Trispecific antibodies targeting PD-1, CTLA-4 and VEGF and uses thereof

Abstract

The present invention relates to trispecific antibodies that specifically bind to PD-1, CTLA-4, and VEGF. The present invention also relates to compositions comprising the trispecific antibodies and their use in the treatment of cancer, autoimmune diseases, infectious diseases, or angiogenesis-related diseases.

Description

[Technical Field]

[0001] The present invention is in the field of biomedicine, and specifically relates to a trispecific antibody targeting PD-1, CTLA-4 and VEGF for the treatment of tumors and its use. [Background technology]

[0002] In clinical studies, immunotherapy has significantly enhanced the ability to treat diseases (such as cancer), but many patients do not respond adequately to monospecific therapies due to the complexity of their disease. The tumor microenvironment is composed of cells, such as tumor cells, immune cells, and vascular cells, as well as noncellular components in the extracellular matrix. Interactions between these components limit the host's antitumor immunity and control of tumor growth. Researchers hope to achieve more favorable therapeutic outcomes by developing combination therapies that target different cells and different targets. For example, using nivolumab, an antibody that blocks the immune checkpoint PD-1, in combination with ipilimumab, an antibody that blocks CTLA-4, stimulates a stronger immune response in preclinical and clinical trials, resulting in improved therapeutic efficacy compared to blockade of a single immune checkpoint (The efficacy and safety of combined immune checkpoint inhibitors (nivolumab plus ipilimumab): a systematic review and meta-analysis, Jingjie Chen et al., World J Surg Oncol, 2020 Jul 3;18(1):150). However, these combination therapies increase drug costs, enhance multiple side effects, and lead to higher toxicity, which may be related to the clearance of regulatory T cells (Tregs) by ipilimumab (Safety profiles of anti-CTLA-4 and anti-PD-1 antibodies alone and in combination, Celine Boutros et al., Nat Rev Clin Oncol 2016 Aug;13(8):473-86).

[0003] The immune checkpoints PD-1 and CTLA-4 have distinct expression patterns in different immune cells, and the signaling interactions mediated by these two receptors suppress antitumor immunity. Therefore, simultaneous blockade of PD-1 and CTLA-4 can promote antitumor immunity through multiple mechanisms (PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways, Yunlong Zhao et al., Immunity. 2019 Dec 17;51(6):1059–1073 e9). Bispecific antibodies targeting both PD-1 and CTLA-4 not only simultaneously block the PD-1 and CTLA-4 signaling pathways, but also induce endocytosis of PD-1 for degradation. In cells co-expressing PD-1 and CTLA-4 (such as exhausted T cells in the tumor microenvironment), the cells can enhance antitumor immunity by blocking the interaction between CTLA-4 and CD80, demonstrating clinical efficacy in patients refractory to immune checkpoint inhibitors (Development and Preliminary Clinical Activity of PD-1-Guided CTLA-4 Blocking Bispecific DART Molecule, Alexey Berezhnoy et al., Cell Rep Med. 2020 Dec 22;1(9):100-163; Design and Efficacy of a Monovalent Bispecific PD-1 / CTLA4 Antibody That Enhances CTLA-4 Blockade on PD-1). + Activated T Cells, Dovedi SJ et al., Cancer Discovery, 08 Jan 2021, 11(5):1100-1117).

[0004] Angiogenic factors can drive immunosuppression by directly suppressing the function of antigen-presenting cells and immune effector cells and by enhancing the function of immunosuppressive cells such as regulatory T cells (Tregs), while immunosuppressive cells promote angiogenesis by secreting cytokines and other factors. Immune responses and angiogenesis influence each other, and both play important roles in tumor development and growth. Research has shown that immune checkpoint inhibitors can promote vascular normalization in tumor tissue (The Intersection between Tumor Angiogenesis and Immune Suppression, Rahma OE et al., Clin Cancer Res (2019) 25(18):5449-5457), and angiogenesis suppression can also improve antitumor immunity in the tumor microenvironment (Antiangiogenic therapy reverses the immunosuppressive breast cancer microenvironment, Wuzhen Chen et al., Biomark Res. 2021 Jul 22;9(1):59). The combination of the anti-PD-L1 antibodies atezolizumab and bevacizumab has shown remarkable efficacy in unresectable liver cancer (Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma, Richard S Finn et al., N Engl J Med., 2020 May 14;382(20):1894-1905) and has been approved by the FDA for the treatment of unresectable liver cancer.Combinations of other immune checkpoint inhibitors (including anti-PD-1 / PD-L1 antibodies) with anti-angiogenesis inhibitors (including anti-VEGF antibodies and other small molecule inhibitors that block VEGF signaling) have also achieved good results in clinical treatment (Combination of Anti-Angiogenics and Checkpoint Inhibitors for Renal Cell Carcinoma: Is the Whole Greater Than the Sum of Its Parts? Erich Jonasch et al., Cancers (Basel). 2022 Jan 27;14(3):644). Bispecific antibodies that simultaneously block both immune checkpoints and angiogenesis have shown superior efficacy to combinations in preclinical animal models (A Novel Bispecific Antibody Targeting PD-L1 and VEGF With Combined Anti-Tumor Activities, Xiaopei Cui, Front Immunol. 2021 Dec 2;12:778-978). A recent study found that the combination of the anti-PD-L1 antibodies atezolizumab and bevacizumab was less effective in treating liver cancer in patients with a higher ratio of Treg to effector T cells (Molecular correlates of clinical response and resistance to atezolizumab in combination with bevacizumab in advanced hepatocellular carcinoma. Andrew X Zhu et al., Nat Med. 2022 Aug;28(8):1599-1611). This suggests that simultaneously targeting T cells, Treg cells, and angiogenesis may provide new therapeutic insights for the clinic.

[0005] Therefore, multispecific antibodies, such as bispecific and trispecific antibodies, that simultaneously target multiple targets offer great promise for the clinical treatment of complex diseases.However, it has been recognized in the art that simply linking two or more antibodies or proteins together usually does not induce synergistic effects, and may even cause adverse effects.Therefore, bispecific or trispecific antibodies, especially trispecific antibodies, face great challenges in design, and need to consider many variables, including molecular compatibility, antibody affinity, stability and pharmaceutical properties.

[0006] The present invention provides a trispecific antibody that can simultaneously block two major immune checkpoints and one major angiogenesis pathway, thereby solving the above problems and meeting market demand to some extent. The trispecific antibody provided by the present invention simultaneously targets PD-1, CTLA-4, and VEGF in the tumor microenvironment, simultaneously blocking immunosuppression and angiogenesis, thereby regulating immune responses and angiogenesis in the tumor microenvironment, thereby achieving the therapeutic effect of the combination of three antibodies. Summary of the Invention [Means for solving the problem]

[0007] The present invention provides a novel trispecific antibody that simultaneously targets PD-1, CTLA-4, and VEGF, which simultaneously maintains the excellent antigen-binding specificity and selectivity of each antigen-binding site and therefore has excellent biological activity. In vitro efficacy studies have shown that the trispecific antibody of the present invention has immune stimulating activity similar to that of a combination of anti-PD-1 antibody and anti-CTLA-4 antibody, and VEGF-blocking activity similar to that of bevacizumab. When used in vivo in combination with the treatment of various tumors (melanoma, lung cancer, colon cancer, liver cancer, etc.), the trispecific antibody of the present invention has antitumor activity comparable to or even superior to that of anti-PD-1 antibody, anti-CTLA-4 antibody, and bevacizumab. Compared with monotherapy and combination therapy, the trispecific antibody targeting PD-1, CTLA-4, and VEGF has the following advantages: 1. Synergistically inhibit tumor cell proliferation and have a superior therapeutic effect to any single monoclonal antibody; 2. Due to the bivalent antigen-binding sites for each target, they have stronger affinity than multispecific antibodies with monovalent antigen-binding sites; 3. Improving a subject's low immune response to a monoclonal antibody; 4. Reducing or lowering the toxic side effects of anti-CTLA-4 monoclonal antibodies caused by Treg clearance by using FCs without or with weak ADCC function; 5. Increasing the ratio of trispecific antibodies distributed within and around the tumor, reducing toxicity of targeting CTLA-4 and VEGF through PD-1-mediated responses; 6. Degrading PD-1 antigen on the cell surface through CTLA-4-mediated endocytosis; 7. Reducing the number of doses and discomfort caused by coadministration; 8. Has increased stability; and 9. Prolong the effective duration of tumor suppression.

[0008] In general, the trispecific antibody molecule provided by the present invention can recognize three targets, significantly reducing costs compared to the combination of three antibodies, and the two identical antigen-binding sites of the trispecific antibody molecule for each target essentially maintain the binding ability of the corresponding natural bivalent antibody to the target, allowing for localized synergistic effects in tumors. Furthermore, the trispecific antibody provided by the present invention has good purity and thermal stability, which are highly advantageous for downstream development and large-scale production.

[0009] Therefore, the present invention mainly relates to the following aspects: In a first aspect, the present invention provides a trispecific antibody that simultaneously targets PD-1, CTLA-4 and VEGF, wherein the antibody comprises a first, second and third antigen-binding site, wherein the first, second and third antigen-binding sites bind to first, second and third antigens that are different from each other and are independently selected from PD-1, CTLA-4 and VEGF.

[0010] In one embodiment, the trispecific antibody provided by the present invention comprises a dimerized Fc region and one or more of the first, second and third antigen-binding sites are attached to the N-terminus and / or C-terminus of the dimerized Fc region.

[0011] In another embodiment of the trispecific antibody provided by the present invention, the first, second, and third antigen-binding sites can be in the form of a Fab, scFv, or VHH. In a specific embodiment, the antigen-binding site recognizing the antigen CTLA-4 is in the form of an scFc or VHH.

[0012] In certain embodiments of the trispecific antibodies provided by the invention, the antigen-binding site that binds to PD-1 comprises heavy chain CDRs (HCDRs) and / or light chain CDRs (LCDRs) as follows: HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 1, HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 2, and HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 3, and / or LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 4, LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 5, and LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 6.

[0013] In certain embodiments of the trispecific antibodies provided by the invention, the antigen-binding site that binds to PD-1 comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO:7, or comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:7, or consists of the sequence set forth in SEQ ID NO:7, and the light chain variable region comprises the sequence set forth in SEQ ID NO:8, or comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:8, or consists of the sequence set forth in SEQ ID NO:8.

[0014] In certain embodiments of the trispecific antibodies provided by the present invention, the antigen-binding site that binds to CTLA-4 comprises HCDRs and / or LCDRs as follows: HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 17, HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 18, and HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 19, and / or LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 20, LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 21, and LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 22.

[0015] In another particular embodiment of the trispecific antibody provided by the invention, the antigen-binding site that binds to CTLA-4 is a VHH and comprises HCDRs as follows: HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 12, HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 13, and HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 14.

[0016] In certain embodiments of the trispecific antibodies provided by the invention, the antigen-binding site that binds to CTLA-4 comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 15, or comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 15, or consisting of the sequence set forth in SEQ ID NO: 15, and a light chain variable region comprising the sequence set forth in SEQ ID NO: 16, or comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 16, or consisting of the sequence set forth in SEQ ID NO: 16.

[0017] In certain embodiments of the trispecific antibodies provided by the present invention, the antigen-binding site that binds to CTLA-4 comprises a single VH domain (VHH) that comprises the sequence set forth in SEQ ID NO:11, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:11, or consists of the sequence set forth in SEQ ID NO:11.

[0018] In certain embodiments of the trispecific antibodies provided by the present invention, the antigen-binding site that binds to VEGF comprises HCDRs and / or LCDRs as follows: HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 26, HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 27, and HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 28, and / or LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 29, LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 30, and LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 31.

[0019] In certain embodiments of the trispecific antibodies provided by the invention, the antigen-binding site that binds to VEGF comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO:24, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:24, or consists of the sequence set forth in SEQ ID NO:24, and the light chain variable region comprises the sequence set forth in SEQ ID NO:25, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:25, or consists of the sequence set forth in SEQ ID NO:25.

[0020] In one embodiment of the trispecific antibody provided by the invention, the first, second and third antigen-binding sites are 1) an antigen-binding site that binds to PD-1, comprising an HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 1, an HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 2, and an HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 3, as well as an LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 4, an LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 5, and an LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 6; 2) an antigen-binding site that binds to CTLA-4, i) an HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 17, an HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 18, and an HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 19, and an LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 20, an LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 21, and an LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 22; or ii) HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 12, HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 13, and HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 14 an antigen-binding site comprising: 3) An antigen-binding site that binds to VEGF, comprising an HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 26, an HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 27, and an HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 28, as well as an LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 29, an LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 30, and an LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 31. Includes:

[0021] In one embodiment of the trispecific antibody provided by the invention, the first, second and third antigen-binding sites are 1) an antigen-binding site that binds to PD-1, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO:7, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:7, or consists of the sequence set forth in SEQ ID NO:7, and the light chain variable region comprises the sequence set forth in SEQ ID NO:8, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:8, or consists of the sequence set forth in SEQ ID NO:8; 2) an antigen-binding site that binds to CTLA-4, i) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 15, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 15, or consists of the sequence set forth in SEQ ID NO: 15; and the light chain variable region comprises the sequence set forth in SEQ ID NO: 16, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 16, or consists of the sequence set forth in SEQ ID NO: 16; ii) comprising the sequence set forth in SEQ ID NO: 11, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 11, or consisting of the sequence set forth in SEQ ID NO: 11 an antigen-binding site, and 3) an antigen-binding site that binds to VEGF, comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 24, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 24, or consists of the sequence set forth in SEQ ID NO: 24, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 25, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 25, or consists of the sequence set forth in SEQ ID NO: 25. Includes:

[0022] In one embodiment of the trispecific antibody provided by the present invention, the antigen-binding site comprises one or more amino acid substitutions, deletions, or additions, or any combination thereof. In a preferred embodiment, the substitutions occur in the framework regions (FR regions). In a more preferred embodiment, the substitution is G44C in the heavy chain variable region, or Q100C or G100C (according to Kabat numbering) in the light chain variable region.

[0023] In one embodiment of the trispecific antibody provided by the present invention, the trispecific antibody comprises a homodimeric or heterodimeric Fc region, which may be one of immunoglobulins IgG1, IgG2, IgG3, or IgG4 having a native or variant sequence. In a preferred embodiment, the Fc region comprises modification(s), e.g., the Fc region comprises a knob-into-hole structure. In a further preferred embodiment, the Fc region comprises a substitution selected from the group consisting of S228P, S354C, T366W, T366S, L368A, Y394C, Y407V, H435R, Y436F, and K447A (according to the EU numbering system). In one embodiment, the Fc region is derived from the heavy chain constant region sequence set forth in SEQ ID NO: 33, 34, or 35.

[0024] In one embodiment of the trispecific antibody provided by the present invention, the first, second, and third antigen-binding sites are linked to each other by a linker / hinge region, or the first, second, and third antigen-binding sites are linked to the Fc region by a linker / hinge region. In a preferred embodiment, the linker has the amino acid sequence (G4S) n wherein n is an integer equal to or greater than 1. In a preferred embodiment, the linker consists of the amino acid sequence (G4S)3 or (G4S)4. In a preferred embodiment, the linker has the sequence shown in SEQ ID NO:9.

[0025] In one embodiment, the invention provides a trispecific antibody comprising a first, a second, and a third antigen-binding site and consisting of two identical heavy chains and two identical light chains, wherein the heavy and light chains are as follows: 1) a heavy chain having the structure VH-CH1-Fc-VHH-ScFv from the N-terminus to the C-terminus; a light chain having the structure VL-CL from the N-terminus to the C-terminus; 2) a heavy chain having the structure ScFv-VH-CH1-Fc-VHH from the N-terminus to the C-terminus; a light chain having the structure VL-CL from the N-terminus to the C-terminus; 3) a heavy chain having the structure VHH-VH-CH1-Fc-ScFv from the N-terminus to the C-terminus; a light chain having the structure VL-CL from the N-terminus to the C-terminus; 4) a heavy chain having the structure VH-CH1-Fc-ScFv-VHH from the N-terminus to the C-terminus; a light chain having the structure VL-CL from the N-terminus to the C-terminus; 5) a heavy chain having the structure VH-CH1-Fc-ScFv from the N-terminus to the C-terminus; a light chain having a structure of VL-CL-VHH from the N-terminus to the C-terminus; 6) a heavy chain having the structure ScFv-VH-CH1-Fc-ScFv from the N-terminus to the C-terminus; a light chain having the structure VL-CL from the N-terminus to the C-terminus; 7) a heavy chain having the structure VH-CH1-Fc-ScFv from the N-terminus to the C-terminus; a light chain having the structure VHH-VL-CL from N- to C-terminus, or 8) a heavy chain having the structure ScFv-VHH-VH-CH1-Fc from the N-terminus to the C-terminus; Light chains with the structure VL-CL from N- to C-terminus having a structure selected from Fc represents the Fc region of an immunoglobulin heavy chain, and two heavy chains containing the Fc region homodimerize through the Fc region. CH1 represents an immunoglobulin heavy chain CH1 domain, CL represents an immunoglobulin light chain CL domain, VH-CH1 and VL-CL pair together to form Fab, the first, second and third antigen-binding sites are in the form of Fab, VHH and / or ScFv, each binding to a different antigen independently selected from PD-1, CTLA-4 and VEGF; Trispecific antibodies are provided.

[0026] In a preferred embodiment, adjacent antigen binding sites are linked via a linker, preferably the antigen binding sites are linked to the Fc via a linker / hinge region. In another embodiment, the invention provides a method for the production of a medicament comprising administering to a subject a first, second, and third antigen-binding site, the method ... third antigen 1) a first heavy chain comprising the structure VH-CH1-Fc-ScFv from the N-terminus to the C-terminus; 2) a second heavy chain comprising the structure VHH-Fc-ScFv from the N-terminus to the C-terminus; and 3) Light chain containing the VL-CL structure from the N-terminus to the C-terminus a three-chain trispecific antibody having the following structure: Fc is the Fc region of an immunoglobulin heavy chain, which contains a knob-into-hole structure, and two heavy chains containing the Fc region heterodimerize through the Fc region. CH1 represents an immunoglobulin heavy chain CH1 domain, CL represents an immunoglobulin light chain CL domain, VH-CH1 and VL-CL pair together to form Fab, the first, second and third antigen-binding sites are in the form of Fab, VHH and / or ScFv and bind to different antigens independently selected from PD-1, CTLA-4 and VEGF; Trispecific antibodies are provided.

[0027] In a preferred embodiment, adjacent antigen binding sites are linked via a linker, preferably the antigen binding sites are linked to the Fc via a linker / hinge region. In another embodiment, the invention provides a method for the production of a medicament comprising administering to a subject a first, second, and third antigen-binding site, the method ... third antigen 1) a first heavy chain comprising the structure VH-CH1-Fc-ScFv2 from the N-terminus to the C-terminus; 2) a second heavy chain comprising the structure ScFv1-Fc-ScFv2 from the N-terminus to the C-terminus; 3) Light chain containing the VL-CL structure from the N-terminus to the C-terminus a three-chain trispecific antibody having the following structure: Fc is the Fc region of an immunoglobulin heavy chain, which contains a knob-into-hole structure, and two heavy chains containing the Fc region heterodimerize through the Fc region. CH1 represents an immunoglobulin heavy chain CH1 domain, CL represents an immunoglobulin light chain CL domain, VH-CH1 and VL-CL pair together to form Fab, the first, second and third antigen-binding sites are in the form of Fab, ScFv1 and / or ScFv2, each binding to a different antigen independently selected from PD-1, CTLA-4 and VEGF; Trispecific antibodies are provided.

[0028] In a preferred embodiment, adjacent antigen binding sites are linked via a linker, preferably the antigen binding sites are linked to the Fc via a linker / hinge region. In another embodiment, the invention provides a method for the production of a medicament comprising administering to a subject a first, second, and third antigen-binding site, the method ... third antigen 1) a first heavy chain comprising the structure ScFv1-Fc-ScFv2 from the N-terminus to the C-terminus; 2) A second heavy chain comprising the structure VHH-Fc-ScFv2 from the N-terminus to the C-terminus. a trispecific antibody consisting of two chains having the following structure: Fc is the Fc region of an immunoglobulin heavy chain, which contains a knob-into-hole structure, and two heavy chains containing the Fc region heterodimerize through the Fc region. the first, second and third antigen-binding sites are in the form of VHH, ScFv1 and / or ScFv2, each binding to a different antigen independently selected from PD-1, CTLA-4 and VEGF; Trispecific antibodies are provided.

[0029] In a preferred embodiment, adjacent antigen binding sites are linked via a linker, preferably the antigen binding sites are linked to the Fc via a linker / hinge region. In a preferred embodiment, the first, second and third antigen-binding sites of the trispecific antibody are 1) an antigen-binding site that binds to PD-1, comprising an HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 1, an HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 2, and an HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 3, as well as an LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 4, an LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 5, and an LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 6; 2) an antigen-binding site that binds to CTLA-4, i) an HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 17, an HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 18, and an HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 19, and an LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 20, an LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 21, and an LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 22, or ii) HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 12, HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 13, and HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 14 an antigen-binding site comprising: 3) An antigen-binding site that binds to VEGF, comprising an HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 26, an HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 27, and an HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 28, as well as an LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 29, an LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 30, and an LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 31. Includes:

[0030] In a preferred embodiment, the first, second and third antigen-binding sites of the trispecific antibody are 1) an antigen-binding site that binds to PD-1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO:7, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:7, or consists of the sequence set forth in SEQ ID NO:7, and the light chain variable region comprises the sequence set forth in SEQ ID NO:8, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:8, or consists of the sequence set forth in SEQ ID NO:8; 2) an antigen-binding site that binds to CTLA-4, i) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 15, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 15, or consists of the sequence set forth in SEQ ID NO: 15; and the light chain variable region comprises the sequence set forth in SEQ ID NO: 16, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 16, or consists of the sequence set forth in SEQ ID NO: 16; ii) comprising the sequence set forth in SEQ ID NO: 11, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 11, or consisting of the sequence set forth in SEQ ID NO: 11 an antigen-binding site, and 3) an antigen-binding site that binds to VEGF, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 24, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 24, or consists of the sequence set forth in SEQ ID NO: 24, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 25, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 25, or consists of the sequence set forth in SEQ ID NO: 25. Includes:

[0031] In a preferred embodiment, the first, second, and third antigen-binding sites described above comprise substitution(s) in the framework region (FR region). In a more preferred embodiment, the substitution is G44C in the heavy chain variable region, or Q100C or G100C (according to Kabat numbering) in the light chain variable region.

[0032] In a preferred embodiment, the Fc region of the trispecific antibody is derived from the Fc region of IgG1 or IgG4. In a preferred embodiment, the Fc region comprises modification(s), for example, the Fc region comprises a knobs-into-holes structure. In a further preferred embodiment, the Fc region comprises a substitution selected from the group consisting of S228P, S354C, T366W, T366S, L368A, Y394C, Y407V, H435R, Y436F, and K447A (according to the EU numbering system). In a preferred embodiment, the Fc region is derived from the heavy chain constant region sequence set forth in SEQ ID NO: 33, 34, or 35.

[0033] In a preferred embodiment, the linker comprises the amino acid sequence (G4S) n wherein n is an integer equal to or greater than 1. In a preferred embodiment, the linker consists of the amino acid sequence (G4S)3 or (G4S)4. In a preferred embodiment, the linker has the sequence shown in SEQ ID NO:9.

[0034] In a preferred embodiment, the present invention comprises: 1) a heavy chain comprising SEQ ID NO: 37, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 38, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 2) a heavy chain comprising SEQ ID NO: 39, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 3) a heavy chain comprising SEQ ID NO: 41, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 42, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 4) a heavy chain comprising SEQ ID NO: 43, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 44, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 5) a heavy chain comprising SEQ ID NO: 45, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 46, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 6) a heavy chain comprising SEQ ID NO: 47, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 48, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 7) a heavy chain comprising SEQ ID NO:49, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO:50, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 8) a heavy chain comprising SEQ ID NO: 59, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 9) a heavy chain comprising SEQ ID NO: 61, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 62, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 10) a heavy chain comprising SEQ ID NO: 63, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 64, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 11) a heavy chain comprising SEQ ID NO: 65, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 66, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 12) a heavy chain comprising SEQ ID NO: 67, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 68, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 13) a heavy chain comprising SEQ ID NO: 69, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 70, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 14) a heavy chain comprising SEQ ID NO: 71, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 72, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 15) a heavy chain comprising SEQ ID NO: 73, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 74, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 16) A heavy chain comprising an amino acid sequence of SEQ ID NO: 75 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising an amino acid sequence of SEQ ID NO: 76 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. The present invention provides a trispecific antibody comprising:

[0035] In a preferred embodiment, the present invention comprises: 1) a first heavy chain comprising SEQ ID NO: 51, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, a second heavy chain comprising SEQ ID NO: 53, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a light chain comprising SEQ ID NO: 52, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or 2) a first heavy chain comprising SEQ ID NO: 56, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; a second heavy chain comprising SEQ ID NO: 58, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and a light chain comprising SEQ ID NO: 57, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. The present invention provides a trispecific antibody comprising:

[0036] In a preferred embodiment, the present invention provides a trispecific antibody comprising a first heavy chain comprising SEQ ID NO: 54, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a second heavy chain comprising SEQ ID NO: 55, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0037] In a preferred embodiment, the present invention comprises: 1) a heavy chain comprising or consisting of SEQ ID NO: 37 and a light chain comprising or consisting of SEQ ID NO: 38, or 2) a heavy chain comprising or consisting of SEQ ID NO: 39 and a light chain comprising or consisting of SEQ ID NO: 40, or 3) a heavy chain comprising or consisting of SEQ ID NO: 41 and a light chain comprising or consisting of SEQ ID NO: 42, or 4) a heavy chain comprising or consisting of SEQ ID NO: 43 and a light chain comprising or consisting of SEQ ID NO: 44, or 5) a heavy chain comprising or consisting of SEQ ID NO: 45 and a light chain comprising or consisting of SEQ ID NO: 46, or 6) a heavy chain comprising or consisting of SEQ ID NO: 47 and a light chain comprising or consisting of SEQ ID NO: 48, or 7) a heavy chain comprising or consisting of SEQ ID NO: 49 and a light chain comprising or consisting of SEQ ID NO: 50, or 8) a heavy chain comprising or consisting of SEQ ID NO: 59 and a light chain comprising or consisting of SEQ ID NO: 60, or 9) a heavy chain comprising or consisting of SEQ ID NO: 61 and a light chain comprising or consisting of SEQ ID NO: 62, or 10) a heavy chain comprising or consisting of SEQ ID NO: 63 and a light chain comprising or consisting of SEQ ID NO: 64, or 11) a heavy chain comprising or consisting of SEQ ID NO: 65 and a light chain comprising or consisting of SEQ ID NO: 66, or 12) a heavy chain comprising or consisting of SEQ ID NO: 67 and a light chain comprising or consisting of SEQ ID NO: 68, or 13) a heavy chain comprising or consisting of SEQ ID NO: 69 and a light chain comprising or consisting of SEQ ID NO: 70, or 14) a heavy chain comprising or consisting of SEQ ID NO: 71 and a light chain comprising or consisting of SEQ ID NO: 72, or 15) a heavy chain comprising or consisting of SEQ ID NO: 73 and a light chain comprising or consisting of SEQ ID NO: 74, or 16) A heavy chain comprising or consisting of SEQ ID NO: 75 and a light chain comprising or consisting of SEQ ID NO: 76 The present invention provides a trispecific antibody comprising:

[0038] In a preferred embodiment, the present invention comprises: 1) a first heavy chain comprising or consisting of SEQ ID NO: 51, a second heavy chain comprising or consisting of SEQ ID NO: 53, and a light chain comprising or consisting of SEQ ID NO: 52, or 2) a first heavy chain comprising or consisting of SEQ ID NO: 56, a second heavy chain comprising or consisting of SEQ ID NO: 58, and a light chain comprising or consisting of SEQ ID NO: 57 The present invention provides a trispecific antibody comprising:

[0039] In a preferred embodiment, the present invention comprises: a first heavy chain comprising or consisting of SEQ ID NO: 54, and a second heavy chain comprising or consisting of SEQ ID NO: 55 a second heavy chain consisting of The present invention provides a trispecific antibody comprising:

[0040] In a second aspect, the present invention provides polynucleotides encoding the trispecific antibody molecules of the invention, vectors comprising the polynucleotides, and host cells comprising the polynucleotides or vectors of the invention.

[0041] In one embodiment, the vector is preferably an expression vector. In one embodiment, the host cell can be any prokaryotic or eukaryotic cell commonly used in the art.

[0042] In one embodiment, the present invention provides a host cell comprising one or more polynucleotides of the present invention. In some embodiments, a host cell comprising a vector of the present invention is provided. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, and the like. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney cell lines (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), CHO cells, NSO cells, myeloma cell lines such as Y0, NS0, P3X63, and Sp2 / 0, etc. In a preferred embodiment, the host cells are CHO, HEK293, or NSO cells.

[0043] In a third aspect, the present invention provides a method for producing a trispecific antibody of the present invention, the method comprising the steps of (i) culturing a host cell as disclosed in the second aspect of the invention under conditions suitable for expressing the trispecific antibody disclosed in the first aspect of the invention, and optionally (ii) recovering the trispecific antibody of the present invention.

[0044] In a fourth aspect, the present invention provides a pharmaceutical composition comprising a trispecific antibody molecule of the present invention. In one embodiment, the pharmaceutical composition provided by the present invention further comprises other therapeutic agents, and optional excipients, preferably the other therapeutic agents are selected from chemotherapeutic agents, cytotoxic drugs, and the like.

[0045] In a fifth aspect, the present invention provides the use of the trispecific antibodies and pharmaceutical compositions of the invention for treating, preventing and / or diagnosing cancer, an autoimmune disease, an infectious disease or an angiogenesis-related disease.

[0046] In one embodiment, the invention provides the use of an antibody according to the first aspect, any polynucleotide or vector or host cell according to the second aspect, and a pharmaceutical composition according to the fourth aspect in the preparation of a medicament for the treatment, prevention and / or diagnosis of cancer, an autoimmune disease, an infectious disease or an angiogenesis-related disease.

[0047] In one embodiment, the invention provides an antibody of any of the first aspect, a polynucleotide or vector or host cell of any of the second aspect, and a pharmaceutical composition of the fourth aspect for use in therapy, prevention and / or diagnosis.

[0048] In one embodiment, the invention provides an antibody of any of the first aspect, a polynucleotide or vector or host cell of any of the second aspect, and a pharmaceutical composition of the fourth aspect for use in the treatment, prevention and / or diagnosis of cancer, an autoimmune disease, an infectious disease or an angiogenesis-related disease.

[0049] In one embodiment, the cancer is, for example, a solid tumor such as lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), breast cancer, liver cancer, bladder cancer, breast cancer, melanoma, colon cancer, rectal cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic adenocarcinoma, basal cell carcinoma, esophageal cancer, bile duct cancer, head and neck squamous cell carcinoma, thyroid cancer, brain cancer, gastric cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, testicular cancer, multiple myeloma, glioblastoma, glioma, and a hematological tumor such as leukemia, lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma).

[0050] In a sixth aspect, the present invention provides a method for treating, preventing and / or diagnosing cancer, an autoimmune disease, an infectious disease or an angiogenesis-related disease, the method comprising the step of administering a therapeutically effective amount of a trispecific antibody of the invention, or a pharmaceutical composition of the invention, to a patient in need thereof.

[0051] In one embodiment, the cancer is, for example, a solid tumor such as lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), breast cancer, liver cancer, bladder cancer, breast cancer, melanoma, colon cancer, rectal cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic adenocarcinoma, basal cell carcinoma, esophageal cancer, bile duct cancer, head and neck squamous cell carcinoma, thyroid cancer, brain cancer, gastric cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, testicular cancer, multiple myeloma, glioblastoma, glioma, and a hematological tumor such as leukemia, lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma). [Brief explanation of the drawings]

[0052] [Figure 1-1] FIG. 1 shows a schematic diagram of the structure of the trispecific antibody constructed in this application. [Figure 1-2] FIG. 1 shows a schematic diagram of the structure of the trispecific antibody constructed in this application. [Figure 1-3] FIG. 1 shows a schematic diagram of the structure of the trispecific antibody constructed in this application. [Figure 1-4] FIG. 1 shows a schematic diagram of the structure of the trispecific antibody constructed in this application. [Figure 2] Figure 2 shows the activity of the trispecific antibody in binding to cell surface PD-1. The activity of the trispecific antibody in binding to 293T-hPD-1 cells was detected using FACS. Panels A and B are assays FACS#1 and FACS#2, respectively. [Figure 3] Figure 3 shows the activity of the trispecific antibody binding to cell surface CTLA-4. The activity of the trispecific antibody binding to CHO-hCTLA-4 cells was detected using FACS. Panels A and B are assays FACS#3 and FACS#4, respectively. [Figure 4] Figure 4 shows the avidity of the trispecific antibody binding to activated T cells. Panel A shows the avidity of the trispecific antibody and anti-PD-1 antibody binding to T cells, and Panel B shows the avidity of the anti-CTLA-4 antibody binding to T cells. [Figure 5] FIG. 5 shows the activity of the trispecific antibody in binding to free VEGFA after binding to 293T-hPD-1-hCTLA-4 cells (which co-express PD-1 and CTLA-4). [Figure 6] FIG. 6 shows the activity of the trispecific antibody to bind to free VEGFA after binding to activated T cells. [Figure 7] Figure 7 shows the ability of the trispecific antibody to enhance SEB-stimulated IL-2 secretion from PBMCs. Panels A and B show the results of IL-2 secretion by PBMCs from donors Lot#A10Z707023 and Lot#A10Z647018, respectively, after treatment with SEB and antibody for 3 days. [Figure 8] Figure 8 shows that the trispecific antibody promotes IL-2 secretion in an MLR. The trispecific antibody or control antibody was added to an MLR, and IL-2 secretion was measured after 4 days. Panel A: DC donor: Lot#Z0160, PBMC donor: Lot#Z0177. IL-2 secretion was detected after 4 days; Panel B: DC donor: Lot#Z0160, PBMC donor: Lot#Z0182. [Figure 9-1] Figure 9 shows that different concentrations of trispecific antibodies increase IL-2 secretion in MLRs containing different ratios of Tregs. The concentrations of the test antibodies in A-C were 500, 50, and 5 nM, respectively, and IL-2 secretion was measured 4 days after treatment of the MLRs. [Figure 9-2] FIG. 9 shows that different concentrations of trispecific antibody increase IL-2 secretion in MLRs containing different ratios of Tregs. [Figure 10] FIG. 10 shows that the trispecific antibody inhibits VEGFA-induced proliferation of human umbilical vein endothelial cells. [Figure 11] FIG. 11 shows that trispecific antibody HC010-F8 inhibits the growth of human melanoma A375 in human PBMC-humanized mice. [Figure 12] FIG. 12 shows that trispecific antibody HC010-F8 has no effect on mouse body weight in the human PBMC humanized human melanoma A375 mouse model. [Figure 13] Figure 13 shows the binding of the trispecific antibody to VEGFA (Panel A) and CTLA-4 (Panel B) after binding to human PD-1. [Figure 14] FIG. 14 shows the binding of the trispecific antibody to VEGFA (Panel A) and CTLA-4 (Panel B) after binding to cell surface human PD-1. [Figure 15] FIG. 15 shows the binding of the trispecific antibody to PD-1 (Panel A) and VEGFA (Panel B) after binding to cell surface CTLA-4. [Figure 16] FIG. 16 shows that trispecific antibodies HC010-F8 and HC010-F23 inhibit the growth of human melanoma A375 in human PBMC-humanized mice. [Figure 17] FIG. 17 shows that trispecific antibodies HC010-F8 and HC010-F23 have no effect on mouse body weight in the human PBMC humanized human melanoma A375 mouse model. [Figure 18] FIG. 18 shows the activity of the trispecific antibody in inhibiting human non-small cell lung cancer A549 cells in mice. [Figure 19] FIG. 19 shows the activity of the trispecific antibody in inhibiting human non-small cell lung cancer H1299 cells in mice. [Figure 20] FIG. 20 shows the activity of the trispecific antibody in inhibiting human hepatoma Huh7 cells in mice. DETAILED DESCRIPTION OF THE INVENTION

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting. Other features, objects, and advantages of the present invention will be apparent from the description and drawings, and from the appended claims.

[0054] I. Definition The term "about" when used in reference to a numerical value is intended to encompass numerical values ​​within a range between a lower limit of 5% less than the stated numerical value and an upper limit of 5% more than the stated numerical value.

[0055] As used herein, the terms "comprising" or "including" mean the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.

[0056] The term "antibody" is used herein in its broadest sense to refer to a protein that contains an antigen-binding site. The terms "antigen-binding site" and "antigen-binding domain" are used interchangeably and refer to the region of an antibody molecule that actually binds to an antigen. Antigen-binding sites include, but are not limited to, Fv, Fab fragments, Fab', Fab'-SH, F(ab')2, single-chain antibody molecules (e.g., scFv), VHH, and the like.

[0057] The term "immunoglobulin" refers to a protein having the structure of a naturally occurring antibody and is often used interchangeably with the term "antibody" in this application. IgG class immunoglobulins are heterotetrameric glycoproteins composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called the heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called the light chain variable domain, followed by a light chain constant domain (CL). In IgG molecules, the VH-CH1 of the heavy chain usually pairs with the VL-CL of the light chain to form Fab fragments that specifically bind to antigens. Thus, one IgG immunoglobulin essentially consists of two Fab molecules linked by an immunoglobulin hinge region and two dimerized Fc regions. Immunoglobulin heavy chains can be assigned to one of five classes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), based on the type of their constant domain, some of which can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α (IgA1), and α2 (IgA2). Immunoglobulin light chains can also be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of their constant domain.

[0058] The term "variable region" or "variable domain" of an antibody refers to the domain of an antibody heavy or light chain that is responsible for binding the antibody to an antigen. The variable region of an antibody can be further subdivided into hypervariable regions, i.e., complementarity-determining regions (CDRs), and more conserved regions, i.e., framework regions (FRs) intervening between the hypervariable regions. In the case of IgG class immunoglobulins, the heavy chain variable region or light chain variable region comprises, from the N-terminus to the C-terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively. For example, in the case of heavy chain antibodies (also referred to herein as nanobodies) derived from camelids, the antigen-binding site consists of a single VH domain (i.e., a "VHH" domain). The VHH of a natural heavy chain antibody has a structure similar to that of the heavy chain variable region of a natural IgG antibody, i.e., the VHH of a natural heavy chain antibody contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs).

[0059] The term "multispecific antibody" refers to an antibody that has at least two antigen-binding sites, each of which binds to a different epitope on the same antigen or to a different epitope on a different antigen.

[0060] The term "valency" in the context of an antibody refers to the total number of antigen-binding sites in an antibody molecule or the number of antigen-binding sites with the same antigen-binding specificity. For example, a hexavalent antibody means that the antibody molecule contains a total of six antigen-binding sites, regardless of whether the epitopes bound to it are the same. Preferably, in the present invention, the hexavalent antibody has three different antigen-binding specificities, and there are two identical antigen-binding sites for each antigen-binding specificity.

[0061] A "heavy chain constant region domain" or "heavy chain constant region" refers to a constant region obtained from, or derived from, an immunoglobulin heavy chain, comprising the heavy chain constant regions CH1, CH2, CH3, and optionally the heavy chain constant region CH4, covalently linked sequentially from N-terminus to C-terminus. In most cases, the heavy chain constant regions CH1 and CH2 are connected by a heavy chain hinge region, but may be connected by a flexible linker if necessary. In some embodiments of the present invention, the heavy chain constant region of an antibody molecule of the present invention comprises CH1-hinge-CH2-CH3. The heavy chain constant region domain can be selected according to the intended function of the antibody molecule. For example, the constant domain may be an IgA, IgD, IgE, IgG, or IgM domain, particularly an immunoglobulin constant domain of human IgG, such as the constant domain of human IgG1, IgG2, IgG3, or IgG4.

[0062] In the multispecific antibodies of the present invention, the chain containing the Fc domain is a heavy chain, and the chain lacking the Fc domain is a light chain. In some embodiments of the present invention, the antibody molecules of the present invention are composed of two identical heavy chains and two identical light chains. In other embodiments of the present invention, the antibody molecules of the present invention comprise two different heavy chains.

[0063] The term "Fc domain" or "Fc region" refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes naturally occurring Fc regions or mutant Fc regions. Fc domains that can be used in the antibodies of the present invention include, but are not limited to, IgG1, IgG2, IgG3, or IgG4 Fc domains having naturally occurring or mutant sequences. The lysine residue at position 447 (according to the EU numbering system) at the C-terminus of the Fc domain may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system described in Kabat et al., Sequences of Proteins of Immunological Interests, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (also called the EU index).

[0064] Fc domains, fusion proteins (eg, antibodies) containing Fc domains, can be modified using a variety of known methods, such as modifications to reduce immunogenicity, improve stability, solubility, function, and clinical benefit. Such modifications include, but are not limited to, the following: modification of amino acid residue K447 to increase IgG stability, preferably the amino acid substitution K447A; modification of amino acid residue S228 to address the issue of IgG4 heterogeneity, preferably the amino acid substitution S228P; knob-into-hole structures to increase antibody stability, where the knob can be at amino acid residues S354 and T366 and the hole can be at amino acid residues Y394, T366, L368, and Y407, preferably the knob can be the following amino acid substitution: S354C, T366W, and the hole can be the following amino acid substitution: Y394C, T366S, L368A, Y407V; and hole structure modifications to reduce homodimers can be at amino acid residues H435 and Y436, preferably the amino acid substitutions H435R and Y436F.

[0065] "Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" are regions of antibody variable domains that are highly variable in sequence and that form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). The CDRs are primarily responsible for binding to an antigen epitope.

[0066] The term "immune checkpoint" refers to a type of inhibitory signaling molecule present in the immune system, which regulates the duration and intensity of immune responses in peripheral tissues, thereby preventing tissue damage and maintaining tolerance to self-antigens (Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4):252-264). Research has shown that one of the reasons tumor cells are able to evade the body's immune system and proliferate uncontrollably is because they exploit the inhibitory signaling pathway of immune checkpoints to suppress the activity of T lymphocytes, preventing them from effectively killing tumors. Immune checkpoint molecules include, but are not limited to, programmed cell death 1 (PD-1), programmed cell death-ligand 1 (PD-L1), PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3, and TIM-3.

[0067] The term "PD-1" refers to programmed cell death protein 1, which is expressed on the surface of T cells, and its ligands, PD-L1 and PD-L2, are expressed on the surface of various cell types, including many cancer cells. Upon binding to its ligand (PD-L1 or PD-L2), PD-1 suppresses T cell activation by recruiting SHP-2, thereby suppressing T cell proliferation and effector functions such as IFN-γ production and cytotoxic activity. Cancer cells also use this mechanism to evade immune responses by inhibiting T cells from attacking cancer cells via the ligand or PD-L2 on their surface. As used herein, the term "PD-1" includes human PD-1, human PD-1 variants, isoforms, and species homologs.

[0068] The term "human PD-1" refers to the human PD-1 protein encoded by the wild-type human PD-1 gene, such as the human PD-1 disclosed in GenBank Accession No. NM_005018.2.

[0069] The term "CTLA-4," or cytotoxic T-lymphocyte-associated protein 4, suppresses immune responses by binding to its ligands CD80 (also known as B7-1) and CD86 (also known as B7-2). CTLA-4 suppresses immune responses in several ways: for example, 1) it competes with the T-cell costimulatory receptor CD28 for its ligands CD80 and CD86; and 2) it sends negative signals that suppress T-cell activation. CTLA-4 inhibitors can increase the number of T cells that can attack tumor cells by suppressing the CTLA-4 molecule. As used herein, the term "CTLA-4" includes human "CTLA-4," human CTLA-4 variants, isoforms, and species homologs. Human CTLA-4 is disclosed, for example, in GenBank Accession No. AAB59385.

[0070] Both PD-1 and CTLA-4 are immune checkpoints belonging to the CD28 family and are expressed on activated T cells, but they differ somewhat in their expression at different stages of the immune response and on different immune cells. PD-1 inhibitors and CTLA-4 inhibitors have different activating effects on different immune cells. In addition to activating exhausted T cells, PD-1 inhibitors primarily activate cytotoxic T cells and Tregs, while CTLA-4 inhibitors primarily activate Th1 effector T cells and Tfh cells (PMID: 35241833). Similar to the phenotypes of PD-1 knockout and CTLA-4 knockout mice, PD-1 inhibitors tend to be less toxic than CTLA-4 inhibitors, whereas CTLA-4 inhibitors tend to have greater toxicity, which may be related to the elimination of peripheral Tregs from these cells. For example, widespread clinical use of the anti-CTLA-4 antibody ipilimumab for the treatment of advanced melanoma has been limited due to its severe toxic side effects.

[0071] The term "VEGF" refers to vascular endothelial growth factor, also known as vascular permeability factor (VPF), which is a highly specific vascular endothelial cell growth factor that promotes vascular permeability, extracellular matrix modification, endothelial cell migration, proliferation, and angiogenesis, etc. VEGF is a family that includes VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, and placental growth factor (PGF).

[0072] The term "VEGFA (vascular endothelial growth factor A)" refers to a highly conserved 27-kDa dimeric glycoprotein secreted by various cells, including endothelial cells and tumors. Common isoforms of VEGFA produced by alternative splicing include VEGFA121, VEGFA165, VEGFA189, and VEGFA206, which contain 121, 165, 189, and 206 amino acids, respectively. VEGFA includes human VEGFA, e.g., the human VEGFA protein under accession number UniProt NO.:P15692. VEGFA is a key regulator of angiogenesis during solid tumor growth. Therefore, VEGFA-targeted therapy is one of the recent focuses of interest in oncology. Bevacizumab is the first approved VEGFA inhibitor. Throughout this application, VEGF generally refers to VEGFA, e.g., VEGF165 generally refers to VEGFA165.

[0073] The term “EC 50 ", also known as "half maximal effective concentration," is the concentration of a drug, antibody, or toxic agent that induces a response that is 50% between baseline and maximum after a specified exposure time. In the context of this application, EC 50 The unit is "nM".

[0074] The term “IC 50 ', also known as the '50% inhibitory concentration', is the concentration of a drug or substance (inhibitor) that inhibits a biological process (or a specific substance involved in that process, such as an enzyme, cell receptor, or microorganism) by 50%.

[0075] The terms "flexible linker" or "linker" or "connector peptide" are used interchangeably and refer to a short amino acid sequence consisting of amino acids such as glycine (G) and / or serine (S) and / or threonine residues (T), alone or in combination, or derived from the hinge region of an immunoglobulin.

[0076] Linkers that can be used in the present invention can be easily determined by those skilled in the art. For example, the linker may have the amino acid sequence (G4S): n wherein n is an integer equal to or greater than 1. In a preferred embodiment, the linker consists of the amino acid sequence (G4S)3 or (G4S)4. Linkers that can be used in the antibody molecules of the present invention can also have, but are not limited to, the following amino acid sequences: (G3S)2, (G4S)2, (G3S)3, (G4S)3, (G3S)4, (G4S)4, (G3S)5, (G4S)5, (G3S)6, (G4S)6, GGG, DGGGS, TGEKP, GGRR, EGKSSGSGSESKVD, KESGSVSSEQLAQFRSLD, GGRRGGGS, LQRDGERP, LRQKDGGGSERP, and GSTSGSGKPGSGEGSTKG.

[0077] As used herein, the terms "binding" or "specific binding" mean that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0078] "Percentage (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to those in a specific amino acid sequence set forth herein, after aligning the candidate sequence to the specific amino acid sequence set forth herein and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, without considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention having a substantial degree of identity, such as at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more, relative to the amino acid molecules and sequences specifically disclosed herein. Variants may contain conservative modifications.

[0079] In polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions to a polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention. The following eight groups contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.

[0080] The term "host cell" refers to a cell into which an exogenous polypeptide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny. Host cells are any type of cell line that can be used to produce the antibody molecules of the invention, including eukaryotic cells such as mammalian cells, insect cells, yeast cells, and prokaryotic cells such as E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissue.

[0081] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or by an in vitro expression system. Expression vectors include all expression vectors known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which the recombinant polynucleotide has been incorporated.

[0082] The terms "individual" and "subject" are used interchangeably to refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual is a human.

[0083] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival. The terms "tumor" and "cancer" are used interchangeably herein and include both solid and liquid tumors.

[0084] The term "cancer" refers to a physiological disorder in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Cancer can be, for example, and solid tumors such as lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), breast cancer, liver cancer, bladder cancer, breast cancer, melanoma, colon cancer, rectal cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic adenocarcinoma, basal cell carcinoma, esophageal cancer, bile duct cancer, head and neck squamous cell carcinoma, thyroid cancer, brain cancer, gastric cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, testicular cancer, multiple myeloma, glioblastoma, glioma, and hematological tumors such as leukemia, lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma). In certain embodiments, cancers suitable for treatment with the antibodies of the invention include breast cancer, gastric cancer, ovarian cancer, gastroesophageal junction cancer, bladder cancer, small intestine and ampullary cancer, esophageal cancer, lung cancer, and cervical cancer. Metastatic forms of those cancers are also included. In some embodiments, the present invention provides multispecific antibodies useful, inter alia, in the treatment of tumors / cancers and their therapeutic uses in the treatment of said tumors / cancers.

[0085] The term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in the individual being treated. Desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the antibody molecules of the present invention are used to delay the onset of the disease or slow the progression of the disease.

[0086] The term "prevention" includes suppressing the occurrence or progression of a disease or disorder or the symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for a preventive regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0087] The term "effective amount" refers to the amount or dose of an antibody or composition of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration to the patient in single or multiple doses. The effective amount can be easily determined by a physician skilled in the art by taking into consideration various factors such as the species of mammal, body weight, age, and general health of the mammal; the specific disease involved; the extent or severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability profile of the administered formulation; the selected dosing regimen; and the use of any combination therapy.

[0088] The term "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic result at the necessary dosage for the necessary period of time. A therapeutically effective amount of an antibody or antibody fragment or composition may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the antibody or antibody fragment or composition are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, more preferably at least about 50%, 60%, or 70%, and even more preferably at least about 80% or 90% compared to untreated controls. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be assessed in an animal model system predictive of efficacy in human tumors.

[0089] The term "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0090] The term "pharmaceutical composition" refers to a composition that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0091] II. Production and Purification of Antibodies of the Invention To produce a trispecific antibody of the invention, each polypeptide chain of the antibody of the invention is obtained, for example, by solid phase peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinant production methods, and assembled under appropriate conditions.

[0092] For recombinant production, polynucleotide(s) encoding any one and / or more than one polypeptide chain of the antibody are isolated and inserted into one or more vectors for subsequent cloning and / or expression in host cells. The polynucleotide(s) can be readily isolated using conventional methods and verified, for example, by sequencing. In one embodiment, polynucleotide(s) encoding one or more polypeptide chains of a triabody of the invention are provided. In yet another embodiment, the invention provides a vector, preferably an expression vector, comprising one or more polynucleotides of the invention. Thus, in one embodiment, the invention provides a method for producing a triabody of the invention, comprising culturing host cells comprising polynucleotides encoding the polypeptide chains of the triabody under conditions suitable for expression of the polypeptide chains, and allowing the polypeptide chains to assemble into the triabody to produce the antibody.

[0093] Expression vectors can be constructed using methods well known to those skilled in the art, including, but not limited to, viruses, plasmids, cosmids, lambda phage, or yeast artificial chromosomes (YACs).

[0094] Antibodies prepared by the methods described herein can be purified by known and existing techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. Following purification, the purity of the antibodies of the invention can be determined by any of several well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like. The physical / chemical properties and / or biological activity of the antibodies provided herein can be identified, screened, or characterized by various assays known in the art.

[0095] III. Pharmaceutical Compositions, Pharmaceutical Formulations, and Kits In one aspect, the present invention provides compositions, e.g., pharmaceutical compositions, comprising an antibody described herein formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents, and the like. The pharmaceutical compositions of the present invention are suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the antibody of the present invention is the only active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise an antibody described herein and more than one therapeutic agent.

[0096] In another aspect, the invention also provides pharmaceutical formulations comprising an antibody described herein and one or more therapeutic agents. The composition of the present invention may be in various forms.These forms include, for example, liquid, semi-solid, and solid dosage forms, such as solutions (e.g., injections and infusions), suspensions or suspensions, liposomes, and suppositories.The preferred form depends on the intended mode of administration and therapeutic application.Typical preferred compositions are in the form of injections or infusions.

[0097] The pharmaceutical compositions of the present invention may contain a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody of the present invention. A "therapeutically effective amount" is an amount effective to achieve a desired therapeutic result at a necessary dosage for a necessary period of time. A therapeutically effective amount may vary depending on various factors, such as the individual's disease state, age, sex, and weight, and the like. A therapeutically effective amount is an amount in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate, etc.) by at least about 20%, more preferably at least about 40%, more preferably at least about 60%, and even more preferably at least about 80% compared to untreated controls. The ability of the antibodies of the present invention to inhibit a measurable parameter (e.g., tumor volume) can be evaluated in an animal model system predictive of efficacy in human tumors. A "prophylactically effective amount" is an amount effective to achieve a desired prophylactic result at a necessary dosage for a necessary period of time. Typically, because a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0098] In another aspect, the present invention provides a kit comprising the antibody or composition described herein. The kit may also include one or more other components, including, for example, instructions for use; other reagents such as labels or coupling reagents; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject.

[0099] IV. Uses and Treatment Methods of the Antibodies and Compositions of the Invention The PD-1 / CTLA-4 / VEGF-binding trispecific antibodies of the invention are suitable for use as anti-tumor, anti-angiogenic, anti-autoimmune disease, and anti-infective agents. In some embodiments, the trispecific antibodies of the invention are used to treat cancer, such as melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, Hodgkin's lymphoma, head and neck cancer, ovarian cancer, and brain cancer. [Example]

[0100] The following examples are for illustrative purposes only and therefore should not be construed as limiting the present invention in any way. Example 1 Construction, expression, and purification of anti-PD-1 / CTLA-4 / VEGF trispecific antibody 1.1 Triabody Sequence and Structure A trispecific antibody capable of recognizing PD-1, CTLA-4, and VEGF was constructed for this application based on an anti-PD-1 antibody, an anti-CTLA-4 antibody, and an anti-VEGF antibody. The anti-PD-1 antibody is the in-house developed antibody CQ1-3 / 1-11; the anti-CTLA-4 antibody is the disclosed nanobody 202F1 (Application No. CN202111229808.3, SEQ ID NO: 9) or ipilimumab, which blocks CD80 binding to CTLA-4; and the anti-VEGF antibody is bevacizumab, which blocks VEGF binding to VEGFR-2 (KDR). The variable region sequences of each monoclonal antibody are listed in Table 1. In the trispecific antibody, the anti-PD-1 antibody and the anti-VEGF antibody can be in the form of Fab or scFv, and the corresponding sequences are listed in Table 1. Antibody fragments can be linked using a linker sequence commonly used in the art, for example, a (G4S)4(GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9)) linker. The constant region sequences of human hIgG1 or hIgG4 disclosed in the prior art can be used to construct the trispecific antibodies of the present application. For example, the constant region sequences of the trispecific antibodies are the hIgG1 or hIgG4(S228P) sequences shown in Table 2. The structures and sequences of the constructed trispecific antibodies are shown in Table 3.

[0101] [Table 1-1]

[0102] [Table 1-2]

[0103] Table 1-3

[0104]

Table 2

[0105] Table 3-1

[0106] Table 3-2

[0107] Table 3-3

[0108] Table 3-4

[0109] Table 3-5

[0110] Table 3-6

[0111] Table 3-7

[0112] Table 3-8

[0113] Table 3-9

[0114] [Table 3-10]

[0115] [Table 3-11]

[0116] [Table 3-12]

[0117] [Table 3-13]

[0118] [Table 3-14]

[0119] [Table 3-15]

[0120] [Table 3-16]

[0121] [Table 3-17]

[0122] [Table 3-18]

[0123] 1.2 Expression and purification of trispecific antibodies According to the sequences of the trispecific antibodies in Table 3, polynucleotides with the corresponding sequences were synthesized and inserted into the expression vector pCDNA3.1. After the accuracy was confirmed by sequencing, expression was carried out. The specific protocol was as follows: Cell density 3 × 10 6 200 mL of expiCHO cells (Gibco, catalog no. A29129) at 100 μg / mL were co-transfected with 100 μg each of the light and heavy chain expression plasmids encoding the corresponding trispecific antibodies shown in Table 3 and cultured at 37°C in a carbon dioxide incubator for 7 days. The supernatant was collected by centrifugation and filtered through a 0.22 μM filter membrane. The filtered supernatant was transferred to a Protein A chromatography column containing 2 mL of Protein A chromatography packing MabSelect PrismA (Cytiva, catalog no. 17549802) for affinity chromatography, and the column was pre-equilibrated with 5 column volumes of PBS. The sample retention time was at least 2 minutes. The column was then washed with 10-15 column volumes of PBS to remove nonspecifically adsorbed impurities. The target protein was eluted with 4 mL of 20 mM sodium citrate, pH 3.2, elution solution. Finally, the pH of the eluent was adjusted to 5.0-6.0 with 1 M Tris. After centrifugal filtration, the protein concentration was detected by UV spectrophotometry. The relative expression level was calculated based on the concentration. Protein purity was detected by size-exclusion chromatography coupled with high-performance liquid chromatography (Dionex, Ultimate 3000). The corresponding expression levels and purity of each detected trispecific antibody are shown in Table 4.

[0124] The control antibody used in this application is constructed as follows and purified according to the above method. Adjusting the corresponding parameters of specific expression and purification for a particular antibody is within the common knowledge of a person skilled in the art.

[0125] The heavy chain variable region of the anti-PD-1 antibody (CQ1-3 / 1-11) shown in Table 1 was fused to the hIgG4-1 constant region sequence shown in Table 2 to obtain a full-length heavy chain, and the light chain variable region of CQ1-3 / 1-11 was fused to the light chain constant region sequence shown in Table 2 to obtain a full-length light chain. The resulting full-length antibody was named CQ1-3 / 1-11-hIgG4 antibody, or CQ1-3 / 1-11 for short.

[0126] The heavy chain variable region of the anti-CTLA-4 antibody (202F1) shown in Table 1 was fused to the hIgG4-1 constant region sequence shown in Table 2 to obtain a full-length heavy chain, and the light chain variable region of 202F1 was fused to the light chain constant region sequence shown in Table 2 to obtain a full-length light chain. The full-length antibody thus obtained was designated 202F1-hIgG4 antibody.

[0127] The heavy chain variable region of antibody 202F1 shown in Table 1 was fused to the hIgG1 constant region sequence shown in Table 2 and assembled with the corresponding full-length light chain described above to obtain a full-length antibody designated 202F1-hIgG1.

[0128] The variable regions of ipilimumab shown in Table 1 were fused to the hIgG1 constant region sequences shown in Table 2 and assembled with the corresponding full-length light chain to yield ipilimumab. The variable regions of bevacizumab shown in Table 1 were fused to the hIgG1 constant region sequences shown in Table 2 and assembled with the corresponding full-length light chain to yield bevacizumab.

[0129] For the corresponding scFv of each antibody used in the present application, the corresponding expression and purification was carried out by the above-described methods according to the sequences disclosed in Table 1. Adjusting the corresponding parameters of specific expression and purification for a particular antibody is within the routine knowledge of a person skilled in the art.

[0130] [Table 4]

[0131] Example 2 Analysis of the binding activity and physicochemical properties of anti-PD-1 / CTLA-4 / VEGF trispecific antibodies 2.1 Activity of trispecific antibodies that bind to PD-1 Enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) were used to detect the activity of the trispecific antibody to bind to PD-1 protein and PD-1 antigen on the cell surface, respectively.

[0132] ELISA was used to determine the activity of the trispecific antibody in binding to PD-1 protein: 1 μg / mL human PD-1 protein (Acro, catalog no. PD1-H5221) was used to coat a high-binding 96-well ELISA plate and incubated overnight at 4°C. The following day, the plate was washed three times with PBST and blocked with 1% BSA-PBS at 37°C for 1 hour. Simultaneously, a 3-fold gradient dilution of the antibody was prepared in 1% BSA-PBS at a starting concentration of 10 nM. Each gradient antibody concentration was added in triplicate to the blocked 96-well ELISA plate at 100 μL / well and incubated at 37°C for 1 hour. The plate was then washed three times with 0.05% PBST, and 100 μL / well of a 1:5000 diluted HRP-conjugated secondary antibody (Sigma, catalog no. A0170) was added to the ELISA plate and incubated at 37°C for 1 hour. The plate was washed three times with 0.05% PBST and developed with TMB. The absorbance of the 96-well ELISA plate at 450 nm was detected using an ELISA reader. 450 The values ​​were fitted using a four-parameter model to determine the EC values ​​of each antibody binding to PD-1. 50 The values ​​were calculated and the results are shown in Table 5.

[0133] FACS was used to detect the activity of the trispecific antibody binding to cell surface PD-1 antigen: 293T cells overexpressing PD-1 (293T-hPD-1, Kangyuan Bochuang, KC-0204) were digested with trypsin (Gibco, 25200072), and the cells were washed twice with PBS containing 2% fetal bovine serum (FBS) (FACS buffer) and resuspended in FACS buffer at approximately 1 × 10 5 Cells / well were added to a 96-well plate. After centrifugation at 300g for 5 minutes, the supernatant was discarded. The antibody was serially diluted 3-fold in FACS buffer to obtain two gradient series, FACS#1 and FACS#2, with starting concentrations of 200 nM (Table 5, FACS#1) or 100 nM (Table 5, FACS#2, Figure 2), respectively. 100 μL / well was then added in triplicate to the 96-well plate, mixed, and incubated for 60 minutes at 4°C. The plate was centrifuged and washed three times with FACS buffer. 100 μL / well of a 1:800 diluted AF647-anti-hIgG(H+L) (Jackson, catalog number 109-605-003) was added and incubated for 50 minutes at 4°C. After washing the plate three times with FACS buffer, 100 μL / well of FACS buffer was added to resuspend the cells, and the fluorescent signal was detected on a flow cytometer (BECKMAN COULTER cytoFLEX). The binding activity was fitted using a four-parameter model to determine the EC of each antibody binding to PD-1. 50 The values ​​were calculated and the results are shown in Table 5 and Figure 2.

[0134] To compare the performance of the in-house developed antibody CQ1-3 / 1-11 with the corresponding commercially available antibody, the applicant used the same method to detect the binding activity of the antibody CQ1-3 / 1-11-hIgG4 and the antibody pembrolizumab to the cell surface PD-1 antigen. The results showed that the in-house developed antibody CQ1-3 / 1-11 and pembrolizumab had EC values ​​of 4.34 nM and 5.05 nM, respectively. 50 These antibodies were shown to have comparable ability to bind to cell surface PD-1.

[0135] [Table 5]

[0136] 2.2 Binding activity of trispecific antibodies to CTLA-4 The binding activity of the trispecific antibody to CTLA-4 protein and cell surface CTLA-4 antigen was detected by ELISA and FACS methods, respectively, as disclosed in Section 2.1.

[0137] The human CTLA-4 protein used was purchased from Acro, catalog number CT4-H52H9. The results are shown in Table 5. For the FACS assay, CHOK1 cells overexpressing human CTLA-4 (CHO-hCTLA-4, Genomeditech, GM-C18989) were used, and approximately 2 × 10 5 Cells / well were added to a 96-well U-bottom plate. Antibodies were serially diluted 3-fold in FACS buffer, starting at 200 nM. The results are shown in Table 5 and Figure 3.

[0138] 2.3 Binding activity of trispecific antibodies to VEGFA The binding activity of the trispecific antibody to VEGFA protein was detected by the ELISA method described in Section 2.1. The human VEGF165-his protein used was purchased from Acro, catalog number VE5-H5248. The positive control antibody bevacizumab was purchased from Roche, catalog number 0210008H1545. The results are shown in Table 5.

[0139] 2.4 Trispecific antibody binding activity to activated T cells PD-1 and CTLA-4 expression are upregulated on the surface of activated T cells. Human peripheral blood T cells were activated with anti-CD3 and anti-CD28 antibodies to detect the binding activity of the trispecific antibody with activated T cells. The anti-PD-1 antibodies pembrolizumab (Merck, Catalog No. S028905), nivolumab (Biointron, Catalog No. B6924), sintilimab (Biointron, Catalog No. B682101), and camrelizumab (Biointron, Catalog No. B852001) and the anti-CTLA-4 antibody ipilimumab were used as positive controls.

[0140] Human peripheral blood mononuclear cells (PBMCs, Miaoshun (Shanghai) Biotechnology Co., Ltd. Catalog No. A19K154025) were rapidly thawed in a 37°C water bath, resuspended in T cell isolation solution, and counted. After centrifugation at 400g for 10 min, the supernatant was removed and the cells were diluted to 5 × 10 in T cell isolation buffer. 7 T cells were isolated according to the procedure in the T cell isolation kit (Stemcell, Cat. No. 19051), and the resulting T cells were resuspended in culture medium. Cells were counted using a NucleoCounter NC-200, and the T cell density was approximately 1 × 10 6 The activated T cells were adjusted to 1 × 10 cells / mL. Dynabeads containing anti-CD3 and anti-CD28 antibodies (Invitrogen™, Cat. No. 11132D) and 10 ng / mL hIL-2 (Peprotech, Cat. No. 200-02) were added and cultured at 37°C in a 5% CO2 incubator for 3 days. After removing the Dynabeads and supernatant, the activated T cells were resuspended in FACS buffer. The cell density was approximately 1 × 10 6 100 μL of cell solution is adjusted to 1 x 10 cells / mL. 5Cells were added to a 96-well U-bottom plate at 100 μL / well. After centrifugation at 500 g for 5 minutes, the supernatant was discarded, and 100 μL of the trispecific antibody solution of the present application, serially diluted 3-fold with FACS buffer, was added in triplicate to a starting concentration of 200 nM. After mixing, the plate was incubated at 4°C for 60 minutes. After centrifugation and washing three times with FACS buffer, 100 μL / well of PE anti-human IgG Fc (Biolegend, catalog no. 366903) was added and incubated at 4°C for 50 minutes. After washing the plate three times with FACS buffer, 100 μL / well of BV421 anti-human CD3 antibody (OKT3) (Biolegend, catalog no. 317344) was added and incubated at 4°C for 50 minutes. After washing the plate three times with FACS buffer, 70 μL / well of PBS was added to resuspend the cells, and the fluorescent signal on CD3 T cells was detected on a flow cytometer (BD Cellesta). The avidity was fitted using a four-parameter model to determine the EC of each antibody binding to T cells. 50 values ​​were calculated.

[0141] As shown in Figure 4 and Table 6, the commercially available anti-PD-1 antibodies pembrolizumab, nivolumab, sintilimab, and karelizumab can bind to activated T cells in a dose-dependent manner. Furthermore, the commercially available anti-CTLA-4 antibody ipilimumab can also bind to activated T cells in a dose-dependent manner, indicating that T cells activated by the method of this example express PD-1 and CTLA-4 on their surface. Because the mean fluorescence intensity (MFI) of ipilimumab binding to cell surface CTLA-4 is lower than the MFI of anti-PD-1 antibodies binding to cell surface PD-1, this may be related to the low expression of CTLA-4 on the surface of activated T cells.

[0142] As shown in Figure 4 and Table 6, both the trispecific antibodies HC010-F8 and HC010-F23 prepared in this application can bind to activated T cells in a dose-dependent manner, and their binding ability is superior to that of the anti-CTLA-4 antibodies ipilimumab and 202F1.

[0143] [Table 6]

[0144] 2.5 Simultaneous binding activity of trispecific antibodies to PD-1, CTLA-4, and VEGFA 2.5.1 Binding of Trispecific Antibodies to CTLA-4 or VEGFA After Binding to PD-1 Protein A high-binding 96-well ELISA plate was coated with 1 μg / mL human PD1-his (Acro, catalog no. PD1-H5221) and incubated overnight at 4°C. The plate was washed with PBST and then blocked with 300 μL / well of 1% BSA-PBS for 2 hours at 37°C. Simultaneously, 3-fold serial dilutions of the antibody were prepared with 1% BSA-PBS starting at 20 nM. After washing the plate, 100 μL of each gradient antibody solution was added to each well in triplicate and incubated at 37°C for 1 hour. The ELISA plate was then mechanically washed three times with 0.05% PBST. One set of wells was incubated with 100 μL / well of 1 μg / mL biotinylated hCTLA4 (Acro, catalog no. CT4-H82E3) to assess the binding activity of the trispecific antibody to CTLA-4 after binding to PD-1 protein. Another set was incubated with 100 μL / well of 1 μg / mL biotinylated hVEGF165 (Acro, catalog no. VE5-H82Q0) to evaluate the binding activity of the trispecific antibody to VEGF after binding to PD-1 protein. The final set was incubated with hIgG4 as a negative control. After 1 hour of incubation at 37°C, the ELISA plate was washed three times with PBST, and 100 μL / well of 1:5000 diluted streptavidin-HRP (BD pharmingen, catalog no. 554066) was added to the plate and incubated for 45 minutes at 37°C. The ELISA plate was washed three times with PBST, and TMB was used for color development. The absorbance of the 96-well ELISA plate at 450 nm was detected using an ELISA reader, and the EC values ​​of the antibodies were determined. 50 Values ​​were calculated by fitting using a four-parameter method.

[0145] The results are shown in Figure 13. After binding to human PD1, trispecific antibodies HC010-F8 and HC010-F23 still bind to human VEGF (Figure 13A, EC 50 values ​​were 0.049 and 0.054 nM, respectively) or human CTLA-4 (Figure 13B, EC 50The binding activity was dose-dependent (the values ​​were 0.105 and 0.250 nM, respectively).

[0146] 2.5.2 Trispecific Antibodies that Simultaneously Bind CTLA-4 and VEGFA After Binding to Cell Surface PD-1 Protein 293T-hPD-1 cells were obtained by trypsin digestion and washed twice with PBS containing 2% FBS (FACS buffer), at a cell density of approximately 6 × 10 5 The antibody solution was adjusted to 100 cells / mL. 200 μL / well of cells was added to a 96-well U-bottom plate. After centrifugation at 300×g for 5 minutes, the supernatant was discarded, and 100 μL of serially diluted antibody solution in FACS buffer was added to each well in triplicate (starting concentration 200 nM, 3-fold serial dilutions, and the same volume of FACS buffer was used as a negative control). The wells were mixed and incubated for 60 minutes at 4°C. After washing the plate three times with FACS buffer, a mixture consisting of 1 μg / mL biotin-hVEGF165 and 1 μg / mL hCTLA4-mFc (DIMA BIOTECH, catalog no. PME100017) in a 1:1 volume ratio was added to each well at 100 μL / well to detect the simultaneous binding activity of the trispecific antibody to CTLA-4 and VEGF after binding to cell surface PD-1. After 60 minutes of incubation at 4°C, the cells were washed three times with FACS buffer, and a mixture of AF647 donkey anti-mIgG(H+L) (1:800 dilution, Invitrogen, Cat. No. A31571) and PE-streptavidin (1:800 dilution, BD, Cat. No. 554061) was added to each well at 100 μL / well and incubated at 4°C for 50 minutes. After washing the plate three times with FACS buffer, 100 μL / well of PBS was added to resuspend the cells, and the fluorescent signal was detected on a flow cytometer (BECKMAN COULTER cytoFLEX). Binding activity was fitted using a four-parameter model to determine the EC of each antibody. 50 The value was calculated.

[0147] The results are shown in Figure 14. After binding to human PD-1 on the cell surface, trispecific antibodies HC010-F8, HC010-F10, HC010-F22, HC010-F23, and HC010-F24 still inhibited human VEGF (Figure 14A, EC 50 The values ​​were 1.413, 2.835, 2.288, 2.223, and 9.151 nM, respectively.) and human CTLA-4 (Figure 14AB, EC 50 The values ​​were 2.744, 3.323, 5.196, 3.837 and 12,000 nM, respectively. ) and the simultaneous binding activity was dose-dependent.

[0148] 2.5.3 Trispecific Antibodies that Bind to Cell Surface CTLA-4 Protein and Then Simultaneously Bind to PD-1 and VEGFA CHOK1-hCTLA4 cells were obtained by trypsin digestion and washed twice with PBS containing 2% FBS (FACS buffer), at a cell density of approximately 8 × 10 5The antibody solution was adjusted to 100 cells / ml. 200 μL / well of cells was added to a 96-well U-bottom plate. After centrifugation at 300×g for 5 minutes, the supernatant was discarded, and 100 μL of serially diluted antibody solution in FACS buffer was added in triplicate (starting concentration 200 nM, 3-fold serial dilutions, and the same volume of FACS buffer was used as a negative control). The wells were mixed and incubated for 60 minutes at 4°C. After washing the plate three times with FACS buffer, a mixture consisting of 1 μg / mL hPD1-mFc (Acro, catalog no. PD1-H5255) and 1 μg / mL biotin-hVEGF165 in a 1:1 volume ratio was added to each well at 100 μL / well to detect the simultaneous binding activity of the trispecific antibody to VEGFA and PD-1 after binding to cell surface CTLA-4. After incubation at 4°C for 60 minutes, the cells were washed three times with FACS buffer, and a 1:1 volumetric mixture of AF647 donkey anti-mIgG(H+L) (1:800 dilution) and PE streptavidin (1:800 dilution) was added to each well at 100 μL / well and incubated at 4°C for 50 minutes. After washing the plate three times with FACS buffer, 100 μL / well of PBS was added to resuspend the cells, and the fluorescent signal was detected by flow cytometry. The binding activity was fitted using a four-parameter model to determine the EC of each antibody. 50 The value was calculated.

[0149] The results are shown in Figure 15. After binding to human CTLA-4 on the cell surface, trispecific antibodies HC010-F8, HC010-F9, and HC010-F10 still bind to human PD-1 (Figure 15A, EC 50 The values ​​were 1.919, 1.533, and 2.734 nM, respectively.) and human VEGF (Figure 15B, EC 50 The values ​​were 1.435, 1.376 and 1.370 nM, respectively. ) and the simultaneous binding activity was dose-dependent.

[0150] 2.6 Binding activity of the trispecific antibody to free VEGFA after binding to cells co-expressing PD-1 and CTLA-4 In the tumor microenvironment, the trispecific antibody can simultaneously block immunosuppression and angiogenesis by binding to PD-1 and CTLA-4 on immune cells and VEGFA, thereby regulating immune responses and angiogenesis in the tumor microenvironment. In this example, the activity of the trispecific antibody to bind to free VEGFA after binding to cells co-expressing PD-1 and CTLA-4 was separately detected using 293T cells and activated T cells overexpressing PD-1 and CTLA-4.

[0151] 2.6.1 Trispecific Antibody Binding to VEGFA After Binding to Cells Co-Expressing PD-1 and CTLA-4 293T cells overexpressing human PD-1 and human CTLA-4 (293T-hPD-1-hCTLA-4, Genomeditech, GM-C19526) were used to detect the binding activity of the trispecific antibody to VEGFA after binding to cells co-expressing PD-1 and CTLA-4. The expression level of PD-1 on 293T-hPD-1-hCTLA-4 cells was higher than that of CTLA-4. After binding to 293T-hPD-1-hCTLA-4 cells via the PD-1 and CTLA-4 arms, the trispecific antibody binding to 293T-hPD-1-hCTLA-4 cells was detected using biotin-labeled VEGFA. 293T-hPD-1-hCTLA-4 cells were digested with trypsin, and the detached cells were resuspended in FACS buffer at a cell density of approximately 1 × 10 6 Then, 200 μL / well of cells was adjusted to approximately 2 x 10 cells / mL. 5Cells were added to a 96-well U-bottom plate at a cell density of 100 cells / well. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and 100 μL of a 3-fold serially diluted trispecific antibody solution in FACS buffer was added to a starting concentration of 200 nM. The same volume of FACS buffer was added as a negative control. After mixing, the plate was incubated for 60 minutes at 4°C. After centrifugation and washing three times with FACS buffer, 100 μL / well of 2 μg / mL biotin-hVEGF165 was added and incubated for 60 minutes at 4°C. After centrifugation and washing three times with FACS buffer, 100 μL / well of 1:800 diluted PE streptavidin (BD, catalog no. 554061) was added and incubated for 50 minutes at 4°C. After centrifugation and washing three times with FACS buffer, 100 μL / well of FACS buffer was added to resuspend the cells, and the fluorescent signal was detected on a flow cytometer (BECKMAN COULTER cytoFLEX). The binding activity was fitted using a four-parameter model to obtain the EC of each antibody binding. 50 The value was calculated.

[0152] As shown in Figure 5, the trispecific antibodies HC010-F8, HC010-F9, and HC010-F10 are still able to bind to free VEGFA after binding to cells co-expressing PD-1 and CTLA-4. The EC 50 The values ​​were 5.057, 4.204, and 4.904 nM, respectively, indicating that the trispecific antibody still had high binding activity to VEGFA after binding to cells co-expressing PD-1 and CTLA-4.

[0153] 2.6.2 Trispecific Antibody Binding Activity to Free VEGFA After Binding to Activated T Cells The activated T cells from section 2.4 were used, with a cell density of 1 x 10 6 100 μL of cells were added to a 96-well U-bottom plate at 1 × 10 cells / mL. 5After centrifugation at 500g for 5 minutes, the supernatant was discarded, and 100 μL of a 3-fold serially diluted antibody solution in FACS buffer was added in triplicate to a starting concentration of 200 nM. After mixing, the plate was incubated for 60 minutes at 4°C. After centrifugation and washing three times with FACS buffer, 2 μg / mL biotinylated human VEGF165 (Acro, catalog no. VE5-H82Q0) was added at 100 μL / well and incubated for 50 minutes at 4°C. After washing three times with FACS buffer, a mixture of BV421 anti-human CD3 antibody (OKT3) (Biolegend, catalog no. 317344) and PE-streptavidin was added at 100 μL / well and incubated for 50 minutes at 4°C. After washing three times with FACS buffer, 70 μL / well of PBS was added to resuspend the cells, and the fluorescent signal on CD3 T cells was detected on a flow cytometer (BDCellesta). The binding activity was fitted using a four-parameter model, and the EC 50 The value was calculated.

[0154] As shown in Figure 6, the trispecific antibodies HC010-F8 and HC010-F23 are still able to bind to free VEGFA after binding to activated T cells. The EC 50 The values ​​were 0.440 and 0.377 nM, respectively, indicating that the trispecific antibody still had high binding activity to VEGFA after binding to T cells co-expressing PD-1 and CTLA-4, enabling simultaneous blockade of immune checkpoints and angiogenesis in the tumor microenvironment.

[0155] 2.7 Determination of Trispecific Antibody Affinity for Human PD-1, CTLA-4, and VEGFA The affinity of the trispecific antibody for human PD-1, CTLA-4, and VEGFA was detected using Octet® RED96e (ForteBio). The specific method was as follows: the AHC sensor was activated by immersing it in PBST (PBS containing 0.02% Tween) buffer for 10 minutes. The ligand (antibody to be tested) was diluted to 5 μg / mL in PBST buffer. The analytes: human PD1 (ACRO, catalog no. PD1-H5221), human VEGF (ACRO, catalog no. VE5-H4210), and human CTLA4 (ACRO, catalog no. CT4-H52H9) were diluted in PBST buffer to a concentration gradient of 100, 50, 25, 12.5, and 6.25 nM, with the intermediate concentration set as a quality control point and 0 nM set as a blank control point. The diluted ligand and analyte were added to the corresponding wells at 200 μL / well in triplicate. The program parameters were: Loading: 180 s, Association: 180 s, Dissociation: 600 s, Regeneration: Gly-HCl pH 1.5, 30 s, Regeneration 3 times. Data Analysis 11.1r software was used to fit and analyze the curves using a one-to-one model after assuming a blank (concentration point 0) to calculate the experimental results. Curve fit acceptance criteria: Full R^2 ≥ 0.95.

[0156] The results are shown in Table 7. HC010-F8 and HC010-F23 have high affinity for human PD-1, human VEGF, and human CTLA-4, respectively, and are 10 -9 reaches the digits.

[0157] [Table 7]

[0158] Example 3 Blocking activity of anti-PD-1 / CTLA-4 / VEGF trispecific antibodies 3.1 Activity of trispecific antibodies blocking the binding of PD-1 and PD-L1 Competitive ELISA and FACS were used to detect the activity of the trispecific antibody in blocking the binding of PD-1 and PD-L1.

[0159] The activity of the trispecific antibody in blocking the binding of PD-1 and PD-L1 was detected by ELISA. A high-binding 96-well ELISA plate was coated with 1 μg / mL human PD-1 protein (Acro, PD1-H5257) and incubated overnight at 4°C. The following day, the plate was washed three times with PBS and blocked with 1% BSA-PBS at 37°C for 1 hour. A 1:1 mixture of serial antibody dilutions (starting concentration 200 nM, 3-fold serial dilutions) prepared in 1% BSA-PBST diluent and 1 μg / mL biotin-labeled PD-L1 protein (Acro, PD1-H82F3) was mixed in a 1:1 volume ratio to obtain an antibody-protein mixture. 100 μL of the antibody-protein mixture was added to each well of the blocked 96-well ELISA plate and incubated at 37°C for 1 hour. After washing four times with PBST, 100 μL of Strep-HRP (BD, Cat. No. 554066) diluted 1:5000 was added to each well of the ELISA plate and incubated at 37°C for 1 hour. After washing four times with PBST, TMB was used for color development. The absorbance at 450 nm and 630 nm was detected by a microplate reader, and the OD450-OD630 was calculated. The fluorescence intensity was fitted using a four-parameter model to determine the IC of blocking activity of each antibody. 50 The values ​​were calculated and the results are shown in Table 8.

[0160] To compare the performance of the in-house developed antibody CQ1-3 / 1-11 with pembrolizumab, the applicant used the same method to detect the activity of the control in-house developed antibody CQ1-3 / 1-11-hIgG4 in blocking the binding of the cell surface antigen PD-1 to PD-L1. The results showed that the in-house developed antibody CQ1-3 / 1-11 and pembrolizumab had comparable blocking abilities, and the IC 50 The values ​​are shown to be 3.190 nM and 3.569 nM, respectively.

[0161] The activity of the trispecific antibody in blocking the binding of PD-1 to PD-L1 was detected by FACS using 293T cells overexpressing human PD-1 (293T-hPD-1, Kangyuan Bochuang, KC-0204). 293T-hPD-1 cells were obtained by digestion with trypsin, washed twice with PBS containing 2% FBS (FACS buffer), and resuspended in FACS buffer. 1 μg / mL biotin-human PDL1 (Acro, catalog no. PD1-H82F3) was added to the suspended cell solution, and 50 μL / well of cells was cultured at approximately 1 × 10 5 Cells were added to a 96-well U-bottom plate at 100 μL / well. 50 μL of antibody solution, serially diluted 3-fold in FACS buffer, was added in triplicate, with a starting concentration of 200 nM. After mixing, the mixture was incubated at 4°C for 60 minutes. After three washes with FACS buffer, 100 μL / well of a 1:800 dilution of PE-streptavidin was added and incubated at 4°C for 50 minutes. After three washes with FACS buffer, 150 μL / well of PBS was added to resuspend the cells, and the fluorescent signal was detected on a flow cytometer (BECKMAN COULTER cytoFLEX). A four-parameter model was used to fit the binding activity, and the IC of blocking activity of each antibody was calculated. 50 The values ​​were calculated, and the results are shown in Table 8. The trispecific antibody can effectively block the binding of PD-L1 to PD-1 on the surface of 293T cells.

[0162] [Table 8]

[0163] 3.2 Activity of trispecific antibodies blocking CD80 and CTLA-4 binding 3.2.1 Trispecific antibody activity blocking CD80 and CTLA-4 binding CHO-hCTLA-4 cells were used to detect the ability of the trispecific antibody to block binding of human CTLA-4 to human CD80. CHO-hCTLA-4 cells were obtained by trypsin digestion, washed twice with PBS containing 2% FBS (FACS buffer), and resuspended in FACS buffer. 200 μL of cells contained approximately 2 × 10 5 Cells were added to a 96-well U-bottom plate at 100 μL / well. After centrifugation at 300×g for 5 minutes, the supernatant was discarded. A series of antibody gradient dilutions (starting at 200 nM, 3-fold serial dilutions) and 0.6 μg / mL biotin-labeled human CD80 protein (Acro, catalog no. B71-H82F2) were prepared using FACS buffer. 50 μL of antibody and 50 μL of biotin-human CD80 were added to each well of the 96-well plate, mixed well, and incubated for 60 minutes at 4°C. After three washes with FACS buffer, 100 μL / well of a 1:800 dilution of PE streptavidin was added and incubated for 50 minutes at 4°C. After three washes with FACS buffer, 150 μL / well of PBS was added to resuspend the cells, and the fluorescent signal was detected on a flow cytometer (Beckman Coulter cytoFLEX). Fit the binding activity using a four-parameter model and calculate the IC of the blocking activity of each antibody. 50 The values ​​were calculated, and the results are shown in Table 8. The trispecific antibody can effectively block the binding of human CTLA-4 to human CD80.

[0164] 3.2.2 Activity of Trispecific Antibodies to Block CD80 Binding to CTLA-4 on Cells Co-Expressing PD-1 and CTLA-4 PD-1 and CTLA-4 are co-expressed on functionally exhausted T cells in the tumor microenvironment, with PD-1 expression levels higher than CTLA-4. Bispecific antibodies that simultaneously target PD-1 and CTLA-4 can enhance the blocking function of anti-CTLA-4 antibodies on cells co-expressing PD-1 and CTLA-4. We used 293T cells co-overexpressing human PD-1 and human CTLA-4 (293T-hPD-1-hCTLA-4, Genomeditech, GM-C19526, also known as 293T-CTLA-4-PD-1 cells) to detect the ability of the trispecific antibody to block the binding of human CTLA-4 to human CD80. 293T-hPD-1-hCTLA-4 cells were obtained by trypsin digestion, washed twice with PBS containing 2% FBS (FACS buffer), and then resuspended in FACS buffer. 200 μL of cells contains approximately 1.4 x 10 5 Cells were added to a 96-well U-bottom plate at 100 μL / well. After centrifugation at 300×g for 5 minutes, the supernatant was discarded. A series of antibody gradient dilutions (starting at 400 nM, 3-fold serial dilutions) and 2 μg / mL biotin-labeled human CD80 protein were prepared using FACS buffer. 50 μL of antibody and 50 μL of biotin-human CD80 were added to each well of the 96-well plate, mixed well, and incubated at 4°C for 60 minutes. After three washes with FACS buffer, 100 μL / well of a 1:800 dilution of PE streptavidin was added and incubated at 4°C for 50 minutes. After three washes with FACS buffer, 150 μL / well of PBS was added to resuspend the cells, and the fluorescent signal was detected on a flow cytometer (BECKMAN COULTER cytoFLEX). Binding activity was fitted using a four-parameter model to determine the IC value of the blocking activity of each antibody. 50 The value was calculated.

[0165] The results are shown in Table 8. Compared to the parental antibody, the trispecific antibody significantly enhanced the blocking effect on CD80 binding to CTLA-4 on 293T-hPD-1-hCTLA-4 cells.

[0166] 3.3 Activity of trispecific antibodies blocking the binding of VEGFA and KDR (VEGFR2) 3.3.1. The activity of the trispecific antibody to block the binding of VEGFA and KDR (VEGFR2) was detected by competitive ELISA A high-binding 96-well ELISA plate was coated with 1 μg / mL human VEGF165-His (Acro, catalog no. VE5-H5248) and incubated for 2 hours at 37°C. After washing, each well was blocked with 300 μL of 1% BSA-PBS for 1.5 hours at 37°C. 50 μL of serially diluted antibody solution (starting concentration was 400 nM, 3-fold serial dilutions) and 50 μL of 1.72 μg / mL biotin-human KDR (Acro, catalog no. KDR-H82E5) were added to each well of the ELISA plate and incubated for 1 hour at 37°C. After washing three times with PBST, 100 μL / well of a 1:5000 diluted streptavidin-HRP (BD pharmingen, catalog no. 554066) was added to the plate and incubated for 45 minutes at 37°C. After washing three times with PBST, TMB was used for color development, and the absorbance at 450 nm was detected by an enzyme reader and fitted with a four-parameter model. The IC of blocking activity of the antibody was calculated. 50 Values ​​were calculated, and the results are shown in Table 8. The activity of the trispecific antibody in blocking the binding of VEGFA to KDR was comparable to that of bevacizumab.

[0167] 3.3.2 Detection of the Blocking Activity of Trispecific Antibodies against VEGF Binding to KDR by Reporter Gene Assay The blocking activity of the trispecific antibody against the binding of human VEGFR and human KDR was detected using the human VEGFR2-293 reporter cell line (Genomeditech, GM-C09057). The human VEGFR2-293 reporter cell line is a luciferase reporter gene cell line constructed based on the NFAT signaling pathway. After binding to the KDR receptor, VEGF activates the NFAT signaling pathway, thereby activating luciferase expression.

[0168] VEGFR2-293 reporter cells were obtained by trypsin digestion, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. After resuspending in DMEM medium containing 1% FBS (test medium), 0.6 μg / mL of hVEGF165-His was added to the cell suspension, which was then plated in approximately 1 × 10 wells of a 96-well plate with a clear bottom. 4 Three-fold serial dilutions of antibodies were prepared in assay medium (starting at 22.2 nM), added to a 96-well plate, mixed well, and incubated at 37°C for 6 hours. Luciferase buffer (Promega, Cat. No. G7940) was added and incubated in the dark for 5 minutes, and the fluorescent signal was detected on a microplate reader (CLARIOstar Plus).

[0169] The results are shown in Table 8. The trispecific antibody can effectively block the binding of human VEGF to human KDR on the cell surface, with a blocking efficiency comparable to that of the parent antibody bevacizumab.

[0170] Example 4 Function of trispecific antibodies in activating immune responses and suppressing VEGF-induced cell proliferation in vitro 4.1 Trispecific antibodies enhance T cell function 4.1.1. Trispecific antibodies enhance IL-2 secretion by T cells in SEB-stimulated PBMCs Staphylococcal enterotoxin B (SEB) is a superantigen that can activate large numbers of T cells at low concentrations, generating potent immune responses. This superantigen can directly bind to T cell receptors and MHC molecules without being processed into antigenic peptides that activate T cells. Anti-PD-1 and anti-CTLA-4 antibodies can promote SEB-stimulated T cells to express and secrete IL-2.

[0171] This example tested the effect of the trispecific antibody on IL-2 secretion using SEB-stimulated T cells, with irrelevant human anti-HEL hIgG4 (Taizhou Baiying Biotechnology Co., Ltd., Catalog No. B107804) used as a negative control. PBMCs were rapidly thawed in a 37°C water bath and resuspended in RPMI 1640 medium (Gibco, Catalog No. A10491-01) containing 10% FBS (fetal bovine serum, Gibco, Catalog No. 10091-148) and 1% P / S (Pen-Strep, Gibco, Catalog No. 15140122). The cell density of PBMCs was 1 x 10 6 The cells were adjusted to 100 cells / mL, and SEB (Toxin Technology, Cat. No. 92815B) was added to the cell suspension to a final concentration of 200 ng / mL. 100 μL / well of the cell suspension mixed with SEB was added to a flat-bottom 96-well plate. Antibodies and control antibodies, starting at 400 nM, were diluted 3-fold in culture medium and added in triplicate to the 96-well plate at 100 μL / well and mixed with the cells. The cells were cultured for 3 days in a 37°C, 5% CO2 incubator. After 3 days, the supernatant was collected, and IL-2 secretion was detected using a human IL-2 detection kit (CisBio, Cat. No. 62HIL02PEH) according to the manufacturer's instructions. Values ​​were read on a CLARIOstar Plus.

[0172] The results are shown in Figure 7, which shows that trispecific antibodies HC010-F8, HC010-F10, HC010-F22, HC010-F23 and HC010-F24 are all able to enhance the secretion of IL-2 by SEB-stimulated PBMCs.

[0173] 4.1.2. Trispecific antibodies enhance IL-2 secretion by T cells in the mixed lymphocyte reaction (MLR) When mature dendritic cells (DCs) from different donors are incubated with PBMCs, the DCs activate PBMCs (mainly T cells) to promote the expression and secretion of IL-2. PD-1 and CTLA-4 on T cells bind to PD-L1 / PD-L2 and CD80 / CD86, respectively, which are highly expressed on mature DCs, resulting in a decrease in IL-2 cytokine expression. Immune checkpoint inhibitors, such as anti-PD-1 antibodies, can promote cytokine secretion in MLR reactions.

[0174] This study examined the effect of the trispecific antibody on IL-2 secretion in MLR. DCs (Allcells) and PBMCs (Allcells) were rapidly thawed in a 37°C water bath and resuspended in X-VIVO15 (Lonza, Cat. No. 04-418Q) medium. The cell densities of DCs and PBMCs were 1 × 10, respectively. 5 cells / mL and 2 x 10 6 The concentration of DCs and PBMCs was adjusted to 1000 nM per mL. DCs and PBMCs were mixed at a 1:1 volume ratio. After mixing, 200 μL of the cell mixture was added to a round-bottom 96-well plate. The trispecific and control antibodies were diluted 5-fold with X-VIVO15 solution at a starting concentration of 1000 nM, and 50 μL per well was added to the 96-well plate in triplicate and mixed with the cells. The cells were cultured for 4 days in a 37°C, 5% CO2 incubator. After 4 days, the supernatant was collected, and IL-2 secretion was detected using a human IL-2 detection kit according to the manufacturer's instructions. Values ​​were read on a CLARIOstar Plus.

[0175] The results are shown in Figure 8. The trispecific antibodies HC010-F8, HC010-F10, HC010-F22, HC010-F23, and HC010-F24 were all able to enhance IL-2 secretion and expression in MLRs. The activity was superior to that of the parent anti-CTLA-4 antibody 202F1-hIgG4 and comparable to that of the anti-PD-1 antibody CQ1-3 / 1-11 and the combination of 202F1-hIgG4 and CQ1-3 / 1-11.

[0176] Using the same approach, the effects of antibodies CQ1-3 / 1-11 and pembrolizumab on IL-2 secretion were compared in an MLR, and the results showed that CQ1-3 / 1-11 and pembrolizumab had comparable activity.

[0177] 4.1.3. Trispecific antibodies enhance the activity of IL-2-secreting T cells in Treg-containing mixed lymphocyte reactions (MLRs) Blocking PD-1 activity on effector T cells can promote anti-tumor immunity. However, because Treg cells also express high levels of PD-1, blocking PD-1 activity on Tregs can increase the suppressive function of Tregs, thereby suppressing anti-tumor immunity. This may be related to the poor response of some cancer patients to anti-PD-1 therapy. Studies have shown that the combination of anti-PD-1 and anti-CTLA-4 antibodies or bispecific antibodies against PD-1 and CTLA-4 can increase T cell activity in the presence of Tregs. Therefore, in this example, MLRs containing different ratios of Tregs were used to detect the ability of trispecific antibodies to activate immune cells in the presence of Tregs.

[0178] Frozen PBMCs (Allcells), mature DCs (Allcells), and activated Tregs (Allcells) from three different healthy donors were thawed. PBMCs and DCs were cultured at a cell density of 2 × 10 in RPMI 1640 complete medium (Gibco, Cat. No. 61870-036). 6 cells / mL and 0.1 x 10 6 Treg cells were grown in RPMI 1640 complete medium at a cell density of 0.5 × 10 cells / mL and plated at 50 μL per well in a U-bottom 96-well plate. 6 adjusted to 0.25 x 10 cells / mL 6 , 0.125×10 6 and 0.0625 × 10 6Treg cells were serially diluted 2-fold to 1000 cells / mL, and 50 μL of each at different densities was placed into each well of the 96-well plate. Antibodies HC010-F8, HC010-F23, CQ1-3 / 1-11, 202F1, bevacizumab, the combination of CQ1-3 / 1-11 and 202F1-hIgG4, the combination of CQ1-3 / 1-11, 202F1-hIgG4, and bevacizumab, and an irrelevant isotype antibody were serially diluted 4-fold with RPMI 1640 complete medium starting at a concentration of 2000 nM. The serially diluted samples were then diluted 1:10 with culture medium and placed in triplicate in 50 μL per well of the 96-well plate. The samples were incubated at 37°C and 5% CO2 for 4 days. After 4 days, the supernatant was collected by centrifugation and IL-2 release was detected using hIL-2 HTRF (Cisbio, Cat. No. 62HIL02PEH) according to the manufacturer's instructions.

[0179] The results are shown in Figure 9. When a relatively high proportion of Tregs was present, IL-2 release in the MLR was suppressed. The trispecific antibodies HC010-F8 and HC010-F23 were able to increase IL-2 release, and the enhancing effect was essentially equivalent to that of the combination of CQ1-3 / 1-11 and 202F1-hIgG4, and the combination of bevacizumab, CQ1-3 / 1-11, and 202F1-hIgG4.

[0180] 4.2 Trispecific antibodies inhibit VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) Human umbilical vein endothelial cells (HUVECs) express VEGF receptors and are therefore induced to proliferate by VEGF. Human umbilical vein endothelial cells can be used to evaluate the inhibitory effect of anti-VEGF antibodies, such as bevacizumab, on VEGFA-induced angiogenesis. In this example, HUVECs (ATCC, CRL-1730) were also used to detect the inhibition of VEGF function by trispecific antibodies. HUVEC cells were obtained by trypsin digestion and resuspended in F-12K medium (Gibco, catalog number 21127-022) containing 10% FBS. The cell density was 1 × 10 550 μL of the cell solution was added to a 96-well clear-bottom plate, and approximately 5 × 10 cells / mL were added. 3 Cells / well. Serially diluted antibody solutions (starting concentration 200 nM, 3-fold serial dilutions) and 0.4 μg / mL human VEGFA165 protein were prepared in test medium. 25 μL of antibody and 25 μL of human VEGFA165 were added to each well of a 96-well plate, mixed well, and incubated at 37°C for 5 days. After 5 days, 60 μL / well of CellTiter-Glo (Promega, Cat. No. G7572) was added, and the fluorescent signal was detected on a microplate reader (CLARIOstar Plus). The IC of antibody inhibitory activity was calculated. 50 Values ​​were calculated using a four-parameter model.

[0181] As shown in Figure 10, trispecific antibodies HC010-F8 and HC010-F23 could effectively block VEGFA-induced HUVEC cell proliferation, and the blocking activity IC 50 The IC values ​​for blocking activity of the parent antibody bevacizumab were 0.860 and 0.828 nM, respectively. 50 was 0.367 nM. hIgG1 and hIgG4 were negative control antibodies unrelated to the antibodies of the present application.

[0182] Example 5 In vivo efficacy of trispecific antibodies 5.1 Trispecific antibody HC010-F8 induces long-term antitumor effects Human melanoma A375 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). When A375 cells reached a growth density of 60-80%, PBMCs (Allcells, catalog number FPB004F-C) were thawed and resuspended in RPMI 1640 medium at a cell density of 3 × 10 6 The PBMCs were adjusted to 5 × 10 cells / mL and co-cultured with mitomycin C-treated A375 cells. After 5 days of co-culture with A375 cells, freshly digested PBMCs and A375 cells were collected. PBMCs (5 × 10 5cells / mouse) and A375 cells (4 × 10 6 Cells (cells / mouse) were inoculated subcutaneously into the right flank of NCG female mice (purchased from GemPharmatech Co. Ltd.) in a seeding volume of 0.2 mL / mouse containing 50% Matrigel (BD, Cat. No. 354234). On the day of cell inoculation, mice were randomly divided into four groups (5 mice per group) and subcutaneously injected twice weekly for 3 weeks with the trispecific antibody HC010-F8 (1 and 5 mg / kg), the three monoclonal antibodies CQ1-3 / 1-11 (0.65 mg / kg), ipilimumab (0.35 mg / kg) and bevacizumab (0.65 mg / kg), or a negative control (PBS). Tumor volume was measured twice weekly using a Vernier caliper and calculated using the formula V = 0.5 × a × b 2 where a and b represent the longest and widest diameters of the tumor, respectively.

[0183] As shown in Figure 11, antibody HC010-F8 effectively suppressed tumor growth at both 1 mg / kg and 5 mg / kg. After drug administration was discontinued, the two treatment groups (1 and 5 mg / kg) and the combination group with antibody HC010-F8 all delayed tumor recurrence compared to the negative control group, and the tumor-suppressing effect of HC010-F8 (1 and 5 mg / kg) was superior to that of the combination groups of CQ1-3 / 1-11, ipilimumab, and bevacizumab.

[0184] In this example, mice in all treatment groups showed neither abnormal behavior nor weight loss (FIG. 12), indicating that tumor-bearing mice tolerated the drug well at the doses tested. 5.2 Antitumor effects induced by trispecific antibodies HC010-F8 and HC010-F23 Human melanoma A375 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). When the A375 cells reached a growth density of 60–80%, PBMCs were thawed and then resuspended in RPMI 1640 medium at a cell density of 3 × 10 6The PBMCs were adjusted to 5 × 10 cells / mL and co-cultured with mitomycin C-treated A375 cells. After 5 days of co-culture with A375 cells, freshly digested PBMCs and A375 cells were collected. PBMCs (5 × 10 5 cells / mouse) and A375 cells (4 × 10 6 The tumors (cells / mouse) were inoculated subcutaneously into the right flank of NCG female mice in an inoculation volume of 0.2 mL / mouse containing 50% Matrigel. Ten days after tumor inoculation, the average tumor volume was approximately 80 mm. 3 Mice were randomly divided into four groups (six mice per group) and subcutaneously injected twice weekly for three weeks with a combination of three monoclonal antibodies (0.35 mg / kg 202F1-hIgG4, 0.65 mg / kg CQ1-3 / 1-11, and 0.65 mg / kg bevacizumab), HC-010 F8 (1 mg / kg), HC010-F23 (1 mg / kg), or a negative control (PBS). Tumor volumes were measured twice weekly using a Vernier caliper, and tumor volume was calculated according to the formula above.

[0185] The results are shown in Figure 16. Compared with the negative control group, antibodies HC010-F8 (1 mg / kg) and HC010-F23 (1 mg / kg) and the combination group effectively inhibited tumor growth.

[0186] In this example, mice in all treatment groups showed neither abnormal behavior nor weight loss (FIG. 17), indicating that tumor-bearing mice tolerated the drug well at the tested doses. The excellent tumor suppression of the trispecific antibody of the present application, both during the administration period and the withdrawal period, gives this antibody broad application prospects in the field of tumor treatment. The trispecific antibody not only effectively suppresses tumors but also prolongs the tumor suppression period.

[0187] 5.3 Trispecific antibodies target human CD34 + Suppression of growth of non-small cell lung cancer A549 in HSC-humanized mice Human CD34 +HSC-humanized mice (GemPharmatech) are transfected with human hematopoietic cells (hCD34) for immune reconstitution. + HSCs were obtained by transplanting them into irradiated NCG mice. Human non-small cell lung cancer A549 cells were transfected with human CD34 + The HSC-humanized mice were inoculated subcutaneously on the right flank. The inoculation volume was 0.2 mL / mouse containing 30% Matrigel. The mean tumor volume was approximately 100 mm 3 Once established, mice were randomly assigned to groups and subcutaneously injected twice weekly for 4 weeks with the trispecific antibodies HC010-F8 (5 mg / kg), HC010-F23 (5 mg / kg), control antibodies pembrolizumab (3.25 mg / kg) and bevacizumab (3.25 mg / kg), and negative control (PBS). Tumor volume was measured twice weekly using a Vernier caliper, and tumor volume was calculated according to the formula above. Tumor samples were harvested at the end of the experiment, minced with laboratory scissors, and enzymatically dissociated using a Human Tumor Dissociation Kit (Miltenyi Biotech) combined with a gentleMACS dissociator (Miltenyi Biotec) according to the manufacturer's instructions. The cell suspension was filtered through a 70 μm MACS Smart Filter to obtain a single-cell suspension. Immune cell subsets and functional biomarkers were analyzed by flow cytometry.

[0188] HC010-F8 and HC010-F23 significantly inhibited tumor growth compared to the negative control group, and the tumor inhibition rates were comparable to or even better than those of the pembrolizumab or bevacizumab-treated groups. During the drug withdrawal period, the tumor inhibition rates of the antibodies of the present invention were superior to those of the pembrolizumab or bevacizumab-treated groups (Figure 18).

[0189] 5.4 Trispecific antibodies inhibit the growth of human non-small cell lung cancer H1299 in a human PBMC-humanized mouse model Human non-small cell lung cancer H1299 cells were inoculated subcutaneously into the right flank of human PBMC-humanized mice (GemPharmatech) in a seeding volume of 0.2 mL / mouse containing 30% Matrigel. The mean tumor volume was approximately 100 mm. 3Once established, mice were randomly assigned to groups and subcutaneously injected twice weekly for 4 weeks with the trispecific antibodies HC010-F8 (1–10 mg / kg), HC010-F23 (1–10 mg / kg), control antibody (1–10 mg / kg), or negative control (PBS). Tumor volumes were measured twice weekly using a Vernier caliper, and tumor volume was calculated according to the formula above.

[0190] HC010-F8 and HC010-F23 effectively inhibited tumor growth compared with the negative control group. We further inoculate human non-small cell lung cancer H1299 cells into the right side of human PBMC-humanized mice (Biocytogen). Seven days after inoculation, each animal receives 4.5 × 10 6 PBMCs (human peripheral blood mononuclear cells) were injected at a rate of 0.1 mL per cell. The average tumor volume was approximately 120 mm 3 After the treatment, appropriate mice were selected according to tumor volume and body weight. Nine mice were assigned to each group, for a total of seven groups: G1: PBS, G2: pembrolizumab (10 mg / kg), G3: bevacizumab (10 mg / kg), G4: HC010-F8 (15 mg / kg), G5: anti-PD-1 / VEGFA bispecific antibody AK112 (13 mg / kg), G6: ipilimumab (10 mg / kg), and G7: pembrolizumab (10 mg / kg) + bevacizumab (10 mg / kg) + ipilimumab (10 mg / kg). Treatment began on the day of grouping and was administered twice weekly for a total of six doses for each group. During the treatment and observation period, mouse body weights and tumor volumes were measured twice weekly, and tumor growth inhibition rates (TGI) were calculated. TV ) was calculated.

[0191] At the end of the experiment, the mean tumor volume in the G1 vehicle control group (PBS) was 1303 ± 188 mm 3 The mean tumor volumes of the G2 to G7 treatment groups were 640 ± 59 mm 3 , 582±96mm 3 , 407±72mm 3 , 406±44mm 3 , 603±92mm 3 and 363±55mm 3The corresponding TGI for each treatment group was TV The tumor suppression rates were 57.40%, 60.97%, 75.70%, 75.72%, 59.06%, and 79.60%, respectively. Compared with the vehicle control group, the G2 to G7 groups had significant tumor suppression effects (P<0.001). Therefore, the tumor suppression rate of HC010-F8 alone was significantly better than that of pembrolizumab, bevacizumab, or ipilimumab alone, respectively, and was comparable to the efficacy of AK112 and the combination of pembrolizumab, bevacizumab, and ipilimumab (Figure 19).

[0192] 5.5 Trispecific antibodies inhibit the growth of human non-small cell lung cancer NCI-H292 in a human PBMC-humanized mouse model Human non-small cell lung cancer NCI-H292 cells were inoculated subcutaneously into the right flank of human PBMC-humanized mice (GemPharmatech) in a seeding volume of 0.2 mL / mouse containing 30% Matrigel. The mean tumor volume was approximately 100 mm. 3 Once established, mice were randomly assigned to groups and subcutaneously injected twice weekly for 4 weeks with the trispecific antibodies HC010-F8 (1–10 mg / kg), HC010-F23 (1–10 mg / kg), control antibody (1–10 mg / kg), or negative control (PBS). Tumor volumes were measured twice weekly using a Vernier caliper, and tumor volume was calculated according to the formula above.

[0193] HC010-F8 and HC010-F23 effectively inhibited tumor growth compared with the negative control group. 5.6 Trispecific antibodies inhibit the growth of human colorectal cancer HT29 in a human PBMC-humanized mouse model Human rectal cancer HT29 cells were inoculated subcutaneously into the right flank of human PBMC-humanized mice (Shanghai Model Organisms Center, Inc.) at an inoculation volume of 0.1 mL / mouse containing 30% Matrigel. The average tumor volume was approximately 100 mm. 3Once established, mice were randomly assigned to groups and subcutaneously injected twice weekly for 4 weeks with the trispecific antibodies HC010-F8 (1–10 mg / kg), HC010-F23 (1–10 mg / kg), control antibody (1–10 mg / kg), or negative control (PBS). Tumor volumes were measured twice weekly using a Vernier caliper, and tumor volume was calculated according to the formula above.

[0194] HC010-F8 and HC010-F23 effectively inhibited tumor growth compared with the negative control group. The inventors will then further detect the activity and dose dependency of the trispecific antibodies of the present invention.

[0195] Human rectal cancer HT29 cells were inoculated subcutaneously into the right flank of human PBMC-humanized M-NSG mice (Shanghai Model Organisms Center, Inc.) at a seeding volume of 0.1 mL / mouse containing 30% Matrigel. The average tumor volume was approximately 100 mm. 3 Once the mice were in the PBS-treated group, they were randomly assigned to: Group 1: PBS, i.p., BIW; * Group 7 (hereafter referred to as G1), Group 2: pembrolizumab, 3.25 mg / kg, i.p., BIW * 7-dose group (hereafter referred to as G2), group 3: bevacizumab, 3.25 mg / kg, i.p., BIW * Group 7 (hereafter referred to as G3), Group 4: anti-CTLA-4 antibody ipilimumab, 3.25 mg / kg, i.p., BIW * 7-dose group (hereafter referred to as G4), Group 5: HC010-F8, 1 mg / kg, ip, BIW * 7-dose group (hereafter referred to as G5), Group 6: HC010-F8, 5 mg / kg, i.p., BIW * 7-dose group (hereafter referred to as G6), Group 7: HC010-F8, 20 mg / kg, i.p., BIW * Group 7 (hereafter referred to as G7), Group 8: pembrolizumab + bevacizumab, 3.25 mg / kg + 3.25 mg / kg, i.p., BIW * Group 7 (hereafter referred to as G8), Group 9: pembrolizumab + ipilimumab, 3.25 mg / kg + 3.25 mg / kg, i.p., BIW* Group 7 (hereafter referred to as G9) and Group 10: pembrolizumab + ipilimumab + bevacizumab, 3.25 mg / kg + 3.25 mg / kg + 3.25 mg / kg, i.p., BIW * They were divided into groups, including the 7-time group (hereafter referred to as G10).

[0196] When the experiment was terminated on the 17th day after administration, the mean tumor volume of the control group G1 was 644.77 ± 61.57 mm 3 In G2, G4, and G9, the mean tumor volume was 498.68 ± 48.30 mm, respectively. 3 , 638.72±70.69mm 3 and 489.19 ± 47.40 mm 3 The tumor volume inhibition rates (TGI) were 26.72%, 1.03%, and 28.42%, respectively. There was no statistically significant difference compared with the control group (P>0.05). In group G3, the mean tumor volume was 400.50±62.73 mm 3 The tumor volume inhibition rate (TGI) was 44.68%, which was statistically different from the mean tumor volume and tumor volume inhibition rate in the control group (P<0.05). The mean tumor volume was 403.54±45.88mm in G5, G6, G7, G8, and G10, respectively. 3 , 312.48±38.02mm 3 , 270.45±33.25mm 3 , 293.13±36.08mm 3 and 313.65±25.23mm 3 The tumor volume inhibition rates (TGI) were 44.26%, 60.91%, 68.59%, 64.42%, and 60.63%, respectively, which were significantly different from the control group (P<0.01). See Table 9 for the results.

[0197] [Table 9]

[0198] Conclusion: Under the conditions of this example, the test drug HC010-F8 at 1, 5, and 20 mg / kg had significant dose-dependent antitumor effects in the HT-29 colorectal cancer model in PBMC-humanized M-NSC mice. Compared to the vehicle control group (PBS), HC010-F8 showed significant efficacy at doses equal to or greater than 1 mg / kg. The tumor inhibition rate of HC010-F8 at 5 mg / kg (60.91%) was better than that of pembrolizumab (26.72%), bevacizumab (44.68%), or ipilimumab (1.03%). The antitumor effect of HC010-F8 at a dose of 5 mg / kg was better than that of the dual immune combination of pembrolizumab and ipilimumab.

[0199] 5.7 Trispecific antibodies inhibit the growth of human liver cancer HepG2 in a human PBMC-humanized mouse model Human liver cancer HepG2 cells were inoculated subcutaneously into the right flank of human PBMC-humanized mice (Shanghai Model Organisms Center, Inc.) at a seeding volume of 0.2 mL / mouse containing 30% Matrigel. The average tumor volume was approximately 150–200 mm. 3 Once established, mice were randomly assigned to groups and subcutaneously injected twice weekly for 4 weeks with the trispecific antibodies HC010-F8 (1–10 mg / kg), HC010-F23 (1–10 mg / kg), control antibody (1–10 mg / kg), or negative control (PBS). Tumor volumes were measured twice weekly using a Vernier caliper, and tumor volume was calculated according to the formula above.

[0200] HC010-F8 and HC010-F23 effectively inhibited tumor growth compared with the negative control group. 5.8 Trispecific antibodies inhibit the growth of human hepatocarcinoma Huh7 in a human PBMC-humanized mouse model Human hepatoma Huh7 cells were cultured in human PBMC-humanized NCG mice (GemPharmatech) at 5 × 10 6 Cells were inoculated subcutaneously at 100 μL per mouse (1:1 ratio with Corning Matrigel). Mice developed tumors with a mean tumor volume of approximately 77.20 mm. 3Twenty-four mice were randomly divided into three groups according to tumor volume, with eight mice in each group. Mice were administered ip with pembrolizumab (3.25 mg / kg), HC010-F8 (5 mg / kg), or a vehicle PBS control (6 doses) twice weekly and observed for 20 days. Drug efficacy was evaluated based on observational indices such as tumor inhibition rate and body weight.

[0201] The study reached its endpoint on day 20 after the start of drug administration. The mean tumor volumes in the PBS group, pembrolizumab group (3.25 mg / kg), and HC010-F8 group (5 mg / kg) were 2124.08 mm, respectively. 3 , 2099.87mm 3 and 1014.72 mm 3 Compared with the PBS control group, HC010-F8 showed a significant difference in tumor growth inhibition (P<0.0001), while pembrolizumab had little tumor-inhibitory effect. Compared with pembrolizumab, HC010-F8 had a significant anti-tumor effect (P<0.0001) (see Figure 20). No animal deaths related to the toxicity of the test drug occurred during the experiment. Conclusion: In a mouse tumor model using human liver cancer Huh7, HC010-F8 had a significant anti-tumor effect, while pembrolizumab showed no obvious tumor-inhibitory effect. HC010-F8 was well tolerated by animals at a dose of 5 mg / kg.

[0202] 5.9 Efficacy of trispecific surrogate antibodies in a human PD-1 and human CTLA-4 dual-targeted humanized mouse tumor model The trispecific antibody does not recognize mouse PD-1, CTLA-4, or VEGFA. The anti-VEGF sequence in HC010-F8 and HC010-F23 was replaced with the anti-VEGFA sequence (B20.4.1) reported in US20110159009A1 and Wei-Ching Liang et al. A cross-species vascular endothelial growth factor (VEGF)-blocking antibody completely inhibits the growth of human tumor xenografts, measuring the contribution of stromal VEGF. J Biol Chem. 2006 Jan 13;281(2):951-61. doi:10.1074 / jbc.M508199200. Epub 2005 Nov 7. Thus, a surrogate antibody capable of recognizing mouse VEGF was obtained. B20.4.1 has similar binding and blocking activity to bevacizumab, which was used in HC010-F8 and HC010-F23.

[0203] The antitumor activity of the trispecific surrogate antibody was evaluated in the colorectal cancer MC38 tumor model and the liver cancer Hepa1-6 tumor model in humanized mice with dual targeting of human PD-1 and human CTLA-4 (B-hPD-1 / hCTLA4, Biocytogen). The mean tumor volume was approximately 100 mm. 3 Mice were then randomly assigned to groups and received subcutaneous injections twice weekly for 4 weeks of the following antibodies: HC010-F8 surrogate antibody (1-10 mg / kg), HC010-F23 surrogate antibody (1-10 mg / kg), control antibody (1-10 mg / kg), and negative control (PBS).

[0204] The results showed that the HC010-F8 and HC010-F23 surrogate antibodies had good antitumor activity in the colorectal cancer MC38 tumor model and liver cancer Hepa1-6 tumor model in humanized mice with dual targeting of human PD-1 and human CTLA-4 (B-hPD-1 / hCTLA4, Biocytogen).

[0205] Example 6 Trispecific antibody stability studies In the field of antibody preparation, stability issues are often encountered with various antibody derivatives (such as multispecific antibodies) obtained based on the modification of natural antibodies. Providing antibody derivatives with excellent stability is crucial for antibody preparation, transportation, storage, etc. The stability of the trispecific antibodies obtained in this application was examined in the Examples, and the thermal stability of the trispecific antibodies was studied under accelerated conditions at elevated temperatures (40°C). Specifically, each trispecific antibody was exchanged with a formulation buffer containing 20 mM His-HCl, pH 5.5, 230 mM trehalose, and 0.01% Tween 80. The samples were then incubated at 40°C for 0, 1, 2, and 4 weeks, and the results of size exclusion chromatography (SEC) and non-reducing capillary gel electrophoresis (NR-CE-SDS) by high-performance liquid chromatography (Dionex, Ultimate 3000) were analyzed. The results are shown in Table 9. After 4 weeks of incubation at 40°C, the SEC purity of trispecific antibodies HC010-F8 and HC010-F22 changed slightly, with a decrease in the main peak (MP%) of less than 5%, and the decrease in the main peak of HC010-F10 and HC010-F23 was approximately 10%. After 4 weeks of incubation at 40°C, the main peak of the trispecific antibodies in NR-CE-SDS decreased by 6.6% to 13.2%, indicating that the trispecific antibodies constructed and expressed in this application have good stability.

[0206] [Table 10]

Claims

1. A trispecific antibody comprising a first, second, and third antigen-binding site, wherein the first, second, and third antigen-binding sites bind to first, second, and third antigens that are different from one another, and are independently selected from PD-1, CTLA-4, and VEGF.

2. comprising a first, second, and third antigen-binding site and consisting of two identical heavy chains and two identical light chains, wherein the heavy and light chains are as follows: 1) a heavy chain having the structure VH-CH1-Fc-VHH-ScFv from the N-terminus to the C-terminus; a light chain having the structure VL-CL from N-terminus to C-terminus, or 2) a heavy chain having the structure ScFv-VH-CH1-Fc-VHH from the N-terminus to the C-terminus; a light chain having the structure VL-CL from N-terminus to C-terminus, or 3) a heavy chain having the structure VHH-VH-CH1-Fc-ScFv from the N-terminus to the C-terminus; a light chain having the structure VL-CL from N-terminus to C-terminus, or 4) a heavy chain having the structure VH-CH1-Fc-ScFv-VHH from the N-terminus to the C-terminus; a light chain having the structure VL-CL from N-terminus to C-terminus, or 5) a heavy chain having the structure VH-CH1-Fc-ScFv from the N-terminus to the C-terminus; a light chain having the structure VL-CL-VHH from N-terminus to C-terminus, or 6) a heavy chain having the structure ScFv-VH-CH1-Fc-ScFv from the N-terminus to the C-terminus; a light chain having the structure VL-CL from N-terminus to C-terminus, or 7) a heavy chain having the structure VH-CH1-Fc-ScFv from the N-terminus to the C-terminus; a light chain having the structure VHH-VL-CL from the N-terminus to the C-terminus, or 8) a heavy chain having the structure ScFv-VHH-VH-CH1-Fc from the N-terminus to the C-terminus; A light chain having a VL-CL structure from the N-terminus to the C-terminus having a structure selected from Fc represents the Fc region of an immunoglobulin heavy chain, and two heavy chains containing the Fc region homodimerize through the Fc region. CH1 represents an immunoglobulin heavy chain CH1 domain, CL represents an immunoglobulin light chain CL domain, VH-CH1 and VL-CL pair together to form Fab, the first, second, and third antigen-binding sites are in the form of Fab, VHH, or ScFv, each binding to a different antigen independently selected from PD-1, CTLA-4, and VEGF; The trispecific antibody of claim 1.

3. comprising a first, second, and third antigen-binding site, and having the following structure: 1) a first heavy chain comprising the structure VH-CH1-Fc-ScFv from the N-terminus to the C-terminus; 2) a second heavy chain comprising the structure VHH-Fc-ScFv from the N-terminus to the C-terminus; and 3) a light chain comprising a VL-CL structure from the N-terminus to the C-terminus and consists of three chains having the following structure: Fc represents the Fc region of an immunoglobulin heavy chain, and two heavy chains containing the Fc region dimerize through the Fc region; CH1 represents an immunoglobulin heavy chain CH1 domain, CL represents an immunoglobulin light chain CL domain, VH-CH1 and VL-CL pair together to form Fab, the first, second and third antigen-binding sites are in the form of Fab, VHH and / or ScFv, each binding to a different antigen independently selected from PD-1, CTLA-4 and VEGF; The trispecific antibody of claim 1.

4. comprising a first, second, and third antigen-binding site, and having the following structure: 1) a first heavy chain comprising the structure VH-CH1-Fc-ScFv2 from the N-terminus to the C-terminus; 2) a second heavy chain comprising the structure ScFv1-Fc-ScFv2 from the N-terminus to the C-terminus; 3) a light chain comprising a VL-CL structure from the N-terminus to the C-terminus and consists of three chains having the following structure: Fc represents the Fc region of an immunoglobulin heavy chain, and two heavy chains containing the Fc region dimerize through the Fc region; CH1 represents an immunoglobulin heavy chain CH1 domain, CL represents an immunoglobulin light chain CL domain, VH-CH1 and VL-CL pair together to form Fab, the first, second, and third antigen-binding sites are in the form of Fab, ScFv1, or ScFv2, each binding to a different antigen independently selected from PD-1, CTLA-4, and VEGF; The trispecific antibody of claim 1.

5. comprising a first, second, and third antigen-binding site, and having the following structure: 1) a first heavy chain comprising the structure ScFv1-Fc-ScFv2 from the N-terminus to the C-terminus; 2) A second heavy chain comprising the structure VHH-Fc-ScFv2 from the N-terminus to the C-terminus. and Fc represents the Fc region of an immunoglobulin heavy chain, and two heavy chains containing the Fc region dimerize through the Fc region; the first, second, and third antigen-binding sites are in the form of a VHH, ScFv1, or ScFv2, each binding to a different antigen independently selected from PD-1, CTLA-4, and VEGF; The trispecific antibody of claim 1.

6. the first, second and third antigen-binding sites are 1) an antigen-binding site that binds to PD-1, comprising an HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 1, an HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 2, and an HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 3, as well as an LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 4, an LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 5, and an LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 6; 2) an antigen-binding site that binds to CTLA-4, i) an HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 17, an HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 18, and an HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 19, and an LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 20, an LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 21, and an LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 22; or ii) an HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 12, an HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 13, and an HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 14; an antigen-binding site comprising: 3) An antigen-binding site that binds to VEGF, comprising an HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 26, an HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 27, and an HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 28, as well as an LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 29, an LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 30, and an LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO:

31. The trispecific antibody of claims 1 to 5, comprising:

7. 7. The trispecific antibody of claims 1 to 6, wherein the first, second and third antigen-binding sites comprise substitutions in the FR regions, preferably the substitution is G44C in the heavy chain variable region, Q100C or G100C (according to Kabat numbering) in the light chain variable region.

8. the first, second, and third antigen-binding sites are 1) an antigen-binding site that binds to PD-1, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 7, or comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 7, or consists of the sequence set forth in SEQ ID NO: 7, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 8, or comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 8, or consists of the sequence set forth in SEQ ID NO: 8; 2) an antigen-binding site that binds to CTLA-4, i) comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 15, or comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 15, or consists of the sequence set forth in SEQ ID NO: 15, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 16, or comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 16, or consists of the sequence set forth in SEQ ID NO: 16; ii) comprising the sequence set forth in SEQ ID NO: 11, or comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 11, or consisting of the sequence set forth in SEQ ID NO: 11 an antigen-binding site, and 3) An antigen-binding site that binds to VEGF, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence set forth in SEQ ID NO: 24, or comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 24, or consists of the sequence set forth in SEQ ID NO: 24, and the light chain variable region comprises the sequence set forth in SEQ ID NO: 25, or comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 25, or consists of the sequence set forth in SEQ ID NO:

25. The trispecific antibody of claims 1 to 7, comprising:

9. The trispecific antibody of claims 1 to 8, wherein adjacent antigen-binding sites are connected by a linker, and the antigen-binding sites are connected to the Fc by a linker / hinge region.

10. The linker has the amino acid sequence (G ４ S) ｎ wherein n is an integer equal to or greater than 1, and preferably the linker comprises the amino acid sequence (G ４ S) ３ or (G ４ S) ４ 10. The trispecific antibody of claim 9, wherein the linker preferably has the sequence shown in SEQ ID NO: 9 and the hinge region is an immunoglobulin-derived hinge region, preferably an IgG-derived hinge region.

11. 9. The trispecific antibody of claims 1 to 8, wherein the Fc region is derived from the Fc region of immunoglobulin IgG1 or IgG4, preferably the Fc region is derived from the heavy chain constant region sequence shown in SEQ ID NO: 33, 34 or 35, preferably the Fc region comprises a modification, for example the Fc region comprises a knob-into-hole structure.

12. 12. The trispecific antibody of claim 11 , wherein the Fc region comprises a substitution selected from the group consisting of S228P, S354C, T366W, T366S, L368A, Y394C, Y407V, H435R, Y436F and K447A (according to the EU numbering system).

13. 1) a heavy chain comprising SEQ ID NO: 37, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 38, or an amino acid sequence having at least 90% identity thereto; or 2) a heavy chain comprising SEQ ID NO: 39, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 40, or an amino acid sequence having at least 90% identity thereto; or 3) a heavy chain comprising SEQ ID NO: 41, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 42, or an amino acid sequence having at least 90% identity thereto; or 4) a heavy chain comprising SEQ ID NO: 43, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 44, or an amino acid sequence having at least 90% identity thereto; or 5) a heavy chain comprising SEQ ID NO: 45, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 46, or an amino acid sequence having at least 90% identity thereto; or 6) a heavy chain comprising SEQ ID NO: 47, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 48, or an amino acid sequence having at least 90% identity thereto; or 7) a heavy chain comprising SEQ ID NO: 49, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 50, or an amino acid sequence having at least 90% identity thereto; or 8) a heavy chain comprising SEQ ID NO: 59, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 60, or an amino acid sequence having at least 90% identity thereto; or 9) a heavy chain comprising SEQ ID NO: 61, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 62, or an amino acid sequence having at least 90% identity thereto; or 10) a heavy chain comprising SEQ ID NO: 63, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 64, or an amino acid sequence having at least 90% identity thereto; or 11) a heavy chain comprising SEQ ID NO: 65, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 66, or an amino acid sequence having at least 90% identity thereto; or 12) a heavy chain comprising SEQ ID NO: 67, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 68, or an amino acid sequence having at least 90% identity thereto; or 13) a heavy chain comprising SEQ ID NO: 69, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 70, or an amino acid sequence having at least 90% identity thereto; or 14) a heavy chain comprising SEQ ID NO: 71, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 72, or an amino acid sequence having at least 90% identity thereto; or 15) a heavy chain comprising SEQ ID NO: 73, or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 74, or an amino acid sequence having at least 90% identity thereto; or 16) A heavy chain comprising SEQ ID NO: 75 or an amino acid sequence having at least 90% identity thereto, and a light chain comprising SEQ ID NO: 76 or an amino acid sequence having at least 90% identity thereto. The trispecific antibody of claim 1 or 2, comprising:

14. a first heavy chain comprising an amino acid sequence of SEQ ID NO: 51, or an amino acid sequence having at least 90% identity thereto; a second heavy chain comprising SEQ ID NO: 53, or an amino acid sequence having at least 90% identity thereto; and A light chain comprising SEQ ID NO: 52, or an amino acid sequence having at least 90% identity thereto. The trispecific antibody of claim 3, comprising:

15. a first heavy chain comprising an amino acid sequence of SEQ ID NO: 56, or an amino acid sequence having at least 90% identity thereto; a second heavy chain comprising SEQ ID NO: 58, or an amino acid sequence having at least 90% identity thereto; and A light chain comprising SEQ ID NO: 57, or an amino acid sequence having at least 90% identity thereto. The trispecific antibody of claim 4, comprising:

16. a first heavy chain comprising an amino acid sequence of SEQ ID NO: 54, or an amino acid sequence having at least 90% identity thereto; and A second heavy chain comprising SEQ ID NO: 55, or an amino acid sequence having at least 90% identity thereto. The trispecific antibody of claim 5, comprising:

17. 1) a heavy chain comprising or consisting of SEQ ID NO: 37, and a light chain comprising or consisting of SEQ ID NO: 38; or 2) a heavy chain comprising or consisting of SEQ ID NO: 39, and a light chain comprising or consisting of SEQ ID NO: 40; or 3) a heavy chain comprising or consisting of SEQ ID NO: 41, and a light chain comprising or consisting of SEQ ID NO: 42; or 4) a heavy chain comprising or consisting of SEQ ID NO: 43, and a light chain comprising or consisting of SEQ ID NO: 44; or 5) a heavy chain comprising or consisting of SEQ ID NO: 45, and a light chain comprising or consisting of SEQ ID NO: 46; or 6) a heavy chain comprising or consisting of SEQ ID NO: 47, and a light chain comprising or consisting of SEQ ID NO: 48; or 7) a heavy chain comprising or consisting of SEQ ID NO: 49 and a light chain comprising or consisting of SEQ ID NO: 50; or 8) a heavy chain comprising or consisting of SEQ ID NO: 59, and a light chain comprising or consisting of SEQ ID NO: 60; or 9) a heavy chain comprising or consisting of SEQ ID NO: 61, and a light chain comprising or consisting of SEQ ID NO: 62; or 10) a heavy chain comprising or consisting of SEQ ID NO: 63 and a light chain comprising or consisting of SEQ ID NO: 64; or 11) a heavy chain comprising or consisting of SEQ ID NO: 65, and a light chain comprising or consisting of SEQ ID NO: 66; or 12) a heavy chain comprising or consisting of SEQ ID NO: 67, and a light chain comprising or consisting of SEQ ID NO: 68; or 13) a heavy chain comprising or consisting of SEQ ID NO: 69 and a light chain comprising or consisting of SEQ ID NO: 70; or 14) a heavy chain comprising or consisting of SEQ ID NO: 71, and a light chain comprising or consisting of SEQ ID NO: 72; or 15) a heavy chain comprising or consisting of SEQ ID NO: 73 and a light chain comprising or consisting of SEQ ID NO: 74; or 16) A heavy chain comprising or consisting of SEQ ID NO: 75, and a light chain comprising or consisting of SEQ ID NO: 76 14. The trispecific antibody of claim 1, 2 or 13, comprising:

18. a first heavy chain comprising or consisting of SEQ ID NO: 51; a second heavy chain comprising or consisting of SEQ ID NO: 53, and A light chain comprising or consisting of SEQ ID NO: 52 15. The trispecific antibody of claim 1, 3 or 14, comprising:

19. a first heavy chain comprising or consisting of SEQ ID NO: 56; a second heavy chain comprising or consisting of SEQ ID NO: 58, and A light chain comprising or consisting of SEQ ID NO: 57 16. The trispecific antibody of claim 1, 4 or 15, comprising:

20. a first heavy chain comprising or consisting of SEQ ID NO: 54, and A second heavy chain comprising or consisting of SEQ ID NO:

55.

17. The trispecific antibody of claim 1, 5 or 16, comprising:

21. A polynucleotide encoding the trispecific antibody of any one of claims 1 to 20.

22. 22. A vector, preferably an expression vector, comprising the polynucleotide of claim 21.

23. 23. A host cell, e.g. a mammalian cell, comprising the polynucleotide of claim 21 or the vector of claim 22.

24. 21. A method for producing the trispecific antibody of any one of claims 1 to 20, comprising culturing a host cell containing polynucleotides encoding the polypeptide chains of the antibody under conditions suitable for expressing the polypeptide chains, and assembling the polypeptide chains into the antibody under conditions suitable for the assembly of the polypeptide chains into the antibody to produce the antibody.

25. A pharmaceutical composition comprising the trispecific antibody of any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

26. 26. Use of the trispecific antibody of any one of claims 1 to 20 or the pharmaceutical composition of claim 25 in the manufacture of a medicament for treating and / or preventing cancer, an autoimmune disease, an infectious disease or an angiogenesis-related disease in an individual, or in the manufacture of a reagent for diagnosing cancer, an autoimmune disease, an infectious disease or an angiogenesis-related disease.

27. 27. The use according to claim 26, wherein the cancer is selected from the group consisting of solid tumors such as lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), breast cancer, liver cancer, bladder cancer, melanoma, colon cancer, rectal cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic adenocarcinoma, basal cell carcinoma, esophageal cancer, bile duct cancer, head and neck squamous cell carcinoma, thyroid cancer, brain cancer, gastric cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, testicular cancer, multiple myeloma, glioblastoma, glioma, and hematological tumors such as leukemia and lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma).

28. 26. A method for treating, preventing and / or diagnosing cancer, an autoimmune disease, an infectious disease or an angiogenesis-related disease, comprising administering to a patient in need thereof an effective amount of the trispecific antibody of any one of claims 1 to 20, or the pharmaceutical composition of claim 25.

29. 29. The method of claim 28, wherein the cancer is selected from the group consisting of solid tumors such as lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), breast cancer, liver cancer, bladder cancer, melanoma, colon cancer, rectal cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic adenocarcinoma, basal cell carcinoma, esophageal cancer, bile duct cancer, head and neck squamous cell carcinoma, thyroid cancer, brain cancer, gastric cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, testicular cancer, multiple myeloma, glioblastoma, glioma, and hematological tumors such as leukemia and lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma).

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