, and 101-102 in the heavy chain variable domain. In one embodiment, these hybridization hypervariable positions include one or more of positions 24-29, 35-36, 46-49, 56, and 97 in the light chain variable domain. In one embodiment, the antibodies of the invention comprise human variant human subgroup consensus framework sequence modified at one or more hybrid hypervariable positions.

The antibodies of the invention may comprise any suitable human or human consensus light chain framework sequence, so long as the antibodies exhibit the desired biological properties (e.g., the desired binding affinity). In one embodiment, the antibody of the invention comprises at least a portion (or all) of the framework sequence of a human kappa light chain. In one embodiment, an antibody of the invention comprises at least a portion (or all) of a human kappa subgroup I framework consensus sequence.

Methods for humanizing non-human antibodies are well known in the art. As discussed, humanized antibodies typically have one or more amino acid residues introduced into them from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed by substituting rodent CDR or CDR sequences for the corresponding sequences of human antibodies according to the method of Winter and colleagues (Jones et al, nature,321:522-525 (1986); riechmann et al, nature,332:323-327 (1988); verhoeyen et al, science,239:1534-1536 (1988)). Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than the complete human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from similar sites in rodent antibodies.

When the antibody is intended for human therapeutic use, the choice of human variable domains (both light and heavy chains) to be used in the preparation of the humanized antibody is important for reducing antigenicity and HAMA response (human anti-mouse antibodies). Reduction or elimination of HAMA response is an important aspect of the clinical development of suitable therapeutic agents (see, e.g., khaxzaeli et al, j. Natl. Cancer Inst (1988), 80:937; jaffers et al, transduction (1986), 41:572; shawler et al, j. Immunol (1985), 135:1530; search et al, j. Biol. Response mod (1984), 3:138; miller et al Blood (1983), 62:988; hakimi et al, j. Immunol (1991), 147:1352; reichmann et al, nature (1988), 332:323; junghans et al, cancer res (1990), 50:1495). As described herein, the present invention provides humanized antibodies such that HAMA reactions are reduced or eliminated. Variants of these antibodies may be further obtained using conventional methods known in the art, some of which are described further below. The sequence of the variable domain of a rodent antibody is screened against an entire library of known human variable domain sequences according to the so-called "best fit" method. The human V domain sequence closest to rodents was confirmed and the human Framework Region (FR) therein was accepted for humanized antibodies (Sims et al, J.Immunol.151:2296 (1993); chothia et al, J.mol. Biol.,196:901 (1987)). Another approach uses specific framework regions derived from the consensus sequences of all human antibodies of a specific light chain or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al, proc. Natl. Acad. Sci. USA,89:4285 (1992); presta et al, J. Immunol.151:2623 (1993)).

For example, amino acid sequences from antibodies as described herein can be used as diverse starting (parent) sequences for framework and/or hypervariable sequences. The selected framework sequence to which the initial hypervariable sequence is linked is referred to herein as the recipient human framework. Although the recipient human framework may be derived or derived from a human immunoglobulin (VL and/or VH regions thereof), it is preferred that the recipient human framework is derived or derived from a human consensus framework sequence, as such frameworks have been demonstrated to have minimal or no immunogenicity in human patients.

In the case where the recipient is derived from a human immunoglobulin, a human framework sequence may optionally be selected, which is selected based on its homology to the donor framework sequence by aligning the donor framework sequence with various human framework sequences in the collection of human framework sequences, and selecting the most homologous framework sequence as the recipient.

In one embodiment, the human consensus framework herein is derived or derived from VH subgroup III and/or VL kappa subgroup I consensus framework sequences.

While the receptor may be identical in sequence to the selected human framework sequence, whether it is from a human immunoglobulin or a human consensus framework, the present invention contemplates that the receptor sequence may comprise pre-existing amino acid substitutions relative to the human immunoglobulin sequence or the human consensus framework sequence. These pre-existing substitutions are preferably minimal, typically only four, three, two or one amino acid differences relative to the human immunoglobulin sequence or the consensus framework sequence.

Hypervariable region residues of non-human antibodies are incorporated into the VL and/or VH acceptor human framework. For example, residues corresponding to Kabat CDR residues, chothia hypervariable loop residues, abm residues, and/or contact residues may be incorporated. Optionally, extended hypervariable region residues of 24-34 (L1), 50-56 (L2) and 89-97 (L3), 26-35B (H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) are incorporated.

Although "incorporating" hypervariable region residues is discussed herein, it will be appreciated that this can be accomplished in a variety of ways, for example, by mutating a nucleic acid encoding a mouse variable domain sequence to produce a nucleic acid encoding a desired amino acid sequence such that its framework residues are changed to acceptor human framework residues, or by mutating a nucleic acid encoding a human variable domain sequence such that the hypervariable domain residues are changed to non-human residues, or by synthesizing a nucleic acid encoding a desired sequence, and the like.

As described herein, hypervariable region graft variants can be generated by Kunkel mutagenesis of a nucleic acid encoding a human receptor sequence using separate oligonucleotides for each hypervariable region. Kunkel et al, methods enzymol.154:367-382 (1987). Appropriate changes may be introduced within the framework and/or hypervariable regions using conventional techniques to correct and reconstruct the appropriate hypervariable region-antigen interactions.

Phage (particle) display (also referred to herein in some cases as phage display) can be used as a convenient and rapid method of generating and screening a number of different potential variant antibodies in libraries generated by sequence randomization. However, methods of making and screening for altered antibodies are available to the skilled artisan.

Phage (particle) display technology provides a powerful tool for the generation and selection of novel proteins that bind to ligands such as antigens. Display techniques employing phage (particles) allow the generation of large libraries that can rapidly sort protein variants of those sequences that bind to target molecules with high affinity. Nucleic acids encoding variant polypeptides are typically fused to nucleic acid sequences encoding viral coat proteins such as gene III proteins or gene VIII proteins. Monovalent phagemid display systems have been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused to a nucleic acid sequence encoding a portion of a gene III protein. (Bass, S., proteins,8:309 (1990); lowman and Wells, methods: A Companion to Methods in Enzymology,3:205 (1991)). In monovalent phagemid display systems, the gene fusion is expressed at low levels and the wild-type gene III protein is also expressed, so that the infectivity of the particles is preserved. Methods of generating peptide libraries and screening these libraries have been disclosed in a number of patents (e.g., U.S. Pat. No. 5,723,286, U.S. Pat. No. 5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908, and U.S. Pat. No. 5,498,530).

Libraries of antibodies or antigen binding polypeptides have been prepared in a variety of ways, including by altering individual genes by insertion of random DNA sequences or by cloning related gene families. Methods for displaying antibodies or antigen binding fragments using phage (particle) display have been described in U.S. Pat. nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727. Libraries expressing antibodies or antigen binding proteins with the desired properties are then screened.

Methods for substituting selected amino acids into a template nucleic acid are well established in the art, some of which are described herein. For example, methods for introducing modifications into nucleic acid sequences can include the use of various commercially available kits (e.g., quickChange site-directed mutagenesis kit, agilent, SANTA CLARA, CA). As another example, hypervariable region residues may be replaced using the Kunkel method (e.g., kunkel et al, methods enzymol.154:367-382 (1987)).

It is important that antibodies be humanized, retaining high binding affinity for antigens and other favorable biological properties. To achieve this objective, humanized antibodies are prepared according to a preferred method by a process of analyzing a parent sequence and various conceptual humanized products using a three-dimensional model of the parent sequence and humanized sequence. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e. analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected from the receptor and the introduced sequence and combined to obtain the desired antibody properties, such as achieving increased affinity for the target antigen. In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Various forms of humanized anti-PSMA antibodies are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

As an alternative to humanization, human antibodies may be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable of producing a complete human antibody repertoire without endogenous immunoglobulin production after immunization. For example, homozygous deletion of the antibody heavy chain Junction (JH) gene in chimeric and germ-line mutant mice has been described as resulting in complete inhibition of endogenous antibody production. Transfer of an array of human germline immunoglobulin genes into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., jakobovits et al, proc. Natl. Acad. Sci. USA,90:2551 (1993); jakobovits et al, nature,362:255-8 (1993); bruggemann et al, year in Immuno.7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807; and WO 97/17852).

Alternatively, phage display technology (McCafferty et al, nature 348:552-53 (1990)) can be used to generate human antibodies and antibody fragments in vitro from a pool of immunoglobulin variable (V) domain genes from a non-immunized donor. According to this technique, the antibody V domain gene is cloned in-frame into the major or minor coat protein gene of a filamentous phage (e.g., M13 or fd) and displayed as a functional antibody fragment on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of genes encoding antibodies exhibiting these properties. Thus, phages mimic some of the characteristics of B cells. Phage display can be performed in a variety of formats, for example, as reviewed in Johnson, kevin S and Chiswell, david J., current Opinion in Structural Biology3:564-571 (1993). Several sources of V gene segments are available for phage display. Clackson et al, nature,352:624-628 (1991) isolated a variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleen of immunized mice. V gene libraries from non-immunized human donors can be constructed and antibodies to a variety of antigens, including autoantigens, can be isolated essentially according to the techniques described by Marks et al, J.mol. Biol.222:581-97 (1991) or Griffith et al, EMBO J.12:725-34 (1993) (see also U.S. Pat. Nos. 5,565,332 and 5,573,905).

Human antibodies can also be produced by in vitro activated B cells (see, e.g., U.S. Pat. nos. 5,567,610 and 5,229,275).

Thus, in embodiments, transgenic or transchromosomal mice carrying a portion of the human immune system, rather than the mouse system, may be used to generate such human monoclonal antibodies directed against PSMA.

HuMAb Mouse ^TM (Medarex, inc.) contains human immunoglobulin gene miniloci encoding unrearranged human heavy (μ and γ) and kappa light chain immunoglobulin sequences, and targeting mutations that inactivate endogenous μ and kappa chain loci (see, e.g., lonberg et al, (1994) Nature 368 (6474): 856-9). Thus, mice exhibit reduced expression of mouse IgM or kappa, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high affinity human IgG kappa monoclonal antibodies (Lonberg, N.et al (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; lonberg, N.and Huszar, D. (1995) International. Rev. Immunol.13:65-93; and Harding, F. And Lonberg, N. (1995) Ann.N.Y. Acad. Sci.764:536-46). Preparation and use of HuMAb Mouse ^TM, and the modifications carried by such mice, are further described in Taylor, L.et al (1992) Nucleic ACIDS RESEARCH20:6287-6295; chen, J.et al (1993) International Immunology 5:647-656; tuaillon et al (1993) Proc.Natl. Acad.Sci.USA 90:3720-4; choi et al (1993) Nature Genetics 4:117-23; chen, J.et al (1993) EMBO J.12:21-830; tuaillon et al, (1994) J.immunol.152:2912-20; taylor, L.et al (1994) International Immunology 6:579-91; and Fishwild, D.et al (1996) Nature Biotechnology 14:845-51), and the genome carried by such mice, all of which are expressly incorporated by reference in their entirety. See also, U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429, U.S. Pat. Nos. 5,545,807, PCT publications WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, and PCT publication WO 01/14424.

In another embodiment, a Mouse called "KM Mouse ^TM" carrying human immunoglobulin sequences on transgenes and transchromosomes may be used to produce the human antibodies of the present disclosure, as described in detail in PCT publication WO 02/43478.

In another embodiment, an alternative transgenic system known as Xenomouse (Abgenix, inc.) may be used, such mice are described, for example, in U.S. Pat. nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963.

Additional related transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to generate anti-PSMA antibodies of the present disclosure. For example, mice carrying both human heavy chain and human light chain transchromosomes, referred to as "TC mice", can be used, and such mice are described in Tomizuka et al (2000) Proc. Natl. Acad. Sci. USA 97:722-7. As another example, cows carrying human heavy and light chain transchromosomes have been described in the art (e.g., kuroiwa et al (2002) Nature Biotechnology 20:889-94 and PCT application No. WO 2002/092812) and are useful in producing anti-PSMA antibodies of the present disclosure. Other examples of transgenic animals that can be used to produce anti-PSMA antibodies include OmniRat ^TM and OmniMouse ^TM (see, e.g., osborn m.et al (2013) Journal of Immunology 190:1481-90; ma b.et al (2013) Journal of Immunological Methods-401:78-86; references a. Et al (2009) Science325:433, U.S. patent No. 8,907,157; european patent No. 2152880B 1; european patent No. 2336329B 1). Yet another example includes the use ofTechniques (see, e.g., U.S. Pat. nos. 6,596,541, regeneron Pharmaceuticals,). In short, the process is carried out,The technology relates to producing a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces antigen binding proteins, e.g., antibodies, comprising human variable regions and mouse constant regions in response to antigen stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

4. Antibody fragments

Embodiments of the present disclosure encompass antibody fragments.

Various techniques for producing antibody fragments have been developed. Traditionally, these fragments have been obtained via proteolytic digestion of the intact antibody (see, e.g., morimoto et al, journal of Biochemical and Biophysical Methods 24:107-7 (1992); and Brennan et al, science,229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, fv and scFv antibody fragments can all be expressed in and secreted from e.coli, thus allowing for easy production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab') 2 fragments (Carter et al, bio/Technology10:163-7 (1992)). According to another method, the F (ab') 2 fragment may be isolated directly from the recombinant host cell culture. Fab and F (ab') 2 fragments with increased in vivo half-life are described in U.S. Pat. No. 5,869,046, which contain salvage receptor binding epitope residues. Other techniques for generating antibody fragments will be apparent to the skilled artisan. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) (see WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458). Fv and sFv are the only species having a complete binding site lacking a constant region and therefore they are suitable for reduced non-specific binding during in vivo use. The sFv fusion proteins can be constructed to produce fusion of effector proteins at the amino or carboxy terminus of sFv (see Antibody Engineering, borreback et al, supra). An antibody fragment may also be a "linear antibody", for example, as described in U.S. Pat. No. 5,641,870.

In one embodiment, an anti-PSMA antibody-derived scFv is used in a CAR of the present disclosure. Included among the anti-PSMA antibody fragments are portions of anti-PSMA antibodies (and combinations of portions of anti-PSMA antibodies, e.g., scFv) that can be used as targeting arms against PSMA epitopes in one or more CAR-modified immune cells of the disclosure. Such fragments are not necessarily proteolytic fragments, but rather portions of the polypeptide sequence that can confer affinity to the target.

5. Multispecific antibodies

In any aspect of the disclosure, the anti-PSMA antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of PSMA proteins as described herein. Other such antibodies may combine a PSMA binding site with a binding site of another protein. In some examples, the anti-PSMA arm may be combined with an arm that binds to a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD 3) or Fc receptor of IgG (fcγr), such as fcγri (CD 64), fcγrii (CD 32), and fcγriii (CD 16), in order to concentrate and localize the cellular defense mechanisms to PSMA expressing cells. Bispecific antibodies may also be used to localize cytotoxic agents to PSMA-expressing cells. These antibodies have PSMA binding arms and arms that bind to a cytotoxic agent (e.g., saporin, anti-interferon-alpha, vinca alkaloid, ricin a chain, methotrexate, or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab') 2 bispecific antibodies).

Methods for preparing bispecific antibodies are known in the art. Traditional methods of producing full length bispecific antibodies are based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al Nature 305:537-9 (1983)). Due to the random classification of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, which is usually accomplished by an affinity chromatography step, is quite cumbersome and the product yield is low. A similar procedure is disclosed in WO 93/08829 and Traunecker et al, EMBO J.10:3655-3659 (1991).

Other methods of preparing bispecific antibodies are known. One method is a "pestle into mortar" or "protrusion into cavity" method (see, e.g., U.S. Pat. No. 5,731,168). In this method, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each comprise an interface. The interface of one immunoglobulin polypeptide interacts with a corresponding interface on another immunoglobulin polypeptide, thereby associating the two immunoglobulin polypeptides. These interfaces may be engineered such that a "knob" or "protrusion" (these terms are used interchangeably herein) located in the interface of one immunoglobulin polypeptide corresponds to a "socket" or "cavity" (these terms are used interchangeably herein) located in the interface of another immunoglobulin polypeptide. In embodiments, the socket is the same or similar in size as the pestle and is suitably positioned such that when the two interfaces interact, the pestle of one interface can be positioned in the corresponding hole of the other interface. Without being bound by theory, it is believed that this may stabilize the heteromultimer and facilitate the formation of the heteromultimer relative to other substances (e.g., homomultimers). In embodiments, such methods can be used to promote heteromultimerization of two different immunoglobulin polypeptides, thereby producing bispecific antibodies comprising two immunoglobulin polypeptides having binding specificities for different epitopes.

According to various methods, an antibody variable domain (antibody-antigen combining site) with the desired binding specificity is fused to an immunoglobulin constant domain sequence. Fusion is preferably with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Typically, the first heavy chain constant region (CH 1) contains the sites necessary for light chain binding and is present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into a separate expression vector and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in an embodiment when using unequal ratios of the three polypeptide chains in the construction provides the best yield. However, when expressing at least two polypeptide chains in equal ratios yields high yields or when the ratios are of no special significance, the coding sequences of two or all three polypeptide chains may be inserted into one expression vector.

In one embodiment of this method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It has been found that this asymmetric structure facilitates the combined separation of the desired bispecific compound from the undesired immunoglobulin chains, since the presence of only half of the immunoglobulin light chains in the bispecific molecule provides a simple separation method. This method is disclosed in WO 94/04690. For more details on the generation of bispecific antibodies, see, e.g., suresh et al Methods in Enzymology,121:210 (1986).

According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell cultures. One interface comprises at least a portion of the C _H domain of the antibody constant domain. In this approach, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine), a compensating "cavity" is created at the interface of the second antibody molecule that is the same or similar to the size of the large side chain. This provides a mechanism to increase the yield of heterodimers relative to other unwanted end products, such as homodimers.

Bispecific antibodies include cross-linked or "heteroconjugate (heteroconjugate)" antibodies. For example, one of the antibodies in the heteroconjugate may be coupled to avidin and the other antibody may be coupled to biotin. For example, such antibodies have been proposed for targeting immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treating HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Any convenient crosslinking method may be used to prepare the heteroconjugate antibodies. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, a number of crosslinking techniques being disclosed.

Techniques for producing bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al, science,229:81 (1985) describe a procedure in which whole antibodies are proteolytically cleaved to yield the F (ab') ₂ fragment. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the ortho dithiol and prevent intermolecular disulfide formation. The resulting Fab' fragment is then converted to a Thionitrobenzoate (TNB) derivative. One of the Fab ' -TNB derivatives is then reconverted to Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form a bispecific antibody. The bispecific antibodies produced can be used as selective immobilization agents for enzymes. The production of fully humanized bispecific antibody F (ab') ₂ molecules is described by Shalaby et al, J.Exp.Med.,175:217-225 (1992). Each Fab' fragment was secreted separately from escherichia coli (e.coli) and underwent directed chemical coupling in vitro to form bispecific antibodies.

Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture are also described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al, J.Immunol.,148 (5): 1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers are reduced at the hinge region to form monomers, which are then reoxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technique described by Hollinger et al, proc.Natl.Acad.Sci.USA,90:6444-6448 (1993) provides an alternative mechanism for the preparation of bispecific antibody fragments. The fragment comprises a heavy chain variable domain (V _H) linked to a light chain variable domain (V _L) by a linker that is too short to pair two domains on the same chain. Thus, the V _H and V _L domains of one fragment are forced to pair with the complementary V _L and V _H domains of the other fragment, thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments using single chain Fv (sFv) dimers has also been reported. See Gruber et al, J.Immunol,152:5368 (1994).

Another technique for preparing bispecific antibody fragments is "bispecific T cell conjugates" orMethods (see, e.g., WO2004/106381, WO2005/061547, WO2007/042261, and WO 2008/119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain comprises two single chain Fv (scFv) fragments, each having a variable heavy (V _H) and a variable light (V _L) domain separated by a polypeptide linker of sufficient length to enable intramolecular association between the two domains. The single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope and these epitopes may be specific for different cell types such that when each scFv binds to its cognate epitope, cells of two different cell types come close or tether together. One particular embodiment of this method comprises linking an scFv that recognizes a cell surface antigen expressed by an immune cell (e.g., a CD3 polypeptide on a T cell) to another scFv that recognizes a cell surface antigen expressed by a target cell (such as a malignant cell or a tumor cell).

Since it is a single polypeptide, the bispecific T cell conjugate can be expressed using any prokaryotic or eukaryotic cell expression system known in the art (e.g., CHO cell line). However, specific purification techniques (see, e.g., EP 1691833) may be required to separate monomeric bispecific T cell conjugates from other multimeric species that may have biological activity in addition to the expected activity of the monomer. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, followed by elution of the polypeptide with an imidazole concentration gradient. This eluate was further purified using anion exchange chromatography and the polypeptide was eluted using a sodium chloride concentration gradient. Finally, the eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species.

Other related bispecific antibody fragment formats include, but are not limited to, dual affinity re-targeting proteins (DART) and tandem diabodies (TandAbs). DART consists of two Fv fragments which when heterodimerized form two unique antigen binding sites (Holliger et al, proc. Natl. Acad. Sci. USA.90:6444-6448 (1993). Specifically Fv1 consists of VH from antibody "A" and VL from antibody "B", whereas Fv2 consists of VH from antibody "B" and VL from antibody "A", unlike BiTE antibodies linked by polypeptide linkers, this combination allows DART to mimic natural interactions within an IgG molecule. The addition of another cysteine residue at the end of each heavy chain can increase stability by forming a C-terminal disulfide bridge. TandAbs is a tetravalent bispecific antibody, providing two binding sites for each antigen to maintain the affinity of a natural bivalent antibody. Furthermore, the molecular weight of TandAbs exceeds the first pass renal clearance threshold (about 105 kDa), thus allowing for longer half-life compared to smaller antibody constructs (human antibodies, such as used in, and light antibodies, 35:35; 4:35; standard antibodies, such as well as human antibodies, 35:58; and 5:29.58; standard antibodies, and so on, human antibodies, such as well as human antibodies, whereby standard antibodies, such as human antibodies, comprise human antibodies, 35:2016.58:2016.58, and so on).

6. Antibody variants and modifications

A) Substitution, insertion and deletion variants

In addition to the anti-PSMA antibodies described herein, it is contemplated that anti-PSMA antibody variants may be prepared. anti-PSMA antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesizing the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processing of the anti-PSMA antibody, such as altering the number or position of glycosylation sites, or altering membrane anchoring characteristics.

The variation of the anti-PSMA antibodies described herein may be performed, for example, using any of the techniques and guidelines for conservative and non-conservative mutations, such as set forth in U.S. patent No. 5,364,934. A variant may be a substitution, deletion, or insertion of one or more codons encoding an antibody or polypeptide that results in a change in the amino acid sequence as compared to the native sequence antibody or polypeptide. Optionally, the variation is by substitution of at least one amino acid with any other amino acid in one or more domains of the anti-PSMA antibody. By comparing the sequence of an anti-PSMA antibody to the sequence of a homologous known protein molecule, and minimizing the number of amino acid sequence changes made in the highly homologous regions, guidance can be established to determine which amino acid residues can be inserted, substituted or deleted without adversely affecting the desired activity. Amino acid substitutions may be the result of substitution of one amino acid with another amino acid of similar structure and/or chemical nature, such as substitution of serine for leucine, i.e., a conservative amino acid substitution. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The allowable variation can be determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting variants for activity exhibited by full length or mature natural sequences.

Provided herein are anti-PSMA antibody fragments. For example, such fragments may be truncated at the N-or C-terminus, or may lack internal residues, when compared to full length natural antibodies or proteins. Certain fragments lack amino acid residues that are not necessary for the desired biological activity of the anti-PSMA antibody.

Anti-PSMA antibody fragments may be prepared by any of a variety of conventional techniques. The desired peptide fragment can be chemically synthesized. Alternative methods involve the production of antibodies or polypeptide fragments by enzymatic digestion, for example by treating the protein with enzymes known to cleave the protein at sites defined by specific amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying DNA fragments encoding the desired antibodies or polypeptide fragments by Polymerase Chain Reaction (PCR). Oligonucleotides defining the desired ends of the DNA fragments are used at the 5 'and 3' primers in PCR. Preferably, the anti-PSMA antibody fragment shares at least one biological and/or immunological activity with the natural anti-PSMA antibodies disclosed herein.

In particular embodiments, conservative substitutions of interest are shown under the heading of the preferred substitutions in table 1. If such substitutions result in a change in biological activity, then more substantial changes are introduced, i.e., the exemplary substitutions named in Table 1, or as further described below with reference to amino acid groups, and the products are screened.

TABLE 1

Substantial modification of the functional or immunological properties of an anti-PSMA antibody is achieved by selection of substitutions that differ significantly in their effect on maintaining the structure of the polypeptide backbone in (a) the substitution region, e.g., folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chain. Naturally occurring residues fall into several groups according to common side chain characteristics:

(1) Hydrophobic norleucine, met, ala, val, leu, ile;

(2) Cys, ser, thr, neutral hydrophilic;

(3) Acid, asp and glu;

(4) Alkaline asn, gln, his, lys, arg;

(5) Residues influencing the orientation of the chain, gly, pro, and

(6) Aromatic, trp, tyr, phe.

Non-conservative substitutions will require replacement of one member of one of these classes with another class. Such substituted residues may also be introduced at conserved substitution sites, or more preferably, at the remaining (non-conserved) sites.

Variations can be generated using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al, nucleic acids Res.,13:4331 (1986); zoller et al, nucleic acids Res.,10:6487 (1987)), cassette mutagenesis (Wells et al, gene,34:315 (1985)), restriction-selective mutagenesis (Wells et al, phols. Trans. R. Soc. London serA,317:415 (1986)), or other known techniques can be performed on cloned DNA to generate anti-PSMA antibody variant DNA.

Scanned amino acid analysis may also be used to identify one or more amino acids along a continuous sequence. Among the preferred amino acids to scan are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is generally the preferred scanning amino acid in this group because it eliminates side chains other than the beta-carbon and is less likely to alter the backbone conformation of the variant (Cunningham and Wells, science,244:1081-5 (1989)). Alanine is also generally preferred because it is the most common amino acid. In addition, it is often found in buried and exposed locations (Cright on, the Proteins, (W.H. Freeman & Co., N.Y.); chothia, J.mol. Biol.,150:1 (1976)). If alanine substitutions do not result in a sufficient amount of variants, homoleptic amino acids can be used.

Any cysteine residue that does not participate in maintaining the correct conformation of the anti-PSMA antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Instead, cysteine bonds may be added to the anti-PSMA antibody to increase its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

A particularly preferred type of substitution variant involves substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variants selected for further development have improved biological properties relative to the parent antibody from which they were derived. A convenient method of producing such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants so produced are displayed in a monovalent manner from the filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. As disclosed herein, phage-displayed variants are then screened for biological activity (e.g., binding affinity). To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to confirm the point of contact between the antibody and the PSMA polypeptide. Such contact residues and neighboring residues are candidates for substitution according to the techniques detailed herein. Once such variants are produced, the set of variants is subjected to screening as described herein, and antibodies having excellent properties in one or more relevant assays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of anti-PSMA antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of variant or non-variant versions of the anti-PSMA antibodies produced earlier.

B) Modification

Covalent modifications of anti-PSMA antibodies are included within the scope of the invention. One type of covalent modification involves reacting targeted amino acid residues of an anti-PSMA antibody with an organic derivatizing agent capable of reacting with selected side chains or N-terminal or C-terminal residues of the anti-PSMA antibody. Derivatization with bifunctional agents is useful, for example, for cross-linking an anti-PSMA antibody to a water-insoluble support matrix or surface, for use in a method of purifying an anti-PSMA antibody, and vice versa. Commonly used cross-linking agents include, for example, 1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid), homobifunctional imidoesters (including disuccinimide esters such as 3,3' -dithiobis (succinimidyl propionate)), bifunctional maleimides such as bis-N-maleimido-1, 8-octane, and agents such as methyl-3- [ (p-azidophenyl) dithio ] propionyl imidoester.

Other modifications include deamidation of glutamyl and asparagine residues to the corresponding glutamyl and asparagine residues, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of the seryl or threonyl residues, methylation of the alpha-amino groups of the lysine, arginine and histidine side chains, respectively (t.e. Cright on, proteins: structure and Molecular Properties, w.h. freeman & co., san Francisco, pages 79-86 (1983)), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

Another type of covalent modification of anti-PSMA antibodies included within the scope of the invention includes altering the native glycosylation pattern of the antibody or polypeptide. For purposes herein, "altering the native glycosylation pattern" means deleting one or more carbohydrate moieties present in the native sequence anti-PSMA antibody (by removing potential glycosylation sites or deleting glycosylation by chemical and/or enzymatic means) and/or adding one or more glycosylation sites not present in the native sequence anti-PSMA antibody. Furthermore, the phrase includes qualitative changes in glycosylation of the native protein, involving changes in the nature and proportion of the various carbohydrate moieties present.

Glycosylation of antibodies and other polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences used to enzymatically link carbohydrate moieties to asparagine side chains. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to anti-PSMA antibodies is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). Alterations (to the O-linked glycosylation site) may also be made by adding one or more serine or threonine residues to the sequence of the original anti-PSMA antibody or by substitution of the sequence of the original anti-PSMA antibody with one or more serine or threonine residues. The anti-PSMA antibody amino acid sequence may optionally be altered by a change at the DNA level, particularly by mutating the DNA encoding the anti-PSMA antibody at preselected bases, such that codons are generated that will translate to the desired amino acids.

Another method of increasing the number of carbohydrate moieties on an anti-PSMA antibody is by chemical or enzymatic coupling of a glycoside to a polypeptide. Such methods are described in the art, for example in WO 87/05330 published on 9, 11, 1987 and in CRC crit.Rev.biochem, pages 259-306 (1981).

Removal of the carbohydrate moiety present on the anti-PSMA antibody may be accomplished chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that serve as glycosylation targets. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin et al, arch. Biochem. Biophysics, 259:52 (1987) and by Edge et al, anal. Biochem, 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be accomplished by using a variety of endo-and exoglycosidases, as described in Thotakura et al, meth. Enzymol.,138:350 (1987).

C) Variant Fc region

It may be desirable to modify the antibodies of the invention in terms of effector function, for example, in order to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of the antibodies. This can be accomplished by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine residues may be introduced in the Fc region, allowing for inter-chain disulfide bond formation in this region. Homodimeric antibodies so produced may have improved internalization ability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al, J.exp Med.176:1191-5 (1992); shopes, B.J.Immunol.148:2918-22 (1992)). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolff et al CANCER RESEARCH 53:2560-5 (1993). Alternatively, antibodies may be engineered to have a dual Fc region, which may have enhanced complement lysis and ADCC capabilities. See Stevenson et al, anti-Cancer Drug Design 3:219-30 (1989). To increase the serum half-life of antibodies, salvage receptor binding epitopes can be incorporated into antibodies (particularly antibody fragments), for example as described in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., igG1, igG2, igG3, or IgG 4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

D) Cysteine engineering antibodies variants

In certain embodiments, it may be desirable to produce cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are substituted with cysteine residues. In particular embodiments, the substituted residue is present at an accessible site of the antibody. By replacing those residues with cysteines, reactive thiol groups are thereby located at accessible sites of the antibody, and can be used to conjugate the antibody with other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates as described further herein. Cysteine engineered antibodies may be produced as described, for example, in U.S. patent 7,521,541.

E) Immunoconjugates

The presently disclosed subject matter also provides immunoconjugates comprising an antibody disclosed herein conjugated to one or more cytotoxic agents (e.g., chemotherapeutic agents or drugs), growth inhibitory agents, proteins, peptides, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes. For example, an antibody of the disclosed subject matter can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or otherwise) to one or more other binding molecules (e.g., another antibody, antibody fragment, peptide, or binding mimetic).

In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody of the disclosure is conjugated to one or more drugs, including, but not limited to maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064, and european patent EP 0 425 235 Bl); auristatins, such as monomethyl auristatin drug fractions DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or derivatives thereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; hinman et al, cancer Res.53:3336-3342 (1993), and Lode et al, cancer Res.58:2925-2928 (1998)), anthracyclines such as daunomycin or doxorubicin (see Kratz et al, current Med. Chem.13:477-523 (2006), jeffrey et al, bioorganic & Med. Chem. Letters 16:358-362 (2006), torgov et al, bioconj. Chem.16:717-721 (2005), nagy et al, natl. Acad. Sci. USA 97:829-834 (2000)), anthracycline or doxorubicin (see Kratz et al, current Med. Chem.13:477-523 (2006), jeffrey et al, bioorganic & Med. Chem. Letters 16:358-362 (2002), bioconj. Chem. 16:15217, nagy. Scc. Natl. Acad. Sci. 37 (2000), and Methoxel. 35, med. 43, prael. Chetts. 35, and Prael. 35, praeco. 35, praeco. Det. 35, and Praeco. 35. Praeparata. 35, praeparata. 6, praeparata. Praeco. 6. Praemetal, praeco. Praemetal, praemg. PraeXmg. Prae. PraeXmg. And Prae. And PraeXmg. And. A. 35). In certain embodiments, immunoconjugates include antibodies as described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from pseudomonas aeruginosa (Pseudomonas aeruginosa)), ricin a chain, abrin a chain, mo Disu a chain, α -octacocin, aleurone, caryophyllin protein, pokeweed proteins (PAPI, PAPII, and PAP-S), balsam pear inhibitors, curcin, crotonin, saponaria inhibitors, gelonin, mitogens (mitogellin), restrictocins, phenomycin, enomycin, and tricyclodeoxyenoltoxoids.

In certain embodiments, an immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form the radioactive conjugate. A variety of radioisotopes may be used to prepare the radio conjugate. Non-limiting examples include At²¹¹、Ac²²⁵、1¹³¹、1¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹² and radioactive isotopes of Lu. When a radioconjugate is used for detection, it may include a radioactive atom for scintigraphic studies, such as tc99m or I ¹²³, or a spin label for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

Conjugates of antibody fragments and cytotoxic agents may be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC 1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as1, 5-difluoro-2, 4-dinitrobenzene). For example, ricin immunotoxins may be prepared as described in Vitetta et al, science238:1098 (1987). Carbon-14 labeled 1-thiobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid labile linkers, peptidase sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers may be used (Chari et al, cancer Res.52:127-131 (1992); U.S. Pat. No. 5,208,020). Non-limiting examples of linkers are disclosed above. Immunoconjugates disclosed herein expressly encompass, but are not limited to, such conjugates prepared with cross-linking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and SVSB (succinimidyl- (4-vinyl sulfone) benzoate) that are commercially available (e.g., from Pierce Biotechnology, inc., rockford, il., u.s.a.).

F) Antibody fusions

The subject matter disclosed herein also encompasses antibody fusions. For example, proteins may be linked together by chemical or genetic manipulation using methods known in the art. See, e.g., gillies et al, proc.Nat' l Acad.Sci.USA 89:1428-1432 (1992) and U.S. Pat. No. 5,650,150.

In one example, the disclosure encompasses anti-PSMA antibody-cytokine fusion proteins. In principle, an anti-PSMA antibody as disclosed herein is fused to any cytokine via the use of recombinant molecular biology techniques. As an example, an anti-PSMA antibody may be fused to IL-2 (Gillies, S., protein Engineering, DESIGN AND Selection 26 (10): 561-569 (2013); klein, C.et al, oncoImmunology 6:3 (2017)).

In another example, the disclosure encompasses anti-PSMA antibody-T cell conjugate fusion proteins. The anti-PSMA antibody-T cell conjugate fusion proteins discussed herein comprise a fusion between an anti-PSMA antibody and a ligand for a receptor expressed on a T cell. Examples of such ligands include, but are not limited to, CD40L, OX L, 4-1BBL, CD80/86, ICOSL, and the like. In embodiments, the ligand is fused to the Fc portion of an anti-PSMA antibody. In embodiments, the ligand is fused to the C-terminus of the light chain of the anti-PSMA antibody. Such methods are described with respect to 4-1BBL (Dafne m. Et al, journal of Immunotherapy (8): 714-722 (2008)), and similar methods can be used to produce other antibody-T cell conjugate fusion proteins.

B. recombinant methods and compositions

The anti-PSMA antibodies or antigen-binding fragments of the present disclosure may be produced using recombinant methods and compositions, for example, as described in U.S. patent No. 4,816,567. In embodiments, the invention also provides transformed cells and progeny thereof, wherein the nucleic acid molecule encoding the antibody or antigen binding fragment has been introduced by recombinant DNA techniques in vitro, ex vivo, or in vivo. Transformed eukaryotic or prokaryotic cells may be used to produce recombinant antibodies or antibody fragments for purification, or for expression in situ or secretion for various purposes, such as diagnosis or treatment of tumors. The transformed cells may proliferate and the introduced nucleic acids may be transcribed or the encoded proteins may be expressed. It will be appreciated that the daughter cells may not be identical to the parent cell, as mutations may occur during replication. Transformed cells include, but are not limited to, prokaryotic and eukaryotic cells, such as bacterial, fungal, plant, insect, and animal (e.g., mammalian, including human) cells. The cells may be present ex vivo in a culture, cell, tissue or organ or in a subject. In one embodiment, the antibody or antibody fragment is displayed on the surface of a yeast cell, in another embodiment the antibody or antibody fragment is coated on the surface of a nanoparticle, in another embodiment the antibody or antibody fragment is displayed on the surface of a mammalian cell, such as a T cell, NK cell, or other human or other mammalian cell, in another embodiment the antibody or antibody fragment is produced by a yeast, E.coli, or mammalian cell as a secreted protein.

Typically, cell transformation employs a vector. The term "vector" refers to, for example, a plasmid, virus (such as a viral vector), or other vehicle known in the art that can be manipulated by insertion or incorporation of a nucleic acid-genetic manipulation (i.e., a "cloning vector"), or can be used to transcribe or translate an inserted polynucleic acid (i.e., an "expression vector"). Such vectors can be used to introduce nucleic acids, including nucleic acids encoding antibodies or antibody antigen binding fragments operably linked to expression control elements, and express the encoded protein in vitro (e.g., in solution or in solid phase), in cells, or in vivo.

In one embodiment, the expression vector is transferred into a host cell by conventional techniques, and the transfected cell is then cultured by conventional techniques to produce the antibody or antigen-binding fragment of the invention. Thus, the invention includes host cells containing polynucleic acids encoding an antibody of the invention (e.g., an entire antibody, heavy or light chain thereof, or a portion thereof, or a single chain antibody, or fragment or variant thereof) operably linked to a heterologous promoter. In other embodiments, to express the entire antibody molecule, the vectors encoding the heavy and light chains are co-expressed in a host cell to express the entire immunoglobulin molecule.

A variety of host expression vector systems may be utilized to express the antibody molecules of the invention. Such host expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, and also represent cells that can express the antibody molecules of the invention in situ when transformed or transfected with the appropriate nucleic acid coding sequences. These include, but are not limited to, phage particles engineered to express antibody fragments or variants thereof (single chain antibodies), microorganisms such as bacteria (e.g., E.coli, B.subtilis)) transformed with recombinant phage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences, yeasts transformed with recombinant yeast expression vectors containing antibody coding sequences (e.g., saccharomyces, pichia (Pichia)), insect cell systems infected with recombinant virus expression vectors containing antibody coding sequences (e.g., baculovirus), plant cell systems infected with recombinant virus expression vectors containing antibody coding sequences (e.g., cauliflower mosaic virus (cauliflower mosaic virus), caes; tobacco mosaic virus (tobacco mosaic virus), TMV) or transformed with recombinant plasmid expression vectors containing antibody coding sequences (e.g., ti plasmid), or mammalian cell systems containing recombinant expression constructs (e.g., COS, CHO, BHK, 293, 3T3, NSO cells) containing mammalian cell genome, e.g., sulfur promoter (CMV promoter, 37. Promoter, or CMV promoter, 37. Promoter, e.g., promoter, 37 virus promoter. Preferably, bacterial cells such as e.coli are used, and more preferably eukaryotic cells (especially for expression of the whole recombinant antibody molecule) are used to express the recombinant antibody molecule. For example, the binding of mammalian cells, such as chinese hamster ovary Cells (CHO), to vectors, such as major intermediate early Gene promoter elements from human cytomegalovirus, is an efficient antibody expression system (foeking et al, gene 45:101 (1986); cockett et al, bio/Technology 8:2 (11990); B ebbington et al, bio/technologies 10:169 (1992); keen and Hale, cytotechnology 18:207 (1996)). These references are incorporated by reference herein in their entirety.

The vector or host expression vector used to transform the cell typically contains at least one origin of replication for propagation in the cell. Control elements present within the vector (including expression control elements as set forth herein) are included to facilitate transcription and translation. The term "expression control element" is intended to include at least one or more components whose presence may affect expression, and may include components other than promoters or enhancers, such as leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for creating polygenic or polycistronic information, splicing signals for introns, maintenance of the correct reading frame of the gene (allowing in-frame translation of mRNA), polyadenylation signals (providing the correct polyadenylation of the gene transcript of interest), stop codons, and the like.

The vector may comprise a selectable marker. As known in the art, "selectable marker" means a gene that allows selection of cells containing the gene. "Forward selection" refers to the process whereby only cells containing a selectable marker survive exposure to forward selection. Resistance is an example of a positive selection marker, cells containing the marker will survive in the medium containing the selection drug, while cells not containing the marker will die. Such markers include drug resistance genes such as neo conferring resistance to G418, hygr conferring resistance to hygromycin or puro conferring resistance to puromycin, and the like. Other positive selection marker genes include genes that allow identification or screening of cells containing the marker. These genes include fluorescent protein (GFP) genes, lacZ genes, alkaline phosphatase genes, surface markers such as CD8, and the like.

The vector may contain a negative selection marker. "negative selection" refers to the process by which cells containing a negative selection marker are killed upon exposure to an appropriate negative selection agent. For example, cells containing the herpes simplex virus-thymidine kinase (HSV-tk) gene (Wigler et al, cell 11:223 (1977)) are sensitive to the drug Ganciclovir (GANC). Similarly, the gpt gene sensitizes cells to 6-thioxanthines.

Mammalian expression systems further include vectors specifically designed for in vivo and ex vivo expression. Such systems include adeno-associated virus (AAV) vectors (U.S. Pat. No. 5,604,090). AAV vectors have previously been shown to provide expression of factor IX in humans and mice at levels sufficient to produce therapeutic benefit (Kay et al, nat. Genet.24:257 (2000); nakai et al, blood91:4600 (1998)). Adenovirus vectors (U.S. Pat. Nos. 5,700,470, 5,731,172 and 5,928,944), herpes simplex virus vectors (U.S. Pat. No. 5,501,979) and retrovirus (e.g., lentiviral vectors can be used to infect dividing cells as well as non-dividing cells and foamy viruses) vectors (U.S. Pat. Nos. 5,624,820, 5,693,508, 5,665,577, 6,013,516 and 5,674,703 and WIPO publications WO92/05266 and WO 92/14829) and papillomavirus vectors (e.g., human and bovine papillomaviruses) have been used for gene therapy (U.S. Pat. No. 5,719,054). Vectors also include Cytomegalovirus (CMV) based vectors (U.S. Pat. No. 5,561,063). Vectors that are effective in delivering genes to intestinal cells have been developed and can also be used (see, e.g., U.S. Pat. nos. 5,821,235, 5,786,340 and 6,110,456). In yeast, for example, vectors that facilitate integration of foreign nucleic acid sequences into chromosomes via homologous recombination are known in the art and may be used. When the inserted nucleic acid is too large (e.g., greater than about 12 kb) for more conventional vectors, yeast Artificial Chromosomes (YACs) are typically used.

In one embodiment, the phagemid vector used in the invention includes any phagemid vector useful in the art suitable for producing the antibody/antibody template/FR library of the invention and includes phagemid vectors pCB04, pIT1, pIT2, CANTAB 6, pComb 3HS. Filamentous vectors and phagemid construction methods are described, for example, in U.S. patent No. 6,054,312 and U.S. patent No. 6,803,230, each of which is incorporated herein by reference. Phage display systems involving non-filamentous phage vectors (known as cytoplasmic phages or lytic phages) can also be utilized, as described, for example, in U.S. Pat. No. 5,766,905, which is incorporated herein by reference.

Bacterial expression constructs suitable for use in the present invention include, but are not limited to, pCAL、pUC、pET、pETBlue^TM(Novagen)、pBAD、pLEX、pTrcHis2、pSE280、pSE380、pSE420(Invitrogen)、pKK223-2(Clontech)、pTrc99A、pKK223-3、pRIT2T、pMC1871、pEZZ 18(Pharmacia)、pBluescript II SK(Stratagene)、pALTER-Exl、pALTER-Ex2、pGEMEX(Promega)、pFivE(MBI)、pQE(Qiagen) commercially available expression constructs and derivatives thereof, as well as other constructs known in the art. In embodiments of the invention, the construct may also include a virus, plasmid, bacmid, phagemid, cosmid or phage.

The use of liposomes to introduce various compositions, including nucleic acids, into cells is known to those skilled in the art (see, e.g., U.S. Pat. nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282). Carriers comprising natural polymers or derivatives or hydrolysates of natural polymers described in WO 94/20078 and us patent No. 6,096,291 are suitable for mucosal delivery of molecules, such as polypeptides and polynucleic acids, and piperazinyl amphiphilic cationic lipids useful in gene therapy are also known (see e.g. us patent No. 5,861,397). Cationic lipid systems are also known (see, e.g., U.S. Pat. No. 5,459,127). Thus, means for delivering viral and non-viral vectors to cells or tissues (in vitro, in vivo, and ex vivo) are included.

In one embodiment, the nucleic acid sequences may be "operably linked", i.e., positioned, to ensure the function of the expression control sequences. These expression constructs are usually replicable in cells, either as episomes or as an integral part of the chromosomal DNA of the cell, and may contain suitable origins of replication for the corresponding prokaryotic strains to be expressed. Typically, the expression construct contains a selectable marker, such as, for example, tetracycline resistance, ampicillin resistance, kanamycin resistance, or chloramphenicol resistance, to facilitate detection and/or selection of those bacterial cells transformed with the desired nucleic acid sequence (see, e.g., U.S. Pat. No. 4,704,362). However, these markers are not exclusive and many other markers are known to those skilled in the art to be useful. In another embodiment of the invention, the expression construct contains both a positive selection marker and a negative selection marker.

Similarly, a reporter gene may be integrated into the expression construct to facilitate recognition of the transcribed product. Thus, in one embodiment of the invention, the reporter gene utilized is selected from the group consisting of beta-galactosidase, chloramphenicol acetyl transferase, luciferase, and fluorescent protein.

Prokaryotic promoter sequences regulate the expression of the encoded polynucleotide sequences and, in some embodiments of the invention, are operably linked to a polynucleic acid encoding a polypeptide of the invention. In additional embodiments of the invention, these promoters are constitutive or inducible and provide a means for high and low level expression of the polypeptides of the invention, and in some embodiments, for regulating the expression of the various polypeptides of the invention, which in some embodiments are expressed as fusion proteins.

Many well known bacterial promoters may be employed, including the T7 promoter system, lactose promoter system, tryptophan (Trp) promoter system, trc/Tac promoter system, beta-lactamase promoter system, tetA promoter system, arabinose regulated promoter system, phage T5 promoter or promoter system from phage lambda, among others, and constitute embodiments of the present invention. Promoters will generally control expression, optionally with operator sequences, and may include ribosome binding site sequences, for example, for the initiation and completion of transcription and translation. According to additional embodiments, the vector may also contain expression control sequences, enhancers that regulate the transcriptional activity of the promoter, appropriate restriction sites to facilitate cloning of the insert in the vicinity of the promoter, and other necessary information processing sites, such as RNA splice sites, polyadenylation sites, and transcription termination sequences, as well as any other sequences that facilitate expression of the inserted nucleic acid.

C. purification of anti-PSMA antibodies

The form of the anti-PSMA antibody may be recovered from the culture medium or from the host cell lysate. If membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., triton-X100) or by enzymatic cleavage. Cells used to express the anti-PSMA antibody may be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents.

It may be desirable to purify an anti-PSMA antibody from a recombinant cellular protein or polypeptide. The following procedures are examples of suitable purification procedures by fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel or cation exchange resins such as DEAE chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, sephadex G-75, protein A sepharose columns to remove contaminants such as IgG, and metal chelating columns to bind epitope-tagged forms of anti-PSMA antibodies. Various protein purification methods can be used, and such methods are known in the art and described, for example, in Deutscher,Methods in Enzymology,182(1990);Scopes,Protein Purification:Principles and Practice,Springer-Verlag,New York(1982). The purification step chosen will depend, for example, on the nature of the production method employed and the particular anti-PSMA antibody produced.

When recombinant techniques are employed, antibodies may be produced in the intracellular, periplasmic space, or secreted directly into the culture medium. If the antibodies are produced intracellularly, as a first step, the particulate fragments, whether host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al, bio/Technology 10:163-7 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for more than about 30 minutes. Cell debris can be removed by centrifugation. In the case of antibody secretion into the culture medium, the supernatant from such an expression system is typically first concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors, such as PMSF, may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein a as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on the heavy chain of human gamma 1, gamma 2 or gamma 4 (Lindmark et al J.Immunol. Meth.62:1-13 (1983)). Protein G is recommended for all mouse isoforms and human gamma 3 (Guss et al, EMBO J.5:15671575 (1986)). The matrix to which the affinity ligand is attached is typically agarose, but other matrices are also useful. Mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene allow for faster flow rates and shorter processing times than with agarose. Where the antibody comprises a CH3 domain, bakerbond ABX ^TM resin (j.t.baker, philipsburg, NJ) may be used for purification. Other protein purification techniques are also available, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin SEPHAROSE ^TM chromatography, anion or cation exchange resin (e.g., polyaspartic acid column) chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the antibody to be recovered.

After any preliminary purification steps, the mixture comprising the antibody of interest and the contaminant may be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5-4.5 and typically a low salt concentration (e.g., about 0-0.25M salt).

D. Measurement

Antibodies of the invention may be used in any known assay, such as ELISA, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, (1987) Monoclonal Antibodies: A Manual of Techniques, pages 147-158, CRC Press, inc.).

Detection markers can be used to locate, visualize and quantify binding or recognition events. The labeled antibodies of the invention can detect cell surface receptors or antigens. Another use of the detectably labeled antibodies is a bead-based immunocapture method comprising conjugating the beads with a fluorescently labeled antibody and detecting a fluorescent signal upon ligand binding. Similar binding detection methods utilize the Surface Plasmon Resonance (SPR) effect to measure and detect antibody-antigen interactions.

Detection labels such as fluorescent dyes and chemiluminescent dyes (Briggs et al (1997) J.chem. Soc., perkin-Trans. 1:1051-8) provide a detectable signal and are generally suitable for labeling antibodies, preferably with the property that (i) the labeled antibody should produce a very high signal with a low background so that small amounts of antibody can be sensitively detected in cell-free and cell-based assays, and (ii) the labeled antibody should be photostable so that fluorescent signals can be observed, monitored and recorded without significant photobleaching. For applications involving the binding of a labeled antibody to the membrane or cell surface (particularly living cells), the label preferably (iii) has good water solubility to achieve effective conjugate concentration and detection sensitivity, and (iv) is non-toxic to living cells, so as not to disrupt the normal metabolic processes of the cells or cause premature cell death.

Direct quantification of cell fluorescence intensity and counting of fluorescent labeling events (e.g., cell surface binding of peptide-dye conjugates) can be performed in a system that automatically mixes and reads non-radioactive assays performed with living cells or beads8100 HTS SYSTEM, applied Biosystems, foster City, calif.) the use of (Miraglia,"Homogeneous cell-and bead-based assays for high throughput screening using fluorometric microvolume assay technology",(1999)J.of Biomolecular Screening4:193-204). -labeled antibodies also includes cell surface receptor binding assays, immunocapture assays, fluorescent linked immunosorbent assays (FLISA), caspase cleavage (Zheng,"Caspase-3controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo",(1998)Proc.Natl.Acad.Sci.USA 95:618-23;US 6372907)、 apoptosis (Vermes,"A novel assay for apoptosis.Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V"(1995)J.Immunol.Methods 184:39-51), and cytotoxicity assays. Fluorescent microvolume assay techniques can be used to confirm up-or down-regulation of molecules targeted to the cell surface (Swartzman,"A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology",(1999)Anal.Biochem.271:143-51).

The labeled antibodies of the invention can be used as imaging biomarkers and probes by various methods and techniques of biomedical and molecular imaging, such as (i) MRI (magnetic resonance imaging), (ii) MicroCT (computed tomography), (iii) SPECT (single photon emission computed tomography), (iv) PET (positron emission tomography) Chen et al Bioconjugate chem.15:41-9 (2004), (v) bioluminescence, (vi) fluorescence, and (vii) ultrasound. Immunoscintillation is an imaging procedure in which antibodies labeled with a radioactive substance are administered to an animal or human patient and photographs of the site where the antibodies are located in the body are taken (US 6528624). Imaging biomarkers can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions.

Peptide labelling methods are well known (e.g., ,Haugland,2003,Molecular Probes Handbook of Fluorescent Probes and Research Chemicals,Molecular Probes,Inc.;Brinkley,1992,Bioconjugate Chem.3:2;Garman,(1997)Non-Radioactive Labelling:A Practical Approach,Academic Press,London;Means(1990)Bioconjugate Chem.1:2;Glazer et al (1975)Chemical Modification of Proteins.Laboratory Techniques in Biochemistry and Molecular Biology(T.S.Work and E.work code) AMERICAN ELSEVIER Publishing Co., new York, lundblad, R.L. and Noyes, C.M. (1984) CHEMICAL REAGENTS for Protein Modification, volumes I and II ,CRC Press,New York;Pfleiderer,G.(1985)"Chemical Modification of Proteins",Modern Methods in Protein Chemistry,H.Tschesche code, WALTER DEGRYTER, berlin and New York, and Wong(1991)Chemistry of Protein Conjugation and Cross-linking,CRC Press,Boca Raton,Fla.);De Leon-Rodriguez et al (2004) chem.Eur.J.10:1149-1155, lewis et al (2001) Bioconjugate chem.12:320-324, li et al (2002) Bioconjugate chem.13:110-115, mier et al (2005) Bioconjugate chem.16:240-237).

Peptides and proteins labeled with two moieties (fluorescent reporter and quencher) undergo Fluorescence Resonance Energy Transfer (FRET) in close enough proximity. The reporter group is typically a fluorescent dye that is excited by light of a certain wavelength and transfers energy to an acceptor or quencher group with an appropriate Stokes shift to emit at maximum brightness. Fluorescent dyes include molecules with extended aromaticity, such as fluorescein and rhodamine, and their derivatives. The fluorescent reporter may be partially or significantly quenched by a quencher moiety in the intact peptide. Detectable increase in fluorescence can be measured after cleavage of the peptide by a peptidase or protease (Knight,C.(1995)"Fluorimetric Assays of Proteolytic Enzymes",Methods in Enzymology,Academic Press,248:18-34).

The labeled antibodies of the invention may also be used as affinity purificants. In this process, the labeled antibody is immobilized on a solid phase such as Sephadex resin or filter paper using methods well known in the art. Contacting the immobilized antibody with a sample containing the antigen to be purified, after which the support is washed with a suitable solvent, which will remove substantially all but the antigen to be purified in the sample, which antigen to be purified binds to the immobilized polypeptide variant. Finally, the support is washed with another suitable solvent, such as glycine buffer (pH 5.0), which will release the antigen from the polypeptide variant.

1. Activity determination

In one aspect, an assay for identifying an anti-PSMA antibody having biological activity is provided. Biological activity may include, for example, the ability to inhibit cell growth or proliferation (e.g., a "cell killing" activity) or the ability to induce cell death, including programmed cell death (apoptosis). Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, anti-PSMA antibodies are tested for their ability to inhibit cell growth or proliferation in vitro. Assays for inhibiting cell growth or proliferation are well known in the art. Some cell proliferation assays, such as "cell killing" assays, measure cell viability. One such assay is the CellTiter-GloTM luminescent cell viability assay, which is commercially available from Promega (Madison, wis.). The assay determines the number of surviving cells in culture based on the quantification of the presence of ATP, which is an indicator of metabolically active cells. See Crouch et al (1993) J.Immunol. Meth.160:81-8, U.S. Pat. No. 6602677. The assay can be performed in 96-well or 384-well formats, making it suitable for automated High Throughput Screening (HTS) (see Cree et al (1995) ANTICANCER DRUGS 6:398-404). The measurement procedure involves the addition of a single reagentReagents) are added directly to the cultured cells. This results in cell lysis and the generation of a luminescent signal generated by the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of surviving cells present in the culture. The data may be recorded by a luminometer or a CCD camera imaging device. The luminous output is expressed in Relative Light Units (RLU).

Another cell proliferation assay is the "MTT" assay, which is a colorimetric assay that measures the oxidation of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide to formazan by mitochondrial reductase. Similar to the CellTiter-GloTM assay, this assay indicates the number of metabolically active cells present in the cell culture (see, e.g., mosmann (1983) J. Immunol. Meth.65:55-63, and Zhang et al (2005) Cancer Res. 65:3877-82).

In one aspect, anti-PSMA antibodies are tested for their ability to induce cell death in vitro. Assays for inducing cell death are well known in the art. In embodiments, such assays measure, for example, loss of membrane integrity as indicated by uptake of Propidium Iodide (PI), trypan blue (see Moore et al Cytotechnology,17:1-11 (1995)), or 7 AAD. In an exemplary PI uptake assay, cells are cultured in Du's modified eagle Medium (D-MEM) supplemented with 10% heat-inactivated FBS (Hyclone) and 2mM L-glutamine, hamsw F-12 (50:50). Thus, the assay is performed in the absence of complement and immune effector cells. Cells were seeded in 100x 20mm dishes at a density of 3x 10 ⁶ per dish and allowed to attach overnight. The medium was removed and replaced with fresh medium alone or medium containing various concentrations of antibody. Cells were incubated for a period of 3 days. After treatment, the monolayers were washed with PBS and detached by trypsin digestion. The cells were then centrifuged at 1200rpm for 5 minutes at 4 ℃, the pellet resuspended in 3mL cold ca2+ binding buffer (10mM Hepes,pH 7.4,140mM NaCl,2.5mM CaCl2) and aliquoted into 35mm sieve capped 12x 75mm tubes (1 mL per tube, 3 tubes per treatment group) to remove cell clumps. The tube then receives PI (10. Mu.g/mL). Samples were analyzed using a FACSCAN ^TM flow cytometer and FACSCONVERT ^TM CellQuest software (Becton Dickinson). Antibodies that induced statistically significant levels of cell death as determined by PI uptake were thus confirmed.

In one aspect, anti-PSMA antibodies are tested for their ability to induce apoptosis (programmed cell death) in vitro. An exemplary assay for antibodies that induce apoptosis is an annexin binding assay. In an exemplary annexin binding assay, cells are cultured and seeded in a petri dish as discussed in the previous paragraph. The medium is removed and replaced with fresh medium alone or medium containing 0.001 to 10 μg/mL antibody. After a three day incubation period, the monolayers were washed with PBS and detached by trypsin digestion. The cells were then centrifuged, resuspended in ca2+ binding buffer, and aliquoted into tubes as discussed in the previous paragraph. The tube was then subjected to labeled annexin (e.g., annexin V-FITC) (1. Mu.g/mL). Samples were analyzed using a FACSCAN ^TM flow cytometer and FACSCONVERT ^TM CellQuest software (BD Biosciences). Antibodies that induced statistically significant levels of annexin binding relative to control were thus confirmed. Another exemplary assay for antibodies that induce apoptosis is a histone DNA ELISA colorimetric assay for detecting the degradation of genomic DNA between nucleosomes. Such an assay can be performed using, for example, a cell death detection ELISA kit (Roche, palo Alto, calif.).

Cells for use in any of the in vitro assays described above include cells or cell lines that naturally express PSMA or that have been engineered to express PSMA. Such cells include tumor cells that overexpress PSMA relative to normal cells of the same tissue origin. Such cells also include PSMA-expressing cell lines (including tumor cell lines) and cell lines that do not normally express PSMA but have been transfected with a nucleic acid encoding PSMA.

In one aspect, the anti-PSMA antibodies are tested for their ability to inhibit cell growth or proliferation in vivo. In certain embodiments, the anti-PSMA antibodies are tested for their ability to inhibit tumor growth in vivo. Such testing may use in vivo model systems, such as xenograft models. In an exemplary xenograft system, human tumor cells are introduced into a suitable immunocompromised non-human animal (e.g., SCID mouse). The antibodies of the invention are administered to animals. The ability of the antibodies to inhibit or reduce tumor growth was measured. In certain embodiments of the above xenograft systems, the human tumor cells are tumor cells from a human patient. In certain embodiments, the human tumor cells are introduced into a suitably immunocompromised non-human animal by subcutaneous injection or by implantation into a suitable site (e.g., a mammary fat pad).

2. Binding assays and other assays

In one aspect, the anti-PSMA antibodies are tested for antigen-binding activity. For example, in certain embodiments, anti-PSMA antibodies are tested for their ability to bind to PSMA expressed on the surface of a cell. FACS assays can be used for such testing.

In one aspect, the competition assay can be used to identify monoclonal antibodies ：SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24、25/26、27/28、29/30、31/32、33/34 or 35/36 that compete with monoclonal antibodies comprising the following HCVR/LCVR sequence pairs, or monoclonal antibodies ：SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24、25/26、27/28、29/30、31/32、33/34 or 35/36 that compete with monoclonal antibodies comprising six CDRs selected from the following HCVR/LCVR sequence pairs.

In certain embodiments, such competing antibodies bind to ：SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24、25/26、27/28、29/30、31/32、33/34 or 35/36 the same epitope (e.g., linear or conformational epitope) as a monoclonal antibody comprising the following HCVR/LCVR sequence pair, or bind to ：SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24、25/26、27/28、29/30、31/32、33/34 or 35/36 the same epitope as a monoclonal antibody comprising six CDRs selected from the following HCVR/LCVR sequence pair. Exemplary competition assays include, but are not limited to, conventional assays such as those provided in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, cold Spring Harbor, NY). A detailed exemplary method for locating epitopes bound by antibodies is provided in Morris (1996) 'Epitope Mapping Protocols,' volume 66 (Humana Press, totowa, NJ) Methods in Molecular Biology. Two antibodies are considered to bind to the same epitope if they block 50% or more of their binding to each other.

In an exemplary competition assay, immobilized PSMA is incubated in a solution comprising a first labeled antibody that binds PSMA and a second unlabeled antibody that is to be tested for its ability to compete with the first antibody for binding to PSMA. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized PSMA was incubated in a solution containing the first labeled antibody but no second unlabeled antibody. After incubation under conditions that allow the primary antibody to bind to PSMA, excess unbound antibody is removed and the amount of label associated with the immobilized PSMA is measured. If the amount of label associated with immobilized PSMA in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to PSMA. In certain embodiments, the immobilized PSMA is present on the surface of a cell or in a membrane preparation obtained from a cell expressing PSMA on its surface.

In one aspect, the purified anti-PSMA antibodies can be further characterized by a range of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion High Pressure Liquid Chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

E. epitope identification method

In one aspect, the present disclosure provides a method of recognizing an epitope of an anti-PSMA antibody or antigen-binding fragment thereof.

An epitope may comprise at least 3, 4,5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 amino acids in a unique spatial conformation. Epitopes can be formed by contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by consecutive amino acids can generally be retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding can generally be lost upon treatment with denaturing solvents.

Epitope mapping may be performed to confirm linear or nonlinear discontinuous amino acid sequences, i.e., epitopes recognized (e.g., specifically) by an anti-PSMA antibody or antigen-binding fragment thereof. General methods of epitope mapping may require expression of the full-length polypeptide sequence recognized by the antibody or ligand of interest, as well as various fragments, i.e., truncated forms of the polypeptide sequence, typically in heterologous expression systems. These different recombinant polypeptide sequences or fragments thereof (e.g., fused to an N-terminal protein (e.g., GFP)) can then be used to determine whether the antibody or ligand of interest is capable of binding to one or more truncated forms of the polypeptide sequence.

By using repeated truncations and generating recombinant polypeptide sequences with overlapping amino acid regions, it is possible to confirm the region of the polypeptide sequence recognized by the antibody of interest (see, e.g., epitope Mapping Protocols in Methods in Molecular Biology, volume 66, glenn E.Morris (1996)). The method relies on the ability of an agent (e.g., an antibody of interest) to bind to sequences reconstituted from an epitope library (e.g., an epitope library derived from a synthetic peptide array on a membrane support, a combinatorial phage display peptide library). The epitope library then provides a range of possibilities for screening against antibodies. In addition, site-specific mutagenesis or random Ala scanning of one or more residues of the targeted epitope can be performed to confirm the identity of the epitope.

Libraries of epitopes can be created by synthetically designing various portions of PSMA as constructs and expressing them in a suitable system. In other cases, the portion of PSMA may be amplified from total RNA extracted from PSMA expressing cells isolated from normal and/or malignant human tissues.

The host system may be any suitable expression system, such as 293 cells, insect cells or a suitable in vitro translation system. Binding of an anti-PSMA antibody or antigen-binding fragment thereof to one of the epitopes in the library described above can be detected by contacting a labeled PSMA antibody of the present disclosure with the epitopes in the library and detecting a signal from the label.

For epitope localization, computational algorithms have also been developed which have been shown to map conformally discontinuous epitopes. Conformational epitopes can be confirmed by determining the spatial conformation of amino acids using methods including, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. Some epitope localization methods, such as x-ray analysis of crystals of antigen-antibody complexes, can provide atomic resolution of the epitope. In other cases, computational combinatorial approaches for epitope mapping may be employed to model potential epitopes based on the sequence of an anti-PSMA or antigen-binding fragment thereof. In such cases, the antigen binding portion of the antibody is sequenced and a computational model is used to reconstruct and predict the potential binding site of the antibody.

In some cases, the present disclosure provides a method of determining PSMA epitopes comprising (a) preparing a library of epitopes from a PSMA receptor, (b) contacting the library of epitopes with an anti-PSMA antibody, and (c) identifying the amino acid sequence of at least one epitope in the library of epitopes that is bound by the antibody. In one instance, the antibody is attached to a solid support. The library of epitopes may comprise sequences corresponding to contiguous and non-contiguous epitopes of PSMA. In some cases, the library of epitopes comprises fragments from PSMA receptors ranging in length from about 10 amino acids to about 30 amino acids, from about 10 amino acids to about 20 amino acids, or from about 5 amino acids to about 12 amino acids. In some cases, the anti-PSMA antibody or antigen-binding fragment thereof is labeled, and the label is a radioactive molecule, a luminescent molecule, a fluorescent molecule, an enzyme, or biotin.

Phage panning can be used to identify PMSA binding molecules that bind to PMSA or one or more epitopes on PMSA. In embodiments, phage panning using biotinylated recombinant human PSMA protein bound to streptavidin beads is performed on a highly diverse synthetic scFv phage display library using multiple rounds (e.g., 5 rounds) of selection, with decreasing antigen concentrations (e.g., starting from 100pmol to 2 pmol) per round but increasing wash stringency. In embodiments, alternate rounds of selection may be performed with 1e8 psma+ cells or antigens (e.g., 100pmol and 25pmol, respectively) using an alternate panning strategy. Antigen-binding phage can then be pulled down, eluted, amplified and screened using ELISA for binding agent validation and selection. Selected scFv can be reformatted into IgG, expressed, purified and characterized by NGS and/or Sanger clone sequencing.

In embodiments, the PMSA-binding molecule may be produced using an epitope comprising or consisting of human PSMA residues 574-580, 644-649 and 674-686, the residues being numbered according to SEQ ID NO:329 in FIG. 18B. In embodiments, the PMSA-binding molecule may be produced using an epitope comprising or consisting of human PSMA residues 150-161, 167-172 and 256-261, the residues being numbered according to SEQ ID NO:330 in FIG. 18B.

F. chimeric Antigen Receptor (CAR) constructs

Aspects of the invention include nucleic acids encoding CARs, as well as constructs and vectors comprising such nucleic acids. In some cases, the nucleic acid is a heterologous component of, for example, an expression cassette. In embodiments, the nucleic acid is a heterologous component of, for example, a retroviral vector. In embodiments, the nucleic acid is, for example, an αβ or γδ T cell, and preferably a heterologous component of γδ T cells. In embodiments, the nucleic acid is a heterologous component of, for example, gamma ⁺ T cells and/or delta ⁺ T cells. In embodiments, the nucleic acid is a heterologous component of, for example, an α ^- T cell and/or a β ^- cell.

The subject CARs of the present invention comprise an antigen-binding domain capable of specifically binding PSMA. The antigen binding domain can be operably linked to another domain of the CAR, such as a transmembrane domain, a costimulatory domain, and/or an intracellular signaling domain, as described herein. The antigen binding domains described herein can be combined with any transmembrane domain, costimulatory domain, and/or intracellular signaling domain described herein, and/or any other domain described herein that can be included in a CAR of the invention. The subject CARs of the present invention may also include a hinge domain as described herein. The subject CARs of the present invention may also include at least one spacer domain as described herein.

1. Antigen binding domains

The antigen binding domain may include any domain that binds PSMA, and may include, but is not limited to, monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, non-human antibodies, and any fragment thereof. In embodiments, the antigen binding domain portion comprises a mammalian antibody or fragment thereof. The choice of antigen binding domain may depend on the type and amount of antigen present on the surface of the target cell.

In embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), and a single chain variable fragment (scFv).

The present disclosure provides antibodies and CARs having "substantial identity" or "substantial similarity" to the sequences provided herein in CDRs or framework regions. When referring to a nucleic acid or fragment thereof, the term "substantial identity" or "substantially identical" indicates that when optimally aligned with another nucleic acid (or the complementary strand of another nucleic acid), there is nucleotide sequence identity in percent, e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% of the nucleotide bases, as measured by any well known sequence identity algorithm (such as FASTA, BLAST, or GAP), as discussed below. In certain instances, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule may encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

The term "substantial similarity" or "substantially similar" as applied to polypeptides means that when optimally aligned (such as by using the programs GAP or BESTFIT with default GAP weights), two peptide sequences share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity. In some aspects, the different residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is a substitution of one amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions will not substantially alter the functional properties of the protein. In the case of conservative substitutions where two or more amino acid sequences differ from each other, the percentage or degree of similarity may be adjusted upward to correct the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. See, for example, pearson (1994) Methods mol. Biol.24:307-331, which is incorporated herein by reference. Examples of groups of amino acids having side chains of similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine and tryptophan, 5) basic side chains: lysine, arginine and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acid substitutions are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change with positive values in the PAM250 log likelihood matrix disclosed in Gonnet et al (1992) Science 256:1443 45, incorporated herein by reference. A "moderately conservative" permutation is any variation that has a non-negative value in the PAM250 log likelihood matrix.

Sequence identity and/or similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other modifications (including conservative amino acid substitutions) to match similar sequences. For example, GCG software contains programs such as GAP and BESTFIT that can use default parameters to determine sequence homology or sequence identity between closely related polypeptides (such as homologous polypeptides from different organism species), or between wild-type proteins and their mutant proteins. See, e.g., GCG version 6.1. Polypeptide sequences can also be compared using FASTA (program in GCG version 6.1) using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA 3) provide alignment and percent sequence identity (Pearson (2000) supra) of the optimal overlap region between query and search sequences. Sequences can also be compared using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of (gap extension penalty) of 2 and a BLOSUM matrix of 62. When comparing the sequences disclosed herein to a database containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, in particular BLASTP or TBLASTN, using default parameters. See, for example, altschul et al (1990) J.mol.biol.215:403-410and (1997) Nucleic Acids Res.25:3389-3402, each of which is incorporated herein by reference.

Provided herein are anti-PSMACAR comprising variants of any of the HCVR, LCVR, and/or CDR amino acid sequences disclosed herein that have one or more substitutions (e.g., conservative substitutions). For example, the disclosure includes an antibody PSMACAR having an HCVR, LCVR, and/or CDR amino acid sequence having, for example, 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid substitutions relative to any of the HCVR, LCVR, and/or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR 3) amino acid sequences disclosed herein. For example, the anti-PSMACAR can comprise 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 amino acid substitutions (e.g., conservative amino acid substitutions) relative to any of the HCVR, LCVR, and/or CDR (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR 3) amino acid sequences disclosed herein.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds the same epitope as an anti-PSMA binding domain comprising an amino acid sequence selected from the group consisting of any of SEQ ID NOs 1-36, competes with an anti-PSMA binding domain comprising an amino acid sequence selected from the group consisting of any of SEQ ID NOs 1-36, or is an anti-PSMA binding domain comprising an amino acid sequence selected from the group consisting of any of SEQ ID NOs 1-36. In embodiments, the anti-PSMA binding domain binds to the same epitope as an anti-PSMA binding domain comprising an HCVR amino acid sequence selected from the group consisting of, competes with, or is an anti-PSMA binding domain comprising an HCVR amino acid sequence selected from the group consisting of SEQ ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35. In embodiments, the anti-PSMA binding domain binds to the same epitope as an anti-PSMA binding domain comprising an LCVR amino acid sequence selected from the group consisting of, competes with, or is an anti-PSMA binding domain comprising an HCVR amino acid sequence selected from the group consisting of SEQ ID NOs 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36. In embodiments, the anti-PSMA binding domain binds to the same epitope as an anti-PSMA binding domain comprising an LCVR amino acid sequence selected from the group consisting of, competes with, or is an anti-PSMA binding domain comprising an HCVR amino acid sequence selected from the group consisting of any one of SEQ ID NOs 1, 3, 5, 7, 9, 11 and 13. In embodiments, the anti-PSMA binding domain binds to the same epitope as an anti-PSMA binding domain comprising a LCVR amino acid sequence selected from the group consisting of, competes with an anti-PSMA binding domain comprising a LCVR amino acid sequence selected from the group consisting of, or is an anti-PSMA binding domain comprising a HCVR amino acid sequence selected from the group consisting of any one of SEQ ID NOs 2, 4, 6, 8, 10, 12 and 14.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 amino acid sequence selected from the group consisting of SEQ ID NOS: 37-54, a CDR2 amino acid sequence selected from the group consisting of SEQ ID NOS: 55-72, and a HCVR of a CDR3 amino acid sequence selected from the group consisting of SEQ ID NOS: 73-90. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 amino acid sequence selected from the group consisting of SEQ ID NOS: 91-108, a CDR2 amino acid sequence selected from the group consisting of SEQ ID NOS: 109-126, and an LCVR of a CDR3 amino acid sequence selected from the group consisting of SEQ ID NOS: 127-144.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 amino acid sequence selected from the group consisting of SEQ ID NOS: 37-43, a CDR2 amino acid sequence selected from the group consisting of SEQ ID NOS: 55-61, and a HCVR of a CDR3 amino acid sequence selected from the group consisting of SEQ ID NOS: 73-79. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 amino acid sequence selected from the group consisting of SEQ ID NOS: 91-97, a CDR2 amino acid sequence selected from the group consisting of SEQ ID NOS: 109-115, and an LCVR of a CDR3 amino acid sequence selected from the group consisting of SEQ ID NOS: 127-133.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 1 and the LCVR amino acid sequence as set forth in SEQ ID No. 2, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 1 and the LCVR amino acid sequence as set forth in SEQ ID No. 2, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 1 and the LCVR amino acid sequence as set forth in SEQ ID No. 2. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 3 and the LCVR amino acid sequence as set forth in SEQ ID No. 4, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 3 and the LCVR amino acid sequence as set forth in SEQ ID No. 4, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 3 and the LCVR amino acid sequence as set forth in SEQ ID No. 4. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 5 and the LCVR amino acid sequence as set forth in SEQ ID No. 6, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 5 and the LCVR amino acid sequence as set forth in SEQ ID No. 6, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 5 and the LCVR amino acid sequence as set forth in SEQ ID No. 6. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 7 and the LCVR amino acid sequence as set forth in SEQ ID No. 8, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 7 and the LCVR amino acid sequence as set forth in SEQ ID No. 8, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 7 and the LCVR amino acid sequence as set forth in SEQ ID No. 8. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 9 and the LCVR amino acid sequence as set forth in SEQ ID No. 10, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 9 and the LCVR amino acid sequence as set forth in SEQ ID No. 10, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 9 and the LCVR amino acid sequence as set forth in SEQ ID No. 10. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 11 and the LCVR amino acid sequence as set forth in SEQ ID No. 12, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 11 and the LCVR amino acid sequence as set forth in SEQ ID No. 12, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 11 and the LCVR amino acid sequence as set forth in SEQ ID No. 12. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 13 and the LCVR amino acid sequence as set forth in SEQ ID No. 14, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 13 and the LCVR amino acid sequence as set forth in SEQ ID No. 14, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 13 and the LCVR amino acid sequence as set forth in SEQ ID No. 14. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 15 and the LCVR amino acid sequence as set forth in SEQ ID No. 16, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 15 and the LCVR amino acid sequence as set forth in SEQ ID No. 16, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 15 and the LCVR amino acid sequence as set forth in SEQ ID No. 16. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 17 and the LCVR amino acid sequence as set forth in SEQ ID No. 18, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 17 and the LCVR amino acid sequence as set forth in SEQ ID No. 18, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 17 and the LCVR amino acid sequence as set forth in SEQ ID No. 18. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence set forth in SEQ ID NO. 19 and the LCVR amino acid sequence set forth in SEQ ID NO. 20, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence set forth in SEQ ID NO. 19 and the LCVR amino acid sequence set forth in SEQ ID NO. 20, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence set forth in SEQ ID NO. 19 and the LCVR amino acid sequence set forth in SEQ ID NO. 20. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 21 and the LCVR amino acid sequence as set forth in SEQ ID No. 22, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 21 and the LCVR amino acid sequence as set forth in SEQ ID No. 22, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 21 and the LCVR amino acid sequence as set forth in SEQ ID No. 22. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence set forth in SEQ ID NO. 23 and the LCVR amino acid sequence set forth in SEQ ID NO. 24, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence set forth in SEQ ID NO. 23 and the LCVR amino acid sequence set forth in SEQ ID NO. 24, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence set forth in SEQ ID NO. 23 and the LCVR amino acid sequence set forth in SEQ ID NO. 24. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 25 and the LCVR amino acid sequence as set forth in SEQ ID No. 26, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 25 and the LCVR amino acid sequence as set forth in SEQ ID No. 26, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 25 and the LCVR amino acid sequence as set forth in SEQ ID No. 26. in embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 27 and the LCVR amino acid sequence as set forth in SEQ ID No. 28, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 27 and the LCVR amino acid sequence as set forth in SEQ ID No. 28, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 27 and the LCVR amino acid sequence as set forth in SEQ ID No. 28. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence set forth in SEQ ID NO. 29 and the LCVR amino acid sequence set forth in SEQ ID NO. 30, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence set forth in SEQ ID NO. 29 and the LCVR amino acid sequence set forth in SEQ ID NO. 30, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence set forth in SEQ ID NO. 29 and the LCVR amino acid sequence set forth in SEQ ID NO. 30. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 31 and the LCVR amino acid sequence as set forth in SEQ ID No. 32, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 31 and the LCVR amino acid sequence as set forth in SEQ ID No. 32, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 31 and the LCVR amino acid sequence as set forth in SEQ ID No. 32. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 33 and the LCVR amino acid sequence as set forth in SEQ ID No. 34, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 33 and the LCVR amino acid sequence as set forth in SEQ ID No. 34, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 33 and the LCVR amino acid sequence as set forth in SEQ ID No. 34. In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 35 and the LCVR amino acid sequence as set forth in SEQ ID No. 36, competes with an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 35 and the LCVR amino acid sequence as set forth in SEQ ID No. 36, or is an anti-PSMA binding domain comprising the HCVR amino acid sequence as set forth in SEQ ID No. 35 and the LCVR amino acid sequence as set forth in SEQ ID No. 36.

Preferred embodiments include those wherein the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising an HCVR amino acid sequence as shown in SEQ ID NO. 1 and an LCVR amino acid sequence as shown in SEQ ID NO. 2, an HCVR amino acid sequence as shown in SEQ ID NO. 3 and an LCVR amino acid sequence as shown in SEQ ID NO. 4, an HCVR amino acid sequence as shown in SEQ ID NO. 5 and an LCVR amino acid sequence as shown in SEQ ID NO. 6, an HCVR amino acid sequence as shown in SEQ ID NO. 7 and an LCVR amino acid sequence as shown in SEQ ID NO. 8, an HCVR amino acid sequence as shown in SEQ ID NO. 9 and an LCVR amino acid sequence as shown in SEQ ID NO. 10, an HCVR amino acid sequence as shown in SEQ ID NO. 11 and an LCVR amino acid sequence as shown in SEQ ID NO. 12, an LCVR amino acid sequence as shown in SEQ ID NO. 14 and an LCVR amino acid sequence as shown in SEQ ID NO. 14.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:37, a CDR2 sequence as shown in SEQ ID NO:55 and a CDR3 sequence as shown in SEQ ID NO:73, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:91, a CDR2 sequence as shown in SEQ ID NO:109 and a CDR3 sequence as shown in SEQ ID NO: 127.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:38, a CDR2 sequence as shown in SEQ ID NO:56 and a CDR3 sequence as shown in SEQ ID NO:74, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:92, a CDR2 sequence as shown in SEQ ID NO:110 and a CDR3 sequence as shown in SEQ ID NO: 128.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:39, a CDR2 sequence as shown in SEQ ID NO:57 and a CDR3 sequence as shown in SEQ ID NO:75, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:93, a CDR2 sequence as shown in SEQ ID NO:111 and a CDR3 sequence as shown in SEQ ID NO: 129.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:40, a CDR2 sequence as shown in SEQ ID NO:58 and a CDR3 sequence as shown in SEQ ID NO:76, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:94, a CDR2 sequence as shown in SEQ ID NO:112 and a CDR3 sequence as shown in SEQ ID NO: 130.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising the CDR1 sequence as shown in SEQ ID NO:41, the CDR2 sequence as shown in SEQ ID NO:59 and the CDR3 sequence as shown in SEQ ID NO:77, and/or an LCVR comprising the CDR1 sequence as shown in SEQ ID NO:95, the CDR2 sequence as shown in SEQ ID NO:113 and the CDR3 sequence as shown in SEQ ID NO: 131.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO. 42, a CDR2 sequence as shown in SEQ ID NO. 60 and a CDR3 sequence as shown in SEQ ID NO. 78, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO. 96, a CDR2 sequence as shown in SEQ ID NO. 114 and a CDR3 sequence as shown in SEQ ID NO. 132.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO. 43, a CDR2 sequence as shown in SEQ ID NO. 61 and a CDR3 sequence as shown in SEQ ID NO. 79, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO. 97, a CDR2 sequence as shown in SEQ ID NO. 115 and a CDR3 sequence as shown in SEQ ID NO. 133.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising the CDR1 sequence shown as SEQ ID NO:44, the CDR2 sequence shown as SEQ ID NO:62 and the CDR3 sequence shown as SEQ ID NO:80, and/or an LCVR comprising the CDR1 sequence shown as SEQ ID NO:98, the CDR2 sequence shown as SEQ ID NO:116 and the CDR3 sequence shown as SEQ ID NO: 134.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO. 45, a CDR2 sequence as shown in SEQ ID NO. 63 and a CDR3 sequence as shown in SEQ ID NO. 81, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO. 99, a CDR2 sequence as shown in SEQ ID NO. 117 and a CDR3 sequence as shown in SEQ ID NO. 135.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:46, a CDR2 sequence as shown in SEQ ID NO:64 and a CDR3 sequence as shown in SEQ ID NO:82, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:100, a CDR2 sequence as shown in SEQ ID NO:118 and a CDR3 sequence as shown in SEQ ID NO: 136.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:47, a CDR2 sequence as shown in SEQ ID NO:65 and a CDR3 sequence as shown in SEQ ID NO:83, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:101, a CDR2 sequence as shown in SEQ ID NO:119 and a CDR3 sequence as shown in SEQ ID NO: 137.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising the CDR1 sequence as shown in SEQ ID NO:48, the CDR2 sequence as shown in SEQ ID NO:66 and the CDR3 sequence as shown in SEQ ID NO:84, and/or an LCVR comprising the CDR1 sequence as shown in SEQ ID NO:102, the CDR2 sequence as shown in SEQ ID NO:120 and the CDR3 sequence as shown in SEQ ID NO: 138.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising the CDR1 sequence as shown in SEQ ID NO:49, the CDR2 sequence as shown in SEQ ID NO:67 and the CDR3 sequence as shown in SEQ ID NO:85, and/or an LCVR comprising the CDR1 sequence as shown in SEQ ID NO:103, the CDR2 sequence as shown in SEQ ID NO:121 and the CDR3 sequence as shown in SEQ ID NO: 139.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:50, a CDR2 sequence as shown in SEQ ID NO:68 and a CDR3 sequence as shown in SEQ ID NO:86, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:104, a CDR2 sequence as shown in SEQ ID NO:122 and a CDR3 sequence as shown in SEQ ID NO: 140.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:51, a CDR2 sequence as shown in SEQ ID NO:69 and a CDR3 sequence as shown in SEQ ID NO:87, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:105, a CDR2 sequence as shown in SEQ ID NO:123 and a CDR3 sequence as shown in SEQ ID NO: 141.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:52, a CDR2 sequence as shown in SEQ ID NO:70 and a CDR3 sequence as shown in SEQ ID NO:88, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:106, a CDR2 sequence as shown in SEQ ID NO:124 and a CDR3 sequence as shown in SEQ ID NO: 142.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:53, a CDR2 sequence as shown in SEQ ID NO:71 and a CDR3 sequence as shown in SEQ ID NO:89, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:107, a CDR2 sequence as shown in SEQ ID NO:125 and a CDR3 sequence as shown in SEQ ID NO: 143.

In embodiments, the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising a CDR1 sequence as shown in SEQ ID NO:54, a CDR2 sequence as shown in SEQ ID NO:72 and a CDR3 sequence as shown in SEQ ID NO:90, and/or an LCVR comprising a CDR1 sequence as shown in SEQ ID NO:108, a CDR2 sequence as shown in SEQ ID NO:126 and a CDR3 sequence as shown in SEQ ID NO: 144.

Preferred embodiments include those wherein the isolated nucleic acid encodes an anti-PSMA binding domain that binds to the same epitope as an anti-PSMA binding domain comprising, competes with, or comprises an anti-PSMA binding domain comprising the CDR1 sequence shown in SEQ ID NO:37, the CDR2 sequence shown in SEQ ID NO:55 and the CDR3 sequence shown in SEQ ID NO:73, and a HCVR comprising the CDR1 sequence shown in SEQ ID NO:91, LCVR of CDR2 sequence as shown in SEQ ID NO. 109 and CDR3 sequence as shown in SEQ ID NO. 127, HCVR comprising CDR1 sequence as shown in SEQ ID NO. 38, CDR2 sequence as shown in SEQ ID NO. 56 and CDR3 sequence as shown in SEQ ID NO. 74, LCVR comprising CDR1 sequence as shown in SEQ ID NO. 92, CDR2 sequence as shown in SEQ ID NO. 110 and CDR3 sequence as shown in SEQ ID NO. 128, LCVR comprising CDR1 sequence as shown in SEQ ID NO. 39, HCVR comprising the CDR2 sequence shown in SEQ ID NO:57 and the CDR3 sequence shown in SEQ ID NO:75, and LCVR comprising the CDR1 sequence shown in SEQ ID NO:93, the CDR2 sequence shown in SEQ ID NO:111 and the CDR3 sequence shown in SEQ ID NO:129, HCVR comprising the CDR1 sequence shown in SEQ ID NO:40, the CDR2 sequence shown in SEQ ID NO:58 and the CDR3 sequence shown in SEQ ID NO:76, and HCVR comprising the CDR1 sequence shown in SEQ ID NO:94, LCVR comprising the CDR2 sequence shown as SEQ ID NO 112 and the CDR3 sequence shown as SEQ ID NO 130, HCVR comprising the CDR1 sequence shown as SEQ ID NO 41, the CDR2 sequence shown as SEQ ID NO 59 and the CDR3 sequence shown as SEQ ID NO 77, and LCVR comprising the CDR1 sequence shown as SEQ ID NO 95, the CDR2 sequence shown as SEQ ID NO 113 and the CDR3 sequence shown as SEQ ID NO 131, LCVR comprising the CDR1 sequence shown as SEQ ID NO 42, HCVR comprising the CDR2 sequence shown as SEQ ID NO:60 and the CDR3 sequence shown as SEQ ID NO:78, and LCVR comprising the CDR1 sequence shown as SEQ ID NO:96, the CDR2 sequence shown as SEQ ID NO:114 and the CDR3 sequence shown as SEQ ID NO:132, or HCVR comprising the CDR1 sequence shown as SEQ ID NO:43, the CDR2 sequence shown as SEQ ID NO:61 and the CDR3 sequence shown as SEQ ID NO:79, and HCVR comprising the CDR1 sequence shown as SEQ ID NO:97, LCVR of the CDR2 sequence shown as SEQ ID NO. 115 and the CDR3 sequence shown as SEQ ID NO. 133.

Other anti-PSMA binding domains and anti-PSMACAR are known and may be used in accordance with the teachings of the present disclosure. See, for example, ,WO2017180713、WO2019245991A1、WO2002098897、WO2001009192、WO2016179534、WO2019224718、WO2021/188599、WO2016111344、WO2017027325、WO2018098354、WO2017212250、WO 2021/050656 and NNarayan et al, (2022) Nature medicine. Doi:10.1038, the contents of each of which are hereby expressly incorporated by reference in their entirety. In preferred embodiments, such anti-PSMA binding domains are incorporated into CARs as described herein, or known CARs are used as such or modified according to the present disclosure, wherein the CARs are expressed in γδ T cells for use in methods as described herein.

2. Transmembrane domain

The CARs of the disclosure may comprise a transmembrane domain that couples the antigen binding domain of the CAR with one or more intracellular domains of the CAR. The transmembrane domain of a CAR of the present disclosure is a region capable of crossing the plasma membrane of a cell (e.g., γδ T cells). In embodiments, the transmembrane domain is interspersed between the antigen binding domain and the one or more intracellular domains of the CAR.

In embodiments, the transmembrane domain naturally associates with one or more domains in the CAR. In embodiments, the transmembrane domains may be selected or modified by one or more amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

For example and without limitation, the transmembrane domain may be derived from natural or synthetic sources. In the case of natural sources, the domain may be derived from any membrane-bound protein or transmembrane protein. The transmembrane region particularly used in the present invention may be derived from (i.e., at least includes the following transmembrane region) 4-1BB/CD137, activated NK cell receptor, immunoglobulin 、B7-H3、BAFFR、BLAME(SLAMF8)、BTLA、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137 or CD154、CD100(SEMA4D)、CD103、CD160(BY55)、CD18、CD19、CD19a、CD2、CD247、CD27、CD276(B7-H3)、CD28、CD29、CD3δ、CD3ε、CD3γ、CD3ζ、CD30、CD4、CD40、CD49a、CD49D、CD49f、CD69、CD7、CD84、CD8、CD8α、CD8β、CD96(Tactile)、CD11a、CD11b、CD11c、CD11d、CDS、CEACAM1、CRT AM、 cytokine receptor, DAP10, DNAM1 (CD 226), fcgammA receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ig alphA (CD 79A), IL-2RbetA, IL-2 RgammA, IL-7 RalphA, inducible T cell costimulatory factor (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, ligand that specifically binds CD83, LIGHT, LTBR, ly9 (CD 229), lymphocyte function-associated antigen-1 (LFA-1; CD11A/CD 18), MHC class 1 molecules, NKG2C, NKG2D, NKp, NKp44, NKp46, NKp80 (KLRF 1), OX-40, PAG/Cbp, sex death 1 (SLAM 1), SLAM signal (SLAM 1); CD150, IPO-3), SLAMF4 (CD 244;2B 4), SLAMF6 (NTB-A; lyl 08), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, toll ligand receptor, TRANCE/RANKL, VLA1 or VLA-6 or fragments, truncations or combinations thereof. Alternatively, the transmembrane domain may be synthetic, in which case it will predominantly comprise hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan and valine will be present at each end of the synthetic transmembrane domain.

In certain embodiments, the transmembrane domain comprises the transmembrane domain of CD 8. In certain embodiments, the transmembrane domain of CD8 comprises a transmembrane domain that is CD8 a. In certain embodiments, the transmembrane domain of CD8 comprises the amino acid sequence shown in SEQ ID NO. 158. In certain embodiments, the transmembrane domain comprises the transmembrane domain of CD28. In certain embodiments, the transmembrane domain of CD28 comprises the amino acid sequence set forth in SEQ ID NO: 285. In certain embodiments, the transmembrane domain comprises the transmembrane domain of ICOS. In certain embodiments, the transmembrane domain of ICOS comprises the amino acid sequence set forth in SEQ ID NO: 286.

The transmembrane domain described herein can be combined with any antigen binding domain described herein, any intracellular domain described herein, or any other domain described herein that can be included in the subject CAR.

In embodiments, the transmembrane domain further comprises a hinge region. The subject CAR of the present invention may also include a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen binding domain and the transmembrane domain. In embodiments, the domain facilitates proper protein folding of the CAR. The hinge region is an optional component of the CAR. The hinge region may comprise a domain selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, CD8 a hinge, CD8 β hinge, CD28 hinge, 4-1BB hinge, CD7 hinge, artificial hinge made from polypeptides that may be as small as three glycine (Gly), and CHI and CH3 domains of IgG (such as human IgG 4). Naturally occurring hinge domains can be used as wild-type hinge regions, or molecules can be altered.

In embodiments, the subject CARs of the present disclosure include a hinge region coupling an antigen binding domain to a transmembrane domain, which in turn is coupled to one or more intracellular domains. The hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to a target antigen on a target cell (see, e.g., hudecek et al, cancer immunol. Res. (2015) 3 (2): 125-135). In embodiments, the hinge region is a flexible domain, allowing the antigen binding domain to have a structure that optimally recognizes the specific structure and density of a target antigen on a cell, such as a tumor cell (Hudecek et al, previously described). The flexibility of the hinge region allows the hinge region to adopt a number of different conformations. In embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD 8-derived hinge region).

The hinge region can be about 4 amino acids to about 50 amino acids in length, such as about 4aa to about 10aa, about 10aa to about 15aa, about 15aa to about 20aa, about 20aa to about 25aa, about 25aa to about 30aa, about 30aa to about 40aa, or about 40aa to about 50aa. In embodiments, the hinge region may be greater than 5aa, greater than 10aa, greater than 15aa, greater than 20aa, greater than 25aa, greater than 30aa, greater than 35aa, greater than 40aa, greater than 45aa, greater than 50aa, greater than 55aa, or greater in length.

Suitable hinge regions can be readily selected and can be any of a number of suitable lengths, such as 1 amino acid (e.g., gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1,2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

For example, the hinge region includes glycine polymer (G) n, glycine-serine polymer (including, for example, (GS) n, (GSGGS) n (SEQ ID NO: 275) and (GGGS) n (SEQ ID NO: 276), where n is an integer of at least one), glycine-alanine polymer, alanine-serine polymer, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used, and both Gly and Ser are relatively unstructured and therefore can serve as neutral tethers between components. Glycine polymers can be used, glycine is significantly more steric than even alanine can enter phi-psi and is much less restricted than residues with longer side chains (see e.g., scheraga, rev. Computational. Chem. (1992) 2:73-142). Exemplary hinge regions can comprise amino acid sequences including, but not limited to GGSG(SEQ ID NO:278)、GGSGG(SEQ ID NO:279)、GSGSG(SEQ ID NO:280)、GSGGG(SEQ ID NO:281)、GGGSG(SEQ ID NO:282)、GSSSG(SEQ ID NO:283) and the like.

In embodiments, the hinge region is an immunoglobulin heavy chain hinge region. The amino acid sequence of the immunoglobulin hinge region is known in the art, see, e.g., tan et al, proc. Natl. Acad. Sci. USA (1990) 87 (1): 162-166, and Huck et al, nucleic Acids Res (1986) 14 (4): 1779-1789. As non-limiting examples, the immunoglobulin hinge region may comprise one of the following amino acid sequences DKTHT (SEQ ID NO: 287), CPPC (SEQ ID NO: 288), CPEPKSCDTPPPCPR (SEQ ID NO: 289) (see, e.g., glaser et al ,J.Biol.Chem.(2005)280:41494-41503);ELKTPLGDTTHT(SEQ ID NO:290);KSCDKTHTCP(SEQ ID NO:291);KCCVDCP(SEQ ID NO:292);KYGPPCP(SEQ ID NO:293);EPKSCDKTHTCPPCP(SEQ ID NO:294)( human IgGl hinge), ERKCCVECPPCP (SEQ ID NO: 295) (human IgG2 hinge), ELKTPLGDTTHTCPRCP (SEQ ID NO: 296) (human IgG3 hinge), SPNMVPHAHHAQ (SEQ ID NO: 297) (human IgG4 hinge), and so forth.

The hinge region may comprise the amino acid sequence of a human IgG1, igG2, igG3 or IgG4 hinge region. In one embodiment, the hinge region may comprise one or more amino acid substitutions and/or insertions and/or deletions compared to the wild-type (naturally occurring) hinge region. For example, his229 of a human IgG1 hinge may be substituted with Tyr such that the hinge region comprises sequence EPKSCDKTYTCPPCP (SEQ ID NO: 298); see, e.g., yan et al, J.biol. Chem. (2012) 287:5891-5897.

In certain embodiments, the hinge region may comprise an amino acid sequence derived from human CD8 or a variant thereof. In certain embodiments, the CAR comprises a CD8 alpha hinge sequence comprising the amino acid sequence set forth in SEQ ID NO. 156. In certain embodiments, the CAR comprises a hinge and transmembrane domain sequence comprising the amino acid sequence shown in SEQ ID NO. 160.

3. Co-stimulatory domains

In embodiments, a CAR encoded by a nucleic acid may further comprise at least one costimulatory domain, wherein the costimulatory domain comprises a functional costimulatory signaling domain derived from, for example, an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activated NK cell receptor, BTLA, a Toll ligand receptor, or the like. For example, within the scope of the present disclosure, a CAR may comprise 2, 3, 4, or more co-stimulatory domains. It is also within the scope of the present disclosure that when more than one co-stimulatory domain is included, the co-stimulatory domains may be the same, or they may be different. In embodiments, the co-stimulatory domain is derived from one or more of ：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、B7-H3、CEACAM1、CRTAM、CD2、CD3C、CD4、CD7、CD8α、CD8β、CD11a、CD11b、CD11c、CD11d、IL2Rβ、IL2γ、IL7Rα、IL4R、IL7R、IL15R、IL21R、CD18、CD19、CD19a、CD27、CD28、CD29、CD30、CD40、CDS、CD49a、CD49D、CD49f、CD54(ICAM)、CD69、CD70、CD80、CD83、CD84、CD86、CD96(Tactile)、CD100(SEMA4D)、CD103、CD134(OX40)、CD137(4-1BB)、CD152(CTLA-4)、CD160(BY55)、CD162(SELPLG)、CD244(2B4)、CD270(HVEM)、CD226(DNAM1)、CD229(Ly9)、CD278(ICOS)、ICAM-1、LFA-1(CD11a/CD18)、FcR、FcγRI、FcγRII、FcγRIII、LAT、NKG2C、SLP76、TRIM、ZAP70、GITR、BAFFR、LTBR、LAT、GADS、LIGHT、HVEM(LIGHTR)、KIRDS2、ITGA4、ITGA6、ITGAD、ITGAE、ITGAL、ITGAM、ITGAX、ITGB1、ITGB2、ITGB7、NKG2C、NKG2D、IA4、VLA-1、VLA-6、SLAM(SLAMF1、CD150、IPO-3)、SLAMF4、SLAMF6(NTB-A、Ly108)、SLAMF7、SLAMF8(BLAME)、SLP-76、PAG/Cbp、NKp80(KLRF1)、NKp44、NKp30、NKp46、BTLA、JAML、CD150、PSGL1、TSLP、TNFR2 and TRANCE/RANKL or parts thereof and combinations thereof.

In embodiments, the nucleic acid encoding the CAR encodes at least one 4-1BB co-stimulatory domain, and optionally encodes a second co-stimulatory domain selected from the group consisting of 4-1BB, 2B4, ICOS, CD28, OX40 and CD27 co-stimulatory domains or any of the co-stimulatory domains described above. In embodiments, the nucleic acid encodes at least two 4-1BB costimulatory domains, or a combination of at least two 4-1BB costimulatory domains with one, two, three, or four or more costimulatory domains selected from the group consisting of 4-1BB, ICOS, CD, OX40, and CD27, or any of the foregoing costimulatory domains. In embodiments, the 4-1BB co-stimulatory domain comprises the amino acid sequence set forth in SEQ ID NO. 162. In embodiments, the 4-1BB co-stimulatory domain comprises at least one, at least two, or at least three or more modified amino acid sequences having the amino acid sequence of SEQ ID NO. 162. In embodiments, the 4-1BB co-stimulatory domain is substantially similar to the 4-1BB co-stimulatory domain comprising SEQ ID NO. 162.

In embodiments, the nucleic acid encoding the CAR encodes at least one CD27 co-stimulatory domain, and optionally at least one second co-stimulatory domain selected from the group consisting of 4-1BB, ICOS, CD, OX40, 2B4 and CD27 co-stimulatory domains, or any of the co-stimulatory domains described above. In embodiments, the nucleic acid encodes at least one CD27 co-stimulatory domain and a 4-IBB co-stimulatory domain. In embodiments, the nucleic acid encodes two CD27 co-stimulatory domains, and at least one second co-stimulatory domain selected from the group consisting of 4-1BB, ICOS, CD and CD 27. In an embodiment, the CD27 co-stimulatory domain comprises SEQ ID NO 39. In embodiments, the CD27 co-stimulatory domain comprises at least one, at least two, at least three or more modified amino acid sequences having the amino acid sequence of SEQ ID NO. 300. In embodiments, the CD27 co-stimulatory domain is substantially similar to the CD27 co-stimulatory domain comprising SEQ ID NO. 300.

In embodiments, the nucleic acid encoding the CAR encodes at least one CD28 co-stimulatory domain, and optionally encodes a second co-stimulatory domain selected from the group consisting of 4-1BB, 2B4, ICOS, CD28, OX40 and CD27 co-stimulatory domains or any of the co-stimulatory domains mentioned above. In embodiments, the nucleic acid encodes at least two CD28 co-stimulatory domains, or a combination of at least two CD28 co-stimulatory domains with one, two, three, or four or more co-stimulatory domains selected from 4-1BB, ICOS, CD, OX40 and CD27 or any of the above co-stimulatory domains. In an embodiment, the CD28 co-stimulatory domain comprises SEQ ID NO. 254. In an embodiment, the CD28 co-stimulatory domain comprises SEQ ID NO. 301. Included in SEQ ID No. 254 and SEQ ID No. 301 are three subdomains YMNM, PRRP and PYAP, which are capable of modulating signaling pathways. In embodiments, the disclosed CARs comprise a mutation or deletion of one or more of the subdomains (see, e.g., WO 2019010383). In embodiments, the CD28 co-stimulatory domain comprises at least one, at least two, at least three or more modified amino acid sequences having the amino acid sequence of SEQ ID NO:254 or the amino acid sequence of SEQ ID NO:301. In embodiments, the CD28 co-stimulatory domain is substantially similar to the CD28 co-stimulatory domain comprising SEQ ID NO. 254. In embodiments, the CD28 co-stimulatory domain is substantially similar to the CD28 co-stimulatory domain comprising SEQ ID NO. 301.

In embodiments, the nucleic acid encoding the CAR encodes at least one ICOS costimulatory domain, and optionally encodes a second costimulatory domain selected from the group consisting of 4-1BB, 2B4, ICOS, CD28, OX40, and CD27 costimulatory domains, or any of the costimulatory domains described above. In embodiments, the nucleic acid encodes at least two ICOS co-stimulatory domains, or a combination of at least two ICOS co-stimulatory domains with one, two, three, or four, or more co-stimulatory domains selected from 4-1BB, ICOS, CD, OX40 and CD27, or any of the above co-stimulatory domains. In an embodiment, the ICOS co-stimulatory domain comprises SEQ ID NO. 255. In embodiments, the ICOS costimulatory domain comprises at least one, at least two, at least three, or more modified amino acid sequences having the amino acid sequence of SEQ ID NO:255 (see, e.g., US 20170209492). In an embodiment, the ICOS co-stimulatory domain is substantially similar to the ICOS co-stimulatory domain comprising SEQ ID NO. 255.

In embodiments, the nucleic acid encoding the CAR encodes at least one OX40 co-stimulatory domain, and optionally encodes a second co-stimulatory domain selected from the group consisting of 4-1BB, 2B4, ICOS, CD28, OX40 and CD27 co-stimulatory domains or any of the co-stimulatory domains described above. In embodiments, the nucleic acid encodes at least two OX40 co-stimulatory domains, or a combination of at least two OX40 co-stimulatory domains with one, two, three, or four or more co-stimulatory domains selected from 4-1BB, ICOS, CD, OX40 and CD27, or any of the above co-stimulatory domains. In an embodiment, the OX40 co-stimulatory domain comprises SEQ ID NO. 256. In embodiments, the OX40 co-stimulatory domain comprises at least one, at least two, at least three or more modified amino acid sequences having the amino acid sequence of SEQ ID NO. 256. In embodiments, the OX40 co-stimulatory domain is substantially similar to an OX40 co-stimulatory domain comprising SEQ ID NO. 256.

4. Intracellular signaling domains

In embodiments, the nucleic acid encoding the CAR encodes at least one intracellular signaling domain. In embodiments, at least one intracellular signaling domain is outside of one or more co-stimulatory domains. In embodiments, one or more intracellular signaling domains are included to increase proliferation, persistence, and/or cytotoxic activity of host cells, preferably γδ cells, containing a CAR as disclosed herein. For example, in some embodiments, the intracellular signaling domain comprises a cd3ζ, a repeat (e.g., 2-5) DAP10 YINM motif, a signaling domain derived from LFA-1, DAP12, fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, CD79a, CD79b, CD5, CD22, fceri, CD66d, and the like. Within the scope of this disclosure, the intracellular domains of the disclosed CARs can include multiple (e.g., 2,3, 4, or more) intracellular signaling domains. Where more than one intracellular signaling domain is included, the intracellular signaling domains may be the same, or they may be different.

In embodiments, the intracellular signaling domain of the disclosed CARs is or comprises a CD3 zeta signaling domain. In embodiments, the CD3 zeta signaling domain is or comprises the amino acid sequence as set forth in SEQ ID NO. 164, 166 or 167.

5. Additional polypeptides

In embodiments, an isolated nucleic acid encoding a CAR of the invention can further encode one or more polycistronic linker regions configured to facilitate translation of the CAR polypeptide and one or more additional polypeptides. In embodiments, the nucleic acid encoding one or more additional polypeptides and associated linker regions may be located at the 3 'end of the isolated nucleic acid, or at the 5' end of the isolated nucleic acid, or in some instances at the 5 'and 3' ends of the isolated nucleic acid. In some embodiments, the linker region may encode a self-cleaving and/or cleaving polypeptide sequence. In some examples, the self-cleaving sequence is a 2A self-cleaving sequence (e.g., T2A, P2A, E2A, F a) that can induce ribosome jump during translation of the CAR. In embodiments, the cleavage sequence is a furin (furin) sequence. In some examples, the cleavage sequence (e.g., the furin cleavage sequence shown as SEQ ID NO: 242) is the amino terminus of a self-cleavage sequence such as furin-P2A (FP 2A). In embodiments, the polycistronic linker region encodes an internal ribosome entry site. In embodiments, the addition of an optional linker "GSG" or "SGSG" or the like can improve cleavage efficiency. In this way, one or more additional polypeptides can be released from the CAR and directed to the secretory pathway.

In an embodiment, the cleavage sequence is the FP2A amino acid sequence as shown in SEQ ID NO. 236. In embodiments, the cleavage sequence is the P2A amino acid sequence as shown in SEQ ID NO 238 or SEQ ID NO 240-241. In an embodiment, the cleavage sequence is the furin amino acid sequence shown as SEQ ID NO. 242. In embodiments, the cleavage sequence is the F2A amino acid sequence as shown in SEQ ID NO 243. In an embodiment, the cleavage sequence is the E2A amino acid sequence as shown in SEQ ID NO. 244. In an embodiment, the cleavage sequence is a T2A amino acid sequence as set forth in SEQ ID NO. 245. In certain aspects, multiple cleavage and/or self-cleavage sequences can be encoded at the carboxy-terminus of the signaling and/or costimulatory domain and at the amino-terminus of the encoded one or more additional polypeptides. In certain aspects, one or more self-cleaving sequences and one or more sequences cleaved by an endogenous protease are encoded in the constructs described herein. In certain embodiments, the endogenous protease recognition site is encoded at the amino terminus of the self-cleaving sequence.

In embodiments, the polycistronic linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by the nucleotide sequence shown as SEQ ID NO. 246. Another exemplary internal ribosome entry site is encoded by the nucleotide sequence shown as SEQ ID NO. 247. Other suitable internal ribosome entry sites include, but are not limited to, nucleic Acids Res.2010Jan;38 (database stage): D131-6.Doi:10.1093/nar/gkp981.Epub 2009nov 16, those described by iresite. Org, those described in WO2018/215787, sequences described in GenBank accession No. KP019382.1, and IRES elements disclosed in GenBank accession No. LT 727339.1. Additional polycistronic linker regions, including cleavage, self-cleavage and IRES elements, are disclosed in U.S. Pat. No. 8,865,467 and U.S. 2018/0360992.

In embodiments, the one or more additional polypeptides include one or more soluble gamma chain cytokines expressed as isolated polypeptides from the CAR. The one or more soluble common gamma chain cytokines may include, but are not limited to, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IL-23. In embodiments, the common gamma chain cytokine is selected from the group consisting of IL-2, IL-7 and IL-15. In embodiments, the common gamma chain cytokine is IL-15.IL-15 sequences, including codon-optimized nucleic acid sequences encoding soluble IL-15 (sIL-15), are disclosed herein and in WO 2007/037780.

In embodiments, the one or more additional polypeptides include one or more tags or markers, e.g., to facilitate the ability to monitor the expression level of the CAR, to serve as an internal control, etc. In embodiments, the isolated nucleic acid encoding the CAR encodes a fluorescent protein, examples of which include, but are not limited to, green Fluorescent Protein (GFP), red Fluorescent Protein (RFP), enhanced GFP (EGFP), enhanced Cyan Fluorescent Protein (ECFP), enhanced Yellow Fluorescent Protein (EYFP), and the like. Other examples may include, but are not limited to, chloramphenicol acetyl transferase, beta-galactosidase, beta-glucuronidase, beta-lactamase, luciferase, and the like.

In embodiments, one or more additional polypeptides include a protein expressed on the cell surface to facilitate detection and/or isolation of cells expressing the protein, e.g., via Fluorescence Activated Cell Sorting (FACS), or for enrichment by forward selection using antibodies specific for the encoded protein, e.g., using antibodies to purify or enrich cell products on a column or device, or for in vivo binding of antibodies to proteins to enhance or eliminate activity, e.g., to facilitate removal of cells expressing the protein in a patient for safety concerns. Exemplary proteins for these purposes include, for example, CD19, CD20 (rituximab (Rituxumab) recognition domain), RQR8, LNGFR, truncated forms of human epidermal growth factor receptor (EGFRt), and the like. By way of example, EGFRt may be targeted by a clinical stage antibody, wherein such treatment of a patient with the antibody results in the elimination of cells containing an isolated nucleic acid encoding a CAR and/or the CAR as disclosed herein. See, e.g., wang et al, a genetically encoded cell surface polypeptide for selection, in vivo tracking and ablation of engineered cells, blood 2011 118 (5): 1255-63; philip et al, a highly compact epitope-based marker/suicide gene for simpler, safer T cell therapies, blood 2014 124 (8): 1277-87; smith J. Et al, UCART, an allogeneic "ready" adoptive T cell immunotherapy ;DOI:10.1200/jco.2015.33.15_suppl.3069Journal of Clinical Oncology 33,no.15_suppl(May 20,2015)3069-3069;Gouble A. et al, UCART19 for CD19+ B cell leukemia, an in vivo validation of the activity and safety concepts of "ready" adoptive T cell immunotherapy for CD19+ B cell leukemia, blood (2014) 124 (21): 4689, doi.org/10.1182/blood.V124.21.4689.4689, each of which is incorporated by reference in its entirety.

In embodiments, the one or more additional polypeptides include proteins having the function of increasing resistance to depletion and activation-induced apoptosis and/or up-regulating one or more pro-inflammatory cytokines, co-stimulatory molecules, and/or antigen presentation mechanisms. Representative examples include, but are not limited to, lymphotoxin beta receptor (LTBR). LTBR is typically expressed in a subset of myeloid lineage cells, but is not present in lymphocytes. When expressed in T cells, LTBR can induce transcriptional remodeling, conferring one or more of the above-described beneficial functions to T cells (Legut et al, blood. (2021); 138 (1): 1726).

In embodiments, the one or more additional polypeptides include polypeptides that confer upon the host cell the ability to resist tumor antigen-specific cellular immunity, such as cellular immunity mediated by transforming growth factor beta (TGF- β). For example, the isolated nucleic acid may encode a dominant negative receptor for TGF-beta (dnTGF βR2), e.g., as described in Foster et al, J ImmunotherA (2008); 31:500-505, WO2019/173324A1, WO2020/183131A1, and WO2020042647A 1. The incorporation of such TGF-beta dominant negative receptors in the presence of TGF-beta secreting tumors may provide functional advantages, including enhanced anti-tumor activity, relative to control cells lacking such TGF-beta dominant negative receptors.

In some embodiments, the isolated nucleic acid encodes a signal peptide operably linked to facilitate the directing of one or more additional polypeptides to the secretory pathway. Such one or more additional polypeptides may be polypeptides located inside certain organelles, secreted from the host cell, or inserted into the cell membrane. In embodiments, the signal peptide comprises or consists of the amino acid sequence shown as SEQ ID NO. 152. In embodiments, the signal peptide comprises or consists of the amino acid sequence shown as SEQ ID NO. 248. In embodiments, the signal peptide comprises or consists of the amino acid sequence shown as SEQ ID NO. 259. In an embodiment, the signal peptide comprises or consists of the amino acid sequence shown as SEQ ID NO. 263. In an embodiment, the signal peptide comprises or consists of the amino acid sequence shown as SEQ ID NO 267. In an embodiment, the signal peptide comprises or consists of the amino acid sequence shown as SEQ ID NO: 271.

In embodiments, one or more additional polypeptides comprise or consist of the EGFRt amino acid sequence shown as SEQ ID NO: 261. In embodiments, one or more additional polypeptides comprise or consist of the GMCSFR amino acid sequence set forth in SEQ ID NO. 260. In embodiments, a signal peptide comprising or consisting of the amino acid sequence shown as SEQ ID NO. 259 is operably linked to SEQ ID NO. 260. In embodiments, one or more additional polypeptides comprise or consist of the amino acid sequence of dominant negative TGF-beta receptor II (dnTGF. Beta.R2) as set forth in SEQ ID NO. 265. In embodiments, a signal peptide comprising or consisting of the amino acid sequence shown as SEQ ID NO. 263 is operably linked to SEQ ID NO. 265. In embodiments, one or more additional polypeptides comprise the full-length LTBR amino acid sequence shown as SEQ ID NO:269. In embodiments, a signal peptide comprising or consisting of the amino acid sequence shown as SEQ ID NO. 267 is operably linked to SEQ ID NO. 269. In embodiments, one or more additional polypeptides comprise an LNGFR amino acid sequence as set forth in SEQ ID NO. 273. In embodiments, a signal peptide comprising or consisting of the amino acid sequence shown as SEQ ID NO:271 is operably linked to SEQ ID NO:273. In embodiments, one or more additional polypeptides comprise the amino acid sequence of sIL-15 as set forth in SEQ ID NO. 249. In embodiments, a signal peptide comprising or consisting of the amino acid sequence shown as SEQ ID NO. 248 is operably linked to SEQ ID NO. 249.

In embodiments, one or more additional polypeptides include a chimeric switch receptor comprising an extracellular domain of a tgfβ receptor and an intracellular domain of a cytokine receptor for binding to tgfβ (e.g., tgfβri and/or tgfβrii). Chimeric switch receptors can convert tgfβ signaling into cytokine signaling that promotes cytotoxicity. Examples of such chimeric switch receptors include those described in WO2012138858, WO2016122738, WO2018094244, WO2014172584, WO2019109980 and WO2022037562, which are incorporated by reference in their entirety.

In embodiments, the one or more additional polypeptides comprise dominant negative Fas (dnFas). Incorporation of such dominant negative Fas in T cells can provide a functional advantage over control cells lacking such dominant negative Fas, can prevent Fas ligand-induced apoptosis and allow T cells to persist and have anti-tumor efficacy. Examples of dnFas include those described in Yamamoto TN et al, ,T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy,J Clin Invest.2019, 25, 2, and 129 (4): 1551-1565, which are incorporated herein by reference in their entirety.

In embodiments, one or more additional polypeptides include membrane-bound IL-12 (mbiL-12). The incorporation of such mbIL-12 in T cells may provide functional advantages over control cells lacking such mbIL-12, may enhance effector functions of T cells and/or limit systemic toxicity associated with IL-12. Examples of mbIL-12 include those described in Hu j. Et al ,Cell membrane-anchored and tumor-targeted IL-12(attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors,J Immunother Cancer.2022, month 1, 10 (1): e003633, hombach a. Et al ,IL12 integrated into the CAR exodomain converts CD8+T cells to poly-functional NK-like cells with superior killing of antigen-loss tumors,Mol Ther.2022, month 2, day, 30 (2): 593-605, and Lee EH et al ,Antigen-dependent IL-12signaling in CAR T cells promotes regional to systemic disease targeting,bioRxiv.2023, month 1, 7, and 2023.01.06.522784, each of which are incorporated herein by reference in their entirety.

In embodiments, the one or more additional polypeptides include an antibody or fragment thereof that binds to CD70, or a CAR comprising such an antibody or fragment. Incorporation of such a CD70 binding molecule in a T cell may provide a functional advantage over control cells lacking the CD70 binding molecule, and HvG alloreactivity may be reduced by targeting cd70+ activated T cells. Examples of such CD70 binding molecules include those described in PCT/US2023/29047, which is incorporated herein by reference in its entirety.

6. Exemplary CAR

The invention provides nucleic acid molecules encoding one or more CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

In some embodiments, the isolated nucleic acid encodes SEQ ID NO 204, a CAR polypeptide PL805 comprising, in order, a signal peptide, a PSMA binding domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

In an embodiment, the nucleic acid encoding PL805 CAR comprises the sequence of SEQ ID No. 205. Table 2 below provides an annotation of the nucleotide sequence of SEQ ID NO. 205.

In some embodiments, the isolated nucleic acid encodes SEQ ID NO 208, a CAR polypeptide PL880 comprising, in order, a signal peptide, a PSMA binding domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

In an embodiment, the nucleic acid encoding PL880 CAR comprises the sequence of SEQ ID No. 209. Table 3 below provides an annotation of the nucleotide sequence of SEQ ID NO. 209.

In some embodiments, the isolated nucleic acid encodes SEQ ID NO. 212, CAR polypeptide PL1027 comprising, in order, a signal peptide, a PSMA binding domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

In an embodiment, the nucleic acid encoding PL1027 CAR comprises the sequence of SEQ ID NO: 213. Table 4 below provides an annotation of the nucleotide sequence of SEQ ID NO: 213.

In some embodiments, the isolated nucleic acid encodes SEQ ID NO 216, a CAR polypeptide PL1028 comprising, in order, a signal peptide, a PSMA binding domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

In an embodiment, the nucleic acid encoding the PL1028 CAR comprises the sequence of SEQ ID NO: 217. Table 5 below provides an annotation of the nucleotide sequence of SEQ ID NO: 217.

In some embodiments, the isolated nucleic acid encodes SEQ ID NO 220, a CAR polypeptide PL1042 comprising, in order, a signal peptide, a PSMA binding domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

In an embodiment, the nucleic acid encoding PL1042 CAR comprises the sequence of SEQ ID No. 221. Table 6 below provides an annotation of the nucleotide sequence of SEQ ID NO: 221.

In some embodiments, the isolated nucleic acid encodes SEQ ID NO 224, a CAR polypeptide PL1045 comprising, in order, a signal peptide, a PSMA binding domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

In an embodiment, the nucleic acid encoding PL1045 CAR comprises the sequence of SEQ ID No. 225. Table 7 below provides an annotation of the nucleotide sequence of SEQ ID NO: 225.

In some embodiments, the isolated nucleic acid encodes SEQ ID NO 228, a CAR polypeptide PL1049 comprising, in order, a signal peptide, a PSMA binding domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

In an embodiment, the nucleic acid encoding a PL1049 CAR comprises the sequence of SEQ ID No. 229. Table 8 below provides an annotation of the nucleotide sequence of SEQ ID NO: 229.

In some embodiments, the isolated nucleic acid encodes SEQ ID NO 232, a CAR polypeptide PL1062 comprising, in order, a signal peptide, a PSMA binding domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain.

In an embodiment, the nucleic acid encoding the PL1062 CAR comprises the sequence of SEQ ID NO: 233. Table 9 below provides an annotation of the nucleotide sequence of SEQ ID NO: 233.

The CAR and nucleic acids encoding the CAR comprise a specific signal peptide. In embodiments, it may be desirable to replace one signal peptide with another signal peptide in a CAR of the present disclosure. Thus, in embodiments, the isolated nucleic acid comprising SEQ ID NO. 207 encodes SEQ ID NO. 206 comprising PL805 minus a signal peptide, the isolated nucleic acid comprising SEQ ID NO. 211 encodes SEQ ID NO. 210 comprising PL880 minus a signal peptide, the isolated nucleic acid comprising SEQ ID NO. 215 encodes SEQ ID NO. 214 comprising PL1027 minus a signal peptide, the isolated nucleic acid comprising SEQ ID NO. 219 encodes SEQ ID NO. 218 comprising PL1028 minus a signal peptide, the nucleic acid comprising SEQ ID NO. 223 encodes SEQ ID NO. 222 comprising PL1042 minus a signal peptide, the isolated nucleic acid comprising SEQ ID NO. 227 encodes SEQ ID NO. 226 comprising PL1045 minus a signal peptide, the nucleic acid comprising SEQ ID NO. 231 encodes SEQ ID NO. 230 comprising PL1049 minus a signal peptide, or the isolated nucleic acid comprising SEQ ID NO. 235 encodes SEQ ID NO. 234 comprising PL1062 minus a signal peptide.

Any of the above-described isolated nucleic acids encoding a particular CAR polypeptide can further encode one or more additional polypeptides, as discussed herein. For example, but not limited to, any of the above-described nucleic acids encoding a particular CAR can comprise at least one polycistronic linker and a polynucleic acid encoding a dnTGF βr2 polypeptide.

7. Carrier body

The invention encompasses DNA constructs comprising a CAR sequence. Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, for example, by screening libraries from cells expressing the genes, by obtaining the genes from vectors known to contain the genes, or by direct isolation from cells and tissues containing the same using standard techniques. Alternatively, the gene of interest may be synthetically produced rather than cloned.

The present invention provides a vector into which the DNA of the present invention is inserted. Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer, as they allow for long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors offer advantages over vectors derived from tumor retroviruses (e.g., murine leukemia virus) because they transduce non-proliferating cells, such as hepatocytes. They also have the advantage of increased low immunogenicity.

In another embodiment, the vector comprising a nucleic acid encoding a desired CAR of the invention is an adenovirus vector (A5/35). In another embodiment, expression of nucleic acids encoding the CAR can be achieved using transposons (such as sleeping beauty, crisper, CAS9, and zinc finger nucleases).

Briefly, expression of a natural or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide or portion thereof to a promoter and incorporating the construct into an expression vector. The vectors may be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequences.

The expression constructs of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art (e.g., U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety). In another embodiment, the invention provides a gene therapy vector.

Nucleic acids can be cloned into various types of vectors. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe-generating vectors and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors contain an origin of replication that is functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193). Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene transfer systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to cells of the subject in vivo or ex vivo. Many retroviral systems are known in the art. In embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110bp upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is typically flexible so that promoter function is preserved when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50bp before the activity begins to decrease. Depending on the promoter, it appears that individual elements may act synergistically or independently to activate transcription.

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence to which it is operably linked. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV 40) early promoter, mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, moMuLV promoter, avian leukemia virus promoter, ai Baer immediate early promoter, rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of an operably linked polynucleotide sequence when such expression is desired or turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess expression of the CAR polypeptide or portion thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both, to facilitate identification and selection of the expressing cell from a population of cells that are attempted to be transfected or infected by the viral vector. In other aspects, selectable markers may be carried on separate DNA fragments and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue and encodes a polypeptide whose expression is expressed by some readily detectable property (e.g., enzymatic activity). The expression of the reporter gene is determined at a suitable time after the DNA has been introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., ui-Tei et al 2000FEBS Letters479:79-82). Suitable expression systems are well known and may be prepared using known techniques or commercially available. In general, constructs with minimal 5' flanking regions that show the highest expression levels of the reporter gene are identified as promoters. Such promoter regions may be linked to a reporter gene and used to assess the ability of an agent to regulate promoter-driven transcription.

Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors may be readily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York). One method of introducing polynucleotides into host cells is calcium phosphate transfection.

Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammals (e.g., human cells). Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. Nucleic acids associated with a lipid can be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, linked to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing the lipid, mixed with the lipid, combined with the lipid, contained in the lipid as a suspension, contained in a micelle, or complexed with or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector-related composition is not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or have a "collapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include aliphatic droplets naturally occurring in the cytoplasm as well as a class of compounds containing long chain aliphatic hydrocarbons and derivatives thereof (e.g., fatty acids, alcohols, amines, amino alcohols, and aldehydes).

Suitable lipids may be obtained from commercial sources. For example, dimyristoyl phosphatidylcholine ("DMPC") is available from Sigma, st.louis, mo., dimyristoyl phosphate ("DCP") is available from K & K Laboratories (plaiview, n.y.), cholesterol ("Choi") is available from Calbiochem-Behring, dimyristoyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, inc. (Birmingham, AL). A stock solution of lipids in chloroform or chloroform/methanol can be stored at about-20 ℃. Chloroform is used as the only solvent because it evaporates more readily than methanol. "liposomes" is a generic term covering a variety of unilamellar and multilamellar lipid vehicles formed by the creation of a closed lipid bilayer or aggregate. Liposomes can be characterized as having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. Phospholipids spontaneously form when suspended in excess aqueous solution. The lipid module undergoes self-rearrangement before a closed structure is formed and entraps water and dissolved solutes between the lipid bilayers (Ghosh et al, 1991glycobiology 5:505-10). However, compositions having a structure in solution that is different from the normal vesicle structure are also contemplated. For example, the lipid may exhibit a micelle structure, or exist only as heterogeneous aggregates of lipid molecules. Cationic liposome (lipofectamine) -nucleic acid complexes are also contemplated.

Regardless of the method used to introduce exogenous nucleic acid into a host cell or expose the cell to the inhibitors of the invention, a variety of assays can be performed in order to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those of skill in the art, such as Southern and Northern blots, RT-PCR and PCR, "biochemical" assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological methods (ELISA and Western blot) or by assays described herein to confirm agents falling within the scope of the invention.

8. Host cells

The CAR polypeptides of the present disclosure can be expressed in a variety of host cells via their corresponding nucleic acid constructs. In embodiments, the host cell is a mammalian cell. Host cells as described herein may be stored, e.g., cryopreserved, for adoptive cell transfer. In embodiments, the host cell is stored prior to engineering the cell to express the CAR polypeptide. In embodiments, the cells are engineered to express the CAR polypeptide, and then the cells are stored.

Preferred host cells for use with CAD polypeptides and chimeric receptors of the disclosure include immune cells. Such cells may be obtained from the subject to be treated (i.e., autologous), or alternatively an immune cell line or donor immune cell (allogeneic, syngeneic) may be used. Immune cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune cells can be obtained from blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll ^TM isolation. For example, cells in the circulating blood of an individual may be obtained by apheresis. In embodiments, immune cells are isolated from peripheral blood lymphocytes by lysing the erythrocytes and depleting monocytes, e.g., by PERCOLL ^TM gradient centrifugation or by countercurrent centrifugation. Specific subpopulations of immune cells can be further isolated by positive or negative selection techniques. For example, immune cells can be isolated using a combination of antibodies directed against surface markers characteristic of positively selected cells, e.g., by incubation with antibody-conjugated beads, for a time sufficient to positively select for the desired immune cells. Alternatively, enrichment of immune cell populations can be achieved by negative selection using a combination of antibodies directed against surface markers specific for the negative selection cells. Other embodiments of separation and/or enrichment are disclosed herein.

In embodiments, immune cells include any white blood cells that are involved in protecting the body from infectious diseases and foreign substances. For example, the immune cells may include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune cells related to the present disclosure may include, but are not limited to, αβ T cells, γδ T cells, NK cells, NKT cells, γδ NKT cells, B cells, congenital lymphoid cells (ILCs), cytokine-induced killing (CIK) cells, cytotoxic T Lymphocytes (CTLs), lymphokine-activated killing (LAK) cells, regulatory T cells, and the like. In embodiments, preferred immune cells include αβ T cells, γδ T cells, NK cells, NKT cells, γδ NKT cells, and/or macrophages in some examples. In embodiments, preferred immune cells include γδ T cells. In embodiments, immune cells related to the present disclosure include allogeneic cells, autologous cells, or syngeneic cells.

Thus, aspects of the invention include a host cell (in some preferred embodiments, γδ T cells) that functionally expresses the isolated nucleic acids described herein and thereby expresses the CAR on the surface of the cell.

Aspects of the invention may additionally or alternatively include host cells, preferably γδ T cells, having in vitro or in vivo cytotoxic activity against tumor cells exhibiting PSMA cell surface expression.

In some cases, the cytotoxic activity is a congenital activity. In some cases, cytotoxicity is due at least in part, significantly (> about 25%) or entirely to the presence of a CAR construct having a binding domain that specifically binds PSMA expressed on the surface of tumor cells. In some cases, the host cell, preferably a γδ T cell, exhibits a tumor cell killing activity that is greater than the congenital level of tumor cell killing activity in vitro and/or in vivo in a control cell of the same cell type. In some cases, the control cells do not comprise a CAR construct. In some cases, the control cell comprises a CAR construct that lacks the binding domain described herein, the hinge region described herein, the transmembrane domain described herein, the intracellular signaling domain described herein, and/or the co-stimulatory intracellular domain described herein.

In some cases, cytotoxicity is due at least in part, significantly (> about 25%) or entirely to the presence of CAR constructs having a binding domain that specifically binds PSMA or an epitope within PSMA. In some cases, the host cell, preferably a γδ T cell, functionally expresses a PSMA-specific CAR encoded by an isolated nucleic acid described herein.

In embodiments in which the host cell is a γδ T cell, the γδ T cell can exhibit HLA-restricted (e.g., HLA class I-restricted) cytotoxicity. In other embodiments, most (> 50%), substantially all (> 90%), or all cytotoxic activity is not HLA-restricted (e.g., HLA class I-restricted). HLA-restricted cytotoxic activity can be assessed by comparing in vitro cytotoxicity against HLA (e.g., HLA class I) (null) tumor cell lines to in vitro cytotoxicity against hla+ (e.g., HLA class I ⁺) tumor cell lines. In embodiments, HLA-restricted cytotoxic activity is provided at least in part, significantly (> 25%) or entirely through the use of T cell receptor-like binding domains. A T cell receptor-like binding domain is a binding domain that specifically recognizes an antigen when presented in complex with MHC molecules on the cell surface. T cell receptor-like binding domains are further described, for example, in WO 2016/199141.

The host cells described herein, preferably γδ T cells, can exhibit robust and/or sustained tumor cell killing activity. In some cases, the tumor cell killing activity may last for at least about 6 days to 120 days, or at least about 6 days to 180 days, from the first time of contacting the tumor cells. In some cases, the tumor cell killing activity of a host cell, preferably a γδ T cell or its progeny, described herein can last for at least about 6 days to 120 days, or at least about 6 days to 180 days, from the first time of contacting the tumor cell, or from the administration of the host cell. This sustained tumor cell killing activity can be expressed in vitro, in vivo, or both in vitro and in vivo.

Aspects of the invention may additionally or alternatively include host cells, preferably γδ T cells, that proliferate in response to contact with cells exhibiting cell surface expression or overexpression of PSMA. The cells exhibiting PSMA cell surface expression or overexpression may be tumor cells or may be non-tumor cells. In some cases, proliferation is congenital activity. In some cases, the proliferation is due at least in part, significantly (> about 20% or > about 25%) or entirely to the presence of a CAR construct having a binding domain that specifically binds PSMA expressed on the surface of tumor cells. In some cases, host cells, preferably γδ T cells, exhibit higher levels of proliferation in vitro and/or in vivo than control cells of the same type. In some cases, the control cells do not comprise a CAR construct. In some cases, the control cell comprises a CAR construct that lacks the binding domain described herein, the hinge region described herein, the transmembrane domain described herein, the intracellular signaling domain described herein, and/or the co-stimulatory intracellular domain described herein.

The host cells, preferably γδ T cells, as described herein, exhibit robust and/or sustained proliferation in host organisms comprising cells (e.g., tumor cells) that exhibit cell surface expression or overexpression of PSMA. In some cases, proliferation may last for at least about 6 to 120 days, or for at least about 6 to 180 days, from the first time of contacting the tumor cells, or from the day of administration of the host cells, preferably γδ T cells, to the host organism. In some cases, the proliferation of host cells, preferably γδ T cells or progeny thereof, described herein in a host organism comprising cells exhibiting PSMA cell surface expression or overexpression may last for at least about 6 days to about 120 days, or for at least about 6 days to about 180 days, from the first time of contacting the PSMA expressing cells, or from the first time of administration of the host cells, preferably γδ T cells, to the host organism. In some cases, proliferation in the host organism is due at least in part, significantly (> about 20% or > about 25%) or entirely to the presence of a CAR construct having a binding domain that specifically binds PSMA or an epitope within PSMA. In some cases, a host organism comprising a cell exhibiting PSMA cell surface expression exhibits proliferation, preferably γδ T cells functionally express a PSMA-specific CAR encoded by an isolated nucleic acid described herein.

In embodiments, the host cells described herein, preferably γδ T cells, express or persist to express a pro-inflammatory cytokine, such as tumor necrosis factor α or interferon γ, upon contact with PSMA expressing cells. In embodiments, a host cell described herein, or a progeny thereof, expresses or continues to express a pro-inflammatory cytokine, such as tumor necrosis factor α or interferon gamma, upon contact with a PSMA expressing cell (e.g., in a host organism comprising a PSMA expressing cell).

In embodiments, the γδ T cells or pharmaceutical compositions containing γδ T cells exhibit substantially no or no graft versus host response when introduced into an allogeneic host. In embodiments, the γδ T cells or pharmaceutical compositions containing the γδ T cells exhibit clinically acceptable levels of graft versus host response when introduced into an allogeneic host. In embodiments, the clinically acceptable level is an amount of graft versus host response that does not require cessation of γδ T cell therapy to achieve a therapeutically effective treatment. In embodiments, the clinically acceptable level of graft versus host response (GvHD) is less severe than grade C acute response according to the applicable IBMTR classification scale. The severity of the acute graft versus host response is determined by assessing the extent of involvement of the skin, liver and gastrointestinal tract. The stages involved in each organ combine to produce an overall score, which is prognostic. Grade I (a) GvHD is characterized by mild disease, grade II (B) GvHD by moderate, grade III (C) by severe, and grade IV (D) life threatening. IBMTR the grading system defines the severity of acute GvHD as follows (Rowlings et al =, br J Haematol 1997; 97:855):

● Class a-stage 1 only skin involvement (maculopapules on <25% of the body), no liver or gastrointestinal involvement

● B-stage-2 stage cutaneous involvement, intestinal or liver involvement 1-2 stage

● Grade C-stage 3 of any organ system involvement (systemic erythroderma; bilirubin 6.1-15.0 mg/dL; diarrhea 1500-2000 mL/day)

● Grade D-stage 4 of any organ system involvement (systemic erythroderma with bullous formation; bilirubin >15mg/dL; diarrhea >2000 mL/day or pain or ileus).

See also Schoemans et al, volume Bone Marrow Transplantation, volume 53, pages 1401-1415 (2018), e.g., at tables 1 and 2, which references disclose criteria for assessing and grading acute GvHD.

In embodiments, the γδ T cells or pharmaceutical compositions comprising the γδ T cells exhibit a reduced or significantly reduced graft versus host response when introduced into an allogeneic host as compared to the graft versus host response exhibited by a control αβ T cell or a control pharmaceutical composition comprising the control αβ T cell administered to the allogeneic host. In some cases, the control αβ T cells are allogeneic non-engineered control αβ T cells. In some cases, the control αβ T cells do not contain a CAR or do not contain the same CAR as the reference γδ T cells.

In embodiments, the host cells described herein, preferably γδ T cells, can be modified to comprise one or more gene edits. The gene editing discussed herein is a genetic engineering in which nucleotides/nucleic acids are inserted, deleted and/or substituted in a DNA sequence (such as the genome of γδ T cells). Targeted gene editing can insert, delete and/or replace at preselected sites in the target cell genome. When editing an endogenous gene sequence, for example by deleting, inserting or substituting nucleotides/nucleic acids, the endogenous gene comprising the affected sequence may be knocked out or knocked down due to sequence changes. Thus, targeted editing can be used to disrupt endogenous gene expression. "disrupted gene" as discussed herein refers to a gene comprising an insertion, deletion or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. As used herein, "disruption gene" refers to a method of inserting, deleting, or substituting at least one nucleotide/nucleic acid in an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. Methods of disrupting genes are known to those skilled in the art and are described, for example, in U.S. patent No. 11254912, which is incorporated herein by reference in its entirety.

In embodiments, targeted gene editing of T cells may be performed using nuclease-dependent methods. This nuclease-dependent approach allows targeted editing by specific introduction of Double Strand Breaks (DSBs) by specific endonucleases. This nuclease-dependent targeted editing utilizes DNA repair mechanisms, such as non-homologous end joining (NHEJ), which occurs in response to DSBs. Repair of DNA by NHEJ typically results in random insertions or deletions (indels) of small amounts of endogenous nucleotides. In contrast to NHEJ-mediated repair, repair can also occur through Homology Directed Repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material. Useful endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided CRISPR-Cas9 nucleases (CRISPR/Cas 9; clustered regularly interspaced short palindromic repeat related 9). It is discussed herein that a CRISPR system or CRISPR nuclease system can include a non-coding RNA molecule (e.g., guide RNA) that binds DNA and a Cas protein (e.g., cas 9) with nuclease functionality (Sander et al, nature Biotechnology (2014); 32:347-355; hsu et al, cell (2014); 157 (6): 1262-1278).

In embodiments, the host cell, preferably a γδ T cell, comprises one or more disrupted genes. For example, the one or more genes whose expression is disrupted may include the adenosine A2a receptor (ADORA), CD276, a V-group domain containing T-cell activation inhibitor 1 (VTCN 1), B and T lymphocyte-associated (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA 4), indoleamine 2, 3-dioxygenase 1 (IDO 1), a killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR 3DL 1), lymphocyte activation gene 3 (LAG 3), programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR 2), a killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR 3DL 1), lymphocyte activation gene 3 (LAG 3), T cell activated V domain immunoglobulin inhibitor (VISTA), natural killer cell receptor 2B4 (CD 244), cytokine induced SH 2-containing protein (CISH), hypoxanthine phosphoribosyl transferase 1 (HPRT), adeno-associated virus integration site AAVSSITE (e.g., AAVS1, AAVS2, etc.), or chemokine (C-C motif) receptor 5 (gene/pseudogene) (CCR 5), CD160 molecule (CD 160), T cell immunoreceptor (TIGIT) with Ig and ITIM domains, CD96 molecule (CD 96), and, Cytotoxic and regulatory T cell molecules (CRTAM), leukocyte-associated immunoglobulin-like receptor 1 (LAIR 1), sialic acid binding Ig-like lectin 7 (SIGLEC 7), sialic acid binding Ig-like lectin 9 (SIGLEC 9), tumor necrosis factor receptor superfamily member 10B (TNFRSF 10B), tumor necrosis factor receptor superfamily member 10A (TNFRSF 10A), caspase 8 (CASP 8), caspase 10 (CASP 10), caspase 3 (CASP 3), caspase 6 (CASP 6), tumor necrosis factor receptor superfamily member 10B (TNFRSF 10A), and method of producing same, Caspase 7 (CASP 7), fas-related death domain (FADD), fas cell surface death receptor (FAS), transforming growth factor beta receptor II (TGFBRII), transforming growth factor beta receptor I (TGFBR 1), SMAD family member 2 (SMAD 2), SMAD family member 3 (SMAD 3), SMAD family member 4 (SMAD 4), SKI protooncogene (SKI), SKI-like protooncogene (SKIL), TGFB induction factor homeobox 1 (TGIF 1), interleukin 10 receptor subunit alpha (IL 10 RA), Interleukin 10 receptor subunit beta (IL 10 RB), heme oxygenase 2 (HMOX 2), interleukin 6 receptor (IL 6R), interleukin 6 signal transducer (IL 6 ST), c-src tyrosine kinase (CSK), phosphoprotein membrane anchor 1 with glycosphingolipid microdomains (PAG 1), signaling threshold modulating transmembrane adapter 1 (SIT 1), fork pocket P3 (FOXP 3), PR domain 1 (PRDM 1), basic leucine zipper transcription factor, ATF-like (BATF), soluble guanylate cyclase 1 alpha 2 (GUCY 1A 2), amino acid sequence, and combinations thereof, Soluble guanylate cyclase 1α3 (GUCY 1 A3), soluble guanylate cyclase 1β2 (GUCY 1B 2), soluble guanylate cyclase 1β3 (GUCY 1B 3), cytokine-induced SH 2-containing proteins (CISH), prolyl hydroxylase domains (PHD 1, PHD2, PHD 3) protein family, or Cbl protooncogene B (Cbl-B), zinc finger protein 91 (ZFP 91), roquin, CD58, ICAM-1, or any combination thereof.

In embodiments, the gene whose expression is disrupted is CISH, a negative regulator of TCR signaling. Disruption of the CISH gene may provide a functional advantage over control cells with intact CISH genes, improve sensitivity to certain cytokines (e.g., IL-2/IL-15), increase T cell proliferation, and/or limit T cell depletion. In some examples, the CISH gene may be disrupted by the method described in Daher M et al, TARGETING A cytokine checkpoint ENHANCES THE FITNESS of armored cord blood CAR-NK cells, blood.2021, month 2, 4; 137 (5): 624-636, which is incorporated herein by reference in its entirety. In some examples, the CISH gene is disrupted by gene editing using an RNA-directed nuclease system comprising one or more guide RNAs comprising the sequences of any one of SEQ ID NOS 250-253 and 315-316.

In embodiments, the gene whose expression is disrupted is a negative regulator of CBL-B, T cell activation. Disruption of the CBL-B gene may provide a functional advantage in enhancing T cell activation relative to control cells with the complete CBL-B gene. In some examples, the CBL-B gene may be disrupted by the method described in Augustin R.et al, TARGETING CBL-B in cancer immunotherapy, J Immunother cancer.2023, month 2, 11 (2): e006007; hooper K.et al ,Knockout of CBLB Greatly Enhances Anti-Tumor Activity of CAR T Cells,Blood(2018)132(Supplement 1):338; and Guo X.et al, month ,CBLB ablation with CRISPR/Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy,J Immunother Cancer.2021, 9 (3): e001975, which are incorporated herein by reference in their entirety. In one example, the CBL-B gene is disrupted by gene editing using a CRISPR-Cas system comprising one or more guide RNAs comprising the sequence of any one of SEQ ID NOs 317-320.

In embodiments, the gene whose expression is disrupted is Roquin (e.g., roquin-1). Disruption of the Roquin gene may provide a functional advantage in increasing T cell proliferation and enhancing antitumor activity relative to control cells with the complete Roquin gene. In some examples, the Roquin gene may be disrupted by the method described in Mai D et al ,Combined disruption of T cell inflammatory regulators Regnase-1 and Roquin-1 enhances antitumor activity of engineered human T cells,Proc Natl Acad Sci U S A.2023, 21, 120 (12): e2218632120, which is incorporated herein by reference in its entirety.

In embodiments, the gene whose expression is disrupted is ZFP91. Disruption of the ZFP91 gene may provide a functional advantage in improving T glycolytic fitness and effector function relative to control cells with the complete ZFP91 gene. In some examples, the ZFP91 gene may be disrupted by the method described in Wang F. Et al, J Clin invest.2021, 10 month 1, 131 (19): e144318, which is incorporated herein by reference in its entirety.

In embodiments, the gene whose expression is disrupted is CD58. In embodiments, the gene whose expression is disrupted is ICAM-1. In embodiments, both CD58 and ICAM-1 are destroyed. Disruption of the CD58 and/or ICAM-1 genes may provide functional advantages in disrupting T cell adhesion and costimulatory interactions to reduce host versus graft allograft cytotoxicity relative to control cells with intact CD58 and/or ICAM-1 genes. In one example, the ICAM-1 gene can be disrupted as described in Teo HY et al IL12/18/21Preactivation Enhances the Antitumor Efficacy of ExpandedγδT Cells and Overcomes Resistance to Anti-PD-L1 Treatment,Cancer Immunol Res.2023, 7, 11 (7): 978-999, which is incorporated herein by reference in its entirety. In one example, the ICAM-1 gene is disrupted by gene editing using an RNA-guided nuclease system (e.g., a CRISPR-Cas or CRISPR-Mad7 system), wherein the RNA-guided nuclease system comprises one or more guide RNAs comprising the sequence of any one of SEQ ID NOs 325-326. In one example, the CD58 gene is disrupted by gene editing using an RNA-guided nuclease system (e.g., a CRISPR-Cas or CRISPR-Mad7 system), wherein the RNA-guided nuclease system comprises one or more guide RNAs comprising the sequence of any one of SEQ ID NOs 327-328.

In embodiments, the host cells of the present disclosure, preferably γδ T cells, can be modified to include a nucleic acid construct encoding a protein that confers a desired function to the host cell. For example and without limitation, such nucleic acids can encode chimeric DAP10 adapter polypeptides, as described in U.S. provisional application No. 63/272,613 and U.S. provisional application No. 63/347,194, the contents of each of which are hereby expressly incorporated by reference in their entirety. In embodiments, the chimeric DAP10 adapter polypeptide is capable of associating with a chimeric antigen receptor of the disclosure, e.g., a CAR of the disclosure can comprise a DAP10 interaction domain. In such examples, the chimeric DAP10 adapter polypeptide can also be associated with one or more additional endogenous or exogenous polypeptides (e.g., endogenous or exogenous NKG 2D). In embodiments, the chimeric DAP10 adapter polypeptide may not be associated with a CAR of the disclosure, but may interact with an endogenous or exogenous polypeptide (e.g., NKG 2D) that includes a DAP10 interaction domain.

G. Diagnostic and detection methods and compositions

It will be appreciated by those skilled in the art that the antibodies, antigen-binding fragments, or compositions provided herein may be used in diagnostic or therapeutic procedures.

One embodiment provided herein relates to a method of diagnosing the presence of a tumor or cancer growth in a subject. In one embodiment, the method comprises determining the presence of PSMA in a sample suspected of containing a PSMA polypeptide. The sample may contain cells suspected of PSMA expression (which may be cancer cells). In embodiments, the method comprises exposing the sample to an antibody or antigen-binding fragment as disclosed herein, whereby specific binding of the antibody or antigen-binding fragment to the sample is indicative of the presence of a tumor or cancer growth in the subject. The antibodies used in the methods may optionally be detectably labeled, attached to a solid support, or the like. The detectable label may include, but is not limited to, a photoactivator, a fluorophore, a radioisotope, a bioluminescent protein, a bioluminescent peptide, a fluorescent tag, a fluorescent protein, or a fluorescent peptide. Exemplary assays for detecting signals from the labels include assays conventionally used in the art, such as, but not limited to, flow cytometry, ELISA, western blot, immunohistochemistry, membrane assays, and microscopic imaging.

H. Imaging of disorders associated with abnormal PSMA expression

In one embodiment, a composition comprising an antibody or antigen-binding fragment thereof of the invention is administered to a subject suffering from a disease involving inappropriate expression of a target antigen, protein, or other molecule. For example, in one embodiment, a composition comprising an antibody or antibody fragment that binds PSMA is administered to detect the presence, abundance, location, or combination thereof of PSMA in a subject. Within the scope of the present invention, this is meant to include diseases and conditions characterized by abnormal proteins, e.g. due to the amount of protein present, protein localization, post-translational modifications, conformational states, changes in the presence of mutant or pathogen proteins, etc. Similarly, a disease or condition may be characterized by alterations in molecules including, but not limited to, polysaccharides and gangliosides. The excess may be caused by any cause, including but not limited to overexpression at the molecular level, prolonged or cumulative appearance of the site of action, or increased protein activity relative to normal. This definition includes diseases and conditions characterized by protein reduction. Such a reduction may be caused by any reason, including but not limited to, reduced expression at the molecular level, shortened or reduced appearance of the site of action, a mutated form of the protein or reduced activity of the protein relative to normal. Such an excess or reduction of protein may be measured relative to the normal expression, appearance or activity of the protein, and such measurements may play an important role in the development and/or clinical testing of the antibodies of the invention.

In one embodiment, the antibody or antibody fragment of the invention binds to an antigen expressed on tumor cells (such as prostate cancer cells) when administered to a subject, and in another embodiment, the antibody or antibody fragment of the invention administered to a subject binds to an antigen expressed on a neovasculature of a solid tumor, such as a tumor with PSMA-positive neovasculature, including, but not limited to, lung cancer, liver cancer, pancreatic cancer, colon cancer, gastric cancer, breast cancer, ovarian cancer, renal cancer, prostate cancer, bladder cancer, melanoma, glioma, and the like.

The detection method used to identify binding may be direct or indirect and may include, for example, measuring light emissions, radioisotopes, calorimetric dyes, and fluorescent dyes. Direct detection includes methods that operate without intermediate or secondary measurement procedures to assess the amount of antigen or ligand bound. Such methods typically employ ligands that are themselves labeled with, for example, radioactive, light-emitting or fluorescent moieties. In contrast, indirect detection includes methods that operate through intermediate or secondary measurement procedures. These methods typically employ molecules that react specifically with the antigen or ligand and can themselves be labeled directly or detected by secondary reagents. For example, antibodies specific for a ligand can be detected using a secondary antibody capable of interacting with a primary antibody specific for the ligand, again using the detection methods described above for direct detection. The indirect method may additionally employ enzyme-labeled detection. Furthermore, for specific examples of screening catalytic antibodies, the disappearance of the substrate or the appearance of the product may be used as an indirect measure of binding affinity or catalytic activity.

I. Pharmaceutical composition

The antibodies, antigen binding fragments thereof, and/or engineered cells of the present disclosure may be administered to the subject itself, or in the form of a pharmaceutical composition in admixture with a suitable carrier or excipient.

The antibodies and/or engineered cells of the invention may be administered by any route suitable for the condition to be treated. Administration is typically parenteral, i.e., infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

Therapeutic formulations comprising anti-PSMA antibodies for use according to the invention for storage (Remington's Pharmaceutical Sciences th edition, osol, code a. 1980)) are prepared by mixing antibodies of the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers, which are in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as acetate, tris, phosphate, citrate and other organic acids, antioxidants including ascorbic acid and methionine, preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethyl diammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin, chelating agents such as EDTA, tonicity modifiers such as sugars and sodium chloride, sugars such as sucrose, mannitol, trehalose or sorbitol, surface active agents such as Zn-ion complexing agents such as sodium or non-ionic complexing agents such as Zn-ion surface complexing agents such as Zn-ion or non-ionic complexing agents such as Zn-complexing agents Or polyethylene glycol (PEG). Pharmaceutical formulations to be used for in vivo administration are generally sterile. This is easily accomplished by filtration through sterile filtration membranes.

The active ingredient may also be encapsulated in microcapsules, such as prepared by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16 th edition, osol, article a (1980).

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamate, nondegradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON(Injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprorelin acetate) and poly-D- (-) -3-hydroxybutyric acid. Although polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow release of molecules for more than 100 days, certain hydrogels release proteins for a shorter period of time. When encapsulated immunoglobulins remain in the body for a long period of time, they may denature or aggregate as a result of exposure to moisture at 37 ℃, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, a reasonable stabilization strategy may be devised. For example, if the aggregation mechanism is found to be the formation of intermolecular S-S bonds through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

The antibodies can be formulated in any form suitable for delivery to the target cells/tissues. For example, antibodies can be formulated as immunoliposomes. A "liposome" is a vesicle composed of various types of lipids, phospholipids, and/or surfactants that can be used to deliver a drug to a mammal. The components of liposomes are typically arranged in bilayer form, similar to the lipid arrangement of biological membranes. Antibody-containing liposomes are prepared by methods known in the art, as described in Epstein et al, proc.Natl. Acad.Sci.USA 82:3688 (1985), hwang et al, proc.Natl Acad.Sci.USA 77:4030 (1980), U.S. Pat. Nos. 4,485,045 and 4,544,545, and WO 97/38031 published 10/23 1997. Liposomes with extended circulation times are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be produced by reverse phase evaporation methods using lipid compositions comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter defining a pore size to produce liposomes having a desired diameter. The Fab' fragments of the antibodies of the invention can be conjugated to liposomes via disulfide exchange reactions as described in Martin et al, J.biol.chem.257:286-8 (1982). The chemotherapeutic agent is optionally contained within liposomes (see Gabizon et al, J. National Cancer Inst 81 (19): 1484 (1989)).

Other pharmaceutical formulations suitable for use in The compositions for administration in The methods of The invention are known in The art (see, e.g., remington's Pharmaceutical Sciences (1990) 18 th edition, mack Publishing Co., easton, pa.; the Merck Index (1996) 12 th edition, merck Publishing Group, whitehouse, N.J., and Pharmaceutical Principles of Solid Dosage Forms, technonic Publishing company, lancaster, pa., (1993)). Pharmaceutical formulations can be packaged in dosage unit form for ease of administration and uniformity of dosage. As used herein, "unit dosage form" refers to physically discrete units suitable as unitary dosages for subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with a pharmaceutical carrier or excipient.

Pharmaceutical compositions suitable for use in the context of some embodiments of the present invention include compositions containing an active ingredient therein in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of an active ingredient (anti-PSMA antibody, antigen-binding fragment thereof, CAR modified immune cells) effective to prevent, ameliorate or improve the symptoms of a disease (e.g., cancer) or prolong survival of a subject being treated.

Determination of a therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any formulation used in the methods of the invention, a therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, dosages may be formulated in animal models to achieve a desired concentration or titer. The information can be used to more accurately determine useful doses in humans. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell culture or in experimental animals. The data obtained from in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage may be selected by the individual physician according to the patient's condition. (see, e.g., fingl, et al 1975,in"The Pharmacological Basis of Therapeutics", chapter 1, page l).

Depending on the severity and responsiveness of the condition being treated, the administration may be performed in a single or multiple administrations, with the course of treatment lasting from days to weeks, or until cure is achieved or a reduction in the disease state is achieved.

Of course, the amount of composition administered will depend on the subject being treated, the severity of the disease, the mode of administration, the judgment of the prescribing physician, and the like.

J. Therapeutic method

As described herein, pharmaceutical compositions comprising an anti-PSMA antibody, antigen-binding fragment thereof, or CAR modified immune cells comprising the same, can be administered for prophylactic and/or therapeutic treatment.

One embodiment of the invention provides a method of treating a disorder associated with aberrant PSMA expression (e.g., PSMA-expressing cancer) in a subject. The methods can include targeting PSMA-expressing cells, e.g., PSMA-expressing tumor cells, with a therapeutically effective amount of the antibody or antigen-binding fragment.

In certain embodiments, the methods comprise administering to a subject a composition comprising a therapeutically effective amount of an anti-PSMA antibody, antigen-binding fragment thereof, or CAR-modified immune cell comprising the same. Preferably, the CAR-modified immune cells are CAR-modified γδ T cells. In one embodiment, the anti-PSMA antibody is used in the form of an ADC. For example, an anti-PSMA antibody may be conjugated with a bioactive agent or combination of such agents, including but not limited to radioisotopes, toxins, and the like.

In one embodiment, a method is provided for treating a solid tumor with aberrant PSMA expression, such as a prostate cancer or a solid tumor with high PSMA expression in a neovasculature. Such methods can include the step of targeting high PSMA expressing cells with a therapeutically effective amount of an anti-PSMA antibody, antigen-binding fragment thereof, or CAR modified immune cells comprising the same. Preferably, the CAR-modified immune cells are CAR-modified γδ T cells. Thus, embodiments include administering an anti-PSMA antibody, antigen-binding fragment thereof, or CAR modified immune cells comprising the same to a subject having a tumor associated with aberrant PSMA expression. In embodiments, the anti-PSMA antibody is used in the form of an ADC.

In one embodiment, a method of inhibiting cell growth or proliferation is provided, the method comprising exposing a PSMA-expressing cell to an anti-PSMA antibody, or antigen-binding fragment thereof, or a CAR-modified immune cell comprising the same, under conditions that allow the antibody to bind to PSMA. Preferably, the CAR-modified immune cells are CAR-modified γδ T cells. By "inhibiting cell growth or proliferation" is meant reducing the growth or proliferation of a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, and may include inducing cell death. In embodiments, the PSMA-expressing cell is a tumor cell.

In one embodiment, an anti-PSMA antibody or antigen-binding fragment, or CAR modified immune cell comprising the same, is used to treat or prevent a cell proliferative disorder. Preferably, the CAR-modified immune cells are CAR-modified γδ T cells. In certain embodiments, the cell proliferative disorder is associated with aberrant expression and/or activity of PSMA. For example, in certain embodiments, a cell proliferative disorder is associated with increased expression of PSMA on the surface of a cell. In certain embodiments, the cell proliferative disorder is a tumor or cancer.

Pharmaceutical compositions comprising engineered host cells expressing a CAR and/or mixtures thereof may be administered for prophylactic and/or therapeutic treatment. In a preferred embodiment, the pharmaceutical composition comprises γδ T cells engineered to express a PSMA-targeted CAR. The mixture may comprise the same or different types of non-engineered cells. Additionally or alternatively, the mixture may comprise the same type of engineered cells expressing different PSMA-targeted CARs. Additionally or alternatively, the mixture may comprise different types of engineered cells expressing the same or different PSMA-targeted CARs. For example, and without limitation, the mixture can comprise a γδ T cell population engineered to express a PSMA-targeted CAR, as well as a non-engineered γδ T cell population. As another example, but not by way of limitation, the mixture can comprise a population of γδ T cells engineered to express a PSMA-targeted CAR, as well as a population of engineered or non-engineered cells (e.g., NK cells, NKT cells, γδ cells, αβ cells, etc.). In therapeutic applications, the composition may be administered to a subject already suffering from a disease or condition in an amount sufficient to reduce at least one sign or symptom associated with the disease or condition. In embodiments, the amount is sufficient to cure the disease or condition.

The population of engineered host cells and/or mixtures thereof may also be administered to reduce the likelihood of developing a condition, an infectious condition, or worsening of a condition. The effective amount of the population of engineered host cells, non-engineered host cells, and/or mixtures thereof for therapeutic use may vary depending on the severity and course of the disease or disorder, previous therapies, the health status, weight, and/or response of the subject to various drugs, and/or the discretion of the treating physician.

In embodiments, one or more of the engineered host cell populations, non-engineered cells, and/or mixtures thereof of the present disclosure can be used to treat a subject in need of treatment of a disorder. Examples of such disorders include, but are not limited to, cancer, infectious diseases, and autoimmune disorders. The subject may be human, non-human primate such as chimpanzees and other apes and monkey species, farm animals such as cattle, horses, sheep, goats, pigs, domestic animals such as rabbits, dogs and cats, laboratory animals including rodents such as rats, mice and guinea pigs, and the like. The subject may be of any age. The subject may be, for example, an elderly person, an adult, a adolescent, a pre-pubertal child, a young child, an infant.

Methods of treating a condition (e.g., affliction) in a subject may comprise administering to the subject a therapeutically effective amount of one or more engineered host cell populations, preferably engineered γδ T cells, non-engineered cells, and/or mixtures thereof. One or more populations of engineered host cells, non-engineered cells, and/or mixtures thereof can be administered according to various protocols (e.g., time, concentration, dosage, treatment interval, and/or formulation). The subject may also receive preconditioning, such as chemotherapy, radiation therapy, or a combination of both, prior to receiving a therapeutically effective amount of one or more populations of engineered host cells, non-engineered cells, and/or mixtures thereof. As part of the treatment, one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof may be administered to the subject according to a first regimen, and the subject may be monitored to determine whether the treatment of the first regimen meets a given level of therapeutic efficacy. In some cases, one or more populations of engineered host cells, non-engineered cells, and/or mixtures thereof may be administered to a subject according to a second regimen based on information collected from providing the first regimen to the subject.

In embodiments, a pharmaceutical composition comprising at least one host cell, preferably γδ T cells, engineered to express a CAR of the present disclosure can be administered in the first embodiment. The subject may be monitored, for example, by a healthcare provider (e.g., a therapist or nurse). In some examples, the subject is monitored to determine or measure the efficacy of the engineered host cell in treating the subject's condition. In some cases, the subject may also be monitored to determine in vivo expansion of the engineered host cell population in the subject. Another pharmaceutical composition comprising at least one host cell engineered to express a CAR of the present disclosure may be administered to a subject in a second regimen. The pharmaceutical composition administered in the second regimen may comprise the same type of host cell expressing the same CAR as administered to the subject in the first regimen. However, it is within the scope of the present disclosure that the pharmaceutical composition administered in the second regimen may comprise a different type of host cell, which optionally expresses a different CAR. In some examples, the second regimen is not performed, such as where the first regimen is found to be effective (e.g., a single round of administration may be sufficient to treat the condition). In embodiments, the population of engineered host cells can be administered to a variety of subjects (e.g., wherein the host cells have universal donor characteristics).

One or more populations of engineered host cells, non-engineered cells, and/or mixtures thereof having cytotoxic activity against PSMA-expressing cells, such as tumor cells, may be administered to a subject in any order or simultaneously. If administered simultaneously, the engineered host cells of the present disclosure and/or mixtures thereof may be provided in a single, unified form (such as intravenous injection), or in multiple forms (e.g., as multiple intravenous infusions, subcutaneous injections, or pellets). One or more populations of engineered host cells, non-engineered cells, and/or mixtures thereof of the present disclosure can be packaged together or separately in a single package or multiple packages. One or all of the one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof of the present disclosure may be administered in multiple doses. If not administered simultaneously, the timing between doses may vary by up to about one week, one month, two months, three months, four months, five months, six months, or about one year. In some cases, the engineered host cells, preferably engineered γδ T cells, of the present disclosure can proliferate in vivo within the body of a subject after administration to the subject. One or more populations of engineered host cells, non-engineered cells, and/or mixtures thereof of the present disclosure can be frozen to provide cells for multiple treatments with the same cell preparation. One or more populations of engineered host cells, non-engineered cells, and/or mixtures thereof of the present disclosure, and pharmaceutical compositions comprising the same, can be packaged as a kit. The kit may include instructions (e.g., written instructions) for use of the one or more engineered host cell populations, the non-engineered cells, and/or mixtures thereof, and pharmaceutical compositions comprising the same.

One or more of the engineered host cell populations, non-engineered cells, and/or mixtures thereof described herein can be administered before, during, or after the occurrence of a disease or condition, and the timing of administration of the pharmaceutical composition containing the engineered host cell populations can vary. For example, one or more populations of engineered host cells, non-engineered cells, and/or mixtures thereof can be used as a prophylactic agent, and can be continuously administered to a subject having a disorder or predisposition to a disorder, in order to reduce the likelihood of occurrence of the disorder or disorder. Initial administration may be via any practical route, such as by any route described herein using any of the formulations described herein. In some examples, the administration of the one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof of the present disclosure is intravenous administration. One or more doses of one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof can be administered as soon as practicable after onset of a particular condition (e.g., cancer), and for a length of time necessary to treat the disease/condition, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. In embodiments, one or more doses of one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof may be administered years after onset of a disease/condition (e.g., cancer) and before or after other treatments.

In embodiments, one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof of the present disclosure are administered simultaneously or sequentially with one or more methods of increasing a common gamma chain cytokine. As used herein, "one or more methods of increasing a common gamma chain cytokine" refers to a method or combination of methods that alters the physiological state of a subject such that the level of at least one common gamma chain cytokine in the subject is increased. In embodiments, the methods increase the level of one or more common gamma chain cytokines selected from the group consisting of IL-2, IL-4, IL-7, IL-15, and IL-21 in a subject. In embodiments, the method comprises lymphocyte depletion. In embodiments, the method comprises administering to the subject one or more common gamma chain cytokines. In some cases, IL-2, IL-4, IL-7, IL-15 and/or IL-21 is administered. In embodiments, the method comprises secreting a common gamma chain cytokine from the administered engineered host cell. In some cases, IL-2, IL-4, IL-7, IL-15 and/or IL-21 are secreted.

In embodiments, the method of administering one or more elevated shared gamma chain cytokines comprises performing lymphocyte depletion prior to introducing one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof of the present disclosure. In embodiments, the method of administering one or more elevated common gamma chain cytokines comprises administering simultaneously with or sequentially with the introduction of the one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof, or an amount of common gamma chain cytokine sufficient to increase proliferation, cytotoxic activity, persistence, or combinations thereof of the introduced one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof. The amount of the co-administered gamma chain cytokine may be an amount effective to increase proliferation, cytotoxic activity, persistence, or a combination thereof of one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof. Exemplary amounts of IL-15 include, but are not limited to, 0.01 to 10 μg/kg/dose of IL-15 per 24 hours. Exemplary amounts of IL-2 include, but are not limited to, about 3X 10 ⁶ to about 22X 10 ⁶ units every 8-48 hours. For example, the regimen for IL2 administration in RCC is 600,000 International units/kg (0.037 mg/kg) IV for 48hr, with infusion over 15 minutes for up to 14 doses.

In embodiments, the method of administering one or more elevated common gamma chain cytokines comprises performing lymphocyte depletion prior to administering the one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof, and administering simultaneously with introducing the one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof, or sequentially with administering an amount of common gamma chain cytokine sufficient to increase proliferation, cytotoxic activity, persistence, or combinations thereof of the introduced one or more engineered host cell populations, non-engineered cells, and/or mixtures thereof.

In embodiments, increasing the shared gamma chain cytokine is achieved at least in part via an engineered host cell, wherein the shared gamma chain cytokine is expressed by a CAR construct as disclosed herein. In such instances, it is within the scope of the present disclosure to additionally administer one or more additional gamma chain cytokines in a manner that elevates the additional gamma chain cytokines.

The γδ T cells of the invention can also be advantageously administered to a subject in combination (e.g., before, simultaneously or after) with any number of relevant therapeutic modalities (including, e.g., chemotherapy, radiation therapy, or immunotherapy) (e.g., for treating cancer). The patient may also be preconditioned with a therapeutically effective amount of γδ T cells prior to receiving chemotherapy, radiation therapy, or immunotherapy (e.g., cell therapy). Immunotherapy suitable for use in combination with γδ T cells includes autologous and allogeneic cell therapies, engineered T and NK cells, immunoconjugates, fusion proteins or other immune tumor agents.

In embodiments, γδ T cells can be administered in combination with an appropriate cellular immunotherapy (e.g., CAR T or CAR NK cells or Treg therapy) for treating a disease such as cancer. For example, a therapeutic method according to the invention (e.g., for treating cancer) may include a regulatory step, such as a preconditioning step, of administering a therapeutically effective amount of γδ T cells to a subject concurrently or sequentially with administration of cellular immunotherapy for a disease (e.g., cancer). In embodiments, cellular immunotherapy may include further administration of engineered T cell or NK cell therapies that include CARs that bind to any tumor-associated antigen of interest. In embodiments, the subject γδ T cells can comprise a dual CAR that binds to CD70 and PMSA.

In embodiments, γδ T cells of the invention can be advantageously administered in combination with Adoptive Cell Therapy (ACT) (for reviews of HSCT and adoptive cell therapy methods, see Rager and Porter, ther Adv Hematol (2011) 2 (6) 409-428; roddie and Peggs, expert opin. Biol. Ter. (2011) 11 (4): 473-487; wang et al int. J. Cancer (2015) 136,1751-1768; and Chang, y. J. And x. J. Huang, blood Rev,2013.27 (1): 55-62), each of which is incorporated herein by reference in its entirety. Such adoptive cell therapies include, but are not limited to, allogeneic and autologous hematopoietic stem cell transplantation, donor leukocyte (or lymphocyte) infusion (DLI), adoptive transfer of tumor-infiltrating lymphocytes, or adoptive transfer of T cells or NK cells (including recombinant cells, e.g., CAR T, CAR NK). In addition to the need for donor-derived cells to reconstitute hematopoietic function following radiotherapy and chemotherapy, immune reconstitution of metastatic cells is important for the elimination of residual tumor cells. ACT efficacy as a therapeutic choice for malignant tumors is affected by a number of factors including the source, composition and phenotype of the donor cells (lymphocyte subpopulation, activated state), underlying disease, pre-transplant modulation regimen and post-transplant immune support (e.g., IL-2 therapy), and graft-versus-tumor (GVT) effects mediated by the donor cells within the graft. In addition, these factors can be balanced with the mortality associated with transplantation, often caused by regulatory regimens and/or excessive immune activity of donor cells within the host (i.e., graft versus host disease, cytokine release syndrome, etc.).

K. articles of manufacture and kits

Another embodiment of the invention is an article of manufacture comprising a material useful for treating, preventing and/or diagnosing a disease or disorder associated with PSMA expression. A disease associated with PSMA expression is selected from proliferative diseases such as cancer or malignant tumor or pre-cancerous disorders such as prostate cancer, or other solid tumors that highly express PSMA on tumor cells or neovasculature, or non-cancer related indications associated with PSMA expression, wherein the solid tumors include malignant epithelial tumors, lymphomas, blastomas, sarcomas (including liposarcoma), neuroendocrine tumors, mesothelioma, schwannoma, meningioma, malignant adenoma, melanoma, and leukemia or malignant lymphoproliferative disorders, such as, in particular, sarcomas, ovarian cancer, breast cancer, glioblastoma, gastric cancer, colon cancer, colorectal cancer, lung cancer, liver cancer, thyroid cancer, lymphomas, nasopharyngeal carcinoma, maxillary sinus cancer, renal cancer, prostate cancer, bladder cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma.

The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container contains a composition effective to treat, prevent, and/or diagnose a disorder associated with PSMA expression, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The at least one active agent in the composition is an anti-PSMA antibody of the invention, or an antigen-binding fragment thereof, or a CAR-modified immune cell of the invention, or a nucleic acid of the invention. Optionally, the composition further comprises a carrier, such as a pharmaceutically acceptable carrier. The label or package insert indicates that the composition is used to treat a disorder associated with PSMA expression, such as cancer. The label or package insert will also include instructions for administering the antibody composition to a patient. In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. It may further include other materials, including other buffers, diluents, filters, needles and syringes, as desired from a commercial and user perspective.

Kits useful for various purposes, such as killing assays for PSMA-expressing cells, for purifying or immunoprecipitation of PSMA polypeptides from cells, are also provided. To isolate and purify PSMA polypeptides, a kit may contain an anti-PSMA antibody coupled to a bead (e.g., an agarose bead). Kits may be provided that contain antibodies for in vitro detection and quantification of PSMA polypeptides, e.g., in ELISA or western blots. As with the articles of manufacture, the kit includes a container and a label or package insert on or associated with the container. The container contains a composition comprising at least one anti-PSMA antibody, or antigen-binding fragment thereof, of the invention. Additional containers containing, for example, diluents and buffers, control antibodies, may be included. The label or package insert may provide a description of the composition as to the intended in vitro or assay use.

For example, a kit may comprise a first container comprising a composition comprising one or more PSMA antibodies or CAR-modified immune cells of the invention, and a second container comprising a buffer. The buffer may be pharmaceutically acceptable.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is degrees celsius, and pressure is at or near atmospheric pressure.

Example 1.

Example 1 binding profile of anti-PSMA antibodies to PSMA expressing and knockout cell lines

Dilutions of purified anti-PSMA antibodies (including positive and negative controls) were prepared in FACS buffer (2% fbs in PBS without Ca ²⁺ and Mg ²⁺) in 96-well plates. The PSMA-expressing 22Rv1 cells (ATCC) and the PSMA-encoding 22Rv1 cells (22 Rv 1-hPSMA-KO) from which the gene encoding PSMA was knocked out were stained with the above-described anti-PSMA antibody. Cells were washed with PE conjugated goat anti-human IgG-Fc secondary antibody (bioleged) and stained for detection by flow cytometry. The average fluorescence intensity of each antibody against both cell lines as detected in the PE channel was determined using FlowJo software. As shown in fig. 1, the anti-PSMA antibodies had a broad binding affinity for PSMA-expressing 22Rv1 cell lines, with some binding intensities comparable to or higher than reference antibody J591. J591 The amino acid sequence of HCVR is shown as SEQ ID NO. 303, and the amino acid sequence of J591 LCVR is shown as SEQ ID NO. 304. The CDRs of the J591 antibody are listed herein as follows for ：HCDR1(SEQ ID NO:305)、HCDR2(SEQ ID NO:306)、HCDR3(SEQ ID NO:307)、LCDR1(SEQ ID NO:308)、LCDR2(SEQ ID NO:309)、LCDR3(SEQ ID NO:310). anti-PSMA antibodies, which are also specific for binding, but not for 22Rv1-hPSMA-KO cell lines.

EXAMPLE 2 binding Profile of anti-PSMA antibodies to recombinant human PSMA protein

The EC50 of the anti-PSMA antibodies to recombinant human PSMA protein was determined by ELISA. Briefly, 96-well plates were coated with 1 μg/mL recombinant human PSMA (Acro Biosciences), blocked with 1% BSA (Sigma Aldrich) in PBS, then treated with anti-PSMA antibodies at an initial concentration of 10 μg/mL and diluted 3-fold to obtain a 12-point μ dilution series, followed by treatment with HRP conjugated anti-human IgG Fc antibodies (Invitrogen) at a 1:3000 dilution. After addition of TMB substrate and stop solution, plates were read at 450nm on a Cytation plate reader (Biotek). EC50 was found to be in the two digit picomolar to unit digit nanomolar range. The EC50 of the highest affinity antibody was similar to that of reference antibody J591. The results are summarized in fig. 2A.

The Kd of the anti-PSMA antibody was determined by biolayer interferometry (Octet Red384, sartorius). Briefly, antibodies were loaded onto an anti-hIgG Fc capture (AHC) biosensor (Sartorius) at a concentration of 1 μg/mL for 300 seconds, followed by an association step in recombinant human PSMA (Acro Biosystems) at different concentrations for 200 seconds, and a dissociation step in 1 XOctet kinetic buffer (Sartorius) for 600 seconds. Data were processed using ForteBio "data analysis" software (version 11.0) and fitted to a 1:1 binding model. Recombinant human PSMA proteins were run on blue native PAGE gels (4-12%, invitrogen) to identify the oligomeric state of the native protein. As shown in fig. 2B, recombinant human PSMA protein appears to be a mixture of dimers and monomers, and anti-PSMA antibodies bind to dimers or monomers, as indicated by their similar Kd but different Rmax values.

Example 3 general method for identifying the leader PSMACAR

Briefly, phage display technology is used to screen binding agents. Screening for recombinant lines (e.g., domains comprising post-translational modifications (PTMs) known to be associated with a particular cancer type) including recombinant proteins, cancer cell lines with variable target densities, and recombinant lines expressing full-length and unique domains of interest. To minimize "on-target, tumor-free" binding, reverse screening was performed using target null cell lines and cell lines expressing related family members.

Phage panning using biotinylated recombinant human PSMA protein (Acro Biosciences) bound to streptavidin beads was performed on highly diverse synthetic scFv phage display libraries (Twist Bio) using 5 rounds of selection with decreasing antigen concentrations per round (starting from 100pmol to 2 pmol) but increasing wash stringency. Alternate rounds of selection were also performed with 1e8 PSMA+ cells or antigens (100 pmol and 25pmol, respectively) using an alternate panning strategy. Antigen-binding phage are pulled down, eluted, amplified and screened using ELISA for binding agent validation and selection. After NGS and sanger clone sequencing, scFv was reformatted into IgG, expressed, purified and characterized.

The lead binding agent is reformatted into a suitable isotype of human immunoglobulin (e.g., igG) or some CAR replacement form (e.g., CAR diabody) to further characterize and evaluate the various potential trends. This is called the "molecular evaluation method" and aims at optimizing the biophysical and chemical properties of the molecules at an early stage prior to CAR development. Evaluation criteria included aggregation habit, solubility level, fragmentation propensity, deamidation, oxidation, isomerization, cyclization, or other chemical modification of key residues, disulfide reorganization, saccharification, and the like. In addition, some trends were assessed via design selection of screening libraries for phage display. In some cases, protein microarray screening was used to evaluate cross-reactivity of fully formatted IgG. By using a select phage display library and an assessment of formatted antibodies, a number of potential trends associated with specific binding agents are assessed early in the process prior to CAR manufacture.

The anti-target antibodies may be further screened/characterized for physical/chemical properties and/or biological activity by various assays. This matrix-based multiparameter approach is used to generate the final leads and rank according to PSMA targets. For example, for biochemical/biophysical characterization, the antigen binding activity of a panel of antibodies is tested by known methods, including but not limited to Biological Layer Interferometry (BLI) and Surface Plasmon Resonance (SPR), on an appropriate instrument using soluble formulations of recombinant full-length or labeled human proteins. In this way, kinetic constants (K _on and K _off) and dissociation constants (K _D) of the reformatted antibodies and (where appropriate) the reference antibodies (J591 disclosed herein) were generated. Biochemical assays relied upon include, but are not limited to, ELISA, western blot, and/or flow cytometry assays for generating relative affinity data (e.g., EC 50) and maximum specific binding (B _max) for the group and reference antibodies.

In some cases, the epitopes to which the disclosed antibodies and reference antibodies bind are also located using specific assays, e.g., in ELISA format, via the use of overlapping peptides across the target. Where appropriate, recombinant cell lines expressing full-length targets or domains known to have specific epitopes are prepared for screening and classification. To further characterize the lead epitope, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used.

The lead binders were reformatted into heavy-light (HL) and LH (light-heavy) oriented CARs and these CARs were further screened in Jurkat Lucia NFAT cells to assess CAR cell surface expression, tonic signaling, and activation in a targeted stimulation setting (in the form of recombinant proteins expressing PSMA targets or cancer cell lines). CARs exhibiting robust activation in Jurkat cells were transduced into vδ1γδ T cells and potency against target expressing cell lines was assessed using a stringent 120 hour in vitro cytotoxicity assay.

Example 4 in vitro cytotoxicity against PSMACAR engineered γδ T cells

Cytotoxic potential of non-transduced γδ T cells and anti-PSMACAR transduced γδ T cells derived from the same healthy donor were evaluated against Nuc NEAR INFRARED (NIR) modified cells (Sartorius), PSMA expressing 22Rv1 target cells, and PC3 cells engineered to express PSMA (PC 3-PSMA) at low E: T ratios of 1:1 (fig. 3A, 3E) or 1:2 (fig. 3C, 3G). Corresponding negative controls included a 22Rv1 with PSMA expression knocked out and a parent PC3 cell line that did not express PSMA, E: T ratio of 1:1 (fig. 3B, 3F) or 1:2 (3D, 3H). A baseline CAR PL744 in which the binding domain was derived from a J591 antibody as described herein was included as a comparative CAR control. Briefly, the nucleic acid encodes SEQ ID NO 313, a CAR polypeptide PL744 comprising, in order, a signaling CAR polypeptide PL744 comprising a PSMA binding domain, a CD8 hinge and transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta signaling domain. The nucleic acid encoding PL744 CAR comprises the sequence of SEQ ID No. 314. Table 10 below provides an annotation of the nucleotide sequence of SEQ ID NO. 314.

EXAMPLE 5 "bolt-on" modified in vitro cytotoxicity against PSMACAR engineered gamma delta T cells

The cytotoxic potential of one of the γδ T cells resistant to PSMACAR transduction and the same CAR construct modified to express "bolting" (i.e., dominant negative tgfβ receptor II (dnTGF βrii)) was evaluated against Nuc NEAR INFRARED (NIR) modified cells, 22Rv1 target cells, and 22Rv1-PSMA-KO cell lines at a low E: T ratio of 1:1. Viability of Nuc NIR expression targets was monitored every 2 hours over 120 hours using the IncuCyte living cell analysis system (Sartorius), reported as cytotoxicity index (calculated by dividing the total NIR subject area at each time point by the time 0 hour value). The lower the cytotoxicity index, the stronger the potency of the construct. As shown in fig. 4A-4B, the "bolt-on" modified CAR construct is highly functional, exhibiting cytotoxicity comparable to the naked CAR construct in vitro. Both constructs were specific and did not show cytotoxicity against PSMA KO cell lines.

EXAMPLE 6 "bolt-on" modified anti-PSMACAR expression of CAR, dnTGF beta RII, CD103 and pSMAD2/3 in gamma delta T cells engineered

V delta 1T cells modified with the anti PSMACAR construct were counted by Annexin-DAPI on Novocyte (Agilent). Approximately 1x10 ⁵ cells were stained using the cell viability dye Zombie Aqua (bioleged). Cells were further surface stained for vδ1 (bioleged), dnTGF βrii (APC anti-human TGF- β receptor II antibody, bioleged) and CD103 (bioleged), and CAR expression was detected using biotinylated protein L (Acro Biosystems) and PE-streptavidin (bioleged). Cells were fixed prior to collection on Novocyte (Agilent). As shown in fig. 5A-B, CAR expression remained essentially unchanged in the "bolt-fastened" modified anti PSMACAR engineered γδ T cells compared to the naked CAR construct, and dnTGF βrii expression was detected only in the "bolt-fastened" modified CAR construct. As shown in fig. 5C, anti PSMACAR transduced γδ T cells containing dnTGF βrii had reduced CD103 expression compared to the control without the dnTGF βrii construct.

Anti-PSMACAR transduced γδ T cells, with or without modification with dnTGF βrii construct, were serum starved at 37 ℃ for 2 hours at 5% CO ₂ to reduce pSMAD2/3 background levels. Cells were stained with Zombie Aqua vital dye (Biolegend) and plated into round bottom wells of ULA 96-well plates. 10ng/mL of human recombinant TGF-beta 1 (R & D Systems) was added at 37℃with 2% CO ₂ for 15 minutes. Cells were immediately fixed by adding an equal volume of pre-warmed BD Cytofix buffer (BD Biosciences) to the cell suspension. Cells were permeabilized using BD ^TM Phosflow Perm buffer III (BD Biosciences) and incubated for 30 min on ice protected from light. After washing with staining buffer (2% FBS 1 XPBS), anti-pSMAD 2/3 antibody conjugated to PE (BD Biosciences) was added to the cells and incubated for 1 hour at room temperature in the dark. Cells were washed twice with staining buffer and collected on Novocyte. As shown in fig. 5D, anti PSMACAR transduced γδ T cells containing dnTGF βrii had reduced levels of pSMAD2/3 compared to the control without the dnTGF βrii construct.

Example 7 expansion of anti-PSMACAR engineered γδ T cells

1X 10 ⁶/mL of human PBMC in X-Vivo 15 medium (Lonza) containing 10% heat-inactivated FBS (HyClone) were activated for 5 days in the presence of IL-2 (100U/mL) on plates pre-coated with Vd1 activating antibody (Adicet Bio). On day 5, cell cultures were transduced with a gamma-retrovirus construct encoding anti-PSMACAR in the presence of fibronectin. Starting on day 6, the cells were further expanded by feeding and IL-2 replacement as needed. On day 17, cells were harvested and usedThe kit (Miltenyi Biotec) depletes αβ T cells. Purity and transduction efficiency of γδ cell populations were assessed by flow cytometry at Novocyte (Agilent). As shown in fig. 6, anti-PSMACAR engineered γδ T cells exhibit a robust cell expansion curve. The dashed line represents the number of cells from previously expanded CD20CAR engineered γδ T cells.

Example 8 in vivo efficacy of anti PSMACAR engineered γδ T cells

The in vivo efficacy of anti PSMACAR engineered vδ1t cells was evaluated in a subcutaneous prostate cancer model using 22Rv1 cells (ATCC) sorted for homogeneous PSMA expression (22 Rv1 clone E7). Male NSG mice (The Jackson Laboratory) were subcutaneously implanted with 1X 10 ⁶ 22Rv1 clones E7 cells mixed with Matrigel (1:1 ratio) (Corning). When tumor volume reached an average of 100mm ³ animals were randomly divided into 5 groups and IV (tail vein) injections were performed with 5x 10 ⁶, or 15x 10 ⁶ vδ1PSMACAR T cells. The positive control group was treated with baseline vδ 1j591 CAR T cells at a dose of 5x 10 ⁶ car+ cells, and also included a separate tumor group (untreated). Human IL-2 (Proleukin) was administered intraperitoneally prior to treatment, followed by three times per week. Tumor volumes were measured twice weekly with calipers. As shown in fig. 7A-7B, a robust tumor response was observed at both doses using one of the pilot anti PSMACAR engineered vδ1t cells (PL 805). Efficacy is comparable to or better than baseline CAR (PL 744).

EXAMPLE 9 Gene editing by gamma delta T cells

To knock out CISH genes in vδ1t cells, ribonucleoprotein (RNP) complexes consisting of CRISPR-Cas9 (INTEGRATED DNA Technologies) and CISH SGRNA (Synthego Corporation) were combined with activated vδ1t cells from PBMCs (P3 primary cell 4D-Nucleofector X kit L, lonza). RNP complexes were delivered to V.delta.1T cells by electroporation (Lonza 4D-Nucleofector). Following electroporation, cells were transferred to T-flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery and expansion in a 37℃CCO2 incubator (ThermoFisher). To evaluate CISH KO efficiency, cell pellets were collected after αβ T cell depletion (StemCell Technologies) on day 7 after gene editing and genomic DNA was extracted using NucleoSpin tissue kit (MACHEREY-NAGEL). The editing region of the CISH gene was amplified by PCR and sanger sequencing was performed (Sequetech). Gene knockout efficiency was determined using ICE analysis tool (Synthego). As shown in FIG. 8, efficient knockout of CISH gene was observed from V.delta.1T cells.

Following CISH gene knockout, V delta 1T cell enrichment in PBMCs was monitored over time by flow cytometry (anti-vdelta 1, biolegend). As shown in fig. 9A, CISH gene knockout did not affect vδ1T cell enrichment compared to non-edited control cells. Similar purity was achieved for CISH gene-edited V.delta.1T cells after TCRαβ depletion (StemCell Technologies) compared to control cells. Cell viability of vδ1T cell enriched PBMCs was monitored following CISH gene knockout (Countess II, thermoFisher). As shown in fig. 9B, CISH KO cells maintained high cell viability similar to non-edited control cells.

Example 10 binding profiles of exemplary anti-PSMA antibodies to PSMA expressing and knockout cell lines

Dilutions of purified anti-PSMA antibodies (including positive and negative controls) were prepared in FACS buffer (2% fbs in PBS without Ca ²⁺ and Mg ²⁺) in 96-well plates. Psma+22Rv1 cells (ATCC), PSMA-encoding gene knockout 22Rv1 cells (22 Rv 1-hPSMA-KO), psma+c4-2B cells (ATCC), and PC3 lines engineered to express PSMA (PC 3-PSMA) were stained with anti-PSMA antibodies. Cells were washed with PE conjugated goat anti-human IgG-Fc secondary antibody (bioleged) and stained for detection by flow cytometry. The average fluorescence intensity of each antibody against both cell lines as detected in the PE channel was determined using FlowJo software. As shown in fig. 10A, the anti-PSMA antibodies had a broad binding affinity for PSMA-expressing 22Rv1 cell lines, with some binding intensities comparable to or higher than reference antibody J591. The binding of the anti-PSMA antibodies was also specific and showed no binding to the 22Rv1-hPSMA-KO cell line. The lead PSMA antibodies showed binding to PCa cell lines expressing different levels of PSMA (fig. 10B). A reference antibody J591 was included as a positive control in these assays.

Example 11 binding Profile of exemplary anti-PSMA antibodies to recombinant human PSMA protein

The EC50 of the anti-PSMA antibodies to recombinant human PSMA protein was determined by ELISA. Briefly, 96-well plates were coated with 1 μg/mL recombinant human PSMA (Acro Biosciences), blocked with 1% BSA (Sigma Aldrich) in PBS, then treated with anti-PSMA antibodies at an initial concentration of 10 μg/mL and diluted 3-fold to obtain a 12-point μ dilution series, followed by treatment with HRP conjugated anti-human IgG Fc antibodies (Invitrogen) at a 1:3000 dilution. After addition of TMB substrate and stop solution, plates were read at 450nm on a Cytation plate reader (Biotek). EC50 was found to be in the two digit picomolar to unit digit nanomolar range. The EC50 of the highest affinity antibody was similar to that of reference antibody J591. The results are summarized in fig. 11A.

The Kd of the anti-PSMA antibody was determined by biolayer interferometry (Octet Red384, sartorius). Briefly, antibodies were loaded onto an anti-hIgG Fc capture (AHC) biosensor (Sartorius) at a concentration of 1 μg/mL for 300 seconds, followed by an association step in recombinant human PSMA (Acro Biosystems) at different concentrations for 200 seconds, and a dissociation step in 1 XOctet kinetic buffer (Sartorius) for 600 seconds. Data were processed using ForteBio "data analysis" software (version 11.0) and fitted to a 1:1 binding model. Recombinant human PSMA proteins were run on blue native PAGE gels (4-12%, invitrogen) to identify the oligomeric state of the native protein. As shown in fig. 11B, recombinant human PSMA protein appears to be a mixture of dimers and monomers, and anti-PSMA antibodies bind to dimers or monomers, as indicated by their similar Kd but different Rmax values.

Example 12 in vitro cytotoxicity against PSMACAR engineered γδ T cells

The cytotoxic potential of untransduced γδ T cells and anti-PSMACAR transduced γδ T cells derived from the same healthy donor was evaluated against Nuc NEAR INFRARED (NIR) modified cells (Sartorius), PSMA expressing 22Rv1 target cells, and PC3 cells engineered to express PSMA (PC 3-PSMA) at a low E: T ratio of 1:1. Viability of Nuc NIR expression targets was monitored every 2 hours over 120 hours using the IncuCyte SX5 system (Sartorius), reported as cytotoxicity index (calculated by dividing the total NIR subject area at each time point by the time 0 hour value). The lower the cytotoxicity index, the stronger the potency of the construct. As shown in fig. 12, several anti-PSMA V delta 1 CARs exhibited robust cytotoxicity, some of which exhibited cytotoxicity comparable to or better than the baseline CAR construct.

Example 13 in vitro cytotoxicity of "bolt-on" modified anti-PSMA CAR engineered γδ T cells in the presence of TGF-beta 1

The cytotoxic potential of one of the γδ T cells resistant to PSMACAR transduction and the same CAR construct modified to express "bolting" (i.e., dominant negative tgfβ receptor II (dnTGF βrii)) was evaluated against Nuc NEAR INFRARED (NIR) modified PC3-PSMA cells at an E: T ratio of 3:1. TGF-beta 1 was added under the respective co-culture conditions at a final concentration of 20ng/mL. The first stimulation was monitored for about 3 days, and then fresh target cells were plated in another 96-well plate. Effector cells from the first stimulus are then transferred to plates with fresh target cells. The second stimulation was monitored for an additional 3 days. Every 4 hours useSX5 monitoring of the co-cultures. Cytotoxicity index was calculated based on the remaining live target tumor cells by dividing the total NIR subject area at each time point by the value of each stimulus at time 0. As shown in fig. 13, lead 1 with dnTF βrii "bolting" maintained a higher cytotoxic activity against PC3-PSMA in the presence of tgfβ1 after a second stimulation with tumor cells when compared to the no tgfβ1 condition. In the presence of tgfβ1, naked lead 1CAR vδ1γδt cells were associated with minimal tumor control after the second stimulation due to the lack of defenses to mitigate the inhibitory effects of tgfβ1.

EXAMPLE 14 "bolt-on" modified anti-PSMACAR expression of CAR, dnTGF beta RII, CD103 and pSMAD2/3 in gamma delta T cells engineered

V delta 1T cells modified with the anti PSMACAR construct were counted by Annexin-DAPI on Novocyte (Agilent). Approximately 1x10 ⁵ cells were stained using the cell viability dye Zombie Aqua (bioleged). Cells were further surface stained for vδ1 (bioleged), dnTGF βrii (APC anti-human TGF- β receptor II antibody, bioleged) and CD103 (bioleged), and CAR expression was detected using biotinylated protein L (Acro Biosystems) and PE-streptavidin (bioleged). Cells were fixed prior to collection on Novocyte (Agilent). As shown in fig. 14A-14B, CAR expression remained essentially unchanged in the "bolt-up" modified anti PSMACAR engineered γδ T cells compared to the naked CAR construct, and dnTGF βrii expression was only detected in the "bolt-up" modified CAR construct. As shown in fig. 14C, anti PSMACAR transduced γδ T cells containing dnTGF βrii had reduced CD103 expression compared to the control without the dnTGF βrii construct. anti-PSMACAR transduced γδ T cells, with or without modification with dnTGF βrii construct, were serum starved at 37 ℃ for 2 hours at 5% CO ₂ to reduce pSMAD2/3 background levels. Cells were stained with Zombie Aqua vital dye (Biolegend) and plated into round bottom wells of ULA 96-well plates. 10ng/mL of human recombinant TGF-beta 1 (R & D Systems) was added at 37℃with 2% CO ₂ for 15 minutes. Cells were immediately fixed by adding an equal volume of pre-warmed BD Cytofix buffer (BD Biosciences) to the cell suspension. Cells were permeabilized using BD ^TM Phosflow Perm buffer III (BD Biosciences) and incubated for 30min on ice protected from light. After washing with staining buffer (2% FBS1 XPBS), anti-pSMAD 2/3 antibody conjugated to PE (BD Biosciences) was added to the cells and incubated for 1 hour at room temperature in the dark. cells were washed twice with staining buffer and collected on Novocyte. As shown in fig. 14D, anti PSMACAR transduced γδ T cells containing dnTGF βrii had reduced levels of pSMAD2/3 compared to the control without the dnTGF βrii construct.

Example 15 expansion of anti-PSMACAR engineered γδ T cells

1X 10 ⁶/mL of human PBMC in X-Vivo 15 medium (Lonza) containing 10% heat-inactivated FBS (HyClone) were activated for 5 days in the presence of IL-2 (100U/mL) on plates pre-coated with Vd1 activating antibody (Adicet Bio). On day 5, cell cultures were transduced with a gamma-retrovirus construct encoding anti-PSMACAR in the presence of fibronectin. Starting on day 6, the cells were further expanded by feeding and IL-2 replacement as needed. On day 17, cells were harvested and usedThe kit (Miltenyi Biotec) depletes αβ T cells. Purity and transduction efficiency of γδ cell populations were assessed by flow cytometry at Novocyte (Agilent). As shown in fig. 15, anti PSMACAR engineered γδ T cells with and without "bolting" exhibited robust cell expansion curves in all 3 donors. The dashed line represents expected fold expansion based on historical data.

EXAMPLE 16 in vivo efficacy of anti PSMACAR engineered γδ T cells

The in vivo efficacy of anti-PSMACAR engineered vδ1t cells was evaluated in a subcutaneous prostate cancer model using 22Rv1 cells (ATCC) sorted for intermediate levels of PSMA expression (22 Rv1 clone E7), depicted in fig. 16A. Male NSG mice (The Jackson Laboratory) were subcutaneously implanted with 2X 10 ⁶ 22Rv1 clones E7 cells mixed with Matrigel (1:1 ratio) (Corning). When tumor volume reached an average of 100mm ³ animals were randomly divided into 5 groups and IV (tail vein) injections were performed with 13x 10 ⁶ vδ1PSMACAR T cells. The positive control group was treated with baseline vδ 1j591 CAR T cells at a dose of 13x 10 ⁶ car+ cells, and also included a separate tumor group (untreated). Human IL-2 (Proleukin) was administered intraperitoneally prior to treatment, followed by three times per week. Tumor volumes were measured twice weekly with calipers. As shown in fig. 16B-16C, robust tumor responses were observed for both lead anti PSMACAR engineered vδ1t cells expanded in all 3 donors. Efficacy in the same donor is equivalent to or better than baseline CAR.

EXAMPLE 17 in vivo efficacy of defensive anti PSMACAR engineered γδ T cells

In the subcutaneous prostate cancer model, the in vivo efficacy of anti PSMACAR engineered vδ1T cells with and without dnTGF βrii "bolting" was assessed using PC3-PIP cells expressing high levels of PSMA and secreting tgfβ1. Male NSG mice (The Jackson Laboratory) were subcutaneously implanted with 1X 10 ⁶ PC3-PIP cells mixed with Matrigel (1:1 ratio) (Corning). When tumor volume reached an average of 100mm ³ animals were randomly divided into 5 groups and IV (tail vein) injections were performed with 1x 10 ⁶, or 5x 10 ⁶ vδ1PSMACAR T cells. Also included are individual tumor groups (untreated). Human IL-2 (Proleukin) was administered intraperitoneally prior to treatment, followed by three times per week. Tumor volumes were measured twice weekly with calipers. As shown in fig. 17A-17B, at higher doses, both V delta 1T cells engineered with and without "bolting" of the lead antibody PSMACAR observed robust tumor responses, while at suboptimal doses, only the lead antibody PSMACAR with "bolting" exhibited significant tumor control compared to the non-defensive lead antibody PSMACAR that lost tumor control at suboptimal doses.

Example 18 epitope mapping of binding agent in lead antibody PSMACAR

Epitope mapping of the lead binding agent was performed using a cross-linked mass spectrometry method (CovalX). First, internally generated antibodies and human PSMA-Fc (Acro Biosciences) were individually characterized using Autoflex II MALDI-ToF mass spectrometry (Bruker) and characterized as cross-linked complexes. Next, human PSMA-Fc was reduced/alkylated and proteolyzed using trypsin, chymotrypsin, asp-N, elastase and thermolysin, followed by nLC-Q-Exactive MS/MS analysis to characterize peptide mass fingerprints. To elucidate the epitopes of the 2 lead antibodies, protein complexes were incubated with deuterated cross-linkers and subjected to the same multi-enzymatic cleavage as applied to human PSMA-Fc alone, followed by nLC-Q-Exactive MS/MS analysis to detect cross-linked peptides in each complex. Fig. 18A locates the regions of human PSMA dimer corresponding to conformational epitopes of lead 1 and lead 2, respectively. In contrast, the linear predictive epitope of reference antibody J591 is also outlined. Fig. 18B outlines human PSMA sequences corresponding to epitopes of two lead anti-PSMA binders.

Example 19 enhancement of CAR V delta 1T cell effector function Using Membrane-bound IL-12 (mbiL-12)

The CAR vector consisting of an anti-CD 19 (clone FMC 63) scFv (as described in Kochendefer JN et al ,Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells,Blood.2010, 11/11; 116 (19): 3875-86), CD 8. Alpha. Hinge and transmembrane domains, and 4-1BB and CD3 zeta signaling domains, followed by a self-cleaving P2A sequence for isolation of the mbiL-12 molecule (as described in Lee et al ,Antigen-dependent IL-12signaling in CAR T cells promotes regional to systemic disease targeting,bioRxiv.2023, 7/1; 2023.01.06.522784) was constructed by cloning (integrating DNA technology) DNA fragments containing all the CAR domains into a self-inactivating (SIN) Moloney murine leukemia virus (Moloney Murine Leukemia Virus, MMLV) gamma retrovirus plasmid. The construct designs are shown in FIG. 19 (construct A) CAR (FMC 63) construct, (construct B) CAR-mbiL-12 construct and (construct C) mbiL-12 construct.

Healthy donor Peripheral Blood Mononuclear Cells (PBMC) are used to activate, expand and engineer vδ1γδ T cells to express the CD19 CAR-mbIL-12 transgene. The results are shown in FIGS. 20A-20D. (FIG. 20A) V.delta.1γ.delta.T cells (post transduction) were sampled at various time points during the expansion process to determine cell counts. There was no difference in total vδ1γδ T cell count between CAR and CAR-mbIL-12, indicating that mbIL-12 has no negative effect on the expansion of vδ1γδ T cells. Cells were harvested, stained for CAR and mbIL-12 using anti-FMC 63 antibody conjugated to PE (Acrobio Systems, newark, DE) and anti-IL 12 conjugated to APC (Miltenyi Biotec), respectively, and were acquired on Novocyte flow cytometry (Agilent Technologies) to determine the percentage of CAR and mbIL-12 positive vδ1γδ T cells. (figure 20B) at the end of the process, no% CAR difference was observed between the CAR and the vδ1γδ T cells of CAR-mbIL-12 to further support that mbIL-12 did not affect CAR surface expression. (FIG. 20C) the% mbiL-12 expressed on the cell surface of V.delta.1γ.delta.T cells decreased over time during the expansion process. (FIG. 20D) CAR-mbiL-12 V.delta.1γ.delta.T cells were co-cultured for 18 hours with and without CD19+ Raji cells. Upon stimulation with target cells, mbIL-12 increased on the cell surface of vδ1γδ T cells compared to the unstimulated conditions, suggesting that surface expression of mbIL-12 is dependent on CAR activation.

To demonstrate the function of CAR-mbIL-12, cytolytic activity against CD19 positive cell lines using CAR, CAR-mbIL-12 and mbIL-12 transduced vδ1γδ T cells was evaluated in an in vitro replicate stimulation assay. Raji-Nuc NIR fluorescence labeled cell lines and effector cells were added to 96-well plates at E:T ratios of 5:1, 2.5:1, and 1.25:1. The first stimulation was monitored for 3 days, and then fresh target cells were plated in another 96-well plate. Effector cells from the first stimulus are then transferred to plates with fresh target cells. The second stimulus was monitored for an additional 3 days and the third stimulus was subjected to the same procedure. Every 4 hours useSX5 (Satorius) monitoring the co-cultures. Cytotoxicity index was calculated based on the remaining live target tumor cells by dividing the total NIR subject area at each time point by the value of each stimulus at time 0. The lower the cytotoxicity index, the better the cytotoxicity potential. After the second and third stimuli, in the tested E:T ratio, CAR-mbiL-12V delta 1 gamma delta T cells were associated with higher tumor cell killing compared to CAR and mbiL 12V delta 1 gamma delta T cells. Co-culture conditions with cytotoxicity index >1 (indicating incomplete tumor control) were not analyzed in the subsequent stimulation. The results are shown in figure 21 and demonstrate the function of CAR-mbIL-12 expressed by vδ1γδ T cells to maintain cytolytic killing of tumor cells.

In the Raji cell subcutaneous xenograft tumor model, suboptimal doses of CAR-mbIL-12vδ1γδ T cells of 2.5e6 car+ live cells were evaluated. Raji cell subcutaneous xenografts were established by inoculating 1E6 cells/mouse into the right posterior flank of female NSG mice. When the tumor volume was about 200mm ³, the mice were randomized into study cohorts (n=5 mice per group). Vδ1γδ T cells expressing CAR and CAR-mbIL-12 were administered in a single bolus dose on day 0. Human IL-2 was supplemented three times a week during the study. Tumor volume and mouse weight were monitored throughout the experiment. The results are shown in fig. 22. Compared to the suboptimal therapeutic dose of CAR vδ1γδ T cell group, CAR-mbIL-12vδ1γδ T cells were associated with significantly higher inhibition of tumor cell growth. The data support enhanced function of vδ1γδ T cells expressing CAR-mbIL-12.

Example 20 CISH KO enhances function of V.delta.1T cells

Healthy donor Peripheral Blood Mononuclear Cells (PBMCs) are used to activate, expand, CRISPR gene edit, and engineer vδ1γδ T cells to express CARs. To knock out CISH genes in vδ1T cells, ribonucleoprotein (RNP) complexes comprising CRISPR-Cas9 (INTEGRATED DNA Technologies) or-MAD 7 (Aldevron) and CISH SGRNA were combined with activated vδ1T cells from PBMCs (P3 primary cell 4D-Nucleofector X kit L, lonza). RNP complexes were delivered to V.delta.1T cells by electroporation (Lonza 4D-Nucleofector). Following electroporation, cells were transferred to T-flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery, then transduced with a gamma retroviral vector encoding a CAR, and then expanded. To evaluate CISH KO efficiency, cell pellets were collected and genomic DNA was extracted using NucleoSpin tissue kit (MACHEREY-NAGEL). The editing region of the CISH gene was amplified by PCR and sanger sequencing was performed (Sequetech). Gene knockout efficiency was determined using ICE analysis tool (Synthego). To evaluate KO of CISH protein in CAR vδ1T cells, WT (not edited) and CISH KO CAR vδ1T cells were incubated with and without IL-2 (Peprotech) for 5 hours. Cell lysates were generated for western blot detection of CISH proteins using anti-CISH rabbit mAb (cell signaling). Primary antibodies were detected using IRDye 800CW goat anti-rabbit IgG secondary antibody (LI-COR) and presence of IRDye was imaged using Odyssey imaging system (LI-COR). The results are shown in FIGS. 23A-23B. (FIG. 23A) WT CAR V.delta.1T cells showed increased levels of CISH protein when exposed to IL-2. In contrast, CISH KO CAR V delta 1T cells had significantly reduced CISH protein levels in the presence of IL-2 supporting CISH KO. (FIG. 23B) to demonstrate the enhanced function of CISH KO CAR transduced and non-transduced V.delta.1T cells, cytolytic activity against the B7-H6+HCT-15 cell line (ATCC) was evaluated using an in vitro replicate stimulus assay. HCT-15NIR fluorescence labeled cell lines and effector cells were added to 96-well plates at a 5:1 E:T ratio, with or without varying concentrations of IL-2. Each stimulus was monitored for 3 days, and then fresh target cells were plated in another 96-well plate. Effector cells from each stimulus are then transferred to a plate containing fresh target cells. Every 4 hours useSX5 (Satorius) monitoring the co-cultures. Cytotoxicity index was calculated based on the remaining live target tumor cells by dividing the total NIR subject area at each time point by the value of each stimulus at time 0. The lower the cytotoxicity index, the better the cytotoxicity potential. After the second stimulation, CISH KO non-transduced vδ1t cells were associated with higher tumor cell killing than WT non-transduced vδ1t cells only in the presence of IL-2. In the absence of IL-2, CISH KO CAR vδ1T cells had better tumor control than WT CAR vδ1T cells. The results indicate that CISH KO enhances cytotoxicity of vδ1 cells in vitro.

CAS9 CISH sgRNA:TGTACAGCAGTGGCTGGTGG(AGG)(SEQ ID NO:315)

MAD7 CISH sgRNA:(TTTA)GGTGTACAGCAGTGGCTGGTG(SEQ ID NO:316)。

Example 21 CBL-BKO enhances the function of V.delta.1T cells

Healthy donor Peripheral Blood Mononuclear Cells (PBMCs) are used to activate, expand, CRISPR gene edit, and engineer vδ1γδ T cells to express CARs. To knock out the CBL-B gene in vδ1T cells, ribonucleoprotein (RNP) complexes comprising CRISPR-Cas9 (INTEGRATED DNA Technologies) and CBL-B sgrnas were combined with activated vδ1T cells from PBMCs (P3 primary cell 4D-Nucleofector X kit L, lonza). RNP complexes were delivered to V.delta.1T cells by electroporation (Lonza 4D-Nucleofector). Following electroporation, cells were transferred to T-flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery, then transduced with a gamma retroviral vector encoding a CAR, and then expanded. The results are shown in FIGS. 24A-24B. (FIG. 24A) to determine CBL-B KO efficiency, genomic DNA was extracted from cell pellets using the Nucleospin tissue kit (MACHARY-NAGEL). The editing region was amplified by PCR using the previously internally optimized primers. The PCR reaction was run on agarose gel and the prominent bands excised and the gel was extracted using Nucleospin gel and PCR Clenup kit (MACHARY-NAGEL). The extracted DNA was sequenced by sanger sequencing (Sequetech). The% indels were determined using the ICE analysis tool (Synthego). (FIG. 24B) to demonstrate the enhanced function of CBL-B KO CAR transduced and non-transduced V.delta.1T cells, cytolytic activity was assessed against PSMA+PC3 cell lines using an in vitro replicate stimulation assay. PC3-PSMA NIR fluorescent labeled cell lines and effector cells were added to 96-well plates at an E:T ratio of about 3:1, with or without IL-2. Each stimulus was monitored for 3 days, and then fresh target cells were plated in another 96-well plate. Effector cells from each stimulus are then transferred to a plate containing fresh target cells. Every 4 hours useSX5 (Satorius) monitoring the co-cultures. Cytotoxicity index was calculated based on the remaining live target tumor cells by dividing the total NIR subject area at each time point by the value of each stimulus at time 0.

Cas9 multi-guide sgRNA sequence:

AAGACUCUUUAAAGAAGGCA(SEQ ID NO:317)

AGUACUCAUUCUCACUGAGU(SEQ ID NO:318)

CGUAAAUGCUGAUAUGUAUC(SEQ ID NO:319)

Cas9 single guide TAATCTGGTGGACCTCATGA (AGG) (SEQ ID NO: 320).

Example 22 Roquin-1 KO enhances function of V.delta.1T cells

Healthy donor Peripheral Blood Mononuclear Cells (PBMCs) are used to activate, expand, CRISPR gene edit, and engineer vδ1γδ T cells to express CARs. To knock out Roquin-1 gene in vδ1T cells, ribonucleoprotein (RNP) complexes comprising CRISPR-Cas9 (INTEGRATED DNA Technologies) and Roquin-1 sgrnas were combined with activated vδ1T cells from PBMCs (P3 primary cell 4D-Nucleofector X kit L, lonza). RNP complexes were delivered to V.delta.1T cells by electroporation (Lonza 4D-Nucleofector). Following electroporation, cells were transferred to T-flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery, then transduced with a gamma retroviral vector encoding a CAR, and then expanded. The results are shown in FIGS. 25A-25B. (FIG. 25A) to determine Roquin KO efficiency, nucleospin tissue kit (MACHARY-NAGEL) was used to extract genomic DNA from cell pellets. The editing region was amplified by PCR using the previously internally optimized primers. The PCR reaction was run on agarose gel and the prominent bands excised and the gel was extracted using Nucleospin gel and PCR Clenup kit (MACHARY-NAGEL). The extracted DNA was sequenced by sanger sequencing (Sequetech). The% indels were determined using the ICE analysis tool (Synthego). Editing was first performed using 3 gRNA sequences in a 1:1:1 ratio to demonstrate the ability to edit genes in Vd 1T cells, followed by single guide optimization to identify the grnas with highest KO efficiency. (FIG. 25B) to demonstrate Roquin-1 KO CAR transduced and non-transduced V.delta.1T cells enhanced function, cytolytic activity was evaluated against PSMA+PC3 cell lines using an in vitro replicate stimulation assay. PC3-PSMA NIR fluorescent labeled cell lines and effector cells were added to 96-well plates at an E:T ratio of about 3:1, with or without IL-2. Each stimulus was monitored for 3 days, and then fresh target cells were plated in another 96-well plate. Effector cells from each stimulus are then transferred to a plate containing fresh target cells. Every 4 hours useSX5 (Satorius) monitoring the co-cultures. Cytotoxicity index was calculated based on the remaining live target tumor cells by dividing the total NIR subject area at each time point by the value of each stimulus at time 0.

Multi-guide sgRNA sequences:

AAGCCCAUCAGUUUGGGUUG(SEQ ID NO:321)

UGUACAAGCUCCACAAUGGA(SEQ ID NO:322)

CAAAUGGGCAAGCCUUGCGG(SEQ ID NO:323)

internally optimized single gRNA sequence CCTGAATAAACTCCACCGCA (AGG) (SEQ ID NO: 324)

Example 23 ICAM-1 and CD58 KO enhanced in vitro cell survival of CAR V.delta.1T cells in the presence of allogeneic PBMC

Healthy donor Peripheral Blood Mononuclear Cells (PBMCs) are used to activate, expand, CRISPR gene edit, and engineer vδ1γδ T cells to express CARs. To knock out CISH gene in V.delta.1T cells, ribonucleoprotein (RNP) complexes consisting of CRISPR-Cas9 (INTEGRATED DNA Technologies) or-MAD 7 (Albevron) and ICAM-1 or CD58 sgRNA were combined with activated V.delta.1T cells from PBMC (P3 primary cell 4D-nucleic actor X kit L, lonza). RNP complexes were delivered to V.delta.1T cells by electroporation (Lonza 4D-Nucleofector). Following electroporation, cells were transferred to T-flasks (Corning) containing X-Vivo15 (Lonza) for cell recovery, then transduced with a gamma retroviral vector encoding a CAR, and then expanded. To determine the KO efficiencies of ICAM-1 and CD58, WT (unedited), ICAM-1KO, and CD58 KO CAR vδ1γδ T cells were stained with anti-ICAM-1, anti-CD 58, or isotype control antibodies conjugated to PE (BioLegend) and acquired using Novocyte flow cytometry (Agilent Technologies).

The results are shown in FIGS. 26A-26B. (FIG. 26A) the KO efficiencies of ICAM-1 and CD58 KO were both observed to be more than 80% in CAR V.delta.1γ.delta.T cells. (FIG. 26B) to evaluate whether ICAM-1 and CD58 enhance survival of CAR V.delta.1T cells in the presence of allogeneic PBMC, a 10 day mixed lymphocyte reaction assay (MLR) was performed. WT (unedited), CISH KO, ICAM-1KO and CD58 KO CAR vδ1γδ T cells (target cells) were co-cultured with allogeneic PBMCs (effector cells) in the presence of IL-2 at an E: T ratio of 10:1. During co-cultivation, half of the medium was replaced with fresh medium containing IL-2 every 2-3 days. On days 3, 6 and 10, cells were harvested from co-culture and stained with B7-H6 conjugated with APC (Adicet Bio produced) for car+ and vδ1+γδt cells for CAR detection and with anti-vδ1 antibody conjugated with PE-CY7 (Adicet Bio produced) for vδ1T cell detection. Cells were obtained using Novocyte flow cytometry to obtain absolute counts of double positive car+vδ1+t cells. To determine the% target cell change at each time point, the absolute count of WT car+vδ1+γδt cells was compared to KO car+vδ1+γδt cell count conditions using the following formula [ (WT car+vδ1+γδt cell count-KO car+vδ1+γδt cell count)/(WT car+vδ1+γδt cell count) ] x 100. Based on the target cell change relative to WT car+vδ1+γδ T cell count, only ICAM-1KO and CD58 KO car+vδ1+γδ T cells had increased target change, suggesting that disruption of ICAM-1 and CD58 reduced in vitro killing of allogeneic PBMCs, thereby improving cell survival.

CAS9 ICAM-1sgRNA:AAAGUCAUCCUGCCCCGGGG(SEQ ID NO:325)

MAD7 ICAM-1sgRNA:TTTG/AATAGCACATTGGTTGGCTAT(SEQ ID NO:326)

CAS9 CD58 sgRNA:AAAUAUAUGGUGUUGUGUAU(SEQ ID NO:327)

MAD7 CD58 sgRNA:TTTA/GACACTGTGTCAGGTAGCCTC(SEQ ID NO:328)

The embodiments and examples described above are illustrative only and not limiting. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific compounds, materials, and procedures. All such equivalents are considered to be within the scope and coverage of the following claims.

Claims (51)

1. An affinity binding entity comprising an antigen binding domain that specifically binds to a Prostate Specific Membrane Antigen (PSMA), wherein the antigen binding domain comprises:

a heavy chain variable region/light chain variable region (HCVR/LCVR) sequence pair ：SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24、25/26、27/28、29/30、31/32、33/34 and 35/36 selected from the group consisting of, or six CDR：SEQ ID NO:1/2、3/4、5/6、7/8、9/10、11/12、13/14、15/16、17/18、19/20、21/22、23/24、25/26、27/28、29/30、31/32、33/34 and 35/36 selected from the group consisting of HCVR/LCVR sequence pairs.

2. The affinity binding entity of claim 1, wherein said antigen binding domain comprises:

The HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NO 1/2, 3/4, 5/6, 7/8, 9/10, 11/12 and 13/14, or the six CDRs of the HCVR/LCVR sequence pair selected from the group consisting of SEQ ID NO 1/2, 3/4, 5/6, 7/8, 9/10, 11/12 and 13/14.

3. The affinity binding entity of claim 1 or claim 2, wherein said affinity binding entity is an antibody or antibody fragment, optionally wherein said affinity binding entity is selected from the group consisting of scFv, fab, fab ', fv, F (ab') ₂, dsFv, dAb, and any combination or plurality thereof.

4. The affinity binding entity of claim 3, wherein said antibody or antibody fragment is bispecific or monoclonal.

5. The affinity binding entity of claim 3 or claim 4, wherein said antibody or antibody fragment is chimeric, humanized or human.

6. A Chimeric Antigen Receptor (CAR), wherein the CAR comprises the affinity binding entity of any one of claims 2-5.

7. The CAR of claim 6, wherein the CAR further comprises a hinge domain, optionally wherein the hinge domain comprises a glycine polymer, a glycine-serine polymer, a glycine-alanine polymer, an alanine-serine polymer, an immunoglobulin heavy chain hinge, or a receptor-derived hinge.

8. The CAR of claim 7, wherein the receptor-derived hinge is a CD8 a hinge domain, optionally wherein the CD8 a hinge domain comprises the amino acid sequence set forth in SEQ ID No. 156.

9. The CAR of any one of claims 6-8, further comprising A Transmembrane (TM) domain; optionally wherein the TM domain comprises the TM region 4-1BB/CD137, an activated NK cell receptor, an immunoglobulin 、B7-H3、BAFFR、BLAME(SLAMF8)、BTLA、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137 or CD154、CD100(SEMA4D)、CD103、CD160(BY55)、CD18、CD19、CD19a、CD2、CD247、CD27、CD276(B7-H3)、CD28、CD29、CD3δ、CD3ε、CD3γ、CD3ζ、CD30、CD4、CD40、CD49a、CD49D、CD49f、CD69、CD7、CD84、CD8、CD8α、CD8β、CD96(Tactile)、CD11a、CD11b、CD11c、CD11d、CDS、CEACAM1、CRT AM、 cytokine receptor, DAP10, DNAM1 (CD 226), an Fc gammA receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ig alphA (CD 79A), IL-2 RbetA, IL-2 RgammA, IL-7 RalphA, an inducible T cell costimulatory factor (ICOS), an integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, A ligand that specifically binds CD83, LIGHT, LTBR, ly9 (CD 229), lymphocyte function-associated antigen-1 (LFA-1; CD1A/CD 18), MHC class 1 molecules, NKG2C, NKG2D, NKp, NKG 44, NKp46, NKp80 (KLRF 1), OX-40, PAG/Cbp, programmed death-1 (PD-1), PD-62, CD 62, SLF-6 or A combination thereof, or A combination thereof.

10. The CAR of claim 9 wherein said TM domain comprises the TM domain of CD8, preferably wherein said CD8 TM domain is the TM domain of CD8 alpha, optionally wherein said TM domain comprises the amino acid sequence shown as SEQ ID NO 158.

11. The CAR of any one of claims 6-10, further comprising at least one co-stimulatory domain, optionally wherein the co-stimulatory domain comprises co-stimulatory domain ：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、B7-H3、CEACAM1、CRTAM、CD2、CD3C、CD4、CD7、CD8α、CD8β、CD11a、CD11b、CD11c、CD11d、IL2Rβ、IL2γ、IL7Rα、IL4R、IL7R、IL15R、IL21R、CD18、CD19、CD19a、CD27、CD28、CD29、CD30、CD40、CDS、CD49a、CD49D、CD49f、CD54(ICAM)、CD69、CD70、CD80、CD83、CD84、CD86、CD96(Tactile)、CD100(SEMA4D)、CD103、CD134(OX40)、CD137(4-1BB)、CD152(CTLA-4)、CD160(BY55)、CD162(SELPLG)、CD244(2B4)、CD270(HVEM)、CD226(DNAM1)、CD229(Ly9)、CD278(ICOS)、ICAM-1、LFA-1(CD11a/CD18)、FcR、FcγRI、FcγRII、FcγRIII、LAT、NKG2C、SLP76、TRIM、ZAP70、GITR、BAFFR、LTBR、LAT、GADS、LIGHT、HVEM(LIGHTR)、KIRDS2、ITGA4、ITGA6、ITGAD、ITGAE、ITGAL、ITGAM、ITGAX、ITGB1、ITGB2、ITGB7、NKG2C、NKG2D、IA4、VLA-1、VLA-6、SLAM(SLAMF1、CD150、IPO-3)、SLAMF4、SLAMF6(NTB-A、Ly108)、SLAMF7、SLAMF8(BLAME)、SLP-76、PAG/Cbp、NKp80(KLRF1)、NKp44、NKp30、NKp46、BTLA、JAML、CD150、PSGL1、TSLP、TNFR2 or TRANCE/RANKL, or a portion or combination thereof.

12. The CAR of claim 11 wherein the costimulatory domain is a 4-1BB costimulatory domain, optionally wherein the 4-1BB costimulatory domain comprises the amino acid sequence depicted as SEQ ID NO. 162.

13. The CAR of any one of claims 6-12, further comprising one or more intracellular signaling domains, preferably wherein the intracellular signaling domain is a CD3 zeta intracellular signaling domain, optionally wherein the CD3 zeta intracellular signaling domain comprises an amino acid sequence as shown in SEQ ID No. 164, 166 or 167.

14. The CAR of any one of claims 6-13, further comprising a signal peptide, optionally wherein the signal peptide comprises the amino acid sequence set forth in SEQ ID No. 152.

15. An isolated polynucleotide comprising a nucleic acid sequence encoding the affinity binding entity of any one of claims 1-5.

16. An expression vector comprising the polynucleotide of claim 15 operably linked to a cis-acting regulatory element.

17. A cell comprising the affinity binding entity of any one of claims 1-5, the isolated polynucleotide of claim 15, and/or the expression vector of claim 16.

18. An isolated polynucleotide comprising a nucleic acid sequence encoding the CAR of any one of claims 6-14.

19. The isolated polynucleotide of claim 18, further comprising a nucleic acid sequence encoding at least one polycistronic linker region, optionally a polycistronic region encoding a cleavage sequence and/or an Internal Ribosome Entry Site (IRES).

20. The isolated polynucleotide of claim 19, wherein the cleavage sequence is selected from T2A, F2A, P2A, E a, furin and furin-P2A (FP 2A).

21. The isolated polynucleotide of any one of claims 18-20, further comprising a nucleic acid sequence encoding one or more additional polypeptides.

22. The isolated polynucleotide of claim 21, wherein the one or more additional polypeptides are selected from the group consisting of lymphotoxin beta receptor (LTBR), low affinity nerve growth factor receptor (LNGFR), dominant negative (dn) receptor of TGF-beta or Fas, truncated forms of human epidermal growth factor receptor (EGFRt), membrane bound IL-12 (mbiL-12), fluorescent proteins, gamma chain cytokines, CD19, CD20, CD 70-binding CAR, and any combination thereof.

23. The isolated polynucleotide of claim 22, wherein the one or more additional polypeptides is dnTGF βr2, optionally wherein the dnTGF βr2 comprises the amino acid sequence set forth in SEQ ID No. 265.

24. The isolated polynucleotide of claim 22, wherein the additional polypeptide is an LTBR, wherein the LTBR comprises an amino acid sequence set forth in SEQ ID No. 267.

25. The isolated polynucleotide of claim 22, wherein the additional polypeptide is EGFRt, wherein the EGFRt comprises the amino acid sequence of SEQ ID No. 261.

26. The isolated polynucleotide of claim 22, wherein the additional polypeptide is an LNGFR, wherein the LNGFR comprises the amino acid sequence set forth in SEQ ID No. 273.

27. The isolated polynucleotide of any of claims 22-26, wherein the one or more additional polypeptides are operably linked to a nucleic acid sequence encoding a signal peptide, optionally wherein the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:259, SEQ ID NO:263, SEQ ID NO:267, SEQ ID NO:271, and SEQ ID NO: 248.

28. The isolated polynucleotide of any one of claims 18-27, comprising the nucleic acid sequence of SEQ ID NO 205, 209, 213, 217, 221, 225, 229 or 233.

29. An expression vector comprising the isolated polynucleotide of any one of claims 18-28 operably linked to a cis-regulatory element.

30. A γδ T cell comprising:

(a) A nucleic acid sequence encoding a Chimeric Antigen Receptor (CAR) comprising an affinity binding domain that specifically binds to a Prostate Specific Membrane Antigen (PSMA), and/or

(B) A polypeptide comprising a CAR, the CAR comprising an amino acid sequence encoded by the nucleic acid of (a);

Wherein the γδ T cells functionally express the binding domain of the polypeptide or nucleic acid encoded CAR on the surface of the γδ T cells.

31. The γδ T-cell of claim 30, wherein the CAR comprises an affinity binding entity of any one of claims 2-5, or

Wherein the nucleic acid sequence comprises the isolated polynucleotide of any one of claims 18-28, or the expression vector of claim 29.

32. A modified immune cell comprising the CAR of any one of claims 6-14, the polynucleotide of any one of claims 18-28, or the expression vector of claim 29.

33. The modified immune cell of claim 32, wherein the modified immune cell is a γδ T cell, γδ NKT cell, αβ T cell, NK cell, NKT cell, or macrophage.

34. The modified immune cell of claim 33, wherein the modified immune cell is a γδ T cell, optionally wherein the γδ T cell is a δ1, δ2, δ3 or δ4γδ T cell, preferably δ2 ^- γδ T cell, more preferably δ1γδ T cell.

35. The modified immune cell of any one of claims 32-34, or the γδ T-cell of claim 30 or claim 31, wherein the modified immune cell or the γδ T-cell exhibits in vitro and/or in vivo cell killing activity against tumor cells exhibiting PSMA cell surface expression.

36. The modified immune cell or γδ T-cell of claim 35, wherein the modified immune cell or γδ T-cell proliferates in response to contact with the tumor cell exhibiting PSMA cell surface expression, optionally wherein the modified immune cell or γδ T-cell proliferates in a host organism comprising a tumor cell exhibiting PSMA cell surface expression.

37. The modified immune cell of any one of claims 32-36, or the γδ T-cell of any one of claims 30-31 or 35-36, wherein the modified immune cell or the γδ T-cell expresses a pro-inflammatory cytokine upon contact with a tumor cell exhibiting PSMA cell surface expression.

38. The modified immune cell of any one of claims 32-37, or the γδ T-cell of any one of claims 30-31 or 35-37, further comprising at least one disrupted gene, optionally wherein the at least one disrupted gene is a cytokine-induced SH 2-containing protein (CISH), cbl-B protooncogene B (Cbl-B), zinc finger protein 91 (ZFP 91), CD58, ICAM-1, or any combination thereof.

39. A method of making the modified immune cell of any one of claims 32-38 or the γδ T cell of any one of claims 30-31 or 35-38, wherein the method comprises transfecting the immune cell or γδ T cell with the expression vector of claim 29, optionally wherein the cell has at least one disrupted gene.

40. The method of claim 39, wherein the method comprises retroviral transduction.

41. The method of claim 39 or 40, wherein the method comprises ex vivo expansion of the immune cells or γδ T cells, wherein the ex vivo expansion is performed before and/or after transfection of the expression vector.

42. An antibody-drug conjugate (ADC) comprising the affinity binding entity of any one of claims 1-5.

43. A pharmaceutical composition comprising the affinity binding entity of any one of claims 1-5 or the ADC of claim 42 and a pharmaceutically acceptable carrier.

44. A pharmaceutical composition comprising a plurality of modified immune cells of any one of claims 32-38, or a plurality of γδ T cells of any one of claims 30-31 or 35-38, optionally wherein the plurality comprises a composition of at least 60%, 80% or about 60% or 80% to about 90% or 95% δ1, δ2, δ3 or δ4γδ T cells, preferably δ1 or δ2γδ T cells, more preferably δ2- γδ T cells, most preferably δ1γδ T cells, and a pharmaceutically acceptable carrier.

45. The pharmaceutical composition of claim 44, wherein the plurality comprises at least about 10 ⁷ modified immune cells or γδ T cells, respectively, preferably about 10 ⁸ modified immune cells or γδ T cells to about 10 ¹¹ modified immune cells or γδ T cells, respectively.

46. A method of inhibiting the growth of a cell exhibiting PSMA cell surface expression, comprising contacting the cell with the affinity binding entity of any one of claims 1-5, the modified immune cell of any one of claims 32-38, the γδ T cell of any one of claims 30-31 or 35-38, the ADC of claim 42, or the pharmaceutical composition of any one of claims 43-45.

47. A method of killing a tumor cell that exhibits PSMA cell surface expression, comprising contacting the tumor cell with a therapeutically effective amount of the affinity binding entity of any one of claims 1-5, the modified immune cell of any one of claims 32-38, the γδ T cell of any one of claims 30-31 or 35-38, the ADC of claim 42, or the pharmaceutical composition of any one of claims 43-45.

48. The method of claim 47, wherein the method comprises introducing a therapeutically effective amount of the affinity binding entity, the modified immune cell, the γδ T cell, the ADC, or the pharmaceutical composition into a host organism comprising the tumor cell.

49. The method of claim 48, further comprising

Optionally wherein said administering one or more methods of increasing a common gamma chain cytokine comprises administering an amount of a common gamma chain cytokine simultaneously or sequentially, depletion of lymphocytes prior to introduction of said modified immune cells or said γδ T cells, and/or secretion of one or more common gamma chain cytokines from said introduced modified immune cells or γδ T cells.

50. The method of any one of claims 46-49, wherein the host organism is a human and the method is a method of treating cancer in a subject in need thereof.

51. Use of an affinity binding entity of any one of claims 1-5, a modified immune cell of any one of claims 32-38, a γδ T cell of any one of claims 30-31, 35-38, an ADC of claim 42, or a pharmaceutical composition of any one of claims 43-45 in the manufacture of a medicament for treating cancer.