Anti-PD-1 / CTLA-4 / VEGF antibodies and their use

Abstract

The present invention provides a PD-1 antibody, a trispecific antibody that specifically binds to PD-1, CTLA-4, and VEGF, or an antigen-binding fragment thereof, a polynucleotide encoding the antibody or its antigen-binding fragment, and a method for preparing and using the same.

Description

[Technical Field]

[0001] The present invention relates to an anti-PD-1 antibody, a trispecific antibody that specifically binds to PD-1, CTLA-4, and VEGF, or an antigen-binding fragment thereof, a polynucleotide encoding the antibody or its antigen-binding fragment, and a method for preparing and using the same. [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 suppressing T cell activation and proliferation and promoting T cell depletion (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. Preclinical and clinical studies have shown that PD-1 inhibition enhances T cell activity and promotes anti-tumor immune responses (Chen et al., 2021; B. Zhao, Zhao and Zhao, 2020). PD-1 inhibitors, such as nivolumab (Opidivo®) and pembrolizumab (Keytruda®), are approved by the FDA for use in 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] Vascular endothelial growth factor (VEGF) is a protein that plays a crucial role in angiogenesis (the process of forming new blood vessels). Because angiogenesis is important 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, including tumor cells (Melicovici et al., 2018; Simons, Gordon; and Cleasson-Welsh, 2016). Preclinical and clinical studies have shown that VEGF inhibition interferes with tumor angiogenesis and reduces 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 inhibiting downstream signal conduction and the formation of new blood vessels, are approved by the FDA for use in 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 modulates T cell function. As the first clinically targeted immune checkpoint receptor expressed on T cells, CTLA-4 shares the same ligands as CD28, CD80 / B7-1 and CD86 / B7-2. CTLA-4 is constitutively expressed on Foxp3+ regulatory T cells (Tregs) and upregulated by other activated T cells (Jago, Yates, C226 mara, Lechler and Lombardi, 2004; Sledzinska, Menger, Bergerhoff, Peggs and Quezada, 2015). The mechanism of CTLA-4 is thought to suppress T cell activation by crossing CD28 in binding to CD80 / B7-1 and CD86 / B7-2 (Chikuma, 2017). CTLA-4 inhibitors, such as ipilimumab, are approved by the FDA for use in the treatment of several types of cancer, including melanoma and prostate cancer.

[0005] As the most well-known anti-CTLA4 antibody, ipilimumab was approved in 2011 for the treatment of advanced melanoma. Ipilimumab has shown clinically potent and broad-spectrum cancer immunotherapeutic activity, both as monotherapy (Hodi et al., 2010) and as part of a 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). In particular, when used in combination with nivolumab, systemic activation of T cells due to blockade of the B7-CTLA-4 pathway led to decreased antibody resistance in patients (Bertrand, Kostine, Barnetche, Truchetet, and Schaeverbeke, 2015; Hodi, 2010). Nevertheless, CTLA-4 remains an important immunotherapeutic target because it can induce sustained immunity in cancer patients (Maio et al., 2015; Schadendorf et al., 2015). The main challenges in producing CTLA-4 antibodies are improving their safety and efficacy. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Liu, J., Chen, Z., Li, Y., Zhao, W., Wu, J., & Zhang, Z. (2021). PD-1 / PD-L1 Checkpoint Inhibitors in Tumor Immunotherapy. Front Pharmacol, 12, 731798. doi:10.3389 / fphar.2021.731798 [Non-Patent Document 2] Sharpe , AH , & Pauken , KE (2018). The diverse functions of the PD1 inhibitory pathway. Nat Rev Immunol, 18(3), 153-167. doi:10.1038 / nri.2017.108

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Summary of the Invention

Problems to be Solved by the Invention

[0007] Simply put, PD-1, CTLA-4, and VEGF are important targets in cancer treatment, and their inhibition has shown promising results in preclinical and clinical studies. PD-1 and CTLA-4 inhibitors enhance T-cell activity and promote anti-tumor immune responses, while VEGF inhibitors disrupt 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 treatments. [Means for solving the problem]

[0008] (Summary of the invention) The present invention provides an antibody or antigen-binding fragment that binds to PD-1 (programmed cell death protein 1), comprising a heavy chain antibody variable region (VHH) including complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region includes an amino acid sequence having at least 80% identity with SEQ ID NO:1, the VHH CDR2 region includes an amino acid sequence having at least 80% identity with SEQ ID NO:2, and the VHH CDR3 region includes an amino acid sequence having at least 80% identity with SEQ ID NO:3.

[0009] In one embodiment, VHH comprises CDRs 1, 2, and 3 having 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.

[0010] In one embodiment, the antibody or antigen-binding fragment specifically binds to PD-1.

[0011] In one embodiment, the antibody or antigen-binding fragment is a humanized antibody or its antigen-binding fragment.

[0012] The present invention also provides isolated antibodies or antigen-binding fragments comprising the above-mentioned antibodies or antigen-binding fragments thereof, comprising VHH CDRs 1, 2, and 3.

[0013] In one embodiment, the antibody or antigen-binding fragment comprises two or more heavy-chain antibody variable regions.

[0014] The present invention also provides an isolated antibody or antigen-binding fragment that cross-competes with the antibody or antigen-binding fragment described above.

[0015] The present invention also provides an isolated multispecific antibody or its antigen-binding fragment comprising a first domain that specifically binds to PD-1 and a second domain that specifically binds to CTLA-4. In one embodiment, the antibody or its antigen-binding fragment further comprises one or more additional domains that specifically bind to antigens other than PD-1 and CTLA-4. In one embodiment, the antibody or its antigen-binding fragment further comprises a domain that specifically binds to VEGF.

[0016] In one embodiment, an anti-PD-1 / CTLA-4 / VEGF trispecific antibody or its antigen-binding fragment is provided, comprising a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and a third domain that specifically binds to VEGF.

[0017] In one embodiment, the first domain that specifically binds to PD-1 includes a heavy chain variable region (VH1) comprising heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), which are SEQ ID NO: 1, 2, and 3, respectively.

[0018] In one embodiment, the first domain that specifically binds to PD-1 includes a heavy chain variable region (VH1) having 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 includes the heavy chain variable region (VH1) of SEQ ID NO:4, or consists of the heavy chain variable region (VH) of SEQ ID NO:4.

[0019] In one embodiment, the second domain that specifically binds to CTLA-4 includes a heavy chain variable region (VH2) comprising heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), which are SEQ ID NO: 7, 8, and 9, respectively.

[0020] In one embodiment, the second domain that specifically binds to CTLA-4 includes a heavy chain variable region (VH2) having an amino acid sequence that is at least 80% identical to SEQ ID NO:10. In one embodiment, the second domain that specifically binds to CTLA-4 includes the heavy chain variable region (VH2) of SEQ ID NO:10, or consists of the heavy chain variable region (VH) of SEQ ID NO:10.

[0021] In one embodiment, the third domain that specifically binds to VEGF includes a heavy chain variable region (VH3) comprising heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), which are SEQ ID NO: 13, 14, and 15 respectively, and a light chain variable region (VL) comprising light chain complementarity determination regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3), which are SEQ ID NO: 19, 20, and 21 respectively.

[0022] In one embodiment, the third domain that specifically binds to VEGF includes or consists of a heavy chain variable region (VH3) with SEQ ID NO:16 and a light chain variable region (VL) with SEQ ID NO:22.

[0023] In one embodiment, the third domain that specifically binds to VEGF includes or consists of a heavy chain with SEQ ID NO:17 and a light chain with SEQ ID NO:23. In one embodiment, the third domain that specifically binds to VEGF includes or consists of a full-length antibody or its antigen-binding fragment, which includes two heavy chains and two light chains linked together by disulfide bonds. In one embodiment, the antigen-binding fragment of the full-length antibody is a Fab fragment or an scFv fragment.

[0024] 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 having the Leu234 Ala / Leu235 Ala mutation).

[0025] In one embodiment, the antibody or antigen-binding fragment according to the present invention belongs to an IgG1, IgG2, IgG3, or IgG4 isotype, optionally containing one, two, three, four, five, six, seven, eight, nine, or ten substitutions in the Fc region.

[0026] In one embodiment, the first domain that specifically binds to PD-1 is located at the N-terminus of the Fc region.

[0027] In one embodiment, the second domain that specifically binds to CTLA-4 is located at the N-terminus of the Fc region.

[0028] In one embodiment, the third domain that specifically binds to VEGF is located at the N-terminus of the Fc region.

[0029] In one embodiment, the first domain that specifically binds to PD-1 is located at the C-terminus of the Fc region.

[0030] In one embodiment, the second domain that specifically binds to CTLA-4 is located at the C-terminus of the Fc region.

[0031] In one embodiment, the third domain that specifically binds to VEGF is located at the C-terminus of the Fc region.

[0032] In one embodiment, a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, a third domain that specifically binds to VEGF, and an Fc region are interconnected directly or via one or more linkers. In one embodiment, the linkers are the same or different. In one embodiment, the linkers are flexible connections. In one embodiment, the linkers are peptide linkers. In one embodiment, the linkers are GGGGSGGGGS.

[0033] 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 by the following formula from the N-terminus to the C-terminus. Third domain - Hinge region - Fc - Linker - Second domain - Linker - First domain

[0034] In one embodiment, the third domain that specifically binds to VEGF is a full-length antibody. In one embodiment, the first domain that specifically binds to PD-1 is optionally linked via a linker to the C-terminus of one or two heavy chains of the full-length antibody, and the second domain that specifically binds to CTLA-4 is optionally linked via a linker to the C-terminus of the first domain that specifically binds to PD-1. In one embodiment, the second domain that specifically binds to CTLA-4 is optionally linked via a linker to the C-terminus of one or two heavy chains of the full-length antibody, and the first domain that specifically binds to PD-1 is optionally linked via a linker to the C-terminus of the second domain that specifically binds to CTLA-4. In one embodiment, the first domain that specifically binds to PD-1 and the second domain that specifically binds to CTLA-4 are linked in the same or different order to the C-terminuses of two heavy chains of the full-length antibody. In one embodiment, two second domains that specifically bind to CTLA-4 are each linked to the C-terminus of the heavy chain of a full-length antibody via linkers, 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 linkers.

[0035] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention comprises or consists of a heavy chain with SEQ ID NO:27 and a light chain with SEQ ID NO:23.

[0036] In one embodiment, the antibody according to the present invention comprises or consists of one or two heavy chains and one or two light chains. In one embodiment, the antibody according to the present invention comprises or consists of two heavy chains and two light chains. In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention comprises a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and a third domain that specifically binds to VEGF. In one embodiment, the first domain that specifically binds to PD-1 includes a heavy chain variable region (VH1) containing heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), respectively, which are SEQ ID NO: 1, 2, and 3; the second domain that specifically binds to CTLA-4 includes a heavy chain variable region (VH2) containing heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), respectively, which are SEQ ID NO: 7, 8, and 9; and the third domain that specifically binds to VEGF includes a heavy chain variable region (VH3) containing heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), respectively, which are SEQ ID NO: 13, 14, and 15, respectively, and SEQ ID The domain includes a light chain variable region (VL) containing light chain complementarity determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3), which are NO:19, 20, and 21, respectively. In one embodiment, the first domain that specifically binds to PD-1 includes or consists of the heavy chain variable region (VH1) of SEQ ID NO:4, the second domain that specifically binds to CTLA-4 includes or consists of the heavy chain variable region (VH2) of SEQ ID NO:10, and the third domain that specifically binds to VEGF includes 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. In one embodiment, the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO:27 and the light chain of SEQ ID NO:23.

[0037] The present invention also provides a pharmaceutical composition comprising the antibody or its antigen-binding fragment, and a pharmaceutically acceptable carrier.

[0038] The present invention also provides polynucleotides encoding the heavy chain variable region (VH) or heavy chain of the antibody or antigen-binding fragment of the present invention.

[0039] The present invention also provides polynucleotides encoding the antibody or its antigen-binding fragment.

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

[0041] The present invention also provides host cells containing the vector of the present invention.

[0042] The present invention also provides a method for producing the antibody of the present invention, comprising culturing host cells of the present invention under conditions that express the antibody or an antigen-binding fragment thereof, and recovering the antibody produced by the host cells.

[0043] The present invention also provides a method for treating a target cancer, comprising administering a therapeutically effective amount of an isolated antibody or its antigen-binding fragment to a target for a period of time sufficient to treat the cancer.

[0044] The present invention also provides the use of the isolated antibody or its antigen-binding fragment in the preparation of pharmaceuticals for treating a target cancer of interest.

[0045] The present invention also provides isolated antibodies or antigen-binding fragments thereof for treating target cancers of interest. [Brief explanation of the drawing]

[0046] [Figure 1] Figure 1 shows a schematic diagram of the structure of the triply specific antibody GBD214-33-03 (anti-PD1 / CTLA4 / VEGF triply specific antibody) according to the present invention. [Figure 2]Figure 2 shows the binding specificity of different antibodies to the human PD1 protein. The results showed that GBD214-33-03 bound to PD1 in HEK293-PD1 cells. GBD214-33-03 showed strong binding to HEK293-PD1 cells with a median fluorescence intensity (MFI) similar to that of its PD1 parent GBD002-hS019-WS (anti-PD1 antibody) and pembrolizumab. [Figure 3] Figure 3 shows the performance of different antibodies in a PD1 reporter assay. The PD1 reporter assay showed that GBD214-33-03 had a similar maximum luminescence signal to pembrolizumab and AK104 (Akesobio, Inc.'s anti-PD1 / CTLA4 bispecific antibody). This result indicates that GBD214-33-03 can block the interaction between PD1 and its ligand, PD-L1. [Figure 4] Figure 4 shows the performance of different antibodies in a mixed lymphocyte reaction (MLR) assay. The 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. [Figure 5] Figure 5 shows the VEGF binding of different antibodies. The results showed that GBD214-33-03 has similar VEGF binding to bevacizumab. [Figure 6] Figure 6 shows the VEGF blockade of different antibodies. The results showed that GBD214-33-03 can suppress the binding of VEGFR2 to VEGF165, similar to bevacizumab. [Figure 7] Figure 7 shows the performance of different antibodies in the VEGF reporter assay. The results showed that GBD214-33-03, similar to bevacizumab, can suppress VEGF165-induced downstream NFAT signaling by blocking the binding of VEGFR2 to VEGF165. [Figure 8a]Figures 8a and 8b show the binding specificity of different antibodies to the human CTLA4 protein. The results show that in CHOK1-CTLA4 cells, GBD214-33-03 had a lower MFI than ipilimumab, suggesting that GBD214-33-03 binds to CTLA4 in CHOK1-CTLA4 cells. The CTLA4 parent GBD008-hS005-3-2 (anti-CTLA4 antibody) had weaker CTLA4 binding ability than ipilimumab. In CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed stronger binding compared to the PD1 parent GBD002-hS019-WS. [Figure 8b] Figures 8a and 8b show the binding specificity of different antibodies to the human CTLA4 protein. The results show that in CHOK1-CTLA4 cells, GBD214-33-03 had a lower MFI than ipilimumab, suggesting that GBD214-33-03 binds to CTLA4 in CHOK1-CTLA4 cells. The CTLA4 parent GBD008-hS005-3-2 (anti-CTLA4 antibody) had weaker CTLA4 binding ability than ipilimumab. In CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed stronger binding compared to the PD1 parent GBD002-hS019-WS. [Figure 9a] Figures 9a-9d show the blocking of CD80 and CD86 binding to CTLA4 by different antibodies. The results showed that GBD214-33-03 partially suppressed the binding of CD80 / CD86 to CTLA4 in CHOK1-CTLA4 cells and CHOK1-PD1-CTLA4 cells. In both CHOK1-CTLA4 cells and CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed weaker blocking activity compared to ipilimumab. [Figure 9b]Figures 9a-9d show the blocking of CD80 and CD86 binding to CTLA4 by different antibodies. The results showed that GBD214-33-03 partially suppressed the binding of CD80 / CD86 to CTLA4 in CHOK1-CTLA4 cells and CHOK1-PD1-CTLA4 cells. In both CHOK1-CTLA4 cells and CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed weaker blocking activity compared to ipilimumab. [Figure 9c] Figures 9a-9d show the blocking of CD80 and CD86 binding to CTLA4 by different antibodies. The results showed that GBD214-33-03 partially suppressed the binding of CD80 / CD86 to CTLA4 in CHOK1-CTLA4 cells and CHOK1-PD1-CTLA4 cells. In both CHOK1-CTLA4 cells and CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed weaker blocking activity compared to ipilimumab. [Figure 9d] Figures 9a-9d show the blocking of CD80 and CD86 binding to CTLA4 by different antibodies. The results showed that GBD214-33-03 partially suppressed the binding of CD80 / CD86 to CTLA4 in CHOK1-CTLA4 cells and CHOK1-PD1-CTLA4 cells. In both CHOK1-CTLA4 cells and CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed weaker blocking activity compared to ipilimumab. [Figure 10] Figure 10 shows the downregulation of different antibodies against the PD1 receptor in CHOK1-PD1-CTLA4 cells. The results indicate that the downregulation of PD1 in the cells is due to CTLA4 internalization. The percentage of downregulated PD1 was measured. GBD214-33-03 showed similar PD1 internalization compared to AK104. [Figure 11a]Figures 11a and 11b show the in vivo antitumor effects of different antibodies in the A375 PBMC mouse model. The results showed that in the A375 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) was more effective than 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 a combination of ipilimumab-LALA (ipilimumab with Leu234Ala / Leu235Ala mutations) (15 mg / kg + 15 mg / kg + 15 mg / kg). Mean body weight of mice began to decrease from day 7 in all treatment groups, but the decrease was less than 15%. [Figure 11b] Figures 11a and 11b show the in vivo antitumor effects of different antibodies in the A375 PBMC mouse model. The results showed that in the A375 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) was more effective than 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 a combination of ipilimumab-LALA (ipilimumab with Leu234Ala / Leu235Ala mutations) (15 mg / kg + 15 mg / kg + 15 mg / kg). Mean body weight of mice began to decrease from day 7 in all treatment groups, but the decrease was less than 15%. [Figure 12a]Figures 12a and 12b show the in vivo antitumor effects of different antibodies in an HT29 PBMC mouse model. The results showed that in the HT29 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) was more effective than AK112 (1 mg / kg, 5 mg / kg, and 20 mg / kg), AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), pembrolizumab, and the combination of bevacizumab and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg). The mean body weight of the mice remained relatively stable across all treatment groups, with a decrease of less than 15%. [Figure 12b] Figures 12a and 12b show the in vivo antitumor effects of different antibodies in an HT29 PBMC mouse model. The results showed that in the HT29 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) was more effective than AK112 (1 mg / kg, 5 mg / kg, and 20 mg / kg), AK104 (1 mg / kg, 5 mg / kg, and 20 mg / kg), pembrolizumab, and the combination of bevacizumab and ipilimumab-LALA (15 mg / kg + 15 mg / kg + 15 mg / kg). The mean body weight of the mice remained relatively stable across all treatment groups, with a decrease of less than 15%. [Modes for carrying out the invention]

[0047] All publications cited herein, including but not limited to patents and patent applications, are incorporated herein by reference, as they are enumerated in their entirety.

[0048] It should be understood that the terms used herein are used solely for the purpose of describing specific embodiments and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains.

[0049] Any methods and materials similar or equivalent to those described herein may be used in carrying out the tests of the present invention, but this specification describes exemplary materials and methods. The following terms are used in describing and claiming the present invention:

[0050] As used herein and in the appended claims, the singular forms “one,” “one kind,” and “this / the foregoing” include plural referents unless the context specifically specifies otherwise. Thus, for example, the reference to “one cell” is by analogy to include combinations of two or more cells, etc.

[0051] "Specific binding," "specific binding," or "binding" means that an antibody binds to an antigen or epitope on an antigen with higher affinity than other antigens. Typically, an antibody binds to an antigen with an affinity of approximately 1 × 10⁻⁶. -8 Less than M, for example, approximately 1 × 10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10- 11 M or less, or approximately 1 × 10 -12 Equilibrium dissociation constants (K) less than or equal to M D ) and usually K is bound to a nonspecific antigen (e.g., BSA, casein). D In comparison, K is up to 1 / 100 D Antibodies bind to an antigen or an epitope in an antigen. The dissociation constant can be measured using a standard program. However, antibodies that specifically bind to an antigen or an epitope in an antigen may cross-react with the same antigen (homolog) from other related antigens, such as humans or monkeys from other species, such as cynomolgus monkeys (Macaca fascicularis, cynomolgus, cyno), chimpanzees (Pan troglodytes, chimpanzee, chimp), or common marmosets (Callithrix jacchus, common marmoset, marmoset). A monospecific antibody specifically binds to one antigen or one epitope, while a bispecific antibody specifically binds to two different antigens or two different epitopes.

[0052] In a broad sense, "antibody" refers to and includes monoclonal antibodies (including mouse, human, humanized, and chimeric monoclonal antibodies), antigen-binding fragments, bispecific or multispecific antibodies, dimeric antibodies, tetrameric or multimeric antibodies, single-chain antibodies, domain antibodies, and immunoglobulin molecules including any other modified structures of immunoglobulin molecules containing a desired specific antigen-binding site. "Full-length antibody" includes two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and its multimer (e.g., IgM). Each heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region (including domain CH1, hinges CH2 and CH3). Each light chain includes a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions may be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) that alternate with framework regions (FRs). Each VH and VL contains three CDR segments and four FR segments arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino group terminus to the carboxyl group terminus.

[0053] 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 (HCDR1, HCDR2, HCDR3) are located in the VH, and three (LCDR1, LCDR2, LCDR3) are located in the VL) are due to sequence changes (Wu and Kabat, (1970) J Exp Med 132:211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) The “highly variable region,” “HVR,” or “HV” (three (H1, H2, H3) in VH and three (L1, L2, L3) in VL) refers to a region within the antibody variable domain that is structurally hypervariable, as defined by Chothia and Lesk (Chothia and Lesk, (1987) Mol Biol 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 described in Lefranc et al., (2003) Dev Comparat Immunol 27:55-77. The terms “CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2,” and “LCDR3” as used herein include CDRs as defined by any of the above methods, Kabat, Chothia, or IMGT, unless otherwise specified herein.

[0054] Immunoglobulins can be classified into five major categories—IgA, IgD, IgE, IgG, and IgM—based on the amino acid sequence of their heavy chain constant domain. IgA and IgG are further subdivided into isotypes such as IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be classified into one of two distinct types, kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0055] "Isolated antibody" means an antibody or antibody fragment that substantially does not contain other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to PD-1 substantially does not contain 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 present invention, the trispecific antibody specifically binds to PD-1, CTLA-4, and VEGF and substantially does not contain antibodies that specifically bind to antigens other than PD-1, CTLA-4, and VEGF. "Isolated antibody" includes antibodies isolated to a higher purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure antibodies.

[0056] "Recombinant" refers to antibodies and other proteins prepared, expressed, produced, or isolated by recombinant means.

[0057] An "epitope" refers to a portion of an antigen to which an antibody specifically binds. Epitopes generally consist of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groupings, such as amino acids or polysaccharide side chains, and can possess specific three-dimensional structural and charge-to-specific properties. Epitopes can be composed of continuous and / or discontinuous amino acids that form conformational units. In the case of discontinuous epitopes, amino acids from different parts of the linear sequence derived from the antigen approach each other in three-dimensional space through protein molecule folding. Antibody "epitopes" depend on the method used to identify them.

[0058] "Multispecific" refers to an antibody that specifically binds to at least two different antigens or two different epitopes on an antigen (e.g., three, four, or five different antigens or epitopes). A "triple-specific" antibody refers to an antibody that specifically binds to three different antigens or three different epitopes on the same antigen. Triple-specific antibodies may cross-react with other related antigens, such as the same antigen (homolog) from humans or other species, such as cynomolgus monkeys (Macaca fascicularis, cynomolgus, cyno), chimpanzees (Pan troglodytes, chimpanzee, chimp), or common marmosets (Callithrix jacchus, common marmoset, marmoset), or may bind to epitopes shared between two or more different antigens.

[0059] A “vector” refers to a polynucleotide that can replicate within a biological system or move between such systems. Vector polynucleotides typically contain elements such as replication origins, polyadenylation signals, or selection markers that function to facilitate the replication or maintenance of these polynucleotides within the biological system. Examples of such biological systems include cells, viruses, animals, plants, and reconstituted biological systems that utilize biological elements capable of replicating vectors. The polynucleotide containing the vector may be a DNA or RNA molecule or a hybrid thereof.

[0060] An "expression vector" refers to a vector that can be used in a biological system or a reconstituted biological system to guide the translation of a polypeptide encoded by the polynucleotide sequence present in the expression vector.

[0061] A "polynucleotide" refers to a synthetic molecule containing nucleotide chains covalently linked by a sugar-phosphate ester skeleton or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

[0062] The terms "overexpress," "overexpressed," and "overexpressing" are used interchangeably and refer to samples, such as cancer cells, malignant cells, or cancerous tissue, that have measurably higher levels of PD-1, CTLA-4, or VEGF or their ligands compared to a reference sample. Overexpression is caused by gene amplification or by increased transcription or translation. Protein expression and overexpression in a sample can be measured in live or lysed cells using well-known assays, such as ELISA, immunofluorescence, flow cytometry, or radioimmunoassays. Polynucleotide expression and overexpression in a sample can be measured using, for example, fluorescence in cis hybridization, Southern blotting, or PCR techniques. A protein or polynucleotide is overexpressed if the level of that protein or polynucleotide in the sample is at least 1.5 times higher or statistically significant compared to a reference sample. The selection of the reference sample is known.

[0063] "Sample" refers to a fluid, cell, or tissue similar to that isolated from the subject, and a collection of fluids, cells, or tissues similar to that present in the subject. Exemplary samples include biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, teardrops, feces, sputum, mucosal secretions from secretory tissues and organs, vaginal secretions, ascites (e.g., ascites associated with non-solid cancers), fluids from the pleural cavity, pericardial cavity, peritoneal cavity, abdominal cavity and other body cavities, fluids collected by bronchial lavage, solutions that have come into contact with the subject or biosource (e.g., cell and organ culture media including cell or organ acclimatization media, lavage solutions, etc.), tissue biopsy, fine-needle aspiration, or surgically excised tumor tissue.

[0064] "Cancer cells" or "tumor cells" refer to cancer cells, precancerous cells, or transformed cells that exhibit spontaneous or induced phenotypic changes in vivo, in vitro, or in tissue culture. These changes are not necessarily related to the uptake of new genetic material. Transformation can result from infection with transforming viruses and the uptake of novel genomic nucleic acids, or from the uptake of exogenous nucleic acids, but it can also appear spontaneously or after exposure to carcinogens to mutate endogenous genes. Transformations / cancer are exemplified by morphological changes in vitro, in vivo, and in vitro, cell immortalization, control of abnormal proliferation, fossilization, growth, malignancy, regulation of tumor-specific marker levels, invasiveness, and tumor growth in a suitable animal host (e.g., nude mouse) (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd edition, 1994)).

[0065] "Approximately" means that a portion of it is within an acceptable margin of error, depending on how a particular value is measured or determined, as determined by those skilled in the art, i.e., on the limits of the measuring system. Unless otherwise explicitly indicated in the examples or other parts of the specification, "approximately" in the context of a particular assay, result, or embodiment means within the range of standard deviations practiced in the art, or within 5%, whichever is greater.

[0066] "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" refers to a molecule that includes at least one binding domain that specifically binds to PD-1, at least one binding domain that specifically binds to CTLA-4, and at least one binding domain that specifically binds to VEGF. The domains that specifically bind to PD-1, CTLA-4, and VEGF are usually VH / VL pairs or VH only. The trispecific anti-PD-1 / CTLA-4 / VEGF antibody can be monovalent or bivalent in terms of binding to PD-1, CTLA-4, or VEGF.

[0067] "Valency" refers to the presence of a specific number of antigen-specific binding sites in a molecule. Thus, the terms "monovalent", "bivalent", "tetravalent", and "hexavalent" refer to the presence of 1, 2, 4, and 6 antigen-specific binding sites in a molecule, respectively.

[0068] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment that contains the V H and V L domains in a single polypeptide chain. Generally, the Fv polypeptide further includes a polypeptide linker between the V H and V L domains such that the scFv can form the structure necessary for antigen binding. For an overview of scFv, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 269-315. See also International Publication No. WO 88 / 01649 and U.S. Patents Nos. 4,946,778 and 5,260,203. In one embodiment, the scFv includes, from the N-terminus to the C-terminus, the V H region, the peptide linker, and the V L region (VH-VL format). In another embodiment, the scFv includes, from the N-terminus to the C-terminus, the VL Region, peptide linker and V H Includes area (VL-VH format).

[0069] As used herein, the term “diabody” refers to a double antibody with a light chain variable domain (V) on the same polypeptide chain. L ) connected to the heavy chain variable domain (V H )(V H -V L or V L -V H This refers to a small antibody fragment containing two antigen-binding sites. By using a linker that is too short to allow pairing between the two domains on the same chain, pairing of these two domains with a complementary domain on another chain is forced, generating two antigen-binding sites. Biantibodies are described in detail, for example, EP 404,097, WO 93 / 11161, and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448. For an overview of modified antibody variants, see generally Holliger and Hudson (2005) Nat. Biotechnol. 23:1126-1136.

[0070] A "Fab" typically comprises heavy chain VH and CH1 regions and light chain VL and CL regions, which are linked by disulfide bonds and have a single antigen-binding site. According to this disclosure, the VH, CH1, VL, and CL regions in a Fab can be arranged in various ways to confer antigen-binding ability. For example, the VH and CH1 regions may be on a single polypeptide, and the VL and CL regions may be on separate polypeptides. Alternatively, the VH, CH1, VL, and CL regions may all be located on the same polypeptide and may be arranged in any different order.

[0071] “Antigen-specific CD4 + or CD8 + "T cells" are CD4 cells that have been activated by a specific antigen or its immunostimulatory epitope. + or CD8+ This refers to T cells.

[0072] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, including mammals and non-mammals such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" and "subject" are used interchangeably.

[0073] "Treat" or "treatment" refers to therapeutic treatment aimed at slowing (reducing) an undesirable physiological change or disease (e.g., the development or spread of a tumor or tumor cells) or providing a beneficial or desirable clinical outcome during treatment. Beneficial or desirable clinical outcomes include reduction of detectable or undetectable symptoms, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delayed or slowed disease progression, absence of metastasis, improvement or mitigation of the disease state, and remission (partial or overall). "Treatment" can also mean an extension of survival beyond the expected survival if the subject were not treated. Subjects requiring treatment include those who already have an undesirable physiological change or disease, and those who are prone to developing physiological changes or disease.

[0074] "Therapeutic effective dose" refers to the amount effective in achieving the desired therapeutic outcome in the required dose and duration. The therapeutic effective dose of the antibody of the present invention may vary depending on factors such as the disease state, the individual's age, sex, and weight, and the ability of the antibody of the present invention to elicit a desired response in the individual. Exemplary indicators of an effective treatment or combination of treatments include, for example, improved patient health, reduced tumor burden, cessation or delay of tumor growth, and / or the absence of metastasis of cancer cells to other locations in the body.

[0075] Throughout this specification, unless otherwise specified, the numbering of amino acid residues in the constant region of antibodies follows 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).

[0076] This specification uses the conventional one-letter and three-letter amino acid codes shown in Table 1. [Table 1]

[0077] Composition of matter The present invention provides anti-PD-1 antibodies and anti-PD-1 / CTLA-4 antibodies, and in particular, triply specific antibodies or antigen-binding fragments thereof that specifically bind to PD-1, CTLA-4, and VEGF. The present invention also provides polynucleotides or complementary nucleic acids, vectors, host cells, and methods for producing and using the antibodies or antigen-binding fragments of the present invention.

[0078] In the context of this disclosure, the antibodies of the present invention include their antigen-binding fragments, where applicable. For example, an isolated anti-PD-1 antibody or its antigen-binding fragment may simply be referred to as "isolated anti-PD-1 antibody."

[0079] antibody

[0080] The present invention comprises a heavy chain antibody variable region (VHH) including complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VHH CDR1 region includes 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, and the VHH CDR2 region includes 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 VHH The CDR3 region provides an isolated antibody or antigen-binding fragment that binds to PD-1 (programmed cell death protein 1), containing an amino acid sequence that has 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:3.

[0081] 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.

[0082] In one embodiment, VHH includes 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 includes or consists of the amino acid sequence of SEQ ID NO:4.

[0083] In one embodiment, the antibody or antigen-binding fragment that binds to PD-1 comprises a heavy chain containing 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.

[0084] 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.

[0085] The present invention also provides isolated antibodies or antigen-binding fragments comprising the above-mentioned antibodies or antigen-binding fragments VHH CDR1, 2, and 3.

[0086] In one embodiment, the antibody or its antigen-binding fragment comprises VHH CDRs 1, 2, and 3 having amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, 2, and 3, respectively.

[0087] In one embodiment, the antibody or antigen-binding fragment comprises two or more heavy-chain antibody variable domains. In one embodiment, the antibody or antigen-binding fragment comprises two, three, four, five, six, seven, eight, nine, or ten VHHs.

[0088] The present invention also provides isolated antibodies or antigen-binding fragments that cross-compete with the antibodies or antigen-binding fragments described above. In one embodiment, the antibodies or antigen-binding fragments cross-compete with antibodies or antigen-binding fragments containing VHH CDRs 1, 2, and 3, each containing amino acid sequences 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. In one embodiment, the antibody or its antigen-binding fragment cross-compete with the antibody or its antigen-binding fragment containing a VHH 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.

[0089] The present invention also provides an isolated multispecific antibody or its antigen-binding fragment comprising a first domain that specifically binds to PD-1 and a second domain that specifically binds to CTLA-4. In one embodiment, the antibody further comprises one or more additional domains that specifically bind to antigens other than PD-1 and CTLA-4. In one embodiment, the additional domains that specifically bind to antigens other than PD-1 and CTLA-4 include a third domain that specifically binds to VEGF.

[0090] In one embodiment, an isolated anti-PD-1 / CTLA-4 / VEGF trispecific antibody or its antigen-binding fragment is provided, comprising a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and a third domain that specifically binds to VEGF.

[0091] In some embodiments, the antibody or antigen-binding fragment of the present invention is antigen-specific CD4 + or CD8 + It enhances the activation of T cells.

[0092] 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.

[0093] In some embodiments, the antibody or antigen-binding fragment of the present invention inhibits the binding of CTLA-4 to CD80 and CD86.

[0094] In some embodiments, the antibody or antigen-binding fragment of the present invention inhibits the binding of VEGF to VEGFR1 and / or VEGFR2.

[0095] In some embodiments, the antibody or antigen-binding fragment of the present invention induces the internalization of PD-1 on the cell surface.

[0096] In one embodiment, a first domain that specifically binds to PD-1 includes or comprises a heavy chain variable region (VH1), the VH1 including heavy chain complementarity determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), each containing 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.

[0097] In one embodiment, the first domain that specifically binds to PD-1 includes or comprises a heavy chain variable region (VH1) which includes heavy chain complementarity determining regions 1 (HCDR 1), 2 (HCDR 2), and 3 (HCDR 3), respectively, which are SEQ ID NO: 1, 2, and 3.

[0098] In one embodiment, a second domain that specifically binds to CTLA-4 includes or comprises a heavy chain variable region (VH2), the VH2 including heavy chain complementarity determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), each containing amino acid sequences 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.

[0099] In one embodiment, the second domain that specifically binds to CTLA-4 includes or comprises a heavy chain variable region (VH2) containing heavy chain complementarity determination regions 1 (HCDR 1), 2 (HCDR 2), and 3 (HCDR 3), which are SEQ ID NO: 7, 8, and 9, respectively.

[0100] In one embodiment, a third domain that specifically binds to VEGF includes or comprises a heavy chain variable region (VH3) and a light chain variable region (VL), wherein the VH3 includes heavy chain complementarity determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), each containing 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, and the VL comprises SEQ ID The light chain complementarity determination regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) each contain amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to NO:19, 20, and 21, respectively.

[0101] In one embodiment, the third domain that specifically binds to VEGF includes or comprises a heavy chain variable region (VH3) containing heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), which are SEQ ID NO: 13, 14, and 15 respectively, and a light chain variable region (VL) containing light chain complementarity determination regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3), which are SEQ ID NO: 19, 20, and 21 respectively.

[0102] In one embodiment, the first domain that specifically binds to PD-1 includes or comprises the heavy chain variable region (VH1) of SEQ ID NO:4, where VH1 optionally has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen conserved amino acid substitutions. Optionally, no substitutions are present in the CDR.

[0103] In one embodiment, the first domain includes or comprises a heavy chain variable region (VH1) containing 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, the CDR has no differences from the sequence of SEQ ID NO:4.

[0104] In one embodiment, the second domain that specifically binds to CTLA-4 includes or comprises the heavy chain variable region (VH2) of SEQ ID NO:10, where VH2 optionally has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen conserved amino acid substitutions. Optionally, no substitutions are present in the CDR.

[0105] In one embodiment, the second domain includes or comprises a heavy chain variable region (VH2) containing 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, the CDR has no differences from the sequence of SEQ ID NO:10.

[0106] In one embodiment, the third domain that specifically binds to VEGF includes or consists of a heavy chain variable region (VH3) with SEQ ID NO:16 and a light chain variable region (VL) with SEQ ID NO:22, where VH3 and VL each optionally have one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen conserved amino acid substitutions. Optionally, no substitutions are present in the CDR.

[0107] In one embodiment, the third domain includes, or comprises, a heavy chain variable region (VH3) containing 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 a light chain variable region (VL) containing 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. There are no differences in the CDR between the sequences of SEQ ID NO:16 and / or SEQ ID NO:22.

[0108] In one embodiment, the first, second, and third domains are independently a chimeric antibody, a humanized antibody, a human antibody, a single-chain antibody, Fv, Fab, F(ab')2, Fd, a single-chain Fv molecule (scFv), a biantibody, or a single-domain antibody (dAb).

[0109] In one embodiment, the first, second, and third domains are humanized independently of each other. In one embodiment, the first and second domains are humanized VHH independently of each other.

[0110] In one embodiment, the antibody according to the present invention comprises only one heavy chain. In one embodiment, the antibody according to the present invention comprises one heavy chain and one light chain. In one embodiment, the antibody according to the present invention comprises two heavy chains. In one embodiment, the antibody according to the present invention comprises two heavy chains and two light chains.

[0111] In one embodiment, the antibody according to the present invention consists of one heavy chain. In one embodiment, the antibody according to the present invention consists of one heavy chain and one light chain. In one embodiment, the antibody according to the present invention consists of two heavy chains. In one embodiment, the antibody according to the present invention consists of two heavy chains and two light chains.

[0112] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody comprises 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 that specifically binds to PD-1 includes a heavy chain variable region (VH1) containing heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), respectively, which are SEQ ID NO: 1, 2, and 3; a second domain that specifically binds to CTLA-4 includes a heavy chain variable region (VH2) containing heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), respectively, which are SEQ ID NO: 7, 8, and 9; and / or a third domain that specifically binds to VEGF includes a heavy chain variable region (VH3) containing heavy chain complementarity determination regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), respectively, which are SEQ ID NO: 13, 14, and 15, and SEQ ID It includes a light chain variable region (VL) which contains light chain complementarity determination regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3), which are NO:19, 20, and 21, respectively.

[0113] In one embodiment, a first domain that specifically binds to PD-1 includes or consists of the heavy chain variable region (VH1) of SEQ ID NO:4, a second domain that specifically binds to CTLA-4 includes or consists of the heavy chain variable region (VH2) of SEQ ID NO:10, and / or a third domain that specifically binds to VEGF includes 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.

[0114] In one embodiment, the third domain that specifically binds to VEGF includes or consists of a heavy chain with SEQ ID NO:17 and a light chain with SEQ ID NO:23. In one embodiment, the third domain that specifically binds to VEGF includes or consists of a full-length antibody or its antigen-binding fragment, which includes two heavy chains and two light chains interconnected by disulfide bonds. In one embodiment, the antigen-binding fragment of the full-length antibody is a Fab fragment or an scFv fragment.

[0115] 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. The term "LALA" refers to the introduced amino acid substitution L234A / L235A (Kabat nomenclature), as is well known in the art.

[0116] 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.

[0117] 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.

[0118] In one embodiment, a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, a third domain that specifically binds to VEGF, and an Fc region are directly connected to each other or connected via one or more linkers (e.g., flexible linkers). In one embodiment, the linkers are the same or different. In one embodiment, the linker is a peptide linker, most preferably a peptide linker lacking protein hydrolysis cleavage sites. 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.

[0119] 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 by the following formula from the N-terminus to the C-terminus. Third domain - Hinge region - Fc - Linker - Second domain - Linker - First domain

[0120] 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 by the following formula from the N-terminus to the C-terminus. Third domain - Hinge region - Fc - Linker - First domain - Linker - Second domain

[0121] 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 by the following formula from the N-terminus to the C-terminus. Second domain - Hinge region - Fc - Linker - Third domain - Linker - First domain

[0122] 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 by the following formula from the N-terminus to the C-terminus. First domain - Hinge region - Fc - Linker - Third domain - Linker - Second domain

[0123] 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 by the following formula from the N-terminus to the C-terminus. Second domain - Hinge region - Fc - Linker - First domain - Linker - Third domain

[0124] 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 by the following formula from the N-terminus to the C-terminus. First domain - Hinge region - Fc - Linker - Second domain - Linker - Third domain

[0125] In one embodiment, the third domain that specifically binds to VEGF is a full-length antibody. In one embodiment, the first domain that specifically binds to PD-1 is optionally linked via a linker to the C-terminus of one or two heavy chains of the full-length antibody, and the second domain that specifically binds to CTLA-4 is optionally linked via a linker to the C-terminus of the first domain that specifically binds to PD-1. In one embodiment, the second domain that specifically binds to CTLA-4 is optionally linked via a linker to the C-terminus of one or two heavy chains of the full-length antibody, and the first domain that specifically binds to PD-1 is optionally linked via a linker to the C-terminus of the second domain that specifically binds to CTLA-4. In one embodiment, the first domain that specifically binds to PD-1 and the second domain that specifically binds to CTLA-4 are linked in the same or different order to the C-terminuses of two heavy chains of the full-length antibody. 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 linkers, 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 linkers (see Figure 1).

[0126] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention comprises or consists of a heavy chain containing one amino acid sequence of SEQID NO:27 and a light chain containing one amino acid sequence of SEQID NO:23.

[0127] In one embodiment, the anti-PD-1 / CTLA-4 / VEGF trispecific antibody according to the present invention comprises or consists of a heavy chain containing two amino acid sequences of SEQID NO:27 and a light chain containing two amino acid sequences of SEQID NO:23.

[0128] In one embodiment, the heavy chain includes 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 includes 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.

[0129] The terms “identity” or “homology” between two amino acid sequences or nucleotide sequences are determined by sequence alignment. If the two sequences being compared differ in length, sequence alignment preferably relates to the percentage of amino acid or nucleotide residues in the shorter sequence being the same as that in the longer sequence. Sequence alignment can be determined using conventional computer programs. Deviations arising in the comparison between a given sequence and the sequences described herein may be caused, for example, by additions, deletions, substitutions, insertions, or recombinations.

[0130] In some embodiments, the CDR sequence of the antibody of the present invention may include conservation modifications.

[0131] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding properties of an antibody, including its amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. Conservative substitutions are those in which an amino acid is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), 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), and amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, cysteine). This includes amino acids with aromatic side chains (e.g., phenylalanine, tryptophan), 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 may be substituted with alanine as previously described with respect to 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 in the antibodies of the present invention can be performed by known methods, such as PCR mutagenesis (U.S. Patent No. 4,683,195). Alternatively, a mutant library can be generated using known methods with random codons (NNK) or non-random codons, such as the DVK codon encoding 11 different amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody mutants can be characterized using the assays described herein.

[0132] Generation of a single-specific antibody according to the present invention

[0133] In some embodiments, the antibody of the present invention is a human antibody.

[0134] In some embodiments, the antibodies of the present invention are humanized.

[0135] The single-specific antibodies of the present invention described herein (e.g., antibodies that specifically bind to PD-1, CTLA-4, or VEGF) can be produced using a variety of techniques. For example, the hybridoma method described by Kohler and Milstein, Nature 256:495 1975, is used for the production of monoclonal antibodies. In the hybridoma method, mice or other host animals (e.g., 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 (e.g., the extracellular domain of PD-1, CTLA-4, or VEGF), and then spleen cells from the immunized animals 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 derived from a single immortalized hybridoma cell are screened for antibody production that possesses desired properties such as binding specificity, cross-reactivity or lack thereof, and affinity for the antigen.

[0136] Various host animals are used to generate antibodies according to the present invention. For example, Balb / c mice are used to generate mouse anti-human PD-1, CTLA-4, or VEGF antibodies. Alpacas are used to generate anti-human PD-1 or CTLA-4 VHH antibodies. Antibodies prepared in Balb / c mice and other non-human animals are humanized using various techniques to generate more human-like sequences.

[0137] Exemplary humanization techniques, including the selection of human receptor frameworks, are known and include CDR transplantation (U.S. Patent No. 5,225,539), SDR transplantation (U.S. Patent No. 6,818,749), surface remodeling (Padlan, (1991) Mol Immunol 28:489-499), specific determination residue surface remodeling (U.S. Patent Publication No. 2010 / 0261620), human framework adaptation (U.S. Patent No. 8,748,356), or superhumanization (U.S. Patent No. 7,709,226). These methods transfer the CDR of a parent antibody to a human framework that can be selected based on overall homology with the parent framework, by similarity of CDR length, identity of canonical structure, or a combination thereof.

[0138] Humanized antibodies can be further optimized to improve their selectivity or affinity for a desired antigen by incorporating modified frame-supporting residues to preserve binding affinity (reverse mutations), such as those described in International Patent Publications WO1090 / 007861 and WO1992 / 22653, or by introducing mutations into any CDR to improve antibody affinity.

[0139] Transgenic animals, such as mice or rats, that possess the human immunoglobulin (Ig) gene locus in their genome can be used to produce human antibodies against target proteins, for example, U.S. Patent No. 6,150,584, International Patent Publication No. WO99 / 45962, International Patent Publication Nos. WO2002 / 06630, WO2002 / 43478, WO2002 / 043478 and WO1990 / 04036, 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 These are described in 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, and Bruggemann and Taussig (1997) Curr Opin Biotechnol 8:455-458. In such animals, the endogenous immunoglobulin locus is disrupted or deleted, and at least one complete or partial human immunoglobulin locus can be inserted into the animal's genome using homologous or non-homologous recombination, transchromosome, or minigene.Companies such as Regeneron (http: / / www.regeneron.com), Harbour Antibodies (http: / / www.harbourantibodies.com), Open Monoclonal Technology, Inc. (OMT) (http: / / www.omtinc.net), KyMab (http: / / www.kymab.com), Trianni (http: / / www.trianni.com), and Ablexis (http: / / www.ablexis.com) can be commissioned to provide human antibodies against selected antigens using the technologies described above.

[0140] Human antibodies are selected from phage display libraries in which phages are modified 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 et al., (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 antibody of the present invention can be isolated from a phage display library expressing, for example, the heavy and light chain variable regions of the antibody, as a fusion protein with the bacterial phage pIX coat protein, as described by Shi et al., (2010) J Mol Biol 397:385-96 and International Patent Publication No. WO09 / 085462. The library may be screened for phage binding with human and / or cynomolgus monkey PD-1, CTLA-4, or VEFG, and the resulting positive clones may be further characterized, and Fab may be isolated from the cloned lysate and expressed as full-length IgG. Such phage display methods for isolating human antibodies are described, for example, in U.S. Patent Publications 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.

[0141] The preparation of immunogenic antigens and the generation of monoclonal antibodies can be carried out using any suitable technique, such as recombinant protein synthesis. Immunogenic antigens can be administered to animals in the form of purified proteins or protein mixtures containing whole cells or cell or tissue extracts, or the antigens can be de novo formed in the animal's body from nucleic acids encoding the antigen or a portion thereof.

[0142] The anti-PD-1 antibody GBD002-hS019-WS obtained from alpaca in this disclosure has the amino acid sequence and nucleotide sequence shown in Table 2.

[0143] [Table 2]

[0144] The anti-CTLA-4 antibody GBD008-hS005-3-2 obtained from alpaca in this disclosure has the amino acid sequence and nucleotide sequence shown in Table 3.

[0145] [Table 3]

[0146] The anti-VEGF antibody in this disclosure, which is a variant of bevacizumab, has the amino acid sequence and nucleotide sequence shown in Table 4.

[0147] [Table 4-1] [Table 4-2]

[0148] Generation of multispecific antibodies according to the present invention

[0149] The bispecific anti-PD-1 / CTLA-4 antibodies of the present invention are generated by combining a PD-1-binding VH (or VH / VL) domain and a CTLA-4-binding VH (or VH / VL) domain, as defined herein. Alternatively, bispecific anti-PD-1 / CTLA-4 antibodies are modified by using the VH (or VH / VL) domain of a publicly available monospecific anti-PD-1 or anti-CTLA-4 antibody, and / or by mixing and matching 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. Triple-specific or quadruple-specific antibodies, etc., are generated in a similar manner.

[0150] For example, the triple-specific anti-PD-1 / CTLA-4 / VEGF antibody of the present invention is generated by combining the isolated and characterized PD-1-binding VH (or VH / VL) domain, CTLA-4-binding VH (or VH / VL) domain, and VEGF-binding VH (or VH / VL) domain as described herein. Alternatively, the triple-specific anti-PD-1 / CTLA-4 / VEGF antibody is modified by using the VH (or VH / VL) domain of a publicly available single-specific anti-PD-1, anti-CTLA-4, or anti-VEGF antibody, and / or by mixing and matching the PD-1, CTLA-4, or VEGF-binding VH (or VH / VL) domain identified herein with a publicly available PD-1, CTLA-4, or VEGF-binding VH (or VH / VL) domain.

[0151] Exemplary anti-PD-1 antibodies that can be used to modify triple-specific anti-PD-1 / CTLA-4 / VEGF antibodies include, for example, pembrolizumab and nivolumab. Exemplary anti-CTLA-4 antibodies that can be used to modify triple-specific anti-PD-1 / CTLA-4 / VEGF antibodies include, for example, ipilimumab. Exemplary anti-VEGF antibodies that can be used to modify triple-specific anti-PD-1 / CTLA-4 / VEGF antibodies include, for example, bevacizumab. Exemplary single-specific domains can also be derivatized from bispecific antibodies such as cadnilimab and MEDI5752, which are anti-PD-1 × CTLA-4 bispecific antibodies, AK104, which is an anti-PD-1 / CTLA-4 bispecific antibody, and AK112, which is an anti-PD-1 / VEGF bispecific antibody.

[0152] The generated triple-specific anti-PD-1 / CTLA-4 / VEGF antibody binds to PD-1, CTLA-4, and VEGF, as well as to the antigen specificity of CD4. + and CD8 + The methods described herein can be used to test for desired functional properties, such as enhanced T cell activation.

[0153] Typically, a molecule (e.g., the constant region of an antibody) is mutated at the DNA level using standard methods.

[0154] The triple-specific PD-1 / CTLA-4 / VEGF antibody GBD214-33-03 constructed in this disclosure has the amino acid sequence and nucleotide sequence shown in Table 5.

[0155] [Table 5-1] [Table 5-2]

[0156] Polynucleotides, vectors, and host cells

[0157] The present invention also provides antibodies or antigen-binding fragments thereof in which VH and / or VL are encoded by polynucleotides. The polynucleotides are complementary deoxypentose nucleic acids (cDNA) that can be codon-optimized for expression in a suitable host. Codon optimization is a well-known technique.

[0158] The present invention also provides isolated polynucleotides encoding the VH of the antibody of the present invention, the VL of the antibody of the present invention, the heavy chain of the antibody of the present invention, and / or the light chain of the antibody of the present invention.

[0159] The present invention also provides isolated polynucleotides encoding VH, VL, or VH and VL of the antibody of the present invention.

[0160] The present invention also provides isolated polynucleotides encoding VH for SEQ ID NO: 4, 10, and / or 16.

[0161] The present invention also provides isolated polynucleotides encoding the heavy and / or light chains of the antibody of the present invention.

[0162] In one embodiment, the present invention provides an isolated polynucleotide encoding a heavy chain of SEQ ID NO: 5 or 11. In one embodiment, the present invention provides an isolated polynucleotide encoding a heavy chain of SEQ ID NO: 17 or 27 and / or a light chain of SEQ ID NO: 23.

[0163] 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 one embodiment, the present invention provides an isolated polynucleotide comprising a nucleotide sequence of SEQ ID NO: 6 or 12.

[0164] In one embodiment, the present invention provides isolated polynucleotides 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: 18 or 28, and / or isolated polynucleotides 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: 24. In one embodiment, the present invention provides an isolated polynucleotide comprising a nucleotide sequence of SEQ ID NO: 18 or 28 and / or a nucleotide sequence of SEQ ID NO: 24.

[0165] The VH or VL of the antibody of the present invention or its antigen-binding fragment, or a polynucleotide sequence encoding the heavy or light chain of the antibody of the present invention, is operably attached to one or more regulatory elements (e.g., promoters or enhancers) to enable the expression of the nucleotide sequence in a desired host cell. The polynucleotide may be cDNA.

[0166] 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-mediated vectors, or any other vectors suitable for introducing the synthetic polynucleotides of the present invention into a given organism or genetic background by any means. For example, polynucleotides encoding the light and / or heavy chain variable regions (optionally connected to a constant region) of the antibody of the present invention are inserted into an expression vector. The light and / or heavy chains are cloned into the same or different expression vectors. DNA segments encoding immunoglobulin chains are operably connected to control sequences in the expression vector to ensure the expression of the immunoglobulin polypeptide. Such control sequences include signal sequences, promoters (e.g., innate correlation promoters or heterologous promoters), enhancer elements, and transcription termination sequences, and are selected to be compatible with host cells selected to express the antibody. 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 polynucleotides.

[0167] Appropriate expression vectors can typically be replicated in a host organism as episomes or as components of host chromosomal DNA. Generally, expression vectors include a selection marker, 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.

[0168] Appropriate 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, the cytomegalovirus immediate early promoter, the herpes simplex virus thymidine kinase promoter, the SV40 early and late promoters, promoters present in long terminal repeats of reverse transcription viruses, the mouse metallothionein-I promoter, and various known tissue-specific promoters. The selection of appropriate vectors and promoters is within the capabilities of those skilled in the art.

[0169] Examples of vectors that can be used include the bacterial pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA), pTrc99 A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden), and the eukaryotic pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia), pEE6.4 (Lonza), and pEE12.4 (Lonza).

[0170] The present invention also provides host cells containing 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 expected to refer not only to a specific target cell, but also to the offspring of such cells, and stable cell lines generated from a specific target cell. Some modifications may occur in subsequent generations due to mutation or environmental influences, so such offspring may differ from the parent cell, but are still included within the scope of the term “host cell” as used herein. Such host cells may be eukaryotic cells, prokaryotic cells, plant cells, or archaeal cells. Examples of prokaryotic host cells include Escherichia coli, Bacillus (e.g., Bacillus subtilis), and other Enterobacteriaceae (e.g., Salmonella, Serratia, and various Pseudomonas species). Other microorganisms (e.g., yeast) may also be used for expression. Yeast (e.g., budding yeast (S. cerevisiae)) and the genus Pichia are examples of suitable yeast host cells. Exemplary eukaryotic cells can belong to mammalian, insect, bird, or other animal origins. Mammalian eukaryotic cells include immortalized cell lines such as hybridoma or myeloma cell lines, such as SP2 / 0 (USA Spawn Culture Collection Center (ATCC), Manassas, VA, CRL-1581), NS0 (European Cell Culture Collection Center (ECACC), Salisbury, Wiltshire, UK, ECACC number 85110503), FO (ATCC CRL-1646), and Ag653 (ATCC CRL-1580) mouse cell line. 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 (Lonza Biologics, Walkersville, MD), Potelligent® CHOK2SV (Lonza), CHO-K1 (ATCC CRL-61), or DG44.

[0171] The present invention also provides a method for producing the antibody of the present invention, comprising culturing host cells of the present invention under conditions that express the antibody, and recovering the antibody produced by the host cells. Methods for preparing and purifying antibodies are well known in the art. Once synthesized (whether chemically or recombinantly), the complete antibody, its dimers, a single light chain and / or heavy chain, or other antibody fragments (e.g., VH and / or VL) may be purified by standard procedures including ammonium sulfate precipitation, affinity columns, column chromatography, high-performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. (see Scopes, Protein Purification (Springer-Verlag, NY, (1982))). The antibody of interest may be substantially pure, e.g., at least about 80%–85% pure, at least about 85%–90% pure, at least about 90%–95% pure, or at least about 98%–99% or more pure, and free from contaminants such as cell debris, macromolecules, etc., other than the antibody of interest.

[0172] The polynucleotide sequence of the present invention is incorporated into a vector using standard molecular biological methods. Host cell transformation, culture, antibody expression, and purification are performed using well-known methods.

[0173] Another embodiment of the present invention is a method for generating an antibody or antigen-binding fragment thereof, The polynucleotide encoding the antibody or antigen-binding fragment of the present invention is incorporated into the expression vector. Transforming host cells using an expression vector, Culturing host cells in culture medium under conditions in which VH, VL, heavy chain and / or light chain are expressed and antibodies are formed, and A method comprising recovering an antibody or its antigen-binding fragment from host cells or culture medium.

[0174] Pharmaceutical composition / administration

[0175] The present invention provides a pharmaceutical composition comprising the antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier. For therapeutic use, the antibody or its antigen-binding fragment can be prepared as a pharmaceutical composition containing an effective amount of the antibody as the active ingredient in a pharmaceutically acceptable carrier. "Carrier" means a diluent, adjuvant, excipient, or medium used for administering the antibody of the present invention. Such a medium may be a liquid such as water and oil, containing oils of animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. For example, 0.4% saline solution and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They are sterilized by conventionally known sterilization techniques (e.g., filtration). The composition may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants. The concentration of the antibody or antigen-binding fragment of the present invention in such pharmaceutical formulations may vary from less than about 0.5% by weight (typically) to at least about 1% by weight to a maximum of 15 or 20% by weight, and may be selected mainly based on the desired dose, fluid volume, viscosity, etc., depending on the specific mode of administration selected. Suitable media and formulations containing other human proteins (e.g., human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, Troy, DB ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pages 691-1092, with particular reference to pages 958-989.

[0176] The mode of administration for the therapeutic use of the antibody or antigen-binding fragment of the present invention may be any suitable route for delivery of the antibody or antigen-binding fragment to the host, such as intradermal, intramuscular, intraperitoneal, intravenous, or parenteral administration such as subcutaneous, pulmonary, or transmucosal (oral, intranasal, vaginal, rectal) using tablets, capsules, solutions, powders, gels, or granules, or by being contained in syringes, implantable devices, osmotic pumps, drug solution cartridges, micropumps, or other means known to those skilled in the art. Site-specific administration can be achieved, for example, by intratumor, intraarticular, intrabronchial, intraperitoneal, intrasacral, intracartilage, intracavitary, intrabody cavity, intracerebellar, intraventricular, intracolon, intracervix, intrastomach, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostate, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal cord, intrasynovial, intrathoracic, intrauterine, intravascular, intrabladder, intrafocal, vaginal, rectal, oral, sublingual, intranasal, or percutaneous delivery.

[0177] The antibody or antigen-binding fragment of the present invention can be administered to a target by any suitable route, for example, parenteral, intramuscular, subcutaneous, or intraperitoneal by intravenous (iv) infusion or bolus infusion. Intravenous infusion can be administered 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.

[0178] The dose administered to the subject is sufficient to alleviate or at least partially suppress the disease being treated ("therapeutic effective dose") and may be, in some cases, 0.005 mg to about 100 mg / kg, for example, 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 also 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.

[0179] Fixed unit doses, such as 50, 100, 200, 500, or 1000 mg, may also be administered, or the dose may be based on the patient's body surface area, such as 500, 400, 300, 250, 200, or 100 mg / m². 2 The decision is based on the following: Typically, 1 to 8 doses (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 doses) can be administered to treat a patient, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may be administered.

[0180] Administration of the antibody or antigen-binding fragment of the present invention can be repeated 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 more. The treatment process can also be repeated, as can long-term administration. Repeated administrations may be at the same dose or different doses. For example, the antibody or antigen-binding fragment of the present invention may be administered at 8 mg / kg or 16 mg / kg once a week for 8 weeks, followed by re-administration at 8 mg / kg or 16 mg / kg every 2 weeks for 16 weeks, followed by intravenous infusion at 8 mg / kg or 16 mg / kg every 4 weeks.

[0181] For example, the antibody or antigen-binding fragment of the present invention may be administered in single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, on at least one day among the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after the start of treatment, or instead on 1, 2, 3, 4, 5, 6, 7, 8, 9 For at least one week among weeks 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, the daily dose may be approximately 0.1 to 100 mg / kg, for example, 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.

[0182] The antibodies or antigen-binding fragments of the present invention may also be administered prophylactically to reduce the risk of cancer progression, delay the onset of events during cancer progression, and / or reduce the risk of recurrence when cancer is in remission.

[0183] The antibody or antigen-binding fragment of the present invention may be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective for conventional protein formulations, and well-known lyophilization and reconstitution techniques can be employed.

[0184] Method and Use

[0185] The antibodies or antigen-binding fragments of the present invention have usefulness in in vitro and in vivo diagnosis, as well as in therapeutic and prophylactic applications. For example, the antibodies or antigen-binding fragments of the present invention can be administered in vitro or in vitro to cultured cells, or to subjects to whom various diseases such as cancer and infectious diseases are being treated, prevented, and / or diagnosed.

[0186] The present invention provides a method for modifying an immune response in a subject, comprising administering the antibody or its antigen-binding fragment for a sufficient period of time to modify the immune response in the subject.

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

[0188] In some embodiments described herein, the subject is a human patient.

[0189] In some embodiments described herein, the subjects are human patients who require enhancement of the immune response.

[0190] In some embodiments, the subjects are immunocompromised individuals.

[0191] In some embodiments, subjects are at risk of immunodeficiency. Immunodeficient subjects are currently receiving or have previously received chemotherapy or radiation therapy.

[0192] In some embodiments, the subjects are immune to the infection or are at risk of having their immunity impaired.

[0193] The antibody or antigen-binding fragment of the present invention is suitable for treating subjects with diseases that can be treated by enhancing T cell-mediated immune responses.

[0194] In some embodiments, the antibody or its antigen-binding fragment used in the method of the present invention is an antibody as defined herein.

[0195] In some embodiments, the triple-specific 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, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment of the present invention over a period of time sufficient to inhibit tumor cell growth in the target.

[0197] The present invention also provides a method for treating cancer by administering the antibody or antigen-binding fragment of the present invention to a target requiring it for a sufficient amount of time to treat the cancer.

[0198] Cancer can be a condition or disease characterized by excessive growth, a solid tumor, a hematological malignancy, a soft tissue tumor, or a metastatic lesion.

[0199] "Cancer" is intended to include all types of cancerous growth or carcinogenic processes, metastatic tissue or malignant transformed cells, tissues or organs, regardless of histopathological type or stage of invasion. Examples of cancer include solid tumors, hematological malignancies, soft tissue tumors, and metastatic lesions. Exemplary solid tumors include, for example, sarcomas and carcinomas (including adenocarcinomas and squamous cell carcinomas), malignancies of various organ systems such as the liver, lungs, mammary glands, lymph nodes, gastrointestinal tract (e.g., colon), urogenital tract (e.g., kidneys, urothelial cells), prostate, and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancers, renal cell carcinomas, liver cancers, non-small cell lung cancers, small intestine cancers, and esophageal cancers. Squamous cell carcinomas include, for example, malignancies of the lungs, esophagus, skin, head and neck, oral cavity, anus, and cervix.

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

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

[0202] Exemplary cancers whose growth can be suppressed or reduced using the antibodies or antigen-binding fragments of the present invention include cancers that respond to immunotherapy. Such exemplary cancers include melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, gastrointestinal cancer, gastric cancer, esophageal cancer, lung cancer, metastatic malignant melanoma, clear cell carcinoma, hormone-resistant prostate cancer, non-small cell lung cancer, or head and neck cancer. Refractory or recurrent malignancies can be treated using the antibodies or antigen-binding fragments of the present invention as described herein.

[0203] Other exemplary cancers that can be treated with the antibody or antigen-binding fragment 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, 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, carcinoma in situ, kidney cancer, laryngeal cancer, liver cancer, small cell lung cancer, neuroblastoma, oral cancer (e.g., lips, tongue, mouth, and pharynx), nasopharyngeal cancer, retinoblastoma, rhabdomyosarcoma, respiratory system cancer, sarcoma, thyroid cancer, and urinary system cancer. Examples include cancer, liver cancer, anal cancer, fallopian tube cancer, vaginal cancer, vulvar cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, neovascularization of tumors, axial tumors of the spinal cord, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, Merkel cell carcinoma, epidermal carcinoma, squamous cell carcinoma, environmentally induced cancers (including cancers induced by asbestos), and other cancers and sarcomas, as well as combinations of the aforementioned cancers.

[0204] Exemplary hematological malignancies that can be treated with the antibody or antigen-binding fragment of the present invention include leukemia, lymphoma, and myeloma, such as progenitor 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 prelymphoblastic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL) (including low-grade, intermediate-grade, and high-grade FL), cutaneous follicular central lymphoma, and extranodal marginal zone B-cell lymphoma. These include tumors (MALT type, lymphadenopathy, and splenic type), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), plasmacytoma, multiple myeloma (MM), plasmacytosis, post-transplant lymphoproliferative disorders, Valdenström macroglobulinemia, plasma cell disorders, anaplastic large cell lymphoma (ALCL), T-cell acute lymphoblastic leukemia, primary systemic amyloidosis (e.g., light chain amyloidosis), prolymphocytic / myeloid leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), large granular lymphocyte (LGL) leukemia, NK cell leukemia, and Hodgkin lymphoma.

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

[0206] Exemplary B-cell non-Hodgkin lymphomas include lymphomatoid granulomatosis, primary exudative lymphoma, intravascular large B-cell lymphoma, mediastinal large B-cell lymphoma, heavy chain disease (including gamma, muco, and a diseases), immunosuppressant-induced lymphomas (e.g., cyclosporine-induced lymphoma), and methotrexate-induced lymphoma.

[0207] In some embodiments, the subjects have tumors that express PD-L1.

[0208] In some embodiments, the subjects have tumors that express CTLA-4.

[0209] In some embodiments, the subjects have tumors that express VEGF.

[0210] In some embodiments, subjects were treated with anti-PD-1 antibodies.

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

[0212] In some embodiments, the subjects have recurrent tumors after treatment with anti-PD-1 antibodies.

[0213] In some embodiments, subjects were treated with an anti-PD-1 antibody (e.g., KEYTRUDA® (pembrolizumab)).

[0214] In some embodiments, subjects were treated with an anti-PD-1 antibody (e.g., OPDIVO® (nivolumab)).

[0215] In some embodiments, the subjects are refractory to treatment with anti-PD-1 antibodies (e.g., KEYTRUDA® (pembrolizumab)).

[0216] In some embodiments, the subject is refractory to treatment with anti-PD-1 antibodies (e.g., OPDIVO® (nivolumab)).

[0217] In some embodiments, the subjects have recurrent tumors after treatment with an anti-PD-1 antibody (e.g., KEYTRUDA® (pembrolizumab)).

[0218] In some embodiments, the subjects have recurrent tumors after treatment with an anti-PD-1 antibody (e.g., OPDIVO® (nivolumab)).

[0219] In some embodiments, subjects have been treated with, or are being treated with, an anti-PD-L1 antibody (e.g., MEDI-4736, MDX-1105, avelumab, or atezolizumab).

[0220] In some embodiments, the subjects are refractory to treatment with anti-PD-L1 antibodies (e.g., MEDI-4736, MDX-1105, avelumab, or atezolizumab).

[0221] In some embodiments, subjects have recurrent tumors after treatment with an anti-PD-L1 antibody (e.g., MEDI-4736, MDX-1105, avelumab, or atezolizumab).

[0222] In some embodiments, subjects are treated with anti-PD-L2 antibodies, or are treated with anti-PD-L2 antibodies.

[0223] In some embodiments described herein, the subjects are refractory to treatment with anti-PD-L2 antibodies.

[0224] In some embodiments, the subjects have recurrent tumors after treatment with anti-PD-L2 antibodies.

[0225] Various qualitative and / or quantitative methods are used to determine the recurrence or refractory nature of the disease. Symptoms that may be associated with recurrence or resistance include, for example, a decline or plateau in the patient's health, or the re-establishment or worsening of various symptoms associated with solid tumors, and / or the in vivo spread of cancer cells from one site to other organs, tissues, or cells.

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

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

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

[0229] Combination therapy for cancer treatment

[0230] The antibody of the present invention is administered in combination with a second therapeutic agent.

[0231] The antibody or antigen-binding fragment of the present invention is administered in combination with one, two, three, four, five, or six additional therapeutic agents.

[0232] "In combination with..." means that the antibody of the present invention and at least one second therapeutic agent are administered simultaneously as a single agent, or sequentially as single agents in any order. Generally, each agent is administered in a dose and / or schedule determined for that agent.

[0233] In some embodiments, the second therapeutic agent modulates the activity of molecules involved in the cancer immune cycle, which are involved in stimulating or inhibitory pathways that function in, for example, the release of cancer cell antigens, cancer antigen presentation, T cell behavior and activation, T cell migration to tumors, T cell infiltration into tumors, T cell recognition of cancer cells, and the killing of cancer cells. The cancer immune cycle is described by Chen and Mellman (2013) in Immunity 39:1-10. In some embodiments, the second therapeutic agent modulates the activity of regulatory T cell (Treg) activity-regulating molecules, co-stimulatory or co-inhibitory ligands expressed in tumors, activating or inhibitory receptors in natural killer (NK) cells, or immunosuppressive factors in the tumor microenvironment. Combination cancer immunotherapy is described by Manoney et al. (2015) in Nature Reviews 14:561-584.

[0234] The second therapeutic agent typically enhances the activity of stimulating molecules and suppresses the activity of inhibitory molecules, as is well known. Therefore, "modulation" means the enhancement of the immune response by the second therapeutic agent, regardless of whether the drug itself is an agonist or antagonist of a particular molecule.

[0235] The effects of the combinations described herein can be tested in animal models known in the art. [Examples]

[0236] The present invention will be described with reference to the following specific non-limiting embodiments.

[0237] Example 1: Production of anti-PD-1 antibody, anti-CTLA-4 antibody, and anti-VEGF antibody Anti-human PD-1 antibody and anti-human CTLA-4 antibody were generated by immunizing alpacas with recombinant human PD-1 and human CTLA-4 extracellular domain (ECD) proteins, respectively. Total RNA of PBMC was extracted, cDNA was synthesized and amplified. The framework region of the alpaca VH gene was replaced with the human framework by CDR transplantation technology and cloned into an expression vector to generate the corresponding humanized antibody clone. The obtained anti-PD-1 antibody GBD002-hS019-WS had a heavy chain variable region of SEQ ID NO:4. The obtained anti-CTLA-4 antibody had a heavy chain variable region (GBD008-hS005-3-2) of SEQ ID NO:10.

[0238] The amino acids of the heavy and light chain variable regions of the anti-VEGF antibody can refer to the commercially available antibody bevacizumab (Avastin), which are shown in SEQ ID NO:16 and SEQ ID NO:22, respectively. The synthesis of the anti-VEGF antibody, a mutant of bevacizumab, was commissioned to Biointron, and its sequence is shown in Table 4. This anti-VEGF antibody contains a heavy chain with two amino acid sequences of SEQ ID NO:17 and a light chain with two amino acid sequences of SEQ ID NO:23. In this anti-VEGF antibody, the Fc fragment is different from that of bevacizumab.

[0239] Example 2: Generation of GBD214-33-03 antibody Construction of trispecific antibody GBD214-33-03: A DNA sequence encoding a heavy chain containing the heavy chain of an anti-VEGF antibody (SEQ ID NO: 17), the variable region of the heavy chain of an anti-CTLA-4 antibody (SEQ ID NO: 10), and the variable region of the heavy chain of an anti-PD-1 antibody (SEQ ID NO: 4) from the N-terminus to the C-terminus, and a DNA sequence encoding the light chain of an anti-VEGF antibody (SEQ ID NO: 23) were each cloned into a pcDNA3.3 expression vector to obtain a trispecific antibody. Linker 1 and / or Linker 2 were added as flexible connections to the heavy chain of GBD214-33-03. The resulting trispecific antibody was named "GBD214-33-03". A schematic diagram of the structure of GBD214-33-03 is shown in Figure 1.

[0240] Example 3: FACS assay for the binding specificity of a PD-1 antibody to human PD1 protein on the cell surface HEK293-PD1 cells (1×10 5 / well) were washed twice with FACS buffer (PBS + 2% BSA), resuspended in 100 μl of FACS buffer containing serially diluted (1:5) anti-PD1 mAb, and incubated at 4°C for 1 hour. Then, the cells were washed twice with FACS buffer, and the bound antibody was detected by incubating with APC anti-human IgG Fc (Biolegend, catalog number 410712) at 4°C for 1 hour. Thereafter, the cells were washed twice with FACS buffer, and then the cells were collected and analyzed using a Fortessa flow cytometer (BD Bioscience). The results are shown in Figure 2. As a result, the binding of GBD214-33-03 to PD1 in HEK293-PD1 cells was shown. GBD214-33-03 showed strong binding to HEK293-PD1 cells with a median fluorescence intensity (MFI) similar to that of its PD1 parent GBD002-hS019-WS and pembrolizumab.

[0241] Example 4: PD1 reporter assay (ELISA method) in Jurkat-NFAT-Luc2-PD1 cells CHOK1-PD-L1 cells (4×10 4Harvest the cells (5 × 10¹⁶ cells / well), suspend them in PRMI 1640 medium (Gibco, catalog no. 31800022) containing 10% FBS (Gibco, catalog no. 10099-141), and grow overnight in a 96-well plate at 37°C. The next day, discard the supernatant and place Jurkat-NFAT-Luc2-PD1 cells (5 × 10¹⁶ cells / well) in each well. 4 Serially diluted antibodies were added to the cells (per well). The cells were incubated at 37°C for 5 hours. Then, Bio-Glo luciferase assay buffer (Promega, catalog no. 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 plate reader (PerkinElmer). The results are shown in Figure 3. According to the PD1 reporter assay, GBD214-33-03 had a similar maximum luminescence signal compared to pembrolizumab and AK 104. As a result, it was found that GBD214-33-03 can block the interaction between PD1 and its ligand PD-L1.

[0242] Example 5: Antibody mixed lymphocyte reaction (MLR) assay CD14+ cells were isolated from PBMCs using an isolation kit (Stem Cell, catalog number 17858) according to the manufacturer's instructions. Subsequently, the CD14+ cells were divided into 1 × 10⁶ cells. 6 Cells / ml, 6-well plate (3-5 x 10 6 3-5 ml of cells were seeded in medium supplemented with 50 ng / ml IL-4 and 50 ng / ml GMCSF in a well. Cytokines were refreshed every 2-3 days. On day 6, 1 μg / ml LPS (Sigma-Aldrich, catalog number L6529) was added to the medium to induce mature DCs, and the cells were cultured at 37°C for 24 hours. On day 7, mature DC cells were collected for the MLR assay. For the MLR assay, fresh T cells were isolated according to the manufacturer's instructions (Stem Cell, catalog number 17951), and the cells were 2 × 10⁶ 5 Cells were seeded into assay plates at a rate of 1 × 10⁶ cells / well. Serially diluted antibodies were added to the assay plates, followed by mature DC cells at a rate of 1 × 10⁶ cells. 4Cells were seeded in assay plates at a rate of one cell / well. The assay plates were incubated at 37°C for 5 days, and the supernatant was collected and used in an HTRF kit (Cisbio, catalog no. 62HIFNGPEH) to detect IFNγ according to the manufacturer's instructions. The 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. The results indicate that GBD214-33-03 has potent immunogenicity similar to pembrolizumab.

[0243] 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 antibodies were added to each well. The plate was incubated at 37°C for 1 hour, 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. Then, 100 μl TMB solution was added to each well, the plate was incubated at room temperature for 5 minutes, and then 50 μl TMB solution was added. The optical density (OD) of each pore was measured using the EnVision 2105 multimode plate reader (PerkinElmer). 450 The following was measured. The results are shown in Figure 5. The results showed that GBD214-33-03 has similar VEGF binding properties to bevacizumab.

[0244] Example 7: VEGF blockade 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 and VEGFR2-mFc-biotin (Kactus, catalog number: VGF-HM3R2B) were added to each well. The plate was incubated at 37°C for 1 hour, followed by 6 washes 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. Next, 100 μl of TMB solution was added to each well, and the plate was incubated at room temperature for 5 minutes, after which 50 μl of TMB solution was added. The light density (OD) of each well was measured using an EnVision 2105 multimode plate reader (PerkinElmer). 450 The following was measured. The results are shown in Figure 6. The results showed that GBD214-33-03 inhibits the binding of VEGFR2 to VEGF165, similar to bevacizumab.

[0245] Example 8: Antibody VEGF reporter assay (ELISA method) HEK293-NFAT-KDR-Luc cells (4 × 10 4 Harvest the cells (1 / well) and resuspend them in PRMI 1640 medium (Gibco, catalog no. 31800022) containing 10% FBS (Gibco, catalog no. 10099-141), then 4 × 10⁶ cells. 4Cells were seeded per well and grown overnight in a 96-well white plate at 37°C. The following day, the supernatant was discarded, and VEGF165 (15 ng / ml) (Kactus, catalog no. VEG-HM065) and serially diluted antibody were added to each well. Cells were incubated at 37°C for 5 hours. Subsequently, Bio-Glo luciferase assay buffer (Promega, catalog no. G7940) was added to each well. After incubating the plate at room temperature for up to 10 minutes, readings were performed using an EnVision 2105 multimode plate reader (PerkinElmer). The results are shown in 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.

[0246] Example 9: FACS assay of the binding specificity of CTLA4 antibody to human CTLA4 protein on the cell surface. CHOK1-CTLA4 cells (1 x 10 5 / well) and CHOK1-PD1-CTLA4 cells (1×10 5Cells were harvested and collected (per well) and resuspended in 100 μl of FACS buffer (PBS + 2% BSA). Serially diluted anti-CTLA4 antibody was added to the plate, and the cells were incubated at 4°C for 1 hour. The cells were then washed twice with FACS buffer, and the bound antibody was detected by incubation with APC anti-human IgG Fc (Biolegend, catalog no. 410712) at 4°C for 1 hour. After washing the cells twice with FACS buffer, they were collected, and the cells were analyzed using a Fortessa flow cytometer (BD Bioscience). The results are shown in Figures 8a and 8b. As a result, in CHOK1-CTLA4 cells, GBD214-33-03 had a lower MFI than ipilimumab, suggesting that GBD214-33-03 partially binds to CTLA4 in CHOK1-CTLA4 cells. The CTLA4 parent of GBD214-33-03, GBD008-hS005-3-2, exhibited weaker binding affinity to CTLA4 than ipilimumab. In CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed stronger binding compared to its PD1 parent, GBD002-hS019-WS.

[0247] Example 10: FACS assay of CTLA4 antibody blocking the binding of CD80 and CD86 to CTLA4. CHOK1-CTLA4 cells or CHOK1-PD1-CTLA4 cells (1 × 10⁻¹⁰ 5Cells were harvested and collected from each well, and resuspended in 100 μl of FACS buffer containing serially diluted anti-CTLA4 antibody and CD80-biotin (SinoBiological, catalog no. 10698-H49H-B) or CD86-biotin (SinoBiological, catalog no. 10699-H03H-B). Cells were incubated at 4°C for 30 minutes and then washed twice with FACS buffer. Bound antibodies were detected by incubation with APC anti-human IgG Fc (Biolegend, catalog no. 410712) at 4°C for 30 minutes. After washing twice with FACS buffer, cells were collected and then analyzed using a Fortessa flow cytometer (BD Bioscience). Results are shown in Figures 9a-9d. The results showed that GBD214-33-03 partially inhibited the binding of CD80 / CD86 to CTLA4 in CHOK1-CTLA4 cells and CHOK1-PD1-CTLA4 cells. In both CHOK1-CTLA4 cells and CHOK1-PD1-CTLA4 cells, GBD214-33-03 showed weaker blocking activity than ipilimumab.

[0248] Example 11: Affinity of antibodies against 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 for protein interactions (Octect RH-16, Sartorius). 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 Koff / Kon ratio. The results are shown in Tables 6, 7, and 8. The results in Table 6 show that all antibodies bound to the CTLA-4 antigen, the results in Table 7 show that all antibodies bound to the PD1 antigen, and the results in Table 8 show that all antibodies bound to the VEGF antigen.

[0249] [Table 6]

[0250]

Table 7

[0251]

Table 8

[0252] Example 12: Measurement of downregulation of PD-1 receptor in CHOK1-PD1-CTLA4 cells by flow cytometry. CHOK1-PD1-CTLA4 cells were harvested and the cell density was adjusted to 5×10 6 / ml. The cells were added to F12 K + 10% FBS medium in a 96-well U-bottom plate at 50 μl / well. Serial dilutions of antibodies were added to the cells. The cells were incubated at 4 °C for 30 minutes and then washed twice with FACS buffer at 4 °C. Thereafter, all procedures including pipetting and centrifugation were performed at 4 °C. Next, a non-competing PD-1-AF647 antibody with an operating concentration of 10 μg / ml was added to the plate and the cells were re-incubated at 4 °C for 30 minutes. After the cells were washed twice with FACS buffer at 4 °C, they were fixed with 4% paraformaldehyde (100 μL / well) for 10 minutes at room temperature. After fixation, the cells were washed once with FACS buffer and analyzed using a Fortessa flow cytometer (BD Bioscience). The results are shown in Figure 10. As a result, it was shown that PD1 in the cells was downregulated by CTLA4 internalization. The percentage of downregulated PD1 was measured. GBD214-33-03 cells showed similar PD1 internalization compared to AK 104.

[0253] Example 13: In vivo antitumor effect of GBD214-33-03 antibody in A375 PBMC mouse model. hPBMC (6×10 5 cells) pre-mixed with A375 human melanoma cancer cells (5×10 6Cells were used to subcutaneously inoculate NSG mice (n=6 per group, 4 weeks old). At the time of tumor formation (approximately 220 mm) 3 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 by intraperitoneal injection. Treatment was performed twice a week for a total of five sessions. 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 Figures 11a and 11b. As a result, in the A375 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) demonstrated superior efficacy 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). The mean body weight of mice began to decrease from day 7 in all treatment groups, but the decrease was less than 15%.

[0254] Example 14: In vivo antitumor effect of GBD214-33-03 antibody in an HT29 PBMC mouse model. hPBMC(6×10 5 HT29 human colon cancer cells (5 × 10) pre-mixed with cells 6 Cells were used to subcutaneously inoculate NSG mice (n=6 per group, 4 weeks old). At the time of tumor formation (approximately 250 mm) 3Pembrolizumab (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 by intraperitoneal injection. Treatment was performed twice a week for a total of five sessions. 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 Figures 12a and 12b. As a result, in the HT29 PBMC model, GBD214-33-03 (1 mg / kg, 5 mg / kg, and 20 mg / kg) demonstrated superior efficacy 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). The mean body weight of mice remained relatively stable in all treatment groups, with a decrease of less than 15%.

[0255] In short, the present invention has developed a triple-specific antibody that specifically binds to PD-1, CTLA-4, and VEGF and blocks checkpoints expressed on T cells. This TsAb has potent receptor binding and ligand blocking activity for PD-1 and VEGF, while simultaneously partially blocking the interaction between CTLA-4 and its ligand. The designed partial blocking activity for CTLA-4 is intended to reduce peripheral irAE toxicity. In a mouse model, the triple-specific antibody of the present invention showed potent antitumor activity.

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Claims

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

3.

2. The antibody or antigen-binding fragment according to claim 1, wherein the VHH comprises CDR1, 2, and 3 having amino acid sequences shown in SEQ ID NO: 1, 2, and 3, respectively.

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

4.

5. An antibody or antigen-binding fragment according to any one of claims 1 to 4, which specifically binds to PD-1.

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

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

8. An antibody or antigen-binding fragment according to any one of claims 1 to 7, comprising two or more heavy-chain antibody variable regions.

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

10. A multispecific antibody or its antigen-binding fragment comprising a first domain that specifically binds to PD-1 and a second domain that specifically binds to CTLA-4.

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

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

10.

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

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

15. A triplicate antibody or antigen-binding fragment of an anti-PD-1 / CTLA-4 / VEGF antibody or the antigen-binding fragment thereof, comprising a first domain that specifically binds to PD-1, a second domain that specifically binds to CTLA-4, and a third domain that specifically binds to VEGF, as described in any one of claims 10 to 14.

16. a) The first domain that specifically binds to PD-1 includes a heavy chain variable region (VH1) which comprises heavy chain complementarity determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), respectively, with SEQ ID NO: 1, 2, and 3. b) The second domain that specifically binds to CTLA-4 includes a heavy chain variable region (VH2) comprising heavy chain complementarity determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), which are SEQ ID NO: 7, 8, and 9, respectively, and / or c) The multispecific antibody or antigen-binding fragment according to claim 15, wherein the third domain that specifically binds to VEGF comprises a heavy chain variable region (VH3) including heavy chain complementarity-determining regions 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3), which are SEQ ID NO: 13, 14, and 15 respectively, and a light chain variable region (VL3) including light chain complementarity-determining regions 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3), which are SEQ ID NO: 19, 20, and 21 respectively.

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

10.

18. a) The first domain that specifically binds to PD-1 contains or consists of the heavy chain variable region (VH) of SEQ ID NO:

4. b) The second domain that specifically binds to CTLA-4 contains or consists of the heavy chain variable region (VH) of SEQ ID NO: 10, and / or c) The multispecific antibody or antigen-binding fragment according to claim 16, wherein the third domain that specifically binds to VEGF comprises or consists of a heavy chain variable region (VH) with SEQ ID NO: 16 and a light chain variable region (VL) with SEQ ID NO:

22.

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

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

21. The antibody or antigen-binding fragment according to claim 20, wherein the linkers are the same or different, and / or the linkers are flexible linkers, and / or the linkers are peptide linkers.

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

23.

23. The antibody or its antigen-binding fragment according to any one of claims 1 to 22, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 isotype, which optionally contains one, two, three, four, five, six, seven, eight, nine, or ten substitutions in the Fc region.

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

26. A vector comprising the polynucleotide described in claim 25.

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

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

29. A method for treating a target cancer, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment according to any one of claims 1 to 23, or a pharmaceutical composition according to claim 24, to a target requiring treatment for a period of time sufficient to treat the cancer.

30. The method according to claim 29, wherein the cancer is a solid tumor or a hematological malignancy.

31. The method according to 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 an antibody or antigen-binding fragment according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 24 in the preparation of a pharmaceutical for treating a target cancer of a required nature.

33. The use according to claim 32, wherein the cancer is a solid tumor or a hematological 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. An antibody or antigen-binding fragment according to any one of claims 1 to 23, or a pharmaceutical composition according to claim 24, for treating a target cancer of a particular nature.

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

37. The antibody or antigen-binding fragment or pharmaceutical composition for use 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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