CN119698295A - Cancer treatment approaches using anti-TIGIT antibodies - Google Patents

Abstract

Methods of treating cancer or increasing, enhancing or stimulating an immune response with antibodies and antigen binding fragments thereof that specifically bind to TIGIT (T cell immune receptor with Ig and ITIM domains WUCAM or Vstm 3) in combination with anti-PD 1 antibodies are provided.

Description

Methods of cancer treatment using anti-TIGIT antibodies

Technical Field

The present application relates to the combination of antibodies that specifically bind to TIGIT (T cell immune receptor with Ig and ITIM domains) with anti-PD 1 antibodies for the treatment of cancer.

Background

TIGIT (T cell immunoglobulins and ITIM domains) are type I transmembrane proteins, members of the CD28 family of proteins, which play an important role in inhibiting T cell and NK cell mediated functional activity in anti-tumor immunity (Boles KS, et al 2009Eur.J Immunol,39:695-703; stanietsky N, et al 2009pna 106:17858-63; yu X, et al 2009nat. Immunol, 10:48-57).

Genes and cdnas encoding TIGIT were cloned and characterized in mice and humans. The full-length human TIGIT has a sequence of 244 amino acids in length (SEQ ID NO: 26), wherein the first 21 amino acids consist of a signal peptide. The amino acid sequence of mature human TIGIT contains 223 amino acid (aa) residues (NCBI accession number: NM-173799). The extracellular domain (ECD) of mature human TIGIT consists of 120 amino acid residues (amino acids 22-141 corresponding to SEQ ID NO: 26) with a V-type Ig-like domain (amino acids 39-127 corresponding to SEQ ID NO: 26) followed by a transmembrane sequence of 21 aa and a cytoplasmic domain of 82 aa with an immunoreceptor tyrosine based inhibitory motif (ITIM) (Yu X, et al 2009Nat. Immunol,10:48-57; stengel KF, et al 2012PNA 109:5399-04). In ECD, human TIGIT shares only 59% and 87% amino acid sequence identity with mice and cynomolgus monkeys, respectively.

TIGIT is expressed on T cells (including activated T cells, memory T cells, regulatory T (Treg) cells, and follicular T helper (Tfh) cells) and NK cells (Boles KS, et al, 2009Eur J Immunol,39:695-703; joller N, et al, 2014Immunity 40:569-81; levin SD, et al, 2011Eur J Immunol,41:902-15; stanietsky N, et al, 2009pnas106:17858-63; yu X, et al, 2009nat. Immunol, 10:48-57).

To date, two TIGIT ligands have been identified, CD155 (also known as poliovirus receptor or PVR) and CD112 (also known as poliovirus receptor associated 2, pvrl2, nectin-2). These ligands are expressed predominantly on APCs (such as dendritic cells and macrophages) and tumor cells (Casado JG, et al, 2009Cancer Immunol Immunother58:1517-26; levin SD, et al, 2011Eur.J Immunol,41:902-15; mendelsohn CL et al, 1989:855-65; stanietsky N, et al, 2009PNAS 106:17858-63; yu X, et al 2009Nat. Immunol, 10:48-57). TIGIT, as an immune "checkpoint" molecule, initiates inhibitory signaling in immune cells upon binding to its ligands CD155 and CD 112. It is yet to be determined whether TIGIT has a functional correlation in mediating inhibitory signals with respect to its binding affinity (Kd: about 1 nM) for CD155 that is much higher than for CD 112. The co-stimulatory receptor CD226 (DNAM-1) binds with lower affinity to the same ligand (Kd: about 100 nM), but delivers a positive signal (Bottino C, et al, 2003J Exp Med 198:557-67). In addition, CD96 (TACTILE), a "TIGIT-like" receptor, also exerts a similar inhibitory effect in the same pathway (Chan CJ, et al, 2014Nat.Immunol 15:431-8).

Upregulation of TIGIT expression in Tumor Infiltrating Lymphocytes (TIL) and Peripheral Blood Mononuclear Cells (PBMCs) has been reported to occur in many types of cancers, such as lung Cancer (Tassi, et al, cancer res.2017:851-861), esophageal Cancer (Xie J, et al, oncotarget 20167:63669-63678), breast Cancer (Gil Del Alcazar CR, et al 2017Cancer discover), acute Myelogenous Leukemia (AML) (Kong Y et al, CLIN CANCER res.2016 22:3057-66) and melanoma (Chauvin JM, et al, J Clin invest.2015:2046-2058). Increased expression of TIGIT in AML is associated with a poor prognosis for patient survival outcome (Kong Y et al CLIN CANCER res.2016 22:3057-66). Upregulation of TIGIT signaling plays an important role not only in immune tolerance to cancer but also in immune tolerance to chronic viral infection. During HIV infection TIGIT expression on T cells is significantly higher and is positively correlated with viral load and disease progression (Chew GM, et al 2016PLoS Pathog.12:e1005349). In addition, blockade of TIGIT receptors, alone or in combination with other blockades, can rescue functional "depleted" T cells in vitro and in vivo (Chauvin JM, et al, J Clin invest.2015 125:2046-2058; chew GM, et al, 2016PLoS Pathog.12:e1005349;Johnston RJ, et al CANCER CELL 2014:923-937). In the case of cancer and viral infections, activation of TIGIT signaling promotes immune cell dysfunction, resulting in cancer proliferation or prolonged viral infection. Inhibition of TIGIT-mediated inhibitory signaling by therapeutic agents may restore functional activity of immune cells, including T cells, NK cells, and Dendritic Cells (DCs), and thus enhance immunity against cancer or chronic viral infection.

Thus, anti-TIGIT antibodies with enhanced effector function may induce an effective immune response in the treatment of cancer or chronic viral infection.

Disclosure of Invention

The present disclosure relates to methods of treating cancer by administering anti-TIGIT antibodies.

A method of cancer treatment, the method comprising administering to a subject an effective amount of a pH-dependent anti-TIGIT antibody or antigen-binding fragment thereof.

The method, wherein the anti-TIGIT antibody binds to the TIGIT protein at amino acid histidine 76.

The method, wherein the anti-TIGIT antibody binds to the TIGIT protein at histidine 76 and leucine 73.

The method, wherein the method comprises administering to the subject an effective amount of a pH-dependent antibody or antigen-binding fragment thereof that specifically binds to human TIGIT and comprises a heavy chain variable region comprising HCDR (heavy chain complementarity determining region) 1 of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, and HCDR3 of SEQ ID NO:3, and a light chain variable region comprising LCDR (light chain complementarity determining region) 1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and LCDR3 of SEQ ID NO: 6.

The method, wherein the anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising SEQ ID No. 7 and a light chain variable region (VL) comprising SEQ ID No. 8.

The method, wherein the anti-TIGIT antibody has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

The method, wherein the anti-TIGIT antibody has reduced fucosylation.

The method, wherein the anti-TIGIT antibody has Fc amino acid changes at S239D and I332E (EU numbering).

The method, wherein the anti-TIGIT antibody has an Fc amino acid change at S239D, I E and a330L (EU numbering).

The method of any one of the above claims, wherein the anti-TIGIT antibody has increased binding affinity for fcyriiia-V158 and fcyriiia-F158.

The method of any one of the above claims, wherein the anti-TIGIT antibody has enhanced ADCC in T-regulatory (Treg) cells.

The method of any one of the above claims, wherein the anti-TIGIT antibody activates Natural Killer (NK) cells.

The method of any one of the above claims, wherein the anti-TIGIT antibody has increased cell gnawing (trogocytosis).

The above method, wherein the method further comprises administering an anti-PD 1 antibody that specifically binds human PD1 and comprises a heavy chain variable region comprising HCDR1 of SEQ ID NO. 15, HCDR2 of SEQ ID NO. 16 and HCDR3 of SEQ ID NO. 17, and a light chain variable region comprising LCDR1 of SEQ ID NO. 18, LCDR2 of SEQ ID NO. 19 and LCDR3 of SEQ ID NO. 20.

The above method, wherein the anti-PD 1 antibody or antigen-binding fragment thereof specifically binds human PD1 and comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO. 21 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO. 22.

The method, wherein the anti-PD 1 antibody comprises an IgG4 constant domain comprising SEQ ID NO. 23.

The method wherein the cancer is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, gastric cancer, renal cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, or sarcoma.

The method, wherein the cancer is non-small cell lung cancer.

The method, wherein the head and neck cancer is nasopharyngeal cancer.

The method, wherein the esophageal cancer is Esophageal Squamous Cell Carcinoma (ESCC).

The method, wherein the cancer is uterine cancer.

The method, wherein the gastric cancer is a gastric or gastroesophageal junction cancer.

The method wherein the cervical cancer is recurrent or metastatic cervical cancer.

The method, wherein the cancer is renal cancer.

The method further comprises administering chemotherapy.

The method, wherein the chemotherapy is chemoradiotherapy.

The method, wherein the anti-PD 1 antibody is administered at 200mg every three weeks.

Drawings

FIGS. 1A-D show the crystal structure of A1217 (European-Perot Li Shan anti (Ociperlimab)) Fab which binds to human TIGIT. FIG. 1A is the crystal structure of A1217Fab which binds to human TIGIT, wherein the A1217 Heavy (HC) and Light (LC) chain regions are colored in black and gray, and TIGIT is represented by a white surface. Figure 1B shows the crystal structure of a1217Fab that binds to human TIGIT superimposed with the known PVR/TIGIT composite structure (PDB 3 UDW), showing that a1217 and PVR in the gray band have spatial conflict and competitive binding to TIGIT. FIG. 1C is an atomic interaction on the binding surface of the A1217/TIGIT complex, identifying certain key residues of A1217 (paratope residues as shown by the lines) and certain key residues of TIGIT (epitope residues as shown by the underlined bars). For clarity, the non-CDR regions of a1217 are removed and TIGIT is represented by a white transparent surface. Boxed residues were identified as functionally important epitope residues by alanine scanning mutagenesis. FIG. 1D shows the surrounding residues of HIS76 _TIGIT in A1217 to emphasize the interaction between HIS76 _TIGIT and ASP103 _HCDR3, which is critical for pH-dependent binding to TIGIT. HIS76 _TIGIT and ASP103 _HCDR3 are highlighted with underlined text.

Figures 2A-C show a blocking comparison between a1217 (olprivet Li Shan anti) Fab and tirelizumab (Tiragolumab) Fab. FIG. 2A shows the superposition of the European style Li Shan anti-Fab/TIGIT and tirelimumab Fab/TIGIT complexes. The osppero Li Shan anti-Fab and tirelizumab Fab were colored black and white, respectively. TIGIT is shown in a white surface representation. Figure 2B shows epitopes of the osprex Li Shan anti-Fab or tirelimumab Fab. The overlapping epitopes between the two antibodies are marked with underlined black text. The unique epitope of the osprex Li Shan anti-Fab is colored black, with white text, while the unique epitope of the tirelizumab Fab is colored gray, with black text. Fig. 2C is fig. 2B (rotated 90 °) from a side view.

Figures 3A-B depict atomic interactions on the binding surface of the ospperot Li Shan anti-Fab/TIGIT and tirelizumab Fab/TIGIT complexes. Figure 3A depicts the binding interface between the ospperot Li Shan anti-Fab and TIGIT. HCDR1, HCDR2 and HCDR3 of the heavy chain are marked with bold italic black text, black text and underlined black text, respectively. LCDR1, LCDR2 and LCDR3 of the light chain are marked with bold italic grey text, grey text and underlined grey text, respectively. Fig. 3B depicts the binding interface between tirelimumab Fab and TIGIT. HCDR2 and HCDR3 of the heavy chain are marked with black text and underlined black text, respectively. LCDR1 and LCDR3 of the light chain are marked with gray text and underlined gray text, respectively. TIGIT is marked with bold white text in both (a) and (b).

FIG. 4 is a table summarizing afucosylated A1217 antibody production.

FIGS. 5A-B FIG. 5A shows binding of the A1217AF antibody to FcgammaRIIIA-F158, while FIG. 5B depicts binding of the A1217AF antibody to FcgammaRIIIA-V158.

FIGS. 6A-D FIGS. 6A-B are comparisons of A1217WT with A1217AF and mutant effector variant antibodies (including Fc silencing antibodies) and how they bind to FcgammaRIIIA-V158 and FcgammaRIIIA-F158. FIG. 6C-D depicts binding of an A1217 antibody to FcgammaRIIIA-V158 and FcgammaRIIIA-F158 expressed on HEK293 cells.

FIG. 7 shows that A1217, A1217DE and A1217DEL have comparable binding to TIGIT over-expressing HEK293 cells.

FIG. 8 indicates that A1217AF and A1217 show comparable binding to C1 q.

FIGS. 9A-B show that A1217AF, A1217DEL and A1217DE have enhanced ADCC activity when compared to the A1217 wild type.

Figures 10A-C show that a1217AF has enhanced ADCC activity against tregs.

FIGS. 11A-B indicate activation of NK cells by A1217 AF.

FIG. 12 shows that TIGIT is down-regulated by A1217 AF.

Fig. 13 shows the cell biting property of a 1217.

FIG. 14 is a mouse model of A1217 in combination with an anti-PD 1 antibody.

Fig. 15A-C are mouse models of a1217 in combination with anti-PD 1 antibody and Treg reduction.

Detailed Description

Definition of the definition

Conservative amino acid substitutions of amino acids are well known in the art. In general, conservative amino acid substitutions mean that one amino acid residue is replaced with another amino acid residue having a similar side chain.

Unless defined otherwise herein, all other 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.

As used herein, including the appended claims, the singular forms of words (such as "a," "an," and "the") include the plural forms corresponding thereto, unless the context clearly dictates otherwise.

The term "or" is used to mean, and is used interchangeably with, the term "and/or" unless the context clearly dictates otherwise.

In this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated amino acid sequence, DNA sequence, step or group thereof but not the exclusion of any other amino acid sequence, DNA sequence, step. As used herein, the term "comprising" may be replaced with the terms "including", "comprising" or sometimes "having".

The term "TIGIT" includes various mammalian isoforms, e.g., human TIGIT, orthologs of human TIGIT, and analogs comprising at least one epitope within TIGIT. The amino acid sequence of TIGIT (e.g., human TIGIT) and the nucleotide sequences encoding it are known in the art (see Genbank AAI 01289). Human TIGIT sequence (SEQ ID NO: 26)

When applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, the terms "administration", "treatment" and "treatment" as used herein mean the contact of an exogenous drug, therapeutic, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent with a cell, as well as contact of a reagent with a fluid, wherein the fluid is in contact with the cell. The term "administering" or "treatment" also includes in vitro and ex vivo treatment of a cell, e.g., by an agent, diagnostic agent, binding compound, or by another cell. The term "subject" refers herein to any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit), and most preferably a human.

The term "antibody" is used herein in its broadest sense and specifically covers antibodies (including full length monoclonal antibodies) and antibody fragments as long as they recognize an antigen, e.g., TIGIT. Antibodies are generally monospecific, but may also be described as individual-specific, xenogenic or multispecific. The antibody molecule binds to a specific epitope or epitope on the antigen through a specific binding site.

The term "monoclonal antibody" or "mAb" is meant herein to refer to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprised in the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a plurality of different antibodies having different amino acid sequences in their variable domains, particularly their Complementarity Determining Regions (CDRs), which are typically specific for different epitopes. The modifier "monoclonal" refers to the characteristic of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies (mabs) may be obtained by methods known to those skilled in the art. See, for example, kohler G et al, nature 1975 256:495-497, U.S. Pat. No. 4,376,110;Ausubel FM et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1992, harlow E et al, ANTIBODIES: ALABORATORY MANUAL, cold spring Harbor Laboratory 1988, and Colligan JE et al, CURRENT PROTOCOLS IN IMMUNOLOGY 1993. The mabs disclosed herein can be of any immunoglobulin class, including IgG, igM, igD, igE, igA and any subclass thereof. The mAb-producing hybridomas can be cultured in vitro or in vivo. High titers of mAbs can be obtained by in vivo production, wherein cells from an individual hybridoma are injected intraperitoneally into mice, such as naive (prine-prime) Balb/c mice, to produce ascites fluid containing the desired mAb in high concentrations. The isotype IgM or IgG MAb can be purified from such ascites fluid or from culture supernatants using column chromatography methods well known to those skilled in the art.

Typically, the basic antibody structural units comprise tetramers. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Generally, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are generally classified as α, δ, ε, γ, or μ, and the isotypes of antibodies are defined as IgA, igD, igE, igG and IgM, respectively. In the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, wherein the heavy chain further comprises a "D" region of about 10 or more amino acids.

The variable region of each light chain/heavy chain (VL/VH) pair forms an antibody binding site. Thus, typically an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are typically identical.

Typically, the variable regions of both the heavy and light chains comprise three hypervariable regions, also known as "Complementarity Determining Regions (CDRs)", which are located between relatively conserved Framework Regions (FR). CDRs are typically arranged by framework regions so as to be able to bind to a particular epitope. Typically, the light chain variable domain and the heavy chain variable domain comprise, in order from the N-terminus to the C-terminus, FR-1 (or FR 1), CDR-1 (or CDR 1), FR-2 (FR 2), CDR-2 (CDR 2), FR-3 (or FR 3), CDR-3 (CDR 3) and FR-4 (or FR 4). Amino acids are typically assigned to each domain according to the definition of the following immunologically interesting protein sequences, kabat, et al National Institutes of Health, bethesda, md.; 5 th edition; NIH publication No. 91-3242 (1991); kabat (1978) adv. Prot. Chem.32:1-75; kabat, et al, (1977) J.biol. Chem.252:6609-6616; chothia, et al, (1987) J mol. Biol.196:901-917 or Chothia, et al, (1989) Nature 342:878-883.

The term "hypervariable region" means the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region comprises amino acid residues from "CDRs" (i.e., VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable domain and VH-CDR1, VH-CDR2 and VH-CDR3 in the heavy chain variable domain). See Kabat et al (1991) Sequences of Proteins of Immunological Interest, public HEALTH SERVICE, national Institutes of Health, bethesda, md (CDR regions of antibodies are defined by sequence), and Chothia and Lesk (1987) J.mol. Biol.196:901-917 (CDR regions of antibodies are defined by structure). The term "framework" or "FR" residues means those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

Unless otherwise indicated, "antibody fragment" or "antigen-binding fragment" means an antigen-binding fragment of an antibody, i.e., an antibody fragment that retains the ability to specifically bind to an antigen that binds to a full-length antibody, e.g., a fragment that retains one or more CDR regions. Examples of antigen binding fragments include, but are not limited to, fab ', F (ab') 2, and Fv fragments, diabodies, linear antibodies, single chain antibody molecules such as single chain Fv (ScFv), nanobodies formed from antibody fragments, and multispecific antibodies.

Antibodies that bind specifically to a given target protein are also described as specifically binding to a particular target protein. This means that antibodies exhibit preferential binding to the target compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered "specific" for its intended target if binding of the antibody is a determinant of the presence of the target protein in the sample, e.g., does not produce undesirable results, such as false positives. Antibodies or binding fragments thereof useful in the present invention will bind to a target protein with an affinity that is at least twice, preferably at least 10-fold, more preferably at least 20-fold, most preferably at least 100-fold, the affinity for a non-target protein. An antibody herein is considered to specifically bind to a polypeptide comprising a given amino acid sequence (e.g., the amino acid sequence of a mature human TIGIT molecule) if it binds to the polypeptide but not to a protein lacking that sequence.

The expressions "pH dependent binding", "pH dependent target binding" and "pH dependent antigen binding" are interchangeable in the present disclosure, indicating that the antibodies of the application bind to their target/antigen, i.e. human TIGIT, in a pH dependent manner. In particular, antibodies of the application exhibit higher binding affinity and/or binding signal to their antigen at mildly acidic pH (e.g., pH 6.0) than at physiological pH (e.g., pH 7.4), which is typically found in tumor microenvironments. Methods for determining the binding affinity and/or binding signal strength of the antibodies of the application are well known in the art, including but not limited to surface plasmon resonance (Biacore) or similar techniques. More specifically, the antibodies of the application have a K _D ratio at pH 7.4/pH 6.0 of greater than 2,3, 4, 5,6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater as measured by surface plasmon resonance (Biacore) or similar techniques. Alternatively or additionally, the antibodies of the application have a Rmax (RU) value at pH 6.0 that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold greater than Rmax at pH 7.4, as measured by surface plasmon resonance (Biacore) or similar techniques. The binding affinity of an antibody can be measured at 25 ℃ or 37 ℃. Tumor microenvironments have been found to exhibit a relatively more acidic pH than physiological conditions or normal tissue (Zhang et al Focus on molecular Imaging 2010; tandock and Rotin et al CANCER RES 1989). Thus, the antibodies of the application having the pH-dependent binding described above are advantageous as anti-TIGIT therapeutics for selectively targeting TIGIT positive lymphocytes in a tumor microenvironment and having lower toxicity associated with peripheral activation of lymphocytes.

The term "human antibody" is intended herein to mean an antibody comprising only human immunoglobulin protein sequences. Human antibodies may contain murine carbohydrate chains if produced in mice, mouse cells, or hybridomas derived from mouse cells. Similarly, "mouse antibody" or "rat antibody" means an antibody comprising only mouse or rat immunoglobulin sequences, respectively.

The term "humanized antibody" means a form of antibody that contains sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequences derived from non-human immunoglobulins. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. When it is desired to distinguish between humanized antibodies and parent rodent antibodies, the prefix "hum", "Hu" or "h" is added to the antibody clone designation. Humanized forms of rodent antibodies will typically comprise the same CDR sequences as the parent rodent antibody, but may include certain amino acid substitutions to increase affinity, increase stability of the humanized antibody, or for other reasons.

The antibodies of the application have potential therapeutic use in the treatment of cancer. The term "cancer" or "tumor" herein means or describes a physiological condition in a mammal that is generally characterized by unregulated cell growth. Examples of cancers include, but are not limited to, lung cancer (including small cell lung cancer or non-small cell lung cancer), adrenal cancer, liver cancer, stomach cancer, cervical cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, leukemia, bladder cancer, bone cancer, brain cancer, endometrial cancer, head and neck cancer, lymphoma, ovarian cancer, skin cancer, thyroid tumor, or metastatic lesions of cancer.

Furthermore, the antibodies of the application have potential therapeutic use in controlling viral infections and other human diseases mechanically involved in immune tolerance or "exhaustion". In the context of the present application, the term "depletion" refers to the process of depleting the ability of immune cells to respond during cancer or chronic viral infection.

The term "therapeutically effective amount" as used herein refers to an amount of an antibody sufficient to affect a disease, disorder or condition when administered to a subject to treat the disease or disorder or at least one clinical symptom of the disease or disorder. The "therapeutically effective amount" may vary with the antibody, the disease, the disorder, and/or the symptoms of the disease or disorder, the disease, the disorder, and/or the severity of the symptoms of the disease or disorder, the age of the subject to be treated, and/or the weight of the subject to be treated. The appropriate amount may be readily apparent to one of ordinary skill in the art in any given situation, or may be determined by routine experimentation. In the case of combination therapies, "therapeutically effective amount" refers to the total amount of active agents contained in a combination for effective treatment of a disease, disorder, or condition.

As used herein, a "subject" is a mammal, e.g., a rodent or primate, preferably a higher primate, e.g., a human (e.g., a patient suffering from or at risk of suffering from a disorder described herein).

Anti-TIGIT antibodies

The present disclosure provides antibodies and antigen binding fragments thereof that specifically bind to human TIGIT. Furthermore, the present disclosure provides antibodies that have desirable pharmacokinetic characteristics and other desirable properties, and thus may be used to reduce the likelihood of cancer or treat cancer. The disclosure also provides pharmaceutical compositions comprising antibodies and methods of making and using such pharmaceutical compositions for the prevention and treatment of cancer and related disorders.

The present disclosure provides antibodies or antigen binding fragments thereof that specifically bind to TIGIT. Antibodies or antigen binding fragments of the present disclosure include, but are not limited to, antibodies or antigen binding fragments thereof produced as described in table 1 below.

TABLE 1A 1217 (European Perot Li Shan antibody) sequences

The present disclosure provides antibodies or antigen-binding fragments that specifically bind to TIGIT, wherein the antibodies or antibody fragments (e.g., antigen-binding fragments) comprise a VH domain with the amino acid sequence of SEQ ID No. 7. The present disclosure also provides antibodies or antigen-binding fragments that specifically bind TIGIT, wherein the antibodies or antigen-binding fragments comprise VH CDRs having the amino acid sequences of any one of the VH CDRs provided herein. In one aspect, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to TIGIT, wherein the antibody comprises (or alternatively consists of) one, two, three or more VH CDRs having the amino acid sequences of any one of the VH CDRs provided in the present disclosure.

The present disclosure provides antibodies or antigen-binding fragments that specifically bind to TIGIT, wherein the antibodies or antigen-binding fragments comprise a VL domain having the amino acid sequence of SEQ ID No. 8. The present disclosure also provides antibodies or antigen-binding fragments that specifically bind to TIGIT, wherein the antibodies or antigen-binding fragments comprise VL CDRs having the amino acid sequences of any one of the VL CDRs listed herein. In particular, the present disclosure provides antibodies or antigen-binding fragments that specifically bind to TIGIT comprising (or alternatively, consisting of) one, two, three, or more VL CDRs having the amino acid sequences of any one of the VL CDRs of the present disclosure.

Other antibodies of the disclosure, or antigen binding fragments thereof, comprise mutated amino acids, but have at least 60%, 70%, 80%, 90%, 95% or 99% percent identity in CDR regions to CDR regions depicted in the sequences described herein. In some aspects, it comprises a mutant amino acid sequence, wherein no more than 1,2, 3, 4, or 5 amino acids have been mutated in the CDR regions when compared to the CDR regions disclosed in the provided sequences.

Other antibodies of the disclosure include antibodies in which the amino acid or nucleic acid encoding the amino acid has been mutated, but have at least 60%, 70%, 80%, 90%, 95% or 99% percent identity to the sequences set forth in table 1. In some aspects, it comprises a mutated amino acid sequence, wherein no more than 1,2, 3, 4, or 5 amino acids have been mutated in the variable region when compared to the variable region depicted in the sequences described herein, while maintaining substantially the same therapeutic activity.

Further alterations of the framework of the Fc region

In other aspects, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids may be substituted with a different amino acid residue such that the antibody has an altered affinity for the effector ligand, but retains the antigen binding capacity of the parent antibody. The effector ligand for which affinity is altered may be, for example, an Fc receptor or the C1 component of complement. This method is described, for example, in both U.S. Pat. nos. 5,624,821 and 5,648,260 to Winter et al.

In another aspect, one or more amino acid residues may be replaced with one or more different amino acid residues such that the antibody has altered C1q binding and/or reduced or eliminated Complement Dependent Cytotoxicity (CDC). This method is described, for example, in U.S. Pat. No. 6,194,551 to Idusogie et al.

In another aspect, one or more amino acid residues are altered, thereby altering the ability of the antibody to fix complement. This method is described, for example, in PCT publication WO 94/29351 to Bodmer et al. In a particular aspect, for the IgG1 subclass and kappa isotype, one or more amino acids of the antibodies of the disclosure, or antigen binding fragments thereof, are replaced with one or more allotype amino acid residues. Allotype amino acid residues also include, but are not limited to, the constant regions of the heavy chains of the subclasses IgG1, igG2, and IgG3, as well as the constant regions of the light chains of the kappa isotype as described in Jefferis et al, MAbs.1:332-338 (2009).

In another aspect, the Fc region is modified by modifying one or more amino acids to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for fcγ receptors. This method is described, for example, in PCT publication WO00/42072 to Presta. In addition, binding sites for FcgammaRI, fcgammaRII, fcgammaRIII and FcRn have been mapped on human IgG1 and variants with improved binding have been described (see Shields et al, J.biol. Chem.276:6591-6604, 2001).

In another aspect, glycosylation of the antibody is modified. For example, an aglycosylated antibody (i.e., an antibody lacking or having reduced glycosylation) may be prepared. Glycosylation can be altered, for example, to increase the affinity of an antibody for an "antigen". Such carbohydrate modification may be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions may be made resulting in elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for the antigen. Such a process is described, for example, in U.S. Pat. nos. 5,714,350 and 6,350,861 to Co et al.

Additionally or alternatively, antibodies with altered glycosylation patterns, such as low fucosylation antibodies with reduced fucose residues or antibodies with increased bisecting GlcNac structure, can be prepared. Such altered glycosylation patterns have been demonstrated to increase the ADCC capacity of antibodies. Such carbohydrate modification may be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation mechanism. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant antibodies are expressed to produce antibodies with altered glycosylation. For example, EP1,176,195 to Hang et al describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase such that antibodies expressed in such a cell line exhibit low fucosylation. PCT publication WO 03/035835 to Presta describes a variant CHO cell line, lecl cells, which have a reduced ability to attach fucose to Asn (297) -linked carbohydrates, also resulting in low fucosylation of antibodies expressed in the host cells (see also Shields et al, (2002) J.biol. Chem. 277:26733-26740). PCT publication WO 99/54342 to Umana et al describes cell lines engineered to express glycoprotein modified glycosyltransferases (e.g., beta (1, 4) -N-acetylglucosamine transferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisected GlcNac structure, which results in increased ADCC activity of the antibodies (see also Umana et al, nat. Biotech.17:176-180, 1999).

On the other hand, if it is desired to reduce ADCC, many previous reports have shown that human antibody subclass IgG4 has only modest ADCC and little CDC effector function (Moore G.L. et al 2010MAbs, 2:181-189). On the other hand, native IgG4 was found to be less stable under stress conditions, such as in acidic buffers or at elevated temperatures (Angal 1993Mol Immunol,30:105-108; dall' acqua, et al, 1998biochemistry,37:9266-9273; aalbrese et al, 2002Immunol, 105:9-19). Reduced ADCC may be achieved by operably linking antibodies to IgG4 engineered with each altered combination to have reduced or ineffective fcγr binding or C1q binding activity, thereby reducing or eliminating ADCC and CDC effector function. Given the physicochemical properties of antibodies as biopharmaceuticals, one of the less desirable inherent properties of IgG4 is that its two heavy chains dynamically separate in solution to form half antibodies, which results in the production of bispecific antibodies in vivo via a process called "Fab arm exchange" (Van der Neut Kolfschoten et al, 2007science, 317:1554-157). Mutation of serine to proline at position 228 (EU numbering system) appears to be inhibitory for IgG4 heavy chain separation (Angal 1993Mol Immunol,30:105-108; aalbrese et al, 2002immunol, 105:9-19). Some amino acid residues in the hinge and γFc regions are reported to have an effect on the interaction of antibodies with the Fcγ receptor (Chappel et al, 1991Proc. Natl. Acad. Sci. USA,88:9036-9040; mukherjee et al, 1995FASEB J,9:115-119; armour et al, 1999Eur J Immunol,29:2613-2624; clynes et al, 2000Nature Medicine,6:443-446;Arnold 2007Annu Rev immunol,25:21-50). In addition, some rare occurrence of IgG4 isotypes in the human population can also cause different physicochemical properties (Brusco et al, 1998Eur J Immunogenet,25:349-55; aalbrese et al, 2002immunol, 105:9-19). To generate TIGIT antibodies with low ADCC, CDC and instability, the hinge and Fc regions of human IgG4 can be modified and a number of changes introduced. These modified IgG4 Fc molecules can be found disclosed in SEQ ID NOS: 83-88 of U.S. Pat. No.8,735,553.

Antibody production

Anti-TIGIT antibodies and antigen-binding fragments thereof may be produced by any method known in the art, including but not limited to recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers, whereas full-length monoclonal antibodies may be obtained by, for example, hybridoma or recombinant production. Recombinant expression may be from any suitable host cell known in the art, such as mammalian host cells, bacterial host cells, yeast host cells, insect host cells, and the like.

The disclosure also provides polynucleotides encoding antibodies described herein, e.g., polynucleotides encoding heavy or light chain variable regions or fragments comprising complementarity determining regions as described herein. In some aspects, the polynucleotide encoding the heavy chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity to a polynucleotide encoding the polypeptide of SEQ ID NO. 7. In some aspects, the polynucleotide encoding the light chain variable region has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity to a polynucleotide encoding the polypeptide of SEQ ID NO. 8.

The polynucleotides of the present disclosure may encode variable region sequences of anti-TIGIT antibodies. They may also encode both the variable and constant regions of an antibody. Some polynucleotide sequences encode polypeptides comprising variable regions of both the heavy and light chains of one of the exemplified anti-TIGIT antibodies. Some other polynucleotides encode two polypeptide fragments that are substantially identical to the variable regions of the heavy and light chains, respectively, of one of the murine antibodies.

The present disclosure also provides expression vectors and host cells for producing anti-TIGIT antibodies. The choice of expression vector depends on the intended host cell in which the vector is to be expressed. Typically, expression vectors contain promoters and other regulatory sequences (e.g., enhancers) operably linked to a polynucleotide encoding an anti-TIGIT antibody chain or antigen-binding fragment thereof. In some aspects, an inducible promoter is employed to prevent expression of the inserted sequence except under the control of induction conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters or heat shock promoters. Cultures of transformed organisms may be expanded under non-inducing conditions without departing from the population of coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other regulatory elements may be required or desired for efficient expression of the anti-TIGIT antibody or antigen-binding fragment thereof. These elements typically include an ATG initiation codon and adjacent ribosome binding sites or other sequences. Furthermore, expression efficiency can be enhanced by including enhancers appropriate for the cell system used (see, e.g., scharf et al, results Probl. Cell differ.20:125,1994; and Bittner et al, meth. Enzymol.,153:516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.

The host cell used to carry and express the anti-TIGIT antibody chain may be prokaryotic or eukaryotic. Coli (e.coli) is a prokaryotic host useful for cloning and expressing the polynucleotides of the present disclosure. Other suitable microbial hosts for use include bacilli, such as bacillus subtilis, and other enterobacteriaceae, such as salmonella, serratia, and various pseudomonas species. In these prokaryotic hosts, expression vectors may also be prepared, which typically contain expression control sequences (e.g., origins of replication) compatible with the host cell. In addition, there will be any number of a variety of well known promoters, such as lactose promoter system, tryptophan (trp) promoter system, beta-lactamase promoter system or promoter system from phage lambda. Promoters typically optionally control expression with operator sequences, and have ribosome binding site sequences and the like, for initiation and completion of transcription and translation. Other microorganisms, such as yeast, may also be used to express the anti-TIGIT polypeptide. Combinations of insect cells with baculovirus vectors may also be used.

In other aspects, mammalian host cells are used to express and produce the anti-TIGIT antibodies of the present disclosure. For example, they may be hybridoma cell lines expressing endogenous immunoglobulin genes or mammalian cell lines carrying exogenous expression vectors. These cells include any normal dead or normal or abnormal immortalized animal or human cells. For example, many suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various COS cell lines, HEK 293 cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue cell cultures to express polypeptides is generally discussed in, for example, winnacker, from Genes to Clones, VCH Publishers, NY, n.y., 1987. Expression vectors for mammalian host cells may include expression control sequences such as origins of replication, promoters and enhancers (see, e.g., queen et al, immunol. Rev.89:49-68,1986), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type specific, stage specific and/or regulatable. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPSV promoters, tetracycline inducible CMV promoters (such as human immediate early CMV promoters), constitutive CMV promoters, and promoter-enhancer combinations known in the art.

Detection and diagnostic methods

The antibodies or antigen binding fragments of the present disclosure may be used in a variety of applications, including but not limited to methods for detecting TIGIT. In one aspect, the antibody or antigen binding fragment can be used to detect the presence of TIGIT in a biological sample. The term "detection" as used herein includes quantitative or qualitative detection. In certain aspects, the biological sample comprises a cell or tissue. In other aspects, such tissues include normal and/or cancerous tissues that express TIGIT at higher levels relative to other tissues.

In one aspect, the present disclosure provides a method of detecting the presence of TIGIT in a biological sample. In certain aspects, the method comprises contacting the biological sample with an anti-TIGIT antibody under conditions that allow the antibody to bind to the antigen, and detecting whether a complex is formed between the antibody and the antigen. Biological samples may include, but are not limited to, urine or blood samples.

Also included are methods of diagnosing disorders associated with TIGIT expression. In certain aspects, the methods comprise contacting a test cell with an anti-TIGIT antibody, determining the expression level of TIGIT in the test cell by detecting binding of the anti-TIGIT antibody to a TIGIT polypeptide (quantitative or qualitative), and comparing the expression level in the test cell to the expression level of TIGIT in a control cell (e.g., a normal cell or a non-TIGIT expressing cell having the same tissue origin as the test cell), wherein a higher TIGIT expression level in the test cell compared to the control cell indicates the presence of a disorder associated with TIGIT expression.

Therapeutic method

The antibodies or antigen binding fragments of the present disclosure may be used in a variety of applications, including but not limited to methods for treating TIGIT-related disorders or diseases. In one aspect, the TIGIT-related disorder or disease is cancer.

In one aspect, the present disclosure provides a method of treating cancer. In certain aspects, the method comprises administering to a patient in need thereof an effective amount of an anti-TIGIT antibody or antigen-binding fragment. Cancers may include, but are not limited to, breast cancer, head and neck cancer, gastric cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, ovarian cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, and sarcoma.

The antibodies or antigen binding fragments of the invention may be administered by any suitable means, including parenteral, intrapulmonary and intranasal administration, and if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing regimens including, but not limited to, single or multiple administrations, bolus administrations, and pulse infusion at various points in time are contemplated herein.

The antibodies or antigen binding fragments of the invention will be formulated, administered and administered in a manner consistent with good medical practice. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the time of administration, and other factors known to practitioners. Antibodies need not be, but are optionally formulated with one or more agents currently used to prevent or treat the condition in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dose and with the route of administration as described herein, or about 1% to 99% of the dose described herein, or at any dose and by any route empirically/clinically determined to be appropriate.

For the prevention or treatment of a disease, the appropriate dosage of the antibodies or antigen-binding fragments of the invention will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapies, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitable for administration to a patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 100mg/kg of antibody may be the initial candidate dose for administration to the patient, whether by one or more separate administrations, or by continuous infusion, for example. A typical daily dosage range may be about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the conditions, the treatment will generally be maintained until the desired inhibition of disease symptoms occurs. Such doses may be administered intermittently, e.g., weekly or every three weeks (e.g., such that the patient receives about two to about twenty, or e.g., about six doses of antibody). An initial higher loading dose may be administered followed by one or more lower doses. However, other dosage regimens are also useful. The progress of this treatment is readily monitored by conventional techniques and assays.

Combination therapy

In one aspect, TIGIT antibodies of the disclosure may be used in combination with other therapeutic agents, such as anti-PD 1 antibodies. Other therapeutic agents that may be used with TIGIT antibodies of the present disclosure include, but are not limited to, chemotherapeutic agents (e.g., paclitaxel or paclitaxel agents, (e.g.,) Docetaxel; carboplatin, topotecan, cisplatin, irinotecan, doxorubicin, lenalidomide, 5-azacytidine, ifosfamide, oxaliplatin, pemetrexed disodium, cyclophosphamide, etoposide, decitabine, fludarabine, vincristine, bendamustine, chlorambucil, busulfan, gemcitabine, melphalan, pravastatin, mitoxantrone, pemetrexed disodium), tyrosine kinase inhibitors (e.g., EGFR inhibitors (e.g., erlotinib), multi-kinase inhibitors (e.g., MGCD265, RGB-286638), CD-20 targets (e.g., rituximab, ofatuzumab, RO5072759, LFB-R603), CD52 targets (e.g., alemtuzumab), prednisolone, albendamustine, lenalidomide, bcl-2 inhibitors (e.g., sodium), aurora kinase inhibitors (e.g., N8237, TAK proteins), TAK inhibitors (e.g., CB-1, such as well as inhibitors, e.g., mFabryurt) 2), inhibitors (e.g., mFabryurt) inhibitors (e.g., mK-35), mF-35, such as inhibitors (e.g., mF-35), mF-35, F-20, such as inhibitors (e.g., mF-35) as inhibitors, BI 672).

TIGIT antibodies of the present disclosure may be used in combination with other therapeutic agents, such as anti-PD 1 antibodies. anti-PD 1 antibodies may include, but are not limited to, tirelimumab, pembrolizumab, or nivolumab. Tirelimumab is disclosed in US 8,735,553 and in table 2 below.

Table 2. -tirelib bead mab sequences

Pembrolizumab (previously referred to as MK-3475) as disclosed by Merck in US 8,354,509 and US 8,900,587 is a humanized lgG4-K immunoglobulin that targets the PD1 receptor and inhibits the binding of the PD1 receptor ligands PD-L1 and PD-L2. Pembrolizumab has been approved for the indications of metastatic melanoma and metastatic non-small cell lung cancer (NSCLC), and is being used in clinical studies to treat Head and Neck Squamous Cell Carcinoma (HNSCC) and refractory hodgkin's lymphoma (cHL). Nawuzumab (as disclosed by Bristol-Meyers Squibb) is a fully human lgG4-K monoclonal antibody. Nivolumab (clone 5C 4) is disclosed in U.S. Pat. No. 8,008,449 and WO 2006/121168. Nivolumab is approved for the treatment of melanoma, lung cancer, renal cancer, and hodgkin's lymphoma.

Pharmaceutical composition and formulation

Also provided are compositions, including pharmaceutical formulations, comprising an anti-TIGIT antibody or antigen-binding fragment, or a polynucleotide comprising a sequence encoding an anti-TIGIT antibody or antigen-binding fragment. In certain embodiments, the composition comprises one or more antibodies or antigen binding fragments that bind to TIGIT, or one or more polynucleotides comprising sequences encoding one or more antibodies or antigen binding fragments that bind to TIGIT. These compositions may also comprise suitable carriers, such as pharmaceutically acceptable excipients, including buffers well known in the art.

Pharmaceutical formulations of TIGIT antibodies or antigen-binding fragments as described herein are prepared by mixing such antibodies or antigen-binding fragments of the desired purity with one or more optional pharmaceutically acceptable carriers in the form of lyophilized formulations or aqueous solutions (Remington' sPharmaceutical Sciences th edition, osol, editions (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethyldiammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, or benzyl alcohol, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, sugar, or sorbitol, salt forming ions such as sodium, metal complexes (e.g., zn-and/or non-complexing agents such as PEG).

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations comprising histidine-acetate buffer.

Can be prepared into sustained release (Sustained-release) preparation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

Formulations to be used for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

Pharmaceutical compositions and kits

In some aspects, the present disclosure provides compositions, e.g., pharmaceutically acceptable compositions, comprising an anti-TIGIT antibody described herein formulated with at least one pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, isotonic and absorption delaying agents and the like that are physiologically compatible. The vehicle may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., by injection or infusion).

The compositions herein may take a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The appropriate form depends on the intended mode of administration and therapeutic application. Typical suitable compositions are in the form of injectable or infusible solutions. One suitable mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In some embodiments, the antibody is administered by intravenous infusion or injection. In certain embodiments, the antibody is administered by intramuscular or subcutaneous injection.

Examples

EXAMPLE 1 production of anti-TIGIT monoclonal antibodies

Anti-TIGIT monoclonal antibodies (mabs) were generated based on minor modifications of conventional hybridoma fusion techniques (de St Groth and SHEIDEGGER,1980J Immunol Methods 35:1;Mechetner,2007Methods Mol Biol 378:1). Mabs with high binding activity in enzyme-linked immunosorbent assay (ELISA) and Fluorescence Activated Cell Sorting (FACS) assays were selected for further characterization.

Cloning and sequence analysis of TIGIT antibodies

Murine hybridoma clones were harvested to prepare total cellular RNA using the Ultrapure RNA kit (catalog number 74104, QIAGEN, germany) according to the manufacturer's protocol. The 1 st strand cDNA was synthesized using a cDNA synthesis kit (catalog No. 18080-051) from Invitrogen, and PCR amplification of nucleotide sequences encoding the heavy chain variable region (Vh) and the kappa chain variable region (Vk) of the murine mAb was performed using a PCR kit (catalog No. CW0686, CWBio, beijing, china). The oligonucleotide primers for antibody cDNA clones of Vh and Vk were synthesized by Invitrogen (Beijing, china) based on previously reported sequences (Brocks et al, 2001Mol Med 7:461). The PCR product was then subcloned into a pEASY-Blunt cloning vector (catalog number C B-101-02, transGen, china) and sequenced by Genewiz (Beijing, china). The amino acid sequences of the Vh region and the Vk region were deduced from the DNA sequencing results. Mu1217 was identified as a specific clone of interest.

Humanization of murine anti-human TIGIT mAb mu1217

For humanization of mu1217, sequences with high homology to the cDNA sequence of the mu1217 variable region in the human germline IgG gene were searched by comparison with the human immunoglobulin gene database in IMGT. Human IGVH and human IGVH were selected to be present at high frequency in the human antibody repertoire (GLANVILLE et al, PNAS106:20216-20221 2009) and to be highly homologous to mu1217The gene is used as a humanized template.

Humanization was performed by CDR-grafting (Methods in Molecular Biology, volume 248: antibody Engineering, methods and Protocols, humana Press) and humanized antibody (A1217) was engineered into human IgG1MF form using an internally developed expression vector, and the sequences of the antibodies are shown in Table 1.

Example 2 binding Activity, structure and function of A1217 on TIGIT

To better understand how the a1217 antibody can have high affinity for TIGIT, robust blocking of TIGIT-PVR interactions, and in particular pH-dependent binding to TIGIT, while exhibiting pharmacokinetic and molecular evaluation properties, the crystal structure of a1217 complexed with TIGIT is determined as described in detail below. Mutagenesis experiments of TIGIT interface were also performed to identify functional epitope residues, especially HIS76 for pH dependent binding of TIGIT.

TIGIT and Fab expression, purification and crystallization

Human TIGIT residues 23-128 with a C-terminal HIS tag were expressed as inclusion bodies in e.coli BL21 (DE 3) pLysS using pET21a vector (Novagen). Site-directed mutagenesis of TIGIT was introduced using Q5 DNA polymerase (NEW ENGLAND Biolabs) using a QuickChange-based program (Xia et al, nucleic Acids Res,2015.43 (2): page e 12). Protein expression in BL21 (DE 3) pLysS host strain was induced with 1mM IPTG at OD600 of 0.6-1.0 for 4h at 37 ℃. Cells were collected by centrifugation and resuspended in lysis buffer (50 mM sodium phosphate pH 7.0, 300mM sodium chloride). Cells were lysed under sonication on ice. The inclusion bodies were recovered by centrifugation (at 20,000rpm for 30min at 4 ℃) and dissolved in 8M urea, 20mM Tris pH 8.0, 200mM NaCl, 1mM DTT followed by stirring overnight. After removal of undissolved precipitate by centrifugation (30 min at 20,000rpm at 4 ℃) the dissolved fraction was applied to a Ni-Penta ^TM affinity column (MARVELGENT BIOSCIENCES inc.) and washed with 10 column volumes of wash buffer (8M urea, 20mM Tris pH 8.0, 200mM NaCl, 5mM imidazole). The proteins were then eluted with elution buffer (8M urea, 20mM Tris pH 8.0, 200mM NaCl, 200mM imidazole). Eluted proteins were refolded by dialysis against buffer containing 20mM Tris pH 8.0, 200mM NaCl, 0.4M L-arginine, 1mM oxidized glutathione, 5mM reduced glutathione and further purified by gel filtration in buffer (20 mM Tris pH 8.0, 100mM NaCl) using a HiLoad 16/600Superdex ^TM pg column (GE HEALTHCARE LIFE SCIENCES). TIGIT mutants were purified similarly to the wild type protein.

The DNA sequence of Fab fragment of a1217 was synthesized in mammalian cells by codon optimization. The heavy and light chain sequences of Fab were cloned into pMAX vectors with the C-terminal 6xHIS tag of the heavy chain, respectively. Plasmids carrying the heavy and light chains of Fab were transiently co-transfected into HEK293G cells for protein expression. The supernatant containing secreted Fab was purified by TALON affinity resin (Clontech Laboratories) followed by further purification using HiLoad 16/600Superdex ^TM 75pg column (GE HEALTHCARE LIFE SCIENCES). Similarly, plasmids of full length heavy and light chains of a1217 were transiently co-transfected into HEK293G cells for protein expression. The full length antibody was purified by Mab Select SuRe ^TM affinity resin (GE HEALTHCARE) followed by further polishing with a HiLoad 16/600Superdex ^TM 200pg column (GE HEALTHCARE LIFE SCIENCES) (polished).

The Fab of A1217 was concentrated to about 10mg/ml in 20mM Tris pH 8.0, 100mM NaCl for initial crystallization screening. The A1217Fab was incubated with a 1.5 molar excess of TIGIT on ice for 30 minutes and purified by Superdex ^TM 75Increate 10/300GL column (GE HEALTHCARE) in 20mM Tris pH 8.0, 100mM NaCl. The combined fractions were concentrated to about 10mg/ml and used for initial crystal screening. Crystals of A1217Fab/TIGIT were grown in 0.1M citrate pH4.6, 1M lithium chloride, 7% PEG 6000. Crystals that were freeze-protected with stepwise 5% glycerol to a final 20% concentration were flash frozen in liquid nitrogen. X-ray diffraction data were collected at beam line BL45XU of Spring-8 (Japan Synchrotron Radiation Research Institute). For comparison, tirelimumab Fab was produced by a similar method.

Data collection and structure parsing

Diffraction data for the A1217 Fab/TIGIT and tirelimumab Fab/TIGIT complexes were collected in BL45XU of Spring-8 (Japan) using an automated data collection system ZOO (Hirata et al, acta Crystallogr D Struct Biol,2019.75 (Pt 2): pages 138-150) and processed by KAMO addition (Yamashita et al, acta Crystallographica Section D, structural biology,2018.74 (Pt 5): pages 441-449). Molecular substitution using PHASER (McCoy et al J Appl Crystallogr,2007.40 (Pt 4): pages 658-674) employs a rigid body search model from internally resolved Fab and TIGIT (PDB: 3 UCR). The structure refinement was performed using the programs REFMAC (Murshudov et al, acta Crystallogr DBiol Crystallogr,1997.53 (Pt 3): pages 240-55) and PHENIX (Adams et al, acta Crystallogr D Biol Crystallogr,2010.66 (Pt 2): pages 213-21) and manual model building in COOT (Emsley et al, acta Crystallogr D Biol Crystallogr,2004.60 (Pt 12Pt 1): pages 2126-32). The crystallographic data statistics are summarized in table 3. All molecular patterns were made by pyromol (Schrodinger, LLC, the PyMOL Molecular GRAPHICS SYSTEM, version 1.8.2015).

TABLE 3 data collection and refinement statistics

^a The values in brackets are the values of the highest resolution shell.

^b Calculated from about 5% of the reflections left during refinement

^c R.m.s.d., root mean square deviation

Example 3 Structure of A1217 binding to human TIGIT

A1217 Fab complexed with TIGIT crystallized in the P2 21 21 space group, had four complexes in asymmetric units, and was diffracted toThe overlap of four individual complexes in an asymmetric unit shows only minor variations in backbone positioning between the individual copies. The structure of a1217 (fig. 1A) in combination with human TIGIT shows the spatial interface of a1217 in combination with PVR (fig. 1B). The buried surface area between A1217 and TIGIT is aboutThe TIGIT epitope of a1217 consists of a number of discrete regions. A1217 Eighteen residues (paratopes) of Fab and fifteen residues (epitopes) of TIGIT are involved in paratope-epitope formation (fig. 1C). The interaction at the a1217/TIGIT interface is primarily nonpolar in nature, with a total of eleven hydrogen bonds and three salt bridges. The paratope of A1217 is composed of TYR33 of HCDR1, THR52, LYS53, GLY54, GLY56, SER57, TYR59 of HCDR2, ASN101, TYR102, ASP103, PHE104 of HCDR3, THR31, SER32 of LCDR1, TYR49, TRP50 of LCDR2, TYR91, SER92, TYR94 of LCDR3 (FIG. 1C). The A1217 epitope of TIGIT contains GLN56, GLU60, ASP63, GLN64, LEU65, ILE68, ASN70, LEU73, GLY74, TRP75, HIS76, SER78, PRO79, SER80, LYS82 (FIG. 1C). The hydrogen bond between TIGIT and a1217 involves the side chain atom of GLN56_TIGIT、ASP63_TIGIT、ASN70_TIGIT、HIS76_TIGIT、SER80_TIGIT、LYS82_TIGIT、TYR33_HCDR1、THR52_HCDR2、ASP103_HCR3、THR31_LCDR1、TRP50_LCDR2、SER92_LCDR3、TYR94_LCDR3 and the main chain atom of LEU65_TIGIT、LEU73_TIGIT、LYS53_HCDR2、GLY54_HCDR2、GLY56_HCDR2、ASN101_HCDR3、TYR102_HCDR3, whereas two salt bridges are formed between HIS76 _TIGIT and ASP103 _HCDR3, and one is found between GLU60 _TIGIT and LYS53 _HCDR2. Furthermore, residues of TIGIT that are involved in only van der waals interactions are GLN64, ILE68, GLY74, TRP75, SER78, PRO79, while residues of A1217 that are involved in these interactions are heavy chain SER57, TYR59, PHE104, light chain SER32, TYR49, TYR91.

The crystal structure of tirelimumab was also produced by a method similar to that described above. Epitope mapping of tirelimumab Fab to TIGIT shows a number of break-off regions. Thirteen residues (paratopes) and ten residues (epitopes) of TIGIT of tirizumab Fab are involved in paratope formation withIs used for calculating the cut-off distance of the (C). The paratope of tirelimumab Fab consists of ARG56, PHE57, LYS58 and TYR60 of HCDR2, TYR106, ASP107, LEU108 and LEU109 of HCDR3, TYR31 and TYR38 of LCDR1, and TYR98, SER99 and THR100 of LCDR 3. Thus, HCDR1 and LCDR2 of tirelib monoclonal antibody Fab are not directly involved in epitope-paratope interactions. The tirelib bead mab epitope consists of GLN56, ASN58, GLU60, HIS76, ILE77, SER78, PRO79, SER80, LYS82 and HIS 111. eight hydrogen bonds, four salt bridges and van der waals forces contribute to the formation of the bonding interface. The hydrogen bond between TIGIT and tirelimumab Fab involves the side chain atoms ASN58_TIGIT、GLU60_TIGIT、HIS76_TIGIT、SER80_TIGIT、LYS82_TIGIT、LYS58_HCDR2、TYR60_HCDR2、ASP107_HCDR3 and THR100 _LCDR3 and the main chain atoms PRO79 _TIGIT、ARG56_HCDR2 and THR100 _LCDR3, whereas two salt bridges are formed between GLU60 _TIGIT and LYS58 _HCDR2, two salt bridges are formed between LYS82 _TIGIT and ASP107 _HCDR3, and one salt bridge is formed between HIS76 _TIGIT and ASP107 _HCDR3. Furthermore, residues of TIGIT that are involved in only van der waals interactions are GLN56, ILE77, SER78 and HIS111, while residues of tirelimumab Fab that are involved in this interaction are PHE57, TYR106, LEU108 and LEU109 of the heavy chain and TYR31, TYR38, TYR98 and SER99 of the light chain. Amino acids ARG56, PHE57, LYS58 and TYR60 of HCDR2 in Tirilizumab Fab cover the exposed hydrophobic surfaces formed by THR55, GLN56, ASN58, GLU60, ASP63, GLN64, LEU65, ALA67, ILE68, ILE109 and HIS111 of TIGIT. LEU109 of HCDR3 in tirelimumab Fab was inserted into the hydrophobic pocket formed by ALA67, ILE68, HIS76, ILE77 and SER78 of TIGIT. A1217 Comparative data between interactions of (osprey Li Shan antibody) and tirelimumab with TIGIT are shown in figures 2A-C and 3A-B.

Based on the crystal structure of the a1217/TIGIT complex, residues of TIGIT that contact a1217 (i.e., epitope residues of TIGIT that bind a 1217) and residues of a1217 that contact TIGIT (i.e., paratope residues of a1217 that contact TIGIT) were determined. Tables 4 and 5 below show the residues of TIGIT and the light or heavy chain residues of a1217 to which they are exposed, e.g., usingIs evaluated by the contact distance severity of (c), the van der waals (nonpolar) interaction forces are highest at this point.

TABLE 4 epitope residues of TIGIT and corresponding paratope residues on the light chain of A1217

TIGIT	Residue numbering	A1217 light chain	Residue numbering
				ILE	68	TRP	50
ASN	70	TRP	50
				LEU	73	TYR	49
		TRP	50
				GLY	74	TRP	50
TRP	75	TRP	50
				HIS	76	THR	31
		SER	32
						TRP	50
PRO	79	TYR	91
						SER	92
SER	80	TYR	94
				LYS	82	SER	92

TABLE 5 epitope residues of TIGIT and corresponding paratope residues on the heavy chain of A1217

TIGIT	Residue numbering	A1217 heavy chain	Residue numbering
				GLN	56	ASN	101
		TYR	102
				GLU	60	LYS	53
ASP	63	TYR	33
						THR	52
		GLY	54
						GLY	56
GLN	64	TYR	33
						SER	57
LEU	65	TYR	33
				ILE	68	ASP	103
ASN	70	TYR	102
				HIS	76	ASP	103
		PHE	104
				SER	78	PHE	104
SER	80	TYR	59

Alanine scans of the human TIGIT interface were also performed, in which the alanine mutations of TIGIT residues were performed on LEU73/HIS76, ILE68, ASP63, LEU73, PRO79, LEU65, gla 56, ASN70, GLU60, LYS82, SER80, gla 64 (table 6). The capture immobilization strategy in SPR was used to test the binding kinetics of a1217 to TIGIT variants. In this experiment, mutations at TIGIT residues LEU73/HIS76, ILE68 reduced a1217 binding to less than a factor of 10 (table 6), and HIS76 mutation almost abrogated a1217 binding, indicating that this residue is critical for interaction. The LEU73A/HIS76A double mutant completely abrogated a1217 binding, consistent with structural observations that LEU73 and HIS76 of TIGIT sandwich TRP50 of LCDR2 with strong hydrophobic interactions and are supplemented with several hydrogen bonds (H76 _TIGIT-D103_HCDR3、H76_TIGIT-T31_LCDR1、L73_TIGIT-W50_LCDR2) and salt bridges (H76 _TIGIT-D103_HCDR3). The LEU73A mutation in TIGIT has a similar association rate with WT but a slightly faster dissociation rate, resulting in a decrease in binding affinity to a1217 to about 7-fold. Furthermore, ILE68A mutations in TIGIT have similar association rates compared to WTs, but dissociate much faster, resulting in a decrease in binding affinity to a1217 to about 30-fold. The complex structure reveals that ILE68 of TIGIT is located in the hydrophobic core of the IgV domain and interacts extensively with HCDR3 of a 1217. In addition, the ASP63A mutation showed a reduction in binding affinity to a1217 to about 7-fold, as ASP63 of TIGIT formed three hydrogen bonds with THR52, GLY54, and GLY56 of HCDR2 in a 1217. Furthermore, PRO79A mutations in TIGIT have a slower association rate and faster dissociation rate than WT, resulting in binding affinity to a1217 as low as about 3-fold. The composite structure suggests that PRO79 of TIGIT forms a strong hydrophobic interaction with PHE104 of HCDR3 and TYR91, SER92 and TYR94 of LCDR3 in A1217. Mutations in TIGIT at SER80, GLN64 did not affect a1217 binding (table 6). This analysis is consistent with a crystal structure analysis, in which TIGIT residues that most affect a1217 binding were found to interact with a1217 in the structure.

For tirelimumab interactions, the LYS82A mutant, which has a broad hydrophobic interaction with TYR31/TYR 38/TYR 98 of the light chain and forms a strong hydrogen bond/salt bridge with ASP107 of the heavy chain, completely abrogates tirelimumab binding. ILE68A mutants formed strong hydrophobic interactions with LEU108 and LEU109 of HCDR3 in tirelimumab, resulting in binding affinities to tirelimumab as low as about 40-fold (table 6), a site that also affected a1217 binding. The LEU65A mutation in TIGIT resulted in binding affinity to tirelimumab as low as about 25-fold, resulting in a slower association rate and faster dissociation rate compared to WT TIGIT. The HIS76A mutant had a slight effect on tirelimumab binding, whereas the mutation almost abrogated a1217 binding. Similarly, the P79A/S80A single mutant, which forms hydrogen bonds with THR100, had only a slight effect on tirelizumab binding. Mutagenesis experiments showed that a1217 and tirelimumab have different epitopes when bound to TIGIT, meaning that the antagonism mechanism of the two antibodies may be different.

TABLE 6 kinetics of binding of tigit to A1217 and tirelizumab

^a HB/SB represents hydrogen bonds and salt bridges, respectively.

A1217 has the property of pH-dependent binding to TIGIT

Based on our SPR data, we observed an approximately 17-fold increase in binding affinity (KD) for TIGIT for a1217 when pH was reduced from 7.4 to 6.0 (table 7). The binding affinity between a1217 and TIGIT was measured using an amine coupling method at different pH. In our composite structure, ASP103 _HCDR3 faces HIS76 _TIGIT toThis forms a strong electrostatic interaction (fig. 1D). In tumor microenvironments, the imidazole ring of HIS76 _TIGIT may be mostly protonated and carry a positive charge in this acidic environment, which enhances electrostatic interactions with ASP103 _HCDR3. However, under physiological conditions, HIS76 _TIGIT will carry a less positive charge and attenuate this interaction. In the A1217/TIGIT composite structure, ASP103 _HCDR3 is arranged relative to HIS76 _TIGIT Is well positioned and forms a strong electrostatic interaction, while such an advantageous structure surrounding HIS76 _TIGIT is not observed in tirelimumab/TIGIT. We found that when pH was reduced from 7.4 to 6.0, the binding affinity (KD) of a1217 to TIGIT increased approximately 17-fold, whereas tirelizumab showed no significant pH-dependent binding to TIGIT (table 7). In summary, pH sensitive antibodies can better alleviate the balance between efficacy on tumor cells and safety on normal cells than non-pH sensitive antibodies.

TABLE 7 kinetics of binding of TIGIT to tirelizumab at different pH compared to A1217

Example 4 production of reduced fucosylated anti-TIGIT antibodies

Removal of core fucose from N-glycans attached to human IgG1 significantly enhanced the antibody-dependent cellular cytotoxicity (ADCC) response (Shields, et al, (2002) J Biol Chem277, 26733-26740); shinkawa et al, (2003) J Biol Chem 278, 3466-3473). There are many ways to reduce core fucosylation. The work presented herein utilizes a method of fucosyl transferase (FUT) inhibitor 2F-holoacetose.

A1217 P5-2 3G11 is a stably transfected CHO-K1 cell line expressing the A1217 antibody. A1217P 5-2 3G11 cells were cultured in shake flasks, maintained at passage every 3-4 days, and inoculated at 5X10 ⁵ cells/mL in Hyclone ActiPro ^TM medium with or without the addition of 1.25% Glycosylation Adjust (Gal+) (Sigma-Aldrich, 14701C 0). 2F-holoacetofucose was added to the cultures at concentrations of 0, 50, 100, 150, 200. Mu.M. Cell supernatants were harvested on day 14 and filtered through a 0.2 μm filter for further analysis.

The a1217 antibody was purified under platform conditions using a protein a chromatographic capture step and glycan profile was analyzed by HILIC-UPLC oligosaccharides (fig. 4). Glycan analysis showed that the inhibitors reduced total fucosylation to 11% at 50. Mu.M 2F-holoacetose and to less than 7% at 100, 150 or 200. Mu.M 2F-holoacetose.

Example 5 production of anti-TIGIT effector variants

The combination of Fc mutations of S239D, I332E or S239D, I E, A330L (EU numbering) was produced on a plasmid containing a DNA fragment encoding the heavy chain of A1217 according to the method provided by Fast MultiSite Mutagenesis System ^TM (FM 201-01,Transgen biotech). Variant A1217DE contains the amino acid change S239D, I E (DE). Variant A1217DEL contains the amino acid change S239D, I332,332, 332E, A330L (DEL). The Expi-CHO cells were transfected with the corresponding plasmids of the A1217 effector variants (A1217 DE or A1217 DEL) and cultured at 30℃and 5% CO2 for 10 days. The supernatant was then harvested and the protein purified using a MabSelect SuRe ^TM (17543802, cytova).

Example 6 binding affinity of tigit antibodies to FcgammaR

To determine fcyri binding, protein a was coupled to an activated CM5 biosensor chip (catalog No. BR100530, GE LIFE SCIENCES), and 30nM of a1217 or a1217 variant was flowed through the chip and captured by protein a. A range of concentrations of fcyri (from 0.0586nM to 15 nM) were injected into SPR running buffer (10 mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween20, ph 7.4) at 30 μl/min. Binding response of a1217 or mutant to fcyri was calculated by subtracting RU from the reference flow cell without injection of a1217 or mutant. To determine fcyri binding affinity, kon and koff were calculated using a one-to-one Langmuir binding model and KD was calculated as the ratio of koff/kon.

Anti-human kappa antibodies were coupled to activated CM5 biosensor chips (catalog No. BR100530, GE LIFE SCIENCES), allowing a1217 or a1217 variant to flow through the chip and be captured by the anti-human kappa surface. A series of concentrations of different FcgammaR (FcgammaRIIA, fcgammaRIIB, fcgammaRIIIA) were injected at 30 μl/min into SPR running buffer (10 mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween20, pH 7.4). Binding response of a1217 or mutant to fcγr was calculated by subtracting RU from the reference flow cell without injection of a1217 or mutant. To determine fcyriia, fcyriib, and fcyriiia binding affinities, steady state affinity models were used to fit data from all concentrations.

As shown in Table 8, A1217AF showed an approximately 10-fold increase in binding affinity to FcgammaRIIIA-V158 and FcgammaRIIIA-F158 over A1217. A1217DEL and A1217DE also increased binding affinity to FcgammaRIIIA-V158 and FcgammaRIIIA-F158 compared to A1217. In addition, a1217DEL and a1217DE increased binding affinity to fcγri and fcγrii.

TABLE 8 binding affinity of TIGIT antibodies to FcgammaR as measured by SPR

Example 7 binding of reduced fucosylated TIGIT antibodies to FcgammaRIII

HEK293 cells overexpressing fcyriiia-V158 or fcyriiia-F158 were first incubated with a1217 or a1217AF followed by staining with secondary antibody Alexa Fluor ^TM 647 anti-human IgG Fc, bioleged, ref# 409320). Cell samples were washed and fixed with 1% paraformaldehyde in DPBS. UsingImmunofluorescence was detected by flow cytometry (ACEA) and analyzed using the Guava Soft ^TM.1.1 software. The results of this analysis are shown in fig. 5A-B. A1217AF showed increased binding to HEK293 cells overexpressing FcgammaRIIIA-V158 or FcgammaRIIIA-F158 compared to A1217, indicating enhanced Fc effector function of A1217AF by both variants of FcgammaRIIIA compared to A1217.

Example 8 binding of anti-TIGIT Effector variants to FcgammaRIII

HEK293 cells overexpressing FcgammaRIIIA-V158 or FcgammaRIIIA-F158 were first incubated with A1217, A1217DE or A1217DEL followed by the secondary antibody AlexaThe 488F (ab ') ₂ fragment goat anti-human IgG (F (ab') ₂ fragment specific, jackson ImmunoResearch, catalog number 109-546-097) was stained. Cell samples were washed and fixed with 1% paraformaldehyde in DPBS. Immunofluorescence was detected using the Guava easyCyte ^TM HT (Merck-Millipore, usa) and analyzed using the Guava Soft ^TM 3.1.1 software. The results are shown in fig. 6A-D, a1217DE and a1217DEL showed increased binding to HEK293 cells overexpressing fcyriiia-V158 or fcyriiia-F158 compared to wild-type a1217, indicating enhanced Fc effector function of a1217DE and a1217DEL through both variants of fcyriiia compared to a 1217.

Example 9 binding of anti-TIGIT effector variants to TIGIT

BW5147.3 cells overexpressing TIGIT were first incubated with A1217, A1217DE or A1217DEL followed by the secondary antibodies AlexaThe 488F (ab ') ₂ fragment goat anti-human IgG [ F (ab') ₂ fragment specific, jackson ImmunoResearch, catalog number 109-546-097 ]. Cell samples were washed and fixed with 1% paraformaldehyde in DPBS. Immunofluorescence was detected using the Guava easyCyte ^TM HT (Merck-Millipore, USA) and analyzed using the Guava Soft 3.1.1 software. This data is shown in fig. 7. In this result, a1217DE and a1217DEL showed comparable binding to HEK293 cells overexpressing TIGIT, thus indicating that the deletion did not produce a change in TIGIT binding via FACS.

Example 10 binding of reduced fucosylated anti-TIGIT antibodies to complement C1q

The C1q binding activity of a1217 or a1217AF was determined by sandwich ELISA. Briefly, serial dilutions of either the a1217 or a1217AF variants shown were plated on MaxiSorp immune plates. C1q binding was tested by incubating human C1q with antibody coated wells. After washing, bound C1q was detected with anti-C1 q monoclonal antibody, followed by HRP conjugated secondary antibody. The binding signal was measured by absorbance at 450nm using TMB (3, 3', 5' -tetramethylbenzidine) substrate. The results are shown in fig. 8, where both a1217AF and a1217 show comparable binding to C1 q.

Example 11 anti-TIGIT antibodies with reduced fucosylation and effector variants with enhanced ADCC

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism used to kill target cells. The antibody binds to a target antigen on the surface of a target cell. When the Fc portion of the target binding antibody also binds to fcγriiia receptors on the cell surface of effector cells such as NK cells, cross-linking and activation of fcγriiia results in ADCC, hogarth and pietercz, (2012) Nat Rev Drug Discov, 311-331. The human fcyriiia gene exhibits a binary property at the position encoding amino acid residue 158. Fcyriiia variants with valine (V158) at amino acid residue 158 have high affinity for IgG1 Fc portions, and fcyriiia variants with phenylalanine (F158) have lower affinity for IgG1 Fc portions. NK cell killing of target cells is often used as a readout of ADCC activity in classical ADCC assays. These cells can be highly variable in response due to variability of different donors and polymorphism of fcyriiia. To use fcyriiia activation as an alternative readout for ADCC, we monitored fcyriiia activation in a more simplified and stable setting. Briefly, in an alternative ADCC assay, jurkat cells are engineered to stably express fcyriiia-V158 or fcyriiia-F158 variants, and NFAT response elements (NFAT-reporter-luciferase) that drive firefly luciferase expression. The resulting Jurkat/NFAT-reporter-luciferase/FcgammaRIIIA-V158 or Jurkat/NFAT-reporter-luciferase/FcgammaRIIIA-F158 cells were used as effector cells. BW5147.3 cells engineered to stably express TIGIT (BW 5147.3/TIGIT cells) were used as target cells. The biological activity of the antibodies in the surrogate ADCC assay was quantified by luciferase as a result of NFAT pathway activation. Luminescence readings are used to quantify luciferase activity in effector cells.

(A) Jurkat/NFAT-reporter-luciferase/FcgammaRIIIA-V158 cells (1X 10 ⁴ cells/well) were co-cultured overnight as effector cells with BW5147.3/TIGIT cells (1X 10 ⁴ cells/well). NFAT reporter activity was detected using One-Step ^TM luciferase assay system BPS Bioscience.

This experiment shows that a1217AF, a1217DEL and a1217DE have enhanced ADCC activity when compared to the a1217 wild type (fig. 9A).

(B) Jurkat/NFAT-reporter-luciferase/FcgammaRIIIA-F158 cells (5 x10 ⁴ cells/well) were co-cultured overnight with BW5147.3/TIGIT cells (1 x10 ⁴ cells/well). NFAT reporter activity was detected using One-glo ^TM luciferase assay system (Promega). A1217AF, A1217DEL and A1217DE had enhanced ADCC activity when compared to the A1217 wild type (FIG. 9B).

Example 12 reduced fucosylation against TIGIT and effector variants with enhanced ADCC in Treg

TIGIT ⁺ tregs represent a functionally diverse subset of tregs with high immune suppression (Joller et al, (2014) Immunity 40, 569-581)). Since TIGIT expression on intratumoral tregs is higher than on effector T cells and PBMC-derived tregs on cancer patients are higher than on PBMC-derived tregs in healthy donors (Preillon et al, (2021) Mol CANCER THER, 121-131.), it is reasonably speculated that Fc-functional TIGIT antibody a1217 can induce ADCC in TIGIT ⁺ Treg cells.

To measure a1217 or a1217 AF-induced ADCC activity against T cells, in particular tregs, PBMCs from lung cancer patients were used as target cells. NK cells isolated from PBMCs from healthy donors were used as effector cells. A1217MF contained a silent Fc that reduced the amount of ADCC (SEQ ID NO: 12). Anti TIGIT antibodies a1217, a1217AF or a1217MF were incubated overnight with target cells (5 x10 ⁴ cells/well) and NK effector cells (5 x10 ⁴ cells/well, purified using NK cell separation kit, miltenyi Biotec, catalog No. 130-092-657) in 96-well plates. Flow cytometry analysis was performed on the cell samples.

The result is that a1217AF enhanced ADCC activity against tregs when compared to a1217 wild type, as shown in figures 10A-C. As shown in fig. 10A, treg frequency in CD3 ⁺ T cells was significantly reduced in a dose-dependent manner after a1217 or a1217AF treatment, while the percentage remained largely unchanged in the a1217MF treatment group. A1217AF induced significantly higher ADCC against tregs compared to a1217 wild type. On the other hand, none of the a1217 wild type, a1217MF, or a1217AF treatments altered the frequency of effector cd4+ T cells or cd8+ T cells (fig. 10B and 10C). The results clearly demonstrate that a1217AF is able to induce stronger ADCC against Treg cells in PBMCs derived from cancer patients compared to a1217 wild type. Enhanced Fc function may enhance TIGIT antibody activity in anti-tumor immune responses through Treg reduction.

Example 13 activation of NK cells by anti-TIGIT antibodies with reduced fucosylation

TIGIT is constitutively expressed on Natural Killer (NK) cells, and the interaction between TIGIT and its ligands PVR and PVR-L2 inhibits NK cell mediated cytotoxicity (STANIETSKY et al, (2009) Proc NATL ACAD SCI U S a106,17858-17863; wang et al, (2015) Eur J Immunol 45, 2886-2897). The dominant fcγr expressed on NK cells is fcγriiia (Bruhns, (2012) Blood 119, 5640-5649). The additive and/or synergistic effects of the co-junctions of TIGIT and fcyriiia on NK cells may be concentrated downstream, resulting in a stronger signal. To confirm whether optimal NK activation by TIGIT antibodies requires the ability of fcγr co-conjugation, and whether Fc-enhanced a1217 can further promote NK cell activation, purified primary NK cells were co-cultured with human breast cancer cell line SK-BR-3 (ATCC HTB-30) expressing high levels of PVR in the presence of TIGIT antibodies with different Fc forms. NK cell activation was determined by measuring NK cell degranulation marker CD107a via flow cytometry. The result was that a1217AF had greater activation of NK cells when compared to a1217 wild type or a1217MF (fig. 11A-B).

In the co-culture assay, anti-TIGIT antibody a1217 wild type, a1217AF or a1217MF was added to a co-culture of SK-BR-3 (5 x10 ⁴ cells/well) and primary NK cells (5 x10 ⁴ cells/well) isolated from PBMCs of healthy donor origin in 96-well plates overnight. NK cells were pre-stimulated overnight with 25U/mL recombinant human IL-2 (Novoprotein, china, catalog number C013) prior to co-culture assay. CD107a expression on NK cells was determined by FACS. When compared to the a1217 wild type, a1217AF significantly increased NK activation, indicating that fcγr co-conjugation enhanced by afucosylation of a1217 induced optimal NK cell activation in the presence of tumor cells.

Example 14 Cytometric Properties of anti-TIGIT antibodies with reduced fucosylation

The cell gnawing effect is the process of transferring cell surface molecules from donor cells to recipient cells (Beum et al, (2008) J Immunol 181,8120-8132; joly and Hudrisier, (2003) Immunity 40,569-581; machlenkin et al, (2008) CANCER RES 68,2006-2013; rossi et al, (2013) Blood 122, 3020-3029). The cell-biting action mediated by antibodies to fcγreceptor (fcγr) causes down-regulation of the receptor on the cell surface (Taylor and Lindorfer, (2015) Blood 125, 762-766). Down-regulation of target receptors by cytoplasmic action can lead to attenuated signaling. To investigate whether Fc-functional TIGIT antibodies can induce fcγr mediated cell-biting to remove TIGIT from the cell surface, and whether binding of Fc to fcγr enhanced by DE/DEL mutation or reduced fucosylation can further promote cell-biting, a cell-biting assay was performed.

Jurkat/TIGIT/DNAM-1 cells (2X 10 ⁴ cells/well) were used as donor cells and CFSE (Invitrogen, cat. No. C34554) labeled HEK293 cells expressing different FcgammaR (4X 10 ⁴ cells/well) were used as recipient cells. Donor cells were pre-incubated with 10 μg/mL CF 633-labeled a1217 wild-type, a1217AF or a1217MF for 30 minutes and washed. Donor cells were then incubated with recipient cells overnight in 96-well plates. The change in TIGIT (CF 633) Mean Fluorescence Intensity (MFI) on donor cells was measured by FACS.

As shown in fig. 12, a1217AF significantly induced TIGIT down-regulation on Jurkat/TIGIT/DNAM-1 cells when the recipient cells expressed fcyriiia-F158 and fcyriiia-V158 when compared to a1217 wild-type, suggesting that Fc-enhancement of a1217 by afucosylation may induce maximum TIGIT down-regulation by fcyriiia binding-dependent cell-biting. Interestingly, when the receptor cells expressed fcyri, a1217AF induced a level of cytoplasmic gnawing comparable to a1217 wild type, consistent with comparable binding affinities of a1217AF and a1217 for fcyri.

Example 15 Cytomentogenic Properties of anti-TIGIT antibody mutants

Jurkat/TIGIT/DNAM-1 (2X 10 ⁴ cells/well) cells were used as donor cells and CFSE (Invitrogen, cat. No. C34554) labeled HEK293 cells expressing different FcgammaR (4X 10 ⁴ cells/well) were used as recipient cells. Donor cells were pre-incubated with 10 μg/mL CF 633-labeled a1217 wild-type, a1217DE, a1217DEL or a1217MF for 30 minutes and washed. Donor cells were then incubated with recipient cells overnight in 96-well plates. The change in TIGIT (CF 633) MFI on donor cells was measured by FACS.

The results for the a1217DE and DEL effector variants were similar to those for a1217AF with reduced fucosylation. On cells expressing fcγriiia-F158 or fcγriiia-V158, the cell gnawing effect of a1217DE and a1217DEL is greater when compared to the a1217 wild type. Fc silence a1217MF had little or no activity as expected, and this data is graphically shown in fig. 13.

Example 16A 1217 antibody with reduced fucosylation in combination with anti-PD 1 antibody in mouse tumor model

For this experiment, 2x10 ⁵ Renca cells were subcutaneously implanted in the right flank of humanized TIGIT mice in a BABL/c background. Renca cells are a murine model of renal adenocarcinoma. The mice were then given PBS as a control, 3mg/kg mPD-1Ab, 10mg/kg A1217 wild type or 10mg/kg A1217AF as a monotherapy. For combination therapy, 3mg/kg mPD-1 antibody was administered to mice in combination with 10mg/kg A1217 wild type or 3mg/kg mPD-1Ab with 10mg/kg A1217 AF. The treatment was administered intraperitoneally on day 5 post tumor cell inoculation. Tumor volume was determined twice weekly using the following formula v=0.5 (a x b ²) where a and b are the long and short diameters of the tumor, respectively. * p <0.05.

This data is graphically represented in fig. 14. There was little difference when comparing a1217 wild type and a1217AF when administered as a single dose. However, when administered in combination with an anti-PD 1 antibody, the a1217AF antibody had improved tumor reduction compared to the a1217 wild-type administered in combination with an anti-PD 1 antibody.

Example 17A 1217 antibody with reduced fucosylation in combination with anti-PD 1 antibody induces Treg reduction in mouse tumor model

To investigate whether a1217AF antibody with reduced fucosylation combined with an anti-PD-1 antibody in Renca model reduced Treg cells, 30 ten thousand Renca tumor cells were subcutaneously implanted in right flank of female hTIGIT Balb/c mice (6-8 weeks old) on day 0, and the mice were randomly divided into 8 groups according to body weight and tumor volume when the average tumor volume reached 100-200 cm ³. Mice were treated with anti-TIGIT antibody a1217 (10 mg/kg) with different Fc forms as a single agent, with murine PD-1 blocking antibody Ch15mt (3 mg/kg) as a single agent, or with a combination of both antibodies at the indicated doses. Tumor samples were collected 48 hours post-dose for tumor-infiltrating lymphocyte (TIL) isolation and FACS analysis. Treg cells were gated with CD45 ⁺CD4⁺FOXP3⁺. As shown in fig. 15A-C, there was no significant difference when administered as a single dose when comparing a1217 wild type and a1217 AF. However, when administered in combination with an anti-PD 1 antibody, the a1217AF antibody significantly reduced intratumoral tregs compared to the a1217 wild-type and anti-PD 1 antibody administration, suggesting that afucosylation of a1217 may induce a more significant anti-tumor effect in combination with the PD-1 antibody by reducing Treg cells in the tumor microenvironment.

Claims (27)

1. A method for cancer treatment, comprising administering to a subject an effective amount of a pH-dependent anti-TIGIT antibody or an antigen-binding fragment thereof. 2. The method of claim 1, wherein the anti-TIGIT antibody binds to the TIGIT protein at amino acid histidine 76. 3. The method of claim 2, wherein the anti-TIGIT antibody binds to the TIGIT protein at histidine 76 and leucine 73. 4. The method of claim 3, wherein the method comprises administering to the subject an effective amount of a pH-dependent antibody or an antigen-binding fragment thereof, wherein the pH-dependent antibody or antigen-binding fragment thereof specifically binds to human TIGIT and comprises: a heavy chain variable region comprising HCDR (heavy chain complementarity determining region) 1 of SEQ ID NO: 1, HCDR2 of SEQ ID NO: 2, and HCDR3 of SEQ ID NO: 3; and a light chain variable region comprising LCDR (light chain complementarity determining region) 1 of SEQ ID NO: 4, LCDR2 of SEQ ID NO: 5, and LCDR3 of SEQ ID NO: 6. 5. The method of claim 4, wherein the anti-TIGIT antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising SEQ ID NO: 7, and a light chain variable region (VL) comprising SEQ ID NO: 8. 6. The method of claim 1, wherein the anti-TIGIT antibody has enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity. 7. The method of claim 6, wherein the anti-TIGIT antibody has reduced fucosylation. 8. The method of claim 6, wherein the anti-TIGIT antibody has Fc amino acid changes at S239D and I332E (EU numbering). 9. The method of claim 6, wherein the anti-TIGIT antibody has Fc amino acid changes at S239D, I332E, and A330L (EU numbering). 10. The method of any one of claims 7 to 9, wherein the anti-TIGIT antibody has increased binding affinity for FcγRIIIA-V158 and FcγRIIIA-F158. 11. The method of any one of claims 7 to 9, wherein the anti-TIGIT antibody has enhanced ADCC in T-regulatory (Treg) cells. 12. The method of any one of claims 7 to 9, wherein the anti-TIGIT antibody activates natural killer (NK) cells. 13. The method of any one of claims 7 to 9, wherein the anti-TIGIT antibody has increased cytotoxicity. 14. The method of claim 1, wherein the method further comprises administering an anti-PD1 antibody that specifically binds to human PD1 and comprises: a heavy chain variable region comprising HCDR1 of SEQ ID NO: 15, HCDR2 of SEQ ID NO: 16, and HCDR3 of SEQ ID NO: 17; and a light chain variable region comprising LCDR1 of SEQ ID NO: 18, LCDR2 of SEQ ID NO: 19, and LCDR3 of SEQ ID NO: 20. 15. The method of claim 14, wherein the anti-PD1 antibody or antigen-binding fragment thereof specifically binds to human PD1 and comprises: a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 21, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 22. 16. The method of claim 14 or 15, wherein the anti-PD1 antibody comprises: an IgG4 constant domain comprising SEQ ID NO: 23. 17. The method of claim 1, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, pancreatic cancer, head and neck cancer, stomach cancer, kidney cancer, liver cancer, small cell lung cancer, non-small cell lung cancer, esophageal cancer, ovarian cancer, uterine cancer, cervical cancer, skin cancer, mesothelioma, lymphoma, leukemia, myeloma, or sarcoma. 18. The method of claim 17, wherein the cancer is non-small cell lung cancer. 19. The method of claim 17, wherein the head and neck cancer is nasopharyngeal carcinoma. 20. The method of claim 17, wherein the esophageal cancer is esophageal squamous cell carcinoma (ESCC). 21. The method of claim 17, wherein the cancer is uterine cancer. 22. The method of claim 17, wherein the gastric cancer is gastric or gastroesophageal junction cancer. 23. The method of claim 17, wherein the cervical cancer is recurrent or metastatic cervical cancer. 24. The method of claim 17, wherein the cancer is renal cancer. 25. The method of claim 14, further comprising administering chemotherapy. 26. The method of claim 20, wherein the chemotherapy is chemoradiotherapy. 27. The method of claim 1, wherein the anti-PD1 antibody is administered at 200 mg every three weeks.