Bispecific antibody and its use

Abstract

FIELD: biotechnology.SUBSTANCE: bispecific antibody targeting PD-1 and VEGF and a pharmaceutical composition containing it are proposed. A method for treating a malignant tumor is also provided, comprising administering said antibody to a subject. Also provided are a polynucleotide encoding said antibody, an expression vector, and a host cell for expressing the bispecific antibody.EFFECT: designed bispecific antibody targeting PD-1 and VEGF has high stability, can target the accumulation of VEGF with high expression in the tumor area, and has good medical efficacy and safety.47 cl, 8 dwg, 18 tbl, 14 ex

Description

REGION

[0001] The present invention relates to the field of antibodies, and in particular, to a bispecific antibody targeting PD-1 and VEGF and its use. BACKGROUND

[0002] Programmed death protein receptor-1 (PD-1) belongs to the CD28 / B7 immunoglobulin superfamily and is an immunosuppressive receptor. Binding of PD-1 to programmed death receptor ligand 1 (PD-L1) leads to phosphorylation of phosphatidylinositol 3-kinase, subsequent activation of protein kinase B, activation of T-cell stimulatory signaling pathways, glucose metabolism, and interferon secretion. This leads to the blocking of subsequent T-cell activation signals, thereby effectively inhibiting T-cell transcription and, in turn, inhibiting T-cell immune function. This process plays a key role in the regulation of negative immune responses. The broad-spectrum antitumor efficacy of anti-PD-1 antibodies has led to the launch of numerous blockbuster drugs, such as O (trade name: Opdivo; generic name: Nivolumab), which was approved in the United States on December 22, 2014.Its initial indication was melanoma and it was subsequently approved in the US for indications such as non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck squamous cell carcinoma, colorectal cancer, hepatocellular carcinoma, urothelial cancer, colorectal cancer, hepatocellular carcinoma, esophageal cancer, esophageal squamous cell carcinoma, pleural mesothelioma, esophageal adenocarcinoma, gastroesophageal junction cancer, gastric cancer, etc. However, an analysis of existing clinical data including 28,304 patient records from 160 clinical trials (Ref. 1: Efficacy of PD-1 / PD-L1 blockade monotherapy in clinical trials, Bin Zhao, Hong Zhao, et al., Ther Adv Med Oncol. 2020, Vol. 12, pp. 1-22.) indicates that only 4,747 patients out of 22,165 patients who received PD-1 / PD-L1 monotherapy experienced an overall treatment effect [objective response rate (ORR), 20.21%; 95% confidence interval (CI), 18.34-22.15%].A total of 862 complete responses (CRs) were observed in 19,418 cancer patients. The overall CR rate was 3.85% (95% CI, 3.06-4.73%). The ORR varied significantly across tumor types and PD-L1 expression status. This suggests that the efficacy of PD-1 / PD-L1 monotherapy varies significantly across tumor types and PD-L1 expression status. Therefore, there remains a significant unmet clinical need.

[0003] Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a highly specific factor that promotes vascular permeability, extracellular matrix degradation, endothelial cell migration, proliferation, angiogenesis, etc. Data show that VEGF plays a significant role in pathological angiogenesis and can induce the occurrence and progression of various pathologies, including tumor growth and metastasis, macular degeneration, diabetic retinopathy, inflammatory processes (such as rheumatoid arthritis), ischemic processes (myocardial ischemia), preeclampsia, etc. (Ref. 2: Molecular and functional diversity of vascular endothelial growth factors. Yamazaki Y., Morita T., Mol. Divers. November 2006, Vol. 10, No. 4, pp. 515-527.).The high-affinity receptor that specifically binds vascular endothelial growth factor is called the vascular endothelial growth factor receptor (VEGFR). It is a tyrosine kinase receptor that plays a key role in multiple signaling pathways required for angiogenesis and cell migration. VEGF binds to VEGFR, initiating a signaling cascade and stimulating angiogenesis. Thus, VEGF / VEGFR is a highly important target because they are almost exclusively expressed in endothelial cells and are highly activated in many types of tumor endothelium. The use of anti-VEGF / VEGFR therapies to block angiogenesis in malignant tumors and other pathological processes is currently considered extremely important.Inhibition of vascular endothelial growth factor / vascular endothelial growth factor receptor is currently the mainstay of treatment for certain types of malignant tumors, including renal cell carcinoma, hepatocellular carcinoma, etc.

[0004] Currently, numerous clinical trials are investigating the safety and efficacy of vascular endothelial growth factor / vascular endothelial growth factor receptor (VEGF) inhibitors in combination with immune checkpoint inhibitors in the treatment of cancer. Some preliminary studies have shown that, compared with VEGFR inhibition or immunosuppression alone, the combined use of immune checkpoint inhibitors and VEGFR inhibitors may lead to better therapeutic outcomes in patients (https: / / www.obroncology.com / article / combining-immune-checkpoint-and-vegf-inhibitors-improves-survival-in-pretreated-nsclc). However, potential risks associated with this favorable outcome remain unknown (Ref. 3: Combining Immune Checkpoint and VEGFR Inhibition in Favorable Risk and Elderly Patients with Metastatic Renal Cell Carcinoma. Varkaris A, Xu W, Davis RB, Healy B, McDermott DF. Clin Genitourin Cancer. June 2020, Vol. 18, No.3, pp. 179-184.). In some clinical trials of dual VEGFR / ICI blockade (e.g., pembrolizumab / pazopanib combination group, nivolumab / sunitinib combination group, nivolumab / pazopanib combination group, etc.), the results showed promising therapeutic effects in early stages of clinical trials (Ref. 4: A Phase I / II Study to Assess the Safety and Efficacy of Pazopanib and Pembrolizumab Combination Therapy in Patients with Advanced Renal Cell Carcinoma. Chowdhury S, Infante JR, et al. Clin Genitourin Cancer. 2021 rh October, Vol. 19, No. 5, pp. 434-446; Ref. 5: Nivolumab (anti-PD-1; BMS-936558, ONO-4538) in combination with sunitinib or pazopanib in patients (pts) with metastatic renal cell carcinoma (mRCC). Amin, A. et al., Journal of Clinical Oncology. 32, (suppl.), May 20, 2014, Abstract 5010). However, severe toxicity was observed in the subsequent high-dose group.Grade 3 or greater severe treatment-related toxicity occurred in 73% and 60% of patients in the nivolumab / sunitinib combination group and the nivolumab / pazopanib combination group, respectively. Additionally, 23% of patients in the nivolumab / sunitinib combination group and 20% of patients in the nivolumab / pazopanib combination group discontinued treatment due to adverse events. Similarly, high-dose hepatotoxicity occurred in the pembrolizumab / pazopanib combination group. Therefore, these combinations were deemed ineligible for testing in the phase 3 trial and were therefore discontinued. Furthermore, positive efficacy data have been obtained in other clinical trials of dual VEGFR / ICI blockade, such as the phase III clinical trial of bevacizumab in combination with atezolizumab (Ref. 6: IMmotion151: a randomized phase III study of atezolizumab plus bevacizumab vs sunitinib in untreated metastatic renal cell carcinoma (mRCC), Motzer, RJ et al.Journal of Clinical Oncology. 36, abstr. 578 (2018). This trial increased the remission rate in patients. However, according to MSKCC standards, only 20% of patients participating in this clinical trial were considered to be at acceptable risk. The main reason was that combination therapy also increased toxic side effects in patients.

[0005] In addition to the study of combination therapy, there are also a number of bispecific antibodies targeting VEGF / VEGF receptor and PD-1 that are still in the complex research stage, such as AK112 developed by Akeso Biomedical, Inc., Zhongshan (see Chinese Patent Application CN109053895). However, to date, no bispecific antibody targeting VEGF / VEGF receptor and immune checkpoints has been approved for sale worldwide. Significant clinical needs and development challenges remain. SUMMARY OF THE INVENTION

[0006] Through the research within the scope of the invention, the inventors developed a bispecific antibody targeting PD-1 and VEGF, in which the variable domain of the heavy chain of a heavy chain-only antibody (VHH) targeting VEGF, which was obtained after several rounds of screening and optimization, is combined with an anti-PD-1 antibody as a modular component of the binding functional region. The bispecific antibody contains:

[0007] (a) a first binding functional region targeting PD-1; and

[0008] (b) a second binding functional region targeting VEGF;

[0009] where:

[0010] the first binding functional region targeting PD-1 is an anti-PD-1 antibody (Ab) or an antigen-binding fragment thereof (Ab'); and

[0011] The second VEGF-targeting functional binding region contains complementarity-determining regions (CDRs) 1-3 of the VHH domain, defined by the following amino acid sequences: CDR1 SEQ ID NO:1 CDR2 SEQ ID NO: 2 represents a suitable manufacturer's active ingredient CDR3 SEQ ID NO: 3

[0012] In addition, the amino acid mutation in CDR2 is at position 58 and / or position 65.

[0013] In addition, the amino acid mutation in CDR2 is N58Y and / or D65G mutation.

[0014] Preferably, the amino acid mutation in CDR2 is N58Y and D65G mutations.

[0015] Preferably, the amino acid sequence of CDR2 with the mutation is shown as SEQ ID NO: 24.

[0016] Preferably, the combination of CDRs 1-3 of the VHH domain of the second binding functional region targeting VEGF is: CDR1 SEQ ID NO:1 CDR2 SEQ ID NO: 2 CDR3 SEQ ID NO: 3 or CDR1 SEQ ID NO:1 CDR2 SEQ ID NO: 24 CDR3 SEQ ID NO: 3

[0017] In addition, the VHH domain is a humanized VHH domain.

[0018] Furthermore, the second binding functional region targeting VEGF further comprises: 1) a first framework domain FR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, with or without one or two amino acid mutations; and / or 2) a second framework domain FR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, with or without one amino acid mutation; and / or 3) a third framework domain FR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, with or without 1-5 amino acid mutations; and / or 4) a fourth framework domain FR4 comprising the amino acid sequence set forth in SEQ ID NO: 7, with or without one amino acid mutation.

[0019] In addition, one or two amino acid mutations in FR1 are at position 1 and / or position 5; preferably, Q1E and / or Q5L mutations.

[0020] In addition, one amino acid mutation in FR2 is at position 49; the preferred mutation is A49S.

[0021] Furthermore, 1-5 amino acid mutations in FR3 are at positions selected from the group consisting of positions 74, 82B, 83, 84, 89 and a combination thereof; preferably, 1-5 amino acid mutations are selected from the group consisting of D74S, V(82B)S, K83R, P84A, M89V and a combination thereof.

[0022] In addition, one amino acid mutation in FR4 is at position 108; preferably Q108L.

[0023] Furthermore, the second binding functional region targeting VEGF further comprises: 1) a first framework domain FR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 9; and / or 2) a second framework domain FR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 10; and / or 3) a third framework domain FR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 11; and / or 4) a fourth framework domain FR4 comprising the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12.

[0024] In addition, the second VEGF-targeting functional binding region contains a combination of framework regions defined by the following amino acid sequences: FR1 SEQ ID NO: 4 FR2 SEQ ID NO:5 FR3 SEQ ID NO:6 FR4 SEQ ID NO: 7 or FR1 SEQ ID NO:9 FR2 SEQ ID NO: 10 FR3 SEQ ID NO: 11 FR4 SEQ ID NO:12 or FR1 SEQ ID NO:9 FR2 SEQ ID NO:5 FR3 SEQ ID NO: 11 FR4 SEQ ID NO:12

[0025] Furthermore, the second binding functional region targeting VEGF comprises the amino acid sequence set forth in SEQ ID NO: 8, with or without 1-11 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) amino acid mutations. In addition, the amino acid mutation is at a position selected from the group consisting of positions 1, 5, 49, 58, 65, 74, 82B, 83, 84, 89, 108 and a combination thereof. In addition, the amino acid mutation is selected from the group consisting of Q1E, Q5L, A49S, N58Y, D65G, D74S, V(82B)S, K83R, P84A, M89V, Q108L and a combination thereof.

[0026] More preferably, the amino acid mutation is selected from the following combinations of mutations: Комбинация мутаций Q1E+Q5L+A49S+N58Y+D65G+D74S+V(82B)S+K83R+P84A+M89V+Q108L Комбинация мутаций Q1E+Q5L+N58Y+D65G+D74S+V(82B)S+K83R+P84A+M89V+Q108L

[0027] More preferably, the second binding functional region targeting VEGF comprises the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 13, or SEQ ID NO: 14. Table 1. Sequences of each structural region of VHH strain #6 (Kabat system) CDR1 (SEQ ID NO: 1) TSTMS CDR2 (SEQ ID NO: 2) FITSAGATTNEW VKD CDR3 (SEQ ID NO: 3) LVTLWNVY FR1 (SEQ ID NO: 4) QVQLQESGGGLVQPGGSLRLSCAASGFTFS FR2 (SEQ ID NO: 5) WYRQAPGKERELVA FR3 (SEQ ID NO: 6) RFTMSRDNDKNTVYLQMNVLKPEDTAMYYCRA FR4 (SEQ ID NO: 7) WGQGTQVTVSS VHH sheet #6 (SEQ ID NO: 8) QVQLQESGGGLVQPGGSLRLSCAASGFTFSTSTMSSWYRQAPGKERELVAFITSAGATTNYADSVKDRFTMSRDNDKNTVYLQMNVLKPEDTAMYYCRALVTLWNVYWGQGTQVTVSS Table 2 Sequences of each structural region of VHH strain #48 (Kabat system) CDR1 (SEQ ID NO: 1) TSTMS CDR2 (SEQ ID NO: 24) FITSAGATTYYADSVKG CDR3 (SEQ ID NO: 3) LVTLWNVY FR1 (SEQ ID NO: 9) EVQLLESGGGLVQPGGSLRLSCAASGFTFS FR2 (SEQ ID NO: 10) WYRQAPGKERELVS FR3 (SEQ ID NO: 11) RFTMSDNSKNTVYLQMNSLRAEDTAVYYCRA FR4 (SEQ ID NO: 12) WGQGTLVTVSS VHH штамма #48 (SEQ ID NO: 13) EVQLLESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYRQAPGKERELVSFITSAGATTYYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCRALVTLWNVYWGQGTLVTVSS Table 3 Sequences of each structural region of VHH strain #49 (Kabat system) CDR1 (SEQ ID NO: 1) TSTMS CDR2 (SEQ ID NO: 24) FITSAGATTYYADSVKG CDR3 (SEQ ID NO: 3) LVTLWNVY FR1 (SEQ ID NO: 9) EVQLLESGGGLVQPGGSLRLSCAASGFTFS FR2 (SEQ ID NO: 5) WYRQAPGKERELVA FR3 (SEQ ID NO: 11) RFTMSDNSKNTVYLQMNSLRAEDTAVYYCRA FR4 (SEQ ID NO: 12) WGQGTLVTVSS VHH штамма #49 (SEQ ID NO: 14) EVQLLESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYRQAPGKERELVAFITSAGATTYYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCRALVTLWNVYWGQGTLVTVSS

[0028] Furthermore, the anti-PD-1 antibody (Ab) or antigen-binding fragment thereof (Ab') is selected from the group consisting of a humanized antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, and a human antibody or antigen-binding fragment thereof.

[0029] In addition, the anti-PD-1 antibody (Ab) or its antigen-binding fragment (Ab') is an IgG antibody.

[0030] In addition, the anti-PD-1 antibody (Ab) or its antigen-binding fragment (Ab') is IgG1, IgG2, or IgG4.

[0031] In addition, the first binding functional region targeting PD-1 contains a light chain and / or heavy chain CDR amino acid sequence identical to that of Nivolumab (Opdivo) or pembrolizumab (Keytruda).

[0032] In addition, the first binding functional region targeting PD-1 contains a heavy chain variable region amino acid sequence identical to that of Nivolumab (Opdivo) or pembrolizumab (Keytruda).

[0033] In addition, the first binding functional region targeting PD-1 contains a light chain variable region amino acid sequence identical to that of Nivolumab (Opdivo) or pembrolizumab (Keytruda).

[0034] Furthermore, the first binding functional region targeting PD-1 comprises a heavy chain portion, wherein the heavy chain portion further comprises a domain selected from the group consisting of a heavy chain constant region CH1 (CH1), a hinge region (Hinge), a heavy chain constant region CH2 (CH2), a heavy chain constant region CH3 (CH3), and a combination thereof.

[0035] In addition, the first binding functional region targeting PD-1 further contains a light chain (CL) constant region.

[0036] In addition, the anti-PD-1 antibody comprises a heavy chain portion and a light chain portion, wherein the heavy chain portion comprises, from the N-terminus to the C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 (CH1), a hinge region (Hinge), a heavy chain constant region CH2 (CH2), and a heavy chain constant region CH3 (CH3), and the light chain portion comprises, from the N-terminus to the C-terminus, a light chain variable region (VL) and a light chain constant region (CL).

[0037] In addition, the Fc domain (i.e., the hinge region, CH2 of the heavy chain constant region, and CH3 of the heavy chain constant region) of the anti-PD-1 antibody is the Fc domain of IgG1 or its silent mutation of IgG1.

[0038] Furthermore, the Fc domain comprises the amino acid sequence set forth in SEQ ID NO: 15, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 15. Table 4 Amino acid sequence of the Fc domain

[0039] Furthermore, the first binding functional region targeting PD-1 has a combination of a light chain and a heavy chain selected from the following amino acid sequences: 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 16 and / or a light chain comprising the amino acid sequence set forth in SEQ ID NO: 17; or 2) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 18 and / or a light chain comprising the amino acid sequence set forth in SEQ ID NO: 19. Table 5. Preferred amino acid sequences of the first binding functional region

[0040] In addition, the second binding functional region is connected to the C-terminus or N-terminus of the first binding functional region.

[0041] In addition, the VHH domain is directly or via a peptide linker connected to the C-terminus or N-terminus of a heavy chain portion of an antibody (Ab) or an antigen-binding fragment thereof (Ab').

[0042] In addition, the peptide linker is a flexible peptide linker.

[0043] In addition, the peptide linker contains one or more amino acids.

[0044] In addition, the peptide linker contains at least 5 amino acids.

[0045] Preferably, the peptide linker comprises the amino acid sequence (GGGGS) n , where n is 1, 2, 3 or 4.

[0046] In addition, the bispecific antibody is a divalent, trivalent or tetravalent bispecific antibody.

[0047] Furthermore, the bispecific antibody comprises a combination of a heavy chain as set forth in SEQ ID NO: 20 and a light chain as set forth in SEQ ID NO: 21, or comprises a combination of a heavy chain as set forth in SEQ ID NO: 22 and a light chain as set forth in SEQ ID NO: 23. Table 6. Preferred amino acid sequences of bispecific antibodies #10 Heavy chain (SEQ ID NO: 20): Light chain (SEQ ID NO: 21): #16 Heavy chain (SEQ ID NO: 22): Light chain (SEQ ID NO: 23):

[0048] Furthermore, the present invention relates to a VHH domain targeting VEGF, wherein the VHH domain comprises CDRs 1-3 defined by the following amino acid sequences: CDR1 SEQ ID NO: 1 CDR2 SEQ ID NO: 2 CDR3 SEQ ID NO: 3

[0049] In addition, the amino acid mutation in CDR2 is at position 58 and / or position 65.

[0050] In addition, the amino acid mutation in CDR2 is N58Y and / or D65G mutation.

[0051] In addition, the VHH domain is a humanized VHH domain.

[0052] Furthermore, the VHH domain further comprises: 1) a first framework domain FR1 comprising the amino acid sequence set forth under SEQ ID NO: 4, with or without one or two amino acid mutations; and / or 2) a second framework domain FR2 comprising the amino acid sequence set forth under SEQ ID NO: 5, with or without one amino acid mutation; and / or 3) a third framework domain FR3 comprising the amino acid sequence set forth under SEQ ID NO: 6, with or without 1-5 amino acid mutations; and / or 4) a fourth framework domain FR4 comprising the amino acid sequence set forth under SEQ ID NO: 7, with or without one amino acid mutation.

[0053] In addition, one or two amino acid mutations in FR1 are at position 1 and / or position 5; preferably, Q1E and / or Q5L mutations.

[0054] In addition, one amino acid mutation in FR2 is at position 49; preferably, the A49S mutation.

[0055] Furthermore, 1-5 amino acid mutations in FR3 are at positions selected from the group consisting of positions 74, 82B, 83, 84, 89 and a combination thereof; preferably, 1-5 amino acid mutations are selected from the group consisting of D74S, V(82B)S, K83R, P84A, M89V and a combination thereof.

[0056] In addition, one amino acid mutation in FR4 is at position 108; preferably Q108L.

[0057] Furthermore, the VHH domain targeting VEGF further comprises: 1) a first framework domain FR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 9; and / or 2) a second framework domain FR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 10; and / or 3) a third framework domain FR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 11; and / or 4) a fourth framework domain FR4 comprising the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12.

[0058] In addition, the VHH domain contains a combination of framework regions defined by the following amino acid sequences: FR1 SEQ ID NO: 4 FR2 SEQ ID NO: 5 FR3 SEQ ID NO: 6 FR4 SEQ ID NO: 7 or FR1 SEQ ID NO: 9 FR2 SEQ ID NO: 10 FR3 SEQ ID NO: 11 FR4 SEQ ID NO: 12 or FR1 SEQ ID NO: 9 FR2 SEQ ID NO: 5 FR3 SEQ ID NO: 11 FR4 SEQ ID NO: 12

[0059] Furthermore, the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 8, with or without 1-11 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acid mutations. Furthermore, the amino acid mutation is at a position selected from the group consisting of positions 1, 5, 49, 58, 65, 74, 82B, 83, 84, 89, 108, and a combination thereof. Furthermore, the amino acid mutation is selected from the group consisting of Q1E, Q5L, A49S, N58Y, D65G, D74S, V(82B)S, K83R, P84A, M89V, Q108L, and a combination thereof. Furthermore, preferably, the amino acid mutation is selected from the following combinations of mutations: Mutation combination 1 Q1E+Q5L+A49S+N58Y+D65G+D74S+V(82B)S+K83R+P84A+M8V+Q108L Mutation combination 2 Q1E+Q5L+N58Y+D65G+D74S+V(82B)S+K83R+P84A+M89V+Q108L

[0060] Furthermore, preferably, the VHH domain comprises the amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 13, or SEQ ID NO: 14.

[0061] Furthermore, the present invention relates to the use of a VHH domain for constructing a recombinant protein.

[0062] Furthermore, the present invention relates to a recombinant protein containing a VHH domain targeting VEGF.

[0063] Furthermore, the recombinant protein is selected from the group consisting of a bispecific antibody, a multispecific antibody, and an antibody-drug conjugate.

[0064] Furthermore, the present invention relates to a polynucleotide encoding any of the above-mentioned bispecific antibody, multispecific antibody, recombinant protein or VHH domain.

[0065] Furthermore, the present invention relates to an expression vector comprising any of the above-mentioned polynucleotides.

[0066] Furthermore, the present invention relates to a host cell comprising any of the above-mentioned expression vectors or having integrated any of the above-mentioned polynucleotides.

[0067] Furthermore, the present invention relates to a pharmaceutical composition comprising any of the above-mentioned VHH domain, bispecific antibody, multispecific antibody or recombinant protein, and a pharmaceutically acceptable carrier.

[0068] Furthermore, the present invention relates to the use of any of the above-mentioned bispecific antibody, multispecific antibody, recombinant protein or VHH domain for the production of a medicament for the treatment of a malignant tumor.

[0069] Furthermore, the present invention relates to a method for treating a malignant tumor, comprising administering to an individual in need thereof an effective amount of any of the above-mentioned VHH domain, bispecific antibody, multispecific antibody or recombinant protein.

[0070] Furthermore, in any of the above-mentioned uses or methods, the malignant tumor is colorectal cancer.

[0071] The beneficial effects of the present invention are as follows. The VHH domain targeting VEGF, which was screened and optimized according to the present invention, exhibits a stable structure, high thermal stability, and strong binding activity to both human and mouse VEGF-A. This enables immediate testing in animal models, thereby increasing the likelihood of successful drug development. Furthermore, it has the characteristics of high protein expression and easy purification.The bispecific antibody developed based on the VHH domain of the present invention has a unique bispecific antibody structure, a high degree of humanization, does not contain artificially added redundant sequences, and can potentially locally concentrate in tumors with high VEGF expression, thereby providing a more targeted tumor inhibition effect, achieving a more effective tumor inhibition effect and solving the problems of insufficient efficacy of PD-1 / PD-L1 inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS.

[0072] Figure 1 shows the binding of antibodies to VEGF after multiple rounds of combined mutations.

[0073] Figure 2 shows a schematic diagram of the structure of the bispecific antibody.

[0074] Fig. 3 shows the level of interleukin 2 (IL-2) released by using the test antibodies in the MLR reaction.

[0075] Fig. 4 shows the level of interferon-γ (IFN-γ) released by using the test antibodies in the MLR reaction.

[0076] Figure 5 shows the relative inhibition rate of HUVEC cell proliferation by the tested antibodies.

[0077] Figure 6 shows the inhibition of HUVEC cell migration by the tested antibodies.

[0078] Figure 7 shows the amount of antibody remaining after binding to magnetic microcarriers with VEGF, which was quantified by ELISA.

[0079] Fig. 8 shows the tumor volume in tumor-bearing mouse models administered with the test antibodies.DETAILED DESCRIPTION[Definitions]

[0080] Unless otherwise specifically indicated, the present invention will be practiced using conventional techniques of virology, immunology, microbiology, molecular biology, and recombinant DNA technology within the technical scope of the art or as generally understood by those skilled in the art to which the present invention pertains. Many of these are described below for illustrative purposes, and such techniques are fully described in the literature.

[0081] As used herein, the term "antibody" refers to an antibody in its substantially complete form, as opposed to an antibody fragment. In particular, the "antibody" provided herein is preferably an antibody in its complete form, which has a symmetrical structure with four polypeptide chains, two heavy chains (H chains) and two light chains (L chains), wherein the chains are linked by disulfide bonds and non-covalent bonds to form a monomer molecule consisting of four polypeptide chains.

[0082] As used herein, the term "antigen-binding fragment" includes a portion of an intact antibody, preferably the antigen-binding region and / or the variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, etc.

[0083] As used herein, the term "humanized antibody or antigen-binding fragment" refers to a human immunoglobulin (antibody receptor) in which the HVR residues of the receptor are replaced by residues of a non-human species (donor antibody) with the desired specificity, affinity, and / or characteristics, such as mice, rats, rabbits, or non-human primates. In some cases, the framework ("FR") residues of the human immunoglobulin are replaced by the corresponding non-human residues. Furthermore, humanized antibodies or antigen-binding fragments may include residues that are absent from the receptor antibody or donor antibody. These modifications can be made to further improve antibody characteristics, such as binding affinity.In general, humanized antibodies or antigen-binding fragments comprise at least one, and typically substantially all, of two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulin sequences and all or substantially all of the FR regions are those of human immunoglobulin sequences, but the FR regions may include one or more individual FR residues that improve antibody characteristics such as binding affinity, isomerization, immunogenicity, etc. Humanized antibodies may optionally further comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321: 522–525 (1986); Riechmann et al., Nature 332: 323–329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593–596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105–115 (1998); Harris, Biochem. Soc. Transactions 23: 1035–1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428–433 (1994); and U.S. Patents 6,982,321 and 7,087,409.

[0084] As used herein, the term "human antibody" refers to an antibody comprising an amino acid sequence corresponding to an antibody produced by a human and / or obtained using any of the methods for producing human antibodies. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of methods known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) can also be used to produce human monoclonal antibodies. Boerner et al., J. Immunol., 147 (1): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001).Human antibodies can be produced by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigen challenge, but whose endogenous loci have been disrupted, such as immunized heterologous mice (see, e.g., U.S. Patents 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557–3562 (2006), regarding human antibodies produced using human B-cell hybridoma technology.

[0085] As used herein, the term "CDR" (i.e., complementarity-determining region) is used to refer to the hypervariable region as defined using the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Other definitions of CDR exist, such as IMGT, Chothia, AbM, Contact, etc.

[0086] Unless otherwise indicated, the numbering of immunoglobulin residues given in this description is the numbering according to Kabat.

[0087] As used herein, the term "Fc domain" generally includes the hinge region, CH2 of the heavy chain constant region, and CH3 of the heavy chain constant region (i.e., hinge region-CH2-CH3). The Fc domain forms a dimer, usually through a disulfide bond. Currently, the most commonly used fusion partner is the Fc fragment of IgG immunoglobulin. The Fc of an antibody is a part of the constant region of the antibody (including the hinge region-CH2-CH3, but not the CH1 part of the constant region of the antibody). After fusion with the Fc fragment, the fused molecule has increased stability and a prolonged half-life in vivo due to the increased molecular weight and the recycling mechanism mediated by FcRn. Meanwhile, the Fc fragment can be used to mediate various biological functions such as ADCC and CDC.However, this type of fusion protein lacks the variable region of an antibody, and its pharmacology and efficacy depend primarily on the functional molecule fused to the Fc. There are four subtypes of human immunoglobulin G, and the biological activities of the different subtypes of immunoglobulin G vary. Currently, IgG1 antibodies are the most widely used. In recent years, with the expansion of new indications and the emergence of antibody drugs with new mechanisms of action, attention has been paid to the IgG2 and IgG4 subtypes, which have less cytotoxicity. As used herein, the "Fc domain" preferably refers to the Fc domain of an IgG1 antibody or the Fc domain of an IgG4 antibody.

[0088] As used herein, "specificity" refers to an antigen-binding protein or antibody that selectively recognizes a specific epitope of an antigen. For example, natural antibodies are monospecific. As used herein, "bispecific" or "multispecific" means that the antigen-binding protein or antibody has two or more antigen-binding sites, at least two of which bind to different antigens or different epitopes of the same antigen.

[0089] As used herein, the terms "bivalent," "trivalent," and "quadrivalent" are related to "valency," which refers to a specific number of binding sites present in an antigen-binding protein or antibody molecule. Thus, the terms "bivalent," "trivalent," and "quadrivalent" refer to the presence of two binding sites, three binding sites, and four binding sites, respectively, in an antigen-binding protein or antibody molecule.

[0090] The "VHH domain" (heavy chain variable domain of a heavy-chain antibody), also known as VHH or VHH fragment, is originally derived from the variable domain of an antigen-binding immunoglobulin, which is a "heavy-chain antibody" (hcAb, i.e., "antibody lacking a light chain") (C. Hamers-Casterman, T. Atarhouch, et al. Naturally occurring antibodies devoid of light chains. Nature, June 3, 1993, Vol. 363, pp. 446-448). The term "VHH domain" is used to distinguish this type of variable domain from the heavy chain variable domain (referred to herein as the "VH domain") present in conventional antibodies and the light chain variable domain (referred to herein as the "VL domain") present in conventional antibodies consisting of two light chains and two heavy chains (hereinafter referred to as conventional antibodies).The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (unlike the VH or VL domains in conventional antibodies, in which the VL and VH domains are combined to recognize the epitope). The VHH domain is an antigen recognition unit formed by a single immunoglobulin domain.

[0091] In the present description, the terms "single domain antibody (sdAb)", "VHH domain", "VHH", "VHH antibody fragment", "VHH antibody", "nanobody" and "nanobody domain" are used interchangeably.

[0092] As used herein, the term "identity" is the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity, but not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of amino acid sequence identity can be performed by various methods available to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software.Those skilled in the art can determine appropriate parameters for performing the alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0093] As used herein, the term "treatment" refers to a clinical intervention intended to alter the natural course of a clinical pathology in a treated individual or cell. The desired therapeutic effect includes a reduction in the rate of disease progression, amelioration or alleviation of the disease state, and a mitigation or improvement of the prognosis. For example, alleviation or elimination of one or more symptoms associated with the disease or condition being treated (such as a malignancy, inflammation, or an autoimmune disease).

[0094] As used herein, an "effective amount" refers to an amount of an agent or drug that effectively treats a disease or condition in an individual. In the case of a malignant tumor, an effective amount of the binding fragment of the single-domain anti-VEGF antibody, the bispecific antibody, the multispecific antigen-binding construct, the pharmaceutical composition, and the immunoconjugate provided by the present invention can reduce the number of malignant cells, reduce the size of the tumor, inhibit (i.e., slow down to a certain extent and preferably prevent) the infiltration of cancer cells into peripheral organs, suppress (i.e., slow down to a certain extent and preferably prevent) the metastasis of malignant tumor cells, inhibit tumor growth to a certain extent, and / or alleviate to a certain extent one or more symptoms associated with the malignant tumor.As is understood in clinical practice, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" can be considered in the context of administering one or more therapeutic agents, and in the case of combination with one or more other agents, it can be considered that one agent administered in an effective amount can achieve the desired result.

[0095] As used herein, the "individual" is preferably a mammal, including, but not limited to, humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human. [Specific Embodiments]

[0096] Embodiments of the present invention will be described in detail below along with examples, but those skilled in the art will understand that the following examples are used only to illustrate the present invention and should not be construed as limiting the scope of the present invention. Example 1 Screening and development of VEGF-targeted VHH

[0097] 500 μg of human and mouse VEGF-A proteins were mixed with an equal volume of adjuvant to produce an immunogen. Two-year-old female alpacas were immunized with the immunogen four times, each time at two-week intervals. After immunization, hyperimmune serum was collected, peripheral blood mononuclear cells were isolated, and RNA was extracted. The variable region gene of the nanobody was amplified by RT-PCR. A library of VEGF-A immune antibodies was generated by enzymatic digestion, ligation, and electroporation.

[0098] The resulting anti-VEGF-A immune antibody library was subjected to two rounds of panning using solid-phase panning on immunotubes and liquid-phase screening with magnetic beads, and individual clones were selected for detection by ELISA. Positive clones were sequenced, yielding a total of 81 different antibody genes. VHH-Fc DNA fragments were constructed for all 81 resulting strains. After sequencing, 293T cells were transfected for eukaryotic expression, and the expression supernatant was collected for specific detection by ELISA. After testing, strain 6 (#6) was found to be the only antibody with excellent binding ability to both human and mouse VEGF-A (see Tables 7 and 8). Table 7. Binding ability of #6 to human VEGF-A VHH number EC50 (ng / ml) #6 9,331 Bevacizumab 36,56 Table 8 Ability of #6 to bind to mouse VEGF-A VHH number EC50 (ng / ml) #6 6,46 Bevacizumab -

[0099] Strain 6 (#6) was sequenced, and its amino acid sequence is as follows:SEQ ID No: 8 (CDRs 1-3 (Kabat numbering) are in bold and underlined)QVQLQESGGGLVQPGGSLRLSCAASGFTFS TSTMS WYRQAPGKERELVA FITSAGATTNYADSVKD RFTMSRDNDKNTVYLQMNVLKPEDTAMYYCRA LVTLWNVY WGQGTQVTVSSTable 9 Position number for amino acid sequence #6 (Kabat numbering) Example 2 Humanization and Stability Optimization of the Original Anti-VEGF VHH Sequence

[00100] To further enhance the thermal stability and humanization of the VHH of strain 6 in combination with protein structure mimicry based on structural biology, five rounds of sequence optimization and modification were performed to obtain two VHH sequences with good thermal stability and high humanization.#6 (SEQ ID NO: 8):QVQLQESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYRQAPGKERELVAFITSAGATTNYADSVKDRFTMSRDNDKNTVYLQMNVLKPEDTAMYYCRALVTLWNVYWGQGTQVTVSS#48 (SEQ ID NO: 13):E VQL L ESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYRQAPGKERELV S FITSAGATT Y YADSVK G RFTMSRDN S KNTVYLQMN S L RA EDTA V YYCRALVTLWNVYWGQGT L VTVSS#49 (SEQ ID NO: 14): E VQL L ESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYRQAPGKERELVAFITSAGATT Y YADSVK G RFTMSRDN S KNTVYLQMN S L RA EDTA V YYCRALVTLWNVYWGQGT L VTVSSTable 10 Amino acid mutation sites after multiple rounds of sequence optimization (using the Kabat system position in Table 9) № Mutation site relative to VHH #6 sequence Sequence identity of VHH #6 #48 Q1E+Q5L+A49S+N58Y+D65G+D74S+V(82B)S+K83R+P84A+M89V+Q108L 106 / 117(91%) #49 Q1E+Q5L+N58Y+D65G+D74S+V(82B)S+K83R+P84A+ M89V+Q108L 107 / 117(91%)

[00101] For sequence #48, Q1E+Q5L is in FR1 region, A49S is in FR2 region, N58Y+D65G is in CDR2 region, D74S+V(82B)S+K83R+P84A+M89V is in FR3 region and Q108L is in FR4 region.

[00102] For sequence #49, Q1E+Q5L is in FR1 region, N58Y+D65G is in CDR2 region, D74S+V(82B)S+K83R+P84A+M89V is in FR3 region and Q108L is in FR4 region.

[00103] The results of VEGF binding ability and thermal stability analysis show that both #48 and #49 showed good VEGF binding ability, comparable to #6 (See Fig. 1). The results of thermal stability analysis show that the thermal stability of the sequences with the combined mutation increased by almost 10°C, with Tm values ​​of 64.1°C and 64.7°C, respectively.Example 3 Screening of Molecular Structures of a Candidate Bispecific Antibody and Construction of Control Structures

[00104] Based on VHH #48 and fragments of the antigen-binding region of the anti-PD-1 antibody Nivolumab (Opdivo) or pembrolizumab (Keytruda), various bispecific antibody structures were evaluated and screened. After a comprehensive evaluation of activity, expression, stability, etc., the bispecific antibody structure shown in Fig. 2 was selected. Bispecific antibody molecules #10 and #16 were designed for further evaluation. The valency of both cases was bivalent+bivalent (2+2), and the Fc was IgG1 Fc with a functionally silent mutation (AEASS). The amino acid sequences of the designed molecules are presented in Table 11.Table 11 Amino acid sequence of the designed molecule. #10 Heavy chain (SEQ ID NO: 20): QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYY 60 ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS 120 VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS 180 VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEA EGAPSVFLFP 240 PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS 300 VLTVLHQDWL NGKEYKCKVS NKALPSSIEK TISKAKGQPR EPQVYTLPPS REEMTKNQVS 360 LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS 420 CSVMHEALHN HYTQKSLSLS PGEVQLLESG GGLVQPGGSL RLSCAASGFT FSTSTMSWYR 480 QAPGKERELV SFITSAGATT YYADSVKGRF TMSRDNSKNT VYLQMNSLRA EDTAVYYCRA 540 LVTLWNVYWG QGTLVTVSS 559 Легкая цепь (SEQ ID NO: 21): EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA 60 RFSGSGSGTD FTLTISSLEP EDFAVYCQQ SSNWPRTFGQ GTKVEIKRTV AAPSVFIFPP 120 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180 LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC 214 #16 Тяжелая цепь (SEQ ID NO: 22): QVQLVQSGVE VKKPGASVKV SCKASGYTFT NYYMYWVRQA PGQGLEWMGG INPSNGGTNF 60 NEKFKNRVTL TTDSSTTTAY MELKSLQFDD TAVYYCARRD YRFDMGFDYW 120 GQGTTVTVSS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300 STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPSSIEKTIS KAKGQPREPQ VYTLPPSREE 360 MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420 QQGNVFSCSV MHEALHNHYT QKSLSLSPGE VQLLESGGGL VQPGGSLRLS CAASGFTFST 480 STMSWYRQAP GKERELVSFI TSAGATTYYA DSVKGRFTMS RDNSKNTVYL QMNSLRAEDT 540 AVYYCRALVT LWNVYWGQGT LVTVSS 566 Легкая цепь (SEQ ID NO: 23): EIVLTQSPAT LSLSPGERAT LSCRASKGVS TSGYSYLHWY QQKPGQAPRL LIYLASYLES 60 GVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRDLPL TFGGGTKVEI KRTVAAPSVF 120 IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS 180 STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 218

[00105] As a control, a bispecific antibody analogue of AK112, targeting PD-1 and VEGF, was used, which was independently obtained based on the amino acid sequence of the heavy chain of SEQ ID NO: 25 and the amino acid sequence of the light chain of SEQ ID NO: 26.SEQ ID NO:25:EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGR FTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSMSASVGDRVTFCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKSEQ ID NO: 26:DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAYEKHKVYACEVTHQGLSSPVTKSFNRGECPrimer 4 Связывание с Human PD-1 and VEGFAntibody binding to human PD-1 was determined by ELISA. 1 μg / mL human PD-1 (ACRO, His-tag) was coated on an ELISA plate overnight and blocked. Gradient dilutions of the bispecific antibody and control antibody were then added to the plate, incubated, and washed. HRP-labeled anti-human IgG Fc secondary antibody was then added to the plate, incubated, and washed. After adding TMB solution for color development, the OD450 value was read on a microplate reader (see Table 12). Antibody binding to human VEGF was detected by ELISA. 50 ng / mL human VEGF (ACRO, His-tag) was coated on an ELISA plate overnight and blocked. Gradient dilutions of the test sample and control antibody were then added to the plate, incubated, and washed. HRP-labeled anti-human IgG Fc secondary antibody was then added to the plate, incubated, and washed.After adding TMB solution for color development, the OD450 value was read using a microplate reader (see Table 13). From the ELISA detection results, it can be seen that the binding ability of #10 and #16 to human PD-1 was comparable to that of Opdivo and Keytruda, but with a higher peak signal and stronger binding ability than the control analog AK112. The binding pattern of #10 and #16 to VEGF was highly unique. Compared with the binding ability of #48 to VEGF, the binding ability of #10 and #16 to VEGF was slightly reduced, and the highest absorbance value was approximately half of that of #48, indicating that structural biology-based optimization changed the binding state of #10 and #16 to VEGF, thereby achieving greater drug accumulation. The antibody fragment bevacizumab was used as the anti-VEGF portion of the AK112 analogue, which exhibitedsimilar characteristics to bevacizumab in terms of VEGF binding (all controls were generated by in-house expression and purification). Table 12 Binding of test antibodies to human PD-1 Read more Molecular weight (kDa) EC50 (nM) HIGHEST OD #10 169,3 0,114 2,64 #16 171,9 0,114 2,579 Opdivo 143 0,1 2,271 Keytruda 146 0,102 2,324 Analogue of AK112 201,5 0,528 2,19 Bevacizumab 146,29 - 0,151 Table 13 Binding of tested antibodies to human VEGF Protein molecules Molecular weight (kDa) EC50 (nM) HIGHEST OD #10 169,3 0,366 1,752 #16 171,9 0,579 1,745 #48 76,46 0,608 3,091 Opdivo 143 - 0,048 Analogue of AK112 201,5 0,64 2,683 Bevacizumab 146,29 0,374 3,157 Example 5 Dual Binding Ability of Human VEGF and Human PD-1

[00107] To further test the dual binding ability of #10, #16, and AK112 analog to human VEGF and human PD-1, a 96-well ELISA plate was coated with 50 ng / mL human VEGF (Sino Biological, unlabeled) overnight, washed, and blocked for 1 hour. Gradient dilutions of the test antibody were then added to the plate, incubated for 2 hours, followed by the addition of 1 μg / mL human PD-1 (ACRO, His-tag), incubated for 2 hours, washed, and incubated with HRP-labeled anti-His antibody for 1 hour. After the addition of TMB solution for color development, the optical density OD450 nm was read on a microplate reader. The results are presented in Table 14. The single-targeting antibody was unable to bind to two targets simultaneously.All of #10, #16, and the AK112 analog had dual binding activity to human VEGF and human PD-1 with EC50 values ​​of 0.279 nM, 0.326 nM, and 0.254 nM, respectively.

[00108] The order of binding to VEGF and PD-1 was reversed for detection by dual ELISA. A 96-well ELISA plate was coated with 1 μg / mL human PD-1 (ACRO, His-tag) overnight, washed, and blocked for 1 hour. Gradient dilutions of specific antibodies were then added to the plate, incubated for 2 hours, followed by the addition of 50 ng / ml biotin-labeled human VEGF (ACRO, biotinylated protein), incubated for 2 hours, washed, and incubated with HRP-labeled streptavidin for 1 hour. TMB solution was added for color development, and the optical density (OD450 nm) was read on a microplate reader. The results are presented in Table 14. The single-targeting antibody was unable to bind to two targets simultaneously.All #10, #16, and the AK112 analog exhibited dual binding to human PD-1 and human VEGF with EC50 values ​​of 0.114 nM, 0.117 nM, and 0.634 nM, respectively. Based on the comparison of EC50 values, the dual binding affinity of #10 and #16 to human PD-1 and human VEGF was 5-6 times stronger than that of the competitor analog AK112. Table 14. Dual binding of tested antibodies to human PD-1 and human VEGF. Protein molecules Molecular weight (kDa) EC50 (nM) HIGHEST OD #10 169,3 0,114 3,571 #16 171,9 0,117 3,649 #48 76,46 - 0,061 Opdivo 143 - 0,048 Analogue of AK112 201,5 0,634 3,415 Bevacizumab 146,29 - 0,067 Example 6. Binding of Antigens from a Different Species

[00109] Detection of binding of candidate molecules and competitors to antigens from a different species was performed using ELISA. The sequence of monkey VEGF was completely identical to that of human VEGF, and the candidate antibody had the same binding ability to monkey VEGF as to human VEGF. Detection of antibody binding to monkey PD-1 was performed using ELISA. An ELISA plate was coated with 1 μg / ml monkey PD-1 (ACRO, His-tag) overnight and blocked. Gradient dilutions of the test sample and control antibody were then added to the plate, incubated, and washed. HRP-labeled anti-human IgG Fc secondary antibody was then added to the plate, incubated, and washed. After adding the TMB solution for color development, the optical density OD450 value was read on a microplate reader (see Table 15).Consistent with the human PD-1 binding results, the binding affinity of #10 and #16 to monkey PD-1 was comparable to that of Opdivo and Keytruda and stronger than that of the competitor AK112. Table 15 Binding of tested antibodies to monkey PD-1. Protein molecules Molecular weight (kDa) EC50 (nM) HIGHEST OD #10 169,3 0,229 3,359 #16 171,9 0,171 3,376 Opdivo 143 0,153 2,987 Keytruda 146 0,187 3,078 Analogue of AK112 201,5 1,704 2,561 Bevacizumab 146,29 - 0,127 Example 7. Ability to Block PD-1 and PD-L1 Binding

[00110] Next, the ability of the tested antibodies to block PD-1 and PD-L1 binding was detected. An ELISA plate was coated with 1 μg / ml human PD-1 (ACRO, Fc-tag) overnight, washed, and blocked. A gradient dilution mixture of the antibody and human PD-L1 (Sino Biological, His-tag) was then added to the plate and incubated for 2 hours. HRP-labeled anti-His antibody was then added to the plate and incubated for 1 hour. After adding TMB solution for color development, the OD450 value was read on a microplate reader (see Table 16). As the results showed, with the exception of #48, which bound only to VEGF, the remaining antibodies could block the binding of PD-1 and PD-L1, and the blocking activity was comparable. Table 16 Detection of the ability of the tested antibodies to block the binding of human PD-1 to human PD-L1 Protein molecules IC50 (nM) #10 2,419 #16 2,529 #48 - Opdivo 1,947 Keytruda 2,13 Analogue of AK112 2,898 Example 8 Ability to Block VEGF Binding to VEGFR1 and VEGFR2

[00111] The ability of the tested antibodies to block VEGF binding to VEGFR1 and VEGFR2 was detected. ELISA plates were coated with 2 μg / ml human VEGFR1 and 5 μg / ml human VEGFR2 (in-house engineered and expressed, Fc tag) overnight, washed, and blocked, respectively. A gradient dilution mixture of antibodies and human VEGF (ACRO, biotinylated protein) was then added to the plates, incubated for 2 hours, and washed. HRP-labeled streptavidin was then added to the plates and incubated for 1 hour. After adding TMB solution for color development, the optical density OD450 nm was read on a microplate reader.The results show that #10 and #16 exhibited stronger blocking ability for VEGF and VEGFR1 binding, with IC50 values ​​of 0.491 nM and 0.507 nM, respectively, which was better than the blocking ability of #48, demonstrating the benefits of altering the spatial structure of the engineered bispecific antibodies. The blocking ability of the competitive analog AK112 was comparable to that of bevacizumab, with IC50 values ​​of 4.610 nM and 5.647 nM, respectively. Furthermore, the results show that #10 and #16 had stronger blocking ability for VEGF and VEGFR2 binding, with IC50 values ​​of 3.643 nM and 3.249 nM, respectively, which was better than the blocking ability of #48 with an IC50 value of 5.841 nM, demonstrating the benefits of altering the spatial structure of bispecific antibodies. The blocking ability of the competitive analog AK112 was comparable to that of bevacizumab, with IC50 values ​​of 6.481 nM and 6.637 nM, respectively.Example 9 T-cell response after PD-1 blockade tested by mixed lymphocyte reaction (MLR)

[00112] The functions of candidate molecules and the competitive analogue AK112 were further compared at the cellular level. T-cell response after PD-1 blockade was tested by mixed lymphocyte reaction (MLR). 2×10. 4 DC from one donor and 2×10 5CD4+ T cells from another donor were mixed in equal volumes, gradient dilutions of test antibodies were added, and the cells were cultured for 72 hours. The cell culture supernatant was collected, and the content of IL-2 (Fig. 3) and IFNγ (Fig. 4) released in the cell supernatant was measured using a quantitative ELISA kit. The results show that the levels of IL-2 and IFNγ released by CD4+ T cells in the MLR response stimulated by #10 and #16 were higher than those of the competitive analog AK112, indicating that #10 and #16 had a stronger effect on T cells. Example 10 The ability of the tested antibodies to inhibit the proliferation of VEGF-stimulated human umbilical vein endothelial cells (HUVEC) confirmed by the HUVEC

[00113] 5×10 proliferation inhibition assay 3HUVEC cells / well were added to a 96-well cell plate in medium containing 1% FBS and cultured overnight in an incubator at 37°C with 5% carbon dioxide. The following day, the medium was removed and 70 ng / ml VEGF and gradient-diluted test antibodies, as well as the competitive analogue AK112, were added. The reaction plate was placed in an incubator at 37°C with 5% carbon dioxide for 4 days. After adding CTG reagent to the plate, the relative fluorescence signal was read on a microplate reader. The fluorescence value of the ECM medium containing 1% FBS and the VEGF-stimulated cell group (without antibodies) was defined as an inhibition rate of 0, and the fluorescence value of the PBS group (without ECM medium containing 1% FBS and without VEGF and antibody stimulation) was defined as an inhibition rate of 100%. The relative inhibition rate was transformed into a curve. As shown in Fig.5, the maximum inhibition rate of #10 and #16 was approximately 60%, while the maximum inhibition rate of the AK112 analog was approximately 40%. The inhibition rate of #10 and #16 was higher than that of the competitive analog AK112. Example 11 Ability to Inhibit HUVEC Cell Migration

[00114] The ability of the tested antibodies to inhibit HUVEC cell migration was determined using a Transwell chamber with a well size of 8 μm. The Transwell consists of an upper and a lower chamber. 5×10. 3HUVEC cells / well were added to the upper chamber and incubated at 37°C with 5% carbon dioxide for 30 minutes. 200 ng / ml VEGF and various concentrations of test antibodies or the competitive analogue AK112 were added to the lower chamber, with three replicate wells for each group. The cells were incubated at 37°C with 5% carbon dioxide for 24 hours, then fixed with 4% paraformaldehyde and stained with crystal violet. After photographing, the number of cells migrating to the back of the upper chamber was counted. The lower the cell count, the higher the inhibition rate. The results in Fig. 6 show that the inhibition rate of #10 and #16 was higher than that of the competitive analogue AK112. Example 12: Improved antibody accumulation properties

[00115] Structural biological modeling shows that the conformation of #10 can change the binding mode of the anti-VEGF nanobody to VEGF, and the antibodies can accumulate due to the VEGF dimeric proteins.To test the antibody accumulation enhancement properties of #10, human VEGF protein-coupled magnetic beads (ACRO) were diluted to 250 ng / mL, 125 ng / mL, and 50 ng / mL in PBS supplemented with 10 nM #10, #16, and the competitive analog AK112, incubated for 1 hour, and then placed on a magnet for 2 minutes. The supernatant was collected for protein quantification by ELISA. The results are shown in Fig. 7. The amount of remaining #10 and #16 antibodies was lower than that of the competitive analog AK112, indicating that VEGF-containing magnetic beads accumulated more bispecific antibodies. This quantitative ELISA experiment was aimed at testing the degree of cross-linking between antibodies and VEGF. Antibodies were first cross-linked with VEGF magnetic microcarriers, and the remaining amount of uncross-linked antibodies was determined by ELISA.The results showed that the degree of cross-linking of #10 and #16 with VEGF was higher than that of the competitive analogue AK112, which was mutually confirmed by the results of structural biological modeling. This property will allow anti-VEGF antibodies to locally accumulate in the tumor due to its high local expression, demonstrating the different characteristics and therapeutic potential of bispecific antibodies #10 and #16. Example 13: Efficacy in an Animal Model

[00116] To evaluate the efficacy of the tested antibodies in a mouse model, Balb / c mice transformed with human PD-1 were selected and inoculated with wild-type CT26 tumor cells (colon cancer cells). Since neither bevacizumab nor the AK112 analogue bound to mouse VEGF, these two groups were not added. A VEGF trap (a fusion protein consisting of the second domain of VEGFR1, the third domain of VEGFR2, and human IgGFc) that can bind to both human and mouse VEGF was selected as a control. A total of six groups were created.The experimental design, the amount administered, and the route of administration are presented in Table 17.Table 17. Design of experiments to evaluate efficacy in an animal model. Test group Medicine Number of mice Amount administered (mg / kg) Frequency of administration Route of administration 1 Empty vector (PBS) 8 0 D0, D4, D7, D11 v / b 2 Opdivo 8 7,5 D0, D4, D7, D11 v / b 3 VEGF trap 8 5 D0, D4, D7, D11 v / b 4 Opdivo+VEGF-trap 8 7,5+5 D0, D4, D7, D11 v / b 5 #10 8 8,8 D0, D4, D7, D11 v / b 6 #16 8 8,9 D0, D4, D7, D11 v / b

[00117] As shown in Fig. 8, the tumor inhibition rates in the tumor-bearing mice administered with #10 and #16 were 57% and 62%, respectively, which were higher than the tumor inhibition rate (20%) in the Opdivo monotherapy group and the tumor inhibition rate (45%) in the VEGF Trap monotherapy group, and comparable to the tumor inhibition rate (56%) in the Opdivo and VEGF Trap combination therapy group. On day 14, the average tumor volume in the #10 and 16 administration groups was slightly smaller than that in the combination therapy group. In addition, the changes in the body weight of the mice followed the trend of tumor growth. On day 14, the change in body weight in the #10 and #16 groups was within 10%. As can be seen, both #10 and #16 had good efficacy and safety.Example 14 Study of the synergistic effect of #10 in a subcutaneous transplantation model of human immune-reconstituted melanoma

[00118] To evaluate the synergistic effect of #10 in a human immune-reconstituted melanoma model, B-NDG immunodeficient mice were inoculated with human PD-L1-transformed A375 tumor cells (melanoma cells) and inoculated with human peripheral blood mononuclear cells (huPBMCs) to reconstitute the immune system of tumor-bearing mice. The anti-PD-1 drug Opdivo (drug O), the anti-VEGF drug bevacizumab, and #48 were selected as single-target controls, and AK112 analog (AK112 analog expressed and prepared in-house) was selected as the control drug of #10. A total of 8 groups were created. The experimental design, the administered amount and the route of administration are presented in Table 18.Table 18. Experimental design of the synergistic study in a subcutaneous transplantation model of human melanoma with restored immunity. Test group Medicine Number of mice Amount administered (mg / kg) Frequency of administration Route of administration 1 Empty vector 9 -- D0, D3, D7, D10, D14, D17 intravenously 2 #10 9 5,7 D0, D3, D7, D10, D14, D17 intravenously 3 Bevacizumab 9 5 D0, D3, D7, D10, D14, D17 intravenously 4 #48 9 2,6 D0, D3, D7, D10, D14, D17 intravenously 5 Opdivo 9 4,9 D0, D3, D7, D10, D14, D17 intravenously 6 Bevacizumab + Opdivo 9 5+4,9 D0, D3, D7, D10, D14, D17 в / в 7 #48 + Опдиво 9 2,6+4,9 D0, D3, D7, D10, D14, D17 в / в 8 Аналог AK112 9 6,8 D0, D3, D7, D10, D14, D17 в / в

[00119] Detection was performed on day 21. In this experimental system, the Opdivo monotherapy group had no antitumor effect, the tumor inhibition rate #10 was TGI=73%, demonstrating a significantly higher tumor inhibition rate than the bevacizumab monotherapy group (TGI=53%), the #48 monotherapy group (TGI=57%), and the Opdivo and #48 combination treatment groups (TGI=50%), and was superior to the Opdivo and bevacizumab combination treatment group (TGI=68%, based on which the #10 tumor inhibition rate increased by 7%) and the AK112 analog (TGI=64%, based on which the relative tumor inhibition rate #10 increased by 14%). The results indicate that the bispecific antibodies of the present invention exert a synergistic effect and have significant advantages over the control molecules.

[00120] Only some of the embodiments of the present invention have been disclosed above, which do not limit the present invention in any way. Those skilled in the art will understand that the present invention is not limited to each specific embodiment. For those skilled in the art, some improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications are also included within the scope of protection of the claims of the present invention. ---> <?xml version="1.0" encoding="ISO-8859-1"?><!DOCTYPE ST26SequenceListing SYSTEM "ST26SequenceListing_V1_3.dtd"PUBLIC "- / / WIPO / / DTD Sequence Listing 1.3 / / EN"> . <st26sequencelisting productiondate="2023-12-27" softwareversion="2.3.0" softwarename="WIPO Sequence" filename="AAO.xml" dtdversion="V1_3"> <applicationidentification> <ipofficecode> CN < / ipofficecode> <applicationnumbertext / > <filingdate / > < / applicationidentification> <applicantfilereference> 2023AAO0041 < / applicantfilereference> <earliestpriorityapplicationidentification> <ipofficecode> CN < / ipofficecode> <applicationnumbertext> CN202211722833.X < / applicationnumbertext> <filingdate> 2022-12-30< / filingdate> < / earliestpriorityapplicationidentification> <applicantname languagecode="en"> RemeGen (Shanghai) Co.,Ltd. < / applicantname> <applicantnamelatin> RemeGen (Shanghai) Co., Ltd. < / applicantnamelatin> <inventorname languagecode="en"> LI DONG < / inventorname> <inventornamelatin> LI DONG < / inventornamelatin> <inventiontitle languagecode="en"> BISPECIFIC ANTIBODY AND APPLICATIONTHEREOF < / inventiontitle> <sequencetotalquantity> 26< / sequencetotalquantity> <sequencedata sequenceidnumber="1"> <insdseq><INSDSeq_length>5< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..5< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q11"> <INSDQualifier_name>organism< / 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insdqualifier> <insdqualifier id="q15"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence>WYRQAPGKERELVA< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="6"> <insdseq><INSDSeq_length>32< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..32< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q16"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / 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<INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q33"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence> QVQLVESGGGVVQPGRSLRLDKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAEGAPSVFLFPPKKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="17"> <insdseq><INSDSeq_length>214< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..214< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q35"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence>EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="18"> <insdseq><INSDSeq_length>449< / INSDSeq_length><INSDSeq_moltype> AA< / INSDSeq_moltype><INSDSeq_division> PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..449< / INSDFeature_location><INSDFeature_quals> <insdqualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q37"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence> QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="19"> <insdseq><INSDSeq_length>218< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..218< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q39"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence>EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="20"> <insdseq><INSDSeq_length>559< / INSDSeq_length><INSDSeq_moltype> AA< / INSDSeq_moltype><INSDSeq_division> PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..559< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q41"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence>< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="21"> <insdseq><INSDSeq_length>214< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..214< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q43"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence>EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="22"> <insdseq><INSDSeq_length>566< / INSDSeq_length><INSDSeq_moltype> AA< / INSDSeq_moltype><INSDSeq_division> PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..566< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value>< / insdqualifier> <insdqualifier id="q45"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence>< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="23"> <insdseq><INSDSeq_length>218< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..218< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q47"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence>EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="24"> <insdseq><INSDSeq_length>17< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..17< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q49"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence>FITSAGATTYYADSVKG< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="25"> <insdseq><INSDSeq_length>717< / INSDSeq_length><INSDSeq_moltype> AA< / INSDSeq_moltype><INSDSeq_division> PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..717< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q54"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence>< / INSDSeq_sequence>< / insdseq> < / sequencedata> <sequencedata sequenceidnumber="26"> <insdseq><INSDSeq_length>214< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table> <insdfeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..214< / INSDFeature_location><INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q55"> <INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / insdqualifier> < / INSDFeature_quals>< / insdfeature> < / INSDSeq_feature-table><INSDSeq_sequence>DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>< / insdseq> < / sequencedata> < / st26sequencelisting> <---

Claims

1. A bispecific antibody targeting programmed death protein 1 (PD-1) and vascular endothelial growth factor (VEGF), containing: (a) a first binding region targeting PD-1; and (b) a second binding region targeting VEGF; Where the first binding region targeting PD-1 comprises an anti-PD-1 antibody or an antigen-binding fragment thereof; and the second binding region targeting VEGF comprises the variable domain of a heavy chain-only antibody (VHH) containing complementarity determining regions (VHH-CDRs) 1-3, where 1) VHH-CDR1 contains the amino acid sequence of SEQ ID NO: 1; VHH-CDR2 comprises the amino acid sequence of SEQ ID NO: 2; and VHH-CDR3 comprises the amino acid sequence of SEQ ID NO: 3; or 2) VHH-CDR1 contains the amino acid sequence of SEQ ID NO: 1; VHH-CDR2 comprises the amino acid sequence of SEQ ID NO: 24; and VHH-CDR3 contains the amino acid sequence of SEQ ID NO:

3.

2. The bispecific antibody of claim 1, wherein the VHH domain comprises: VHH-CDR1 comprises the amino acid sequence of SEQ ID NO: 1; VHH-CDR2 comprises the amino acid sequence of SEQ ID NO: 2; and VHH-CDR3 contains the amino acid sequence of SEQ ID NO:

3.

3. The bispecific antibody according to claim 1 or 2, wherein the VHH comprises the amino acid sequence of SEQ ID NO:

8.

4. The bispecific antibody of claim 1, wherein the VHH domain comprises: VHH-CDR1, which contains the amino acid sequence of SEQ ID NO: 1; VHH-CDR2 comprises the amino acid sequence of SEQ ID NO: 24; and VHH-CDR3 contains the amino acid sequence of SEQ ID NO:

3.

5. The bispecific antibody according to claim 1 or 4, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO:

13.

6. The bispecific antibody according to claim 1 or 4, wherein the VHH domain comprises the amino acid sequence of SEQ ID NO:

14.

7. The bispecific antibody of any one of claims 1 to 6, wherein the VHH domain is a humanized VHH domain.

8. The bispecific antibody of any one of claims 1 to 7, wherein the first binding region targeting PD-1 comprises a variable heavy chain (VH) comprising the variable heavy chain complementarity determining region 1 (VH-CDR1), the variable heavy chain complementarity determining region 2 (VH-CDR2) and the variable heavy chain complementarity determining region 3 (VH-CDR3), as they are present for the VH of the antibody nivolumab; and a variable light chain (VL) region comprising the variable light chain complementarity determining region 1 (VL-CDR1), the variable light chain complementarity determining region 2 (VL-CDR2) and the variable light chain complementarity determining region 3 (VL-CDR3), as they are present for the VL antibody nivolumab.

9. The bispecific antibody of claim 8, wherein VH comprises the VH of the antibody nivolumab, and VL comprises the VL of the antibody nivolumab.

10. Bispecific antibody according to claim 9, where: (a) the VH of the first binding region targeting PD-1 comprises the VH of the antibody nivolumab, and the VL of the first binding region targeting PD-1 comprises the VL of the antibody nivolumab; and (b) The VHH of the second binding region targeting VEGF comprises the amino acid sequence of SEQ ID NO:

8.

11. Bispecific antibody according to claim 9, where: (a) the VH of the first binding region targeting PD-1 comprises the VH of the antibody nivolumab, and the VL of the first binding region targeting PD-1 comprises the VL of the antibody nivolumab; and (b) The VHH of the second binding region targeting VEGF comprises the amino acid sequence of SEQ ID NO:

13.

12. Bispecific antibody according to claim 9, where: (a) the VH of the first binding region targeting PD-1 comprises the VH of the antibody nivolumab, and the VL of the first binding region targeting PD-1 comprises the VL of the antibody nivolumab; and (b) The VHH of the second binding region targeting VEGF comprises the amino acid sequence of SEQ ID NO:

14.

13. The bispecific antibody of any one of claims 8-12, wherein the first binding region targeting PD-1 comprises a heavy chain comprising the heavy chain of the antibody nivolumab and a light chain comprising the light chain of the antibody nivolumab.

14. The bispecific antibody of claim 13, wherein the first binding region targeting PD-1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 16, and a light chain comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:

17.

15. The bispecific antibody of claim 14, wherein the first binding region targeting PD-1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 16 and a light chain comprising the amino acid sequence of SEQ ID NO:

17.

16. The bispecific antibody of any one of claims 1-7, wherein the first binding region targeting PD-1 comprises a variable heavy chain (VH) region comprising the variable heavy chain complementarity determining region 1 (VH-CDR1), the variable heavy chain complementarity determining region 2 (VH-CDR2) and the variable heavy chain complementarity determining region 3 (VH-CDR3), as present in the VH of the antibody pembrolizumab; and a variable light chain (VL) region comprising the variable light chain complementarity determining region 1 (VL-CDR1), the variable light chain complementarity determining region 2 (VL-CDR2) and the variable light chain complementarity determining region 3 (VL-CDR3), as they are present for the VL antibody pembrolizumab.

17. The bispecific antibody of claim 16, wherein VH comprises the VH of the antibody pembrolizumab, and VL comprises the VL of the antibody pembrolizumab.

18. Bispecific antibody according to claim 17, where: (a) the VH of the first binding region targeting PD-1 comprises the VH of the antibody pembrolizumab, and the VL of the first binding region targeting PD-1 comprises the VL of the antibody pembrolizumab; and (b) The VHH of the second binding region targeting VEGF comprises the amino acid sequence of SEQ ID NO:

8.

19. Bispecific antibody according to claim 17, where: (a) the VH of the first binding region targeting PD-1 comprises the VH of the antibody pembrolizumab, and the VL of the first binding region targeting PD-1 comprises the VL of the antibody pembrolizumab; and (b) The VHH of the second binding region targeting VEGF comprises the amino acid sequence of SEQ ID NO:

13.

20. Bispecific antibody according to claim 17, where: (a) the VH of the first binding region targeting PD-1 comprises the VH of the antibody pembrolizumab, and the VL of the first binding region targeting PD-1 comprises the VL of the antibody pembrolizumab; and (b) The VHH of the second binding region targeting VEGF comprises the amino acid sequence of SEQ ID NO:

14.

21. The bispecific antibody of any one of claims 16-20, wherein the first binding region targeting PD-1 comprises a heavy chain that contains the heavy chain of the antibody pembrolizumab, and a light chain that contains the light chain of the antibody pembrolizumab.

22. The bispecific antibody of claim 21, wherein the first binding region targeting PD-1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:

19.

23. The bispecific antibody of claim 22, wherein the first binding region targeting PD-1 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18, and a light chain comprising the amino acid sequence of SEQ ID NO:

19.

24. The bispecific antibody of any one of claims 8-23, wherein the anti-PD-1 antibody or antigen-binding fragment thereof comprises a humanized antibody or antigen-binding fragment thereof, a chimeric antibody or antigen-binding fragment thereof, or a human antibody or antigen-binding fragment thereof.

25. The bispecific antibody of claim 24, wherein the anti-PD-1 antibody is an IgG antibody.

26. The bispecific antibody of claim 25, wherein the anti-PD-1 antibody is IgG1, IgG2, or IgG4.

27. The bispecific antibody of any one of claims 8-26, wherein the anti-PD-1 antibody comprises an Fc domain and wherein the Fc domain is an IgG1 Fc domain or a silent mutation thereof of IgG1.

28. The bispecific antibody of claim 27, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:

15.

29. The bispecific antibody of claim 28, wherein the Fc domain comprises the amino acid sequence of SEQ ID NO:

15.

30. The bispecific antibody of any one of claims 8-29, wherein the second binding region targeting VEGF is connected to the first binding region targeting PD-1 via a peptide linker.

31. The bispecific antibody of claim 30, wherein the peptide linker is a flexible peptide linker.

32. The bispecific antibody of claim 31, wherein the peptide linker comprises one or more amino acids.

33. The bispecific antibody of claim 32, wherein the peptide linker comprises at least 5 amino acids.

34. The bispecific antibody of any one of claims 31-33, wherein the peptide linker comprises the amino acid sequence (GGGGS)n, where n is 1, 2, 3 or 4.

35. The bispecific antibody of any one of claims 8-34, wherein the second binding region targeting VEGF is linked to the C-terminus or N-terminus of the first binding region targeting PD-1.

36. The bispecific antibody of claim 35, wherein the second binding region targeting VEGF is linked to the N-terminus of the first binding region targeting PD-1.

37. The bispecific antibody of claim 35, wherein the second binding region targeting VEGF is linked to the C-terminus of the first binding region targeting PD-1.

38. The bispecific antibody of claim 37, wherein the first binding region targeting PD-1 is an IgG antibody, and wherein the C-terminus of each heavy chain of the IgG antibody is connected to a second binding region targeting VEGF.

39. The bispecific antibody of claim 37 or 38, wherein the bispecific antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 20, and / or a light chain that comprises the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:

21.

40. The bispecific antibody of claim 39, wherein the bispecific antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 20 and a light chain that comprises the amino acid sequence of SEQ ID NO:

21.

41. The bispecific antibody of claim 37 or 38, wherein the bispecific antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 22, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 22, and a light chain that comprises the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:

23.

42. The bispecific antibody of claim 41, wherein the bispecific antibody comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO: 22, and a light chain that comprises the amino acid sequence of SEQ ID NO:

23.

43. A polynucleotide encoding a bispecific antibody according to any one of claims 1-42.

44. An expression vector containing the polynucleotide according to claim 43.

45. A host cell for expressing a bispecific antibody, containing the expression vector according to claim 44 or having the polynucleotide according to claim 43 integrated into its genome.

46. ​​A pharmaceutical composition for treating a malignant tumor, comprising a bispecific antibody according to any one of claims 1-42 and a pharmaceutically acceptable carrier.

47. A method for treating a malignant tumor, comprising administering to a subject in need thereof an effective amount of a bispecific antibody according to any one of claims 1-42.

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