HK1247625A1 - Masking chimeric antigen receptor t cells for tumor-specific activation - Google Patents

Description

Masked chimeric antigen receptor T cells for tumor specific activation

Government rights

The invention was made with government support under grant numbers CA170820, EB017206 and CA132681 awarded by the National Institutes of Health. The government has certain rights in this invention.

Technical Field

The present invention relates to activatable chimeric antigen receptors and cells genetically engineered using the chimeric antigen receptors. The activatable mCAR is inactive when masked and active when unmasked.

Background

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be helpful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Adoptive transfer of T cells, particularly Chimeric Antigen Receptor (CAR) -engineered T cells, has emerged as a promising approach in cancer immunotherapy. CARs are synthetic receptors consisting of an extracellular single-chain variable fragment (scFv) that specifically recognizes a tumor-associated antigen (TAA), a hinge, a transmembrane domain, and an intracellular signaling and co-stimulatory domain. Unlike naturally occurring T cell receptors, CARs can directly recognize their target antigen without the limitations imposed by Major Histocompatibility Complex (MHC) molecules and can potentially mediate high levels of cell killing activity.

CAR-modified T cell (CAR-T) therapy has shown significant success in treating B cell malignancies by targeting the B cell specific receptor CD19 in multiple clinical trials. This has re-stimulated significant interest in exploiting CAR-T technology for the treatment of solid tumors, and several ongoing clinical trials are aimed at testing this treatment modality. However, one challenging aspect of such a switch is the identification of solid tumor antigens that are ideally restricted to tumor cells. Although many solid tumor antigens have been identified, most of them are also expressed at low levels in normal tissues. It is this low level of antigen expression in healthy cells that can cause activation of CAR-T cells and lead to "on-target off-tumor" toxicity. For example, infusion of human epidermal growth factor receptor 2(HER2) -specific CAR-T cells in one patient caused lethal inflammatory cytokine release due to HER2 expression in lung tissue. Given the challenges of identifying ideal tumor antigens, one strategy to ameliorate the undesirable on-target but off-tumor effects is to engineer tumor-selective mechanisms into CAR structures to allow better differentiation between target antigens in the tumor microenvironment and those in normal tissues.

T cell immunotherapy is a powerful treatment that can lead to long-term cures in patients with melanoma, B cell lymphoma, and other cancers. One commonly used approach is to genetically engineer T cells ex vivo to express Chimeric Antigen Receptors (CARs) that recognize a target antigen without MHC presentation. These CAR-T cells have the potential to generate extremely high levels of anti-tumor activity, but they can also display enhanced off-target cell killing. Therefore, there is a need in the art to minimize such side effects. Described herein are compositions that reduce off-target cell killing of CAR-T cells.

Summary of The Invention

The following embodiments and aspects thereof are described and illustrated in conjunction with systems, compositions, and methods, which are meant to be exemplary and illustrative, not limiting in scope.

T cells expressing chimeric antigen receptors have the potential to produce extremely high levels of anti-tumor activity, but they may also exhibit enhanced off-target cell killing. To minimize this side effect, a Chimeric Antigen Receptor (CAR) has been designed that contains an N-terminal masking peptide that blocks the ability of the CAR to bind to its target, the Epidermal Growth Factor Receptor (EGFR), a tumor-associated antigen that is highly expressed in a wide variety of tumors. The masking peptide may be cleaved by, for example, a protease that is normally active in the tumor microenvironment, thereby allowing the mCAR to recognize only its target antigen at the tumor site.

Provided herein are compositions comprising a masked chimeric antigen receptor (mCAR), wherein the CAR is inactive when masked and active when the mask is cleaved. In one embodiment, the mCAR comprises, consists of, or consists essentially of: the sequence shown in Table 1 and/or SEQ ID NO: 29. As described herein, the masking peptide comprises a mask that prevents premature binding of the antigen-specific targeting domain in the mCAR to its target, a cleavage site that may be a substrate for a protease, a linker sequence that links the mask to the cleavage site, and a linker sequence that links the cleavage site to the car.

In one embodiment, the structural arrangement of mCAR from N-terminus to C-terminus is mask-linker-cleavage site-linker-CAR when the mask is not cleaved. In one embodiment, the structural arrangement of mCAR from N-terminus to C-terminus is a linker-CAR when the mask is cleaved.

In one embodiment, the structural arrangement of mCAR from N-terminus to C-terminus when the mask is not cleaved comprises, consists of, or consists essentially of: mask-linker-cleavage site-linker-antigen specific targeting domain-transmembrane domain-costimulatory domain-intracellular signaling domain. Additional sequences may be present between each domain, for example to provide further flexibility and stability to the mCAR.

In one embodiment, the structural arrangement of mCAR from N-terminus to C-terminus when the mask is not cleaved comprises, consists of, or consists essentially of: mask-linker-cleavage site-linker-antigen specific targeting domain-extracellular spacer domain-transmembrane domain-costimulatory domain-intracellular signaling domain. Additional sequences may be present between each domain, for example to provide further flexibility and stability to the mCAR.

In one embodiment, the mCAR specific for EGFR comprises a masking peptide, wherein the mask in the masking peptide comprises, consists or consists essentially of: a sequence that is at least 100%, 99%, 98%, 97%, 96% 95%, 94%, 93%, 92%, 91%, 90%, 85% or 80% identical to CISPRGCPDGPYVMY (SEQ ID NO: 1). In another embodiment, the mCAR specific for Her2 comprises a masking peptide, wherein the mask in the masking peptide comprises, consists or consists essentially of: a sequence that is at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85% or 80% identical to LLGPYELWELSH (SEQ ID NO: 17). In other embodiments, the mCAR specific for GD2 comprises a masking peptide, wherein a mask in the masking peptide comprises or consists essentially of: a sequence that is at least 100%, 99%, 98%, 97%, 96% 95%, 94%, 93%, 92%, 91%, 90%, 85% or 80% identical to RCNPNMEPPRCWAAEGD (SEQ ID NO:22) or a sequence that is at least 100%, 99%, 98%, 97%, 96% 95%, 94%, 93%, 92%, 91%, 90%, 85% or 80% identical to (VCNPLTGALLCSAAEGD) (SEQ ID NO: 23). In further embodiments, the mCAR specific for carbonic anhydrase 9(CA-IX) comprises a masking peptide, wherein the mask in the masking peptide comprises or consists of or consists essentially of: a sequence that is at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, or 80% identical to LSTAFARV (SEQ ID NO:24) or a sequence that is at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, or 80% identical to ALGPGREYRAL (SEQ ID NO: 25).

In one embodiment, the mCAR comprises a masking peptide, wherein the cleavage site in the masking peptide comprises, consists of, or consists essentially of: a sequence that is at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85% or 80% identical to LSGRSDNH (SEQ ID NO: 2).

In one embodiment, the mCAR comprises an antigen-specific targeting domain that specifically binds and inhibits (EGF R). In one embodiment, the EGFR inhibitor comprises, consists of, or consists essentially of: a variable light chain sequence at least 100%, 99%, 98%, 97%, 96% 95%, 94%, 93%, 92%, 91%, 90%, 85% or 80% identical to QILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKR (SEQ ID NO:3) and a variable heavy chain sequence at least 100%, 99%, 98%, 97%, 96% 95%, 94%, 93%, 92%, 91%, 90%, 85% or 80% identical to QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSS (SEQ ID NO: 4).

In some embodiments, the mCAR comprises, consists of, or consists essentially of: a sequence that is at least 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85% or 80% identical to the sequence shown in Table 1 and/or SEQ ID NO: 29.

Also provided herein are methods for generating a plurality of T cells expressing a masked chimeric antigen receptor. The methods comprise transfecting T cells with a vector encoding a masked chimeric antigen receptor described herein and stimulating one or more T cells with cells expressing an antigen targeted by an antigen-specific targeting domain or with a recombinant antigen specific for the ASTD of mCAR, or a combination thereof. In some embodiments, stimulation of the transfected cells causes T cells to proliferate, thereby producing a large number of T cells.

Also provided herein are methods for treating, inhibiting, alleviating the symptoms of, or delaying/slowing the progression of a disease in a subject. The method comprises administering to the subject an effective amount of a composition comprising mCAR described herein. In one embodiment, the mCER in the composition comprises the sequence shown in Table 1 and/or SEQ ID NO: 29. In one embodiment, the mCAR specifically binds EGFR. In one embodiment, the disease is any disease that can be treated by inhibiting EGFR. In one embodiment, the disease is cancer. In some embodiments, the cancer is any one or more of: lung cancer, colorectal cancer, breast cancer, head and neck cancer, melanoma, glioblastoma, pancreatic cancer, ovarian cancer. In one embodiment, the mCAR specifically binds GD2 ganglioside and the cancer is neuroblastoma, melanoma, small cell lung cancer, osteosarcoma, or soft tissue sarcoma. In one embodiment, the mCAR specifically binds GD2 ganglioside and the cancer is neuroblastoma. In yet another embodiment, the mCAR specifically binds carbonic anhydrase 9 and the cancer is renal cell carcinoma, superficial bladder cancer, or invasive urothelial cancer. In one embodiment, the mCAR specifically binds carbonic anhydrase 9 and the cancer is renal cell carcinoma.

Also provided herein are methods for treating, inhibiting, alleviating the symptoms of, or delaying/slowing the progression of lung cancer in a subject. The method comprises administering to the subject an effective amount of a composition comprising mCAR described herein. In one embodiment, the mCAR in the composition comprises, consists of, or consists essentially of: the sequence shown in Table 1 and/or SEQ ID NO: 29. In one embodiment, the mCAR specifically binds EGFR.

Brief Description of Drawings

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

Figure 1 depicts a schematic of an unmasked, masked and NSUB form of an anti-EGFR CAR construct according to one embodiment of the invention (a) a schematic of a masked CAR to improve tumor selectivity in a tumor microenvironment in the presence of a protease, a masking peptide is cleaved and exposes a previously blocked antigen binding site of a single chain variable fragment (scFv) (b) a schematic of various anti-EGFR mCAR constructs scFv sequences derived from the monoclonal antibody cetuximab scFv in-frame fusion with a CD8 α hinge and transmembrane domain followed by a CD28/41BB/CD3 zeta signaling domain and then cloned into a retroviral vector to produce an unmasked CAR.

Figure 2 depicts the binding ability to EGFR protein of anti-EGFR CAR transduced Jurkat cells according to one embodiment of the present invention. Jurkat cells were transduced with lentiviral vectors encoding EGFR CARs or masking EGFR CARs, respectively. CAR-Jurkat cells were stained with recombinant human EGFR-Fc, and goat-anti-human Fc antibody was then used as secondary antibody (black) or secondary antibody against background only (grey).

FIG. 3 depicts CAR-Jurkat-NFAT-GFP reporter cells activated by co-culture with corresponding target cells K562-EGFR or K562-CD19 cells, according to one embodiment of the invention. Jurkat reporter cells were transduced with lentiviral vectors encoding EGFR CAR, masked EGFRCAR, or CD19CAR, respectively. CAR-Jurkat reporter cells were co-cultured with their target cells. CAR-Jurkat reporter cell activation was assessed by GFP expression. Masking the attenuation of activation of EGFR CARs but partial recovery after cleavage of protease uPA (50 nM).

FIG. 4 depicts CAR-Jurkat-NFAT-GFP reporter cells activated by coculture with breast cancer cells MDA-MB-231 according to one embodiment of the invention. Endogenous secretion of proteases from MDA-MB-231 cells may partially activate the masked EGFR CAR, while treatment with uPA (50nM) may enhance the activation signal.

FIG. 5 depicts titration of uPA concentration to restore activation of masked EGFR CAR-Jurkat reporter cells, according to one embodiment of the invention. Treatment with higher concentrations of uPA produced a more activated masked EGFR CAR-Jurkat reporter population, assessed by its GFP expression following stimulation with target K562-EGFR cells.

Figure 6 depicts the expression of various CARs and their binding capacity to the target antigen EGFR in human T cells according to one embodiment of the present invention. Human PBMCs were activated and transduced with retroviral vectors encoding unmasked, masked and NSUB anti-EGFR CARs and ex vivo expanded for 10 days. (a) Three sets of CAR-T cells were stained with biotinylated protein L followed by APC-conjugated streptavidin to detect CAR expression on the cell surface. (b) CAR-T cells were incubated with recombinant human EGFR-Fc protein, followed by staining with PE-conjugated goat anti-human Fc antibody to assess the binding ability of CARs to their target antigen human EGFR.

Figure 7 depicts the binding of various CARs to the target antigen EGFR after protease treatment, according to one embodiment of the present invention. Unmasked, masked and NSUB anti-EGFR CAR-T cells were treated with uPA at increasing concentrations (0nM, 100nM and 400nM) and then stained with recombinant human EGFR-Fc (rhEGFR-Fc) and goat anti-human Fc antibody to evaluate the effect of protease treatment on CAR binding to antigen.

Figure 8 depicts intracellular cytokine staining of various CAR-T cells stimulated with different target cells according to an embodiment of the invention. (a) On day 10 after ex vivo activation and expansion, unmasked, masked and NSUB CAR-T cells were co-cultured with K562, K562-EGFR, MDA-MB-231 or NCI-H292 cells for 6 hours with a GolgiPlug inhibitor. Unstimulated CAR-T cells were used as negative controls, while CAR-T cells stimulated with anti-CD 3/CD28 antibodies were used as positive controls. Interferon gamma (IFN- γ) production was measured by intracellular staining. CD8⁺T cells are shown in each figure. IFN-gamma secreting CD8T cells are gated and account for total CD8⁺The percentage of T cells is shown in each scatter plot. (b) The summarized statistics are shown as bar graphs (n-3, mean ± SEM; ns, not significant;, P<0.05；**，P<0.01；***，P<0.001, one-way ANOVA with Tukey multiple comparison).

Figure 9 depicts the in vitro cytotoxicity of various CAR-T cells against different target cell lines, according to one embodiment of the invention. Unmasked, masked and NSUB CAR-T cells were co-cultured with different target cell lines. (a) CAR-T cells were co-cultured with K562-EGFR cells at effector to target cell ratios of 1:1, 3:1 or 10:1 for 4 hours and cytotoxicity against K562-EGFR was measured and shown in the figure. (b) CAR-T cells were co-cultured with NCI-H292 cells at effector to target cell ratios of 1:1, 3:1, or 10:1 for 18 hours and cytotoxicity was measured. (c) CAR-T cells were co-cultured with MDA-MB-231 cells at effector to target cell ratios of 1:1, 2.5:1, 5:1, or 10:1 for 18 hours, and cytotoxicity was measured.

Figure 10 depicts the anti-tumor efficacy of CAR-T cells in a human lung cancer xenograft model, according to one embodiment of the invention. (a) Schematic of CAR-T treatment protocol in vivo. NCI-H292 cells were injected into the right flank of NSG mice on day 0. Mice were randomized into 4 groups (n ═ 8 per group) and treated with 4 million unmasked, masked, or NSUB CAR-T cells on days 13 and 26; untransduced T cells were included as controls. Tumor size was measured twice weekly with calipers. (b) Tumor growth curves in each group are shown as mean ± SEM (ns, not significant;, P < 0.05;, P < 0.01). (c) Mouse survival curves were calculated using the Kaplan-Meier method.

Detailed Description

All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al, Dictionary of Microbiology and Molecular Biology 3 rd edition, J.Wiley & Sons (New York, NY 2001); march, Advanced Organic Chemistry Reactions, mechanics and Structure 5 th edition, j.wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3 rd edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) provide one of skill in the art with a general guide to many of the terms used in this application.

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For the purposes of the present invention, the following terms are defined below.

The invention described herein provides Chimeric Antigen Receptors (CARs) that can be activated or masked (mCARS). Chimeric antigen receptors are engineered receptors that transplant immunospecificity onto genetically engineered cells. The masking peptide is unique to each antigen binding site on the CAR and renders the CAR inactive until activated, for example by cleavage of a linker connecting the CAR to the masking peptide.

As used herein, a "masking peptide" (MP) refers to a peptide that inhibits binding of the ASTD of the CAR to an antigen on a target cell when the MP is in an uncleaved state. The MP is attached to the CAR via a cleavable linker moiety. The MP comprises a mask (peptide) and a cleavage site that prevents the CAR from binding to an antigen on the target cell. In some embodiments, the linker sequence separates the masking peptide from the cleavage site and the Masking Peptide (MP) has a structural mask-linker-cleavage site. The cleavage site comprises an amino acid recognized by a protease. The length of the mask may be 5-50 amino acids. The MP, in the cleaved state, does not interfere with the binding of the CAR to the antigen on the target cell. In some embodiments, the structural arrangement of the masked car in the uncleaved state (mcar) from N-terminus to C-terminus is MP-L-ASTD-ESD-TM-CSD-ISD. In some embodiments, the structural arrangement of the mCAR in an uncleaved state, from N-terminus to C-terminus, is MP-L-ASTD-TM-CSD-ISD. In various embodiments, the masking peptide is unique to each ASTD.

As used herein, "antigen-specific targeting domain" (ASTD) refers to a domain/region of a CAR that targets a specific antigen. The mCAR may include one or more ASTDs. ASTD is extracellular and may comprise an antibody or functional equivalent thereof or a fragment or derivative thereof. The targeting domain/region can comprise a full-length heavy chain, a Fab fragment, a single chain fv (scfv) fragment, a bivalent single chain antibody, or a diabody, each of which is specific for a target antigen. As will be appreciated by those skilled in the art, in some embodiments, any molecule that binds a given antigen with high affinity may be used as an ASTD, e.g., a linked cytokine (which results in recognition of a cell bearing a cytokine receptor), an affibody, a ligand binding domain from a naturally occurring receptor, a soluble protein/peptide ligand for a receptor (e.g., on tumor cells), a peptide, and a vaccine, to elicit an immune response.

As used herein, "chimeric antigen receptor" or "CAR" refers to an engineered receptor that specifically transplants an antigen onto a cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immunoreceptors. The CAR comprises one or more antigen-specific targeting domains, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. In one embodiment, if the CAR targets two different antigens, the antigen specific targeting domains may be arranged in tandem and separated by a linker sequence.

As used herein, "co-stimulatory domain" (CSD) refers to a portion of a CAR that enhances proliferation, survival, and/or development of a memory cell. The CAR may comprise one or more co-stimulatory domains. Each co-stimulatory domain comprises a co-stimulatory domain of any one or more of: for example, members of the TNFR superfamily, CD28, CD137(4-1BB), CD134(OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1(CD11a/CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, or combinations thereof. Other co-stimulatory domains (e.g., from other proteins) will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention.

As used herein, "extracellular spacer domain" (ESD) refers to a hydrophilic region between an antigen-specific targeting domain and a transmembrane domain. In some embodiments, the mCAR of the present invention may or may not include an extracellular spacer domain. Extracellular spacer domains include, but are not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof.

As used herein, "intracellular signaling domain" (ISD) or "cytoplasmic domain" refers to the portion of the CAR that transduces effector function signals and directs the cell to perform its specialized function examples of domains that transduce effector function signals include, but are not limited to, the zeta chain of the T cell receptor complex or any homolog thereof (e.g., η chain, Fc epsilon R1 gamma and β chain, MB1(Ig α) chain, B29(Ig β) chain, etc.), human CD3 zeta chain, CD3 polypeptides (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 CD 28.

As used herein, a "linker" (L) or "linker domain" or "linker region" refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links any domains/regions of the CAR together and the CAR to the masking peptide. The linker may be composed of flexible residues like glycine and serine so that adjacent protein domains move freely with respect to each other. Longer linkers can be used when it is desired to ensure that two adjacent domains do not interfere spatially with each other. The linker may be cleavable or non-cleavable. In some embodiments, the masking peptide is attached to the CAR cleavable. Examples of cleavable linkers include a 2A linker (e.g., T2A), a 2A-like linker, or functional equivalents thereof, and combinations thereof. In some embodiments, the linker comprises a picornavirus 2A-like linker, a porcine teschovirus (P2A), a CHYSEL (SEQ ID NO:5) sequence of the Choristoneura littoralis (Thosea asigna) virus (T2A), or a combination, variant, and functional equivalent thereof. In other embodiments, the linker sequence may comprise Asp-Val/Ile-Glu-X-Asn-Pro-Gly^(2A)–Pro^(2B)Motifs (SEQ ID NO:6 and SEQ ID NO:7) which cause cleavage between 2A glycine and 2B proline. Other linkers will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention.

As used herein, "transmembrane domain" (TMD) refers to a region of a CAR that spans the plasma membrane. The transmembrane domain of the CAR of the invention is a transmembrane region of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof.

As used herein, "B cell-associated disease" includes B cell immunodeficiency, autoimmune diseases, and/or excessive/uncontrolled cell proliferation associated with B cells (including lymphoma and/or leukemia). Examples of such diseases in which the mCAR of the invention may be used in a method of treatment include, but are not limited to, Systemic Lupus Erythematosus (SLE), diabetes, Rheumatoid Arthritis (RA), reactive arthritis, Multiple Sclerosis (MS), pemphigus vulgaris, celiac disease, Crohn's disease, inflammatory bowel disease, ulcerative colitis, autoimmune thyroid disease, X-chromosome-linked agammaglobulinemia, pre-B acute lymphoblastic leukemia, systemic lupus erythematosus, common variant immunodeficiency diseases, chronic lymphocytic leukemia, diseases associated with selective IgA deficiency and/or IgG subclass deficiency, B-lineage lymphoma (Hodgkin's lymphoma) and/or non-Hodgkin's lymphoma), immunodeficiency with thymoma, transient hypogammaglobulinemia and/or hyper IgM syndrome, and B-cell mediated diseases such as EBV-mediated lymphoproliferative tissue (e B v-mediated lymphoproliferative tissue) Ill) and chronic infections in which B cells are involved in pathophysiological processes.

"beneficial results" can include, but are in no way limited to, lessening or lessening the severity of a disease condition, preventing the worsening of a disease condition, curing a disease condition, preventing the development of a disease condition, reducing the chances of a patient developing a disease condition, and extending the life span or life expectancy of a patient.

"cancer" and "cancerous" refer to or describe the physiological condition in mammals that is generally characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, B cell lymphoma (hodgkin's lymphoma and/or non-hodgkin's lymphoma), brain tumors, breast cancer, colon cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, brain cancer, and prostate cancer, including, but not limited to, androgen-dependent prostate cancer and androgen-independent prostate cancer.

As used herein, "condition," "disease," and "disease state" include physiological states in which diseased cells can be targeted with a CAR of the invention, e.g., an antibody expressed against a specific antigen on the diseased cells. Examples of antigens that can be targeted include, but are not limited to, antigens expressed on B cells; antigens expressed on carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas; antigens expressed on various immune cells; and antigens expressed on cells associated with various hematologic, autoimmune and/or inflammatory diseases. In an exemplary embodiment, the antigen targeted is EGFR.

"Effector function" refers to a specialized function of a differentiated cell. The effector function of a T cell may be, for example, cytolytic activity or helper activity, including secretion of cytokines.

As used herein, "genetically modified cell," "redirected cell," "genetically engineered cell," or "modified cell" refers to a cell that expresses a mCAR of the invention. Genetically modified cells include, but are not limited to, genetically modified T cells, NK cells, hematopoietic stem cells, pluripotent embryonic stem cells, or embryonic stem cells. Genetically modified cells express the mCAR of the invention, which is activatable and can target any antigen expressed on the surface of a target cell

As used herein, "immune cell" refers to a cell of the mammalian immune system, including but not limited to antigen presenting cells, B cells, basophils, cytotoxic T cells, dendritic cells, eosinophils, granulocytes, helper T cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells, and T cells.

As used herein, "immune response" refers to immunity including, but not limited to: innate immunity, humoral immunity, cellular immunity, inflammatory response, adaptive immunity, autoimmunity, and/or hyperactive immunity.

As used herein, "mammal" refers to any member of the class mammalia, including, but not limited to, humans and non-human primates, such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or gender. Thus, adult and newborn and fetal subjects, whether male or female, are intended to be included within the scope of this term.

"polynucleotide" as used herein includes, but is not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microrna), genomic DNA, synthetic RNA, and/or tRNA.

"naked DNA" as used herein refers to DNA encoding a CAR cloned in a suitable expression vector in the appropriate orientation for expression. Viral vectors that may be used include, but are not limited to, SIN lentiviral vectors, retroviral vectors, foamy viral vectors, adeno-associated viral (AAV) vectors, hybrid vectors, and/or plasmid transposons (e.g., sleeping beauty transposon systems) or integrase-based vector systems. Other carriers that may be used in conjunction with alternative embodiments of the present invention will be apparent to those skilled in the art.

As used herein, a "single chain variable fragment," "single chain antibody variable fragment," or "scFv" antibody refers to a form of antibody that comprises only the heavy and light chain variable regions connected by a linker peptide.

"target cell" as used herein refers to a cell involved in a disease and that can be targeted by the genetically modified cells of the invention (including but not limited to genetically modified T cells, NK cells, hematopoietic stem cells, pluripotent stem cells, and embryonic stem cells). Other target cells will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention.

The terms "T cell" and "T lymphocyte" are used interchangeably and synonymously herein. Examples include, but are not limited to, natural T cells, central memory T cells, effector memory T cells, or combinations thereof.

As used herein, "therapeutic agent" refers to an agent that is used, for example, to treat, inhibit, prevent, lessen the impact of, reduce the severity of, reduce the likelihood of developing, slow the progression of, and/or cure a disease. Diseases targeted by therapeutic agents include, but are not limited to, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas, antigens expressed on various immune cells, and antigens expressed on cells associated with various hematologic, autoimmune, and/or inflammatory diseases.

"transduction" as used herein refers to the introduction of an exogenous nucleic acid into a cell using a viral vector.

"transfection" as used herein refers to the introduction of exogenous nucleic acid into a cell using recombinant DNA techniques. The term "transformation" means the introduction of an "exogenous" (i.e., external or extracellular) gene, DNA or RNA sequence into a host cell such that the host cell will express the introduced gene or sequence to produce a desired substance, such as a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be referred to as a "cloned" or "foreign" gene or sequence, and may include regulatory or control sequences, such as initiation, termination, promoter, signal, secretion, or other sequences used by the cytogenetic entity. The gene or sequence may include non-functional sequences or sequences with unknown function. Host cells that receive and express the introduced DNA or RNA have become "transformed" and are "transformants" or "clones". The DNA or RNA introduced into the host cell may be from any source, including cells of the same species or species as the host cell or cells of a different species or species.

As used herein, "Treatment" and "treating" refer to both therapeutic Treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a target pathological condition, prevent a pathological condition, seek or obtain a beneficial result, or reduce the chance of an individual developing a condition, even if the Treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those for which the condition is to be prevented.

As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

As used herein, "vector," "cloning vector," and "expression vector" refer to a vehicle whereby a polynucleotide sequence (e.g., a foreign gene) can be introduced into a host cell in order to transform the host and facilitate expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.

T cells expressing chimeric antigen receptors have the potential to produce extremely high levels of anti-tumor activity, but they may also exhibit enhanced off-target cell killing. To minimize this side effect, the inventors describe CARs with N-terminal masking peptides that block the ability of the CAR to bind prematurely to its target.

Described herein are masked/activatable chimeric antigen receptors (mcars) comprising a chimeric antigen receptor specific for one or more antigens and a masking peptide. The masking peptide blocks premature binding of the antigen-specific binding region on the CAR to the antigen on the target cell. In general embodiments, the invention relates to mcars, nucleic acid sequences encoding mcars, vectors comprising nucleic acids encoding mcars, viruses comprising nucleic acid sequences encoding mcars, host cells (e.g., genetically modified cells) that express mcars and use mcars as therapeutic agents. The mcars of the invention are constructed so that they can be expressed in cells that, in turn, proliferate in response to the presence of at least one molecule (e.g., an antigen) that interacts with at least one antigen-specific targeting domain. Specifically, the interaction between the antigen and the antigen binding domain promotes the proliferation of cells expressing mCAR. Other factors (e.g., cytokines in the microenvironment, binding affinity, presence of regulatory cells, etc.) may also promote proliferation of cells expressing mCAR.

The mCAR described herein can be synthesized as a single polypeptide chain and comprises a masking peptide, one or more antigen-specific targeting domains, an extracellular spacer domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. In this embodiment, the masking peptides are N-terminal to the antigen-specific targeting domain and are arranged in tandem and separated by a linker peptide. The antigen-specific targeting domain is linked to an extracellular spacer domain, which is linked to a transmembrane domain. The transmembrane domain is linked to a costimulatory domain. The costimulatory domain is linked to an intracellular signaling domain at the C-terminus. If more than one co-stimulatory domain is used, multiple co-stimulatory domains may be arranged in tandem with the transmembrane domain at its N-terminus and the intracellular signaling domain at its C-terminus. Polynucleotides encoding these polypeptides may further comprise an N-terminal signal sequence that directs mCAR to the cell surface as a type I transmembrane protein. The antigen-specific targeting domain may be oriented extracellularly, while the intracellular signaling domain may be cytoplasmic.

FIG. 1 shows a schematic representation of a masked chimeric antigen receptor of the present invention.

Masking peptides

The masking peptide comprises a mask and a cleavage site. In various embodiments, the masking peptide is specific for each antigen-specific targeting domain of the CAR. For example, each scFv specific for a target antigen will have a unique masking peptide sequence. The mask is attached to the cleavage site by a linker. The mask is specific/unique to the antigen specific targeting domain of the CAR and blocks binding of the CAR to the antigen on the target cell until the mCAR is activated by cleavage of the masking peptide at the cleavage site. An exemplary embodiment of a masked CAR is described in Table 1 and/or SEQ ID NO: 29.

The masking peptide may be 5 to 50 amino acids in length. In various embodiments, the masking peptide (comprising the mask, linker, and cleavage site) is 2-5, 5-10, 5-15, 10-25, 15-20, 15-25, 20-30, 25-35, 30-35, 35-40, 35-45, 40-45, or 45-50 amino acids in length. In some embodiments, the masking peptide is 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In one embodiment, the mask in the masking peptide is specific for each antigen-specific targeting domain of the CAR. In some embodiments, the length of the mask may be 2-5, 5-10, 5-15, 10-25 amino acids.

In an exemplary embodiment, the ASTD in the mCAR is specific for EGFR and the mask comprises or consists of or consists essentially of: sequence CISPRGCPDGPYVMY (SEQ ID NO: 1). In another exemplary embodiment, the ASTD in the mCAR is specific for EGFR and the mask comprises or consists of or consists essentially of: sequence QGQSGQCISPRGCPDGPYVMY (SEQ ID NO: 8). In another exemplary embodiment, the ASTD in the mCAR is specific for Her2 and the mask comprises or consists essentially of: sequence LLGPYELWELSH (SEQ ID NO: 17). In other exemplary embodiments, the ASTD in the CAR is specific for GD2 ganglioside and the mask comprises or consists essentially of: sequence RCNPNMEPPRCWAAEGD (SEQ ID NO:22) or (VCNPLTGALLCSAAEGD) (SEQ ID NO: 23). In other exemplary embodiments, the ASTD in the CAR is specific for carbonic anhydrase 9(CA-IX) and the mask comprises or consists essentially of: sequence LSTAFARV (SEQ ID NO:24) or ALGPGREYRAL (SEQ ID NO: 25).

Methods for screening for masking in a masking peptide will be apparent to those skilled in the art. In some embodiments, a combinatorial approach is used to design masking peptides. The combination method includes combining a known mask (e.g., a peptide that has been shown to bind to an antibody or other antigen binding domain) with a known antigen binding domain (e.g., a single chain antibody or other protein binding domain). In some embodiments, de novo screening methods are used to design masking peptides by screening peptides in, for example, bacterial display peptide libraries for their ability to mask antigen-specific binding domains in existing CARs. Suitable masking moieties are identified using any of a variety of known techniques. For example, the peptide masking moiety is identified using the method described in U.S. patent application publication No. 2009/0062142 to Daugherty et al, the contents of which are hereby incorporated by reference in their entirety. With bacterial display screening, for example, a library of masking peptides is displayed on the cell surface by fusion with a bacterial membrane protein such as OmpX. The transformed cells are incubated with a fluorophore-labeled antibody corresponding to the antigen-binding domain of the CAR, and the bound cells are isolated by fluorescence activated cell sorting. The bound cells were expanded and the selection process was repeated for several rounds. Candidate masking peptide sequences were identified by sequencing of selected clones. Other screening methods include Phage display, such as the Barbas, Carlos F. et al phase display: a laboratory manual. CSHL Press,2004 teaching; mRNA displays, as taught in Wilson, David S., Anthony D.Keefe and Jack W.Szostak, "The use of mRNA display of choice high-affinity protein-binding peptides," Proceedings of The national academy of Sciences 98.7(2001): 3750-3755; and Yeast displays, such as those taught in Boder, Eric T. and K.Dane Wittrup. "Yeast surface display for screening combinatorial genetic polypeptides," Nature biotechnology 15.6(1997): 553-.

In various embodiments, the cleavage site comprises a sequence that includes a substrate for a protease, e.g., a protease that is co-localized with the target antigen at the treatment site of the subject. In some embodiments, the cleavage site in the masking peptide is 2-5, 5-10, 5-15, 10-15, 5,8, 10, 12, or 15 amino acids in length. In an exemplary embodiment, the cleavage site comprises or consists essentially of: sequence LSGRSDNH (SEQ ID NO: 2); and is specific for uPA protease. In another exemplary embodiment, the cleavage site comprises or consists of or consists essentially of: sequence LSGRSDNHGSSGT (SEQ ID NO: 9); and is specific for uPA protease. Methods for selecting suitable cleavage sites for use with mCAR described herein will be apparent to those skilled in the art. In some embodiments, the cleavage site is a substrate for a protease such as uPA, MT-SPl, legumain. In some embodiments, the cleavage site is a substrate for a protease, such as a Matrix Metalloproteinase (MMP). Examples of Matrix Metalloprotease (MMP) cleavable linker sequences include, but are not limited to, protease MMP-1 substrate VLVPMAMMAS (SEQ ID NO:26), MMP-2 and/or MMP-9 substrates GPLGIAGQ (SEQ ID NO:27), or PVGLIG (SEQ ID NO: 28).

Desirable characteristics of the cleavage site include, but are not limited to, non-toxicity to the subject, stability during systemic circulation in the subject, insensitivity to circulating proteases (e.g., thrombin, plasmin, etc.), and activity at the intended treatment site of the subject. Suitable substrates are identified using any of a variety of known techniques. For example, peptide substrates are identified using the method described in U.S. patent No. 7,666,817 to Daugherty et al, the contents of which are hereby incorporated by reference in their entirety. (see also Boulware et al, "evolution optimization for peptide substrates for protease infected amplified hydrolysis kinetics," Biotechnol Bioeng.106.3(2010): 339-46).

In an exemplary embodiment, the linker between the mask and the cleavage site comprises or consists essentially of: sequence GSSGGSGGSGGSG (SEQ ID NO:10), and the linker that connects the masking peptide to the CAR comprises or consists essentially of: the sequence GSSGT (SEQ ID NO: 11).

In some embodiments, the masked CAR (mcar) reduces the ability of the CAR to bind to the target antigen at the treatment site of the subject such that the dissociation constant (Kd) of the CAR when attached to a masking peptide against the target antigen is at least 20-fold greater than the Kd of the CAR when not attached to a masking peptide against the antigen. In some embodiments, the masked CAR (mcar) reduces the ability of the CAR to bind to the target antigen at the treatment site of the subject such that the dissociation constant (Kd) of the CAR when attached to a masking peptide against the target antigen is at least 100-fold greater than the Kd of the CAR when not attached to a masking peptide against the antigen. In some embodiments, the masked CAR (mcar) reduces the ability of the CAR to bind to the target antigen at the treatment site of the subject such that the dissociation constant (Kd) of the CAR when attached to a masking peptide against the target antigen is at least 1000-fold greater than the Kd of the CAR when not attached to a masking peptide against the antigen. In some embodiments, the masked CAR (mcar) reduces the ability of the CAR to bind to the target antigen at the treatment site of the subject such that the dissociation constant (Kd) of the CAR when attached to a masking peptide against the target antigen is at least 1000-fold greater than the Kd of the CAR when not attached to a masking peptide against the antigen. In an exemplary embodiment, the masked CAR (mcar) reduces the ability of the EGFR specific CAR to bind to EGFR at the treatment site of the subject such that the dissociation constant (Kd) of the EGFR specific CAR, when attached to a masking peptide against EGFR, is any of at least 20, 50, 100, or 10,000 times the Kd of the EGFR specific CAR, when not attached to a masking peptide against the antigen.

In some embodiments, the masking peptide reduces the ability of the CAR to bind the target antigen in the presence of the target antigen by at least 95%, 90%, 85%, 80%, 75%, 70%, or 65% compared to when the cleavage site is not cleaved when the cleavage site is cleaved, as determined in vitro using a target displacement assay, as described, for example, in PCT publication nos. WO 2009/025846 and WO 2010/081173. In an exemplary embodiment, in the presence of EGFR, the masking peptide described in table 1 and/or seq id NO:29 reduces the ability of an EGFT-specific CAR to bind EGFR by at least 95%, 90%, 85%, 80%, 75%, 70% or 65% when the cleavage site (LSGRSDNH) as shown in table 1is not cleaved compared to when the cleavage site is cleaved.

Antigen-specific targeting domains of chimeric antigen receptors

The mCAR of the invention may target one or more antigens. The antigen targeted by the ASTD of mCAR may be an antigen on a single diseased cell (e.g., a cancerous B cell) or an antigen expressed on separate cells each contributing to the disease. The antigen targeted by mCAR is an antigen that is directly or indirectly involved in the disease. Antibodies to ASTD comprising mCAR may be specific for any antigen chosen. The antibody specific for the antigen may be a Fab fragment of the antibody or a single chain variable fragment (scFv) of the antibody.

For example, FIG. 1 shows an embodiment of the present invention depicting a masking peptide upstream (i.e., N-terminal) of an ASTD (e.g., scFv) using methods well known to those skilled in the art, as long as the target antigen is expressed on Cells that can be targeted by genetically modified Cells as described below, And further, an scFv specific to an antigen can be cloned upstream (i.e., N-terminal) of a CD28TM-41 CSD-CD3 ISD domain, in another embodiment, an scFv specific to an antigen can be cloned upstream (i.e., N-terminal) of a CD8 α hinge-CD 8TM-CD28CSD-41BBCSD-CD3 ζ ISD (FIG. 1) domain, as long as the target antigen is expressed on Cells that can be targeted by genetically modified Cells as described below (see, protein, Culture et al, "Cell Culture J. (198J), see: Culture J.1994, Molecular J. (see: Culture J., science, plant et al., Culture J. (see, plant J.) (see, plant et al., Culture J. (see, publication No. 5. 1985, publication No. 5. mineral J. (see: Culture, publication No. 5, publication No. Cell J.) (see, publication No. 5, publication No. Cell J. (see (publication No. 5, publication (publication No. 5, No. publication No. 5, publication No. 5, No.

In one embodiment, the antigen specific targeting domain comprises a full length IgG heavy chain (specific for the target antigen) with V_HCH1, hinge, and CH2 and CH3(Fc) Ig domains, if V_HA domain alone is sufficient to confer antigen specificity ("single domain antibody"). Full-length IgG heavy chains can be linked via appropriate transmembrane domainsTo the costimulatory domain and the intracellular signaling domain. In one embodiment, the extracellular spacer domain may be linked between the antigen-specific binding domain and the transmembrane domain.

In another embodiment, each antigen-specific targeting domain of mCAR comprises one or more single chain antibody variable fragments (scFv). If more than one scFV is present in the mCAR, then each scFV is specific for a different target antigen. One of the variable domains (V) has been developed_HOr V_L) Is linked to another via a polypeptide linker (respectively V)_LOr V_H) Without significantly disrupting antigen binding or binding specificity. (Chaudhary et al, aromatic single-chain immunotoxin compounded of anti-Tac variable regionsand a truncated diphythia toxin.1990Proc. Natl. Acad. Sci.,87: 9491; Bedzyk et al, Immunological and structural characterization of a high affinity anti-fluoro single-chain antibody.1990 J.biol.Chem.m., 265: 18615). The joint is V_HN-terminal of (5) and V_LIs connected to the C-terminus of or connects V_HC-terminal of (A) and V_LAre linked at the N-terminus. These scfvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody. In some embodiments, the scFv is specific for at least two different antigens and is arranged in series and linked to the co-stimulatory domain and the intracellular signaling domain via a transmembrane domain. In an optional embodiment, an extracellular spacer domain may be linked between the antigen-specific binding region and the transmembrane domain.

In another aspect, each scFv fragment can be fused to all or a portion of the constant domain of the heavy chain. The resulting antigen-specific targeting domain is linked to the co-stimulatory domain and the intracellular signaling domain via the transmembrane domain. In an optional embodiment, the extracellular spacer domain may be linked between the antigen-specific binding domain and the transmembrane domain.

In yet another embodiment, one or more antigen-specific targeting domains of mCARComprising a bivalent (or bivalent) single-chain variable fragment (di-scFv ). In mCAR containing a di-scFV, two scFv specific for the antigen are generated by using two V_HAnd two V_LThe regions are linked together to produce a single peptide chain, thereby producing tandem scfvs. (Xiong, Cheng-Yi; Natarajan, A; Shi, XB; Denadro, GL; Denadro, SJ (2006). "Development of structural targeting anti-MUC-1 multimers: effects of di-scFv unpaired localization on PEGylation and machinery binding". Protein Engineering Design and Selection 19(8):359 367)). mCAR comprising one or more antigen-specific targeting domains will express two scfvs specific for each of the two antigens. The resulting antigen-specific targeting domain is linked to the co-stimulatory domain and the intracellular signaling domain via the transmembrane domain. In an optional embodiment, the extracellular spacer domain may be linked between the antigen-specific binding domain and the transmembrane domain.

In another embodiment, each antigen-specific targeting domain of mCAR comprises a diabody. In diabodies, a scFv is generated with a linker peptide that is too short for the two variable regions to fold together, thereby promoting scFv dimerization. Shorter linkers (one or two amino acids) also lead to trimer formation, so-called triabodies or trisomies. Tetraantibodies may also be used.

To generate the mCAR of the invention, antigen-specific targeting domains are linked to each other covalently or non-covalently on a single protein molecule. Oligopeptide or polypeptide linkers, Fc hinges or membrane hinge regions may be used to link these domains to each other.

Co-stimulatory domains of masked chimeric antigen receptors

The mCAR of the invention may also comprise a costimulatory domain. This domain can enhance cell proliferation, cell survival and development of memory cells. The mCAR of the invention may also comprise one or more costimulatory domains. Each co-stimulatory domain comprises a co-stimulatory domain of any one or more of: for example, a member of the TNFR superfamily, CD28, CD137(4-1BB), CD134(OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-1, TNFR-II, Fas, CD30, CD40, or a combination thereof. Co-stimulatory domains from other proteins may also be used with the CARs of the invention. Additional co-stimulatory domains will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention. If the mCAR comprises more than one co-stimulatory domain, these domains may be arranged in tandem, optionally separated by a linker.

Masked extracellular spacer domain of chimeric antigen receptor

The extracellular spacer domain may include, but is not limited to, an Fc fragment of an antibody or fragment or derivative thereof, a hinge region of an antibody or fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof examples of extracellular spacer domains include, but are not limited to, a CD8 α hinge, an artificial spacer made from a polypeptide such as Gly3, or a CH1, CH3 domain of an IgG (such as human IgG 4). specifically, the extracellular spacer domain may be (i) a hinge region of IgG 6338, a CH2 region, and a CH3 region, (ii) a hinge region of IgG4, (iii) a hinge and CH2 region of IgG4, (iv) a hinge region of CD8 α, (v) a hinge region of IgG1, a CH 6866 region, and a CH 35 3 region, (ii) a hinge region of IgG 5966 region of IgG4, and/or alternative to the hinge region of IgG1 and/or alternative IgG spacer regions of the present invention may be used in combination with the present invention.

Masked transmembrane domains of chimeric antigen receptors

The mCAR of the present invention may also comprise a transmembrane domain. The transmembrane domain may comprise transmembrane sequences from any protein having a transmembrane domain, including any of the type I, type II or type III transmembrane proteins. The transmembrane domain of the mCAR of the present invention may also comprise an artificial hydrophobic sequence. The transmembrane domain of the mCAR of the present invention may be selected so as not to dimerize. Additional transmembrane domains will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention.

Masked intracellular signaling domains of chimeric antigen receptors

Examples of intracellular signaling domains include, but are not limited to, the zeta chain of the T cell receptor or any homolog thereof (e.g., η chain, fcepsilonr 1 gamma and β chain, MB1(Ig α) chain, B29(Ig β) chain, etc.), CD3 polypeptides (Δ, δ 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. specifically, the intracellular signaling domain may be a human CD3 ξ chain, fcyri, the cytoplasmic tail of the Fc receptor, an immunotyrosine receptor-based activation motif (ITAM) carrying cytoplasmic receptors, or a combination thereof.

Joint in mCAR

In some embodiments, two or more components of the mCAR of the invention are separated by one or more linkers. In some embodiments, the components of the masking peptide (mask and cleavage site) are separated by a linker sequence. In some embodiments, the masking peptide is linked to the CAR via a linker sequence. A linker is an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any domains/regions of the CAR of the invention. In some embodiments, the linker can be, for example, 5 to 12 amino acids long, 5 to 15 amino acids long, or 5 to 20 amino acids long. The linker may be composed of flexible residues like glycine and serine so that adjacent protein domains move freely with respect to each other. Longer linkers (e.g., linkers longer than 100 amino acids) may be used in conjunction with alternative embodiments of the invention, and may be selected, for example, to ensure that two adjacent domains do not sterically interfere with each other. Examples of linkers that can be used in the present invention include, but are not limited to, 2A linkers (e.g., T2A), 2A-like linkers, or functional equivalents thereof.

As described above, the mCAR of the present invention can be synthesized as a single polypeptide chain. In one embodiment, the single polypeptide chain encoding the uncleaved mCAR has the following structural arrangement from N-terminus to C-terminus: mask-linker-cleavage site-linker-antigen specific targeting domain-transmembrane domain-costimulatory domain-intracellular signaling domain. Additional sequences may be present between each domain, for example to provide further flexibility and stability to the mCAR.

In one embodiment, the single polypeptide chain encoding the uncleaved mCAR has the following structural arrangement from N-terminus to C-terminus: mask-linker-cleavage site-linker-antigen specific targeting domain-extracellular spacer domain-transmembrane domain-costimulatory domain-intracellular signaling domain. Additional sequences may be present between each domain, for example to provide further flexibility and stability to the mCAR.

Targets for antigen-specific targeting domains of chimeric antigen receptors

In some embodiments, the antigen-specific targeting domain of mCAR targets antigens specific for: cancer, inflammatory disease, neuronal disorder, diabetes, cardiovascular disease, infectious disease, or a combination thereof. Examples of antigens that may be targeted by the mCAR of the invention include, but are not limited to, antigens expressed on B cells; antigens expressed on carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, blastomas; antigens expressed on various immune cells; and antigens expressed on cells associated with various hematologic, autoimmune and/or inflammatory diseases. The mCAR of the invention is capable of redirecting effector functions of an expressing cell to either of two target antigens. This feature of the construct may overcome the problem of escape variants of antigen loss when using a single antigen to specifically target, for example, genetically unstable B cell lineage malignancies.

Antigens specific for cancer that may be targeted by the mCARs described herein include, but are not limited to, any one or more of 4-1BB, 5T4, adenocarcinoma antigens, α -alpha-fetoprotein, BAFF, B-lymphoma cells, C242 antigens, CA-125, carbonic anhydrase 9(CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23(IgE receptor), CD28, CD30(TNFRSF8), CD33, CD4, CD44v 4, CD4, CEA, CETO, CTLA-4, DR 4, EGFR, EpCAM, CD4, FAP, fibronectin extra domain-B, GD 4, glycoprotein 4, ganglioside, glycoprotein 75, TRAIL B, HER 72, HER-VEGFR-4, VEGF-13, VEGF-binding to human VEGF receptor specific for human insulin receptor, VEGF-receptor, VEGF-4, VEGF-receptor-PGL, VEGF-4, VEGF-receptor-3, VEGF-receptor-2, VEGF-receptor-IgG-3, and other receptor specific for human insulin receptor-mouse, VEGF-mouse, mouse receptor-mouse, mouse receptor, mouse.

Antigens specific for inflammatory diseases that may be targeted by the mCARs described herein include, but are not limited to, any one or more of AOC3(VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basic immunoglobulin), CD154(CD40L), CD2, CD20, CD23(IgE receptor), CD25(α chain of IL-2 receptor), CD3, CD4, CD5, IFN- α, IFN- γ, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17A, IL-22, IL-4, IL-5, IL-6 receptor, integrin α, IL α β, llama (Lamamglasca), LFA-1(CD11, MEDI-528, MUDI-3540, TGF- β, TGF-7, TGF-11, TGF-7, VEGF-3, and alternative to other inflammatory diseases as will be apparent to those of the present invention.

Antigens specific for neuronal disorders that may be targeted by the mCAR described herein include, but are not limited to, any one or more of β amyloid or MABT5102A other antigens specific for neuronal disorders will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the invention.

Antigens specific to diabetes that may be targeted by the mCAR described herein include, but are not limited to, any one or more of L-1 β or CD3 other antigens specific to diabetes or other metabolic disorders will be apparent to those of skill in the art and may be used in conjunction with alternative embodiments of the present invention.

Antigens specific for cardiovascular disease that may be targeted by the mCER described herein include, but are not limited to, any one or more of C5, cardiac myosin, CD41 (integrin α -IIb), fibrin II, β chain, ITGB2(CD18), and sphingosine-1-phosphate other antigens specific for cardiovascular disease will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention.

Antigens specific for infectious diseases that may be targeted by the mCER described herein include, but are not limited to, any one or more of anthrax toxin, CCR5, CD4, aggregation factor A, cytomegalovirus glycoprotein B, endotoxins, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, and TNF- α other antigens specific for infectious diseases will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the present invention.

Other examples of target antigens that can be targeted by the mCAR include, but are not limited to, surface proteins found on cancer cells in a specific or amplified manner (e.g., IL-14 receptor, CD19, CD20, and CD40 for B-cell lymphoma, Lewis Y and CEA antigens for a variety of carcinomas, Tag72 antigen for breast and colorectal cancers, EGF-R for lung cancer, folate binding protein and HER-2 protein that are often amplified in human breast and ovarian cancers), or viral proteins (e.g., gp120 and gp41 envelope proteins of HIV, envelope proteins of hepatitis B and C viruses, glycoprotein B of human cytomegalovirus and other envelope glycoproteins, envelope proteins from oncogenic viruses such as Kaposi's sarcoma associated herpes virus). Other potential targets of the CARs of the invention include CD4, wherein the ligand is HIV gp120 envelope glycoprotein; and other viral receptors, such as ICAM, which is a receptor for human rhinovirus, and related receptor molecules for poliovirus.

Additional targets for mcars of the invention include antigens involved in B cell-related diseases. Still further targets for mcars of the invention will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the invention.

Genetically engineered cells

The invention also provides genetically engineered cells comprising and stably expressing mCAR as described herein. The mCAR expressed by the genetically engineered cell comprises a masking peptide, at least one antigen-specific targeting domain, optionally an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. The polynucleotide sequence encoding the mCAR may also comprise an N-terminal signal sequence.

The genetically engineered cells express mCAR as described herein. In one embodiment, the antigen-specific targeting domain comprises a target-specific antibody or a functional equivalent or fragment or derivative thereof. The antigen-specific antibody may be a Fab fragment of an antibody or a single chain variable fragment (scFv) of an antibody.

Genetically engineered cells that may comprise and express the mCAR of the invention include, but are not limited to, T-lymphocytes (T cells), natural T cells (T cells)_N) Memory T cells (e.g., central memory T cells (T)_CM) Effector memory cells (T)_EM) For example, individual T cells of the invention can be CD4+/CD8-, CD4-/CD8+, CD4-/CD8-, or CD4+/CD8 +. T cells can be a mixed or monoclonal population of CD4+/CD 8-and CD4-/CD8+ cells⁺T cells, when co-cultured with target cells in vitro, can lyse antigen-specific target cells. In some embodiments, the T cell can be any one or more of: CD45RA⁺CD62L⁺Native cell, CD45RO⁺CD62L⁺Central memory cell, CD62L^-Effector memory cells or combinations thereof (Berger et al, adjuvant transfer of video-specific and tumor-specific T cell immunity. curr Opin immunity 200921 (2) 224-.

Genetically modified cells can be produced by stably transfecting cells with DNA encoding mCAR as described herein. Viral vectors are commonly used to carry heterologous genes into cells (e.g., T cells). Examples of viral vectors that can be used to generate the genetically modified cells include, but are not limited to, a SIN lentiviral vector, a retroviral vector, a foamy viral vector, an adeno-associated viral (AAV) vector, and/or a plasmid transposon (e.g., the sleeping beauty transposon system).

Various methods may produce stable transfectants that express mCAR of the invention. In one embodiment, the method of stably transfecting and redirecting cells is achieved by electroporation using naked DNA. By using naked DNA, the time required to produce redirected cells can be significantly reduced. Additional methods for genetically engineering cells using naked DNA encoding a mCAR of the invention include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes, and/or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer, and/or hydrodynamic delivery), and/or gene deliveryMethods for particles (e.g., puncture transfection (immunoperfection), use of a gene gun, and/or magnetic transfection). Transfected cells showing the presence of a single integrated non-rearranged vector and expression of mCAR may be expanded ex vivo. In one embodiment, the cell selected for ex vivo expansion is CD8⁺And exhibits the ability to specifically recognize and lyse antigen-specific target cells.

Viral transduction methods may also be used to generate redirected cells that express mCAR of the invention. Cell types that may be used to generate genetically modified cells expressing mCAR of the present invention include, but are not limited to, T-lymphocytes (T cells), natural killer cells, hematopoietic stem cells, and/or pluripotent embryos/induced stem cells capable of producing therapeutically relevant progeny.

Stimulation of T cells by antigens under appropriate conditions results in proliferation (expansion) of the cells and/or production of IL-2. Cells comprising mCAR of the invention will be quantitatively amplified in response to the binding of one or more antigens to the antigen-specific targeting domain of mCAR. The invention also provides a method for preparing and amplifying mCAR expressing cells. The method includes transfecting or transducing a cell with a vector expressing mCAR and stimulating the cell with a cell expressing a target antigen, a recombinant target antigen, or an antibody directed against a receptor to cause cell proliferation, thereby preparing and expanding T cells. In one embodiment, the cell may be any one or more of: t-lymphocytes (T cells), Natural Killer (NK) cells, Hematopoietic Stem Cells (HSCs), or pluripotent embryos/induced stem cells capable of producing a treatment-related progeny.

Method of treatment

Provided herein are methods for treating a disease associated with an antigen targeted by an mCAR described herein in a subject in need thereof. The method comprises providing a composition comprising mCAR as described herein and administering an effective amount of the composition so as to treat an antigen-associated disease in a subject.

Provided herein are methods for treating, inhibiting, slowing progression of, and/or preventing cancer spread in a subject in need thereof. The methods comprise providing a composition comprising mCAR described herein and administering an effective amount of the composition so as to treat, inhibit, slow progression of, and/or prevent cancer spread in a subject.

In some embodiments, the composition comprises a polynucleotide encoding mCAR, a protein comprising mCAR, or a genetically modified cell comprising mCAR. In another embodiment, the genetically modified cells of the composition are T-lymphocytes (T cells), natural T cells (T cells)_N) Memory T cells (e.g., central memory T cells (T)_CM) Effector memory cells (T)_EM) Natural Killer (NK) cells, Hematopoietic Stem Cells (HSCs), or pluripotent embryos/induced stem cells capable of producing treatment-related progeny, which express the mCAR of the invention. The compositions of the present invention may be administered alone or in combination with existing therapies. If used in conjunction with other therapies, the compositions of the present invention may be administered simultaneously or sequentially with other existing therapies.

In some embodiments, treating, inhibiting, slowing the progression of, and/or preventing cancer spread in a subject in need thereof comprises administering an effective amount of a composition comprising mCAR described herein in combination with an existing therapy. In various embodiments, a composition comprising mCAR may be administered sequentially or concurrently with an existing therapy. Examples of existing cancer treatments include, but are not limited to, active monitoring, observation, surgical intervention, chemotherapy, immunotherapy, radiotherapy (such as external beam radiation, stereotactic radiosurgery (gamma knife), and Fractionated Stereotactic Radiotherapy (FSR)), focal therapy, systemic therapy, vaccine therapy, viral therapy, molecular targeted therapy, or combinations thereof.

Examples of chemotherapeutic agents include, but are not limited to, albumin-bound paclitaxel (nab-paclitaxel), actinomycin, adrertino (alitretinin), all-trans retinoic acid, azacitidine, Azathioprine (Azathioprine), bevacizumab, bexarotene (bexatene), bleomycin, bortezomib, carboplatin, capecitabine, cetuximab, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, erlotinib, etoposide, fluorouracil, gefitinib, gemcitabine, hydroxyurea, idarubicin, imatinib, YIPLICAZIMAIMIUM, irinotecan, mechlorethamine, tramazuren, mercaptopurine, methotrexate, mitoxantrone, Ocrelizumab (Ocrilizumab), ofatumumab, oxaliplatin, paclitaxel, panitumumab, rituximab, pemetrexendin, rituximab, and the like, Tafluposide (Tafluposide), teniposide, thioguanine, topotecan, tretinoin, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, vorinostat, Romidepsin (Romidepsin), 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), cladribine, clofarabine, floxuridine, fludarabine, pentostatin, mitomycin, ixabepilone, estramustine or combinations thereof

In various embodiments, an effective amount of a composition comprising mCAR described herein is any one or more of: about 0.01 to 0.05. mu.g/kg/day, 0.05 to 0.1. mu.g/kg/day, 0.1 to 0.5. mu.g/kg/day, 0.5 to 5. mu.g/kg/day, 5 to 10. mu.g/kg/day, 10 to 20. mu.g/kg/day, 20 to 50. mu.g/kg/day, 50 to 100. mu.g/kg/day, 100 to 150. mu.g/kg/day, 150 to 200. mu.g/kg/day, 200 to 250. mu.g/kg/day, 250 to 300. mu.g/kg/day, 300 to 350. mu.g/kg/day, 350 to 400. mu.g/kg/day, 400 to 500. mu.g/kg/day, 500 to 600. mu.g/kg/day, 600 to 700. mu.g/kg/day, 700 to 800. mu.g/kg/day, 800 to 900. mu.g/kg/day, 900 to 1000. mu.g/kg/day, 0.01 to 0.05 mg/kg/day, 0.05-0.1 mg/kg/day, 0.1 to 0.5 mg/kg/day, 0.5 to 1 mg/kg/day, 1 to 5 mg/kg/day, 5 to 10 mg/kg/day, 10 to 15 mg/kg/day, 15 to 20 mg/kg/day, 20 to 50 mg/kg/day, 50 to 100 mg/kg/day, 100 to 200 mg/kg/day, 200 to 300 mg/kg/day, 300 to 400 mg/kg/day, 400 to 500 mg/kg/day, 500 to 600 mg/kg/day, 600 to 700 mg/kg/day, 700 to 800 mg/kg/day, 800 to 900 mg/kg/day, 900 to 1000 mg/kg/day, or combinations thereof. Typical dosages of effective amounts of mCAR described herein may be within the manufacturer's recommended ranges where known therapeutic compounds are used and are also indicated by the skilled artisan by in vitro responses or responses in animal models. Such dosages can generally be reduced by up to about an order of magnitude in concentration or amount without loss of the relevant biological activity. The actual dosage may depend on the judgment of the physician, the condition of the patient, and the effectiveness of the treatment based on, for example, the in vitro response of the relevant cultured cells or tissue cultured tissue samples (e.g., biopsy malignancies), or the response observed in an appropriate animal model. In various embodiments, a composition of the invention comprising a mCAR described herein may be administered once daily (SID/QD), twice daily (BID), three times daily (TID), four times daily (QID), or more, such that an effective amount of mCAR is administered to a subject, wherein an effective amount is any one or more of the doses described herein.

Pharmaceutical composition

In various embodiments, the present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of mCAR described herein. The mCAR in the composition may be any one or more of: a polynucleotide encoding mCAR, a protein comprising mCAR, or a genetically modified cell comprising mCAR. By "pharmaceutically acceptable excipient" is meant an excipient suitable for use in preparing pharmaceutical compositions which is generally safe, non-toxic and desirable, and includes excipients acceptable for veterinary use and for human pharmaceutical use. Such excipients may be solid, liquid, semi-solid, or in the case of aerosol compositions, gaseous.

In various embodiments, the pharmaceutical compositions according to the present invention may be formulated for delivery via any route of administration. The "route of administration" may refer to any route of administration known in the art, including, but not limited to, aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical administration, capsule, and/or injection.

The pharmaceutical composition according to the invention may also contain any pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle involved in carrying or transporting a compound of interest from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissue or organ with which it may come into contact, which means that it must not be at risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that greatly outweighs its therapeutic benefit.

The pharmaceutical compositions according to the invention may also be encapsulated, tableted or prepared in emulsions or syrups for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, ethanol and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

Pharmaceutical formulations are prepared according to conventional techniques of pharmacy, including grinding, mixing, granulating, tableting as required for tablets, or grinding, mixing and filling as well for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or aqueous or non-aqueous suspension. Such liquid preparations may be administered directly orally or filled into soft gelatin capsules.

The pharmaceutical composition according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition which will produce the most effective result in terms of efficacy of the treatment in a given subject. This amount will vary depending upon a variety of factors including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, type and stage of disease, general physical condition, response to a given dose, and type of drug), the nature of the pharmaceutically acceptable carrier(s) in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount according to routine experimentation, for example, by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly. For further guidance, see Remington, The Science and Practice of pharmacy (Gennaro, 20 th edition, Williams & Wilkins PA, USA) (2000).

Examples

The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. The particular materials mentioned are for illustration only and are not intended to limit the invention. Those skilled in the art can develop equivalent methods or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1

Experimental methods

Cell line construction: the K562-EGFR cell line was generated by stable transduction of K562 cells with VSVg pseudotyped lentiviral vectors. Cdna of human EGFR (ge healthcare) was amplified and cloned into lentiviral plasmid FUW to yield FUW-EGFR. Lentiviral vectors were then generated and used to transduce K562 cells. Transduced cells were stained with PE/Cy7 anti-human EGFR antibody (Biolegend) and sorted to give a population of K562 cells expressing EGFR. A similar approach was used to generate the K562-CD19 cell line.

Plasmid construction: the lentiviral vector pCCW-EGFR CAR was constructed by Gibson assembly of the EGFR scFv sequence (Gibson assembling EGFR scFvsequence), the CD28 transmembrane domain-41 BB-CD3z costimulatory domain, and the backbone pCCW vector. Construction of plasmid pCCW-masked EGFR CAR based on pCC W-EGFR CAR, in which a DNA sequence optimized to express masking peptides and protease substrate sequences flanked by GS linkers is added in the N-terminus of the EGFR sc Fv region (QGQSGQ-CISPRGCPDGPYVMY-GSSGGS GGSGGSG-GSSGT (SEQ ID NO: 12)). The masking peptide is underlined; the bottom region is dashed down.

Lentiviral vector production: lentiviral vectors were prepared by transient transfection of 293T cells using standard calcium phosphate precipitation protocols. 293T cells cultured in 6-cm tissue culture dishes were transfected with 5. mu.g of the lentiviral backbone plasmid pCCW-EGFR CAR or pCCW-masked EGFR CAR along with 2.5. mu.g of the envelope plasmid VSVG and the packaging plasmids pMDLg/pRRE and pRSV-Rev. Viral supernatants were harvested 48 hours post transfection and filtered through 0.45- μm filters (Corning).

Lentiviral vector transduction of cell lines: jurkat or Jurkat-NFAT-GFP reporter cells (1E 5 per well) were seeded in 24-well dishes and spun-infected with freshly harvested virus supernatant (2 ml per well) at 2,500rpm and 25 ℃ for 90 min. The supernatant was then replaced with fresh medium and incubated at 37 ℃ and 5% CO2 for 3 days. Expression of CAR was measured by flow cytometry.

Flow cytometry: to detect CAR expression, CAR-Jurkat cells were washed twice in PBS (wash buffer) containing 4% bovine serum albumin, with 2. mu.g/ml recombinant human EGFR-Fc (R) at 4 ℃&D Systems) for 30min, washed twice, and then stained with R-phycoerythrin Affinipure F (ab')₂Fragment goat anti-human IgG, Fc gamma fragment specificity (Jackson ImmunoResearch) at 4 ℃ staining 30 min. Cells were washed twice and resuspended in PBS. Fluorescence was assessed using a Miltenyi Biotec flow cytometer and data was analyzed with FlowJo software.

Masking proteolytic activation of EGFR CARs: protease-mediated activation of masked EGFR CARs was achieved by incubating 1e5 masked EGFR CAR-Jurkat reporter cells with different concentrations of protease uPA (urokinase-type plasminogen activator, R & D Systems) in PBS for 1 hour at room temperature. Cells were washed twice with PBS and used in other experiments.

CAR-Jurkat reporter cell co-culture with target cells: 1e5 EGFR CAR/masked EGFR CAR-Jurkat reporter cells were seeded with 2e5 target cells K562-EGFR or MDA-MB-231 in 200. mu.l C10 medium in round bottom 96-well plates. CD19CAR-Jurkat reporter cells were co-cultured with K562-CD19 cells. The cell mixture was incubated at 37 ℃ for 5 hours. Cells were washed and resuspended in PBS. GFP fluorescence was analyzed by flow cytometry.

The inventors prepared anti-EGFR mCAR having the sequence shown in table 1 below. Table 1: the mask is bold and the cleavage site is underlined ("Desnoyers, L.R. et al Tumor-specific activation of an EGFR-targeting pro-body enhances thermal index. Sci Transl Med5,207 ra144 (2013)).

Jurkat cells were transduced with anti-EGFR mCAR and analyzed for the ability of anti-EGFR mCAR to bind to EGFR protein. As shown in figure 2, the binding capacity to rhEGFR was greatly reduced in masked EGFR CARs compared to the parental anti-EGFR CARs, but could be restored after cleavage of the protease uPA.

Jurkat-NFAT-GFP reporter cells were transduced with anti-EGFR mCAR and analyzed for activation of anti-EGFR mCAR (and thus GFP expression in reporter cells). As shown in figure 3, the activation of the masked EGFR CAR was attenuated compared to the parental anti-EGFR CAR and partially restored after cleavage of the protease uPA.

As shown in figure 4, partial activation of EGFR CAR was masked in the presence of endogenous protease secreting breast cancer cells MDA-MB-231, and treatment with uPA further enhanced activation.

As shown in figure 5, treatment with higher concentrations of uPA resulted in masking more activation of the EGFR CAR.

Example 2

Masked HER-specific CARs using combinatorial design and de novo screening

And (3) combined design: masked Her 2-targeted CAR was generated by coupling trastuzumab-binding peptide LLGPYELWELSH (SEQ ID NO:17) (Jiang, B. et al A novel peptide isolated from a phase displayability plasmid with a trastuzumab peptide antisense epitope of HER-2.J biol chem 280,4656-4662(2005)) was ligated to the Tumor-specific cleavage sequence LSGRSDNH (Desnoyers, L.R. et al Tumor-specific activation of an EGFR-targeting promoter engineering thermal expression Sci Trans. Med5,207 ra144 (2013.) this cleavable mask was fused to the N-terminus of a single-chain antibody derived from a trastuzumab antibody (Zhao, Y. et al A Receptor-Based modifier with signal promoter with the activity of these T Receptor cells cultured in culture medium of the T Receptor trap 3. T trap subunit of the target of this activity assay and T Receptor trap 3. T trap 3. co-cultured cells were tested with the target of the activity of the T trap molecule trap motif of T3. 9. T3. et al.

De novo screening: masking peptides are selected from the bacterial display peptide library _ ENREF _4 (Rice, J.J., Schohn, A. Bessette, P.H., Boulware, K.T. Daugherty, P.S. Bacterial display peptide protein kinase, Science 2006, fifth ray, etc.) Based on their ability to bind to trastuzumab-Based Her 2-specific CAR (Zhao, Y. et al A Receptor-Based nucleic acid Receptor with modified signalling Domains to Enhanced overview peptide, Activity, the Journal of Immunology183,5563-5574 (2009)). Positive selection for Jurkat T cell lines stably expressing Her 2-specific CARs was followed by negative selection for the basal Jurkat cell line. Isolated clones were sequenced and unique peptides were cloned into the N-terminus of Her2CAR through a cleavable linker. Masking CARs were selected by activity against Her2+ target cells in the presence of tumor specific proteases and lack of activity in the absence of proteases.

Example 3

Masked chimeric antigen receptor (mCARR) for tumor specific activation

Epidermal Growth Factor Receptor (EGFR) is an attractive target for cancer therapy because of its widespread overexpression in many epithelial tumors and the inverse correlation between EGFR expression and clinical outcome. Considerable success has been achieved through the development of small molecule inhibitors and monoclonal antibodies targeting EGFR despite the observed handling toxicity in the skin, kidney and gastrointestinal system due to EGFR expression in these healthy tissues. For example, cetuximab, a human mouse chimeric monoclonal antibody against human EGFR, has been approved for use in colon and head and neck cancers, but rash and diarrhea are the most common side effects caused by expression of endogenous EGFR in epithelial tissues. One approach to increasing the therapeutic index of cetuximab is to develop a precursor, an antibody-based prodrug that remains unresponsive in a healthy environment but is activated in tumors by tumor-associated proteases.

Herein, EGFR-specific CARs were constructed using sequences from precursors derived from cetuximab. This masked car (mcar) contains an N-terminal masking peptide capable of blocking the antibody binding site to EGFR and a linker sensitive to a tumor-associated protease. This design enables CAR-T cells to remain inert after encountering antigen in healthy tissue, but become activated in the tumor microenvironment by exposing the antigen binding site through proteolytic cleavage, thereby allowing recognition and killing of tumor cells.

Experimental methods

Construction of plasmid based on MP71 retroviral vector (Engels, B. et al (2003) retroviruses for high-level expression in T lymphocytes. hum Gene Ther14:1155-1168) construction of a retroviral vector encoding an unmasked EGFR CAR (RV-umCAR). RV-umCAR vector consists of, in frame, from the 5 'end to the 3' end, an MP71 retroviral backbone, a NotI site, an anti-EGFR scFv light chain variable region, a GS linker, an anti-EGFR scFv heavy chain variable region, a hinge region and transmembrane region of CD8 α molecule, cytoplasmic domains of CD28 and 4-1BB (CD137), a CD3 signaling domain, and an EcoRI site.

The anti-EGFR scFv portion of the unmasked CAR is derived from the amino acid sequence of cetuximab. The corresponding DNA sequence of the scFv was codon optimized for its optimal expression in human cells using an online codon optimization tool and synthesized by integrated DNA technology (Coralville, IA). All fragments were gibbsin assembled to generate unmasked EGFR CAR sequences by using the gibbsin assembly cloning kit from New England Biolab (Ipswich, MA) and then ligated into the MP71 backbone vector via NotI and EcoRI. The primers used in gibson assembly were as follows: forward primer (NotI), tta cGC GGC CGC gcc acc atg gct ctg cct gt; reverse primer (EcoRI), tta GAATTC tca tct tgg tgg cag agc ctg c. The upper case represents the target sequence for restriction enzyme digestion.

The masked and NSUB EGFR C AR constructs were cloned based on the unmasked EGFR CAR construct. The coding sequence (underlined) flanked by the GS linker masking peptide and the protease substrate sequence (dashed line) (amino acid sequence:QGQSGQCISPRGCPDGPYVMY-GS SGGSGGSGGSG-GSSGT (SEQ ID NO:12)) as derived from the previous report (Desnoyers, LR et al (2013) Tumor-specific activity of anti-EGFR-targeting promoter enzymes therapeutic index. S ci Transl Med5:207ra144) was codon optimized and then cloned into the N-terminus of the scFv region in the unmasked C AR construct. For the NSUB CAR construct, the protease substrate sequence was replaced with the GS linker sequence (dashed lower line) ((r))QGQSGQCISPRGCPDGPYVMY-GSSGGSGGSGGSG-GSSGT (SEQ ID NO:12)), and then clonedInto the N-terminus of the unmasked CAR construct.

An envelope plasmid (pGALV) encoding gibbon leukemia virus Glycoprotein (GALV) was constructed by the following procedure. GALV cDNA was PCR amplified from genomic DNA of PG13 cell line (Ghani, K et al (2009). effective human hematopoietic cell transformation using RD114-and GALV-pseudotyped transcriptional vectors produced in the culture and serum-free medium. hum Gene Ther 20: 966-. The primers used for cloning were the forward primer (EcoRI), tat GAA TTCgcc acc atg gta ttg ctg cct ggg tcc (SEQ ID NO: 18); and a reverse primer (EcoRI), gcg GAATTC tta aag gtt acc ttc gtt ctc tag ggc (SEQ ID NO: 19). The resulting PCR fragment was then cloned into the pHCMV plasmid backbone from addge (Cambridge, MA) via the EcoRI site.

A lentiviral vector encoding human EGFR (FUW-EGFR) was generated by inserting PCR-amplified cDNA of human EGFR into the pENTR plasmid via SalI and XbaI, and then the EGFR gene was cloned into lentiviral vector FUW via LR reaction using the Gateway cloning kit from Thermo Fisher Scientific (Grand Island, NY). The primers used for cloning were the forward primer (SalI), tat GTC GAC atg cga ccc tcc ggg acg gcg GAA TTC tta aaggtt acc ttc gtt ctc tag ggc (SEQ ID NO: 20); and a reverse primer (XbaI), tcg TCT AGA ccttca ctg tgt ctg caa atc tgc c (SEQ ID NO: 21).

Cell lines and culture media. Cell lines K562, 293T and MDA-MB-231 were obtained from ATCC. The lung cancer cell line NCI-H292 was friendly supplied by Ite Laird-Offringa doctor (University of Southern California, Los Angeles, Calif.). The K562-EGFR cell line was generated by transducing parental K562 cells with lentiviral vector FUW-EGFR. Transduced K562 cells were stained with anti-human EGFR antibody (BioLegend, San Diego, CA) and sorted to give a population of K562 cells overexpressing EGFR.

K562 and K562-EGFR cells were maintained in R10 medium consisting of RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 2mM L-glutamine, 10mM HEPES, 100U/ml penicillin and 100. mu.g/ml streptomycin. 293T and MDA-MB-231 cells were cultured in D10 medium consisting of DMEM medium supplemented with 10% FBS, 2mM L-glutamine, 100U/ml penicillin and 100. mu.g/ml streptomycin. All cell culture media and additives described above were purchased from Hyclone (Logan, UT). Human Peripheral Blood Mononuclear Cells (PBMC) were cultured in T cell culture medium (TCM) consisting of X-Vivo15 medium (Lonza, Walkersville, Md), supplemented with 5% human AB serum (GemCell, West Sacramento, Calif.), 1% HEPES (Gibco, Grand Island, NY), 1% Pen-strep (Gibco), 1% GlutaMax (Gibco) and 0.2% N-acetylcysteine (Sigma-Aldrich, St. Louis, Mo.).

Retroviral vector production. Retroviral vectors were prepared by transient transfection of 293T cells using standard calcium phosphate precipitation protocols. 293T cells cultured in 15-cm tissue culture dishes were transfected with 37.5. mu.g of retroviral backbone plasmid together with 18.75. mu.g of envelope plasmid pGALV and 30. mu.g of packaging plasmid encoding gag-pol. Viral supernatants were harvested 48 and 72 hours post-transfection and filtered through 0.45- μm filters (Corning, NY) prior to use.

T cell transduction and expansion. Frozen human Peripheral Blood Mononuclear Cells (PBMCs) were obtained from AllCells (Alameda, CA). PBMCs were thawed in TCM and left overnight. PBMC were activated by incubation with 50ng/mL OKT3, 50ng/mL anti-CD 28 antibody and 10ng/mL recombinant human IL-7and IL-15(PeproTech, Rocky Hill, N.J.) for 2 days prior to retroviral transduction. For transduction, freshly harvested retroviral supernatants were loaded onto non-tissue culture-treated 12-well plates coated with 15 μ g retronectin/well (Clontech Laboratories, Mountain View, Calif.) by centrifugation at 2000 Xg for 2 hours at 32 ℃. The spin-loading of the vector was repeated once with fresh virus supernatant (Kochenderfer, JN et al (2009). Construction and preclinical evaluation of antisense-CD 19 polymeric antigen receptor. J Immunother 32: 689-702). Activated PBMC at 5X 10⁵The concentration of/mL was resuspended with fresh TCM supplemented with 10ng/mL recombinant human IL-7and IL-15 and added to the vehicle-loaded plates. The plate was rotated at 1000 Xg for 10 minutes at 32 ℃ and at 37 ℃ and 5% CO₂Incubate overnight. The same transduction procedure was repeated the next day. During ex vivo expansion, medium was replenished and cell density was adjusted to 5 × 10 every two days⁵And/ml. In this study, it should be noted that the combination of cytokines IL-7and IL-15 was used to expand CAR-T cells ex vivo, rather than IL-2, which is used more extensively in experimental and clinical protocols. This cytokine condition was chosen because genetically engineered T cells cultured in the presence of IL-7and IL-15 were reported to result in improved engraftment in NSG mice (Cieri, N et al (2013), IL-7and IL-15 induced the generation of human memory T cells from systemic protectants. blood 121: 573-584; Alcantar-Orozco et al (2013), Potential utilization of the NSG human induced mouse a model system to optimal human Gene therapy for human Gene therapy 24: 310-320).

Surface immunostaining and flow cytometry. To detect EGFR CAR expression on the cell surface, cells were stained with protein L or recombinant human EGFR-Fc fusion protein (rhEGFR-Fc). Before FACS staining, 1X 10 were harvested⁶The cells were washed three times with FACS buffer (PBS containing 4% bovine serum albumin fraction V). For Protein L staining, cells were stained with 0.5 μ g biotinylated Protein L (GeneScript, Piscataway, NJ) for 30 minutes at 4 deg.C (Zheng, ZL et al (2012.) Protein L: a novel reagent for the detection of the Clinical Antibiotic Receptor (CAR) expression by flow cytometry. journal of relative Medicine10: 29). Cells were washed three times with FACS buffer, then incubated with 0.1 μ g APC-conjugated streptavidin (BioLegend, San Diego, CA) in FACS buffer for 10 min at 4 ℃ and washed three times. For rhEGFR-Fc staining, cells were stained with 2. mu.g/ml recombinant human EGFR-Fc (R) in FACS buffer at 4 ℃&D Systems, Minneapolis, MN) for 30min, washed twice, and then treated with goat anti-human IgG PE-AffiniPure F (ab') in FACS buffer at 4 ℃₂Fragments (Jackson ImmunoResearch, West Grove, Pa.) were stained for 30 min. Cells were washed twice and resuspended in PBS. Fluorescence was assessed using a Miltenyi Biotec flow cytometer and all FACS data were analyzed with FlowJo software.

Proteolytic activation of masked EGFR CARs: masked protease-mediated activation of EGFR CAR by incubation at room temperature with 1X 10⁶Individual masked EGFR CAR-T cells with varying concentrations of urokinase-type plasminogen activator protease (uPA, R)&D Systems) was incubated in PBS for 1 hour. Cells were then washed twice with PBS and used for subsequent binding and staining experiments.

Intracellular cytokine staining. At 37 ℃ and 5% CO₂T cells (1X 10)⁶) Co-cultured with target cells at a ratio of 1:1 in a 96-well round bottom plate for 6 hours using GolgiPlug (BD Biosciences, San Jose, CA). PE-Cy5.5-anti-CD 3 antibody, APC-Cy 7-anti-CD 4 antibody, Pacific blue CD8 antibody and PE-anti-IFN-gamma were used for immunostaining. All antibodies were purchased from BioLegend. The Cytofix/Cytoperm fixation and permeation kit (BD Biosciences) was used to permeate cell membranes and perform intracellular staining according to the manufacturer's instructions.

Specific cell lysis assay. Lysis of the target cell K562-EGFR was measured by comparing the survival of the target cell K562-EGFR relative to the survival of the negative control cell K562. This method has been previously described (Kochenderfer, JN et al (2009), Construction and preliminary evaluation of an anti-CD19 mathematical receiver. jimmunator 32: 689-). 702). K562 cells were plated by passing them at 1.5X 10⁶Individual cells/mL were labeled suspended in R10 medium with 5 μ M fluorescent dye CMTMR (Invitrogen, Carlsbad, CA). Cells were incubated at 37 ℃ for 30 minutes, then washed twice and suspended in fresh R10 medium. K562-EGFR cells were cultured by passing them at 1X 10⁶Individual cells/mL were labeled suspended in PBS + 0.1% BSA with 5 μ M fluorescent dye CFSE. Cells were incubated at 37 ℃ for 30 minutes. After incubation, the same volume of FBS was added to the cell suspension, followed by incubation at room temperature for 2 minutes. The cells were then washed twice and suspended in fresh R10 medium. Equal numbers of K562 and K562-EGFR cells (5X 10 each)⁴Two) were pooled in the same well for each culture with effector CAR-T cells. Cocultures were plated in triplicate with the following effector cellsRatios to target cells were set in round bottom 96-well plates: 1:1, 3:1 and 10: 1. The cultures were incubated at 37 ℃ for 4 hours followed by 7-AAD labeling according to the manufacturer's instructions (BD Biosciences). Flow cytometric analysis was performed to quantify remaining viable (7-AAD-negative) target cells. For each co-culture, the percent survival of K562-EGFR was determined by dividing the percent of live K562-EGFR by the percent of live K562 cells. In wells containing only target cells and negative control cells without effector cells, the ratio of the percentage of K562-EGFR to the percentage of K562 cells was calculated and used to correct for changes in initial cell number and spontaneous cell death. Cytotoxicity was determined in triplicate and presented as mean ± SEM.

For NCI-H292 and MDA-MB-231 target cells, specific cell lysis was determined as described above, with the following differences. Target cells were labeled with CFSE. Target cells (5X 10) were incubated at 37 deg.C⁴) Incubated with effector cells for 18 hours. The percent survival of target cells is determined by dividing the number of viable target cells with effector cells by the number of viable target cells without effector cells.

Anti-tumor efficacy of CAR-T cells in a non-small cell lung cancer xenograft mouse model. Animal experiments were performed according to Animal protocols approved by the Institutional Animal Care and Use Committee (IACUC) for USC experiments. Six to eight weeks old female NOD.Cg-Prkdc^scidIL2Rg^tm1WjlThe/sz (nsg) mouse (Jackson Laboratory, Farmington, CT) was used in this study. On day 0, 6X 10 suspended in a total of 150. mu.L matrigel (Corning, New York, NY) diluted 1:1 in RPMI medium⁶One NCI-H292 cell was injected into the right flank of each NSG mouse. When the mean tumor size reached 120mm on day 12³All mice were randomized based on tumor size and assigned to 4 groups (n-8). Mice were treated with 4 million CAR-T cells in 150 μ Ι _ PBS and administered twice intravenously via tail vein injection on day 13 and day 26, respectively. CAR expression was normalized to 30% in all CAR groups by addition of donor-matched non-transduced T cells. Tumor growth was monitored twice weekly. Measured by calipersTumor size was quantified and calculated by the following formula: l × W × H/2. When mice exhibited significant weight loss, tumor ulceration or greater than 1000mm³The tumors of (2) were euthanized at size.

And (5) carrying out statistical analysis. Statistical analysis was performed in GraphPad Prism, version 5.01. One-way ANOVA and Tukey multiple comparisons were performed to assess differences in different groups in vitro assays. Tumor growth curves were analyzed using one-way ANOVA and repeated measures (Sidak multiple comparison). Mouse survival curves were evaluated by Kaplan-Meier analysis (log rank test and Bonferroni correction). P values less than 0.05 were considered statistically significant. The significance found was defined as: ns is insignificant, P > 0.05; p < 0.05; p < 0.01; p < 0.001.

Design and generation of masked CAR

Given the known anti-apoptotic effects of the 4-1BB endodomain and the potent cytotoxicity of the CD28 endodomain to the desired CAR function (van der Stegen et al (2015). Thermatology of second-generation genetic engineering receptors nat Rev Drug Discov 14: 499-509; Rice, JJ et al (2006). bacterioldisplay using circulating outer membrane protein OmpX derivatives high affinity peptide Science 15:825 836), various forms of third generation CAR molecules targeting human EGFR were constructed for this study (FIGS. 1a, b) these CAR molecules consist of a single-chain variable fragment derived from monoclonal antibody Cetuximab, the CD8 α transmembrane and scFv domains, the CD28 and 4-1BB costimulatory zeta domains, and the signaling receptor domains of the CD3 receptor.

For mCER, a masking peptide is introduced upstream of the scFv domain using the amino acid sequence from the precursor of cetuximab, together with a cleavable linker sequence (Desnoyers, LR et al (2013). Selection of masking peptides to bind to the antigen recognition domain of cetuximab by Bacterial display (Rice, JJ et al (2006). Bacterial display using circular peripheral membrane protein ompX yields high affinity peptide ligands. protein Science 15:825-836), thereby blocking its ability to bind to EGFR. Linker sequences were determined by using a library of peptide substrates (CLiPS) (Boulware, KT and Daugherty, PS (2006) Protease specificity determination by using Cellular Libraries (CLiPS). Proc Natl Acad Sci USA 103:7583-7588) for their reactivity to a number of Tumor-associated proteases including urokinase-type plasminogen activator (uPA), membrane-type serine Protease 1(MT-SP 1/proteolytic enzyme) and legumain, all of which are upregulated in the Tumor microenvironment of numerous human tumors (Desnyers, LR et al (2013), Medium-specificity of EGFR-targeting pro-coding peptides therapeutic index 5: 144). Conventional unmasked CARs (no masking peptide and linker sequence) were constructed as controls. A control CAR construct containing the same masking peptide but lacking a linker sensitive to protease cleavage was also generated and is denoted as NSUB (b: (b) (r))Is free ofProtease enzymeBottomSubstance sequence) CAR. The NSUB CAR reverses a GS-rich linker that is not cleavable. All of these CARs were cloned into retroviral vectors for delivery to human T cells.

Engineering of this novel mCAR is based on the integration of a safety switch such that 1) the CAR binding site is blocked by a masking peptide, resulting in the rest of CAR-T cells in the systemic circulation or normal tissue environment; but 2) the masking peptide is cleaved by a protease that is locally active in the tumor environment when these CAR-T cells are transported into the tumor, thereby enabling the activated CAR-T cells to recognize the target antigen at the tumor site.

Attenuated binding of mCAR to recombinant EGFR

To test the functional expression of various designed CARs, human Peripheral Blood Mononuclear Cells (PBMCs) were stimulated with anti-CD 3/CD28 antibodies and then transduced with retroviral vectors encoding unmasked, masked or NSUB forms of anti-EGFR CARs. CAR expression was detected using a Protein L staining protocol (Zheng, ZL et al (2012). Protein L: a novel reagent for the detection of a nucleic acid antibody Receptors (CAR) expression by flow cytometry. journal of comparative Medicine10:29) and analyzed by flow cytometry at 8 days post transduction. Similar levels of surface expression of CARs were detected for all three forms of EGFR CARs (approximately 30-35% CAR positive T cells, fig. 2a), and they were observed to expand similarly under T cell culture conditions.

To assess the binding capacity of the different CAR constructs to their target antigen EGFR, CAR-T cells were incubated with recombinant human EGFR fused to Fc (denoted rhEGFR-Fc), followed by staining with anti-human IgG Fc antibody. Approximately 35% of unmasked CAR-T cells showed binding to EGFR, which corresponded well to the percentage of CAR positive cells detected by protein L, while masked and NSUB CAR-T cells showed significantly reduced binding to the target antigen, so that only 3% and 0.4% binding, respectively, were detected (fig. 2 b). Thus, the masking peptide can effectively block the antigen recognition ability of EGFR CAR-T cells.

Restoration of mCRA antigen binding by proteolysis

To explore whether protease-sensitive linkers can be cleaved to expose binding sites and restore CAR binding, CAR-T cells were treated with various concentrations of uPA (a common protease active in a variety of human cancers) prior to incubation with rhEGFR (Ulisse, S et al (2009). As expected, the binding capacity of the unmasked or NSUB CARs did not differ significantly in the presence or absence of uPA treatment (fig. 3, upper and lower panels). However, binding of the masked CAR to EGFR was largely restored by proteolytic cleavage of the protease-sensitive linker after uPA treatment. This protease-mediated activation of binding of masked CAR-T cells was found to be dose-dependent; the percentage of masked CAR-T cells that could bind to rhEGFR increased from 6% without uPA treatment to 23% and 29% with 100nM and 400nM protease, respectively (figure 3, middle panel).

The activity of masked CAR-T cells is blocked in the absence of proteases, but is activated upon in vitro stimulation with protease-secreting tumor cells

Given that the masking peptide attenuates masking and the binding of the NSUB CAR to EGFR, it is hypothesized that the masking peptide may also prevent CAR-T cells from targeting EGFR in the absence of proteases⁺And (4) activating the cells. To test this hypothesis, a target cell line, K562-EGFR, was generated to stably overexpress human wild-type EGFR. The parent K562 cell line is a human chronic myelogenous leukemia cell line that is free of EGFR expression (Ghosh, G et al (2010), Quantifying the presenting properties of EGF receptor (EGFR) tyrosine kinase inhibitors in drug resistant non-small cell regulating cancer (NSCLC) cells using hydrogel-based peptide array.biosensors bioelectronic 26: 424) but has a very low level of uPA expression (Antonyak, H et al (2001), Regulation of expression of the components of expression activity system in the leukaemic cells 23: 253-259). Two previously studied Cancer cell lines, namely The breast Cancer cell line MDA-MB-231 and The lung Cancer cell line NCI-H292, both of which have a high surface expression of wild-type EGFR (Subik, K et al (2010) The expression signatures of ER, PR, HER2, CK5/6, EGFR, Ki-67and AR by immunological analysis in Breast Cancer cell lines (Breast Cancer Auckl)4: 35-41; Raben, D et al (2005) The expression of Cancer cells and in binding with radiation and/or tumor in lung Cancer cell R.2001: 795. and tumor-associated proteases such as upA and protease endogenous secretion of expression of protein kinase (Xas, and expression RH) (1999 of expression of protein kinase J52. expression of protein kinase J. S. A. and expression of protein kinase J MAP kinase and preservation cancer in MDA-MB-231 breakthrough cancer cells J Cell Sci 114: 3387-3396; liu, G et al (1995), Co-expression of a uricase, uricase receiver and PAI-1is a novel for optimal expression of a short lumen Cancer cells. int J Cancer60: 501-. To test whether antigen-specific activation of masked CAR-T cells could be achieved in a protease-active environment, EGFR CAR-T cells were conjugated with K562, K562-EGFR, MDA-MB-231 or NCI-H292 cells were co-cultured. Activation of CAR-T cells was measured by their ability to produce proinflammatory cytokine interferon gamma (IFN- γ) via intracellular cytokine staining (fig. 4a, b).

All CAR-T cell groups exhibited background levels of IFN- γ secretion in the absence of K562 cell stimulation or under K562 cell stimulation. All of them presented with CD8 after stimulation with anti-CD 3/CD28 antibody⁺CAR-T cells (about 20%) the same level of IFN- γ secretion. As expected, unmasked CAR-T cells responded at a similar percentage range when co-cultured with different target cell lines (15.4%, 17.8%, 19.3% for K562-EGFR, MDA-MB-231, NCI-H292, respectively). In contrast, NSUB CAR-T cells of all test groups showed an impaired cytokine response compared to unmasked CAR-T cells (1%, 5%, 6% for K562-EGFR, MDA-MB-231, NCI-H292, respectively). Furthermore, only about 0.9% of CD8 in masked EGFR CAR-T cells⁺Cells were able to secrete IFN- γ in response to stimulation by K562-EGFR cells, which was not significantly different from the NSUB CAR-T group. Thus, this finding is consistent with previous binding data, thereby demonstrating that activation of CAR-T cells is largely abolished by the masking peptides of the masking group and the NSUB group. However, the level of activation of masked CAR-T cells following stimulation of MDA-MB-231 or NCI-H292 tumor cells expressing EGFR and producing tumor-associated proteases (16% and 17.2% CD8, respectively)⁺IFN-γ⁺) Largely reverting to a level similar to that of the unmasked group. Taken together, these data indicate that EGFR-specific CAR-T cell function can be selectively activated in a protease-activated environment, but significantly eliminated in a protease-null environment.

Specific cytolytic assays were also performed to test the cytotoxic effects of all CAR-T cell sets on the above target cell lines. Similar to the IFN- γ assay, unmasked CAR-T cells lysed all target cells, including K562-EGFR, NCI-H292, and MDA-MB-231; whereas masked CAR-T cells lysed only H292 and MDA-MB-231 cells at a high effector cell to target cell ratio and showed no killing activity on K562-EGFR cells (fig. 5 a-c).

Masked CAR-T cells show in vivo equivalent anti-tumor efficacy to unmasked cells

After confirming the specificity and functionality of the masked CAR-T cells in response to tumor cells in vitro, it was then sought to investigate their anti-tumor reactivity in vivo. To evaluate the antitumor efficacy of various CAR-T cells, a subcutaneous human lung cancer xenograft model of NSG mice was used (fig. 6 a). NCI-H292 cells (6X 10)⁶) Injected into the right flank of NSG mice. When the mean tumor size reached about 120mm at day 12 after tumor inoculation³At time, all tumor bearing mice were randomized into tumor size grade matched cohorts (n ═ 8 per treatment group) and CAR-T treatment was initiated. Mice were treated with 4 million CAR-T cells via intravenous injection on days 13 and 26, and tumor growth was monitored. Animals in all treatment groups showed tumor progression. Infusion of NSUBCAR-T cells had no effect on slowing tumor growth compared to the untransduced control T cell group (fig. 6 b). However, those mice that received unmasked or masked CAR-T cells produced tumor growth inhibition compared to mice that received untransduced T cells (unmasked: P ═ 0.0294, masked: P ═ 0.0404, one-way ANOVA). Thus, both unmasked and masked CAR-T cells significantly prolonged the survival of mice (fig. 6c, unmasked: P ═ 0.0006, masked: P ═ 0.0036, log rank test). The unmasked and masked groups had median survival of 50.5 days and 49 days after the first treatment compared to the untransduced and NSUB groups, respectively, which had a median survival of 42 days (fig. 6 c).

"on-target off-tumor" toxicity is a serious limitation of the transforming application of CAR-T therapy in solid tumors. This is usually due to low expression levels of the target antigen in normal tissues. Due to the high sensitivity of CAR-T cells to target antigens, on-target toxicity can be particularly severe for CAR-based cell therapy compared to conventional antibody therapy. Thus, target antigens such as EGFR and HER2 that are overexpressed in various tumors but widely expressed in other tissues are generally considered to be "unwergable" targets for CAR-T cells.

Here, it was demonstrated that this limitation can be overcome by re-engineering the CAR molecule into mCAR via the introduction of a cleavable masking peptide to block the antigen recognition site in the scFv domain. Similar to the parental unmasked anti-EGFR CAR-T cells, the masked anti-EGFR CAR-T cells were found to show similar cytokine production and cell killing activity against the EGFR overexpressing breast cancer cell line MDA-MB-231 and lung cancer cell line NCI-H292. Importantly, masked CAR-T cells significantly reduced reactivity to cells that overexpress EGFR and secrete little or no protease. In a human lung cancer xenograft model established in NSG mice, masked CAR-T cells showed the same efficacy in inhibiting tumor growth in vivo as parental CAR-T cells.

Several attempts have been made to explore engineered designs that improve tumor tissue selectivity of CAR engineered T cells (Klebanoff, CA et al (2016. processes for gene-engineered T cell immunology for solid tumors. nat. Med22: 26-36). An elegant strategy utilizes dual targeting of two CARs to obtain selective recognition of tumors rather than normal cells. It was demonstrated that T cells expressing two CARs targeting two different antigens could serve as a logic gate (logic gate) controlling sufficient T cell activation. In one example, two attenuated CARs are designed such that recognition of one antigen target by one CAR induces only suboptimal T cell activation, while activation of a second CAR by a second antigen can provide additional co-stimulatory signals. Thus, activation of two CARs simultaneously generates T cells with all the potential to induce an anti-tumor immune response against tumor tissue expressing both antigens (Kloss, CC et al (2013). composite anti-inflammatory with cellular signaling proteins selected active tumor tissue T cells. nat Biotechnol 31: 71-75. alternatively, the same group introduces two functionally different CARs, one Inhibitory CAR (iCAR) and the other active CAR (acar), into the same T cellWhen the cells are transported into a tumor environment where only the aCAR, but no iCAR antigen is available, CAR-T cells can be sufficiently activated to elicit anti-tumor immunity.³⁵The present mCAR approach provides yet another strategy to generate conditionally active CARs for improving tumor specificity of CAR-T cells.

Another approach to reducing off-target tumor toxicity involves modulating the affinity of the CAR molecules in order to better distinguish antigen targets in tumor tissue from antigens in normal tissue. Recent studies have shown that CAR derived from low Affinity anti-EGFR or anti-HER 2scFv can selectively target tumor cells, while leaving normal tissue with low target antigen expression (Liu, X et al (2015). Affinity-tuned ErbB2or EGFR molecular antigen receptor T cell expression peptide in cancer tissue 75: 3596. 3607; Caruso et al (2015). Tunning sensitivity of CAR to EGFR density sensitivity of non tissue amplification sensitivity cancer cell R75: 350765. cell 3518; Chmielleski, M et al (2004) T cell activation-tissue expression-expression vector tissue of cell expression of expression. This approach can be applied to design effective and safe CARs, but still has potential drawbacks due to the inability to recognize tumor cell populations with relatively low tumor antigen densities. Thus, for tumors with uneven antigen expression levels, these CAR-engineered cells can exclude tumor cells with high antigen expression, but may miss tumors with low antigen expression. Unlike affinity-modulated designs, mCAR can be derived from currently available high affinity antibodies and the selectivity of mCAR is due to spatially controlled receptor activation. Only in the protease-enriched tumor microenvironment, the masked CAR-T cells become responsive to targets in the surrounding environment and trigger downstream cytotoxic responses.

In summary, the present study has provided a starting point for further investigation and development of a masked CAR platform. However, although in vitro with or without a tumor phaseImproved selectivity between protease-related target cells and anti-tumor reactivity was observed in animal studies, but the safety and efficacy of mCAR constructs in humans also required further evaluation and testing in preclinical or phase I pilot trials involving non-human primates. Our CARs were derived from cetuximab, which had minimal cross-reactivity with mouse EGFR, and thus it was difficult to evaluate the reactivity of masked anti-EGFR CAR-T cells to normal tissues in a mouse model. Furthermore, in this study CAR-T was administered as a single agent, which generally had only poor antitumor efficacy, especially in solid tumors. In a clinical setting, combination therapies including CAR-T therapy and chemotherapy or immune checkpoint inhibitors can be used to enhance therapeutic efficacy. In addition, have been previously tested²⁰The protease cleavable linker used in the mCAR design is sensitive to a variety of proteases that are locally active in the tumor microenvironment, rather than proteases in normal tissues such as tissue plasminogen activator (tPA), plasmin and KLK 5. However, an even broader range of evaluation of normal proteases is required prior to clinical studies in humans.

Since EGFR is deregulated in many types of human carcinomas, this study provides a route to designing CARs capable of targeting different types of cancers. This masked CAR strategy can help expand the applicability of CAR-T cells to cancers that lack additional "pharmaceutically acceptable" tumor antigens. It may also enable CAR-T therapy to target tumor antigens such as carbonic anhydrase IX or HER2, where "off-target" side effects are shown to be intolerable and life threatening (Morgan, RA et al (2010) Castaneport of a laser uptake effect following the administration of T cell transformed with a polymeric antigen receptors registering ERBB2.mol Ther 18: 843. Lamers, CH et al (2013) Treatment of metallic cell with CATH IX CAR-engineered T cells: clinical evaluation and management of-target oxidation. mol Ther 21: 904).

The various methods and techniques described above provide a number of ways to implement the present application. Of course, it is to be understood that not necessarily all such objects or advantages may be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods may be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as may be taught or suggested herein. Various alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another or several features, while others specifically exclude one, another or several features, while still others may weaken a particular feature by including one, another or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be used in various combinations by one of ordinary skill in the art to perform such methods in accordance with the principles described herein. Among the various elements, features and steps, some will be specifically included and others will be specifically excluded in different embodiments.

Although the present application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that embodiments of the present application may be extended beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans may employ such variations as appropriate, and that the application may be practiced otherwise than as specifically described herein. Accordingly, many embodiments of the present application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, this application is intended to cover any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or otherwise clearly contradicted by context.

All patents, patent applications, patent application publications, and other materials cited herein, such as articles, books, specifications, publications, documents, contents, and the like, are hereby incorporated by reference in their entirety for all purposes, except as follows: any prosecution history associated with such, any inconsistency or conflict with the present document, or any other entity which has a limiting impact on the broadest scope of a claim presently or later relating to the present document. For example, the description, definition, and/or use of terms in this document shall control if there is any inconsistency or conflict in the description, definition, and/or use of the terms between those associated with any of the incorporated materials and those associated with this document.

It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the present application. Other modifications may be employed within the scope of the present application. Thus, by way of example, and not limitation, alternative configurations of embodiments of the present application may be utilized in accordance with the teachings herein. Thus, embodiments of the present application are not limited to what is specifically shown and described.

Various embodiments of the present invention are described above in the detailed description. While these descriptions directly describe the above embodiments, it is to be understood that modifications and/or variations to the specific embodiments shown and described herein may occur to those skilled in the art. Any such modifications or variations that fall within the purview of this description are intended to be included therein. Unless otherwise indicated, it is the intention of the inventors that the words or phrases in the specification and claims be given the ordinary and customary meaning to those skilled in the art to which this application pertains.

The foregoing description of various embodiments of the invention known to the applicant at the time of filing has been presented and is intended for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in light of the above teaching. The described embodiments are intended to explain the principles of the invention and its practical application to thereby enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out this invention.

While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention.

Sequence listing

<110>UNIVERSITY OF SOUTHERN CALIFORNIA

WANG, Pin

HAN, Xiaolu

BRYSON, Paul

<120> masked chimeric antigen receptor T cells for tumor specific activation

<130>065715-000066WO00

<150>62/185,398

<151>2015-06-26

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<170> PatentIn 3.5 edition

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Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser

180 185 190

Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly

195 200 205

Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn

210 215 220

Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser

225 230 235 240

Gln Val Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile

245 250 255

Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr

260 265 270

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala

275 280 285

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

290 295 300

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

305 310 315 320

Arg Gly Leu Asp Phe Ala Cys Asp Ile Phe Trp Val Leu Val Val Val

325 330 335

Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Tyr Val Ala Phe Ile

340 345 350

Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr

355 360 365

Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln

370 375 380

Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Phe Ser

385 390 395 400

Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro

405 410 415

Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys

420 425 430

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

435 440 445

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu

450 455 460

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

465 470 475 480

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

485 490 495

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

500 505 510

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

515 520 525

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

530 535 540

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

545 550 555

Claims (38)

1. A masked chimeric antigen receptor (mCAR) comprising:

a. a masking peptide;

b. an antigen-specific targeting domain;

c. a transmembrane domain;

d. at least one co-stimulatory domain; and

e. an intracellular signaling domain of a protein that is capable of signaling,

wherein the antigen-specific targeting domain comprises an antigen-specific single chain fv (scFv) fragment.

2. The mCAR of claim 1, further comprising an extracellular spacer domain.

3. The mCAR of claim 1, wherein the masking peptide comprises a mask that specifically binds the antigen-specific targeting domain and a cleavage site.

4. The mCAR of claim 3, wherein the mask and the cleavage site are connected by a linker.

5. The mCAR of claim 1, wherein the masking peptide is linked to the cAR by a linker.

6. The mCAR of claim 1, wherein the mCAR is inactive when masked and active when the mask is cleaved.

7. The mCAR of claim 3, wherein the cleavage site is a protease-specific cleavage site.

8. The mCRAR of claim 5, wherein the masked mCRAR in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: mask-linker-cleavage site-antigen specific targeting domain-transmembrane domain-costimulatory domain-intracellular signaling domain.

9. The mCRAR of claim 5, wherein the masked mCRAR in an uncleaved state comprises the structural arrangement from N-terminus to C-terminus as follows: mask-linker-cleavage site-antigen specific targeting domain-extracellular spacer domain-transmembrane domain-costimulatory domain-intracellular signaling domain

10. The masked chimeric antigen receptor of claim 2, wherein the extracellular spacer domain comprises any one or more of: an Fc fragment of an antibody or a functional equivalent, fragment or derivative thereof; a hinge region of an antibody or a functional equivalent, fragment or derivative thereof; the CH2 region of the antibody; the CH3 region of the antibody; artificial spacer sequences and combinations thereof.

11. The masked chimeric antigen receptor of claim 10, wherein the extracellular spacer domain comprises any one or more of (i) the hinge region, the CH2 region, and the CH3 region of IgG4, (ii) the hinge region of IgG4, (iii) the hinge region of IgG4 and the CH2 region, (iv) the hinge region of CD8 α, (v) the hinge region of IgG1, the CH2 region, and the CH3 region, (vi) the hinge region of IgG1, (vi) the hinge region of IgG1 and the CH2 region, or (vii) a combination thereof.

12. The masked chimeric antigen receptor of claim 1, wherein the transmembrane domain comprises any one or more of: transmembrane regions of type I transmembrane proteins, artificial hydrophobic sequences, and combinations thereof.

13. The masked chimeric antigen receptor of claim 12, wherein the transmembrane domain comprises any one or more of the transmembrane domain of ξ chain of the T cell receptor complex, CD28, CD8 α, and combinations thereof.

14. The masked chimeric antigen receptor of claim 1, wherein the co-stimulatory domain comprises a signaling domain from any one or more of: CD28, CD137(4-1BB), CD134(OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, and combinations thereof.

15. The masked chimeric antigen receptor of claim 1, wherein the intracellular signaling domain comprises a signaling domain of one or more of human CD3 ξ chain, FcyRIII, FceRI, the cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing a cytoplasmic receptor, and combinations thereof.

16. The masked chimeric antigen receptor of claim 1, wherein the antigen specific targeting domain targets an antigen selected from the group consisting of: antigens specific for cancer, inflammatory diseases, neuronal disorders, diabetes, cardiovascular diseases, infectious diseases, autoimmune diseases, and combinations thereof.

17. A masked chimeric antigen receptor according to claim 16, wherein said antigen specific for cancer comprises any one or more of 4-1BB, 5T4, adenocarcinoma antigen, α -alpha fetoprotein, BAFF, B-lymphoma cells, C242 antigen, CA-125, carbonic anhydrase 9(CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23(IgE receptor), CD28, CD30(TNFRSF8), CD33, CD4, CD 3644 v 4, CD4, CEA, CTLA 888, CTLA-4, DR 4, EGFR, EpsCR 4, FAP, fibronectin extra domain-B, folate receptor 1, GD 4, glycoprotein 4, ganglioside, TRAIL 75, NMDCPD-4, IGF-4, VEGF-binding protein receptor-4, VEGF-binding protein receptor, VEGF-4, VEGF-binding protein receptor, VEGF-4, VEGF-receptor-4, VEGF-binding protein receptor, VEGF-4, VEGF-36.

18. The masked chimeric antigen receptor according to claim 16, wherein the antigen specific for an inflammatory disease comprises any one or more of AOC3(VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basal immunoglobulin), CD154(CD40L), CD2, CD20, CD23(IgE receptor), CD25(α chain of IL-2 receptor), CD3, CD4, CD5, IFN- α, IFN- γ, IgE, IgEFc region, IL-1, IL-12, IL-23, IL-13, IL-17A, IL-22, IL-4, IL-5, IL-6 receptor, integrin α, integrin α 4 6867, llama, LFA-1(CD11, MEDI-528, myostatin-3540, SOIL- β, TNF-11, VEGF- β, VEGF-27, and a combination thereof.

19. The masked chimeric antigen receptor of claim 16, wherein the antigen specific for a neuronal disorder comprises any one or more of β amyloid, MABT5102A, and combinations thereof.

20. The masked chimeric antigen receptor of claim 16, wherein the antigen specific for diabetes comprises any one or more of L-1 β, CD3, and combinations thereof.

21. The masked chimeric antigen receptor according to claim 16, wherein the antigen specific for cardiovascular disease comprises any one or more of C5, cardiac myosin, CD41 (integrin α -IIb), fibrin II, β chain, ITGB2(CD18), sphingosine-1-phosphate, and combinations thereof.

22. The masked chimeric antigen receptor of claim 16, wherein the antigen specific for an infectious disease comprises any one or more of anthrax toxin, CCR5, CD4, aggregation factor A, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF- α, and combinations thereof.

23. The mCAR of claim 1, wherein the antigen-specific targeting domain targets EGFR.

24. The mCER of claim 1, wherein said mCER comprises the sequence listed in Table 1 and/or SEQ ID NO: 29.

25. A polynucleotide encoding the masked chimeric antigen receptor of claim 1.

26. A polypeptide encoded by the polynucleotide of claim 25.

27. A vector comprising the polynucleotide of claim 25.

28. A virus comprising the polynucleotide of claim 25.

29. The virus of claim 28 wherein the virus is an RNA virus.

30. The virus of claim 28, wherein the virus is a retrovirus, adenovirus, adeno-associated virus, lentivirus, poxvirus, or herpesvirus.

31. A genetically engineered cell comprising the polynucleotide sequence of claim 25.

32. The genetically engineered cell of claim 31, wherein the cell is a T cell.

33. The genetically engineered cell of claim 32, wherein the cell is a natural T cell, a central memory T cell, an effector memory T cell, or a combination thereof.

34. The genetically engineered cell of claim 31, wherein the cell is a natural killer cell, hematopoietic stem cell, embryonic stem cell, or pluripotent stem cell.

35. A method for generating a plurality of T cells expressing a masked chimeric antigen receptor, the method comprising:

transfecting one or more T cells with the vector of claim 27; and

stimulating the one or more T cells with a cell expressing an antigen targeted by an antigen-specific targeting domain or with a recombinant antigen specific for the ASTD of mCAR, whereby the T cells proliferate to produce a plurality of T cells.

36. A method for treating a disease in a subject in need thereof, the method comprising:

providing a composition according to claim 1; and

administering a therapeutically effective amount of the composition to the subject, so as to treat the disease, wherein the antigen-specific targeting domain is associated with the disease.

37. The method of claim 36, wherein the disease is cancer.

38. The method of claim 36 or 37, wherein the cancer is lung cancer, breast cancer, renal cancer, or neuroblastoma.