Anti-PD-1 / CTLA-4 / VEGF antibodies and uses thereof

Abstract

Provided are anti-PD-1 antibodies and tri-specific antibodies, or antigen-binding fragments thereof, that specifically bind to PD-1, CTLA-4, and VEGF, polynucleotides encoding the antibodies, or antigen-binding fragments thereof, and methods of making and using the same.

Description

Technical Field

[0001] This invention relates to anti-PD-1 antibodies, trispecific antibodies that specifically bind to PD-1, CTLA-4 and VEGF, or antigen-binding fragments thereof, polynucleotides encoding the antibodies or antigen-binding fragments thereof, and methods for preparing and using the above. Background Technology

[0002] PD-1 (programmed cell death protein 1) is a receptor primarily expressed on the surface of T cells. PD-1 modulates T cell function by inhibiting T cell activation and proliferation and promoting T cell exhaustion (Liu et al., 2021; Sharpe and Pauken, 2018). PD-1 is activated by its ligands PD-L1 and PD-L2, which are expressed on certain tumor cells and other antigen-presenting cells. In preclinical and clinical studies, PD-1 inhibition has been shown to enhance T cell activity and promote anti-tumor immune responses (Chen et al., 2021; B. Zhao, Zhao and Zhao, 2020). PD-1 inhibitors, such as nivolumab (Opidivo®) and pembrolizumab (Keytruda®), have been approved by the FDA for the treatment of several cancer types, including lung cancer, melanoma, and bladder cancer. Keytruda is used to treat several types of cancer, including melanoma, non-small cell lung cancer, and head and neck cancer. Nivolumab is used to treat cancers including melanoma, non-small cell lung cancer, and renal cell carcinoma.

[0003] VEGF (vascular endothelial growth factor) is a protein that plays a crucial role in angiogenesis (the process of forming new blood vessels). Because angiogenesis is essential for the growth and metastasis of solid tumors, VEGF is an important target for cancer therapy. VEGF is secreted by cells (including tumor cells) in the tumor microenvironment (TME). The presence of VEGF promotes the proliferation and migration of cells such as tumor cells (Melincovici et al., 2018; Simons, Gordon, and Claesson-Welsh, 2016). In preclinical and clinical studies, VEGF inhibition has been shown to interfere with tumor angiogenesis and reduce tumor growth (Baraniskin et al., 2019; Garcia et al., 2020; Y. Zhao et al., 2022). VEGF inhibitors, such as bevacizumab (Avastin®), which block the binding of VEGF to VEGFR1 and VEGFR2, thereby preventing downstream signaling and angiogenesis, have been approved by the FDA for the treatment of several cancer types, including colorectal cancer, non-small cell lung cancer, and hepatocellular carcinoma.

[0004] Cytotoxic lymphocyte-associated molecule-4 (CTLA-4, CD152) is another immune checkpoint protein that regulates T cell function. As the first clinically targeted immune checkpoint receptor expressed on T cells, CTLA-4 shares the same ligands as CD28, namely CD80 / B7-1 and CD86 / B7-2. CTLA-4 is constitutively expressed on Foxp3+ regulatory T cells (Tregs) and upregulated on other activated T cells (Jago, Yates, Câmara, Lechler, and Lombardi, 2004; Śledzińska, Menger, Bergerhoff, Peggs, and Quezada, 2015). The mechanism of CTLA-4 is thought to be the inhibition of T cell activation by surpassing CD28 in binding to CD80 / B7-1 and CD86 / B7-2 (Chikuma, 2017). CTLA-4 inhibitors (such as ipilimumab) have been approved by the FDA for the treatment of several types of cancer, including melanoma and prostate cancer.

[0005] Ipilimumab, the most well-known anti-CTLA4 antibody, was approved in 2011 for the treatment of advanced melanoma. Ipilimumab has demonstrated potent cancer immunotherapy efficacy in clinical trials, both as monotherapy (Hodi et al., 2010) and as part of combination therapy with nivolumab (Larkin et al., 2015). However, CTLA-4 therapy has shown serious immunotherapy-related adverse events (irAEs) (Calabrese, Calabrese, and Cappelli, 2018), particularly when combined with nivolumab, due to systemic T-cell activation via blocking the B7-CTLA-4 pathway, leading to decreased patient tolerance to the antibody (Bertrand, Kostine, Barnetche, Truchetet, and Schaeverbeke, 2015; Hodi, 2010). Nevertheless, CTLA-4 remains an important immunotherapy target due to its ability to induce durable immunity in cancer patients (Maio et al., 2015; Schadendorf et al., 2015). The main challenge in generating CTLA-4 antibodies lies in improving their safety and efficacy.

[0006] In summary, PD-1, CTLA-4, and VEGF are important targets for cancer therapy, and their inhibition has shown promising results in both preclinical and clinical studies. PD-1 and CTLA-4 inhibitors enhance T cell activity and promote anti-tumor immune responses, while VEGF inhibitors interfere with tumor angiogenesis and reduce tumor growth. However, drugs like ipilimumab have various side effects. Therefore, further research is needed to optimize the use of these inhibitors and explore their potential in combination with other cancer therapies. Summary of the Invention

[0007] This invention provides an isolated antibody or antigen-binding fragment thereof that binds to PD-1 (programmed cell death protein 1), comprising: a heavy chain antibody variable domain (VHH), wherein the VHH includes complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VHHCDR1 region contains an amino acid sequence having at least 80% identity with SEQ ID NO: 1, the VHHCDR2 region contains an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and the VHHCDR3 region contains an amino acid sequence having at least 80% identity with SEQ ID NO: 3.

[0008] In one embodiment, VHH comprises CDRs 1, 2, and 3 having the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively. In one embodiment, VHH comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 4. In one embodiment, VHH comprises, or consists of, the amino acid sequence of SEQ ID NO: 4.

[0009] In one implementation, the antibody or antigen-binding fragment specifically binds to PD-1.

[0010] In one implementation, the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0011] The present invention also provides an isolated antibody or antigen-binding fragment thereof comprising VHH CDR 1, 2 and 3 of the above-mentioned antibody or antigen-binding fragment thereof.

[0012] In one embodiment, the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

[0013] The present invention also provides an isolated antibody or antigen-binding fragment thereof, which cross-competes with the aforementioned antibody or antigen-binding fragment thereof.

[0014] The present invention also provides an isolated multispecific antibody or antigen-binding fragment thereof, comprising a first domain specifically binding to PD-1 and a second domain specifically binding to CTLA-4. In one embodiment, the antibody or antigen-binding fragment thereof further comprises one or more additional domains specifically binding to antigens other than PD-1 and CTLA-4. In one embodiment, the antibody or antigen-binding fragment thereof further comprises a domain specifically binding to VEGF.

[0015] In one embodiment, an anti-PD-1 / CTLA-4 / VEGF trispecific antibody or an antigen-binding fragment thereof is provided, comprising a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to VEGF.

[0016] In one embodiment, the first domain that specifically binds to PD-1 includes a heavy chain variable region (VH1), which includes heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 1, 2, and 3, respectively.

[0017] In one embodiment, the first domain that specifically binds to PD-1 comprises a heavy chain variable region (VH1), said VH1 containing an amino acid sequence having at least 80% identity with SEQ ID NO: 4. In one embodiment, the first domain that specifically binds to PD-1 comprises the heavy chain variable region (VH1) of SEQ ID NO: 4, or is composed of the heavy chain variable region (VH) of SEQ ID NO: 4.

[0018] In one embodiment, the second domain that specifically binds to CTLA-4 includes a heavy chain variable region (VH2), which includes heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 7, 8, and 9, respectively.

[0019] In one embodiment, the second domain that specifically binds to CTLA-4 comprises a heavy chain variable region (VH2) containing an amino acid sequence having at least 80% identity with SEQ ID NO: 10. In another embodiment, the second domain that specifically binds to CTLA-4 comprises, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 10.

[0020] In one embodiment, the third domain that specifically binds to VEGF comprises a heavy chain variable region (VH3) and a light chain variable region (VL), wherein the VH3 comprises heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) as specified in SEQ ID NOs: 13, 14, and 15, respectively; and the VL comprises light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) as specified in SEQ ID NOs: 19, 20, and 21, respectively.

[0021] In one embodiment, the third domain that specifically binds to VEGF comprises, or consists of, the heavy chain variable region (VH3) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO: 22.

[0022] In one embodiment, the third domain that specifically binds to VEGF comprises, or consists of, the heavy chain of SEQ ID NO: 17 and the light chain of SEQ ID NO: 23. In another embodiment, the third domain that specifically binds to VEGF comprises, or consists of, a full-length antibody comprising two heavy chains and two light chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. In one embodiment, the antigen-binding fragment of the full-length antibody is a Fab fragment or an scFv fragment.

[0023] In one embodiment, the isolated antibody or its antigen-binding fragment further comprises the Fc region of IgG. In one embodiment, the Fc region belongs to IgG1. In one embodiment, the Fc region belongs to IgG1 LALA (IgG1 with the Leu234Ala / Leu235Ala mutation).

[0024] In one embodiment, the antibody or antigen-binding fragment according to the invention belongs to the IgG1, IgG2, IgG3 or IgG4 isotype, and optionally contains one, two, three, four, five, six, seven, eight, nine or ten substitutions in the Fc region.

[0025] In one implementation, the first domain that specifically binds to PD-1 is located at the N-terminus of the Fc region.

[0026] In one implementation, the second domain that specifically binds to CTLA-4 is located at the N-terminus of the Fc region.

[0027] In one implementation, the third domain that specifically binds to VEGF is located at the N-terminus of the Fc region.

[0028] In one implementation, the first domain that specifically binds to PD-1 is located at the C-terminus of the Fc region.

[0029] In one implementation, the second domain that specifically binds to CTLA-4 is located at the C-terminus of the Fc region.

[0030] In one implementation, the third domain that specifically binds to VEGF is located at the C-terminus of the Fc region.

[0031] In one embodiment, a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, a third domain specifically binding to VEGF, and the Fc region are directly interconnected or interconnected via one or more linkers. In one embodiment, the linkers may be the same or different. In one embodiment, the linker is a flexible linker. In one embodiment, the linker is a peptide linker. In one embodiment, the linker is GGGGSGGGGS.

[0032] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention is represented from the N-terminus to the C-terminus by the following formula: Third structural domain – hinge area – Fc – joint – second structural domain – joint – first structural domain.

[0033] In one embodiment, the third domain specifically binding to VEGF is a full-length antibody. In one embodiment, the first domain specifically binding to PD-1 is optionally linked via a linker to the C-terminus of any one or both heavy chains of the full-length antibody, and the second domain specifically binding to CTLA-4 is optionally linked via a linker to the C-terminus of the first domain specifically binding to PD-1. In one embodiment, the second domain specifically binding to CTLA-4 is optionally linked via a linker to the C-terminus of any one or both heavy chains of the full-length antibody, and the first domain specifically binding to PD-1 is optionally linked via a linker to the C-terminus of the second domain specifically binding to CTLA-4. In one embodiment, the first domain specifically binding to PD-1 and the second domain specifically binding to CTLA-4 are linked to the C-terminus of two heavy chains of the full-length antibody in the same or different order. In one embodiment, two second domains that specifically bind to CTLA-4 are each linked to the C-terminus of the heavy chain of the full-length antibody via a linker, and two first domains that specifically bind to PD-1 are each linked to the C-terminus of the two second domains that specifically bind to CTLA-4 via a linker.

[0034] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention comprises, or is composed of, the heavy chain of SEQ ID NO: 27 and the light chain of SEQ ID NO: 23.

[0035] In one embodiment, the antibody according to the invention comprises one or two heavy chains and one or two light chains, or is composed of the latter. In one embodiment, the antibody according to the invention comprises two heavy chains and two light chains, or is composed of the latter. In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the invention comprises a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to VEGF. In one embodiment, a first domain specifically binding to PD-1 includes a heavy chain variable region (VH1), which contains heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 1, 2, and 3, respectively; a second domain specifically binding to CTLA-4 includes a heavy chain variable region (VH2), which contains heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 7, 8, and 9, respectively; and a third domain specifically binding to VEGF includes a heavy chain variable region (VH3) and a light chain variable region (VL), where VH3 contains heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 13, 14, and 15, respectively, and VL contains light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (HCDR3) of SEQ ID NOs: 19, 20, and 21, respectively. (LCDR3). In one embodiment, the first domain specifically binding to PD-1 comprises, or is composed of, the heavy chain variable region (VH1) of SEQ ID NO: 4; the second domain specifically binding to CTLA-4 comprises, or is composed of, the heavy chain variable region (VH2) of SEQ ID NO: 10; and the third domain specifically binding to VEGF comprises, or is composed of, the heavy chain variable region (VH3) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO: 22. In one embodiment, the heavy chain comprises, or is composed of, the amino acid sequence of SEQ ID NO: 27 and the light chain of SEQ ID NO: 23.

[0036] The present invention also provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.

[0037] The present invention also provides a heavy chain variable region (VH) or a polynucleotide of the heavy chain encoding the antibody or antigen-binding fragment thereof of the present invention.

[0038] The present invention also provides a polynucleotide encoding the antibody or antigen-binding fragment thereof of the present invention.

[0039] The present invention also provides a vector comprising the polynucleotide of the present invention.

[0040] The present invention also provides a host cell comprising the vector of the present invention.

[0041] The present invention also provides a method for generating the antibodies of the present invention, the method comprising culturing the host cells of the present invention under conditions expressing the antibody or an antigen-binding fragment thereof, and recovering the antibodies generated by the host cells.

[0042] The present invention also provides a method for treating cancer in a subject, the method comprising administering to the subject in need a therapeutically effective amount of an isolated antibody or an antigen-binding fragment thereof for a duration sufficient to treat the cancer.

[0043] The present invention also provides the use of the isolated antibody or antigen-binding fragment thereof of the present invention in the preparation of a medicament for treating cancer in subjects in need.

[0044] The present invention also provides isolated antibodies or antigen-binding fragments thereof for use in treating cancer in subjects in need. Attached Figure Description

[0045] Figure 1 A schematic diagram of the structure of the trispecific antibody GBD214-33-03 (anti-PD1 / CTLA4 / VEGF trispecific antibody) according to the present invention is shown.

[0046] Figure 2 The binding specificity of different antibodies to human PD1 protein was demonstrated. Results showed the binding of GBD214-33-03 to PD1 in HEK293-PD1 cells. GBD214-33-03 exhibited strong binding to HEK293-PD1 cells with a median fluorescence intensity (MFI) similar to its PD1 parent GBD002-hS019-WS (anti-PD1 antibody) and pembrolizumab.

[0047] Figure 3 The performance of different antibodies in the PD1 reporter gene assay was demonstrated. The PD1 reporter gene assay showed that GBD214-33-03 had a similar maximum luminescent signal to pembrolizumab and AK104 (an anti-PD1 / CTLA4 bispecific antibody from Akesobio, Inc.). This result indicates that GBD214-33-03 can block the interaction between PD1 and its ligand PD-L1.

[0048] Figure 4The performance of different antibodies in a mixed lymphocyte reaction (MLR) assay was demonstrated. Results showed that the maximum IFN-γ production of GBD214-33-03 in the MLR assay was similar to that of its parent, GBD002-hS019-WS, and pembrolizumab. This result indicates that GBD214-33-03 possesses potent immunogenicity similar to pembrolizumab.

[0049] Figure 5 The VEGF binding of different antibodies was shown. Results showed that GBD214-33-03 exhibited similar VEGF binding to bevacizumab compared to bevacizumab.

[0050] Figure 6 The study demonstrated the VEGF blocking effects of different antibodies. Results showed that GBD214-33-03 inhibited the binding of VEGFR2 to VEGF165, similar to bevacizumab.

[0051] Figure 7 The performance of different antibodies in VEGF reporter gene assays was demonstrated. Results showed that GBD214-33-03 inhibited VEGF165-induced downstream NFAT signaling by blocking the binding of VEGFR2 to VEGF165, similar to bevacizumab.

[0052] Figure 8a and 8b The binding specificity of different antibodies to human CTLA4 protein was demonstrated. Results showed that in CHOK1-CTLA4 cells, GBD214-33-03 had a lower MFI compared to ipilimumab, suggesting that GBD214-33-03 binds to the CTLA4 portion of CHOK1-CTLA4 cells. The CTLA4 parent GBD008-hS005-3-2 (anti-CTLA4 antibody) had a weaker CTLA4 binding ability compared to ipilimumab. In CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed a stronger binding than its PD1 parent GBD002-hS019-WS.

[0053] Figures 9a to 9d The study demonstrated the blocking effect of different antibodies on the binding of CD80 and CD86 to CTLA4. Results showed that GBD214-33-03 partially inhibited the binding of CD80 / CD86 to CTLA4 in both CHOK1-CTLA4 and CHOK1-PD1-CTLA4 cells. In both CHOK1-CTLA4 and CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed weaker blocking ability compared to ipilimumab.

[0054] Figure 10The study demonstrated the downregulation of the PD1 receptor in CHOK1-PD1-CTLA4 cells by different antibodies. The results showed that the downregulation of PD1 in cells was due to CTLA4 internalization. The percentage of PD1 downregulation was measured. GBD214-33-03 showed similar PD1 internalization compared to AK104.

[0055] Figure 11a and 11b The in vivo antitumor efficacy of different antibodies in the A375 PBMC mouse model was demonstrated. Results showed that in the A375 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) had better efficacy compared to the combination of AK112 (Akesobio, Inc.'s anti-PD1 / VEGF bispecific antibody) (1 mg / kg, 5 mg / kg, and 20 mg / kg), AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), pembrolizumab, bevacizumab, and ipilimumab-LALA (ipilimumab with Leu234Ala / Leu235Ala mutations) (15 mg / kg + 15 mg / kg + 15 mg / kg). For all treatment groups, the mean body weight of mice decreased from day 7, with a decrease not exceeding 15%.

[0056] Figure 12a and 12b The in vivo antitumor efficacy of different antibodies in the HT29 PBMC mouse model was demonstrated. Results showed that GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) had better efficacy in the HT29 PBMC model compared to AK112 (1 mg / kg, 5 mg / kg, and 20 mg / kg), AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), and the combination of pembrolizumab, bevacizumab, and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg). For all treatment groups, the mean body weight of mice remained relatively stable, with a decrease of no more than 15%. Detailed Implementation

[0057] All publications cited in this specification, including but not limited to patents and patent applications, are incorporated herein by reference as if fully explained.

[0058] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless otherwise defined, 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 pertains.

[0059] Although any methods and materials similar to or equivalent to those described herein may be used in practice for testing the invention, exemplary materials and methods are described herein. The following terminology will be used in describing and claiming protection of the invention.

[0060] As used in this specification and the appended claims, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, reference to “a cell” includes a combination of two or more cells, and so on.

[0061] "Specific binding," "specifically binding," or simply "binding" refers to the binding of an antibody to an antigen or an epitope within an antigen with a higher affinity than to other antigens. Typically, antibodies bind to antigens or epitopes within antigens with an affinity of approximately 1 × 10⁻⁶. -8 M or lower, for example, about 1×10 -9 M or lower, approximately 1×10 -10 M or lower, approximately 1×10 -11 M or lower, or approximately 1×10 -12 M or a lower equilibrium dissociation constant (K) D ) combined, where K D It is usually the K+ that binds to non-specific antigens (such as BSA, casein). D At most 1 / 100. The dissociation constant can be determined using standard procedures. However, antibodies that specifically bind to an antigen or an epitope within an antigen may exhibit cross-reactivity with other related antigens, such as with the same antigen (homologous) from other species, such as humans or monkeys, for example, cynomolgus monkeys. Macaca fascicularis , cynomolgus, cyno), chimpanzee ( Pan troglodytes chimpanzee, chimp) or common marmoset ( Callithrix jacchus (common marmoset, marmoset). When a monospecific antibody binds specifically to one antigen or one epitope, a bispecific antibody binds specifically to two different antigens or two different epitopes.

[0062] "Antibody" refers to immunoglobulin molecules in a broad sense, including monoclonal antibodies (including mouse, human, humanized, and chimeric monoclonal antibodies), antigen-binding fragments, bispecific or multispecific antibodies, dimer antibodies, tetramer or multimer antibodies, single-chain antibodies, domain antibodies, and any other modified conformation of an immunoglobulin molecule containing an antigen-binding site of desired specificity. A "full-length antibody" comprises two heavy (H) chains and two light (L) chains linked together by disulfide bonds, as well as its multimer (e.g., IgM). Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (containing domain CH1, hinge CH2, and CH3). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)), which alternate with framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0063] The "complementarity-determining region (CDR)" is the "antigen-binding site" in an antibody. CDRs can be defined using various terms: (i) Complementarity-determining regions (CDRs) (three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3)) based on sequence variability (Wu and Kabat, (1970)). J Exp Med 132:211-50; Kabat et al. , (ii) “Hypervariate region”, “HVR” or “HV” (three in VH (H1, H2, H3) and three in VL (L1, L2, L3)) refers to, as in Chothia and Lesk (Chothia and Lesk, (1987)). Mol Biol The region of highly variable structure within the variable domain of an antibody, as defined in 196:901-17. The International Immunogenetics (IMGT) database (http: / / www.imgt.org) provides standardized numbers and definitions for antigen-binding sites. The correspondence between CDR, HV, and IMGT classifications is discussed in Lefranc et al. , (2003) Dev Comparat ImmunolAs described in 27:55-77. Unless otherwise expressly stated in the specification, the terms “CDR”, “HCDR1”, “HCDR2”, “HCDR3”, “LCDR1”, “LCDR2”, and “LCDR3” as used herein include the CDR as defined by any of the foregoing methods, Kabat, Chothia, or IMGT.

[0064] Immunoglobulins can be classified into five major classes based on the amino acid sequence of their constant domains in the heavy chain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into the following isotypes: IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chains of any vertebrate species can be classified into one of two distinct types based on the amino acid sequence of their constant domains: kappa (κ) and lambda (λ).

[0065] "Isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies with 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 / VEGF trispecific antibody according to the invention, the trispecific antibody specifically binds to PD-1, CTLA-4, and VEGF, and is substantially free of antibodies that specifically bind to antigens other than PD-1, CTLA-4, and VEGF. "Isolated antibody" encompasses antibodies isolated to a high degree of purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure.

[0066] "Recombinant" refers to antibodies and other proteins that are prepared, expressed, generated, or isolated through recombination.

[0067] An epitope is an antigenic motif that an antibody specifically binds to. Epitopes typically consist of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups, such as amino acids or polysaccharide side chains, and may possess specific three-dimensional structural features and specific charge characteristics. Epitopes can be composed of continuous and / or discontinuous amino acids forming a conformational spatial unit. For discontinuous epitopes, amino acids from different parts of the linear antigen sequence are brought close together in three-dimensional space through protein folding. The identification of an antibody epitope depends on the method used.

[0068] "Multispecific" refers to an antibody that specifically binds to at least two different antigens or two different epitopes within an antigen (e.g., three, four, or five different antigens or epitopes). "Trispecific" antibodies refer to antibodies that specifically bind to three different antigens or three different epitopes within the same antigen. Trispecific antibodies may exhibit cross-reactivity with other related antigens, such as cross-reactivity with the same antigen (homologous) from other species, such as humans or monkeys, for example, cynomolgus monkeys. Macaca fascicularis , cynomolgus, cyno), chimpanzee ( Pan troglodytes chimpanzee (chimp) or common marmoset ( Callithrix jacchus (common marmoset, marmoset), or can bind to epitopes shared between two or more different antigens.

[0069] A "vector" is a polynucleotide capable of replicating within or moving between biological systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selection markers that function to facilitate or maintain the replication of the polynucleotide within the biological system. Examples of such biological systems include cells, viruses, animals, plants, and reconstructed biological systems utilizing biological components capable of replicating vectors. The polynucleotide containing the vector can be a DNA or RNA molecule or a hybrid thereof.

[0070] "Expression vector" refers to a vector that can be used in biological systems or reconstructed biological systems to guide the translation of polypeptides encoded by polynucleotide sequences present in the expression vector.

[0071] A "polynucleotide" is a synthetic molecule that consists of chains of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

[0072] “Overexpression,” “overexpressing,” and “overexpressing” are used interchangeably and refer to a sample, such as cancer cells, malignant cells, or cancerous tissue, that has measurably higher levels of PD-1, CTLA-4, or VEGF or their ligands compared to a reference sample. Overexpression can be caused by gene amplification or increased transcription or translation. Protein expression and overexpression in a sample can be measured in live or lysed cells using known assays such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassay. Polynucleotide expression and overexpression in a sample can be measured, for example, using fluorescence in situ hybridization, Southern blotting, or PCR. A protein or polynucleotide is overexpressed when its level is at least 1.5-fold or statistically significant compared to a reference sample. The selection of the reference sample is known.

[0073] "Sample" refers to a similar fluid, cell, or tissue isolated from a subject, or a collection of similar fluids, cells, or tissues present in the subject's body. Exemplary samples are biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, teardrops, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites (such as ascites associated with non-solid tumors), fluids from the pleural cavity, pericardial cavity, peritoneal cavity, abdominal cavity, and other body cavities, fluids collected by bronchial lavage, liquid solutions that have come into contact with the subject or biological sources (e.g., cell and organ culture media, including cell or organ conditioned media, lavage fluid, etc.), tissue biopsies, tumor tissue removed by fine-needle aspiration or surgical resection.

[0074] “Cancer cells” or “tumor cells” refer to cancer cells, precancerous cells, or transformed cells in vivo, in vitro, or in tissue culture that exhibit spontaneous or induced phenotypic changes. These changes do not necessarily involve the uptake of new genetic material. While transformation can result from infection with transforming viruses and the incorporation of new genomic nucleic acids, the uptake of exogenous nucleic acids can also occur spontaneously or after exposure to carcinogens, thereby mutating endogenous genes. Transformation / cancer is exemplified by: morphological changes in vitro, in vivo, and in vitro; cell immortalization; abnormal growth control; lesion formation; proliferation; malignancy; adjustment of tumor-specific marker levels; invasiveness; and tumor growth in suitable animal hosts (such as nude mice) (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd edition, 1994)).

[0075] "About" means within an acceptable range of error for a particular value, as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless otherwise expressly stated elsewhere in the embodiments or specification, in the context of a particular analysis, result, or implementation, "about" means within a standard deviation according to practice in the art, or within a range not exceeding 5%, whichever is greater.

[0076] The terms "anti-PD-1 / CTLA-4 / VEGF trispecific antibody," "PD-1 / CTLA-4 / VEGF trispecific antibody," "PD-1 / CTLA-4 / VEGF antibody," or "trispecific anti-PD-1 / CTLA-4 / VEGF antibody" refer to molecules containing at least one binding domain specifically binding to PD-1, at least one binding domain specifically binding to CTLA-4, and at least one binding domain specifically binding to VEGF. The binding domains specifically binding to PD-1, CTLA-4, and VEGF are typically VH / VL pairs or VH only. Trispecific anti-PD-1 / CTLA-4 / VEGF antibodies can be monovalent or bivalent in their binding to PD-1, CTLA-4, or VEGF.

[0077] "Valence" refers to the specific number of binding sites in a molecule that specifically target antigens. Therefore, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites in a molecule, respectively.

[0078] The term "single-chain Fv" or "scFv" antibody refers to a V antibody containing an antibody. H and V L Antibody fragments containing domains, wherein these domains are present within a single polypeptide chain. Typically, the Fv polypeptide further contains V... H and V L The polypeptide linkers between the domains allow scFv to form the desired antigen-binding structures. For a review of scFv, see Pluckthun (1994). The Pharmacology of Monoclonal Antibodies Volume 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315. See also International Publication No. WO 88 / 01649 and U.S. Patent Nos. 4,946,778 and 5,260,203. In one embodiment, scFv includes V from the N-terminus to the C-terminus. H Region, peptide linker and V L The region (VH-VL form). In another implementation, scFv contains V from the N-terminus to the C-terminus. L Region, peptide linker and VH Zone (VL-VH form).

[0079] As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, which are contained within the same polypeptide chain and a light chain variable domain (V). L ) connected heavy chain variable structural domain (V H (V) H -V L or V L -V H By using a linker that is too short to allow pairing between two domains on the same strand, these two domains are forced to pair with complementary domains on another strand, resulting in two antigen-binding sites. A more complete description of biantibodies can be found 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 Holliger and Hudson (2005). Nat. Biotechnol. 23:1126-1136.

[0080] The “Fab” comprises 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. According to this disclosure, the VH, CH1, VL, and CL regions in the Fab can be arranged in various ways to confer antigen-binding capability. For example, the VH and CH1 regions may be on a single polypeptide chain, while the VL and CL regions may be on separate polypeptide chains. Alternatively, the VH, CH1, VL, and CL regions may all be on the same polypeptide chain, optionally arranged in different orders.

[0081] Antigen-specific CD4 + or CD8 + "T cells" refer to CD4 cells activated by specific antigens or their immune epitopes. + or CD8 + T cells.

[0082] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Unless otherwise stated, the terms "patient" or "subject" are used interchangeably.

[0083] "Treatment" refers to therapeutic treatment aimed at slowing (reducing) undesirable physiological changes or diseases (such as the development or spread of tumors or tumor cells) or providing beneficial or desired clinical outcomes during treatment. Beneficial or desired clinical outcomes include symptom relief, reduction of disease severity, stabilization (i.e., no worsening) of the disease state, delay or slowing disease progression, absence of metastasis, improvement or relief of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also refer to extended survival compared to the expected survival of a subject without treatment. Subjects requiring treatment include those who already have undesirable physiological changes or diseases, and those who are prone to developing such changes or diseases.

[0084] "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome at the necessary dosage and time. The therapeutic effective dose of the antibody of the present invention may vary depending on factors such as disease state, individual age, sex, weight, and the ability of the antibody of the present invention to elicit the desired response in the individual. Exemplary indicators of effective treatment or treatment combinations include, for example, improvement in the patient's health, reduction in tumor burden, cessation or slowing of tumor growth, and / or failure of cancer cells to metastasize to other parts of the body.

[0085] Unless otherwise expressly stated, the amino acid residues in the constant regions of the antibody throughout this specification are numbered according to the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991).

[0086] This article uses the conventional single-letter and three-letter amino acid codes, as shown in Table 1.

[0087] Table 1: Single-letter and three-letter amino acid codes amino acids Three-letter code Single-letter codes alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C glutamic acid Gln E glutamine Glu Q glycine Gly G Histidine His H Isoleucine Ile I Lysine Lys K Methionine Met M Phenylalanine Phe F proline Pro P Serine Ser S threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Composition of matter This invention provides anti-PD-1 antibodies, anti-PD-1 / CTLA-4 antibodies, and particularly trispecific antibodies or antigen-binding fragments thereof that specifically bind to PD-1, CTLA-4, and VEGF. This invention also provides polynucleotides or complementary nucleic acids thereof encoding the antibodies or antigen-binding fragments thereof, vectors, host cells, and methods for preparing and using them.

[0088] In the context of this disclosure, the antibodies of the present invention include, where applicable, their antigen-binding fragments. For example, isolated anti-PD-1 antibodies or their antigen-binding fragments may be simply referred to as "isolated anti-PD-1 antibodies," and so on.

[0089] Antibody This invention provides isolated antibodies or antigen-binding fragments thereof that bind to PD-1 (programmed cell death protein 1), comprising: a heavy chain antibody variable domain (VHH), said VHH comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1; the VHH CDR2 region comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 2; and the VHH CDR3 ... 3. An amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0090] In one embodiment, VHH comprises CDRs 1, 2, and 3, which have the amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively.

[0091] In one embodiment, VHH comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. In one embodiment, VHH comprises, or is composed of, the amino acid sequence of SEQ ID NO: 4.

[0092] In one embodiment, the antibody or antigen-binding fragment thereof that binds to PD-1 comprises a heavy chain having an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5. In another embodiment, the antibody or antigen-binding fragment thereof that binds to PD-1 comprises a heavy chain that comprises or is composed of the amino acid sequence of SEQ ID NO: 5.

[0093] In one embodiment, the antibody or antigen-binding fragment specifically binds to PD-1. In one embodiment, the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0094] The present invention also provides isolated antibodies or antigen-binding fragments thereof, comprising VHH CDR 1, 2 and 3 of the aforementioned antibodies or antigen-binding fragments thereof.

[0095] In one embodiment, the antibody or its antigen-binding fragment comprises VHH CDRs 1, 2, and 3, each comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1, 2, and 3, respectively. In one embodiment, the antibody or its antigen-binding fragment comprises VHH CDRs 1, 2, and 3 as shown in SEQ ID NO: 1, 2, and 3, respectively.

[0096] In one embodiment, the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains. In another embodiment, the antibody or antigen-binding fragment comprises two, three, four, five, six, seven, eight, nine, or ten VHHs.

[0097] The present invention also provides isolated antibodies or antigen-binding fragments thereof that cross-compete with the antibodies or antigen-binding fragments thereof described above. In one embodiment, the antibody or antigen-binding fragment thereof cross-compete with antibodies or antigen-binding fragments thereof comprising VHH CDRs 1, 2, and 3, each of VHH CDRs 1, 2, and 3 comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1, 2, and 3, respectively. In one embodiment, the antibody or antigen-binding fragment thereof cross-compete with antibodies or antigen-binding fragments thereof comprising VHH CDRs 1, 2, and 3 shown in SEQ ID NO: 1, 2, and 3, respectively. In one embodiment, the antibody or its antigen-binding fragment cross-competes with an antibody or its antigen-binding fragment comprising VHH, wherein the VHH comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. In another embodiment, the antibody or its antigen-binding fragment cross-competes with an antibody or its antigen-binding fragment comprising VHH, wherein the VHH comprises or is composed of the amino acid sequence of SEQ ID NO: 4.

[0098] The present invention also provides isolated multispecific antibodies or antigen-binding fragments thereof, comprising a first domain specifically binding to PD-1 and a second domain specifically binding to CTLA-4. In one embodiment, the antibody further comprises one or more additional domains specifically binding to antigens other than PD-1 and CTLA-4. In one embodiment, the additional domains specifically binding to antigens other than PD-1 and CTLA-4 include a third domain specifically binding to VEGF.

[0099] In one embodiment, an isolated anti-PD-1 / CTLA-4 / VEGF trispecific antibody or its antigen-binding fragment is provided, comprising a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to VEGF.

[0100] In some embodiments, the antibody or its antigen-binding fragment of the present invention enhances antigen-specific CD4. + or CD8 + T cell activation.

[0101] In some embodiments, the antibody or antigen-binding fragment of the present invention inhibits the binding of PD-1 to PD-L1 and PD-L2.

[0102] In some embodiments, the antibody or antigen-binding fragment of the present invention inhibits the binding of CTLA-4 to CD80 and CD86.

[0103] In some embodiments, the antibody or its antigen-binding fragment of the present invention inhibits the binding of VEGF to VEGFR1 and / or VEGFR2.

[0104] In some embodiments, the antibody or antigen-binding fragment of the present invention induces PD-1 internalization on the cell surface.

[0105] In one embodiment, the first domain that specifically binds to PD-1 comprises or is composed of a heavy chain variable region (VH1), said VH1 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), wherein each of HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1, 2, and 3, respectively.

[0106] In one embodiment, the first domain that specifically binds to PD-1 includes or is composed of a heavy chain variable region (VH1), said VH1 comprising heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) respectively, SEQ ID NO: 1, 2, and 3.

[0107] In one embodiment, the second domain that specifically binds to CTLA-4 comprises or is composed of a heavy chain variable region (VH2), said VH2 comprising heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), wherein each of HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 7, 8, and 9, respectively.

[0108] In one embodiment, the second domain that specifically binds to CTLA-4 includes or is composed of a heavy chain variable region (VH2), said VH2 comprising heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 7, 8, and 9, respectively.

[0109] In one embodiment, the third domain that specifically binds to VEGF comprises or is composed of a heavy chain variable region (VH3) and a light chain variable region (VL), wherein the VH3 comprises heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), wherein each of HCDR1, HCDR2, and HCDR3 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13, 14, and 15, respectively; and the VL comprises light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3), wherein each of LCDR1, LCDR2, and LCDR3 comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15, respectively. 19, 20 and 21 have amino acid sequences with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0110] In one embodiment, the third domain that specifically binds to VEGF comprises or consists of a heavy chain variable region (VH3) and a light chain variable region (VL), wherein the VH3 comprises heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) as specified in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) as specified in SEQ ID NOs: 19, 20, and 21, respectively.

[0111] In one embodiment, the first domain that specifically binds to PD-1 comprises, or is composed of, the heavy chain variable region (VH1) of SEQ ID NO: 4, wherein VH1 optionally has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen conserved amino acid substitutions. Optionally, any substitution is not within the CDR.

[0112] In one embodiment, the first domain comprises or is composed of a heavy chain variable region (VH1), said VH1 having an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 4. Optionally, any differences in sequence from SEQ ID NO: 4 are not within the CDR.

[0113] In one embodiment, the second domain that specifically binds to CTLA-4 comprises or is composed of the heavy chain variable region (VH2) of SEQ ID NO: 10, wherein VH2 optionally has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen conserved amino acid substitutions. Optionally, any substitution is not within the CDR.

[0114] In one embodiment, the second domain comprises or is composed of a heavy chain variable region (VH2), said VH2 having an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 10. Optionally, any differences in sequence from SEQ ID NO: 10 are not within the CDR.

[0115] In one embodiment, the third domain that specifically binds to VEGF comprises, or consists of, the heavy chain variable region (VH3) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO: 22, wherein VH3 and VL optionally each have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen conserved amino acid substitutions. Optionally, any substitution is not within the CDR.

[0116] In one embodiment, the third domain comprises, or consists of, a heavy chain variable region (VH3) and a light chain variable region (VL), wherein the VH3 has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 16, and the VL has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 22. Optionally, any differences from the sequences of SEQ ID NO: 16 and / or SEQ ID NO: 22 are not within the CDR.

[0117] In one implementation, the first, second, and third domains are independently of each other chimeric antibody, humanized antibody, human antibody, single-chain antibody, Fv, Fab, F(ab′)2, Fd, single-chain Fv molecule (scFv), biantibody, or single-domain antibody (dAb).

[0118] In one implementation, the first, second, and third domains are humanized independently of each other. In another implementation, the first and second domains are humanized independently of each other as VHH.

[0119] In one embodiment, the antibody according to the invention comprises only one heavy chain. In one embodiment, the antibody according to the invention comprises one heavy chain and one light chain. In one embodiment, the antibody according to the invention comprises two heavy chains. In one embodiment, the antibody according to the invention comprises two heavy chains and two light chains.

[0120] In one embodiment, the antibody according to the invention comprises one heavy chain. In one embodiment, the antibody according to the invention comprises one heavy chain and one light chain. In one embodiment, the antibody according to the invention comprises two heavy chains. In one embodiment, the antibody according to the invention comprises two heavy chains and two light chains.

[0121] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody includes a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and / or a third domain that specifically binds to VEGF. In one embodiment, a first domain specifically binding to PD-1 includes a heavy chain variable region (VH1), which contains heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 1, 2, and 3, respectively; a second domain specifically binding to CTLA-4 includes a heavy chain variable region (VH2), which contains heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 7, 8, and 9, respectively; and / or a third domain specifically binding to VEGF includes a heavy chain variable region (VH3) and a light chain variable region (VL), where VH3 contains heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NOs: 13, 14, and 15, respectively, and VL contains light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (HCDR3) of SEQ ID NOs: 19, 20, and 21, respectively. (LCDR3).

[0122] In one embodiment, the first domain specifically binding to PD-1 comprises, or is composed of, the heavy chain variable region (VH1) of SEQ ID NO: 4; the second domain specifically binding to CTLA-4 comprises, or is composed of, the heavy chain variable region (VH2) of SEQ ID NO: 10; and / or the third domain specifically binding to VEGF comprises, or is composed of, the heavy chain variable region (VH3) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO: 22.

[0123] In one embodiment, the third domain that specifically binds to VEGF comprises, or is composed of, the heavy chain of SEQ ID NO: 17 and the light chain of SEQ ID NO: 23. In another embodiment, the third domain that specifically binds to VEGF comprises, or is composed of, a full-length antibody comprising, two heavy chains and two light chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. In one embodiment, the antigen-binding fragment of the full-length antibody is a Fab fragment or an scFv fragment.

[0124] In one embodiment, the antibody or its antigen-binding fragment further comprises the Fc region of IgG. In some embodiments, the antibody or its antigen-binding fragment is an IgG1, IgG2, IgG3, or IgG4 isotype. In one embodiment, the antibody or its antigen-binding fragment is an IgG1 LALA isotype. As is known in the art, the term "LALA" refers to the introduction of an amino acid substitution L234A / L235A (Kabat nomenclature).

[0125] In one embodiment, a first domain that specifically binds to PD-1 is located at the N-terminus of the Fc region. In one embodiment, a second domain that specifically binds to CTLA-4 is located at the N-terminus of the Fc region. In one embodiment, a third domain that specifically binds to VEGF is located at the N-terminus of the Fc region.

[0126] In one embodiment, a first domain that specifically binds to PD-1 is located at the C-terminus of the Fc region. In one embodiment, a second domain that specifically binds to CTLA-4 is located at the C-terminus of the Fc region. In one embodiment, a third domain that specifically binds to VEGF is located at the C-terminus of the Fc region.

[0127] In one embodiment, a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, a third domain specifically binding to VEGF, and the Fc region are directly interconnected or interconnected via one or more linkers (such as flexible linkers). In one embodiment, the linkers may be the same or different. In one embodiment, the linker is a peptide linker, and most preferably, the linker is a peptide linker lacking a proteolytic cleavage site. In some embodiments, the amino acid residues of the linker are selected from G, A, S, P, E, T, D, and K. In some embodiments, the linker is GGGGSGGGGS.

[0128] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention is represented from the N-terminus to the C-terminus by the following formula: Third structural domain – hinge area – Fc – joint – second structural domain – joint – first structural domain.

[0129] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention is represented from the N-terminus to the C-terminus by the following formula: Third structural domain – hinge area – Fc – joint – first structural domain – joint – second structural domain.

[0130] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention is represented from the N-terminus to the C-terminus by the following formula: Second structural domain – hinge area – Fc – joint – third structural domain – joint – first structural domain.

[0131] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention is represented from the N-terminus to the C-terminus by the following formula: First structural domain – hinge area – Fc – joint – third structural domain – joint – second structural domain.

[0132] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention is represented from the N-terminus to the C-terminus by the following formula: Second structural domain – hinge area – Fc – joint – first structural domain – joint – third structural domain.

[0133] In one embodiment, the third domain that specifically binds to VEGF is a Fab fragment or an scFv fragment, and the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention is represented from the N-terminus to the C-terminus by the following formula: First structural domain – hinge area – Fc – joint – second structural domain – joint – third structural domain.

[0134] In one embodiment, the third domain specifically binding to VEGF is a full-length antibody. In one embodiment, the first domain specifically binding to PD-1 is optionally linked via a linker to the C-terminus of any one or both heavy chains of the full-length antibody, and the second domain specifically binding to CTLA-4 is optionally linked via a linker to the C-terminus of the first domain specifically binding to PD-1. In one embodiment, the second domain specifically binding to CTLA-4 is optionally linked via a linker to the C-terminus of any one or both heavy chains of the full-length antibody, and the first domain specifically binding to PD-1 is optionally linked via a linker to the C-terminus of the second domain specifically binding to CTLA-4. In one embodiment, the first domain specifically binding to PD-1 and the second domain specifically binding to CTLA-4 are linked to the C-terminus of two heavy chains of the full-length antibody in the same or different order. In one embodiment, two second domains specifically binding to CTLA-4 are each linked to the C-terminus of the heavy chain of the full-length antibody via a linker, and two first domains specifically binding to PD-1 are each linked to the C-terminus of the two second domains specifically binding to CTLA-4 via a linker (see [link to implementation details]). Figure 1 ).

[0135] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention comprises a heavy chain and a light chain, or is composed of them, wherein the heavy chain contains the amino acid sequence of SEQ ID NO: 27, and the light chain contains the amino acid sequence of SEQ ID NO: 23.

[0136] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention comprises two heavy chains and two light chains, or is composed of them, wherein the heavy chains contain the amino acid sequence of SEQ ID NO: 27, and the light chains contain the amino acid sequence of SEQ ID NO: 23.

[0137] In one embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 27. In one embodiment, the light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 23. In one embodiment, the heavy chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 27, and the light chain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 23.

[0138] The terms "identity" or "homology" between two amino acid or nucleotide sequences are determined by sequence alignment. If the two sequences to be compared are of different lengths, the sequence alignment preferably involves the percentage of amino acid or nucleotide residues in the shorter sequence that are identical to those in the longer sequence. Sequence alignment can be routinely determined using a computer program. Differences arising in comparisons between a given sequence and the sequences described above in this disclosure can be caused by, for example, additions, deletions, substitutions, insertions, or recombinations.

[0139] In some embodiments, the CDR sequence of the antibody of the present invention may contain any conserved modification.

[0140] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding properties of antibodies containing amino acid sequences. Conservative modifications include amino acid substitutions, additions, and deletions. Conservative substitutions are those where amino acids are replaced by amino acid residues with similar side chains. The family of amino acid residues with similar side chains is well-defined and includes amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), amino acids with aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), amino acids with aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amino acids with amides (e.g., asparagine, glutamine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with sulfur-containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide can be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al., Acta Physiol. Scand. Suppl. 643:55-67, 1998; Sasaki et al., Adv. Biophys. 35:1-24, 1998). Amino acid substitutions of the antibodies of the present invention can be performed by known methods, such as by PCR mutagenesis (US Patent No. 4,683,195). Alternatively, variant libraries can be generated using known methods, such as using random codons (NNK) or non-random codons, such as the DVK codons encoding 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.

[0141] The generation of monospecific antibodies in this invention In some embodiments, the antibody of the present invention is human.

[0142] In some embodiments, the antibodies of the present invention are humanized.

[0143] The monospecific antibodies of the present invention described herein (e.g., antibodies that specifically bind to PD-1, CTLA-4, or VEGF) can be generated using a variety of techniques. For example, Kohler and Milstein can be used. Nature256:495, 1975. The hybridoma method for producing monoclonal antibodies. In the hybridoma method, mice or other host animals (such as hamsters, rats, alpacas, or monkeys) are immunized with human or cynomolgus monkey PD-1, CTLA-4, or VEGF, or fragments of PD-1, CTLA-4, or VEGF (such as the extracellular domains of PD-1, CTLA-4, or VEGF). Then, spleen cells from the immunized animal are fused with myeloma cells using standard methods to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Clones generated from a single immortalized hybridoma cell are screened to produce antibodies with desired properties, such as binding specificity, cross-reactivity or lack of cross-reactivity, and affinity for the antigen.

[0144] A variety of host animals can be used to generate the antibodies of this invention. For example, Balb / c mice can be used to generate mouse anti-human PD-1, CTLA-4, or VEGF antibodies. Alpacas can be used to generate anti-human PD-1 or CTLA-4 VHH. Antibodies prepared in Balb / c mice and other non-human animals can be humanized using various techniques to produce more human-like sequences.

[0145] Exemplary humanization techniques, including the selection of human receptor frameworks, are known and include CDR transplantation (US Patent No. 5,225,539), SDR transplantation (US Patent No. 6,818,749), and surface remodeling (Padlan, (1991)). Mol Immunol Methods include 28:489-499), specific determination residue surface remodeling (US Patent Publication No. 2010 / 0261620), human framework adaptation (US Patent No. 8,748,356), or hyperhumanization (US Patent No. 7,709,226). In these methods, the CDR of the parent antibody is transferred to a human framework, which can be selected based on its overall homology with the parent framework, similarity based on CDR length, or canonical structural identity, or a combination thereof.

[0146] Humanized antibodies can be further optimized to improve their selectivity or affinity for desired antigens by incorporating altered framework support residues via techniques such as those described in International Patent Publications Nos. WO1090 / 007861 and WO1992 / 22653 to maintain binding affinity (reverse mutation), or by introducing mutations at any CDR to improve antibody affinity.

[0147] Transgenic animals, such as mice or rats carrying a human immunoglobulin (Ig) locus in their genome, can be used to produce human antibodies against target proteins, and are described, for example, in U.S. Patent No. 6,150,584, International Patent Publication No. WO99 / 45962, International Patent Publication Nos. WO2002 / 066630, WO2002 / 43478, WO2002 / 043478 and WO1990 / 04036, and Lonberg et al. (1994). Nature 368:856-9, Green et al. (1994) Nature Genet. 7:13-21, Green and Jakobovits (1998) Exp. Med. 188:483-95, Lonberg and Huszar (1995) Int Rev Immunol 13:65-93, Bruggemann et al. , (1991) Eur J Immunol 21:1323-1326, Fishwild et al. , (1996) Nat Biotechnol 14:845-851, Mendez et al. , (1997) Nat Genet 15:146-156, Green (1999) J Immunol Methods 231:11-23, Yang et al. , (1999) Cancer Res 59:1236-1243, Brüggemann and Taussig (1997) Curr Opin Biotechnol8:455-458. In such animals, endogenous immunoglobulin loci may be disrupted or deleted, and at least one complete or partial human immunoglobulin locus may be inserted into the animal's genome using homologous or non-homologous recombination, transchromosome insertion, or minigene insertion. Companies such as Regeneron (http: / / www.regeneron.com), HarbourAntibodies (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) may be engaged to provide human antibodies against selected antigens using the techniques described above.

[0148] Human antibodies can be selected from phage display libraries where phages 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 Biol 296:57-86; Krebs et al., (2001) J Immunol Meth 254:67-84; Vaughan people, (1996) Nature Biotechnology 14:309-314; Sheets et al., (1998) PITAS (USA) 95:6157-6162; Hoogenboom and Winter (1991) J Mol Biol 227:381; Marks et al., (1991) J Mol Biol 222:581). The antibodies of the present invention can be isolated, for example, from phage display libraries expressing antibody heavy and light chain variable regions as fusion proteins with phage pIX coat proteins, as described by Shi et al. , (2010) J Mol BiolAs described in 397:385-96 and International Patent Publication No. WO09 / 085462, the library can be screened for phages binding to human and / or cynomolgus monkey PD-1, CTLA-4, or VEGF, and the obtained positive clones can be further characterized, Fab can be isolated from the clone lysate, 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.

[0149] The preparation of immunogenic antigens and the generation of monoclonal antibodies can be carried out using any suitable technique, such as recombinant protein production. Immunogenic antigens can be administered to animals in the form of purified proteins or protein mixtures comprising whole-cell, cellular, or tissue extracts, or the antigen can be de novo formed in an animal from nucleic acids encoding the antigen or a portion thereof.

[0150] The anti-PD-1 antibody GBD002-hS019-WS obtained from alpacas in this disclosure has the amino acid and nucleotide sequences shown in Table 2.

[0151] Table 2. Amino acid and nucleotide sequences of GBD002-hS019-WS antibody The anti-CTLA-4 antibody GBD008-hS005-3-2 obtained from alpacas in this disclosure has the amino acid and nucleotide sequences shown in Table 3.

[0152] Table 3. Amino acid and nucleotide sequences of GBD008-hS005-3-2 antibody The anti-VEGF antibody in this disclosure, as a variant of bevacizumab, has the amino acid and nucleotide sequences shown in Table 4.

[0153] Table 4. Amino acid and nucleotide sequences of anti-VEGF antibodies The generation of multispecific antibodies in this invention The bispecific PD-1 / CTLA-4 antibody of the present invention can be generated by combining the PD-1 binding VH (or VH / VL) domain isolated and characterized herein with the CTLA-4 binding VH (or VH / VL) domain. Alternatively, the bispecific PD-1 / CTLA-4 antibody can be generated using the VH (or VH / VL) domain from a publicly available monospecific anti-PD-1 or anti-CTLA antibody, and / or by engineering the PD-1 or CTLA-4 VEGF binding VH (or VH / VL) domain identified herein with a publicly available PD-1 or CTLA-4 binding VH (or VH / VL) domain. Trispecific, tetraspecific, and other similar antibodies can be generated in a similar manner.

[0154] For example, the trispecific PD-1 / CTLA-4 / VEGF antibody of the present invention can be generated by combining the PD-1-binding VH (or VH / VL) domain, the CTLA-4-binding VH (or VH / VL) domain, and the VEGF-binding VH (or VH / VL) domain isolated and characterized herein. Alternatively, the trispecific PD-1 / CTLA-4 / VEGF antibody can be engineered using the VH (or VH / VL) domain from publicly available monospecific anti-PD-1, anti-CTLA, or anti-VEGF antibodies, and / or by mixing the PD-1, CTLA-4, or VEGF-binding VH (or VH / VL) domain identified herein with publicly available PD-1, CTLA-4, or VEGF-binding VH (or VH / VL) domains.

[0155] Exemplary anti-PD-1 antibodies that can be used to engineer trispecific PD-1 / CTLA-4 / VEGF antibodies include, for example, pembrolizumab and nivolumab. Exemplary anti-CTLA-4 antibodies that can be used to engineer trispecific PD-1 / CTLA-4 / VEGF antibodies include, for example, ipilimumab. Exemplary anti-VEGF antibodies that can be used to engineer trispecific PD-1 / CTLA-4 / VEGF antibodies include, for example, bevacizumab. Exemplary monospecific domains may also be derived from bispecific antibodies, such as the anti-PD-1×CTLA-4 bispecific antibodies catonilimab and MEDI5752, the anti-PD-1 / CTLA-4 bispecific antibody AK104, and the anti-PD-1 / VEGF bispecific antibody AK112.

[0156] The resulting trispecific PD-1 / CTLA-4 / VEGF antibodies can be tested for binding to PD-1, CTLA-4, and VEGF using the methods described herein, as well as for desired functional properties such as enhanced antigen-specific CD4.+ and CD8 + T cell activation.

[0157] Standard methods are typically used to mutate molecules (such as constant domains of antibodies) at the DNA level.

[0158] The trispecific PD-1 / CTLA-4 / VEGF antibody GBD214-33-03 constructed in this disclosure has the amino acid and nucleotide sequences shown in Table 5.

[0159] Table 5. Amino acid and nucleotide sequences of GBD214-33-03 antibody Polynucleotides, vectors and host cells This invention also provides antibodies or antigen-binding fragments thereof, wherein antibody VH and / or VL are encoded by polynucleotides. The polynucleotides may be complementary deoxyribonucleic acid (cDNA) and may be codon-optimized for expression in a suitable host. Codon optimization is a well-known technique.

[0160] The present invention also provides isolated polynucleotides encoding the VH, VL, heavy chain, and / or light chain of the antibody of the present invention.

[0161] The present invention also provides isolated polynucleotides encoding VH, VL, or VH and VL of the antibodies of the present invention.

[0162] The present invention also provides isolated polynucleotides encoding VH of SEQ ID NO: 4, 10 and / or 16.

[0163] The present invention also provides isolated polynucleotides encoding the heavy and / or light chains of the antibodies of the present invention.

[0164] In one embodiment, the present invention provides isolated polynucleotides encoding the heavy chain of SEQ ID NO: 5 or 11. In one embodiment, the present invention provides isolated polynucleotides encoding the heavy chain of SEQ ID NO: 17 or 27 and / or the light chain of SEQ ID NO: 23.

[0165] In one embodiment, the present invention provides an isolated polynucleotide comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6 or 12. In another embodiment, the present invention provides an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 6 or 12.

[0166] In one embodiment, the present invention provides a separate polynucleotide comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence of SEQ ID NO: 18 or 28, and / or a separate polynucleotide comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence of SEQ ID NO: 24. In one embodiment, the present invention provides a separate polynucleotide comprising the nucleotide sequence of SEQ ID NO: 18 or 28 and / or the nucleotide sequence of SEQ ID NO: 24.

[0167] The VH or VL or its antigen-binding fragment encoding the antibody of the present invention, or the polynucleotide sequence of the heavy or light chain encoding the antibody of the present invention, can be operatively linked to one or more regulatory elements (such as promoters or enhancers) to enable the nucleotide sequence to be expressed in the intended host cell. The polynucleotide can be cDNA.

[0168] The present invention also provides vectors comprising the polynucleotides of the present invention. Such vectors may be plasmid vectors, viral vectors, baculovirus expression vectors, transposon-based vectors, or any other vector suitable for introducing the synthetic polynucleotides of the present invention into a given organism or genetic background by any means. For example, a polynucleotide encoding the variable region (optionally linked to a constant region) of the light chain and / or heavy chain of an antibody of the present invention is inserted into an expression vector. The light chain and / or heavy chain may be cloned into the same or different expression vectors. DNA segments encoding immunoglobulin chains may be operatively linked to control sequences in the expression vector, which ensure the expression of immunoglobulin polypeptides. Such control sequences include signal sequences, promoters (e.g., naturally associated promoters or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected to be compatible with the host cells selected for antibody expression. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the protein encoded by the incorporated polynucleotide.

[0169] Suitable expression vectors can typically replicate in a host organism either as a free organism or as part of the host's chromosomal DNA. Typically, expression vectors contain selection markers, such as ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance, to enable the detection of cells transformed with the desired DNA sequence.

[0170] Suitable promoters and enhancer elements are known in the art. Exemplary promoters for expression in eukaryotic cells include: light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus immediate early promoters; herpes simplex virus thymidine kinase promoters; early and late SV40 promoters; promoters present in long terminal repeat sequences from retroviruses; mouse metallothionein-I promoters; and various known tissue-specific promoters. The selection of suitable vectors and promoters is entirely within the capabilities of those skilled in the art.

[0171] Exemplary vectors that can be used are bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden); and eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia), pEE6.4 (Lonza) and pEE12.4 (Lonza).

[0172] This invention also provides host cells comprising one or more vectors of the present invention. "Host cell" refers to a cell into which a vector has been introduced. It should be understood that the term "host cell" is intended not only to refer to a specific object cell, but also to the progeny of such cells, and stable cell lines generated from the specific object cell. Because certain changes may occur in offspring due to mutations or environmental influences, such progeny may differ from the parent cells, but are still included within the scope of the term "host cell" as used herein. Such host cells can be eukaryotic cells, prokaryotic cells, plant cells, or archaea cells. *Escherichia coli* (…) Escherichia coli ), bacilli (such as Bacillus subtilis ( Bacillus subtilisOther Enterobacteriaceae (such as Salmonella, Serratia, and various Pseudomonas species) are examples of prokaryotic host cells. Other microorganisms (such as yeast) can also be used for expression. Yeasts (e.g., Saccharomyces cerevisiae) and Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells can be of mammalian, insect, bird, or other animal origin. Mammalian eukaryotic cells include immortalized cell lines, such as hybridoma cell lines or myeloma cell lines, such as SP2 / 0 (American Type Culture Collection (ATCC), Manassas, VA, CRL-1581), NSO (European Center for Cell Culture Collection (ECACC), Salisbury, Wiltshire, UK, ECACC No. 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) mouse cell lines. An exemplary human myeloma cell line is U266 (ATCC CRL-TIB-196). Other useful cell lines include those derived from Chinese hamster ovary (CHO) cells, such as CHOK1SV (LonzaBiologics, Walkersville, MD), Potelligent® CHOK2SV (Lonza), CHO-K1 (ATCC CRL-61), or DG44.

[0173] The present invention also provides a method for producing the antibodies of the present invention, the method comprising culturing the host cells of the present invention under conditions expressing the antibody, and recovering the antibodies produced by the host cells. Methods for preparing and purifying antibodies are well known in the art. Once synthesized (whether chemically synthesized or recombinantly synthesized), whole antibodies, their dimers, single light chains and / or heavy chains, or other antibody fragments (such as VH and / or VL) can be purified according to standard procedures including ammonium sulfate precipitation, affinity column, column chromatography, high-performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (generally see Scopes, Protein Purification (Springer-Verlag, NY, (1982)). The target antibody can be substantially pure, for example 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, for example free from contaminants other than the target antibody, such as cell debris, macromolecules, etc.

[0174] The polynucleotide sequences of this invention can be incorporated into vectors using standard molecular biology methods. Host cell transformation, culture, antibody expression, and purification are all performed using known methods.

[0175] Another embodiment of the present invention is a method for generating the antibody or antigen-binding fragment thereof, the method comprising: The VH, VL, heavy chain and / or light chain polynucleotides encoding the antibody or its antigen-binding fragment of the present invention are incorporated into the expression vector; Transform host cells using expression vectors; Host cells are cultured in a medium in which VH, VL, heavy chains, and / or light chains are expressed and antibodies or their antigen-binding fragments are formed; and Antibodies or their antigen-binding fragments are recovered from host cells or culture media.

[0176] Pharmaceutical composition / application This invention provides pharmaceutical compositions comprising the antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. For therapeutic use, the antibody or antigen-binding fragment thereof of the present invention can be prepared into a pharmaceutical composition comprising an effective amount of the antibody or antigen-binding fragment thereof as the active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, excipient, excipient, or solvent used to administer the antibody of the present invention. Such solvents can be liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They can be sterilized using conventional, known sterilization techniques (e.g., filtration). The composition may contain desired pharmaceutically acceptable excipients to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants. The concentration of the antibody or its antigen-binding fragment in such pharmaceutical formulations can range from less than about 0.5% by weight (typically) to at least about 1% by weight to up to 15% by weight or 20% by weight, and can be selected based primarily on the required dose, liquid volume, viscosity, and other factors, depending on the specific administration method chosen. Suitable solvents and formulations, including other human proteins (e.g., human serum albumin), are described, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, edited by Troy, DB, Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092, with particular reference to pp. 958-989.

[0177] The therapeutic use of the antibody or its antigen-binding fragment of the present invention can be administered via any suitable route for delivering the antibody or its antigen-binding fragment to the host, such as parenteral administration, for example intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, or transmucosal (oral, intranasal, intravaginal, rectal), in tablet, capsule, solution, powder, gel, or granule formulations; and contained in syringes, implantation devices, osmotic pumps, cartridges, or micropumps; or other methods as known to those skilled in the art. Site-specific administration can be achieved through, for example, the following: intratumoral, intraarticular, intrabronchial, intraperitoneal, intracystic, intracartilaginous, intracavitary, intrabody cavity, intracerebellum, intravenous, intracolonic, intracervical, intrastomal, intrastomal, intrahepatic, intracardiac, intraosseous, intrapelvic, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intrabladder, intralesional, vaginal, rectal, sublingual, intranasal, or percutaneous delivery.

[0178] The antibodies or antigen-binding fragments of the present invention can be administered to subjects via any suitable route, such as intravenous (iv) infusion or bolus injection parenteral administration, intramuscular or subcutaneous or intraperitoneal administration. Intravenous infusion can be given over, for example, 15, 30, 60, 90, 120, 180 or 240 minutes, or over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

[0179] The dose given to the subject is sufficient to relieve or at least partially suppress the disease being treated (“therapeutic effective dose”), 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 about 5 mg 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.

[0180] A fixed unit dose may also be administered, such as 50, 100, 200, 500, or 1000 mg, or the dose may be determined based on the patient's body surface area, such as 500, 400, 300, 250, 200, or 100 mg / m². 2 Patients can usually be treated with 1 to 8 doses (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 doses), but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may also be given.

[0181] The antibody or antigen-binding fragment of the present invention may be administered after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months or longer. Repeated treatment courses are also possible, as is long-term administration. Repeated administration may be at the same dose or at different doses. For example, the antibody or antigen-binding fragment of the present invention may be administered at a weekly interval of 8 mg / kg or 16 mg / kg for 8 weeks, then at a bi-weekly interval of 8 mg / kg or 16 mg / kg for another 16 weeks, and then at a weekly interval of 8 mg / kg or 16 mg / kg by intravenous infusion.

[0182] For example, the antibody or its antigen-binding fragment of the present invention can be used in amounts of about 0.1-100 mg / kg, such as 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. The daily dose of mg / kg is administered daily, on at least one day of days 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, or alternatively, on 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.

[0183] The antibodies or antigen-binding fragments of the present invention can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of cancer progression events, and / or reduce the risk of recurrence when cancer is in remission.

[0184] The antibodies or antigen-binding fragments of the present invention can be lyophilized for preservation and reconstituted in a suitable carrier prior to use. This technique has proven effective for conventional protein formulations and can be employed using known lyophilization and reconstitution techniques.

[0185] Methods and uses The antibodies or antigen-binding fragments thereof of the present invention have diagnostic, therapeutic, and preventative effects both in vitro and in vivo. For example, the antibodies or antigen-binding fragments thereof of the present invention can be administered in vitro or ex vivo to cultured cells, or to subjects seeking treatment, prevention, and / or diagnosis of various conditions, such as cancer and infectious diseases.

[0186] The present invention provides a method for altering the immune response of a subject, the method comprising administering to the subject an antibody of the present invention or an antigen-binding fragment thereof for a duration sufficient to alter the immune response.

[0187] In some implementations, the immune response is enhanced, stimulated, or upregulated.

[0188] In some of the implementation methods described herein, the subjects are human patients.

[0189] In some of the implementation methods described herein, the subjects are patients who require an enhanced immune response.

[0190] In some implementation schemes, the subjects are immunocompromised.

[0191] In some implementation schemes, subjects are at risk of immunodeficiency. Immunodeficiency subjects may be undergoing or have already undergone chemotherapy or radiation therapy.

[0192] In some implementations, the subject is immune-impaired due to infection or at risk of immune-impaired due to infection.

[0193] The antibodies or antigen-binding fragments thereof of the present invention are suitable for use in treating subjects with a condition that can be treated by enhancing a T-cell-mediated immune response.

[0194] In some embodiments, the antibodies or antigen-binding fragments thereof used in the methods of the present invention are those defined in this disclosure.

[0195] In some embodiments, the trispecific PD-1 / CTLA-4 / VEGF antibody used in the method of the present invention is GBD214-33-03. The amino acid and nucleotide sequences of this antibody are shown in Table 5.

[0196] The present invention also provides a method for inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody of the present invention or an antigen-binding fragment thereof, for a duration sufficient to inhibit tumor cell growth.

[0197] The present invention also provides a method for treating cancer by administering a therapeutically effective amount of the antibody of the present invention or its antigen-binding fragment to a subject in need (for a duration sufficient to treat cancer).

[0198] Cancer can be a proliferative condition or disease, a solid tumor, a hematologic malignancy, a soft tissue tumor, or a metastatic lesion.

[0199] The term "cancer" is intended to encompass all types of cancerous growth or carcinogenic processes, metastatic tissues, or malignant transformations of cells, tissues, or organs, regardless of their histopathological type or stage of invasiveness. Examples of cancer include solid tumors, hematologic malignancies, soft tissue tumors, and metastatic lesions. Exemplary solid tumors include malignancies of various organ systems, such as sarcomas and carcinomas (including adenocarcinoma and squamous cell carcinoma), such as those affecting the liver, lungs, breast, lymph nodes, gastrointestinal tract (e.g., colon), genitourinary tract (e.g., kidneys, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, and esophageal cancer. Squamous cell carcinomas include malignancies such as those in the lungs, esophagus, skin, head and neck, oral cavity, anus, and cervix.

[0200] In some implementations, the cancer is melanoma.

[0201] Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and antibodies of the present invention described herein.

[0202] Exemplary cancers whose growth can be inhibited or reduced using the antibodies of the present invention or their antigen-binding fragments include cancers that respond to immunotherapy. Examples of such cancers include melanoma, kidney cancer, prostate cancer, breast cancer, colon cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, lung cancer, metastatic malignant melanoma, clear cell carcinoma, hormone-refractory prostate adenocarcinoma, non-small cell lung cancer, or head and neck cancer. The antibodies of the present invention described herein or their antigen-binding fragments can be used to treat refractory or recurrent malignancies.

[0203] Other exemplary cancers that can be treated with the antibodies or antigen-binding fragments of the present invention include: anal cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer and CNS cancer, fallopian tube cancer, vaginal cancer, vulvar cancer, malignant melanoma of the skin or eye, astroesophageal cancer, testicular cancer, ovarian cancer, pancreatic cancer, rectal cancer, uterine cancer, primary CNS lymphoma; tumors of the central nervous system (CNS), cervical cancer, choriocarcinoma, rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, eye cancer; intraepithelial neoplasia, renal cancer, laryngeal cancer, liver cancer; small cell lung cancer, neuroblastoma, oral cancer (e.g., lip, tongue, mouth and pharynx), nasopharyngeal carcinoma, retinoblastoma, rhabdomyosarcoma, respiratory system cancer, sarcoma, and nail cancer. Cancer of the thyroid gland, urinary system, liver, anal region, fallopian tube, vagina, vulva, small intestine, endocrine system, parathyroid, adrenal, soft tissue sarcoma, urethral, ​​penile, pediatric solid tumors, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, Merkel cell carcinoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers (including asbestos-induced cancers), and other cancers and sarcomas, as well as combinations of the above cancers.

[0204] Exemplary hematologic malignancies that can be treated with the antibodies or antigen-binding fragments of the present invention include leukemia, lymphoma, and myeloma, such as precursor B-cell lymphoblastic leukemia / lymphoma and B-cell non-Hodgkin lymphoma, acute promyelocytic leukemia, acute lymphoblastic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL) (including low-grade, intermediate-grade, and high-grade FL), cutaneous follicular center lymphoma, and marginal zone B-cell lymphoma. Lymphoma (MALT type, lymph node type, and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), plasmacytoma, multiple myeloma (MM), plasma cell leukemia, post-transplant lymphoproliferative disorders, Waldenström macroglobulinemia, plasma cell diseases, anaplastic large cell lymphoma (ALCL), T-cell acute lymphoblastic leukemia, primary systemic amyloidosis (e.g., light chain amyloidosis), prolymphocytic / granulocytic leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), large granular lymphocytic (LGL) leukemia, NK cell leukemia, and Hodgkin lymphoma.

[0205] "Plasma cell diseases" refer to diseases characterized by clonal plasma cells and include multiple myeloma, light chain amyloidosis, and Waldenström macroglobulinemia. Light chain amyloidosis and Waldenström macroglobulinemia can occur independently of multiple myeloma. They can also coexist with multiple myeloma and develop before or after the development of multiple myeloma.

[0206] Exemplary B-cell non-Hodgkin lymphomas include lymphomatoid granulomas, primary exudative lymphomas, intravascular large B-cell lymphomas, mediastinal large B-cell lymphomas, heavy chain diseases (including γ, μ, and a diseases), lymphomas induced by treatment with immunosuppressants (such as cyclosporine-induced lymphomas), and methotrexate-induced lymphomas.

[0207] In some implementations, the subject has a tumor that expresses PD-L1.

[0208] In some implementations, the subject has a tumor that expresses CTLA-4.

[0209] In some implementations, the subject has a tumor that expresses VEGF.

[0210] In some implementations, the subjects have been treated with anti-PD-1 antibodies.

[0211] In some implementations, the subjects are refractory to treatment with anti-PD-1 antibodies.

[0212] In some implementations, the subject has a tumor that has recurred after treatment with an anti-PD-1 antibody.

[0213] In some implementation schemes, the subjects have been given anti-PD-1 antibodies (e.g., KEYTRUDA). ® Treatment with pembrolizumab.

[0214] In some implementation schemes, the subjects have been given anti-PD-1 antibodies (e.g., OPDIVO). ® Treatment with nivolumab.

[0215] In some implementation schemes, subjects use anti-PD-1 antibodies (e.g., KEYTRUDA). ® Treatment with pembrolizumab was refractory.

[0216] In some implementations, the subject responds to an anti-PD-1 antibody (e.g., OPDIVO). ® Treatment with nivolumab was refractory.

[0217] In some implementations, the subject has an anti-PD-1 antibody in use (e.g., KEYTRUDA). ®Tumors that recur after treatment with pembrolizumab.

[0218] In some implementations, the subject has an anti-PD-1 antibody (e.g., OPDIVO) in use. ® Tumors that relapse after treatment with nivolumab.

[0219] In some implementations, the subject has been treated with or is being treated with an anti-PD-L1 antibody (such as MEDI-4736, MDX-1105, Avelumab, or Atezolizumab).

[0220] In some implementations, the subject is refractory to treatment with anti-PD-L1 antibodies (such as MEDI-4736, MDX-1105, avelumab, or atezolizumab).

[0221] In some implementations, the subject has a tumor that has relapsed after treatment with an anti-PD-L1 antibody (such as MEDI-4736, MDX-1105, avelumab, or atezolizumab).

[0222] In some implementations, the subjects have been treated with or are being treated with anti-PD-L2 antibodies.

[0223] In some of the implementation methods described herein, the subjects were refractory to treatment with anti-PD-L2 antibodies.

[0224] In some implementations, the subject has a tumor that has recurred after treatment with an anti-PD-L2 antibody.

[0225] A variety of qualitative and / or quantitative methods can be used to determine whether a disease is relapsed or refractory. Symptoms that may be associated with relapse or resistance include, for example, a decline or stagnation in the patient's health, or the recurrence or worsening of various symptoms associated with solid tumors, and / or the spread of cancer cells from one site to other organs, tissues, or cells in the body.

[0226] The present invention also provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody of the present invention, wherein the subject is being treated with or has been treated with an anti-PD-1 antibody.

[0227] The present invention also provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody of the present invention, wherein the subject is being treated with or has been treated with an anti-PD-L1 antibody.

[0228] The present invention also provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the antibody of the present invention, wherein the subject is being treated with or has been treated with an anti-PD-L2 antibody.

[0229] Combination therapy for cancer treatment The antibody of the present invention can be administered in combination with a second therapeutic agent.

[0230] The antibody or its antigen-binding fragment of the present invention can be administered in combination with one, two, three, four, five or six other therapeutic agents.

[0231] "In combination with" means that the antibody of the present invention is administered simultaneously with at least one second therapeutic agent as a single agent, or as a single agent in any order. Generally, each agent will be administered at a dose and / or schedule determined according to that agent.

[0232] In some implementations, the second therapeutic agent modulates the activity of molecules involved in the cancer immune cycle, such as molecules involved in stimulating or inhibiting pathways that function in the release of cancer cell antigens, cancer antigen presentation, T cell priming and activation, T cell migration to the tumor, T cell infiltration into the tumor, T cell recognition of cancer cells, and killing of cancer cells. The cancer immune cycle is described in Chen and Mellman (2013). Immunity 39:1-10. In some implementations, the second therapeutic agent modulates the activity of molecules involved in regulating the activity of regulatory T cells (Tregs), co-stimulatory or co-inhibitory ligands expressed on tumors, activating or inhibiting receptors on natural killer (NK) cells, or immunosuppressive factors in the tumor microenvironment. Combination cancer immunotherapy is described in Manoney et al. , (2015) Nature Reviews 14:561-584.

[0233] As is well known, second therapeutic agents typically enhance the activity of stimulating molecules and suppress the activity of inhibitory molecules. Therefore, "modulation" refers to the enhancement of the immune response by a second therapeutic agent, whether the agent itself is an agonist or antagonist of a specific molecule.

[0234] The efficacy of the combination described herein can be tested in animal models known in the art. Example

[0235] The present invention will now be described with reference to the following specific, non-limiting embodiments.

[0236] Example 1: Generation of anti-PD-1 antibody, anti-CTLA-4 antibody and anti-VEGF antibody Anti-human PD-1 antibodies and anti-human CTLA-4 antibodies were generated by immunizing alpacas with recombinant human PD-1 and human CTLA-4 extracellular domain (ECD) proteins, respectively. Total RNA was extracted from PBMCs, and cDNA was synthesized and amplified. Using CDR transplantation technology, the framework region of the alpaca VH gene was replaced with the human framework region and cloned into an expression vector to generate the corresponding humanized antibody clones. The resulting anti-PD-1 antibody GBD002-hS019-WS possesses the heavy chain variable region of SEQ ID NO: 4. The resulting anti-CTLA-4 antibody possesses the heavy chain variable region of SEQ ID NO: 10 (GBD008-hS005-3-2).

[0237] The amino acids of the variable regions of the heavy and light chains of the anti-VEGF antibody are referenced to the commercial antibody bevacizumab (Avastin) and are shown in SEQ ID NO: 16 and SEQ ID NO: 22, respectively. An anti-VEGF antibody, a variant of bevacizumab, was synthesized by Biointron and its sequence is shown in Table 4. This anti-VEGF antibody comprises two heavy chains with the amino acid sequence of SEQ ID NO: 17 and two light chains with the amino acid sequence of SEQ ID NO: 23. In this anti-VEGF antibody, the Fc fragment differs from that of bevacizumab.

[0238] Example 2: Generation of GBD214-33-03 antibody Construction of the trispecific antibody GBD214-33-03: The DNA sequences encoding the heavy chain containing the N-terminus to C-terminus of the anti-VEGF antibody (SEQ ID NO: 17), the variable region of the heavy chain of the anti-CTLA-4 antibody (SEQ ID NO: 10), and the variable region of the heavy chain of the anti-PD-1 antibody (SEQ ID NO: 4), and the DNA sequence encoding the light chain of the anti-VEGF antibody (SEQ ID NO: 23) were cloned into the pcDNA3.3 expression vector to obtain the trispecific antibody. Adapter 1 and / or adapter 2 were added as flexible links to the heavy chain of GBD214-33-03. The obtained trispecific antibody was named "GBD214-33-03". A schematic diagram of the structure of GBD214-33-03 is shown below. Figure 1 middle.

[0239] Example 3. FACS analysis of the binding specificity of anti-PD-1 antibody to human PD1 protein on cell surface HEK293-PD1 cells (1×10⁻⁶) 5Cells were washed twice in FACS buffer (PBS + 2% BSA) and resuspended in 100 μl of FACS buffer containing serially diluted (1:5) anti-PD1 mAb, and incubated at 4°C for 1 hour. Cells were then washed twice in FACS buffer, and antibody binding was detected by incubation at 4°C for 1 hour with APC anti-human IgG Fc (Biolegend, catalog number 410712). Subsequently, cells were washed twice in FACS buffer, and then collected and analyzed using a Fortessa flow cytometer (BD Bioscience). Results are shown in... Figure 2 The results showed that GBD214-33-03 bound to PD1 in HEK293-PD1 cells. GBD214-33-03 exhibited strong binding to HEK293-PD1 cells, with a median fluorescence intensity (MFI) similar to its PD1 parent GBD002-hS019-WS and pembrolizumab.

[0240] Example 4. PD1 reporter gene assay in Jurkat-NFAT-Luc2-PD1 cells (ELISA method) Harvest CHOK1-PD-L1 cells (4×10⁻⁶) 4 The cells were suspended in PRMI 1640 medium (Gibco, catalog number 31800022) containing 10% FBS (Gibco, catalog number 10099-141) and grown overnight at 37°C in 96-well plates. On the second day, the supernatant was discarded, and Jurkat-NFAT-Luc2-PD1 cells (5 × 10⁶ cells / well) were added to each well. 4 Cells were incubated with serially diluted antibodies ( / well) and incubated at 37°C for 5 hours. Subsequently, Bio-Glo luciferase assay buffer (Promega, catalog number G7940) was added to the cells, and the cells were incubated at room temperature for up to 10 minutes before being read using an EnVision 2105 multimode microplate reader (PerkinElmer). Results are shown in... Figure 3 In PD1 reporter gene assays, GBD214-33-03 exhibited a similar maximum luminescence signal to pembrolizumab and AK104. These results indicate that GBD214-33-03 can block the interaction between PD1 and its ligand PD-L1.

[0241] Example 5. Assay of Mixed Lymphocyte Reaction (MLR) of Antibodies CD14+ cells were isolated from PBMCs using a separation kit (Stem Cell, catalog number 17858) and according to the manufacturer's instructions. Subsequently, CD14+ cells were cultured at 1 × 10⁻⁶ cells per cell line. 63-5 ml of cells / ml was seeded into 6-well plates (3-5 × 10⁶ cells / ml). 6 Cells were cultured in medium supplemented with 50 ng / ml IL-4 and 50 ng / ml GMCSF ( / well). Cytokines were replaced every 2-3 days. On day 6, to induce mature DCs, 1 μg / ml LPS (Sigma-Aldrich, catalog number L6529) was added to the medium, and the cells were cultured at 37°C for 24 hours. Mature DCs were then collected on day 7 for MLR assay. For MLR assay, fresh T cells were isolated according to the manufacturer's instructions (Stem Cell, catalog number 17951) and cultured at 2 × 10⁶ cells / well. 5 Seed 10 cells / well into the assay plate. Add serially diluted antibody to the assay plate, then seed mature DCs at 1×10⁻⁶ cells / well. 4 Cells / well were seeded into the assay plate. The assay plate was incubated at 37°C for 5 days, and the supernatant was collected for IFNγ detection using the HTRF kit (Cisbio, catalog number 62HIFNGPEH) according to the manufacturer's instructions. Results are shown in... Figure 4 The results showed that the maximum IFN-γ production of GBD214-33-03 in the mixed lymphocyte reaction (MLR) assay was similar to that of its parent GBD002-hS019-WS and pembrolizumab. These results indicate that GBD214-33-03 possesses potent immunogenicity similar to that of pembrolizumab.

[0242] Example 6. VEGF binding assay (ELISA method) VEGF165 protein (1 µl / ml) (Kactus, catalog number VEG-HM065) was coated onto a 96-well flat-bottom microplate at 37°C for 2 hours. The plate was washed 6 times with PBST and blocked at 37°C for 2 hours by adding 300 µl of blocking buffer. The supernatant was aspirated, and the plate was washed 3 times with PBST. Serially diluted antibody was added to each well. The plate was incubated at 37°C for 1 hour and then washed 6 times with PBST. Anti-human Fc (Jackson ImmunoResearch Inc., catalog number 109-035-008) and SA-HRP (Jackson ImmunoResearch Inc., catalog number 016-030-084) were added to the plate, and the plate was incubated at 37°C for 1 hour. Subsequently, 100 µl of TMB solution was added to each well, and the plate was incubated at room temperature for 5 minutes, followed by the addition of 50 µl of TMB solution. The optical density (OD) of each well was measured using an EnVision 2105 multimode microplate reader (PerkinElmer). 450 The results are shown in Figure 5The results showed that GBD214-33-03 had similar VEGF binding to bevacizumab as bevacizumab.

[0243] Example 7. VEGF Blockade Assay (ELISA) VEGF165 protein (1 µl / ml) (Kactus, catalog number: VEG-HM065) was coated onto a 96-well flat-bottom microplate at 37°C for 2 hours. The plate was washed 6 times with PBST and blocked at 37°C for 2 hours by adding 300 µl of blocking buffer. The supernatant was aspirated, and the plate was washed 3 times with PBST. Serially diluted antibody and VEGFR2-mFc-biotin (Kactus, catalog number: VGF-HM3R2B) were added to each well. The plate was incubated at 37°C for 1 hour and washed 6 times with PBST. Anti-human Fc (Jackson ImmunoResearch Inc., catalog number 109-035-008) and SA-HRP (Jackson ImmunoResearch Inc., catalog number 016-030-084) were added to the plate, and the plate was incubated at 37°C for 1 hour. Subsequently, 100 µl of TMB solution was added to each well, and the plate was incubated at room temperature for 5 minutes, followed by the addition of 50 µl of TMB solution. The optical density (OD) of each well was measured using an EnVision 2105 multimode microplate reader (PerkinElmer). 450 The results are shown in Figure 6 The results showed that GBD214-33-03 could inhibit the binding of VEGFR2 to VEGF165, which is similar to that of bevacizumab.

[0244] Example 8. VEGF reporter gene assay for antibodies (ELISA method) Harvest HEK293-NFAT-KDR-Luc cells (4×10⁻⁶) 4 Cells / well) were resuspended in PRMI 1640 medium (Gibco, catalog number 31800022) containing 10% FBS (Gibco, catalog number 10099-141), and the cells were incubated at 4 × 10⁻⁶ cells / well. 4Cells were seeded at 100 cells / well and grown overnight at 37°C in 96-well white plates. On the second day, the supernatant was discarded, and VEGF165 (15 ng / ml) (Kactus, catalog number VEG-HM065) and serially diluted antibody were added to each well. Cells were incubated at 37°C for 5 hours. Next, Bio-Glo luciferase assay buffer (Promega, catalog number G7940) was added to each well. The plates were incubated at room temperature for up to 10 minutes, and then read using an EnVision 2105 multimode microplate reader (PerkinElmer). Results are shown below. Figure 7 The results showed that GBD214-33-03 can inhibit VEGF165-induced downstream NFAT signaling by blocking the binding of VEGFR2 to VEGF165, similar to bevacizumab.

[0245] Example 9. FACS analysis of the binding specificity of anti-CTLA4 antibody to human CTLA4 protein on cell surface Harvest and collect CHOK1-CTLA4 cells (1×10⁻⁶). 5 / well) and CHOK1-PD1-CTLA4 cells (1×10⁶) 5 Cells were resuspended in 100 μl of FACS buffer (PBS + 2% BSA) in each well. Serially diluted anti-CTLA4 antibody was added to the plate, and cells were incubated at 4°C for 1 hour. Cells were then washed twice with FACS buffer, and antibody binding was detected by incubation at 4°C for 1 hour with APC anti-human IgG Fc (Biolegend, catalog number 410712). Cells were washed twice with FACS buffer and then collected. Cells were analyzed using a Fortessa flow cytometer (BD Bioscience). Results are shown in [Figure number missing]. Figure 8a and 8b The results showed that in CHOK1-CTLA4 cells, GBD214-33-03 had a lower MFI compared to ipilimumab, suggesting that GBD214-33-03 partially binds to CTLA4 in CHOK1-CTLA4 cells. The CTLA4 parent GBD008-hS005-3-2 of GBD214-33-03 had a weaker binding affinity to CTLA4 compared to ipilimumab. In CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed a more potent binding affinity compared to its PD1 parent GBD002-hS019-WS.

[0246] Example 10. FACS analysis of anti-CTLA4 antibody blocking the binding of CD80 and CD86 to CTLA4. Harvest and collect CHOK1-CTLA4 cells or CHOK1-PD1-CTLA4 cells (1×10⁻⁶). 5 Cells were resuspended in 100 μl of FACS buffer containing serially diluted anti-CTLA4 antibody and either CD80-biotin (SinoBiological, catalog number 10698-H49H-B) or CD86-biotin (SinoBiological, catalog number 10699-H03H-B). Cells were incubated at 4°C for 30 min and then washed twice with FACS buffer. Antibody binding was detected by incubation at 4°C for 30 min with APC anti-human IgG Fc (Biolegend, catalog number 410712). Cells were then collected after washing twice with FACS buffer and analyzed using a Fortessa flow cytometer (BD Bioscience). Results are shown in [Figure number missing]. Figures 9a to 9d The results showed that GBD214-33-03 partially inhibited the binding of CD80 / CD86 to CTLA4 in CHOK1-CTLA4 and CHOK1-PD1-CTLA4 cells. In both CHOK1-CTLA4 and CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed weaker blocking ability compared to ipilimumab.

[0247] Example 11. Affinity of antibody with PD1, CTLA4 and VEGF (Octet method) The binding kinetics of antibodies to recombinant human PD1, CTLA-4, or VEGF were qualitatively and quantitatively analyzed using the Octet method (Octet RH-16, Sartorius) for protein interactions. Binding rates (Kon) and dissociation rates (Koff) were calculated using a simple one-to-one binding model (Octet evaluation software version 12.2). The equilibrium dissociation constant (kD) was calculated as the ratio of Koff / Kon. The results are shown in Tables 6, 7, and 8. Table 6 shows that all antibodies bound to the CTLA-4 antigen, Table 7 shows that all antibodies bound to the PD1 antigen, and Table 8 shows that all antibodies bound to the VEGF antigen.

[0248] Table 6. Affinity results of antibodies to PD1 Table 7. Affinity results of antibodies to CTLA4 Table 8. Affinity results of antibodies to VEGF Example 12. Downregulation of PD-1 receptor in CHOK1-PD1-CTLA4 cells was determined by flow cytometry.

[0249] Harvest CHOK1-PD1-CTLA4 cells and adjust the cell density to 5×10⁻⁶. 6 / ml. Cells were added at 50 µl / well to F12K+ 10% FBS medium in 96-well U-plates. Serially diluted antibody was added to the cells. Cells were incubated at 4°C for 30 min and then washed twice with FACS buffer at 4°C. All subsequent procedures, including pipetting and centrifugation, were performed at 4°C. Then, a working concentration of 10 µg / ml of non-competitive PD-1-AF647 antibody was added to the plate, and the cells were incubated at 4°C for another 30 min. Cells were washed twice with FACS buffer at 4°C and then fixed with 4% paraformaldehyde (100 µL / well) at room temperature for 10 min. After fixation, cells were washed once with FACS buffer and analyzed using a Fortessa flow cytometer (BDBioscience). Results are shown in Figure 10 The results showed that PD1 was downregulated in cells due to CTLA4 internalization. The percentage of PD1 downregulated was measured. GBD214-33-03 cells showed similar PD1 internalization compared to AK104.

[0250] Example 13. In vivo antitumor efficacy of GBD214-33-03 antibody in A375 PBMC mouse model.

[0251] Used for hPBMC (6×10) 5 Premixed A375 human melanoma cancer cells (5 × 10⁹ cells) 6 NSG mice were subcutaneously inoculated with 1 cell per subcutaneous injection (n=6 per group, 4 weeks old). When tumors established (approximately 220 mm)... 3 At the time of treatment, pembrolizumab (15 mg / kg), bevacizumab (15 mg / kg), ipilimumab-LALA (15 mg / kg), three doses of AK112 (1 mg / kg, 5 mg / kg, and 20 mg / kg), three doses of AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), three doses of GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg), and a combination of pembrolizumab, bevacizumab, and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg) were administered intraperitoneally. Treatment was given twice weekly for a total of five doses. Tumor growth and mouse body weight were monitored every three days and reported as mean tumor volume and mean body weight. Results are shown in... Figure 11a and11b Results showed that in the A375 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) had better potency than AK112 (1 mg / kg, 5 mg / kg, and 20 mg / kg), AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), and the combination of pembrolizumab, bevacizumab, and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg). For all treatment groups, the mean body weight of mice decreased from day 7, with a decrease not exceeding 15%.

[0252] Example 14. In vivo antitumor efficacy of GBD214-33-03 antibody in HT29 PBMC mouse model.

[0253] Used for hPBMC (6×10) 5 Premixed HT29 human colon cancer cells (5 × 10⁶ cells) 6 NSG mice were subcutaneously inoculated with 1 cell per subcutaneous injection (n=6 per group, 4 weeks old). When tumors established (approximately 250 mm)... 3 At the time of treatment, pembrolizumab (15 mg / kg), bevacizumab (15 mg / kg), ipilimumab-LALA (15 mg / kg), three doses of AK112 (1 mg / kg, 5 mg / kg, and 20 mg / kg), three doses of AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), three doses of GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg), and a combination of pembrolizumab, bevacizumab, and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg) were administered intraperitoneally. Treatment was given twice weekly for a total of five doses. Tumor growth and mouse body weight were monitored every three days and reported as mean tumor volume and mean body weight. Results are shown in... Figure 12a and 12b The results showed that in the HT29 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) had better potency than AK112 (1 mg / kg, 5 mg / kg, and 20 mg / kg), AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), and the combination of pembrolizumab, bevacizumab, and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg). For all treatment groups, the mean body weight of mice remained relatively stable, with a decrease of no more than 15%.

[0254] In summary, this invention develops a trispecific antibody that specifically binds to PD-1, CTLA-4, and VEGF to block checkpoints expressed on T cells. This TsAb exhibits potent receptor-binding and ligand-blocking activity against PD-1 and VEGF, while only partially blocking the interaction between CTLA-4 and its ligand. The designed partial blocking activity against CTLA-4 aims to reduce peripheral irAE toxicity. In a mouse model, the trispecific antibody of this invention demonstrated potent antitumor activity.

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Claims

1. An antibody or antigen-binding fragment thereof that binds to PD-1 (programmed cell death protein 1) comprises: a heavy chain antibody variable domain (VHH), said VHH comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 1, the VHH CDR2 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 2, and the VHH CDR3 region comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

3.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VHH comprises CDRs 1, 2 and 3, which have amino acid sequences as shown in SEQ ID NO: 1, 2 and 3, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the VHH comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

4.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the VHH comprises or is composed of the amino acid sequence of SEQ ID NO:

4.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment specifically binds to PD-1.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof.

7. An antibody or an antigen-binding fragment thereof comprising VHH CDR 1, 2 and 3 of the antibody or antigen-binding fragment thereof according to any one of claims 1-6.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody or antigen-binding fragment comprises two or more heavy chain antibody variable domains.

9. An antibody or an antigen-binding fragment thereof, which cross-competes with an antibody or an antigen-binding fragment thereof according to any one of claims 1-8.

10. A multispecific antibody or an antigen-binding fragment thereof, comprising a first domain specifically binding to PD-1 and a second domain specifically binding to CTLA-4.

11. The multispecific antibody or its antigen-binding fragment according to claim 10, wherein... a) The first domain that specifically binds to PD-1 includes a heavy chain variable region (VH1), wherein the VH1 includes complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 1, 2, and 3, respectively; and / or b) The second domain that specifically binds to CTLA-4 includes a heavy chain variable region (VH2), which includes heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 7, 8, and 9, respectively.

12. The multispecific antibody or its antigen-binding fragment according to claim 10 or 11, wherein... a) The first domain that specifically binds to PD-1 comprises, or is composed of, the heavy chain variable region (VH1) of SEQ ID NO: 4; and / or b) The second domain that specifically binds to CTLA-4 includes, or is composed of, the heavy chain variable region (VH2) of SEQ ID NO:

10.

13. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 10-12, wherein the antibody further comprises one or more additional domains that specifically bind to antigens other than PD-1 and CTLA-4.

14. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 10-13, further comprising a domain specifically binding to VEGF.

15. The multispecific antibody or antigen-binding fragment thereof according to any one of claims 10-14, wherein it is an anti-PD-1 / CTLA-4 / VEGF trispecific antibody or antigen-binding fragment thereof, comprising a first domain specifically binding to PD-1, a second domain specifically binding to CTLA-4, and a third domain specifically binding to VEGF.

16. The multispecific antibody or its antigen-binding fragment according to claim 15, wherein... a) The first domain that specifically binds to PD-1 includes a heavy chain variable region (VH1), which includes 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 includes a heavy chain variable region (VH2), which contains 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 VEGF comprises a heavy chain variable region (VH3) and a light chain variable region (VL3), wherein the VH3 comprises heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) of SEQ ID NO: 13, 14, and 15, respectively, and the VL3 comprises light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) of SEQ ID NO: 19, 20, and 21, respectively.

17. The multispecific antibody or antigen-binding fragment thereof according to claim 16, wherein the VH1 comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 4, and / or the VH2 comprises an amino acid sequence having at least 80% identity with SEQ ID NO:

10.

18. The multispecific antibody or its antigen-binding fragment according to claim 16, 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 comprises, 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 VEGF comprises, or is composed of, the heavy chain variable region (VH) of SEQ ID NO: 16 and the light chain variable region (VL) of SEQ ID NO:

22.

19. The antibody or antigen-binding fragment thereof according to any one of claims 1-18, wherein the antibody further comprises the Fc region of IgG.

20. The antibody or antigen-binding fragment thereof according to any one of claims 10-19, wherein the first domain, the second domain, the third domain and / or the Fc region are directly interconnected via one or more adapters.

21. The antibody or antigen-binding fragment thereof according to any one of claims 20, 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.

22. The antibody or antigen-binding fragment thereof according to any one of claims 15-21, wherein the antibody comprises, or is composed of, the heavy chain of SEQ ID NO: 27 and the light chain of SEQ ID NO:

23.

23. The antibody or antigen-binding fragment thereof according to any one of claims 1-22, wherein the antibody is 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.

24. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1-23 and a pharmaceutically acceptable carrier.

25. A polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1-23.

26. A vector comprising the polynucleotide according to claim 25.

27. An isolated host cell comprising the vector according to claim 26.

28. A method for producing an antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell according to claim 27 under conditions expressing the antibody or an antigen-binding fragment thereof, and recovering and purifying the antibody or antigen-binding fragment produced by the host cell.

29. A method of treating a subject with cancer, the method comprising administering to a subject in need a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1-23 or a pharmaceutical composition according to claim 24, for a duration sufficient to treat the cancer.

30. The method of claim 29, wherein the cancer is a solid tumor or a hematologic malignancy.

31. The method of claim 30, 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 or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary cancer, endometriosis, cervical cancer, or metastatic lesions of cancer.

32. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-23 or the pharmaceutical composition according to claim 24 in the preparation of a medicament for treating cancer in a subject of need.

33. The use according to claim 32, wherein the cancer is a solid tumor or a hematologic malignancy.

34. The use according to claim 33, 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 or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary cancer, endometriosis, cervical cancer, or metastatic lesions of cancer.

35. The antibody or antigen-binding fragment thereof according to any one of claims 1-23, or the pharmaceutical composition according to claim 24, for treating cancer in a subject in need.

36. The antibody or antigen-binding fragment thereof or pharmaceutical composition according to claim 35, wherein the cancer is a solid tumor or a hematologic malignancy.

37. The antibody or antigen-binding fragment thereof or pharmaceutical composition according to claim 36, 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 or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary cancer, endometriosis, cervical cancer, or metastatic lesions of cancer.

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