CN121398845A - Anti-PD-1/CTLA-4/TIGIT Trispecific Antibodies and Their Uses - Google Patents

Abstract

An antibody that specifically binds to TIGIT, a trispecific antibody that specifically binds to PD-1, CTLA-4 and TIGIT, a polynucleotide encoding the antibody or an antigen-binding fragment thereof, and a method for preparing and using the above substances.

Description

Anti-PD-1/CTLA-4/TIGIT trispecific antibodies and uses thereof

Technical Field

The present invention relates to antibodies that specifically bind TIGIT, trispecific antibodies that specifically bind PD-1, CTLA-4, and TIGIT, polynucleotides encoding the antibodies or antigen-binding fragments thereof, and methods of making and using the same.

Background

Immune Checkpoint Inhibitors (ICI) have been widely used in clinic for cancer treatment in recent decades, providing more treatment options and a wider clinical benefit for cancer patients due to their different mechanism of action than traditional chemotherapy and targeted therapies.

Programmed cell death protein 1 (PD-1) plays an important role in suppressing immune responses and promoting self-tolerance by modulating T cell activity, activating apoptosis of antigen-specific T cells and inhibiting apoptosis of regulatory T cells. Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is considered a co-inhibitor of the immune response, which can be combined with PD-1 to reduce proliferation of PD-1 positive cells, inhibit their cytokine secretion and induce apoptosis. From these perspectives, the PD-1/PD-L1 axis is responsible for cancer immune escape and has a tremendous impact on cancer treatment (1, 2). anti-PD-1 antibodies, such as pamphlet Li Zhushan antibody and nivolumab, have been widely used in the clinical treatment of various cancers, including melanoma, non-small cell lung cancer, renal cell carcinoma, and gastroesophageal cancer, and exhibit robust anti-tumor efficacy.

Co-blockade of PD-1 and a second co-inhibitory receptor has been demonstrated to boost anti-tumor immunity relative to single PD-1 blockade in preclinical and subsequent clinical studies. Indeed, blockade of both PD-1 and CTLA-4 shows improved clinical efficacy in patients with melanoma (3) and advanced non-small cell lung cancer (4) and hepatocellular carcinoma (5), among others. CTLA-4 is constitutively expressed by tregs, but can also be upregulated upon activation by other T cell subsets, especially cd4+ T cells (6). CTLA-4 mediates immunosuppression by indirectly attenuating signaling via the co-stimulatory receptor CD 28. CTLA-4 can also remove CD80 and CD86 (including its cytoplasmic domain) from the cell surface of antigen presenting cells via transcytosis (trans-endocytosis) (7), thus reducing the availability of these stimulatory receptors to other T cells expressing CD 28.

However, combined PD-1 and CTLA-4 blockade is hindered by high frequency of severe immune-related systemic adverse effects (8), thus other less toxic combinations are urgently needed. T cell immune receptors with Ig and ITIM domains (TIGIT) are novel checkpoint inhibitory molecules that have recently received attention in cancer immunotherapy (9, 10). It is expressed on a variety of immune cells including T cells, regulatory T cells (tregs) and Natural Killer (NK) cells (11). TIGIT competes with its co-stimulatory counterpart CD226 for binding to poliovirus receptor (PVR, also known as CD 155), with higher affinity (12, 13). Co-blocking of TIGIT/CD155 and PD-1/PD-L1 pathways has been shown to be synergistic in both mouse tumor models and clinical trials (14, 15).

However, most studies of CTLA4/CD28 or TIGIT/CD155 and PD-1/PD-L1 pathway co-blocking are based on the combined use of two separate antibody molecules. There is an unmet need for trispecific antibodies that co-block these three pathways together.

Disclosure of Invention

The invention provides isolated anti-PD-1/CTLA-4/TIGIT trispecific antibodies comprising a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and a third domain that specifically binds to TIGIT.

In one embodiment, the first domain that specifically binds to PD-1 comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOs 1,2 and 3, respectively.

In one embodiment, the first domain that specifically binds to PD-1 comprises or consists of the heavy chain variable region (VH) of SEQ ID NO. 4.

In one embodiment, the second domain that specifically binds to CTLA-4 comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOs 7, 8 and 9, respectively.

In one embodiment, the second domain that specifically binds to CTLA-4 comprises or consists of the heavy chain variable region (VH) of SEQ ID NO. 10.

In one embodiment, the third domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOs 13, 14 and 15, respectively.

In one embodiment, the third domain that specifically binds to TIGIT comprises or consists of the heavy chain variable region (VH) of SEQ ID No. 16.

In one embodiment, the isolated anti-PD-1/CTLA-4/TIGIT trispecific antibody further comprises an Fc region of IgG. In one embodiment, the Fc region has IgG1. In one embodiment, the Fc region has an IgG1 LALA.

In one embodiment, an anti-PD-1/CTLA-4/TIGIT trispecific antibody according to the present invention has an IgG1, igG2, igG3 or IgG4 isotype, optionally comprising one, two, three, four, five, six, seven, eight, nine or ten substitutions in the Fc region.

In one embodiment, the first domain that specifically binds to PD-1 is disposed at the N-terminus of the Fc region.

In one embodiment, the second domain that specifically binds to CTLA-4 is disposed at the C-terminus of the Fc region.

In one embodiment, the third domain that specifically binds to TIGIT is placed at the N-terminus of the Fc region.

In one embodiment, the first domain that specifically binds to PD-1 is disposed at the C-terminus of the Fc region.

In one embodiment, the second domain that specifically binds to CTLA-4 is disposed at the C-terminus of the Fc region.

In one embodiment, the third domain that specifically binds to TIGIT is disposed at the C-terminus of the Fc region.

In one embodiment, the first domain that specifically binds to PD-1, the second domain that specifically binds to CTLA-4, the third domain that specifically binds to TIGIT, and the Fc region are linked to each other directly or via one or more linkers. In one embodiment, the linkers are the same or different. In one embodiment, the joint is a flexible connection. In one embodiment, the linker is a peptide linker. In one embodiment, the linker is GGGGSGGGGS (SEQ ID NO: 21).

In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention are shown from N-terminus to C-terminus by the formula:

First domain-linker-third domain-hinge region-Fc-linker-second domain.

In one embodiment, an anti-PD-1/CTLA-4/TIGIT trispecific antibody according to the invention comprises or consists of the heavy chain of SEQ ID NO. 19.

In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention consist of one or two heavy chains. In one embodiment, the heavy chain comprises a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and a third domain that specifically binds to TIGIT. In one embodiment, the first domain that specifically binds to PD-1 comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOS: 1,2 and 3, respectively, the second domain that specifically binds to CTLA-4 comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOS: 7, 8 and 9, respectively, and the third domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOS: 13, 14 and 15, respectively. In one embodiment, the first domain that specifically binds to PD-1 comprises or consists of the heavy chain variable region (VH) of SEQ ID NO. 4, the second domain that specifically binds to CTLA-4 comprises or consists of the heavy chain variable region (VH) of SEQ ID NO. 10, and the third domain that specifically binds to TIGIT comprises or consists of the heavy chain variable region (VH) of SEQ ID NO. 16, or the heavy chain variable region (VH) of SEQ ID NO. 16. In one embodiment, the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO. 19.

The invention also provides isolated anti-TIGIT antibodies or antigen-binding fragments thereof that specifically bind to TIGIT.

In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOs 13, 14 and 15, respectively.

In one embodiment, the anti-TIGIT antibody or antigen-binding fragment thereof comprises or consists of the heavy chain variable region (VH) of SEQ ID No. 16.

In one embodiment, the anti-TIGIT antibody further comprises an Fc region of IgG. In one embodiment, the Fc region has IgG1. In one embodiment, the Fc region has an IgG1 LALA.

In one embodiment, an anti-TIGIT antibody according to the invention has an IgG1, igG2, igG3 or IgG4 isotype, optionally comprising one, two, three, four, five, six, seven, eight, nine or ten substitutions in the Fc region.

In one embodiment, the heavy chain variable region (VH) is disposed at the N-terminus of the Fc region.

In one embodiment, the heavy chain variable region (VH) is disposed at the C-terminus of the Fc region.

In one embodiment, the heavy chain variable region (VH) and the Fc region are connected to each other directly or via one or more linkers. In one embodiment, the linkers are the same or different. In one embodiment, the joint is a flexible connection. In one embodiment, the linker is a peptide linker. In one embodiment, the linker is GGGGSGGGGS (SEQ ID NO: 21).

In one embodiment, the anti-TIGIT antibodies according to the invention are displayed from N-terminus to C-terminus by the formula:

Heavy chain variable region (VH) -linker-Fc.

In one embodiment, an anti-TIGIT antibody according to the invention consists of one or two heavy chains. In one embodiment, the heavy chain comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOs 13, 14 and 15, respectively. In one embodiment, the heavy chain comprises or consists of the heavy chain variable region (VH) of SEQ ID NO. 16.

The invention also provides a pharmaceutical composition comprising an anti-PD-1/CTLA-4/TIGIT trispecific antibody of the invention, or an anti-TIGIT antibody of the invention or antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

The invention also provides polynucleotides encoding the anti-PD-1/CTLA-4/TIGIT trispecific antibodies of the invention or the heavy chain variable region (VH) or heavy chain of the anti-TIGIT antibodies of the invention.

The invention also provides polynucleotides encoding the anti-PD-1/CTLA-4/TIGIT trispecific antibodies of the invention or the anti-TIGIT antibodies of the invention.

The invention also provides vectors comprising the polynucleotides of the invention.

The invention also provides host cells comprising the vectors of the invention.

The invention also provides a method of producing an antibody of the invention comprising culturing a host cell of the invention under conditions that express the antibody, and recovering the antibody produced by the host cell.

The invention also provides a method of treating cancer in a subject comprising administering to a subject in need thereof a therapeutically effective amount of an isolated antibody of the invention, a pharmaceutical composition of the invention, a polynucleotide of the invention, a vector of the invention, or a host cell of the invention for a time sufficient to treat cancer.

The invention also provides an isolated antibody of the invention, a pharmaceutical composition of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention for use in the treatment of cancer in a subject in need thereof.

The invention also provides the use of an isolated antibody of the invention, a pharmaceutical composition of the invention, a polynucleotide of the invention, a vector of the invention or a host cell of the invention in the manufacture of a medicament for treating cancer in a subject.

Drawings

FIG. 1 shows a schematic illustration of the TsAb-GBD209-24-3 hexavalent structure.

FIG. 2 shows binding of TsAb-GBD209-24-3, parent PD-1 (GBD 002-hS 019-WS), CTLA-4 (GBD 008-hS 005-3-2) nanobody or TIGIT (GB 005-10-H0302) to PD-1 single positive cells (A), CTLA-4 single positive cells (B) and PD-1/CTLA-4/TIGIT triple positive cells (C).

FIG. 3 shows luminescence of Jurkat-PD-1-NFAT-luciferase reporter cells co-cultured with CHOK 1-PD-L1.

FIG. 4 shows the inhibition of PD-1 binding to its ligand PD-L2 on HEK293T-hPD-1 cells by TsAb-GBD209-24-3 or parent PD-1 (GBD 002-hS 019-WS) nanobodies.

FIG. 5 shows inhibition of binding of CTLA-4 to its ligand CD80 on CTLA-4 single positive cells (A) and PD-1/CTLA-4/TIGIT triple positive cells (B) by TsAb-GBD209-24-3 or parent CTLA-4 (GBD 008-hS 005-3-2) nanobody.

FIG. 6 shows inhibition of binding of CTLA-4 to its ligand CD86 on CTLA-4 single positive cells (A) and PD-1/CTLA-4/TIGIT triple positive cells (B) by TsAb-GBD209-24-3 or parent CTLA-4 (GBD 008-hS 005-3-2) nanobody.

FIG. 7 shows the binding of TsAb-GBD209-24-3 or parent TIGIT (GB 005-10-H0302) nanobody to hTIGIT single positive cells.

FIG. 8 shows luminescence of Jurkat-TIGIT-NFAT-luciferase reporter cells co-cultured with CHOK1-CD155 TCR.

FIG. 9 shows IFN gamma secretion in Mixed Lymphocyte Reaction (MLR).

FIG. 10 shows the internalization of PD-1 receptors on PD-1/CTLA-4/TIGIT triple positive cells in the presence of TsAb-GBD209-24-3 or parent PD-1 (GBD 002-hS 019-WS) nanobodies.

FIG. 11 shows the change in tumor growth in A375 melanoma cell line xenograft models following TsAb-GBD209-24-3 or reference antibody administration.

Detailed Description

Immune Checkpoint Inhibitors (ICI), based primarily on PD-1/PD-L1 blockade, have become the primary therapy for cancer. Despite the tremendous clinical success of ICI, about 70% of patients still show de novo and adaptive resistance. In ICI, combinations with PD-1/PD-L1 and CTLA-4 or TIGIT inhibitors show promising therapeutic results, and some have been batched for the treatment of certain cancers while others are in clinical trials. In order to improve ICI response rate and reduce immune related adverse events, one approach is to use bispecific antibodies (bsAb) or trispecific antibodies (tsAb) to direct the effect to the tumor. Here, we developed novel anti-PD-1, anti-CTLA-4, anti-TIGIT trispecific antibodies to simultaneously target three cell surface antigens with overlap on T cells in the Tumor Microenvironment (TME). tsAb exhibit in vitro activity similar to the PD-1/PD-L1 and TIGIT/CD155 axes compared to the palboc Li Zhushan and tirelin Li Youshan antibodies, and weaker activity of the CTLA-4 pathway compared to ipilimumab. In addition tsAb showed robust anti-tumor activity in the mouse model compared to PD1x CTLA4 bsAb of ICI in the clinic. These findings suggest that the present invention can specifically bind to PD1, CTLA4 and TIGIT and alleviate immunosuppression of these checkpoints, and thus has good therapeutic application prospects in cancer treatment.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if fully set forth.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice of the testing of the present invention, the exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

"Specifically binds" or "binds" refers to an antibody that binds an antigen or an epitope within an antigen with higher affinity than other antigens. Typically, an antibody binds an antigen or epitope within an antigen with an equilibrium dissociation constant (K _D) of about 1x10 ^-8 M or less, e.g., about 1x10 ^-9 M or less, about 1x10 ^-10 M or less, about 1x10 ^-11 M or less, or about 1x10 ^-12 M or less, typically up to 1/100 of K _D as compared to K _D, which binds to a non-specific antigen (e.g., BSA, casein). The dissociation constant can be measured using standard procedures. However, antibodies that specifically bind to an antigen or an epitope within an antigen may have cross-reactivity with other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, e.g., cynomolgus monkey (Macaca fascicularis) (cynomolgus), cyno), chimpanzee (Pan troglymes) (chimpanzee, chimp), or common marmoset (Callithrix jacchus) (common marmoset (common marmoset), marmoset). Although monospecific antibodies specifically bind to one antigen or one epitope, bispecific antibodies specifically bind to two different antigens or two different epitopes.

"Antibodies" or "Antibodies" are meant in a broad sense and include immunoglobulin molecules, including monoclonal Antibodies, including murine, human, humanized and chimeric monoclonal Antibodies, antigen binding fragments, bispecific or multispecific Antibodies, dimeric, tetrameric or multimeric Antibodies, single chain Antibodies, domain Antibodies, and any other modified configuration of immunoglobulin molecules comprising an antigen binding site of the desired specificity. "full length antibodies" are composed of two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., igM). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (consisting of domains CH1, hinges CH2 and CH 3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with Framework Regions (FR). Each VH and VL is composed of three CDRs and four FR segments arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

"Complementarity Determining Regions (CDRs)" are "antigen binding sites" in an antibody. CDRs may be defined using various terms (i) Complementarity Determining Regions (CDRs), three of the VHs (HCDR 1, HCDR2, HCDR 3) and three of the VL (LCDR 1, LCDR2, LCDR 3) are based on sequence variability (Wu and Kabat, (1970) J Exp Med132:211-50; kabat et al Sequences of Proteins of Immunological Interest th edition Public HEALTH SERVICE, national Institutes of Health, bethesda, md., 1991). (ii) "hypervariable region", "HVR" or "HV", three of VH (H1, H2, H3) and three of VL (L1, L2, L3) refer to regions in the antibody variable domain that are hypervariable in structure, as defined by Chothia and Lesk (Chothia and Lesk, (1987) Mol Biol 196:901-17). The InternationalImMunoGeneTics (IMGT) database (http:// www_ imgt _org) provides standardized numbering and definition of antigen binding sites. The correspondence between CDR, HV and IMGT partitions is described in Lefranc et al, (2003) Dev Comparat Immunol 27:55-77. As used herein, the terms "CDR," "HCDR1," "HCDR2," "HCDR3," "LCDR1," "LCDR2," and "LCDR3" include CDRs defined by any of the methods described above (Kabat, chothia or IMGT) unless the specification clearly indicates otherwise.

Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be assigned to five major classes, igA, igD, igE, igG and IgM. IgA and IgG are further subdivided into isotypes IgA1, igA2, igG1, igG2, igG3 and IgG4. Based on the amino acid sequence of its constant domain, the antibody light chain of any vertebrate species can be assigned to one of two distinct types (i.e., kappa (kappa) and lambda (lambda)).

An "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to PD-1 is substantially free of antibodies that specifically bind to antigens other than PD-1). In the case of the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention, the trispecific antibodies specifically bind to PD-1, CTLA-4 and TIGIT and are substantially free of antibodies specifically binding to antigens other than PD-1, CTLA-4 and TIGIT. An "isolated antibody" encompasses an antibody that is isolated to a higher purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure antibody.

"Recombinant" refers to antibodies and other proteins produced, expressed, produced, or isolated by recombinant means.

An "epitope" refers to a portion of an antigen to which an antibody specifically binds. Epitopes are generally composed of surface groupings of chemically active (e.g., polar, nonpolar, or hydrophobic) moieties such as amino acid or polysaccharide side chains, and may have specific three-dimensional structural properties as well as specific charge properties. Epitopes can be composed of contiguous and/or non-contiguous amino acids that form conformational space units. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are brought close to each other in three dimensions by folding of the protein molecule. An antibody "epitope" depends on the method used to identify the epitope.

"Multispecific" refers to an antibody that specifically binds to at least two different antigens or at least two different epitopes within an antigen (e.g., three, four, or five different antigens or epitopes). "trispecific" refers to antibodies that specifically bind to three different antigens or three different epitopes within the same antigen. The trispecific antibodies may be cross-reactive with other related antigens, such as the same antigen (homolog) from other species, such as humans or monkeys, e.g. cynomolgus monkey (Macaca fascicularis) (cynomolgus), cyno), chimpanzee (Pan troglodes) (chimpanzee ) or common marmoset (Callithrix jacchus) (common marmoset (common marmoset), marmoset), or may bind an epitope shared between two or more different antigens.

"Vector" refers to a polynucleotide capable of replication within a biological system or that can move between such systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selectable markers that function to facilitate replication or maintenance of the polynucleotides in a biological system. Examples of such biological systems may include cellular, viral, animal, plant, and reconstituted biological systems that utilize biological components capable of replicating vectors. The polynucleotide constituting the vector may be a DNA or RNA molecule or a hybrid of these.

An "expression vector" refers to a vector that can be utilized in a biological system or a reconstituted biological system to direct translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

"Polynucleotide" refers to a synthetic molecule comprising nucleotide chains covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

"Overexpressing (Overexpress)", "overexpressing" and "overexpressing" are used interchangeably and refer to samples such as cancer cells, malignant cells or cancer tissues having measurably higher levels of PD-1, CTLA-4 or TIGIT or a ligand thereof when compared to a reference sample. Overexpression may be caused by gene amplification or by increased transcription or translation. Expression and overexpression of proteins in a sample can be measured using well-known assays, using, for example, ELISA, immunofluorescence, flow cytometry, or radioimmunoassay, on living or lysed cells. Expression and overexpression of polynucleotides in a sample can be measured, for example, using fluorescent in situ hybridization, southern blotting, or PCR techniques. Proteins or polynucleotides are over-expressed when the level of the protein or polynucleotide in the sample is at least 1.5-fold or statistically significant when compared to a reference sample. The selection of the reference sample is known.

"Sample" refers to a collection of similar fluids, cells or tissues isolated from a subject, as well as fluids, cells or tissues present within a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cyst fluid, tear drops, stool, sputum, mucous secretions of secretory tissues and organs, vaginal secretions, ascites fluids such as those associated with non-solid tumors, pleural, pericardial, peritoneal and other body cavities, fluids collected by bronchial lavage, liquid solutions in contact with a subject or biological source such as cell and organ culture media (including cell or organ conditioned media), lavages and the like, tissue biopsies, fine needle aspiration or surgical removal of tumor tissue.

"Cancer cell" or "tumor cell" refers to a cancer cell, a precancerous cell, or a transformed cell that has a spontaneous or induced phenotypic change in vivo, ex vivo, or in tissue culture. These changes do not necessarily involve uptake of new genetic material. Although transformation may result from infection by a transforming virus and the incorporation of new genomic nucleic acid, uptake of exogenous nucleic acid, or it may occur spontaneously or after exposure to a carcinogen, thereby mutating the endogenous gene. Transformation/cancer is exemplified by morphological changes in vitro, in vivo and ex vivo, immortalization of cells, abnormal growth control, foci formation, proliferation, malignancy, modulation of tumor-specific marker levels, invasiveness, tumor growth in a suitable animal host such as nude mice, and the like (Freshney, culture of ANIMAL CELLS: A Manual of Basic Technique (3 rd edition, 1994)).

By "about" is meant that within an acceptable error range with respect to a particular value, as determined by one of ordinary skill in the art, it depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise in the examples or elsewhere in the specification in the context of a particular assay, result, or embodiment, "about" means within one standard deviation, or up to 5%, of the range as practiced in the art, whichever is greater.

"Anti-PD-1/CTLA-4/TIGIT trispecific antibody", "PD-1/CTLA-4/TIGIT antibody" or "trispecific anti-PD-1/CTLA-4/TIGIT antibody" etc. refer to a molecule comprising at least one binding domain which specifically binds to PD-1, at least one binding domain which specifically binds to CTLA-4, and at least one binding domain which specifically binds to TIGIT. The domains that specifically bind to PD-1, CTLA-4 and TIGIT are typically VH/VL pairs or VH only. The trispecific anti-PD-1/CTLA-4/TIGIT antibody may be monovalent or bivalent in its binding to PD-1, CTLA-4 or TIGIT.

"Valency" refers to the presence of a specified number of binding sites in a molecule that are specific for an antigen. Thus, the terms "monovalent", "divalent", "tetravalent" and "hexavalent" refer to the presence of one, two, four and six binding sites, respectively, in a molecule that are specific for an antigen.

The term "single chain Fv" or "scFv" antibody refers to an antibody fragment comprising the V _H and V _L domains of the antibody, wherein these domains are present in a single polypeptide chain. Generally, fv polypeptides further comprise a polypeptide linker between the V _H and V _L domains that enables the scFv to form the structure required for antigen binding. For reviews of scFv, see Pluckaphun (1994) The Pharmacology of Monoclonal Antibodies, vol.113, rosenburg and Moore editions, springer-Verlag, new York, pages 269-315. See also International publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. In one embodiment, the scFv comprises, from N-terminus to C-terminus, a V _H region, a peptide linker, and a V _L region (VH-VL form). In another embodiment, the scFv comprises, from N-terminus to C-terminus, a V _L region, a peptide linker, and a V _H region (VL-VH form).

As used herein, the term "diabody" refers to a small antibody fragment having two antigen binding sites, said fragment comprising a heavy chain variable domain (V _H) (V_H-V_L or V _L-V_H) linked to a light chain variable domain (V _L) in the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same strand, the domains are forced to pair with complementary domains of the other strand and two antigen binding sites are created. Diabodies are described more fully in, for example, EP 404,097, WO 93/11161, and Holliger et al (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For reviews of engineered antibody variants, see generally Holliger and Hudson (2005) Nat. Biotechnol.23:1126-1136.

"Fab" consists of the VH and CH1 regions of the heavy chain and the VL and CL regions of the light chain, which are typically linked together by disulfide bonds and have a single antigen binding site. The VH, CH1, VL and CL regions in Fab may be arranged in various ways to confer antigen binding ability in accordance with the present disclosure. For example, the VH and CH1 regions may be on one polypeptide, while the VL and CL regions may be on separate polypeptides. Alternatively, the VH, CH1, VL and CL regions may all be located on the same polypeptide, optionally arranged in a different order.

"Antigen-specific CD4 ⁺ or CD8 ⁺ T cells" refers to CD4 ⁺ or CD8 ⁺ T cells activated by a specific antigen or immunostimulatory epitope thereof.

"Subject" includes any human or non-human animal. "non-human animals" include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. The terms "patient" or "subject" are used interchangeably unless otherwise indicated.

"Treatment" or "treatment" refers to therapeutic treatment in which the purpose is to slow down (alleviate) unwanted physiological changes or diseases, such as the development or spread of tumors or tumor cells, or to provide beneficial or desired clinical results during treatment. Beneficial or desired clinical results include alleviation of symptoms, whether detectable or undetectable, diminishment of extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, lack of metastasis, amelioration or palliation of the disease state, and remission (whether partial or total). "treatment" may also mean an prolongation of survival compared to the expected survival if the subject is not receiving treatment. Those in need of treatment include those subjects already with an undesirable physiological change or disease and those subject prone to have a physiological change or disease.

"Therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result at the necessary dosage and time period. The therapeutically effective amount of the antibodies of the invention may vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the antibodies of the invention to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, an improvement in the health of a patient, a reduction in tumor burden, a cessation or slowing of tumor growth, and/or the absence of metastasis of cancer cells to other locations in the body.

Amino acid residues in the constant region of an antibody throughout the specification are numbered according to the EU index as described in Kabat et al Sequences of Proteins of Immunological Interest th edition Public HEALTH SERVICE, national Institutes of Health, bethesda, MD. (1991), unless explicitly stated otherwise.

Conventional single-letter and three-letter amino acid codes as shown in table 1 are used herein.

TABLE 1 Single letter and three letter amino acid codes

Amino acids	Three letter code	Single letter code
			Alanine (Ala)	Ala	A
Arginine (Arg)	Arg	R
			Asparagine derivatives	Asn	N
Aspartic acid	Asp	D
			Cysteine (S)	Cys	C
Glutamic acid	Gln	E
			Glutamine	Glu	Q
Glycine (Gly)	Gly	G
			Histidine	His	H
Isoleucine (Ile)	Ile	I
			Lysine	Lys	K
Methionine	Met	M
			Phenylalanine (Phe)	Phe	F
Proline (proline)	Pro	P
			Serine (serine)	Ser	S
Threonine (Thr)	Thr	T
			Tryptophan	Trp	W
Tyrosine	Tyr	Y
			Valine (valine)	Val	V

Composition of matter

The present invention provides trispecific antibodies that specifically bind to PD-1, CTLA-4 and TIGIT. The invention provides polynucleotides encoding antibodies of the invention, or their complementary nucleic acids, vectors, host cells, and methods of making and using the same.

Anti-TIGIT antibodies and trispecific anti-PD-1/CTLA-4/TIGIT antibodies

The invention also provides isolated anti-TIGIT antibodies.

The invention also provides isolated trispecific anti-PD-1/CTLA-4/TIGIT antibodies.

The invention also provides isolated trispecific anti-PD-1/CTLA-4/TIGIT antibodies comprising a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and a third domain that specifically binds to TIGIT.

In some embodiments, the trispecific anti-PD-1/CTLA-4/TIGIT antibodies of the invention enhance activation of antigen-specific CD4 ⁺ or CD8 ⁺ T cells.

In some embodiments, the trispecific anti-PD-1/CTLA-4/TIGIT antibodies of the invention inhibit the binding of PD-1 to PD-L1 and PD-L2.

In some embodiments, the trispecific anti-PD-1/CTLA-4/TIGIT antibodies of the invention inhibit binding of CTLA-4 to CD80 and CD 86.

In some embodiments, the trispecific anti-PD-1/CTLA-4/TIGIT antibodies of the invention inhibit the binding of TIGIT to CD 155.

In some embodiments, the trispecific anti-PD-1/CTLA-4/TIGIT antibodies of the invention induce PD-1 internalization on the surface of the cell.

In one embodiment, the first domain that specifically binds to PD-1 comprises or consists of a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOs 1,2 and 3, respectively.

In one embodiment, the second domain that specifically binds to CTLA-4 comprises or consists of a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOs 7, 8 and 9, respectively.

In one embodiment, the third domain that specifically binds to TIGIT comprises or consists of a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOs 13, 14 and 15, respectively.

In one embodiment, the first domain that specifically binds to PD-1 comprises or consists of the heavy chain variable region (VH) of SEQ ID NO. 4, optionally with one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions. Optionally, any substitution is not within a CDR.

In one embodiment, the first domain comprises a heavy chain variable region (VH) having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 4, or the first domain consists of a heavy chain variable region (VH). Optionally, any variation from the sequence of SEQ ID NO. 4 is not within a CDR.

In one embodiment, the second domain that specifically binds to CTLA-4 comprises or consists of the heavy chain variable region (VH) of SEQ ID No. 10, optionally with one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions. Optionally, any substitution is not within a CDR.

In one embodiment, the second domain comprises a heavy chain variable region (VH) having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No.10, or the second domain consists of a heavy chain variable region (VH). Optionally, any variation from the sequence of SEQ ID NO.10 is not within a CDR.

In one embodiment, the third domain that specifically binds to TIGIT comprises or consists of the heavy chain variable region (VH) of SEQ ID No. 16, optionally with one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen conservative amino acid substitutions. Optionally, any substitution is not within a CDR.

In one embodiment, the third domain comprises a heavy chain variable region (VH) having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID No. 16, or the third domain consists of a heavy chain variable region (VH). Optionally, any variation from the sequence of SEQ ID NO. 16 is not within a CDR.

In one embodiment, the first domain, the second domain, and the third domain are, independently of each other, chimeric antibodies, humanized antibodies, human antibodies, single chain antibodies, fv, fab, F (ab') 2, fd, single chain Fv molecules (scFv), diabodies, or single domain antibodies (dAb).

In one embodiment, the first domain, the second domain and the third domain are humanized independently of each other. In one embodiment, the first domain, the second domain and the third domain are, independently of each other, humanized VHHs.

In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention comprise only one heavy chain. In one embodiment, an anti-PD-1/CTLA-4/TIGIT trispecific antibody according to the invention comprises two heavy chains.

In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention consist of one heavy chain. In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention consist of two heavy chains.

In one embodiment, the heavy chain comprises a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and a third domain that specifically binds to TIGIT. In one embodiment, the first domain that specifically binds to PD-1 comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOS: 1,2 and 3, respectively, the second domain that specifically binds to CTLA-4 comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOS: 7, 8 and 9, respectively, and the third domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2) and 3 (HCDR 3) of SEQ ID NOS: 13, 14 and 15, respectively.

In one embodiment, the first domain that specifically binds to PD-1 comprises or consists of the heavy chain variable region (VH) of SEQ ID NO. 4, the second domain that specifically binds to CTLA-4 comprises or consists of the heavy chain variable region (VH) of SEQ ID NO. 10, and the third domain that specifically binds to TIGIT comprises or consists of the heavy chain variable region (VH) of SEQ ID NO. 16, or the heavy chain variable region (VH) of SEQ ID NO. 16.

In one embodiment, the isolated anti-PD-1/CTLA-4/TIGIT trispecific antibody further comprises an Fc region of IgG. In some embodiments, the antibody is an IgG1, igG2, igG3, or IgG4 isotype. In one embodiment, the antibody is an IgG1 LALA isotype. The term "LALA" refers to the amino acid substitution L234A/L235A (Kabat nomenclature) to be introduced, as is well known in the art.

In one embodiment, the first domain that specifically binds to PD-1 is disposed at the N-terminus of the Fc region. In one embodiment, the second domain that specifically binds to CTLA-4 is disposed at the N-terminus of the Fc region. In one embodiment, the third domain that specifically binds to TIGIT is placed at the N-terminus of the Fc region.

In one embodiment, the first domain that specifically binds to PD-1 is disposed at the C-terminus of the Fc region. In one embodiment, the second domain that specifically binds to CTLA-4 is disposed at the C-terminus of the Fc region. In one embodiment, the third domain that specifically binds to TIGIT is disposed at the C-terminus of the Fc region.

In one embodiment, the first domain that specifically binds to PD-1, the second domain that specifically binds to CTLA-4, the third domain that specifically binds to TIGIT, and the Fc region are linked to each other directly or via one or more linkers, such as flexible linkers. In one embodiment, the linkers are the same or different. In one embodiment, the linker is a peptide linker, and most preferably a peptide linker lacking a proteolytic cleavage site. In some embodiments, the amino acid residue in the linker is selected from G, A, S, P, E, T, D and K. In some embodiments, the linker is a (G4S) _n linker, where n is an integer from 1 to 5, preferably from 2 to 4. In some embodiments, the linker is GGGGSGGGGS (SEQ ID NO: 21).

In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention are shown from N-terminus to C-terminus by the formula:

First domain-linker-third domain-hinge region-Fc-linker-second domain.

In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention are shown from N-terminus to C-terminus by the formula:

second domain-linker-third domain-hinge region-Fc-linker-first domain.

In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention are shown from N-terminus to C-terminus by the formula:

First domain-linker-second domain-hinge region-Fc-linker-third domain.

In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention are shown from N-terminus to C-terminus by the formula:

Second domain-linker-first domain-hinge region-Fc-linker-third domain.

In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention are shown from N-terminus to C-terminus by the formula:

Third domain-linker-second domain-hinge region-Fc-linker-first domain.

In one embodiment, the anti-PD-1/CTLA-4/TIGIT trispecific antibodies according to the invention are shown from N-terminus to C-terminus by the formula:

Third domain-linker-first domain-hinge region-Fc-linker-second domain.

In one embodiment, an anti-PD-1/CTLA-4/TIGIT trispecific antibody according to the invention comprises or consists of only one heavy chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO. 19.

In one embodiment, an anti-PD-1/CTLA-4/TIGIT trispecific antibody according to the invention comprises or consists of two heavy chains, wherein the heavy chains comprise the amino acid sequence of SEQ ID NO. 19.

In one embodiment, the heavy chain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO. 19.

The term "identity" or "homology" between two amino acid sequences or nucleotide sequences is determined by sequence alignment. If the two sequences to be compared to each other differ in length, sequence alignment preferably involves the percentage of amino acid or nucleotide residues of the shorter sequence that are identical to the amino acid or nucleotide residues of the longer sequence. Sequence alignment can be determined as usual using computer programs. Deviations that occur in the comparison between a given sequence and the above-described sequences of the present disclosure may be caused, for example, by additions, deletions, substitutions, insertions or recombinations.

In some embodiments, the CDR sequences of the trispecific anti-PD-1/CTLA-4/TIGIT antibodies of the invention can comprise any conservative modifications. In some embodiments, CDR sequences of anti-TIGIT antibodies of the invention may comprise conservative modifications.

"Conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody that contains the amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. Conservative substitutions are those in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). In addition, any of the natural residues in the polypeptide may also be substituted with alanine, as has been previously described with respect to alanine scanning mutagenesis (MACLENNAN et al, acta Physiol. Scan. Sup. L. 643:55-67,1998; sasaki et al, adv. Biophys. 35:1-24,1998). Amino acid substitutions for antibodies of the invention may be made by well known methods, such as by PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, a library of variants may be generated using known methods, for example using random (NNK) or non-random codons, such as DVK codons, which encode 11 amino acids (Ala, cys, asp, glu, gly, lys, asn, arg, ser, tyr, trp). The resulting antibody variants can be tested for their properties using the assays described herein.

Production of monospecific antibodies of the invention

In some embodiments, the antibodies of the invention are human.

In some embodiments, the antibodies of the invention are humanized.

The monospecific antibodies of the invention described herein (e.g., antibodies that specifically bind to PD-1, CTLA-4, or TIGIT) can be generated using a variety of techniques. For example, the hybridoma method of Kohler and Milstein, nature256:495,1975, can be used to generate monoclonal antibodies. In the hybridoma method, a mouse or other host animal, such as hamster, rat, alpaca or monkey, is immunized with human or cynomolgus monkey PD-1, CTLA-4 or TIGIT, or a fragment of PD-1, CTLA-4 or TIGIT, such as the extracellular domain of PD-1, CTLA-4 or TIGIT, followed by fusion of spleen cells from the immunized animal with myeloma cells using standard methods, to form hybridoma cells (Goding, monoclonal Antibodies: PRINCIPLES AND PRACTICE, pages 59-103 (ACADEMIC PRESS, 1986)). Clones originating from a single immortalized hybridoma cell are screened for the production of antibodies having desired properties, such as binding specificity, cross-reactivity or lack thereof, and affinity for antigen.

Various host animals may be used to produce antibodies of the invention. For example, balb/c mice can be used to generate mouse anti-human PD-1, CTLA-4 or TIGIT antibodies. Alpaca can be used to produce anti-human PD-1, CTLA-4 or TIGIT VHH. Antibodies prepared in Balb/c mice and other non-human animals can be humanized using a variety of techniques to generate sequences that are more human-like.

Exemplary humanization techniques including selection of human acceptor frameworks are known and include CDR grafting (U.S. Pat. No. 5,225,539), SDR grafting (U.S. Pat. No. 6,818,749), surface remodeling (Padlan (1991) Mol Immunol 28:489-499), specificity determining residue surface remodeling (U.S. patent publication No. 2010/0261620), human framework adaptation (U.S. Pat. No. 8,748,356), or superhumanization (U.S. Pat. No. 7,709,226). In these methods, the CDRs of the parent antibody are transferred to a human framework, which can be selected based on its overall homology to the parent framework, based on similarity in CDR length, or canonical structural identity, or a combination thereof.

Humanized antibodies can be further optimized to improve their selectivity or affinity for a desired antigen by incorporating altered framework support residues to preserve binding affinity (back mutation) by techniques such as those described in International patent publication No. WO1090/007861 and WO1992/22653, or introducing variations at any CDR, for example, to improve the affinity of the antibody.

Transgenic animals, such as mice or rats, carrying a human immunoglobulin (Ig) locus in their genome can be used to generate human antibodies to a target protein and are described, for example, in the following: U.S. Pat. No. 6,150,584, international patent publication No. WO99/45962, international patent publication No. WO2002/066630, WO2002/43478, WO2002/043478 and WO1990/04036, lonberg et al (1994) Nature368:856-9; green et al (1994) Nature Genet.7:13-21; green & Jakobovits (1998) exp. Med.188:483-95; lonberg and Huszar (1995) Int Rev Immunol13:65-93; bruggemann et al, (1991) Eur J Immunol 21:1323-1326; fishwild et al, (1996) Nat Biohnol 14:845-851; mendez et al, (1997) Nature Genet15:146-156; gren (J Immunol Methods:11-23) et al, (1999) CANCER RES:1233-1233; and Taus455:455.1997). Endogenous immunoglobulin loci in such animals may be disrupted or deleted and at least one complete or partial human immunoglobulin locus may be inserted into the genome of the animal using homologous or non-homologous recombination, using transchromosomes (transchromosomes) or using minigenes. Companies such as Regeneron (http://_www_regeneron_com)、Harbour Antibodies (http://_www_harbourantibodies_com)、Open Monoclonal Technology、Inc. (OMT) (http://_www_omtinc_net)、KyMab(http://_www_kymab_com)、Trianni (http://_www.trianni_com) and Ablexis (http:// _www_ ablexis _com) can accept commitments to provide human antibodies to selected antigens using the techniques described above.

The human antibody may be selected from phage display libraries in which phage are engineered to express human immunoglobulins or portions thereof, such as Fab, single chain antibodies (scFv), or unpaired or paired antibody variable regions (Knappik et al, (2000) J Mol Biol296:57-86; krebs et al, (2001) J Immunol Meth254:67-84; vaughan et al, (1996) Nature Biotechnology14:309-314; lenses et al, (1998) PITAS (USA) 95:6157-6162; hoogenboom and Winter (1991) J Mol Biol227:381; marks et al, (1991) J Mol Biol 222:581). The antibodies of the invention can be isolated as fusion proteins with bacteriophage pIX coat proteins, e.g.from phage display libraries expressing the heavy and light chain variable regions of the antibodies as described in Shi et al, (2010) J Mol Biol397:385-96 and International patent publication No. WO 09/085462. Libraries may be screened for phage binding to human and/or cynomolgus PD-1, CTLA-4 or TIGIT, and the positive clones obtained may be further characterized, fab isolated from clone lysates and expressed as full length IgG. Such phage display methods for isolating human antibodies are described, for example, in U.S. patent nos. 5,223,409、5,403,484、5,571,698、5,427,908、5、580,717、5,969,108、6,172,197、5,885,793;6,521,404;6,544,731;6,555,313;6,582,915 and 6,593,081.

The preparation of the immunogenic antigen and the production of the monoclonal antibody may be performed using any suitable technique, such as recombinant protein production. The immunogenic antigen may be administered to the animal in the form of a purified protein, or a mixture of proteins including whole cells or cell or tissue extracts, or the antigen may be formed de novo in the body of the animal from nucleic acids encoding the antigen or parts thereof.

In a specific embodiment, the parent anti-PD-1 antibody (e.g., GBD002-hS 019-WS), anti-CTLA-4 antibody (e.g., GBD008-hS 005-3-2) and anti-TIGIT antibody (e.g., GB 005-10-H0302) are obtained from alpaca, respectively.

Anti-PD-1 antibodies GBD002-hS019-WS obtained from alpaca in the present disclosure have amino acid sequences and nucleotide sequences as shown in Table 2.

TABLE 2 amino acid and nucleotide sequences of GBD002-hS019-WS antibodies

The anti-CTLA-4 antibody GBD008-hS005-3-2 obtained from alpaca in the present disclosure has an amino acid sequence and a nucleotide sequence as shown in table 3.

TABLE 3 amino acid and nucleotide sequences of GBD008-hS005-3-2 antibody

The anti-TIGIT antibody GB005-10-H0302 obtained from alpaca in the present disclosure has CDR sequences as shown in table 4 and amino acid sequences and nucleotide sequences as shown in table 5.

TABLE 4 CDR sequences of GB005-10-H0302

TABLE 5 amino acid and nucleotide sequences of GB005-10-H0302

Production of the trispecific anti-PD-1/CTLA-4/TIGIT antibodies of the invention

The trispecific anti-PD-1/CTLA-4/TIGIT antibodies of the invention can be generated, isolated and characterized herein by combining a PD-1 binding VH (or VH/VL) domain, a CTLA-4 binding VH (or VH/VL) domain, and a TIGIT binding VH (or VH/VL) domain. Alternatively, a tri-specific anti-PD-1/CTLA-4/TIGIT antibody can be engineered using VH (or VH/VL) domains from publicly available monospecific anti-PD-1, anti-CTLA-4, or anti-TIGIT antibodies and/or by hybrid matching of PD-1, CTLA-4, or TIGIT binding VH (or VH/VL) domains identified herein with publicly available PD-1, CTLA-4, or TIGIT binding VH (or VH/VL) domains.

Exemplary anti-PD-1 antibodies that can be used to engineer trispecific anti-PD-1/CTLA-4/TIGIT antibodies include, for example, palboc Li Zhushan antibodies and nivolumab. Exemplary anti-CTLA-4 antibodies that can be used to engineer the trispecific anti-PD-1/CTLA-4/TIGIT antibodies include, for example, ipilimumab. Exemplary anti-TIGIT antibodies that can be used to engineer the trispecific anti-PD-1/CTLA-4/TIGIT antibodies include, for example, tireli Li Youshan antibody, vitamin Li Shan antibody (MK-7694), and europal Li Shan antibody (BGB-a 1217). Exemplary monospecific domains may also be derived from bispecific antibodies, such as the anti-PD-1 xCTLA-4 bispecific antibody california Li Shan antibody and MEDI5752.

The generated trispecific anti-PD-1/CTLA-4/TIGIT antibodies can be tested using the methods described herein for their binding to PD-1, CTLA-4 and TIGIT, and for their desired functional properties, such as enhanced activation of antigen-specific CD4 ⁺ and CD8 ⁺ T cells.

The constant regions of molecules such as antibodies are typically mutated at the DNA level using standard methods.

Representative trispecific anti-PD-1/CTLA-4/TIGIT antibodies (e.g., tsAb-GBD 209-24-3) constructed in the present disclosure have the amino acid sequences and nucleotide sequences as shown in Table 6.

TABLE 6 amino acid and nucleotide sequences of TsAb-GBD209-24-3

Polynucleotides, vectors and host cells

The invention also provides an anti-TIGIT antibody or a trispecific anti-PD-1/CTLA-4/TIGIT antibody, wherein the VH and/or VL of the antibody are encoded by a polynucleotide. The polynucleotide may be a complementary deoxyribonucleic acid (cDNA) and may be codon optimized for expression in a suitable host. Codon optimisation is a well known technique.

The invention also provides isolated polynucleotides encoding the VH of the antibody of the invention, the VL of the antibody of the invention, the heavy chain of the antibody of the invention or the light chain of the antibody of the invention.

The invention also provides isolated polynucleotides encoding the VH, VL, or VH and VL of the anti-TIGIT antibodies or trispecific anti-PD-1/CTLA-4/TIGIT antibodies of the invention.

The invention also provides isolated polynucleotides encoding the VH of SEQ ID NOs 4, 10 and/or 16.

The invention also provides isolated polynucleotides encoding the heavy and/or light chains of the anti-TIGIT antibodies or trispecific anti-PD-1/CTLA-4/TIGIT antibodies of the invention.

The invention also provides an isolated polynucleotide encoding the heavy chain of SEQ ID NO. 19.

The invention also provides an isolated polynucleotide comprising the polynucleotide sequence of SEQ ID NO. 20.

The polynucleotide sequence encoding the VH or VL of the antibody of the invention, or an antigen-binding fragment thereof, or the heavy or light chain of the antibody of the invention, may be operably linked to one or more regulatory elements, such as promoters or enhancers, which allow for expression of the nucleotide sequence in the intended host cell. The polynucleotide may be a cDNA.

The invention also provides vectors comprising the polynucleotides of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors or any other vector suitable for introducing the synthetic polynucleotide of the invention into a given biological or genetic background by any means. For example, polynucleotides encoding the light chain variable region and/or the heavy chain variable region of an antibody of the invention, optionally linked to a constant region, are inserted into an expression vector. The light and/or heavy chains may be cloned into the same or different expression vectors. The DNA segment encoding the immunoglobulin chain may be operably linked to control sequences in an expression vector that ensure expression of the immunoglobulin polypeptide. Such control sequences include signal sequences, promoters (e.g., naturally-associated or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected to be compatible with the host cell selected to express the antibody. Once the vector has been incorporated into an appropriate host, the host is maintained under conditions suitable for high level expression of the protein encoded by the incorporated polynucleotide.

Suitable expression vectors are typically replicable in host organisms either as episomes or as integrated parts of the host chromosomal DNA. Typically, the expression vector contains a selectable marker such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance or neomycin resistance to allow detection of those cells transformed with the desired DNA sequence.

Suitable promoter and enhancer elements are known in the art. For expression in eukaryotic cells, exemplary promoters include light and/or heavy chain immunoglobulin gene promoters and enhancer elements, cytomegalovirus immediate early promoters, herpes simplex virus thymidine kinase promoters, SV40 early and late promoters, promoters found in long terminal repeats from retroviruses, mouse metallothionein-I promoters, and various known tissue-specific promoters. The selection of an appropriate vector and promoter is well within the level of one of ordinary skill in the art.

Exemplary vectors that may be used are bacterial ：pBs、phagescript、PsiX174、pBluescript SK、pBs KS、pNH8a、pNH16a、pNH18a、pNH46a (Stratagene,La Jolla,Calif.,USA);pTrc99A、pKK223-3、pKK233-3、pDR540 and pRIT5 (Pharmacia, uppsala, sweden). Eukaryotic organisms pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.4 (Lonza) and pEE12.4 (Lonza).

The invention also provides host cells comprising one or more vectors of the invention. "host cell" refers to a cell into which a vector has been introduced. It is understood that the term host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell, as well as to stable cell lines produced from the particular subject cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Such host cells may be eukaryotic, prokaryotic, plant or archaeal (archeal) cells. Coli (ESCHERICHIA COLI), bacillus such as bacillus subtilis (Bacillus subtilis) and other enterobacteriaceae such as Salmonella (Salmonella), serratia (Serratia) and various Pseudomonas (Pseudomonas) species are examples of prokaryotic host cells. Other microorganisms, such as yeast, may also be used for expression. Saccharomyces (e.g., saccharomyces cerevisiae) and Pichia (Pichia) are examples of suitable yeast host cells. Exemplary eukaryotic cells may be of mammalian, insect, avian, or other animal origin. Mammalian eukaryotic cells include immortalized cell lines, such as hybridoma or myeloma cell lines, e.g., SP2/0 (American type culture Collection (ATCC), manassas, VA, CRL-1581), NS0 (European Collection of cell cultures (European Collection of Cell Cultures) (ECACC), salisbury, wiltshire, UK, ECACC accession number 8510503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) murine cell lines. An exemplary human myeloma cell line is U266 (ATTC CRL-TIB-196). Other useful cell lines include those derived from Chinese Hamster Ovary (CHO) cells, such as CHOK1SV (Lonza Biologics, walkersville, MD), ponelligent cube CHOK2SV (Lonza), CHO-K1 (ATCC CRL-61), or DG44.

The invention also provides a method of producing an antibody of the invention comprising culturing a host cell of the invention under conditions that express the antibody, and recovering the antibody produced by the host cell. Methods for preparing antibodies and purifying them are well known in the art. Once synthesized (or chemically or recombinantly), the intact antibody, dimers thereof, individual light and/or heavy chains, or other antibody fragments, e.g., VH and/or VL, may be purified according to standard procedures, including ammonium sulfate precipitation, affinity column, column chromatography, high Performance Liquid Chromatography (HPLC) purification, gel electrophoresis, etc. (see generally scens, protein Purification (Springer-Verlag, n.y., (1982)). Subject antibodies may be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or at least about 98% to 99% or more pure, e.g., free of contaminants other than the subject antibody, e.g., cell debris, macromolecules, etc.

The polynucleotide sequences of the invention may be incorporated into a vector using standard molecular biology methods. Host cell transformation, culture, antibody expression and purification are accomplished using well known methods.

Another embodiment of the invention is a method of producing an anti-TIGIT antibody or a trispecific anti-PD-1/CTLA-4/TIGIT antibody of the invention, comprising:

Incorporating into an expression vector a polynucleotide encoding a VH, VL, heavy chain and/or light chain of an antibody of the invention;

transforming a host cell with an expression vector;

Culturing the host cell in a medium under conditions wherein the VH, VL, heavy chain and/or light chain are expressed and antibodies are formed, and

Recovering the antibody from the host cell or culture medium.

Pharmaceutical composition/administration

The invention provides a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable carrier. For therapeutic use, the antibodies of the invention may be prepared as pharmaceutical compositions containing an effective amount of the antibody as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which an antibody of the invention is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% brine and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, colorants, and the like. The concentration of the antibodies of the invention in such pharmaceutical formulations may generally vary from less than about 0.5% to at least about 1% up to 15 or 20% by weight, and may be selected based primarily on the desired dosage, fluid volume, viscosity, etc., depending on the particular mode of administration selected. Suitable vehicles and formulations include other human proteins such as human serum albumin, for example as described in Remington: THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, troy, D.B. editions, lipincott WILLIAMS AND WILKINS, philadelphia, pa. 2006,Part 5,PharmaceuticalManufacturing, pages 691-1092, see especially pages 958-989.

The mode of administration for therapeutic use of the antibodies of the invention may be any suitable route for delivering the antibodies to the host, e.g., parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, intranasal, intravaginal, rectal), using formulations in tablets, capsules, solutions, powders, gels, granules, and contained in syringes, implant devices, osmotic pumps, drug cartridges, micropumps, or other means known to the skilled artisan, as is well known in the art. Site-specific administration may be achieved by intratumoral, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavity, intracavitary, intracerebellar, intracerebroventricular, intracolic, endocervical, intragastric, intrahepatic, intracardiac, intraosseous, pelvic, intracardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery, for example.

The antibodies of the invention may be administered to a subject by any suitable route, for example by intravenous (i.v.) infusion or bolus injection, parenterally, intramuscularly or subcutaneously or intraperitoneally. The i.v. infusion may be administered within, for example, 15, 30, 60, 90, 120, 180 or 240 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

The dose administered to the subject is sufficient to alleviate or at least partially arrest the disease to be treated ("therapeutically effective amount"), and may sometimes be from 0.005 mg to about 100 mg/kg, for example from about 0.05 mg to about 30 mg/kg or from about 5mg to about 25 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg or about 24 mg/kg, or for example about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg/kg, but may even be higher, for example about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg.

A fixed unit dose, such as 50, 100, 200, 500 or 1000 mg, may also be administered, or the dose may be based on the body surface area of the patient, such as 500, 400, 300, 250, 200 or 100 mg/m ². Typically 1 to 8 doses (e.g., 1,2, 3,4, 5, 6, 7, or 8) can be administered to treat a patient, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses can be administered.

Administration of the antibodies of the invention may be repeated after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer. Repeated treatment procedures are also possible, as are chronic administration. Repeated administration may be at the same dose or at different doses. For example, the antibodies of the invention may be administered by intravenous infusion at weekly intervals at 8 mg/kg or 16 mg/kg for 8 weeks, followed by administration at 8 mg/kg or 16 mg/kg for 16 weeks every two weeks, followed by administration at 8 mg/kg or 16 mg/kg every four weeks.

For example, the antibodies of the invention may be provided as daily doses in an amount of about 0.1-100 mg/kg, e.g., 0.5、0.9、1.0、1.1、1.5、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、40、45、50、60、70、80、90 or 100 mg/kg, per day of at least one of 1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39 or 40 days after initiation of treatment, or alternatively, at least one week of weeks 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, using a single or divided dose every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

The antibodies of the invention may also be administered prophylactically in order to reduce the risk of developing cancer, delay the onset of event occurrence in the progression of cancer, and/or reduce the risk of recurrence of cancer while in remission.

The antibodies of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective for conventional protein formulations and well known lyophilization and reconstitution techniques can be employed.

Method and use

The antibodies of the invention, the pharmaceutical compositions of the invention, the polynucleotides of the invention, the vectors of the invention and the host cells of the invention have diagnostic in vitro and in vivo, as well as therapeutic and prophylactic utility. For example, an antibody of the invention, a pharmaceutical composition of the invention, a polynucleotide of the invention, or a vector of the invention may be administered to cells cultured in vitro or ex vivo, or to a subject to treat, prevent, and/or diagnose various disorders, such as cancer and infectious disorders.

The invention provides a method of modifying an immune response in a subject comprising administering to the subject an antibody of the invention, a pharmaceutical composition of the invention, a polynucleotide of the invention, a vector of the invention, and/or a host cell of the invention for a time sufficient to modify the immune response.

In some embodiments, the immune response is enhanced, stimulated, or upregulated.

In some embodiments described herein, the subject is a human patient.

In some embodiments described herein, the subject is a human patient in need of an enhanced immune response.

In some embodiments, the subject is immunocompromised.

In some embodiments, the subject is at risk of immune injury. Immunocompromised subjects may be undergoing or have undergone chemotherapy or radiation therapy.

In some embodiments, the subject is immunocompromised or at risk of being immunocompromised due to infection.

The antibodies of the invention, the pharmaceutical compositions of the invention, the polynucleotides of the invention, the vectors of the invention and/or the host cells of the invention are suitable for treating a subject suffering from a disorder which can be treated by boosting a T cell mediated immune response.

In some embodiments, the anti-TIGIT antibodies or trispecific anti-PD-1/CTLA-4/TIGIT antibodies used in the methods of the invention are those as defined in the disclosure.

In some embodiments, the anti-TIGIT antibody used in the methods of the invention is GB005-10-H0302. The amino acid sequences and nucleotide sequences of such antibodies are shown in table 5. In some embodiments, the trispecific anti-PD-1/CTLA-4/TIGIT antibody used in the methods of the invention is TsAb-GBD209-24-3. The amino acid sequences and nucleotide sequences of such antibodies are shown in table 6.

The invention also provides a method of inhibiting tumor cell growth in a subject comprising administering to the subject a therapeutically effective amount of an anti-TIGIT antibody of the invention or a trispecific anti-PD-1/CTLA-4/TIGIT antibody, a pharmaceutical composition of the invention, a polynucleotide of the invention, a vector of the invention, and/or a host cell of the invention for a time sufficient to inhibit tumor cell growth.

The invention also provides a method of treating cancer by administering to a subject in need thereof a therapeutically effective amount of an anti-TIGIT antibody of the invention or a trispecific anti-PD-1/CTLA-4/TIGIT antibody, a pharmaceutical composition of the invention, a polynucleotide of the invention, a vector of the invention, and/or a host cell of the invention for a time sufficient to treat cancer.

The cancer may be a hyperproliferative condition or disorder, a solid tumor, a hematological malignancy, a soft tissue tumor, or a metastatic lesion.

"Cancer" is intended to include all types of cancerous growth or oncogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs, regardless of the type of histopathology or stage of invasion. Examples of cancers include solid tumors, hematological malignancies, soft tissue tumors, and metastatic lesions. Exemplary solid tumors include malignant tumors of various organ systems, such as sarcomas and carcinomas (including adenocarcinomas and squamous cell carcinomas), such as those affecting the liver, lung, breast, lymphoid, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., kidney, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignant tumors such as most colon, rectum, renal cell carcinoma, liver, non-small cell lung, small intestine and esophagus cancers. Squamous cell carcinoma includes, for example, malignant tumors in the lung, esophagus, skin, head and neck region, mouth, anus, and cervix.

In some embodiments, the cancer is melanoma.

Metastatic lesions of the cancers described above may also be treated or prevented using the methods and antibodies of the invention described herein.

Exemplary cancers for which antibodies of the invention may be used to inhibit or reduce their growth include cancers that may respond to immunotherapy. Exemplary cancers include melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, lung cancer, metastatic malignant melanoma, clear cell cancer, hormone refractory prostate cancer, non-small cell lung cancer, or head and neck cancer. Refractory or recurrent malignant tumors can be treated with the antibodies of the invention described herein.

Other exemplary cancers that may be treated with the antibodies of the invention include anal, basal cell, biliary, bladder, bone, brain, and CNS cancers, fallopian tube, vaginal, vulvar, cutaneous or intraocular malignant melanoma, gastroesophageal cancer (astro-esophageal cancer), testicular, ovarian, pancreatic, rectal, uterine, primary CNS lymphoma; neoplasms of the Central Nervous System (CNS), cervical cancer, choriocarcinoma, rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, ocular cancer, intraepithelial neoplasms, kidney cancer, laryngeal cancer, liver cancer, small cell lung cancer, neuroblastoma, oral cancer (e.g., lip, tongue, mouth and throat), nasopharyngeal cancer, retinoblastoma, rhabdomyosarcoma, respiratory system cancer, sarcoma, thyroid cancer, urinary system cancer, hepatocellular carcinoma, anal region cancer, fallopian tube cancer, vaginal cancer, vulval cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, childhood solid tumor, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, kaposi's sarcoma, merck cell carcinoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including those induced by asbestos), and other cancers and sarcomas, and combinations of such cancers.

Exemplary hematological malignancies that can be treated with the antibodies of the present invention include leukemias, lymphomas, and myelomas, such as precursor B-cell lymphoblastic leukemia/lymphoma and B-cell non-hodgkin's lymphoma, acute promyelocytic leukemia, acute Lymphoblastic Leukemia (ALL), B-cell Chronic Lymphoblastic Leukemia (CLL)/Small Lymphoblastic Lymphoma (SLL), B-cell acute lymphoblastic leukemia, B-cell promyelocytic leukemia, lymphoplasmacytic lymphoma, mantle Cell Lymphoma (MCL), follicular Lymphoma (FL) (including low, medium, and high grade FL), skin follicular central lymphoma, marginal zone B-cell lymphoma (MALT type, nodular and spleen type), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), burkitt's Lymphoma (BL), plasma cell lymphoma, multiple Myeloma (MM), plasma cell leukemia, post-transplant lymphoproliferative disorder, macroglobulinemia, plasma cell disorder, anaplastic Large Cell Lymphoma (ALCL), T-cell leukemia, primary, lgic lymphoblastic lymphoma (lgic leukemia), and lymphoblastic leukemia (CML), acute lymphoblastic leukemia (lymphoblastic leukemia), and lymphoblastic leukemia (lymphoblastic leukemia).

"Plasma cell disorder" refers to a disorder characterized by clonal plasma cells and includes multiple myeloma, light chain amyloidosis, and Fahrenheit macroglobulinemia. Light chain amyloidosis and Fahrenheit macroglobulinemia can independently originate from multiple myeloma. They may also be present with multiple myeloma and develop before or after multiple myeloma develops.

Exemplary B-cell non-hodgkin's lymphomas are lymphomatoid granulomatosis, primary exudative lymphomas, intravascular large B-cell lymphomas, mediastinal large B-cell lymphomas, heavy chain diseases (including gamma, mu and a disease), lymphomas induced by therapy with immunosuppressants such as cyclosporine-induced lymphomas and methotrexate-induced lymphomas.

In some embodiments, the subject has a tumor that expresses PD-L1.

In some embodiments, the subject has a tumor that expresses CTLA-4.

In some embodiments, the subject has a tumor that expresses TIGIT.

In some embodiments, the subject has been treated with an anti-PD-1 antibody.

In some embodiments, the subject is refractory to treatment with an anti-PD-1 antibody.

In some embodiments, the subject has a tumor that recurs after treatment with an anti-PD-1 antibody.

In some embodiments, the subject has been treated with an anti-PD-1 antibody, e.g., KEYTRUDA ^® (palbociclizumab).

In some embodiments, the subject has been treated with an anti-PD-1 antibody, e.g., OPDIVO ^® (nivolumab).

In some embodiments, the subject is refractory to treatment with an anti-PD-1 antibody, e.g., KEYTRUDA ^® (palbociclizumab).

In some embodiments, the subject is refractory to treatment with an anti-PD-1 antibody, e.g., OPDIVO ^® (nivolumab).

In some embodiments, the subject has a recurrent tumor following treatment with an anti-PD-1 antibody (e.g., KEYTRUDA ^® (palbociclizumab)).

In some embodiments, the subject has a recurrent tumor following treatment with an anti-PD-1 antibody, e.g., OPDIVO ^® (nivolumab).

In some embodiments, the subject has been treated with or is being treated with an anti-PD-L1 antibody (e.g., MEDI-4736, MDX-1105, avistuzumab, or aletuzumab).

In some embodiments, the subject is refractory to treatment with an anti-PD-L1 antibody (e.g., MEDI-4736, MDX-1105, avistuzumab, or alemtuzumab).

In some embodiments, the subject has a recurrent tumor following treatment with an anti-PD-L1 antibody (e.g., MEDI-4736, MDX-1105, avistuzumab, or alemtuzumab).

In some embodiments, the subject has been treated with an anti-PD-L2 antibody or is being treated with an anti-PD-L2 antibody.

In some embodiments described herein, the subject is refractory to treatment with an anti-PD-L2 antibody.

In some embodiments, the subject has a tumor that recurs after treatment with an anti-PD-L2 antibody.

Various qualitative and/or quantitative methods may be used to determine the recurrent or refractory nature of the disease. Symptoms that may be associated with recurrence or resistance are, for example, a decline or plateau in the patient's health, or a re-establishment or worsening of various symptoms associated with solid tumors, and/or the spread of cancer cells from one location to other organs, tissues or cells in the body.

The invention also provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of an anti-TIGIT antibody of the invention or a trispecific anti-PD-1/CTLA-4/TIGIT antibody, wherein the subject is being treated with an anti-PD-1 antibody or has been treated with an anti-PD-1 antibody.

The invention also provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of an anti-TIGIT antibody of the invention or a trispecific anti-PD-1/CTLA-4/TIGIT antibody, wherein the subject is being treated with an anti-PD-L1 antibody or has been treated with an anti-PD-L1 antibody.

The invention also provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of an anti-TIGIT antibody of the invention or a trispecific anti-PD-1/CTLA-4/TIGIT antibody, wherein the subject is being treated with an anti-PD-L2 antibody or has been treated with an anti-PD-L2 antibody.

Combination therapy for cancer treatment

The antibodies of the invention, the pharmaceutical compositions of the invention, the polynucleotides of the invention, the vectors of the invention and/or the host cells of the invention may be administered in combination with a second therapeutic agent.

The antibodies of the invention, the pharmaceutical compositions of the invention, the polynucleotides of the invention, the vectors of the invention and/or the host cells of the invention may be administered in combination with one, two, three, four, five or six additional therapeutic agents.

"In combination with" means that the antibody of the invention and the at least one second therapeutic agent are administered simultaneously as a single agent, or sequentially as a single agent in any order. Generally, each agent is administered at a dosage and/or schedule determined for that agent.

In some embodiments, the second therapeutic agent modulates the activity of a molecule involved in the immune cycle of cancer, e.g., a molecule involved in a stimulatory or inhibitory pathway that functions in the release of cancer cell antigens, cancer antigen presentation, T cell initiation and activation, T cell migration into a tumor, T cell infiltration into a tumor, T cell recognition of cancer cells, and killing of cancer cells. Cancer immune circulation is described in Chen and Mellman (2013) Immunity 39:1-10. In some embodiments, the second therapeutic agent modulates the activity of a molecule involved in the modulation of the activity of regulatory T cells (tregs), a co-stimulatory or co-inhibitory ligand expressed on a tumor, an activating or inhibitory receptor on Natural Killer (NK) cells, or an immunosuppressive factor in the tumor microenvironment. Combination cancer immunotherapy is described in Manoney et al, (2015) Nature Reviews 14:561-584.

The second therapeutic agent generally enhances the activity of the stimulatory molecule and suppresses the activity of the inhibitory molecule, as is well known. Thus, "modulating" refers to the enhancement of an immune response by a second therapeutic agent, whether the agent itself is an agonist or an antagonist of a particular molecule.

The efficacy of the combinations described herein can be tested in animal models known in the art.

Examples

The invention will now be described with reference to the following specific non-limiting examples.

Examples

Example 1 production of anti-PD-1 antibodies, anti-CTLA-4 antibodies and anti-TIGIT antibodies

Anti-human PD-1 antibodies, anti-human CTLA-4 antibodies and anti-human TIGIT antibodies were generated by immunizing alpaca with recombinant human PD-1, human CTLA-4 and human TIGIT extracellular domain (ECD) proteins, respectively. Total RNA of PBMCs was extracted and cDNA was synthesized and amplified. The framework regions of the alpaca VH gene were replaced with human frameworks by CDR grafting techniques and cloned into expression vectors to generate corresponding clones of humanized antibodies. The resulting anti-PD-1 antibody GBD002-hS019-WS has the heavy chain variable region of SEQ ID NO. 4. The resulting anti-CTLA-4 antibody has the heavy chain variable region of SEQ ID NO. 10 (GBD 008-hS 005-3-2). The resulting anti-TIGIT antibody has the heavy chain variable region of SEQ ID NO. 16 (GB 005-10-H0302). All antibodies have human IgG1 constant regions.

Example 2 production of TsAb-GBD209-24-3 antibody

Construction of trispecific antibody TsAb-GBD209-24-3 the DNA sequences encoding the heavy chain variable regions of the anti-PD 1 and anti-TIGIT antibodies (SEQ ID NO: 4 and SEQ ID NO: 16, respectively) on the N-terminus of the Fc region of IgG1 LALA and the heavy chain variable region of the anti-CTLA 4 antibody (SEQ ID NO: 10) on the C-terminus of the Fc region of IgG1 LALA were cloned into pcDNA3.4 expression vectors. Joint 1 and/or joint 2 are added as flexible connections. The resulting trispecific antibody was designated "TsAb-GBD209-24-3". A schematic illustration of the TsAb-GBD209-24-3 structure is shown in FIG. 1.

Example 3 Generation of reference (BMK) antibodies

DNA sequences encoding the variable regions of the anti-PD-1 antibody palbociclizumab (as disclosed on the IMGT website), the anti-PD-1 antibody nivolumab (as disclosed in clone 5C4 of U.S. Pat. No. 5, 9084776B 2), the anti-CTLA-4 antibody ipilimumab (as disclosed in patent document U.S. Pat. No. 5, 20150283234), the anti-PD-1 xCTLA-4 bispecific antibody california Li Shan antibody (as disclosed on the IMGT website), the anti-PD-1 xCTLA-4 bispecific antibody MEDI5752 (as disclosed on the IMGT website), and the anti-TIGIT antibody tireli Li Youshan antibody (as disclosed on the IMGT website) were synthesized and expressed in Biointron or BioMetas, respectively. The obtained anti-PD-1 reference antibodies are named as palbociclizumab analogues and nivolumab analogues respectively. The obtained reference antibody against CTLA-4 is named as ipilimumab analogue. The resulting anti-TIGIT reference antibody was designated as a tireli Li Youshan anti-analog. The resulting anti-PD-1 xCTLA-4 bispecific antibodies were designated as the california Li Shan anti-analog and MEDI5752 analog, respectively.

EXAMPLE 4 binding of TsAb-GBD209-24-3, parent PD-1 (GBD 002-hS 019-WS), CTLA-4 (GBD 008-hS 005-3-2) or TIGIT (GB 005-10-H0302) nanobodies to cell surface antigens

For PD-1 binding, the binding of the antibodies of the disclosure was assessed by incubating HEK293T cells (Kyinno, catalog #KC-0204) overexpressing human PD-1 with serial dilutions of (1:3) antibodies. Cells were washed and binding was detected by flow cytometry (BD LSRFortessa) with APC anti-human IgG Fc antibodies (Jackson lab, catalogue # 309-605-008). The antibodies of the present disclosure show comparable or better binding capacity to HEK293T cell lines overexpressing human PD-1 compared to the baseline (i.e., palbociclizumab analogs) (fig. 2A).

For CTLA-4 binding, antibodies of the disclosure were evaluated for binding by incubating CHOK1 cells overexpressing human CTLA-4 (Kyinno catalog #KC-1406) with serial dilutions (1:5) of antibodies. Similar to the procedure for PD-1 binding, cells were finally analyzed by flow cytometry. The antibodies of the present disclosure show weaker binding capacity to CHOK1 cell lines overexpressing human CTLA-4 compared to baseline (i.e., ipilimumab analog) (fig. 2B).

For PD-1/CTLA-4/TIGIT binding, binding of the antibodies of the disclosure was assessed by incubating HEK293T cells (built-in) that overexpress human PD-1/CTLA-4/TIGIT with serial dilutions of (1:3) antibodies. Similar to the procedure for PD-1 binding, cells were finally analyzed by flow cytometry. The antibodies of the present disclosure show comparable or better binding capacity to HEK293T cell lines overexpressing human PD-1/CTLA-4/TIGIT compared to the parental anti-PD-1 (GBD 002-hS 019-WS) antibodies (fig. 2C).

For TIGIT binding, the binding of the antibodies of the disclosure was assessed by incubating CHOK1 cells overexpressing human TIGIT (Kyinno catalog #kc-2000) with serial dilutions (1:3) of antibodies. Cells were washed and binding was detected by flow cytometry (BD LSRFortessa) with APC anti-human IgG Fc antibodies (Jackson lab, catalogue # 309-605-008). The antibodies of the present disclosure showed comparable or better binding capacity to CHOK1 cell lines overexpressing human TIGIT compared to the baseline (i.e., tiri Li Youshan anti-analog) (fig. 7).

Example 5 PD-1/PD-L1 blocking Activity in Jurkat NFAT-Luc reporter assay

PD-1/PD-L1 Blockade ASSAY SYSTEM (J1252, promega) was used following the instructions provided by the manufacturer. Briefly, CHOK1-PD-L1-TCRa cells were plated at a volume of 100-. Mu.L in a white 96-well plate at 4X 10 ⁴ cells/well and incubated at 37℃for 16-18 hours. Antibodies were prepared in assay buffer (RPMI 1640 medium+1% FBS) at 2 x working concentration and 1:3 serial dilutions. Jurkat-PD1-NFAT-Luc cells were centrifuged and resuspended at 1X 10 ⁶ cells/mL in assay buffer. Then, 2 Xconcentrated antibody and Jurkat-PD1-NFAT-Luc cells were mixed at a volume ratio of 1:1, and incubated at 37℃for 6 hours. To measure luminescence signals, 100 μl of Bio-GloTM Reagent was added to each well at room temperature and the plate was incubated for 4 minutes and then read by Envision (PerkinElmer). Antibodies of the present disclosure significantly blocked the binding of PD-L1 to PD-1 (fig. 3).

Example 6 blocking of PD-1-PD-L2 binding

The blocking of PD-L2 ligand by antibodies of the present disclosure was evaluated by incubating HEK293T cells overexpressing human PD-1 with serial dilutions of (1:3) antibodies. Cells were washed twice and 1 μg/mL human PD-L2 was added to the designated row of assay plates, thoroughly mixed and incubated for 1 hour at 4 ℃. After incubation, cells were washed once and resuspended in 120 μl FACS buffer (BioLegend, catalog # 420201). Assay plates were tested by flow cytometry. The results are shown in fig. 4. Antibodies of the disclosure significantly block the binding of PD-L2 to PD-1.

EXAMPLE 7 blocking CTLA4-CD80/86 binding

The blocking of CD80 or CD86 ligands by antibodies of the present disclosure was evaluated by incubating CHOK1 cells overexpressing human CTLA-4 or HEK293T cells overexpressing human PD-1/CTLA-4/TIGIT with serial dilutions of (1:5) antibodies in the presence of an anti-human CD80 Fc & his & biotinylated tag (Sino Biological, catalog # 10698-H49H-B) or an anti-human CD86 Fc & his & biotinylated tag (Sino Biological, catalog # 10699-H02H). After incubation at 4 ℃ for 30 min, the cells were washed once and resuspended in 120 μl FACS buffer. Assay plates were tested by flow cytometry. The antibodies of the present disclosure only partially block CD80 or CD86-CTLA4 interactions on CHOK1 cells that overexpress human CTLA-4 (fig. 5A, 5B, 6A and 6B), but significantly block CD80 or CD86 binding to CTLA-4 on HEK293T cells that overexpress human PD-1/CTLA-4/TIGIT (fig. 5B and 6B).

Example 8 TIGIT/CD155 blocking Activity in Jurkat NFAT-Luc reporter assay

TIGIT/CD155 Blockade ASSAY SYSTEM (J2201, promega) was used following the instructions provided by the manufacturer. Briefly, CHOK1-CD155-TCRa cells were plated at 5X 10 ⁴ cells/well in a white 96-well plate at a volume of 100-. Mu.L and incubated at 37℃for 16-18 hours. Antibodies were prepared in assay buffer (RPMI 1640 medium+1% FBS) at 2 x working concentration and 1:3 serial dilutions. Jurkat-TIGIT-NFAT-Luc cells were centrifuged and resuspended at 5X 10 ⁵ cells/mL in assay buffer. Then, 2 Xconcentrated antibody and Jurkat-TIGIT-NFAT-Luc cells were mixed at a volume ratio of 1:1, and incubated at 37℃for 6 hours. To measure luminescence signals, 100 μl of Bio-GloTM Reagent was added to each well at room temperature and the plate was incubated for 5-10 minutes before reading through Envision (PerkinElmer). Antibodies of the present disclosure significantly blocked CD155 binding to TIGIT (fig. 8).

EXAMPLE 9 Mixed Lymphocyte Reaction (MLR)

Mixed Lymphocyte Responses (MLR) were used to test the agonism of antibodies to cytokine secretion and proliferation that activate cd4+ T cells. Human Peripheral Blood Mononuclear Cells (PBMCs) were freshly isolated from healthy donors. Human monocytes were isolated using easy sep ^TM human CD14 positive selection kit II (Stem cell, catalog # 17858) according to the manufacturer's instructions. Monocytes were cultured in complete RPMI-1640 medium with 50ng/ml recombinant human GM-CSF (R & D, catalog # 215-GM-500) and 50ng/ml rhIL-4 (R & D, catalog # 204-IL-500) for 5 to 7 days to differentiate into Immature Dendritic Cells (iDCs). Human cd4+ T cells were isolated using the EasySep ^TM human T cell isolation kit (Stemcell, catalog # 17951) according to the manufacturer's protocol. For allogeneic MLR, isolated cd4+ T cells were co-cultured with immature DCs. And different concentrations of antibody (1:10 dilution starting from 100 nM) were added to complete RPMI-1640 medium in 96-well round bottom plates. Plates were incubated at 37 ℃ in a 5% CO ₂ incubator. Ifnγ in the supernatant was quantified at day 5. The antibodies of the present disclosure showed significant T cell activation compared to the baseline as palbociclib analogs (fig. 9).

Example 10 PD-1 receptor Down-regulation assay

To determine the effect of antibody internalization on surface PD-1 levels, HEK 293T-human PD-1/CTLA-4/TIGIT cells were incubated with serial dilutions of control and test antibodies starting at a concentration of 500 nmol/L. Cells were added at 4 x 10 ⁵ cells/well followed by 20 hours incubation at 37 ℃ with 5% CO ₂. After incubation, cells were washed twice with FACS buffer to remove excess antibody and residual surface PD-1 receptor was detected by incubation with 10 μg/mL of non-competitive anti-PD-1 antibody labeled with AF-647 at 4 ℃ for 30 min. After staining, cells were fixed with 4% PFA for 10 min and analyzed using flow cytometry (BD LSRFortessa). Antibodies of the present disclosure showed significant down-regulation of PD-1 receptor (fig. 10).

EXAMPLE 11 efficacy study in A375 xenograft model

Antitumor activity of TsAb-GBD209-24-3 was measured in the A375 melanoma cell line (Cobioer, catalog #CBP 60329) xenograft model. Female nod.cg-Prkdc ^scidIl2rg^em1Smoc mice (Shanghai Model Organisms Center, inc) of 6-8 weeks of age were SC-implanted with 5×10 ⁶ a375 tumor cells each, and 6×10 ⁵ healthy PBMCs were injected intravenously, followed by randomization into a treatment group of 6 mice/group 11 days post-implantation. On days 0, 3, 7, 10 and 14 post-implantation, abs (TsAb-GBD 209-24-3 or control hIgG1-LALA abs) were administered via IP injection in a volume of 200 μl at the noted dose. Tumor measurements were recorded twice weekly for up to 18 days after continuous implantation, after which mice were euthanized. TsAb-GBD209-24-3 showed significant antitumor activity and good tolerability compared to the negative control (human IgG 1-LALA) (FIG. 11A). In addition, tsAb-GBD209-24-3 showed better anti-tumor efficacy in vivo in a dose-dependent manner compared to the MEDI5752 analog (PD-1 XCTLA-4 bsAb developed by AstraZeneca) (FIG. 11B).

Example 12 kinetic parameters of antibodies detected by Octet

Kinetic assays were performed by first capturing mAb using an anti-human Fc (AHC) Octet (Sartorius) biosensor followed by at least two baseline steps of 30 seconds each in HBS-EBT buffer. The biosensor of capture mAb was then immersed in wells containing different concentrations of antigen for 4-6 minutes followed by a dissociation time of 10-15 minutes in HBS-EBT buffer. The sensor of the capture mAb was also immersed in wells containing HBS-EBT buffer to allow single reference subtraction in order to compensate for the natural dissociation of the captured mAb. Binding sensorgrams were collected using a high sensitivity 16-channel detection mode on an Octet HTX biosensor. Unless otherwise indicated, fresh AHC biosensors were used without any regeneration step. Kinetic parameters of the antibodies of the present disclosure are shown in table 7.

TABLE 7 kinetic parameters of antibodies detected by Octet

Other embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Reference to the literature

Claims (22)

1. A trispecific antibody against PD-1/CTLA-4/TIGIT, comprising a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to TIGIT. 2. The anti-PD-1/CTLA-4/TIGIT trispecific antibody according to claim 1, wherein... a) The first domain that specifically binds to PD-1 includes a heavy chain variable region (VH), which contains heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO:1, 2, and 3, respectively. b) The second domain that specifically binds to CTLA-4 contains a heavy chain variable region (VH) comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO:7, 8, and 9, respectively; and/or c) The third domain that specifically binds to TIGIT contains a heavy chain variable region (VH), which includes heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO:13, 14, and 15, respectively. 3. The anti-PD-1/CTLA-4/TIGIT trispecific antibody according to claim 1 or 2, wherein... a) The first domain that specifically binds to PD-1 includes, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 4; b) The second domain that specifically binds to CTLA-4 contains, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 10; and/or c) The third domain that specifically binds to TIGIT contains, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 16. 4. The anti-PD-1/CTLA-4/TIGIT trispecific antibody according to any one of claims 1-3, wherein the antibody further comprises the Fc region of IgG. 5. The anti-PD-1/CTLA-4/TIGIT trispecific antibody according to claim 4, wherein the first domain, the second domain, the third domain and/or the Fc region are directly connected to each other or connected via one or more adapters. 6. The anti-PD-1/CTLA-4/TIGIT trispecific antibody according to claim 5, wherein the linkers are the same or different, and/or wherein the linkers are flexible linkers, and/or wherein the linkers are peptide linkers. 7. The anti-PD-1/CTLA-4/TIGIT trispecific antibody according to any one of claims 1-6, wherein the antibody comprises or is composed of the heavy chain of SEQ ID NO: 19. 8. The anti-PD-1/CTLA-4/TIGIT trispecific antibody according to any one of claims 1-7, wherein the antibody is an IgG1, IgG2, IgG3 or IgG4 isotype, optionally containing one, two, three, four, five, six, seven, eight, nine or ten substitutions in the Fc region. 9. An anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to TIGIT, comprising a heavy chain variable region (VH) including heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 13, 14, and 15, respectively. 10. The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 9, wherein the anti-TIGIT antibody comprises, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 16. 11. The anti-TIGIT antibody or antigen-binding fragment thereof according to claim 9 or 10, wherein the anti-TIGIT antibody further comprises the Fc region of IgG. 12. The anti-TIGIT antibody or its antigen-binding fragment according to claim 11, wherein the heavy chain variable region (VH) and the Fc region are directly connected to each other or connected via one or more adapters. 13. The anti-TIGIT antibody or its antigen-binding fragment according to claim 12, wherein the adapter is the same or different, and/or wherein the adapter is a flexible adapter, and/or wherein the adapter is a peptide adapter. 14. The anti-TIGIT antibody or antigen-binding fragment thereof according to any one of claims 9-13, wherein the anti-TIGIT antibody is an IgG1, IgG2, IgG3 or IgG4 isotype, optionally containing one, two, three, four, five, six, seven, eight, nine or ten substitutions in the Fc region. 15. A pharmaceutical composition comprising an anti-PD-1/CTLA-4/TIGIT trispecific antibody according to any one of claims 1-8, or an anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of claims 9-14, and a pharmaceutically acceptable carrier. 16. A polynucleotide encoding an anti-PD-1/CTLA-4/TIGIT trispecific antibody according to any one of claims 1-8 or an anti-TIGIT antibody according to any one of claims 9-14, or an antigen-binding fragment thereof. 17. A vector comprising the polynucleotide according to claim 16. 18. An isolated host cell comprising the vector according to claim 17. 19. A method for generating an anti-PD-1/CTLA-4/TIGIT trispecific antibody or an anti-TIGIT antibody or an antigen-binding fragment thereof, comprising culturing a host cell according to claim 18 under antibody-expressing conditions, and recovering and purifying the anti-PD-1/CTLA-4/TIGIT trispecific antibody or anti-TIGIT antibody generated from said host cell. 20. A method of treating cancer in a subject, comprising administering a therapeutically effective amount of an anti-PD-1/CTLA-4/TIGIT trispecific antibody according to any one of claims 1-8, an anti-TIGIT antibody according to any one of claims 9-14, a pharmaceutical composition according to claim 15, a polynucleotide according to claim 16, a carrier according to claim 17, or a host cell according to claim 18 to a subject in need of such treatment for a duration sufficient to treat the cancer. 21. The method of claim 20, wherein the cancer is a solid tumor or a hematologic malignancy. 22. The method of claim 21, wherein the solid tumor is melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, stomach cancer, ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, esophageal cancer or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary cancer, endometriosis, cervical cancer, or metastatic cancer lesions.