Tri-specific antibody targeting PD-1, CTLA-4 and VEGF and application thereof

Abstract

The invention relates to a tri-specific antibody specifically binding to PD-1, CTLA-4 and VEGF, and also relates to a composition containing the tri-specific antibody and an application of the composition in treatment of tumors, autoimmune diseases, infectious diseases or angiogenesis-related diseases.

Description

Trispecific antibodies targeting PD-1, CTLA-4, and VEGF and their applications Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a trispecific antibody targeting PD-1, CTLA-4 and VEGF and its application in tumor treatment. Background Art

[0002] Although immunotherapy has significantly enhanced the ability to treat diseases (such as cancer), clinical studies have found that many patients cannot respond adequately to single-specific therapies due to the complexity of the disease. The tumor microenvironment is composed of cells such as tumor cells, immune cells, vascular cells, and non-cellular components in the extracellular matrix. The interactions between these components limit the host's anti-tumor immunity and control of tumor growth. Researchers hope to achieve better therapeutic effects by developing combined therapies targeting different cells and different targets. For example, the combined use of nivolumab, an antibody that blocks the immune checkpoint PD-1, and ipilimumab, an antibody that blocks CTLA-4, can stimulate a stronger immune response in preclinical and clinical settings, and has better therapeutic effects than blocking 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, the above-mentioned combination therapy also increases the cost of medication, increases multiple side effects, and has higher toxicity, which may be related to the clearance of regulatory T cells (Treg) 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] Immune checkpoints PD-1 and CTLA-4 have different expression patterns on different immune cells, and the signal interaction mediated by the two inhibits anti-tumor immunity. Therefore, simultaneous blockade of PD-1 and CTLA-4 can promote anti-tumor 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 that simultaneously target PD-1 and CTLA-4 can not only block the signaling pathways of PD-1 and CTLA-4 at the same time, but also induce endocytosis of PD-1, leading to its degradation. On cells co-expressing PD-1 and CTLA-4 (such as exhausted T cells in the tumor microenvironment), they enhance the mechanism of blocking the interaction between CTLA-4 and CD80 and promote anti-tumor immunity. In addition, they have shown efficacy in patients who are ineffective with 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): 100163, 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 inhibiting the functions of antigen-presenting cells and immune effector cells, and by enhancing the functions of immunosuppressive cells such as regulatory T cells (Tregs), while immunosuppressive cells promote angiogenesis by secreting cytokines. The immune response and angiogenesis interact with each other and play an important role in the occurrence and development of tumors. Studies have found that immune checkpoint inhibitors can promote the normalization of blood vessels in tumor tissues (The Intersection between Tumor Angiogenesis and Immune Suppression, Rahma OE et al., Clin Cancer Res (2019) 25 (18): 5449–5457), and anti-angiogenic therapy can also improve anti-tumor 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 antibody atezolizumab and bevacizumab has shown significant effect in unresectable hepatocellular carcinoma (Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma, Richard S Finn et al., N Engl J Med., 2020 May14; 382(20): 1894-1905) and has been approved by the FDA for the treatment of unresectable hepatocellular carcinoma. The combined use of other immune checkpoint inhibitors (including anti-PD-1 / PD-L1 antibodies) and anti-angiogenesis inhibitors (including anti-VEGF antibodies and other small molecule inhibitors that block VEGF signaling) has also achieved positive results in clinical practice (Combination of Anti-Angiogenics and Checkpoint Inhibitors for Renal Cell Carcinoma: Is the Whole Greater Than the Sum of Its Parts?, Eric Jonasch et al., Cancers (Basel). 2022 Jan 27; 14(3): 644).Bispecific antibodies that simultaneously block immune checkpoints and angiogenesis have shown better efficacy than combined use 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: 778978). Recent studies have found that the combination of anti-PD-L1 antibodies atezolizumab and bevacizumab has a poorer effect in the treatment of 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 ideas for clinical practice.

[0005] Therefore, multispecific antibodies that simultaneously target multiple targets, such as bispecific and trispecific antibodies, 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 generally does not induce synergistic effects and may even have adverse effects. Therefore, the design of bispecific or trispecific antibodies, especially trispecific antibodies, faces enormous challenges and requires consideration of a large number of variable factors, including molecular compatibility, antibody affinity, stability, and pharmaceutical properties.

[0006] The present invention addresses these issues and meets market demand to a certain extent by providing a trispecific antibody that can simultaneously block two major immune checkpoints and a major angiogenesis pathway. The trispecific antibody provided by the present invention can simultaneously target PD-1, CTLA-4, and VEGF in the tumor microenvironment, simultaneously blocking immunosuppression and angiogenesis, and regulating the immune response and angiogenesis in the tumor microenvironment, thereby achieving the therapeutic effect of the three antibodies in combination.

[0007] Summary of the Invention

[0008] The present invention provides a novel trispecific antibody that simultaneously targets PD-1, CTLA-4, and VEGF, and the trispecific antibody simultaneously retains the good antigen binding specificity and selectivity of each antigen binding site and thus has good biological activity. In in vitro efficacy studies, the trispecific antibody of the present invention has similar immune activation activity as anti-PD-1 antibodies and anti-CTLA-4 antibodies used in combination, and has similar VEGF blocking activity as bevacizumab. In the treatment of various tumors in vivo (such as melanoma, lung cancer, colon cancer, liver cancer, etc.), the trispecific antibody of the present invention has an anti-tumor activity that is equivalent to or even better than that used in combination with anti-PD-1 antibodies, anti-CTLA-4 antibodies, and bevacizumab. Compared with monotherapy and combination therapy, the trispecific antibody targeting PD-1, CTLA-4, and VEGF has the following advantages:

[0009] 1. Trispecific antibodies can synergistically inhibit tumor cell proliferation and have better therapeutic effects than any single monoclonal antibody

[0010] 2. They have two valent antigen-binding sites for each target, thus having a stronger affinity than multispecific antibodies with one valent antigen-binding site;

[0011] 3. Improve the low immune response of subjects to monoclonal antibodies;

[0012] 4. Use FC with no or weak ADCC function to reduce or minimize the toxic side effects of anti-CTLA-4 monoclonal antibodies caused by Treg clearance;

[0013] 5. Through PD-1-mediated targeting, the ratio of trispecific antibody distribution in tumors and peripheral blood is increased, and the toxicity of CTLA-4 and VEGF targeting is reduced;

[0014] 6. Trispecific antibodies can degrade PD-1 antigens on the cell surface through CTLA-4-mediated endocytosis;

[0015] 7. Reduce the frequency of administration and reduce the discomfort caused by combined administration;

[0016] 8. Have increased stability; and

[0017] 9. Prolong the effective period of tumor inhibition.

[0018] Overall, the trispecific antibody molecules provided by the present invention can recognize three targets, significantly reducing costs compared to the combined use of three antibodies. The two identical antigen-binding sites of the trispecific antibody molecules for each target essentially retain the target binding ability of the corresponding natural bivalent antibody, resulting in a synergistic effect at the tumor site. Furthermore, the trispecific antibodies provided by the present invention exhibit excellent purity and thermal stability, which greatly facilitates further downstream development and large-scale production.

[0019] Therefore, the present invention mainly relates to the following aspects:

[0020] 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, a second, and a third antigen binding site, wherein the first, the second, and the third antigen binding site bind to different first, second, and third antigens independently selected from PD-1, CTLA-4, and VEGF.

[0021] 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 connected to the N-terminus and / or C-terminus of the dimerized Fc region.

[0022] 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 Fab, scFv, or VHH. In a specific embodiment, the antigen binding site that recognizes the antigen CTLA-4 is in the form of scFv or VHH.

[0023] In a specific embodiment of the trispecific antibody provided by the present invention, the antigen binding site that binds to PD-1 comprises the following heavy chain CDRs (HCDRs) and / or light chain CDRs (LCDRs): HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 1, HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 2, and HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 3; and / or LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 4, LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 5, and LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 6.

[0024] In a specific embodiment of the trispecific antibody provided herein, 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 of SEQ ID NO: 7, 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: 7, or consists of the sequence of SEQ ID NO: 7, and the light chain variable region comprises the sequence of SEQ ID NO: 8, 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: 8, or consists of the sequence of SEQ ID NO: 8.

[0025] In a specific embodiment of the trispecific antibody provided herein, the antigen binding site that binds to CTLA-4 comprises the following HCDRs and / or LCDRs: a HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 17, a HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 18, and a HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 19; and / or a LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 20, a LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 21, and a LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 22.

[0026] In another specific embodiment of the trispecific antibody provided herein, the antigen binding site that binds to CTLA-4 is a VHH and comprises the following HCDRs: a HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 12, a HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 13, and a HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 14.

[0027] In a specific embodiment of the trispecific antibody provided herein, the antigen-binding site that binds to CTLA-4 comprises a heavy chain variable region comprising the sequence of 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 of SEQ ID NO: 15, and a light chain variable region comprising the sequence of 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 of SEQ ID NO: 16.

[0028] In a specific embodiment of the trispecific antibodies provided herein, the antigen-binding site that binds CTLA-4 comprises a single VH domain (VHH), which comprises the sequence of SEQ ID NO: 11, 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: 11, or consists of the sequence of SEQ ID NO: 11.

[0029] In a specific embodiment of the trispecific antibody provided by the present invention, the antigen binding site that binds to VEGF comprises the following HCDRs and / or LCDRs: HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 26, HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 27, and HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 28; and / or LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 29, LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 30, and LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 31.

[0030] In a specific embodiment of the trispecific antibodies provided herein, the antigen binding site that binds to VEGF comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 24, 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: 24, or consisting of the sequence of SEQ ID NO: 24, and / or a light chain variable region comprising the sequence of SEQ ID NO: 25, 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: 25, or consisting of the sequence of SEQ ID NO: 25.

[0031] In one embodiment of the trispecific antibody provided by the present invention, the first, second and third antigen binding sites comprise:

[0032] 1) an antigen-binding site that binds to PD-1, comprising a HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 1, a HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 2, and a HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 3; and a LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 4, a LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 6;

[0033] 2) binds to the antigen binding site of CTLA-4, which has

[0034] i) a HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 17, a HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 18, and a HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 19; and a LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 20, a LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 21, and a LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 22; or

[0035] 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; and

[0036] 3) an antigen binding site that binds to VEGF, comprising a HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 26, a HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 27, and a HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 28; and a LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 29, a LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 30, and a LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 31.

[0037] In one embodiment of the trispecific antibody provided by the present invention, the first, second and third antigen binding sites comprise:

[0038] 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 of SEQ ID NO: 7, 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: 7, or consists of the sequence of SEQ ID NO: 7, and the light chain variable region comprises the sequence of SEQ ID NO: 8, 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: 8, or consists of the sequence of SEQ ID NO: 8;

[0039] 2) an antigen binding site that binds to CTLA-4, comprising

[0040] i) a heavy chain variable region comprising, or comprising, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to, the sequence of SEQ ID NO: 15, and a light chain variable region comprising, or comprising, or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to, the sequence of SEQ ID NO: 16; or

[0041] ii) comprising the sequence shown in SEQ ID NO: 11, 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: 11, or consisting of the sequence shown in SEQ ID NO: 11; and

[0042] 3) an antigen binding site that binds to VEGF, comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 24, 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: 24, or consisting of the sequence of SEQ ID NO: 24, and / or a light chain variable region comprising the sequence of SEQ ID NO: 25, 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: 25, or consisting of the sequence of SEQ ID NO: 25.

[0043] 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 substitution occurs in the framework region (FR region). In a more preferred embodiment, the substitution is G44C in the heavy chain variable region, Q100C or G100C in the light chain variable region (according to Kabat numbering).

[0044] In one embodiment of the trispecific antibodies provided herein, the Fc region comprising a homodimerization or heterodimerization can be an Fc region of an immunoglobulin IgG1, IgG2, IgG3, or IgG4 having a native sequence or a variant sequence. In a preferred embodiment, the Fc region comprises a modification, such as a knob-and-hole structure. In a more preferred embodiment, the Fc region comprises a substitution selected from S228P, S354C, T366W, T366S, L368A, Y394C, Y407V, H435R, Y436F, K447A (according to the EU numbering system). In one embodiment, the Fc region is derived from the heavy chain constant region sequence shown in SEQ ID NO: 33, 34, or 35.

[0045] In one embodiment of the trispecific antibody provided by the present invention, the first, second, and third antigen binding sites, or the first, second, and third antigen binding sites and the Fc region are connected by a linker / hinge region. 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.

[0046] In one embodiment, the present 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 chain and the light chain have a structure selected from the group consisting of:

[0047] 1) A heavy chain comprising the structure of VH-CH1-Fc-VHH-ScFv from N-terminus to C-terminus,

[0048] The light chain, which comprises the VL-CL structure from N-terminus to C-terminus;

[0049] 2) a heavy chain comprising the structure of ScFv-VH-CH1-Fc-VHH from N-terminus to C-terminus,

[0050] The light chain, which comprises the VL-CL structure from N-terminus to C-terminus;

[0051] 3) a heavy chain comprising the structure of VHH-VH-CH1-Fc-ScFv from N-terminus to C-terminus,

[0052] The light chain, which comprises the VL-CL structure from N-terminus to C-terminus;

[0053] 4) a heavy chain comprising a structure of VH-CH1-Fc-ScFv-VHH from N-terminus to C-terminus,

[0054] The light chain, which comprises the VL-CL structure from N-terminus to C-terminus;

[0055] 5) a heavy chain comprising a VH-CH1-Fc-ScFv structure from N-terminus to C-terminus,

[0056] The light chain consists of a VL-CL-VHH structure from N-terminus to C-terminus;

[0057] 6) a heavy chain comprising the structure of ScFv-VH-CH1-Fc-ScFv from N-terminus to C-terminus,

[0058] The light chain, which comprises the VL-CL structure from N-terminus to C-terminus;

[0059] 7) a heavy chain comprising a VH-CH1-Fc-ScFv structure from N-terminus to C-terminus,

[0060] A light chain comprising a VHH-VL-CL structure from N-terminus to C-terminus; or

[0061] 8) a heavy chain comprising a structure of ScFv-VHH-VH-CH1-Fc from N-terminus to C-terminus,

[0062] The light chain, which comprises the VL-CL structure from N-terminus to C-terminus;

[0063] Wherein Fc represents the Fc region of an immunoglobulin heavy chain, wherein the two heavy chains comprising the Fc region homodimerize through the Fc region.

[0064] Wherein CH1 represents the immunoglobulin heavy chain CH1 domain, CL represents the immunoglobulin light chain CL domain,

[0065] Among them, VH-CH1 and VL-CL pair with each other to form Fab,

[0066] The first, second and third antigen binding sites are in the form of Fab, VHH and / or ScFv, and bind to different and independent antigens PD-1, CTLA-4 and VEGF respectively.

[0067] In a preferred embodiment, adjacent antigen binding sites are connected via a linker. Preferably, the antigen binding site is connected to Fc via a linker / hinge region.

[0068] In another embodiment, the present invention provides a trispecific antibody comprising a first, a second, and a third antigen binding site, and consisting of three chains having the following structure:

[0069] 1) A first heavy chain comprising a VH-CH1-Fc-ScFv structure from N-terminus to C-terminus,

[0070] 2) a second heavy chain comprising the structure of VHH-Fc-ScFv from N-terminus to C-terminus, and

[0071] 3) Light chain, which comprises the VL-CL structure from N-terminus to C-terminus,

[0072] Wherein Fc is the Fc region of an immunoglobulin heavy chain comprising a knob-in-hole structure, wherein the two heavy chains comprising the Fc region heterodimerize through the Fc region.

[0073] Wherein CH1 represents the immunoglobulin heavy chain CH1 domain, CL represents the immunoglobulin light chain CL domain,

[0074] Among them, VH-CH1 and VL-CL pair with each other to form Fab,

[0075] The first, second and third antigen binding sites are in the form of Fab, VHH and / or ScFv, and bind to different and independent antigens PD-1, CTLA-4 and VEGF respectively.

[0076] In a preferred embodiment, adjacent antigen binding sites are connected via a linker. Preferably, the antigen binding site is connected to Fc via a linker / hinge region.

[0077] In another embodiment, the present invention provides a trispecific antibody comprising a first, a second, and a third antigen binding site, and consisting of three chains having the following structure:

[0078] 1) A first heavy chain comprising a VH-CH1-Fc-ScFv2 structure from N-terminus to C-terminus,

[0079] 2) a second heavy chain comprising the structure of ScFv1-Fc-ScFv2 from N-terminus to C-terminus,

[0080] 3) Light chain, which comprises the VL-CL structure from N-terminus to C-terminus,

[0081] Wherein Fc is the Fc region of an immunoglobulin heavy chain comprising a knob-in-hole structure, wherein the two heavy chains comprising the Fc region heterodimerize through the Fc region.

[0082] Wherein CH1 represents the immunoglobulin heavy chain CH1 domain, CL represents the immunoglobulin light chain CL domain,

[0083] Among them, VH-CH1 and VL-CL pair with each other to form Fab,

[0084] The first, second and third antigen binding sites are in the form of Fab, ScFv1 and / or ScFv2, and bind to different and independent antigens PD-1, CTLA-4 and VEGF respectively.

[0085] In a preferred embodiment, adjacent antigen binding sites are connected via a linker. Preferably, the antigen binding site is connected to Fc via a linker / hinge region.

[0086] In another embodiment, the present invention provides a trispecific antibody comprising a first, a second, and a third antigen binding site, and consisting of two chains having the following structure:

[0087] 1) A first heavy chain comprising the structure of ScFv1-Fc-ScFv2 from N-terminus to C-terminus,

[0088] 2) a second heavy chain comprising the structure of VHH-Fc-ScFv2 from N-terminus to C-terminus,

[0089] Wherein Fc is the Fc region of an immunoglobulin heavy chain comprising a knob-in-hole structure, wherein the two heavy chains comprising the Fc region heterodimerize through the Fc region.

[0090] The first, second and third antigen binding sites are in the form of VHH, ScFv1 and / or ScFv2, and bind to different and independent antigens PD-1, CTLA-4 and VEGF respectively.

[0091] In a preferred embodiment, adjacent antigen binding sites are connected via a linker. Preferably, the antigen binding site is connected to Fc via a linker / hinge region.

[0092] In a preferred embodiment, the first, second and third antigen binding sites of the trispecific antibody comprise:

[0093] 1) an antigen-binding site that binds to PD-1, comprising a HCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 1, a HCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 2, and a HCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 3; and a LCDR1 comprising or consisting of the sequence set forth in SEQ ID NO: 4, a LCDR2 comprising or consisting of the sequence set forth in SEQ ID NO: 5, and a LCDR3 comprising or consisting of the sequence set forth in SEQ ID NO: 6;

[0094] 2) binds to the antigen binding site of CTLA-4, which has

[0095] i) a HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 17, a HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 18, and a HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 19; and a LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 20, a LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 21, and a LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 22; or

[0096] 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; and

[0097] 3) an antigen binding site that binds to VEGF, comprising a HCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 26, a HCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 27, and a HCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 28; and a LCDR1 comprising or consisting of the sequence shown in SEQ ID NO: 29, a LCDR2 comprising or consisting of the sequence shown in SEQ ID NO: 30, and a LCDR3 comprising or consisting of the sequence shown in SEQ ID NO: 31.

[0098] In a preferred embodiment, the first, second and third antigen binding sites of the trispecific antibody comprise:

[0099] 1) an antigen-binding site that binds to PD-1, comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 7, 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: 7, or consisting of the sequence of SEQ ID NO: 7; and a light chain variable region comprising the sequence of SEQ ID NO: 8, 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: 8, or consisting of the sequence of SEQ ID NO: 8;

[0100] 2) an antigen binding site that binds to CTLA-4, comprising

[0101] i) a heavy chain variable region comprising, or comprising, or consisting of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to, the sequence of SEQ ID NO: 15, and a light chain variable region comprising, or comprising, or consisting of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to, the sequence of SEQ ID NO: 16; or

[0102] ii) comprising the sequence shown in SEQ ID NO: 11, 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: 11, or consisting of the sequence shown in SEQ ID NO: 11; and

[0103] 3) an antigen binding site that binds to VEGF, comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 24, 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: 24, or consisting of the sequence of SEQ ID NO: 24, and a light chain variable region comprising the sequence of SEQ ID NO: 25, 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: 25, or consisting of the sequence of SEQ ID NO: 25.

[0104] In a preferred embodiment, the first, second, and third antigen-binding sites comprise substitutions in the framework regions (FR regions). In a more preferred embodiment, the substitutions are G44C in the heavy chain variable region, Q100C or G100C in the light chain variable region (according to Kabat numbering).

[0105] 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 a modification, for example, the Fc region comprises a knob-in-hole structure. In a more preferred embodiment, the Fc region comprises a substitution selected from S228P, S354C, T366W, T366S, L368A, Y394C, Y407V, H435R, Y436F, 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.

[0106] 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.

[0107] In a preferred embodiment, the present invention provides a trispecific antibody comprising:

[0108] 1) a heavy chain comprising SEQ ID NO: 37, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 38, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0109] 2) a heavy chain comprising SEQ ID NO: 39, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 40, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0110] 3) a heavy chain comprising SEQ ID NO:41, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain comprising SEQ ID NO:42, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0111] 4) a heavy chain comprising SEQ ID NO: 43, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 44, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0112] 5) a heavy chain comprising SEQ ID NO: 45, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 46, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0113] 6) a heavy chain comprising SEQ ID NO: 47, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 48, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0114] 7) a heavy chain comprising SEQ ID NO: 49, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 50, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0115] 8) a heavy chain comprising SEQ ID NO:59, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain comprising SEQ ID NO:60, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0116] 9) a heavy chain comprising SEQ ID NO: 61, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 62, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0117] 10) a heavy chain comprising SEQ ID NO: 63, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 64, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0118] 11) a heavy chain comprising SEQ ID NO: 65, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain comprising SEQ ID NO: 66, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0119] 12) a heavy chain comprising SEQ ID NO: 67, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 68, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0120] 13) a heavy chain comprising SEQ ID NO: 69, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain comprising SEQ ID NO: 70, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0121] 14) a heavy chain comprising SEQ ID NO: 71, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain comprising SEQ ID NO: 72, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or

[0122] 15) a heavy chain comprising SEQ ID NO: 73, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 74, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0123] 16) a heavy chain comprising SEQ ID NO: 75, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a light chain comprising SEQ ID NO: 76, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0124] In a preferred embodiment, the present invention provides a trispecific antibody comprising:

[0125] 1) a first heavy chain comprising SEQ ID NO:51, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, a second heavy chain comprising SEQ ID NO:53, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO:52, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; or

[0126] 2) a first heavy chain comprising SEQ ID NO: 56, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, a second heavy chain comprising SEQ ID NO: 58, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, and a light chain comprising SEQ ID NO: 57, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0127] In a preferred embodiment, the present invention provides a trispecific antibody comprising:

[0128] A first heavy chain comprising SEQ ID NO:54, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and a second heavy chain comprising SEQ ID NO:55, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0129] In a preferred embodiment, the present invention provides a trispecific antibody comprising:

[0130] 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

[0131] 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

[0132] 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

[0133] 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

[0134] 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

[0135] 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

[0136] 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

[0137] 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

[0138] 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

[0139] 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

[0140] 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

[0141] 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

[0142] 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

[0143] 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

[0144] 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

[0145] 16) A heavy chain comprising or consisting of SEQ ID NO: 75, and a light chain comprising or consisting of SEQ ID NO: 76.

[0146] In a preferred embodiment, the present invention provides a trispecific antibody comprising:

[0147] 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

[0148] 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.

[0149] In a preferred embodiment, the present invention provides a trispecific antibody comprising:

[0150] 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.

[0151] In a second aspect, the present invention provides a polynucleotide encoding a trispecific antibody molecule of the present invention; a vector comprising the polynucleotide; and a host cell comprising the polynucleotide or vector of the present invention.

[0152] In one embodiment, the vector is preferably an expression vector.

[0153] In one embodiment, the host cell can be a prokaryotic cell or a eukaryotic cell commonly used in the art.

[0154] 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, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include monkey kidney CV1 lines (COS-7) transformed by SV40, human embryonic kidney lines (HEK293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer 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 YO, NSO, P3X63 and Sp2 / 0, etc. In a preferred embodiment, the host cell is a CHO, HEK293 or NSO cell.

[0155] In a third aspect, the present invention provides a method for producing the trispecific antibody of the present invention, comprising the steps of (i) culturing the host cell disclosed in the second aspect of the present invention under conditions suitable for expressing the trispecific antibody disclosed in the first aspect of the present invention, and optionally, (ii) recovering the trispecific antibody of the present invention.

[0156] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the trispecific antibody molecule of the present invention.

[0157] In one embodiment, the pharmaceutical composition provided by the present invention further comprises other therapeutic agents, and optional pharmaceutical excipients; preferably, the other therapeutic agents are selected from chemotherapeutic agents, cytotoxic agents, and the like.

[0158] In a fifth aspect, the present invention provides uses of the trispecific antibodies and pharmaceutical compositions of the present invention for treating, preventing and / or diagnosing cancer, autoimmune diseases, infectious diseases or angiogenesis-related diseases.

[0159] In one embodiment, the present invention provides the use of any antibody described in the first aspect, any polynucleotide or vector or host cell described in the second aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of a drug for treating, preventing and / or diagnosing cancer, autoimmune diseases, infectious diseases or angiogenesis-related diseases.

[0160] In one embodiment, the present invention provides any antibody of the first aspect, any polynucleotide or vector or host cell of the second aspect, or the pharmaceutical composition of the fourth aspect for use in treatment, prevention and / or diagnosis.

[0161] In one embodiment, the present invention provides any antibody described in the first aspect, any polynucleotide or vector or host cell described in the second aspect, and the pharmaceutical composition described in the fourth aspect for treating, preventing and / or diagnosing cancer, autoimmune diseases, infectious diseases or angiogenesis-related diseases.

[0162] In one embodiment, the cancer is, for example, 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 other solid tumors and leukemia, lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma) and other blood tumors.

[0163] In a sixth aspect, the present invention provides a method for treating, preventing and / or diagnosing cancer, autoimmune diseases, infectious diseases or angiogenesis-related diseases, comprising administering an effective amount of the trispecific antibody of the present invention, or the pharmaceutical composition of the present invention to a patient in need.

[0164] In one embodiment, the cancer is, for example, 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 other solid tumors and leukemia, lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, acute B-cell lymphoma, follicular lymphoma) and other blood tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0165] FIG1 shows a schematic structural diagram of the trispecific antibody constructed in the present application.

[0166] Figure 2 shows the binding activity of the trispecific antibody to cell surface PD-1. The binding activity of the trispecific antibody to 293T-hPD-1 cells was detected by FACS. Panels A and B are experiments FACS #1 and FACS #2, respectively.

[0167] Figure 3 shows the activity of the trispecific antibody binding to cell surface CTLA-4. The binding activity of the trispecific antibody to CHO-hCTLA-4 cells was detected by FACS. Figures A and B are experiments FACS#3 and FACS#4, respectively.

[0168] Figure 4 shows the binding activity of trispecific antibodies to activated T cells. Panel A shows the binding activity of trispecific antibodies and anti-PD-1 antibodies to T cells; Panel B shows the binding activity of anti-CTLA-4 antibodies to T cells.

[0169] Figure 5 shows the binding activity of the trispecific antibody to free VEGFA after binding to 293T-hPD-1-hCTLA-4 cells (co-expressing PD-1 and CTLA-4).

[0170] FIG6 shows the binding activity of the trispecific antibodies to free VEGFA after binding to activated T cells.

[0171] Figure 7 shows the ability of trispecific antibodies to enhance SEB-stimulated IL-2 secretion by 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 antibodies for 3 days.

[0172] Figure 8 shows that trispecific antibodies promote IL-2 secretion in MLRs. Trispecific antibodies or control antibodies were added to the MLRs, and IL-2 secretion was measured 4 days later. Panel A: DC donor: Lot #Z0160, PBMC donor: Lot #Z0177, IL-2 secretion was measured 4 days later; Panel B: DC donor: Lot #Z0160, PBMC donor: Lot #Z0182.

[0173] 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 AC were 500, 50, and 5 nM, respectively, and IL-2 secretion was measured in MLRs after 4 days of treatment.

[0174] FIG10 shows that trispecific antibodies inhibit VEGFA-induced proliferation of human umbilical vein endothelial cells.

[0175] Figure 11 shows that the trispecific antibody HC010-F8 inhibits the growth of human melanoma A375 in human PBMC humanized mice.

[0176] FIG12 shows that the trispecific antibody HC010-F8 does not affect the body weight of mice in the human PBMC humanized human melanoma A375 mouse model.

[0177] Figure 13 shows the binding of the trispecific antibody to human PD-1 followed by VEGFA (Panel A) and CTLA-4 (Panel B).

[0178] Figure 14 shows the binding of the trispecific antibody to cell surface human PD-1 followed by binding to VEGFA (Panel A) and CTLA-4 (Panel B).

[0179] Figure 15 shows the binding of the trispecific antibody to cell surface human CTLA-4 followed by binding to PD-1 (Panel A) and VEGF (Panel B).

[0180] Figure 16 shows that trispecific antibodies HC010-F8 and HC010-F23 inhibit the growth of human melanoma A375 in human PBMC humanized mice.

[0181] FIG17 shows that the trispecific antibodies HC010-F8 and HC010-F23 do not affect the body weight of mice in the human PBMC humanized human melanoma A375 mouse model.

[0182] Figure 18 shows the activity of trispecific antibodies in inhibiting human non-small cell lung cancer A549 cells in mice.

[0183] FIG19 shows the activity of trispecific antibodies in inhibiting human non-small cell lung cancer H1299 cells in mice.

[0184] FIG20 shows the activity of trispecific antibodies in inhibiting human liver cancer Huh7 cells in mice.

[0185] Detailed Description of the Invention

[0186] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are merely illustrative and are not intended to be restrictive. Other features, objects and advantages of the present invention will become apparent from this specification and the accompanying drawings and from the appended claims.

[0187] I. Definition

[0188] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0189] As used herein, the terms "comprise" or "comprising" are meant to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0190] The term "antibody" is used herein in the broadest sense to refer to a protein that contains an antigen binding site.

[0191] The terms "antigen binding site" and "antigen binding domain" are used interchangeably to 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 other formats.

[0192] The term "immunoglobulin" refers to a protein with the structure of a naturally occurring antibody and is generally used interchangeably with the term "antibody" in this application. IgG immunoglobulins are heterotetrameric glycoproteins consisting of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also known as a 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 known as a light chain variable domain, followed by a light chain constant domain (CL). In IgG molecules, the VH-CH1 of the heavy chain is usually paired with the VL-CL of the light chain to form a Fab fragment that specifically binds to the antigen. Therefore, an IgG immunoglobulin is essentially composed of two Fab molecules connected by the immunoglobulin hinge region and two dimerized Fc regions. The heavy chains of immunoglobulins 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 region, some of which can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of immunoglobulins can also be assigned to one of two types, called κ and λ, based on the amino acid sequence of their constant domains.

[0193] The term "variable region" or "variable domain" of an antibody refers to the domain of the antibody heavy or light chain that participates in binding of the antibody to the antigen. The variable region of an antibody can be further divided into hypervariable regions (i.e., complementary determining regions (CDRs)) and relatively conserved regions (i.e., framework regions (FRs)) interposed between the hypervariable regions. In the case of IgG immunoglobulins, the heavy chain variable region or light chain variable region comprises FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively, from N-terminus to C-terminus. In the case of heavy chain antibodies (also referred to herein as nanobodies), for example, heavy chain antibodies from Camelidae, the antigen binding site is composed of a single VH domain (i.e., "VHH" domain). The VHH of a natural heavy chain antibody has a similar structure to the heavy chain variable region of a natural IgG antibody, i.e., comprises four conserved framework regions (FRs) and three complementary determining regions (CDRs).

[0194] The term "multispecific antibody" refers to an antibody that has at least two antigen-binding sites that each binds to a different epitope of the same antigen or to different epitopes of different antigens.

[0195] The term "valent" with respect to antibodies 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 binding epitopes are the same. Preferably, in the present invention, the hexavalent antibody has three different antigen-binding specificities, wherein two identical antigen-binding sites are present for each antigen-binding specificity.

[0196] "Heavy chain constant region domain" or "heavy chain constant region" refers to the constant region domain from, obtained from, or derived from an immunoglobulin heavy chain, including heavy chain constant regions CH1, CH2, CH3, and optionally heavy chain constant region CH4 covalently linked sequentially from the N-terminus to the C-terminus. In most cases, the heavy chain constant regions CH1 and CH2 are connected by a heavy chain hinge region, but when appropriate, they can also be connected by a flexible linker. In some embodiments of the present invention, the heavy chain constant region of the 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 can be an IgA, IgD, IgE, IgG, or IgM domain, in particular an immunoglobulin constant domain of human IgG, for example, a constant domain of human IgG1, IgG2, IgG3, or IgG4.

[0197] In the multispecific antibodies of the present invention, the chain containing the Fc domain is a heavy chain, and the chain without 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.

[0198] 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. The term includes native Fc regions and variant Fc regions. The Fc domains that can be used in the antibodies of the present invention include, but are not limited to, Fc domains of IgG1, IgG2, IgG3, or IgG4 having native or variant sequences. The lysine residue at position 447 of the C-terminus of the Fc domain (according to the EU numbering system) may be present or absent. Unless otherwise indicated herein, the amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0199] Fc domains, fusion proteins comprising Fc domains (eg, antibodies), can be modified using a variety of known methods, for example to reduce immunogenicity, improve stability, solubility, function, and other modifications that may be of clinical benefit. Such modifications include, but are not limited to, the following modifications: the stability of IgG can be increased by modifying the amino acid residue K447, preferably the amino acid substitution K447A; the heterogeneity problem of IgG4 can be solved by modifying the amino acid residue S228, preferably the amino acid substitution S228P; the knobs-into-holes structure to increase antibody stability, wherein the knob can occur at amino acid residues S354 and T366, and the hole can occur at amino acid residues Y394, T366, L368, and Y407, preferably the knob can be the following amino acid substitutions S354C and T366W, and the hole can be the following amino acid substitutions Y394C, T366S, L368A, and Y407V; the hole structure of the homodimer can be reduced, and the modification can occur at amino acid residues H435 and Y436, preferably the amino acid substitutions H435R and Y436F.

[0200] "Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" are regions of an antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen-contacting residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes.

[0201] The term "immune checkpoint" refers to a class of inhibitory signaling molecules present in the immune system that avoid tissue damage by regulating the persistence and intensity of the immune response in peripheral tissues and participate in maintaining tolerance to self-antigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12 (4): 252-264). Studies have found that one of the reasons why tumor cells can escape the immune system in the body and proliferate uncontrolled is that they utilize the inhibitory signaling pathway of the immune checkpoint, thereby inhibiting the activity of T lymphocytes, so that T lymphocytes cannot effectively exert their killing effect on tumors. Immune checkpoint molecules include but are not limited to programmed 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.

[0202] The term "PD-1" refers to programmed cell death protein 1 expressed on the surface of T cells, and its ligands PD-L1 or PD-L2 are expressed on the surface of various cell types including many cancer cells. When PD-1 binds to its ligand (PD-L1 or PD-L2), it inhibits the activation of T cells by recruiting SHP-2, thereby inhibiting T cell proliferation and effector functions, such as IFN-γ production and cytotoxic activity. Cancer cells also use this mechanism to inhibit T cell attacks on them through their surface ligands PD-L1 or PD-L2, thereby producing immune escape. The term "PD-1" used herein includes human PD-1, human PD-1 variants, isotypes and species homologs.

[0203] The term "human PD-1" refers to the human PD-1 protein encoded by the wild-type human PD-1 gene, such as that disclosed in GenBank Accession No. NM_005018.2.

[0204] The term "CTLA-4," or cytotoxic T lymphocyte-associated protein 4, inhibits immune responses by binding to the ligands CD80 (also known as B7-1) and CD86 (also known as B7-2). CTLA-4 inhibits immune responses in a variety of ways: for example, 1) competing with the T cell co-stimulatory receptor CD28 for its ligands CD80 and CD86, thereby blocking co-stimulation; and 2) sending negative signals that inhibit T cell activation. CTLA-4 inhibitors, by inhibiting the CTLA-4 molecule, can cause T cells to proliferate and attack tumor cells. The term "CTLA-4" as used herein includes human "CTLA-4," human CTLA-4 variants, isoforms, and species homologs. Human CTLA-4 is disclosed, for example, in GenBank Accession No. AAB59385.

[0205] PD-1 and CTLA-4 are immune checkpoints belonging to the CD28 family and are both expressed on activated T cells. However, their expression differs at different stages of the immune response and on different immune cells. PD-1 and CTLA-4 inhibitors activate different immune cells differently. While both can activate 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 and CTLA-4 knockout mice, PD-1 inhibitors are less toxic than CTLA-4 inhibitors, which often exhibit greater toxicity. This may be related to their depletion of peripheral Tregs. For example, the anti-CTLA-4 antibody ipilimumab, used to treat advanced melanoma, has severe side effects, limiting its widespread clinical use.

[0206] The term "VEGF" refers to vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF). It is a highly specific vascular endothelial growth factor that promotes vascular permeability, extracellular matrix degeneration, endothelial cell migration and proliferation, and angiogenesis. VEGF is a family of factors that includes VEGFA, VEGFB, VEGFC, VEGFD, VEGFE, and placental growth factor (PGF).

[0207] The term "VEGFA (vascular endothelial growth factor A)" is a highly conserved 27kDa dimeric glycoprotein secreted by a variety of cells, including endothelial cells and tumors. Common isoforms of VEGFA produced by alternative splicing are VEGFA121, VEGFA165, VEGFA189 and VEGFA206, which contain 121, 165, 189 and 206 amino acids, respectively. VEGFA includes human VEGFA, for example, the human VEGFA protein under the accession number UniProt NO.: P15692. VEGFA is a key regulator of angiogenesis during the growth of solid tumors. Therefore, treatment options targeting VEGFA are one of the current focuses of oncology, and bevacizumab is the first VEGFA inhibitor approved for marketing. Throughout this application, VEGF generally refers to VEGFA, for example, VEGF165 generally refers to VEGFA165.

[0208] The term "EC 50 ”, also known as the “half-maximal effective concentration”, is the concentration of a drug, antibody, or toxicant 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".

[0209] The term "IC 50 ", also known as the "half-inhibitory concentration", refers to the concentration of a drug or substance (inhibitor) that inhibits a biological process (or certain substances involved in the process, such as enzymes, cell receptors or microorganisms) by 50%.

[0210] The terms "flexible linker" or "linker" or "connecting 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), used alone or in combination, or from the hinge region of an immunoglobulin.

[0211] The linkers that can be used in the present invention can be easily determined by those skilled in the art. For example, 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. Linkers that can be used in the antibody molecules of the present invention may also be, for example but 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, LRQRDGERP, LRQKDGGGSERP and GSTSGSGK PGSGEGSTKG.

[0212] As used herein, the term "binding" or "specific binding" means 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.

[0213] "Percent (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variants may comprise conservative modifications.

[0214] With respect to polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions to a polypeptide sequence that result in the substitution 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 the polymorphic variants, interspecies homologs, and alleles of the present invention. The following eight groups contain amino acids that are conservative substitutions for each other: 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.

[0215] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the present invention, including eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., E. coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0216] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operatively linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those 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 is incorporated.

[0217] The terms "individual" or "subject" are used interchangeably and 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.

[0218] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival. The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and liquid tumors.

[0219] The term "cancer" refers to a physiological disorder in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. For example, the cancer is 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, biliary tract cancer, head and neck squamous cell carcinoma, thyroid cancer, brain cancer, stomach 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). In certain embodiments, cancers suitable for treatment by the antibodies of the present invention include breast cancer, gastric cancer, ovarian cancer, gastroesophageal junction cancer, bladder cancer, small intestine cancer and ampullary cancer, esophageal cancer, lung cancer, and cervical cancer, including metastatic forms of those cancers. In some embodiments, the present invention provides, inter alia, multispecific antibodies useful for tumor / cancer treatment and their use in treating such tumors / cancers.

[0220] 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 onset 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 development of the disease or to slow the progression of the disease.

[0221] The term "prevent" includes the inhibition of the development or progression of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevent" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0222] The term "effective amount" refers to an amount or dosage 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 a single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as the species of the mammal; weight, age, and general health; 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 characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0223] The term "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount of an antibody, antibody fragment, or composition can vary depending on a variety of factors, such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody, 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%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%, relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that is predictive of efficacy in human tumors.

[0224] The term "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, a prophylactic effective amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0225] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0226] II. Production and Purification of Antibodies of the Invention

[0227] To produce the trispecific antibodies of the present invention, the individual polypeptide chains of the antibodies of the present invention can be obtained, for example, by solid-state peptide synthesis (eg, Merrifield solid-phase synthesis) or recombinant production, and assembled under appropriate conditions.

[0228] In the case of recombinant production, the polynucleotide encoding any one polypeptide chain and / or multiple polypeptide chains of the antibody can be isolated and inserted into one or more vectors so that they can be subsequently cloned and / or expressed in a host cell. Using conventional methods, the polynucleotides can be easily isolated and verified, for example, by sequencing. In one embodiment, a polynucleotide encoding one or more polypeptide chains of the trispecific antibody of the present invention is provided. In another embodiment, the present invention provides a vector comprising one or more polynucleotides of the present invention, preferably an expression vector. Therefore, in one embodiment, the present invention provides a method for producing the trispecific antibody of the present invention, the method comprising: culturing a host cell comprising a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the trispecific antibody; and assembling the polypeptide chains to produce the antibody under conditions suitable for assembly of the polypeptide chains into the trispecific antibody.

[0229] Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0230] The antibodies prepared by the methods described herein can be purified by known prior art techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. After purification, the purity of the antibodies of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activities of the antibodies provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0231] III. Pharmaceutical Compositions, Drug Combinations, and Kits

[0232] In one aspect, the present invention provides compositions, e.g., pharmaceutical compositions, comprising an antibody as described herein formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible. 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 sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may comprise an antibody as described herein and one or more therapeutic agents.

[0233] In another aspect, the present invention also provides pharmaceutical combinations comprising an antibody described herein and one or more therapeutic agents.

[0234] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use. Common preferred compositions are in the form of injectable solutions or infusible solutions.

[0235] The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibodies of the present invention. A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. The therapeutically effective amount may vary depending on a variety of factors, such as the disease state, age, sex, and weight of the individual. A therapeutically effective amount is an amount in which any toxic or deleterious effects are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80% relative to an untreated subject. 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 that is predictive of efficacy in human tumors. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic outcome at the desired dosage and for the desired period of time. Typically, because prophylactic doses are used in subjects before or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

[0236] In another aspect, the present invention provides a kit comprising an antibody or composition described herein. The kit may also include one or more other elements, such as instructions for use; other reagents, such as labels or reagents for coupling; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

[0237] IV. Uses and Treatment Methods of the Antibodies and Compositions of the Invention

[0238] The trispecific antibodies of the present invention that bind to PD-1 / CTLA-4 / VEGF are suitable for use as anti-tumor, anti-angiogenesis, anti-autoimmune disease, and anti-infective drugs. In some embodiments, the trispecific antibodies according to the present invention are used to treat cancers, 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

[0239] The following examples are intended to illustrate the present invention only and therefore should not be construed as limiting the present invention in any way.

[0240] Example 1. Construction, expression and purification of anti-PD-1 / CTLA-4 / VEGF trispecific antibodies

[0241] 1.1 Sequence and structure of trispecific antibodies

[0242] This application constructs a trispecific antibody that can recognize PD-1, CTLA-4, and VEGF based on anti-PD-1 antibody, anti-CTLA-4 antibody, and anti-VEGFA antibody. The anti-PD-1 antibody is the self-developed antibody CQ1-3 / 1-11; the anti-CTLA-4 antibody is the disclosed nanobody 202F1 (application number: CN202111229808.3, sequence number: 9) or ipilimumab, which blocks the binding of CD80 to CTLA-4; the anti-VEGFA antibody is bevacizumab, which blocks the binding of VEGF to VEGFR-2 (KDR). The variable region sequences of each monoclonal antibody are shown in Table 1. In the trispecific antibody, the anti-PD-1 antibody and the anti-VEGFA antibody can be in the form of their Fab or scFv. The corresponding sequences are shown in Table 1. The antibody fragments can be connected using linker sequences commonly used in the art, such as the (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.

[0243] Table 1. Variable region sequences of the anti-PD-1 antibody CQ1-3 / 1-11, the anti-CTLA-4 antibody 202F1, and the anti-VEGFA antibody bevacizumab, and the sequences of the corresponding ScFv structures (bold indicates CDR sequences determined according to the Kabat numbering rules, and underlines indicate corresponding amino acid residue substitutions)

[0244] Table 2. hIgG1 and hIgG4 constant regions

[0245] Table 3. Structure and sequence of trispecific antibodies (underline indicates substitution of corresponding amino acid residues)

[0246] 1.2 Expression and purification of trispecific antibodies

[0247] According to the sequences of the trispecific antibodies in Table 3, polynucleotides of the corresponding sequences were synthesized and inserted into the expression vector pCDNA3.1. After sequencing was correct, expression was performed. The specific method was as follows: 100 μg of each expression plasmid encoding the light chain and heavy chain of the corresponding trispecific antibody shown in Table 3 was co-transfected into 200 mL of cells at a density of 3×10 6 ExpiCHO cells (Gibco, Catalog No. A29129) were cultured at 37°C in a CO2 shaker for 7 days. The supernatant was collected by centrifugation and filtered through a 0.22 μM filter. The filtered supernatant was transferred to a protein A column (pre-equilibrated with 5 column volumes of PBS) containing 2 mL of protein A chromatography medium, MabSelect PrismA (Cytiva, Catalog No. 17549802), for affinity chromatography. The sample was retained for at least 2 minutes, and the column was then washed with 10-15 column volumes of PBS to remove nonspecifically adsorbed impurities. The target protein was then eluted with 4 mL of 20 mM Na-Citrate, pH 3.2, and the pH of the eluate was adjusted to 5.0-6.0 with 1 M Tris. After centrifugation and filtration, protein concentration was measured using a UV spectrophotometer. Relative expression levels were calculated based on the concentrations, and protein purity was determined using size exclusion chromatography coupled with high-performance liquid chromatography (Dionex, Ultimate 3000). The expression levels and purities of the various trispecific antibodies tested are shown in Table 4.

[0248] The control antibodies used in this application were constructed as follows and expressed and purified according to the above methods. Adjusting the corresponding expression and purification parameters for specific antibodies is within the scope of routine knowledge of those skilled in the art:

[0249] The heavy chain variable region of the anti-PD-1 antibody (CQ1-3 / 1-11) in Table 1 was fused with the hIgG4-1 constant region sequence in Table 2 to obtain a full-length heavy chain; and the light chain variable region of CQ1-3 / 1-11 was fused with the light chain constant region sequence in Table 2 to obtain a full-length light chain. The resulting full-length antibody was named CQ1-3 / 1-11-hIgG4 antibody, or simply CQ1-3 / 1-11.

[0250] The heavy chain variable region of the anti-CTLA-4 antibody (202F1) in Table 1 was fused with the hIgG4-1 constant region sequence in Table 2 to obtain a full-length heavy chain; and the light chain variable region of 202F1 was fused with the light chain constant region sequence in Table 2 to obtain a full-length light chain. The full-length antibody thus obtained was named 202F1-hIgG4 antibody.

[0251] The heavy chain variable region of antibody 202F1 in Table 1 was fused with the hIgG1 constant region sequence in Table 2, and assembled with the corresponding full-length light chain described above to obtain a full-length antibody named 202F1-hIgG1.

[0252] The variable region of Ipilimumab in Table 1 was fused to the hIgG1 constant region sequence in Table 2 and assembled with the corresponding full-length light chain to obtain Ipilimumab.

[0253] Bevacizumab was obtained by fusing the variable region of bevacizumab in Table 1 with the hIgG1 constant region sequence in Table 2 and assembling with the corresponding full-length light chain.

[0254] Regarding the corresponding scFv of each antibody used in this application, the corresponding expression and purification were carried out according to the above-mentioned method based on the sequence disclosed in Table 1. Adjusting the corresponding expression and purification parameters for specific antibodies is within the scope of routine knowledge of those skilled in the art.

[0255] Table 4 Expression and purity of the three specific

[0256] Example 2. Binding activity and physicochemical properties analysis of anti-PD-1 / CTLA-4 / VEGF trispecific antibodies

[0257] 2.1 Binding activity of trispecific antibodies to PD-1

[0258] The binding activity of the trispecific antibodies to PD-1 protein and PD-1 antigen on the cell surface was detected by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS), respectively.

[0259] ELISA assays were used to determine the binding activity of the trispecific antibodies to the PD-1 protein: High-adsorption 96-well microtiter plates were coated with 1 μg / mL human PD-1 protein (Acro, Catalog No. PD1-H5221) and incubated overnight at 4°C. The next day, the plates were washed three times with PBST and blocked with 1% BSA-PBS at 37°C for 1 hour. A three-fold serial dilution of the antibodies was prepared in 1% BSA-PBS, starting at a concentration of 10 nM. Each dilution was added to the blocked 96-well microtiter plates at 100 μL / well in triplicate and incubated at 37°C for 1 hour. The plates were then washed three times with 0.05% PBST, and 100 μL / well of a 1:5000 dilution of HRP-conjugated secondary antibody (Sigma, Catalog No. A0170) was added to the plates and incubated at 37°C for 1 hour. The plate was washed three times with 0.05% PBST solution, and TMB was used for color development. The absorbance of the 96-well plate at 450 nm was detected by an enzyme-labeled instrument. The OD 450 The EC values ​​of each antibody binding to PD-1 were fitted and calculated. 50 The results are shown in Table 5.

[0260] FACS assay for the binding activity of the trispecific antibodies to the cell surface PD-1 antigen: 293T cells overexpressing PD-1 (293T-hPD-1, Kangyuan Broad, KC-0204) were digested with trypsin (Gibco, 25200072), washed twice with PBS containing 2% fetal bovine serum (FBS) (FACS buffer), and resuspended in FACS buffer at a concentration of approximately 1×10 5 Cells were added to a 96-well plate at 1:100 per well. After centrifugation at 300 g for 5 minutes, the supernatant was discarded. Antibodies were serially diluted three-fold in FACS buffer to create 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 of the antibody was then added to a 96-well plate in triplicate, mixed, and incubated at 4°C for 60 minutes. After centrifugation and washing the plate three times with FACS buffer, 100 μL / well of AF647-anti-hIgG (H+L) (Jackson, Cat. No. 109-605-003) diluted 1:800 was added and incubated at 4°C for 50 minutes. After washing the plate three times with FACS buffer, the cells were resuspended in 100 μL / well of FACS buffer and fluorescence signals were detected on a flow cytometer (BECKMAN COULTER cytoFLEX). The four-parameter model was used to fit the binding activity and calculate the EC of each antibody binding to PD-1. 50 The results are shown in Table 5 and Figure 2.

[0261] In order to compare the performance of the self-developed antibody CQ1-3 / 1-11 with the corresponding commercial 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 self-developed antibody CQ1-3 / 1-11 and pembrolizumab had comparable ability to bind to the cell surface PD-1. 50 The values ​​were 4.34 nM and 5.05 nM, respectively.

[0262] Table 5 Binding activity of trispecific antibodies to PD-1, CTLA-4 and VEGFA (EC 50 ,nM) N / A not applicable, NT not tested; FACS#1-#4 refer to different experimental batches, hIgG4 (Taizhou Baiying Biotechnology Co., Ltd., Catalog No.: B107804) is a negative control unrelated to the antibody of this application.

[0263] 2.2 Binding activity of trispecific antibodies to CTLA-4

[0264] The binding activity of the trispecific antibody to CTLA-4 protein and cell surface CTLA-4 antigen was detected by ELISA and FACS respectively as described in Section 2.1.

[0265] The human CTLA-4 protein used was purchased from Acro, catalog number: CT4-H52H9. The results are shown in Table 5.

[0266] In FACS detection, CHOK1 cells overexpressing human CTLA-4 (CHO-hCTLA-4, Yoshiman Bio, GM-C18989) were used, with a density of approximately 2x10 5 Cells / well were added to a 96-well U-bottom plate. Antibodies were serially diluted 3-fold in FACS buffer, starting at a concentration of 200 nM. The results are shown in Table 5 and Figure 3.

[0267] 2.3 Binding activity of trispecific antibodies to VEGFA

[0268] The binding activity of the trispecific antibodies to VEGFA protein was determined using an ELISA assay as described in Section 2.1. Human VEGF165-his protein was purchased from Acro, Catalog No. VE5-H5248. The positive control antibody, bevacizumab, was purchased from Roche, Catalog No. 0210008H1545. The results are shown in Table 5.

[0269] 2.4 Binding activity of trispecific antibodies to activated T cells

[0270] Activated T cells upregulate the expression of PD-1 and CTLA-4. Human peripheral blood T cells activated with anti-CD3 and anti-CD28 antibodies were used to test the binding activity of the trispecific antibodies to activated T cells. The anti-PD-1 antibodies permbrolizumab (Merck, Catalog No. S028905), nivolumab (Bio-Innovative, Catalog No. B6924), sintilimab (Bio-Innovative, Catalog No. B682101), and camrelizumab (Bio-Innovative, Catalog No. B852001) and the anti-CTLA-4 antibody ipilimumab served as positive controls.

[0271] Human peripheral blood mononuclear cells (PBMC, Miaoshun (Shanghai) Biotechnology Co., Ltd., catalog number: A19K154025) were rapidly revived in a 37°C water bath, resuspended in T cell separation buffer and counted. After centrifugation at 400 g for 10 minutes, the supernatant was discarded and the cells were resuspended in T cell separation buffer to 5x10 7 cells / mL. Isolate T cells according to the T cell isolation kit (Stemcell, Cat. No. 19051) and resuspend the obtained T cells in culture medium. Count the cells using NucleoCounter NC-200 and adjust the T cell density to approximately 1x10 6 cells / mL. Dynabeads containing anti-CD3 and anti-CD28 antibodies (Invitrogen TM , Catalog No.: 11132D) and 10 ng / mL hIL-2 (Peprotech, Catalog No.: 200-02) were cultured in a 37°C, 5% CO2 incubator for 3 days. The activated T cells were removed from the dynabeads and supernatant and resuspended in FACS buffer. The cell density was adjusted to approximately 1x10 6 cells / mL, 100 μL of cell solution was added to a 96-well U-bottom plate, 1x10 5cells / well. After centrifugation at 500g for 5 minutes, discard the supernatant and add 100μL of the trispecific antibody of the present application diluted 3 times with FACS buffer, with a starting concentration of 200nM, in triplicate. Mix well and incubate at 4°C for 60 minutes. After centrifugation and washing 3 times with FACS buffer, 100μL / well of PE anti-human IgG Fc (Biolegend, Catalog No.: 366903) was added and incubated at 4°C for 50 minutes. After washing 3 times with FACS buffer, 100μL / well of BV421 anti-human CD3 antibody (OKT3) (Biolegend, Catalog No.: 317344) was added and incubated at 4°C for 50 minutes. After washing 3 times with FACS buffer, 70μL / well of PBS solution was added to resuspend the cells, and the fluorescence signal on CD3T cells was detected on a flow cytometer (BDCelesta). The binding activity was fitted using a four-parameter model to calculate the EC value of each antibody binding to T cells. 50 value.

[0272] As shown in Figure 4 and Table 6, the commercial anti-PD-1 antibodies pembrolizumab, nivolumab, sintilimab, and camrelizumab all bind to the activated T cells in a dose-dependent manner. In addition, the commercial anti-CTLA-4 antibody ipilimumab also binds to the activated T cells in a dose-dependent manner, indicating that the T cells activated by the method of this example express PD-1 and CTLA-4 on their surfaces. Since 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.

[0273] As shown in FIG4 and Table 6, the trispecific antibodies HC010-F8 and HC010-F23 prepared in the present application can bind to activated T cells in a dose-dependent manner, and their binding ability is better than that of the anti-CTLA-4 antibodies ipilimumab and 202F1.

[0274] Table 6 Binding activity of specific antibodies to activated T cells N / A Not applicable. hIgG1 and hIgG4 are negative controls not related to the antibodies of this application.

[0275] 2.5 Trispecific Antibody Simultaneous Binding Activity to PD-1, CTLA-4, and VEGFA

[0276] 2.5.1 Trispecific Antibodies Bind to PD-1 Protein and Then to CTLA-4 or VEGFA

[0277] Coat a high-adsorption 96-well microtiter plate with 1 μg / mL human PD1-his (Acro, Catalog No. PD1-H5221) and incubate overnight at 4°C. Wash the plate with PBST and block with 300 μL / well of 1% BSA-PBS at 37°C for 2 hours. Simultaneously, prepare a 3-fold serial dilution of the antibody in 1% BSA-PBS, starting at a concentration of 20 nM. After washing, add 100 μL of each serial dilution of the antibody to each well in triplicate and incubate at 37°C for 1 hour. The plate was then machine-washed three times with 0.05% PBST. A 100 μL / well portion of 1 μg / mL Biotinylated-hCTLA4 (Acro, Catalog No. CT4-H82E3) was added to each well to detect the binding activity of the trispecific antibody to CTLA-4 after binding to the PD-1 protein. A 100 μL / well portion of 1 μg / mL Biotinylated-hVEGF165 (Acro, Catalog No. VE5-H82Q0) was added to each well to detect the binding activity of the trispecific antibody to VEGF after binding to the PD-1 protein. Finally, hIgG4 was added as a negative control. After incubation at 37°C for 1 hour, the plate was machine-washed three times with PBST. A 1:5000 dilution of Streptavidin-HRP (BD pharmingen, Catalog No. 554066) was added to each well at 100 μL per well and incubated at 37°C for 45 minutes. 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 measured using an ELISA reader. The EC value of the antibody was calculated using a four-parameter model. 50 value.

[0278] The results are shown in Figure 13. The trispecific antibodies HC010-F8 and HC010-F23 can still bind to human VEGF after binding to human PD1 (Figure 13A, EC 50 0.049 and 0.054 nM, respectively) or human CTLA-4 (Figure 13B, EC 50 The binding activity of β-actin was 0.105 and 0.250 nM, respectively, and the binding activity was dose-dependent.

[0279] 2.5.2 Trispecific Antibodies Bind to Cell Surface PD-1 and Simultaneously Bind to CTLA-4 and VEGFA

[0280] 293T-hPD-1 cells were obtained by trypsinization and washed twice with PBS containing 2% FBS (FACS buffer) to adjust the cell density to approximately 6×10 5Cells were centrifuged at 300 g for 5 minutes, and 200 μL / well 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 antibody serially diluted in FACS buffer (starting at 200 nM, serially diluted 3-fold; the same volume of FACS buffer was used as a negative control) was added to each well in triplicate. Mix well and incubate at 4°C for 60 minutes. After washing three times with FACS buffer, 100 μL / well of a mixture consisting of 1 μg / mL Biotin-hVEGF165 and 1 μg / mL hCTLA4-mFc (DIMA BIOTECH, Catalog No. PME100017) at a 1:1 volume ratio was added to each well. The activity of the trispecific antibodies in binding to cell surface PD-1 and simultaneously to CTLA-4 and VEGF was tested. After incubation at 4°C for 60 minutes, the cells were washed three times with FACS buffer and a mixture of AF647 donkey anti-mIgG (H+L) (1:800 dilution, Invitrogen, Catalog No.: A31571) and PE Streptavidin (1:800 dilution, BD, Catalog No.: 554061) was added to each well at 100 μL / well and incubated at 4°C for 50 minutes. After washing three times with FACS buffer, 100 μL / well of PBS solution was added to resuspend the cells and the fluorescence signal was detected on a flow cytometer (BECKMAN COULTER cytoFLEX). The binding activity was fitted using a four-parameter model to calculate the EC of each antibody. 50 value.

[0281] The results are shown in Figure 14. The trispecific antibodies HC010-F8, HC010-F10, HC010-F22, HC010-F23 and HC010-F24 can still bind to human VEGF at the same time after binding to human PD-1 on the cell surface (Figure 14A, EC 50 1.413, 2.835, 2.288, 2.223 and 9.151 nM, respectively) and human CTLA-4 (Figure 14AB, EC 50 The binding activity of the two drugs was 2.744, 3.323, 5.196, 3.837 and 12.000 nM, respectively, and the simultaneous binding activity was dose-dependent.

[0282] 2.5.3 Trispecific Antibodies Bind to Cell Surface CTLA-4 and Simultaneously Bind to PD-1 and VEGFA

[0283] CHOK1-hCTLA4 cells were obtained by trypsinization and washed twice with PBS containing 2% FBS (FACS buffer) to adjust the cell density to approximately 8×10 5Cells were plated at 200 μL / well in a 96-well U-bottom plate at 300 g for 5 minutes. The supernatant was discarded and 100 μL of antibody serially diluted in FACS buffer (starting at 200 nM, serially diluted 3-fold; the same volume of FACS buffer was added as a negative control) was added in triplicate. Mix well and incubate at 4°C for 60 minutes. After washing 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 (1:1 volume ratio) was added to each well at 100 μL / well. The activity of the trispecific antibodies in binding to cell surface CTLA-4 and then to VEGF and PD-1 was tested. After incubation at 4°C for 60 minutes, the cells were washed three times with FACS buffer. A mixture of AF647 donkey anti-mIgG (H+L) (1:800 dilution) and PE Streptavidin (1:800 dilution) at a 1:1 volume ratio was added to each well at 100 μL / well and incubated at 4°C for 50 minutes. After washing three times with FACS buffer, 100 μL / well of PBS solution was added to resuspend the cells, and the fluorescence signal was detected on a flow cytometer. The binding activity was fitted using a four-parameter model to calculate the EC of each antibody. 50 value.

[0284] The results are shown in Figure 15. The trispecific antibodies HC010-F8, HC010-F9 and HC010-F10 can bind to human CTLA-4 on the cell surface and can also bind to human PD-1 (Figure 15A, EC 50 1.919, 1.533 and 2.734 nM, respectively) and human VEGF (Figure 15B, EC 50 The binding activity of 1.435, 1.376 and 1.370 nM, respectively, was also dose-dependent.

[0285] 2.6 Binding activity of trispecific antibodies to free VEGFA after binding to cells co-expressing PD-1 and CTLA-4

[0286] In the tumor microenvironment, trispecific antibodies can bind to PD-1 and CTLA-4 on immune cells and to VEGFA, simultaneously blocking immunosuppression and angiogenesis, thereby regulating the immune response and angiogenesis in the tumor microenvironment. This example used 293T cells overexpressing PD-1 and CTLA-4 and activated T cells to test the binding activity of trispecific antibodies to free VEGFA after binding to cells co-expressing PD-1 and CTLA-4.

[0287] 2.6.1 Binding of trispecific antibodies to cells co-expressing PD-1 and CTLA-4 and to VEGFA

[0288] 293T cells overexpressing human PD-1 and human CTLA-4 (293T-hPD-1-hCTLA-4, Yoshiman Bio, GM-C19526) were used to detect the binding activity of trispecific antibodies 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 the trispecific antibodies bound to 293T-hPD-1-hCTLA-4 cells through the anti-PD-1 and CTLA-4 arms, the trispecific antibodies bound to 293T-hPD-1-hCTLA-4 cells were detected with biotin-labeled VEGFA. 293T-hPD-1-hCTLA-4 cells were digested with trypsin, and the separated cells were resuspended in FACS buffer and the cell density was adjusted to approximately 1x10 6 cells / mL, and then 200 μL was added to each well of a 96-well U-bottom plate, with a cell density of approximately 2x10 5 cells / well. Centrifuge at 300g for 5 minutes, discard the supernatant, add 100μL of trispecific antibody diluted 3 times with FACS buffer, with a starting concentration of 200nM, and add the same volume of FACS buffer as a negative control. Mix well and incubate at 4℃ for 60 minutes. Centrifuge and wash 3 times with FACS buffer, add 2μg / mL Biotin-hVEGF165 to 100μL / well, and incubate at 4℃ for 60 minutes. Centrifuge and wash 3 times with FACS buffer, add 1:800 diluted PE Streptavidin (BD, Catalog No.: 554061) to 100μL / well, and incubate at 4℃ for 50 minutes. After centrifugation and washing 3 times with FACS buffer, resuspend the cells with 100μL / well FACS buffer, and detect the fluorescence signal on a flow cytometer (BECKMAN COULTER cytoFLEX). The binding activity was fitted using a four-parameter model to calculate the EC value of each antibody binding. 50 value.

[0289] As shown in Figure 5, the trispecific antibodies HC010-F8, HC010-F9, and HC010-F10 can still bind to free VEGFA after binding to cells co-expressing PD-1 and CTLA-4. The EC values ​​of the binding activity of HC010-F8, HC010-F9, and HC010-F10 to VEGFA on the cell surface are shown in Figure 5. 50 The results were 5.057, 4.204 and 4.904 nM, respectively, indicating that the trispecific antibody still had high binding activity with VEGFA after binding to cells co-expressing PD-1 and CTLA-4.

[0290] 2.6.2 Binding activity of trispecific antibodies to free VEGFA after binding to activated T cells

[0291] Use the activated T cells in section 2.4 and adjust the cell density to 1x10 6 cells / mL, 100 μL of cells were added to a 96-well U-bottom plate, 1x10 5 cells / well. After centrifugation at 500g for 5 minutes, discard the supernatant and add 100μL of antibody diluted 3 times with FACS buffer, with a starting concentration of 200nM, in triplicate. Mix well and incubate at 4°C for 60 minutes. Centrifuge and wash 3 times with FACS buffer, add 2μg / mL Biotinylated Human VEGF165 (Acro, Catalog No.: VE5-H82Q0) to 100μL / well and incubate at 4°C for 50 minutes. After washing 3 times with FACS buffer, add a mixture of BV421 anti-human CD3 antibody (OKT3) (Biolegend, Catalog No.: 317344) and PE Streptavidin, 100μL / well, and incubate at 4°C for 50 minutes. After washing 3 times with FACS buffer, add PBS to 70μL / well to resuspend the cells, and detect the fluorescence signal on CD3T cells on a flow cytometer (BD Celesta). The binding activity was fitted using a four-parameter model to calculate the EC of each antibody. 50 value.

[0292] As shown in Figure 6, the trispecific antibodies HC010-F8 and HC010-F23 can still bind to free VEGFA after binding to activated T cells. The EC of HC010-F8 and HC010-F23 binding activity to VEGFA on the surface of T cells 50 The values ​​were 0.440 and 0.377 nM, respectively, indicating that the trispecific antibody still had high binding activity with VEGFA after binding to T cells co-expressing PD-1 and CTLA-4, and could simultaneously block immune checkpoints and angiogenesis in the tumor microenvironment.

[0293] 2.7 Affinity determination of trispecific antibodies to human PD-1, CTLA-4, and VEGFA

[0294] use The RED96e (ForteBio) assay was used to detect the affinity of trispecific antibodies for human PD-1, CTLA-4, and VEGFA. The specific method was as follows: The AHC sensor was immersed in PBST (PBS containing 0.02% Tween) buffer for 10 minutes to activate the sensor. 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. The intermediate concentrations were set as quality control points, and a blank control point of 0 nM was set. 200 μL / well of each diluted ligand and analyte was added to the corresponding wells in triplicate. Program parameters were set as follows: loading: 180 s, association: 180 s, dissociation: 600 s, regeneration: Gly-HCl pH 1.5, 30 s, regeneration three times. Data Analysis 11.1r software was used to fit the curve using a 1:1 model after subtracting the blank (0 concentration point). Curve fit acceptance criteria: Full R² ≥ 0.95.

[0295] 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, reaching 10 -9 order of magnitude.

[0296] Table 7 Affinity of trispecific antibodies to human PD-1, CTLA-4 and VEGFA NT: Not tested

[0297] Example 3. Blocking activity of anti-PD-1 / CTLA-4 / VEGF trispecific antibody

[0298] 3.1 Trispecific antibodies block the binding activity of PD-1 and PD-L1

[0299] Competitive ELISA and FACS were used to detect the binding activity of trispecific antibodies in blocking PD-1 and PD-L1.

[0300] ELISA was used to detect the binding activity of trispecific antibodies to PD-1 and PD-L1: 1 μg / mL human PD-1 protein (Acro, PD1-H5257) was used to coat a high-adsorption 96-well microtiter plate and incubate overnight at 4°C. The next day, the plate was washed three times with PBS and blocked with 1% BSA-PBS solution at 37°C for 1 hour. A serial dilution of the antibody prepared in 1% BSA-PBST (starting at 200 nM, 3-fold serial dilution) and 1 μg / mL biotin-labeled PD-L1 protein (Acro, PD1-H82F3) were mixed in a 1:1 ratio to obtain an antibody-protein mixture. 100 μL of the antibody-protein mixture was added to each well of the blocked 96-well microtiter plate and incubated at 37°C for 1 hour. After washing 4 times with PBST, 100 μL of Strep-HRP (BD, Cat. No. 554066) diluted 1:5000 was added to the microplate per well and incubated at 37°C for 1 hour. After washing 4 times with PBST, TMB was used for color development. The absorbance at 450 nm and 630 nm was measured using a microplate reader, and the OD450-OD630 was calculated. The fluorescence intensity was fitted using a four-parameter model to calculate the IC50 of each antibody blocking activity. 50 The results are shown in Table 8.

[0301] In order to compare the performance of the self-developed antibody CQ1-3 / 1-11 with pembrolizumab, the applicant used the same method to detect the activity of the control self-developed antibody CQ1-3 / 1-11-hIgG4 in blocking the binding of cell surface antigen PD-1 to PD-L1. The results showed that the self-developed antibody CQ1-3 / 1-11 had comparable blocking ability with pembrolizumab, IC 50 The values ​​were 3.190 nM and 3.569 nM, respectively.

[0302] FACS detection of the binding activity of trispecific antibodies to block PD-1 and PD-L1: 293T cells overexpressing human PD-1 (293T-hPD-1, Kangyuan Bochuang, KC-0204) were used to detect the blocking ability of trispecific antibodies on the binding of human PD-1 and human PD-L1. 293T-hPD-1 cells were obtained by trypsin digestion, and the cells were washed twice with PBS containing 2% FBS (FACS buffer) and resuspended in FACS buffer. 1μg / mL biotin-human PDL1 (Acro, Catalog No.: PD1-H82F3) was added to the suspended cell solution, and 50μL / well was added to a 96-well U-bottom plate, about 1x10 5cells / well. Add 50 μL of antibody diluted 3 times with FACS buffer, with a starting concentration of 200 nM, in triplicate. Mix well and incubate at 4°C for 60 minutes. After washing 3 times with FACS buffer, add 1:800 diluted PE Streptavidin to 100 μL / well and incubate at 4°C for 50 minutes. After washing 3 times with FACS buffer, add 150 μL / well PBS to resuspend the cells and detect the fluorescence signal on a flow cytometer (BECKMAN COULTER cytoFLEX). The binding activity was fitted using a four-parameter model to calculate the IC of each antibody blocking activity. 50 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.

[0303] Table 8 Blocking activity of trispecific antibodies (IC 50 ,nM) NT: not tested; N / A: not applicable; hIgG4 is a negative control.

[0304] 3.2 Trispecific antibodies block the binding activity of CD80 and CTLA-4

[0305] 3.2.1 Trispecific Antibody Blocks CD80-CTLA-4 Binding Activity

[0306] CHO-hCTLA-4 cells were used to test the ability of the trispecific antibodies to block the 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 were added to a 96-well U-bottom plate, and approximately 2x10 5 cells / well. After centrifugation at 300g for 5 minutes, discard the supernatant. Use FACS buffer solution to prepare a series of antibody concentration dilution gradients (starting concentration is 200nM, 3-fold gradient dilution) and 0.6μg / mL biotin-labeled human CD80 protein (Acro, catalog number: B71-H82F2). Add 50μL of antibody and 50μL of biotin-human CD80 to each well of the 96-well plate, mix well, and incubate at 4°C for 60 minutes. After washing 3 times with FACS buffer, add 100μL / well of PE Streptavidin diluted 1:800 and incubate at 4°C for 50 minutes. After washing 3 times with FACS buffer, add 150μL / well of PBS to resuspend the cells, and detect the fluorescence signal on a flow cytometer (BECKMAN COULTER cytoFLEX). The binding activity was fitted using a four-parameter model to calculate the IC of each antibody blocking activity. 50The results are shown in Table 8, which show that the trispecific antibody can effectively block the binding of human CTLA-4 to human CD80.

[0307] 3.2.2 Trispecific Antibody Blocks CD80 Binding Activity to CTLA-4 on Cells Co-expressing PD-1 and CTLA-4

[0308] PD-1 and CTLA-4 are co-expressed on functionally exhausted T cells in the tumor microenvironment, and the expression level of PD-1 is higher than that of 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. 293T cells that simultaneously overexpress human PD-1 and human CTLA-4 (293T-hPD-1-hCTLA-4, Yoshiman Bio, GM-C19526, also known as 293T-CTLA-4-PD-1 cells) were used to detect the blocking ability of trispecific antibodies on the binding of human CTLA-4 and human CD80. 293T-hPD-1-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 were added to a 96-well U-bottom plate, approximately 1.4x10 5 cells / well. After centrifugation at 300g for 5 minutes, discard the supernatant. Use FACS buffer solution to prepare a series of antibody concentration dilution gradients (starting concentration is 400nM, 3-fold gradient dilution) and 2μg / mL biotin-labeled human CD80 protein. Add 50μL of antibody per well and 50μL of biotin-human CD80 per well to the 96-well plate, mix well, and incubate at 4°C for 60 minutes. After washing 3 times with FACS buffer, add 100μL / well of PE Streptavidin diluted 1:800 and incubate at 4°C for 50 minutes. After washing 3 times with FACS buffer, add 150μL / well of PBS to resuspend the cells, and detect the fluorescence signal on a flow cytometer (BECKMAN COULTER cytoFLEX). The binding activity was fitted using a four-parameter model to calculate the IC of each antibody blocking activity. 50 value.

[0309] The results are shown in Figure 8. Compared with the parent antibody, the trispecific antibodies significantly enhanced the blocking effect on the binding of CTLA-4 to CD80 on 293T-hPD-1-hCTLA-4 cells.

[0310] 3.3 Trispecific antibodies block the binding activity of VEGFA and KDR (VEGFR2)

[0311] 3.3.1 Competitive ELISA to detect the binding activity of trispecific antibodies to VEGFA and KDR (VEGFR2)

[0312] A high-adsorption 96-well microtiter plate was coated with 1 μg / mL human VEGF165-His (Acro, Catalog No. VE5-H5248) and incubated at 37°C for 2 hours. After washing, each well was blocked with 300 μL of 1% BSA-PBS at 37°C for 1.5 hours. 50 μL of serially diluted antibody (starting at 400 nM, serially diluted 3-fold) and 50 μL of 1.72 μg / mL biotin-human KDR (Acro, Catalog No. KDR-H82E5) were added to each well of the microtiter plate and incubated at 37°C for 1 hour. After washing three times with PBST, 100 μL / well of a 1:5000 dilution of Streptavidin-HRP (BD pharmingen, Catalog No. 554066) was added to the plate and incubated at 37°C for 45 minutes. After washing three times with PBST, TMB was used for color development. The absorbance at 450 nm was detected using a microplate reader. The IC value of the antibody blocking activity was calculated using a four-parameter model. 50 The results are shown in Table 8. The blocking activity of the trispecific antibody on VEGFA binding to KDR is comparable to that of bevacizumab.

[0313] 3.3.2 Reporter gene assay to detect the blocking effect of trispecific antibodies on the binding of VEGF to KDR

[0314] The ability of the trispecific antibody to block the binding of human VEGFA to human KDR was tested using the human VEGFR2-293 reporter cell line (Jiman Bio, GM-C09057). The human VEGFR2-293 reporter cell line is a luciferase reporter cell line constructed based on the NFAT signaling pathway. When VEGF binds to the KDR receptor, it activates the NFAT signaling pathway, leading to luciferase expression.

[0315] VEGFR2-293 reporter cells were obtained by trypsin digestion and centrifuged at 300g for 5 minutes. The supernatant was discarded. The cells were resuspended in DMEM medium containing 1% FBS (test medium). 0.6 μg / mL hVEGF165-His was added to the cell suspension and the cells were plated into a 96-well transparent plate at a rate of approximately 1x10 4 Prepare a 3-fold dilution series of antibodies in assay medium, starting at a concentration of 22.2 nM. Add to a 96-well plate, mix thoroughly, and incubate at 37°C for 6 hours. Add luciferase buffer (Promega, Cat. No. G7940) and incubate in the dark for 5 minutes. Detect fluorescence signals using a Microplate Reader (CLARIOstar Plus).

[0316] 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, and its blocking efficiency is comparable to that of the parent antibody bevacizumab.

[0317] Example 4: Trispecific Antibodies Activate Immune Responses and Inhibit VEGF-Induced Cell Proliferation in Vitro

[0318] 4.1 Trispecific antibodies enhance T cell function

[0319] 4.1.1 Trispecific Antibodies Enhance T Cell IL-2 Secretion in SEB-Stimulated PBMCs

[0320] Staphylococcal enterotoxin B (SEB) is a superantigen that can activate large numbers of T cells at low concentrations, generating a strong immune response. It can directly bind to T cell receptors and MHC molecules without being processed into antigenic peptides to activate T cells. Anti-PD-1 and anti-CTLA-4 antibodies can promote SEB-stimulated T cell expression and secretion of IL-2.

[0321] This experiment examined the effect of trispecific antibodies on IL-2 secretion by SEB-stimulated T cells. An irrelevant, human anti-HEL hIgG4 (Taizhou Baiying Biotechnology Co., Ltd., Catalog No. B107804) was used as a negative control. PBMCs were rapidly revived 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 adjusted to approximately 1x10 6 cells / mL, add SEB (Toxin technology, Catalog No.: 92815B) to the cell suspension to a final concentration of 200 ng / mL. 100 μL / well of the cell suspension mixed with SEB is added to a flat-bottom 96-well plate. Antibodies and control antibodies are serially diluted 3-fold with culture medium to a starting concentration of 400 nM. 100 μL / well of the 96-well plate is added to the cells and mixed thoroughly in triplicate. Culture in a 37°C, 5% CO2 incubator for 3 days. After 3 days, the supernatant is collected and IL-2 secretion is measured using the Human IL-2 Detection Kit (CisBio, Catalog No.: 62HIL02PEH) according to the manufacturer's instructions. The values ​​are read on a CLARIOstar Plus.

[0322] The results are shown in Figure 7 , and the trispecific antibodies HC010-F8, HC010-F10, HC010-F22, HC010-F23, and HC010-F24 can all enhance the secretion of IL-2 by SEB-stimulated PBMCs.

[0323] 4.1.2 Trispecific Antibodies Enhance T Cell IL-2 Secretion in Mixed Lymphocyte Reactions (MLRs)

[0324] When mature dendritic cells (DCs) from different donors are co-incubated with PBMCs, DCs promote IL-2 expression and secretion by activating PBMCs (primarily T cells). 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, reducing IL-2 cytokine expression. Immune checkpoint inhibitors, such as anti-PD-1 antibodies, can promote cytokine secretion in MLR responses.

[0325] This experiment examined the effect of a trispecific antibody on IL-2 secretion in MLR. DCs (Allcells) and PBMCs (Allcells) were rapidly revived in a 37°C water bath and resuspended in X-VIVO15 (Lonza, Cat. No. 04-418Q) medium. The cell density of DCs and PBMCs was adjusted to 1x10 5 / mL and 2x10 6 DCs and PBMCs were mixed at a 1:1 ratio. After mixing, 200 μL of the cell mixture was added to a round-bottom 96-well plate. Trispecific antibodies and control antibodies were serially diluted 5-fold with X-VIVO 15 solution, starting at a concentration of 1000 nM. 50 μL / well was added to the 96-well plate and mixed with the cells in triplicate. The cells were cultured in a 37°C, 5% CO2 incubator for 4 days. After 4 days, the supernatant was collected and IL-2 secretion was measured using a human IL-2 detection kit according to the manufacturer's instructions. The values ​​were read on a CLARIOstar Plus.

[0326] The results are shown in Figure 8 . The trispecific antibodies HC010-F8, HC010-F10, HC010-F22, HC010-F23, and HC010-F24 can all enhance the secretion and expression of IL-2 in MLR. Their activity is superior to that of the parent anti-CTLA-4 antibody 202F1-hIgG4 and is comparable to that of the anti-PD-1 antibody CQ1-3 / 1-11 and the combination of 202F1-hIgG4 and CQ1-3 / 1-11.

[0327] The same approach was used to compare the effects of antibodies CQ1-3 / 1-11 and pembrolizumab on IL-2 secretion in MLR and found that CQ1-3 / 1-11 and pembrolizumab had comparable activity.

[0328] 4.1.3 Trispecific Antibodies Enhance T Cell IL-2 Secretion in Mixed Lymphocyte Reactions (MLRs) Containing Tregs

[0329] Blocking PD-1 activity on effector T cells can promote anti-tumor immunity. However, since Treg cells also express high levels of PD-1, blocking PD-1 activity on Tregs can increase the inhibitory function of Tregs, thereby inhibiting anti-tumor immunity. This may be related to the poor efficacy of anti-PD-1 treatment in some cancer patients. Studies have found that the combined use of anti-PD-1 antibodies 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, this example uses MLR containing different ratios of Tregs to detect the activation performance of trispecific antibodies on immune cells in the presence of Tregs.

[0330] PBMCs (all cells), mature DCs (all cells), and activated Tregs (all cells) from three different healthy donors were revived. RPMI 1640 complete medium (Gibco, catalog number: 61870-036) was used to adjust the cell density of PBMCs and DCs to 2×10 6 cells / mL and 0.1×10 6 cells / mL, and 50 μL was placed in each well of a U-bottom 96-well plate. The Treg cell density was adjusted to 0.5×10 cells / mL using RPMI1640 complete medium. 6 cells / mL, and then serially diluted 2-fold to 0.25×10 6 , 0.125×10 6 and 0.0625×10 6 Treg cells were plated at different densities (50 μL per well) in the 96-well plates described above. Antibodies HC010-F8, HC010-F23, CQ1-3 / 1-11, 202F1, bevacizumab, a combination of CQ1-3 / 1-11 and 202F1-hIgG4, a combination of CQ1-3 / 1-11 and 202F1-hIgG4 and bevacizumab, and an unrelated isotype antibody were serially diluted 4-fold in RPMI 1640 complete medium, starting at 2000 nM. These serially diluted samples were then diluted 1:10 in culture medium and plated 50 μL per well in triplicate in the 96-well plates described above. The cells were cultured at 37°C, 5% CO₂, for 4 days. After 4 days, the supernatant was centrifuged and IL-2 release was measured using hIL-2 HTRF (Cisbio, Cat. No. 62HIL02PEH) according to the manufacturer's instructions.

[0331] The results, as shown in Figure 9, show that IL-2 release in the MLR is suppressed when a high proportion of Tregs is present. The trispecific antibodies HC010-F8 and HC010-F23 increased IL-2 release, with effects comparable to those seen in the combination of CQ1-3 / 1-11 and 202F1-hIgG4, and in the combination of bevacizumab, CQ1-3 / 1-11, and 202F1-hIgG4.

[0332] 4.2 Trispecific antibodies inhibit VEGF-induced proliferation of human umbilical vein endothelial cells (HUVEC)

[0333] Human umbilical vein endothelial cells (HUVEC) express VEGF receptors and are therefore induced to proliferate by VEGF. They can be used to evaluate the angiogenic effects of anti-VEGF antibodies such as bevacizumab in inhibiting VEGFA. This example also uses HUVEC (ATCC, CRL-1730) to detect the inhibition of VEGF function by trispecific antibodies. HUVEC cells were obtained by trypsinization and resuspended in F-12K medium (Gibco, Cat. No. 21127-022) containing 10% FBS. The cell density was adjusted to 1x10 5 cells / mL, add 50 μL of cell solution into a 96-well transparent bottom plate, about 5x10 3 Cells / well. Use the test culture medium to prepare the gradient dilution of the antibody (starting concentration is 200nM, 3-fold gradient dilution) and 0.4μg / mL human VEGFA 165 protein, add 25μL of antibody per well and 25μL of human VEGFA165 per well to the 96-well plate, mix well, and incubate at 37°C for five days. After five days, 60μL / well of CellTiter-Glo (Promega, catalog number: G7572) was added, and the fluorescence signal was detected on a Microplate Reader (CLARIOstar Plus). The IC of the antibody inhibitory activity was calculated using a four-parameter model. 50 value.

[0334] As shown in Figure 10, the trispecific antibodies HC010-F8 and HC010-F23 can effectively block VEGFA-induced HUVEC cell proliferation, and their blocking activity IC 50 The IC values ​​of the parent antibody bevacizumab for blocking activity were 0.860 and 0.828 nM, respectively. 50 The concentration of hIgG1 and hIgG4 is 0.367 nM. hIgG1 and hIgG4 are negative control antibodies unrelated to the antibodies of the present application.

[0335] Example 5 In vivo efficacy of trispecific antibodies

[0336] 5.1 Trispecific Antibody HC010-F8 Induces Long-Lasting Antitumor Effects

[0337] Human melanoma A375 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). When the A375 cell density reached 60-80%, PBMC (Allcells, Cat. No. FPB004F-C) were resuspended in RPMI1640 medium and the cell density was adjusted to 3×10 6 cells / mL, and co-cultured with A375 cells treated with Mitomycin C. After 5 days of co-culture of PBMC and A375, PBMC and freshly digested A375 cells were collected. 5 cells / mouse, A375 cells 4×10 6 cells / mouse, inoculated subcutaneously on the right side of NCG female mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.), with an inoculation volume of 0.2 mL / mouse, containing 50% Matrigel (BD, Cat. No.: 354234). On the day of cell inoculation, the mice were randomly divided into 4 groups (5 mice per group) and subcutaneously injected with trispecific antibody HC010-F8 (1 and 5 mg / kg), three monoclonal antibody combination drugs CQ1-3 / 1-11 (0.65 mg / kg), ipilimumab (0.35 mg / kg) and bevacizumab (0.65 mg / kg) or negative control (PBS), twice a week for 3 weeks. Tumor volume was measured twice a week using a vernier caliper, and the tumor volume was calculated as V = 0.5 × a × b 2 , a, b represent the long diameter and wide diameter of the tumor, respectively.

[0338] The results, as shown in Figure 11, show that the antibody HC010-F8 effectively inhibited tumor growth at both 1 mg / kg and 5 mg / kg doses. After drug cessation, both HC010-F8 treatment groups (1 and 5 mg / kg) and the combination group delayed tumor regrowth relative to the negative control group. Furthermore, the tumor inhibition effect of HC010-F8 (1 and 5 mg / kg) was superior to that of the combination of CQ1-3 / 1-11, ipilimumab, and bevacizumab.

[0339] In this experiment, mice in all drug-treated groups showed no behavioral abnormalities or weight loss ( FIG. 12 ), indicating that tumor-bearing mice had good tolerance to the drug at the tested dose.

[0340] 5.2 Trispecific antibodies HC010-F8 and HC010-F23 induce anti-tumor effects

[0341] Human melanoma A375 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). When the A375 cell density reached 60-80%, PBMCs were revived and then resuspended in RPMI1640 medium to adjust the cell density of PBMCs to 3×10 6 cells / mL, and co-cultured with A375 cells treated with Mitomycin C. After 5 days of co-culture of PBMC and A375, PBMC and freshly digested A375 cells were collected. 5 cells / mouse, A375 cells 4×10 6 cells / mouse were inoculated subcutaneously on the right side of NCG female mice with an inoculation volume of 0.2 mL / mouse containing 50% Matrigel. On the 10th day after tumor inoculation, when the average tumor volume was approximately 80 mm 3 Mice were randomly divided into four groups (n=6) and subcutaneously injected twice weekly for three weeks with three monoclonal antibody combinations (0.35 mg / kg 202F1-hIgG4, 0.65 mg / kg CQ1-3 / 1-11, and 0.65 mg / kg bevacizumab), HC-010F8 (1 mg / kg), HC010-F23 (1 mg / kg), and a negative control (PBS). Tumor volume was measured twice weekly with a vernier caliper and calculated using the above formula.

[0342] The results are shown in FIG16 . 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.

[0343] In this experiment, mice in all drug-treated groups showed no behavioral abnormalities or weight loss ( FIG. 17 ), indicating that tumor-bearing mice had good tolerance to the drug at the tested dose.

[0344] Whether during the dosing period or the withdrawal period, the excellent tumor inhibition of the trispecific antibody of the present application gives it broad application prospects in the field of tumor treatment. It can not only effectively inhibit tumors but also prolong the tumor inhibition period.

[0345] 5.3 Trispecific Antibodies in Human CD34 + HSCs inhibit the growth of non-small cell lung cancer A549 in humanized mice

[0346] Human hematopoietic cell hCD34 + HSCs were transplanted into irradiated NCG mice for immune reconstitution to obtain human CD34 +HSC humanized mice (Jicui Yaokang). Human non-small cell lung cancer A549 cells were inoculated with human CD34 + HSC humanized mice were inoculated subcutaneously on the right side of the body with a volume of 0.2 mL / mouse containing 30% Matrigel. When the average tumor volume was approximately 100 mm 3 Mice were randomly divided into groups and subcutaneously injected twice weekly for 4 weeks with the trispecific antibodies HC010-F8 (5 mg / kg) and HC010-F23 (5 mg / kg), the control antibodies pembrolizumab (3.25 mg / kg) and bevacizumab (3.25 mg / kg), and a negative control (PBS). Tumor volume was measured twice weekly with a vernier caliper, and tumor volume was calculated according to the above formula. At the end of the experiment, tumor samples were harvested, minced with dissecting laboratory scissors, and enzymatically dissociated using the 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.

[0347] HC010-F8 and HC010-F23 significantly inhibited tumor growth compared to the negative control group, with tumor inhibition rates comparable to or even superior to those in the pembrolizumab or bevacizumab groups. During the recovery period after drug withdrawal, the tumor inhibition rates of the antibodies of the present invention were superior to those in the pembrolizumab or bevacizumab groups ( Figure 18 ).

[0348] 5.4 Trispecific Antibodies Inhibit Growth of Human Non-Small Cell Lung Cancer H1299 in a Humanized PBMC Mouse Model

[0349] Human non-small cell lung cancer H1299 cells were inoculated subcutaneously on the right side of human PBMC humanized mice (Jicui Yaokang) with an inoculation volume of 0.2 mL / mouse containing 30% Matrigel. When the average tumor volume was approximately 100 mm 3 Mice were randomly divided into groups and subcutaneously injected twice weekly for 4 weeks with the trispecific antibody HC010-F8 (1-10 mg / kg), HC010-F23 (1-10 mg / kg), a control antibody (1-10 mg / kg), and a negative control (PBS). Tumor volume was measured twice weekly with a vernier caliper and calculated using the above formula.

[0350] HC010-F8 and HC010-F23 effectively inhibited tumor growth compared with the negative control group.

[0351] The inventors continued to 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 was injected with 4.5×10 6 When the average tumor volume reaches 120 mm 3 At the same time, appropriate mice were selected for enrollment according to the tumor volume and body weight of the mice, with 9 mice in each group, for a total of 7 groups, namely: 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). All groups started to receive medication on the day of grouping, and were dosed twice a week, for a total of 6 times in each group. The body weight and tumor volume of the mice were measured twice a week during the medication and observation period, and the tumor volume growth inhibition rate (TGI) was calculated. TV ).

[0352] At the end of the experiment, the average tumor volume of the G1 vehicle control group (PBS) was 1303 ± 188 mm 3 The average tumor volumes of the G2-G7 treatment groups were 640±59mm 3 、582±96mm 3 、407±72mm 3 、406±44mm 3 、603±92mm 3 and 363±55mm 3 The TGI of each treatment group TV The percentages were 57.40%, 60.97%, 75.70%, 75.72%, 59.06%, and 79.60%, respectively. Compared to the vehicle control group, the G2-G7 groups all showed significant tumor inhibition (P < 0.001). Therefore, the tumor inhibition rate of HC010-F8 alone was significantly superior to that of pembrolizumab, bevacizumab, and ipilimumab alone, and was comparable to that of AK112 and the combination of pembrolizumab, bevacizumab, and ipilimumab (Figure 19).

[0353] 5.5 Trispecific Antibody Inhibits Growth of Human Non-Small Cell Lung Cancer NCI-H292 in a Human PBMC Humanized Mouse Model

[0354] Human non-small cell lung cancer NCI-H292 cells were inoculated subcutaneously on the right side of human PBMC humanized mice (Jicui Yaokang) with an inoculation volume of 0.2 mL / mouse containing 30% Matrigel. When the average tumor volume was approximately 100 mm 3 Mice were randomly divided into groups and subcutaneously injected twice weekly for 4 weeks with the trispecific antibody HC010-F8 (1-10 mg / kg), HC010-F23 (1-10 mg / kg), a control antibody (1-10 mg / kg), and a negative control (PBS). Tumor volume was measured twice weekly with a vernier caliper and calculated using the above formula.

[0355] HC010-F8 and HC010-F23 effectively inhibited tumor growth compared with the negative control group.

[0356] 5.6 Trispecific Antibody Inhibits Growth of Human Colorectal Cancer HT29 in a Humanized PBMC Mouse Model

[0357] Human rectal cancer HT29 cells were inoculated subcutaneously on the right side of human PBMC-humanized mice (Shanghai Model Organisms) at a volume of 0.1 mL / mouse containing 30% Matrigel. When the average tumor volume was approximately 100 mm 3 Mice were randomly divided into groups and subcutaneously injected twice weekly for 4 weeks with the trispecific antibody HC010-F8 (1-10 mg / kg), HC010-F23 (1-10 mg / kg), a control antibody (1-10 mg / kg), and a negative control (PBS). Tumor volume was measured twice weekly with a vernier caliper and calculated using the above formula.

[0358] HC010-F8 and HC010-F23 effectively inhibited tumor growth compared with the negative control group.

[0359] Afterwards, the inventors continued to detect the activity and dose dependency of the trispecific antibody of the present invention.

[0360] Human rectal cancer HT29 cells were inoculated subcutaneously on the right side of human PBMC-humanized M-NSG mice (Shanghai Model Organisms) at a volume of 0.1 mL / mouse containing 30% Matrigel. When the average tumor volume was approximately 100 mm 3The mice were randomly divided into groups, including group 1: PBS, ip, BIW*7times group (hereinafter referred to as G1), group 2: pembrolizumab, 3.25 mg / kg, ip, BIW*7times group (hereinafter referred to as G2), group 3: bevacizumab, 3.25 mg / kg, ip, BIW*7times group (hereinafter referred to as G3), group 4: anti-CTLA-4 antibody Ipilimumab, 3.25 mg / kg, ip, BIW*7times group (hereinafter referred to as G4), group 5: HC010-F8, 1 mg / kg, ip, BIW*7times group (hereinafter referred to as G5), group 6: HC010-F8, 5 mg / kg, ip, BIW*7times group (hereinafter referred to as G6). es group (hereinafter referred to as G6), Group 7: HC010-F8, 20 mg / kg, ip, BIW*7times group (hereinafter referred to as G7), Group 8: Pembrolizumab + Bevacizumab 3.25 mg / kg+3.25 mg / kg, ip, BIW*7times group (hereinafter referred to as G8), Group 9: Pembrolizumab + Ipilimumab, 3.25 mg / kg+3.25 mg / kg, ip, BIW*7times group (hereinafter referred to as G9) and Group 10: Pembrolizumab + Ipilimumab + Bevacizumab, 3.25 mg / kg+3.25 mg / kg+3.25 mg / kg, ip, BIW*7times group (hereinafter referred to as G10).

[0361] The experiment was ended on the 17th day after administration, at which time the average tumor volume of the control group G1 was 644.77±61.57mm 3 The average tumor volumes of G2, G4, and G9 were 498.68±48.30mm 3 , 638.72±70.69mm 3 and 489.19±47.40mm 3 The tumor volume inhibition rates (TGI) were 26.72%, 1.03% and 28.42% respectively. There was no statistical difference compared with the control group (P>0.05). The average tumor volume of the G3 group was 400.50±62.73mm 3 The tumor volume inhibition rate (TGI) was 44.68%, which was statistically significant compared with the control group (P < 0.05). The average tumor volume of G5, G6, G7, G8 and G10 was 403.54 ± 45.88 mm 3 、312.48±38.02mm 3 、270.45±33.25mm 3 、293.13±36.08mm 3and 313.65±25.23mm 3 The tumor volume inhibition rates (TGI) of the two groups were 44.26%, 60.91%, 68.59%, 64.42% and 60.63%, respectively, which were significantly different from those of the control group (P < 0.01).

[0362] Table 9 ns=not significant; *p<0.05, **p<0.01, ***p<0.001

[0363] Conclusion: Under these experimental conditions, the test drug HC010-F8 exhibited significant antitumor effects in the HT-29 colorectal cancer model in humanized PBMC-derived M-NSG mice at doses of 1, 5, and 20 mg / kg in a dose-dependent manner. Compared to the vehicle control (PBS), HC010-F8 demonstrated significant efficacy at doses of 1 mg / kg and above. The tumor inhibition rate of HC010-F8 at 5 mg / kg (60.91%) was superior to that of single-agent pembrolizumab (26.72%), bevacizumab (44.68%), and ipilimumab (1.03%). The antitumor efficacy of HC010-F8 at a dose of 5 mg / kg was superior to that of the dual immunotherapy combination of pembrolizumab and ipilimumab.

[0364] 5.7 Trispecific Antibody Inhibits Growth of HepG2 Hepatocellular Carcinoma in a Humanized Human PBMC Mouse Model

[0365] Human hepatocellular carcinoma HepG2 cells were inoculated subcutaneously on the right side of human PBMC-humanized mice (Shanghai Model Organisms) at a volume of 0.2 mL / mouse containing 30% Matrigel. When the average tumor volume was approximately 150-200 mm 3 Mice were randomly divided into groups and subcutaneously injected twice weekly for 4 weeks with the trispecific antibody HC010-F8 (1-10 mg / kg), HC010-F23 (1-10 mg / kg), a control antibody (1-10 mg / kg), and a negative control (PBS). Tumor volume was measured twice weekly with a vernier caliper and calculated using the above formula.

[0366] HC010-F8 and HC010-F23 effectively inhibited tumor growth compared with the negative control group.

[0367] 5.8 Trispecific Antibody Inhibits Growth of Human Hepatocellular Carcinoma Huh7 in a Humanized PBMC Mouse Model

[0368] Human hepatocellular carcinoma Huh7 cells were inoculated subcutaneously in human PBMC humanized NCG mice (Jicui Yaokang) at a dose of 5×10^6 cells / 100μL / mouse (with Corning Matrigel at a 1:1 ratio). The average tumor volume reached 77.20mm 3 Mice were randomly divided into three groups according to tumor volume, with 8 mice in each group. They were treated with pembrolizumab (3.25 mg / kg), HC010-F8 (5 mg / kg) and solvent PBS control group (ip). The drugs were administered twice a week for a total of 6 times and observed for 20 days. The drug efficacy was evaluated based on observation indicators such as tumor inhibition rate and body weight.

[0369] The trial reached its endpoint on the 20th day after the start of drug administration, and the average tumor volume in the PBS group was 2124.08 mm 3 The average tumor volume in the pembrolizumab group (3.25 mg / kg) was 2099.87 mm 3 The average tumor volume of HC010-F8 (5 mg / kg) was 1014.72 mm 3 Compared with the PBS control group, HC010-F8 showed significant differences in tumor growth inhibition (P<0.0001), while pembrolizumab had almost no tumor inhibitory effect. Compared with pembrolizumab, HC010-F8 had a significant anti-tumor effect (P<0.0001) (see Figure 20). During the trial, no animal deaths related to test substance toxicity occurred. Conclusion: In the mouse tumor model of human hepatocellular carcinoma Huh7, HC010-F8 has a significant anti-tumor effect, while pembrolizumab did not show a significant tumor inhibitory effect. Animals showed good tolerance to HC010-F8 at a dose of 5 mg / kg.

[0370] 5.9 Efficacy of trispecific antibodies as alternative antibodies in a humanized mouse tumor model targeting both human PD-1 and human CTLA-4

[0371] The trispecific antibody does not recognize mouse PD-1, CTLA-4, and VEGFA. The anti-VEGF sequence in HC010-F8 and HC010-F23 was replaced with the anti-VEGF sequence (B20.4.1) reported in (US20110159009A1 and Wei-Ching Liang et al. Cross-species vascular endothelial growth factor (VEGF)-blocking antibodies completely inhibit the growth of human tumor xenografts and measure the contribution of stromal VEGF. J Biol Chem. 2006 Jan 13; 281(2): 951-61. doi: 10.1074 / jbc.M508199200. Epub 2005 Nov 7.), expressing an alternative antibody that can recognize mouse VEGF. B20.4.1 has similar binding and blocking activities to bevacizumab used in HC010-F8 and HC010-F23.

[0372] The anti-tumor activity of the trispecific antibody was evaluated in the colorectal cancer MC38 tumor model and the liver cancer Hepa1-6 tumor model of humanized mice with dual targets of human PD-1 and human CTLA-4 (B-hPD-1 / hCTLA4, Biocytogen). 3 The mice were randomly divided into groups and subcutaneously injected with an alternative antibody to the trispecific antibody HC010-F8 (1-10 mg / kg), an alternative antibody to HC010-F23 (1-10 mg / kg), a control antibody (1-10 mg / kg) and a negative control (PBS), twice a week for 4 weeks.

[0373] The results showed that the alternative antibodies of HC010-F8 and HC010-F23 had good anti-tumor activity in the colorectal cancer MC38 tumor model and the liver cancer Hepa1-6 tumor model of humanized mice with dual targets of human PD-1 and human CTLA-4 (B-hPD-1 / hCTLA4, Biocytogen).

[0374] Example 6 Stability Study of Trispecific Antibodies

[0375] In the field of antibody preparation, various antibody derivatives (such as multispecific antibodies) obtained based on the transformation of natural antibodies often face stability problems. Obtaining antibody derivatives with excellent stability is of great significance in the preparation, transportation, storage, etc. of antibodies. This example detects the stability of the trispecific antibodies obtained in this application, and studies the thermal stability of the trispecific antibodies by accelerating at an elevated temperature (40°C). Specifically, each trispecific antibody was replaced in 20mM His-HCl, pH 5.5, 230mM trehalose, 0.01% tween 80 formulation buffer. The samples were then incubated at 40°C for 0, 1, 2 and 4 weeks, and size exclusion chromatography (SEC) and non-reducing capillary gel electrophoresis (NR-CE-SDS) were detected by high performance liquid chromatography (Dionex, Ultimate 3000). The results are shown in Table 9. After incubation at 40°C for 4 weeks, the SEC purity of the trispecific antibodies HC010-F8 and HC010-F22 showed minimal change, with the main peak (MP%) decreasing by less than 5%. The main peak of HC010-F10 and HC010-F23 decreased by approximately 10%. After incubation at 40°C for 4 weeks, the main peak of the trispecific antibodies in NR-CE-SDS decreased by 6.6% to 13.2%, demonstrating that the trispecific antibodies constructed and expressed in this application have good stability.

[0376] Table 9. Thermal stability study of trispecific antibodies

Claims

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

2. The trispecific antibody of claim 1 , 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 have structures selected from the group consisting of: 1) A heavy chain comprising the structure of VH-CH1-Fc-VHH-ScFv from N-terminus to C-terminus, A light chain comprising a VL-CL structure from N-terminus to C-terminus; or 2) a heavy chain comprising the structure of ScFv-VH-CH1-Fc-VHH from N-terminus to C-terminus, A light chain comprising a VL-CL structure from N-terminus to C-terminus; or 3) a heavy chain comprising the structure of VHH-VH-CH1-Fc-ScFv from N-terminus to C-terminus, A light chain comprising a VL-CL structure from N-terminus to C-terminus; or 4) a heavy chain comprising a structure of VH-CH1-Fc-ScFv-VHH from N-terminus to C-terminus, A light chain comprising a VL-CL structure from N-terminus to C-terminus; or 5) a heavy chain comprising a VH-CH1-Fc-ScFv structure from N-terminus to C-terminus, A light chain comprising a VL-CL-VHH structure from N-terminus to C-terminus; or 6) a heavy chain comprising the structure of ScFv-VH-CH1-Fc-ScFv from N-terminus to C-terminus, A light chain comprising a VL-CL structure from N-terminus to C-terminus; or 7) a heavy chain comprising a VH-CH1-Fc-ScFv structure from N-terminus to C-terminus, A light chain comprising a VHH-VL-CL structure from N-terminus to C-terminus; or 8) a heavy chain comprising a structure of ScFv-VHH-VH-CH1-Fc from N-terminus to C-terminus, The light chain, which comprises the VL-CL structure from N-terminus to C-terminus; Wherein Fc represents the Fc region of the immunoglobulin heavy chain, and the two heavy chains containing the Fc region homodimerize through the Fc region. Wherein CH1 represents the immunoglobulin heavy chain CH1 domain, CL represents the immunoglobulin light chain CL domain, Among them, VH-CH1 and VL-CL pair with each other to form Fab, The first, second and third antigen binding sites are in the form of Fab, VHH or ScFv, and bind to different and independent antigens PD-1, CTLA-4 and VEGF respectively.

3. The trispecific antibody of claim 1, comprising a first, a second, and a third antigen-binding site and consisting of three chains having the following structure: 1) A first heavy chain comprising a VH-CH1-Fc-ScFv structure from N-terminus to C-terminus, 2) a second heavy chain comprising the structure of VHH-Fc-ScFv from N-terminus to C-terminus, and 3) Light chain, which comprises the VL-CL structure from N-terminus to C-terminus, Fc is the Fc region of the immunoglobulin heavy chain, wherein the two heavy chains comprising the Fc region dimerize through the Fc region. Wherein CH1 represents the immunoglobulin heavy chain CH1 domain, CL represents the immunoglobulin light chain CL domain, Among them, VH-CH1 and VL-CL pair with each other to form Fab, The first, second and third antigen binding sites are in the form of Fab, VHH or ScFv, and bind to different and independent antigens PD-1, CTLA-4 and VEGF respectively.

4. The trispecific antibody of claim 1, comprising a first, a second, and a third antigen-binding site and consisting of three chains having the following structure: 1) A first heavy chain comprising a VH-CH1-Fc-ScFv2 structure from N-terminus to C-terminus, 2) a second heavy chain comprising the structure of ScFv1-Fc-ScFv2 from N-terminus to C-terminus, 3) Light chain, which comprises the VL-CL structure from N-terminus to C-terminus, Fc is the Fc region of the immunoglobulin heavy chain, wherein the two heavy chains comprising the Fc region dimerize through the Fc region. Wherein CH1 represents the immunoglobulin heavy chain CH1 domain, CL represents the immunoglobulin light chain CL domain, Among them, VH-CH1 and VL-CL pair with each other to form Fab, The first, second and third antigen binding sites are in the form of Fab, ScFv1 or ScFv2, and bind to different and independent antigens PD-1, CTLA-4 and VEGF respectively.

5. The trispecific antibody of claim 1, comprising a first, a second, and a third antigen-binding site and consisting of two chains having the following structure: 1) A first heavy chain comprising the structure of ScFv1-Fc-ScFv2 from N-terminus to C-terminus, 2) a second heavy chain comprising the structure of VHH-Fc-ScFv2 from N-terminus to C-terminus, Fc is the Fc region of the immunoglobulin heavy chain, wherein the two heavy chains comprising the Fc region dimerize through the Fc region. The first, second and third antigen binding sites are in the form of VHH, ScFv1 or ScFv2, and bind to different and independent antigens PD-1, CTLA-4 and VEGF respectively.

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

31.

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

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

25.

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

10. The trispecific antibody of claim 9, wherein the linker comprises the amino acid sequence (G4S) n , wherein n is an integer equal to or greater than 1, preferably, the linker consists of the amino acid sequence (G4S)3 or (G4S)4, preferably the linker has the sequence shown in SEQ ID NO:9, and the hinge region is a hinge region from an immunoglobulin, preferably a hinge region from an IgG.

11. The trispecific antibody of claim 1-8, 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-in-hole structure.

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, K447A (according to the EU numbering system).

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

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

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

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

17. The trispecific antibody of claim 1, 2 or 13, comprising: 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.

18. The trispecific antibody of claim 1, 3 or 14, comprising: 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.

19. The trispecific antibody of claim 1, 4, or 15, comprising: 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.

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

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

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

23. A host cell comprising the polynucleotide of claim 21 or the vector of claim 22, for example, the host cell is a mammalian cell.

24. A method for producing the trispecific antibody of any one of claims 1 to 20, comprising: Culturing a host cell containing a protein encoding the antibody polypeptide chain under conditions suitable for expressing the antibody polypeptide chain; and assembling the polypeptide chains 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 according to any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

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

27. The method of claim 26, wherein the cancer is selected from 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, stomach cancer, head and neck cancer, head and neck squamous cell carcinoma, kidney cancer, testicular cancer, multiple myeloma, glioblastoma, glioma, and hematologic 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. A method for treating, preventing and / or diagnosing cancer, autoimmune diseases, infectious diseases or angiogenesis-related diseases, comprising administering an effective amount of the trispecific antibody according to any one of claims 1 to 20, or the pharmaceutical composition according to claim 25 to a patient in need thereof.

29. The method of claim 28, wherein the cancer is selected from 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, stomach 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).

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