Bispecific antibodies and their applications

Abstract

The present invention provides a VHH targeting VEGF, a bispecific antibody targeting PD-1 and VEGF developed based on this VHH, and applications thereof. The VHH has various advantageous effects, including high stability and ease of expression and purification. The constructed bispecific antibody targeting PD-1 and VEGF is highly stable, can be targeted and concentrated in areas of tumors with high VEGF expression, has good efficacy and safety, and exhibits excellent therapeutic effects.

Description

[Technical Field]

[0001] The present invention relates to the field of antibodies, and more particularly to bispecific antibodies targeting PD-1 and VEGF and their applications. [Background technology]

[0002] Programmed death receptor-1 (PD-1) is an immunosuppressive receptor belonging to the immunoglobulin CD28 / B7 superfamily. When PD-1 binds to PD-L1, it phosphorylates phosphatidylinositol-3-kinase, further activates protein kinase B, and activates the stimulatory T cell signaling pathway, glucose metabolism, and interferon secretion, thereby inhibiting downstream signaling pathways of T cell activation, effectively suppressing T cell transcription, and ultimately suppressing T cell immune responses. PD-1 thus plays an important role in the downregulation of immune responses. PD-1 antibodies have a broad spectrum of anti-tumor effects, and currently several groundbreaking drugs (blockbusters) have been listed, such as Opdivo (trade name: Opdivo; generic name: nivolumab), which was approved in the US on December 22, 2014. The initial approved indication was melanoma, but it has since been approved in the US for a number of indications, including non-small cell lung cancer, renal cell carcinoma, Hodgkin's lymphoma, head and neck squamous cell carcinoma, colorectal cancer, hepatocellular carcinoma, urothelial carcinoma, colorectal cancer, hepatocellular carcinoma, esophageal cancer, esophageal squamous cell carcinoma, pleural mesothelioma, esophageal adenocarcinoma, esophagogastric junction adenocarcinoma, and gastric cancer. However, an analysis of existing clinical data (including data from 28,304 patients from 160 clinical trials) (Reference 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) found that, overall, of 22,165 patients treated with PD-1 / PD-L1 monotherapy, a total of 4,747 patients demonstrated therapeutic benefit (objective response rate (ORR), 20.21%; 95% confidence interval (CI), 18.34-22.15%). Among 19,418 cancer patients, 862 complete responses (CR) were observed. The overall incidence of CR was 3.85% (95% CI, 3.06-4.73%). Furthermore, significant differences in ORR were observed among different cancer types and PD-L1 expression status.In summary, the therapeutic effects associated with PD-1 / PD-L1 monotherapy vary significantly among different cancer types and PD-L1 expression levels, and there are still many unmet clinical needs.

[0003] Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), is a highly specific vascular endothelial cell growth-promoting factor that increases vascular permeability, modifies the extracellular matrix, and promotes endothelial cell migration, proliferation, and angiogenesis. According to literature, VEGF plays an important role in pathological angiogenesis and may contribute to the onset and progression of diseases such as tumor growth and metastasis, macular degeneration, diabetic retinal lesions, inflammatory processes (e.g., rheumatoid arthritis), ischemic processes (myocardial ischemia), and preeclampsia (Reference 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 to vascular endothelial growth factor (VEGF) is called the vascular endothelial growth factor receptor (VEGFR). It is a receptor tyrosine kinase that plays a key role in many signaling pathways required for angiogenesis and cell migration. Upon binding to VEGF, it initiates a signaling cascade to stimulate angiogenesis. Therefore, VEGF / VEGFR is a very important target site because it is expressed almost exclusively in endothelial cells and is highly upregulated in many tumor endothelial cells. Currently, it is believed that blocking angiogenesis using anti-VEGF / VEGFR therapy will be crucial in cancer and other pathological processes. Inhibition of VEGF / VEGFR is considered a major therapeutic approach for treating certain cancers, such as renal cell carcinoma and hepatocellular carcinoma.

[0004] Currently, various clinical trials are underway to evaluate the safety and efficacy of vascular endothelial growth factor / vascular endothelial growth factor receptor (VEGF)-immune checkpoint inhibitor combinations to prevent cancer. Several early studies have suggested that the combination of immune checkpoint inhibitors and VEGFR inhibitors may offer favorable therapeutic outcomes compared with single VEGFR inhibitors or immune inhibitors (https: / / www.obroncology.com / article / combining-immune-checkpoint-and-VEGF-inhibitors-improves-survival-in-pretreated-NSCLC). However, the risks associated with this favorable outcome remain unclear (Reference 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).Several clinical trials of VEGFR / ICI dual inhibition (e.g., pembrolizumab / pazopanib combination group, nivolumab / sunitinib combination group, nivolumab / pazopanib combination group, etc.) have shown favorable therapeutic effects in the early clinical research stage (Reference 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; Reference 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). Severe toxic effects were observed in subsequent higher-dose groups. Grade 3 or higher treatment-related toxicities were observed in 73% and 60% of patients in the nivolumab / sunitinib and nivolumab / pazopanib combination groups, respectively. Furthermore, 23% of patients in the nivolumab / sunitinib combination group experienced additional adverse events, leading to treatment discontinuation. The nivolumab / pazopanib combination also experienced adverse events in 20% of patients, leading to treatment discontinuation. Similarly, the pembrolizumab / pazopanib combination experienced high-dose-related liver toxicity. These combinations were discontinued in phase III clinical trials due to incompatibility.Additionally, positive treatment efficacy data have been reported in several other clinical trials of VEGFR / ICI dual inhibition (e.g., the combination of bevacizumab and atezolizumab) in Phase III clinical trials (Reference 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).). While this trial demonstrated a moderate increase in patient response rates, the combination therapy also increased the risk of side effects. Therefore, according to the MSKCC criteria, only 20% of patients in this trial were deemed suitable for risk assessment.

[0005] In addition to the investigation of combination therapies, several bispecific antibodies targeting VEGF / VEGF receptors and PD-1 are still being investigated, such as AK112 (Chinese patent application CN109053895) developed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. However, to date, no bispecific antibodies targeting vascular endothelial growth factor / vascular endothelial growth factor receptors and immune checkpoint inhibitors have been approved for sale worldwide, and significant clinical needs and development challenges remain. Summary of the Invention

[0006] The inventors have conducted extensive research and performed multiple rounds of screening and optimization to obtain a VHH that targets VEGF, and have developed a bispecific antibody that targets PD-1 and VEGF by combining the VHH with a PD-1 antibody as a binding functional domain module component. (a) a first binding functional domain that targets PD-1; (b) a second binding functional region that targets VEGF; where: the first binding functional domain targeting PD-1 is an anti-PD-1 antibody (Ab) or antigen-binding fragment (Ab'); The second binding functional region targeting VEGF comprises a VHH domain (variable domain of heavy chain of heavy-chain antibody) of CDR1 to 3 specified by the following amino acid sequence:

[0007] [Table 1]

[0008] Furthermore, the sites of mutation in said CDR2 are at positions 58 and / or 65.

[0009] Furthermore, the mutation in CDR2 is an N58Y mutation and / or a D65G mutation.

[0010] Preferably, the mutations in CDR2 are N58Y and D65G mutations.

[0011] Preferably, the amino acid sequence of the CDR2 after mutagenesis corresponds to the amino acid sequence shown in SEQ ID NO:24.

[0012] Preferably, the combination of CDR1 to CDR3 of the VHH domain of the second binding functional region targeting VEGF is as follows:

[0013] [Table 2]

[0014] Furthermore, said VHH domain is a humanized VHH domain.

[0015] Furthermore, the second binding functional domain that targets VEGF is 1) a framework region domain FR1 that is identical to the amino acid sequence set forth in SEQ ID NO: 4 or has one or two amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 4, and / or 2) a framework region domain FR2 that is identical to the amino acid sequence set forth in SEQ ID NO: 5 or has one amino acid mutation compared to the amino acid sequence set forth in SEQ ID NO: 5, and / or 3) a framework region domain FR3 that is identical to the amino acid sequence set forth in SEQ ID NO: 6 or has 1 to 5 amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 6, and / or 4) It further comprises a framework region domain FR4 which is identical to the amino acid sequence set forth in SEQ ID NO:7 or which has one amino acid mutation compared to the amino acid sequence set forth in SEQ ID NO:7.

[0016] Furthermore, the one or two amino acid mutations in the FR1 domain are at positions 1 and / or 5, and more preferably are Q1E and / or Q5L mutations.

[0017] Furthermore, the single amino acid mutation in the FR2 domain is at position 49, and more preferably is an A49S mutation.

[0018] Furthermore, the 1 to 5 amino acid mutations in the FR3 domain are 1, 2, 3, 4 or 5 selected from positions 74, 82B, 83, 84 and 89, and more preferably 1, 2, 3, 4 or 5 selected from D74S, V(82B)S, K83R, P84A and M89V.

[0019] Furthermore, the single amino acid mutation in the FR4 domain is at position 108, more preferably Q108L.

[0020] Furthermore, the second binding functional domain targeting VEGF may be 1) a framework region domain FR1 corresponding to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 9, and / or 2) a framework region domain FR2 corresponding to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 10, and / or 3) a framework region domain FR3 corresponding to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 11, and / or 4) Further comprising a framework region domain FR4 corresponding to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:12.

[0021] Furthermore, the second binding functional domain targeting VEGF comprises a combination of framework domains identified by the following amino acids:

[0022] [Table 3]

[0023] Furthermore, the second VEGF-targeting binding domain either matches the amino acid sequence set forth in SEQ ID NO: 8 or has 1 to 11 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 8. Furthermore, the mutation sites are preferably selected from positions 1, 5, 49, 58, 65, 74, 82B, 83, 84, 89, and 108. Furthermore, the mutations are preferably 1 to 11 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) selected from Q1E, Q5L, A49S, N58Y, D65G, D74S, V(82B)S, K83R, P84A, M89V, and Q108L.

[0024] [Table 4]

[0025] More preferably, the amino acid sequence of the second binding functional domain that targets VEGF corresponds to the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:13 or SEQ ID NO:14.

[0026] [Table 5]

[0027] [Table 6]

[0028] [Table 7]

[0029] Furthermore, the anti-PD-1 antibody (Ab) or antigen-binding fragment thereof (Ab') is 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.

[0030] Furthermore, the anti-PD-1 antibody (Ab) or antigen-binding fragment (Ab') thereof is an IgG antibody.

[0031] Furthermore, the anti-PD-1 antibody (Ab) or antigen-binding fragment (Ab') thereof is of the IgG1, IgG2, or IgG4 type.

[0032] Furthermore, the first binding functional domain targeting PD-1 has the same light chain and heavy chain CDR amino acid sequences as those of the nivolumab (Opdivo) antibody or the pembrolizumab (Keytruda) antibody.

[0033] Furthermore, the first binding functional domain targeting PD-1 has the same amino acid sequence of the heavy chain variable domain as that of the nivolumab (Opdivo) antibody or the pembrolizumab (Keytruda) antibody.

[0034] The first binding functional domain targeting PD-1 has the same amino acid sequence of the light chain variable domain as that of the nivolumab (Opdivo) antibody or the pembrolizumab (Keytruda) antibody.

[0035] Furthermore, the heavy chain portion of the first binding functional domain that targets PD-1 further comprises one or more domains selected from the heavy chain constant region CH1 domain (CH1), hinge region (Hinge), heavy chain constant region CH2 domain (CH2), and heavy chain constant region CH3 domain (CH3).

[0036] The first binding functional region that targets PD-1 further comprises a light chain constant region domain (CL).

[0037] Furthermore, the anti-PD-1 antibody comprises a heavy chain portion comprising, from the N-terminus to the C-terminus, a heavy chain variable region domain (VH), a heavy chain constant region CH1 domain (CH1), a hinge region (hinge), a heavy chain constant region CH2 domain (CH2), and a heavy chain constant region CH3 domain (CH3); and a light chain portion comprising, from the N-terminus to the C-terminus, a light chain variable region domain (VH) and a light chain constant region domain (CH).

[0038] Furthermore, the Fc domain (i.e., hinge region, heavy chain constant region CH2 domain, heavy chain constant region CH3 domain) of the anti-PD-1 antibody is an IgG1 Fc domain or a silent IgG1 mutation thereof.

[0039] Furthermore, the Fc domain is identical to the amino acid sequence set forth in SEQ ID NO: 15 or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the amino acid sequence set forth in SEQ ID NO: 15.

[0040] [Table 8]

[0041] Furthermore, the first binding functional region that targets PD-1 comprises a combination of a light chain and a heavy chain consisting of the following amino acid sequences: 1) a heavy chain corresponding to the amino acid sequence set forth in SEQ ID NO: 16, and / or a light chain corresponding to the amino acid sequence set forth in SEQ ID NO: 17; or 2) a heavy chain corresponding to the amino acid sequence shown in SEQ ID NO: 18, and / or a light chain corresponding to the amino acid sequence shown in SEQ ID NO: 19.

[0042] [Table 9]

[0043] Furthermore, the second binding functional region is located at the C-terminus or N-terminus of the first binding functional region. Furthermore, the VHH domain is linked directly to the C-terminus or N-terminus of the heavy chain portion of the antibody (Ab) or its antigen-binding fragment (Ab'), or linked via a peptide linker.

[0044] Furthermore, the peptide linker is a flexible peptide linker. Additionally, the peptide linker comprises one or more amino acids. Additionally, the peptide linker comprises at least five amino acids.

[0045] Preferably, the amino acid sequence of the peptide linker is (GGGGS) n (wherein n is 1, 2, 3, or 4). Furthermore, said bispecific antibody is a bivalent, trivalent or tetravalent bispecific antibody.

[0046] Furthermore, the bispecific antibody comprises a combination of a heavy chain shown in SEQ ID NO: 20 and a light chain shown in SEQ ID NO: 21, or a combination of a heavy chain shown in SEQ ID NO: 22 and a light chain shown in SEQ ID NO: 23.

[0047] [Table 10]

[0048] The present invention further relates to a VHH domain that targets VEGF, which comprises CDRs 1 to 3 defined by the following amino acid sequences:

[0049] [Table 11]

[0050] Furthermore, the sites of mutation in said CDR2 are at positions 58 and / or 65. Furthermore, the mutation in CDR2 is an N58Y mutation and / or a D65G mutation. Furthermore, said VHH domain is a humanized VHH domain.

[0051] Furthermore, the VHH domain comprises: 1) a framework region domain FR1 that is identical to the amino acid sequence set forth in SEQ ID NO: 4 or has one or two amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 4, and / or 2) a framework region domain FR2 that is identical to the amino acid sequence set forth in SEQ ID NO: 5 or has one amino acid mutation compared to the amino acid sequence set forth in SEQ ID NO: 5, and / or 3) a framework region domain FR3 that is identical to the amino acid sequence set forth in SEQ ID NO: 6 or has 1 to 5 amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 6, and / or 4) It further comprises a framework region domain FR4 which is identical to the amino acid sequence set forth in SEQ ID NO:7 or which has one amino acid mutation compared to the amino acid sequence set forth in SEQ ID NO:7.

[0052] Furthermore, the one or two amino acid mutations in the FR1 domain are at positions 1 and / or 5, and more preferably are Q1E and / or Q5L mutations. Furthermore, the single amino acid mutation in the FR2 domain is at position 49, more preferably an A49S mutation.

[0053] Furthermore, the 1 to 5 amino acid mutations in the FR3 domain are 1, 2, 3, 4 or 5 selected from positions 74, 82B, 83, 84 and 89, and more preferably 1, 2, 3, 4 or 5 selected from D74S, V(82B)S, K83R, P84A and M89V. Furthermore, the single amino acid mutation in the FR4 domain is at position 108, and is more preferably Q108L.

[0054] Furthermore, the VEGF-targeting VHH domain may comprise: 1) a framework region domain FR1 corresponding to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 9, and / or 2) a framework region domain FR2 corresponding to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 10, and / or 3) a framework region domain FR3 corresponding to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 11, and / or 4) Further comprising a framework region domain FR4 corresponding to the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:12.

[0055] Furthermore, the VHH domain comprises a combination of framework regions identified by the following amino acids:

[0056] [Table 12]

[0057] Furthermore, the amino acid sequence of the VHH domain is identical to the amino acid sequence set forth in SEQ ID NO: 8 or has 1 to 11 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 8. Furthermore, the amino acid mutation sites are preferably selected from positions 1, 5, 49, 58, 65, 74, 82B, 83, 84, 89, and 108. Furthermore, the mutations are preferably 1 to 11 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) selected from Q1E, Q5L, A49S, N58Y, D65G, D74S, V(82B)S, K83R, P84A, M89V, and Q108L.

[0058] [Table 13]

[0059] More preferably, the amino acid sequence of the VHH domain corresponds to the amino acid sequence shown in SEQ ID NO:8, SEQ ID NO:13 or SEQ ID NO:14. The present invention further relates to the use of said VHH domains in the construction of recombinant proteins. The present invention further relates to a recombinant protein comprising the VHH domain that targets VEGF.

[0060] Furthermore, the recombinant proteins include bispecific antibodies, multispecific antibodies, and antibody-drug conjugates. The present invention further relates to polynucleotides encoding the bispecific antibody, multispecific antibody, recombinant protein, or VHH domain according to any one of the preceding claims. The present invention further relates to an expression vector comprising any one of the polynucleotides described above.

[0061] The present invention further relates to a host cell comprising an expression vector according to any one of the preceding claims or having integrated into its genome a polynucleotide according to any one of the preceding claims. The present invention further relates to a pharmaceutical composition comprising the VHH domain, bispecific antibody, multispecific antibody or recombinant protein according to any one of the above and a pharmaceutically acceptable carrier thereof.

[0062] The present invention further relates to the use of a bispecific antibody, a multispecific antibody, a recombinant protein or a VHH domain according to any one of the preceding claims in the preparation of a medicament for the treatment of cancer. The present invention further relates to a method for treating cancer, comprising administering to a subject in need thereof an effective amount of a VHH domain, a bispecific antibody, a multispecific antibody or a recombinant protein according to any one of the preceding claims. Furthermore, in any one of the above uses or methods of treatment, the cancer is colorectal cancer.

[0063] The beneficial effects of the present invention are as follows: The VEGF-targeting VHH domain screened and optimized in the present invention has a stable structure, high thermostability, and high binding activity to both human VEGF-A and mouse VEGF-A, which is advantageous for simultaneous testing in animal models, thereby increasing the success rate of drug development and facilitating high protein expression and purification. Furthermore, the bispecific antibody developed in the present invention based on this VHH domain has a unique bispecific antibody structural form, is highly humanized, does not contain artificially added redundant sequences, and may be locally concentrated in VEGF-high-expressing tumors, thereby exerting more selective and effective tumor-inhibitory effects and resolving the problem of the insufficient efficacy of PD-1 / PD-L1 inhibitors. [Brief explanation of the drawings]

[0064] [Figure 1] The graph shows the binding status of antibodies that have undergone multiple rounds of mutation modification to VEGF. [Figure 2] 1 shows a schematic diagram of the structure of a bispecific antibody. [Figure 3] The figure shows the amount of interleukin 2 (IL-2) released by the test antibody in the MLR reaction. [Figure 4] The figure shows the amount of interferon-γ (IFN-γ) released by the test antibody in the MLR reaction. [Figure 5] The relative inhibition of HUVEC cell proliferation by the test antibodies is shown. [Figure 6] 1 shows the inhibitory effect of test antibodies on HUVEC cell migration. [Figure 7] Quantification of the remaining amount of antibody bound to VEGF magnetic beads by ELISA is shown. [Figure 8] Tumor volumes in tumor-bearing mouse models treated with the test antibodies are shown.

[0065] definition Unless specifically explained to the contrary, the terms used herein have the same meaning as commonly understood by those skilled in the art, employing conventional methods of virology, immunology, microbiology, molecular biology, and recombinant DNA technology within the skill of the art, many of which are described below for illustrative purposes, but which are also fully explained in the literature.

[0066] The term "antibody" as used herein refers to a substantially intact antibody, not an antibody fragment. Specifically, the term "antibody" as used herein preferably refers to an intact antibody, which has a symmetrical structure of four polypeptide chains, each consisting of two heavy chains (H chains) and two light chains (L chains), which are linked via disulfide bonds and non-covalent bonds to form a monomer molecule of four polypeptide chains.

[0067] The term "antigen-binding fragment" as used herein includes a portion of an intact antibody, preferably the antigen-binding region and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments.

[0068] As used herein, the term "humanized antibody or antigen-binding fragment thereof" refers to a human immunoglobulin (acceptor antibody) in which residues of an HVR from an acceptor are replaced by residues from an HVR from a non-human species (donor antibody) having the desired specificity, affinity, and / or capacity, such as mouse, rat, rabbit, or non-human primate. In some cases, framework ("FR") residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies or antigen-binding fragments thereof may also contain residues that are not present in the acceptor or donor antibody. These modifications can be made to further improve antibody properties, such as binding affinity. Generally speaking, humanized antibodies or antigen-binding fragments typically comprise substantially all of two variable domains, including at least one, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR domains are those of a human immunoglobulin sequence, but the FR regions may contain one or more single FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, or immunogenicity. The humanized antibody optionally also can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of 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. Pat. Nos. 6,982,321 and 7,087,409.

[0069] As used herein, the term "human antibody" refers to an antibody that has an amino acid sequence corresponding to an antibody produced by a human and / or is prepared using any of the techniques for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Additionally, human monoclonal antibodies can be produced using methods described in references such as Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and 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 prepared by administering antigen to transgenic animals (e.g., xenogeneic mice) that have been modified to produce such antibodies in response to antigen challenge, but whose endogenous gene loci are prohibited (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also the disclosure of human antibodies produced by human B cell hybridoma technology in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0070] As used herein, the term "CDR" (i.e., complementarity-determining region) refers to a hypervariable region as defined by the Kabat system. See Kabat et al., Sequences of Proteins of Immunological Interest, Version 5, Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Other definitions of CDRs also exist, such as IMGT, Chothia, AbM, and Contact.

[0071] Unless otherwise stated, immunoglobulin residue numbering in the present invention is according to the Kabat numbering system.

[0072] As used herein, the term "Fc domain" generally includes the hinge region, heavy chain constant region CH2 domain, and heavy chain constant region CH3 domain (i.e., hinge-CH2-CH3). Fc domains always form dimers via disulfide bonds. Currently, the most commonly used fusion partner is the Fc fragment of immunoglobulin IgG, where the antibody Fc is a portion of the antibody constant region (including the hinge region-CH2-CH3, but not the CH1 region of the antibody constant region). Fusion with an Fc fragment increases the molecular weight and the FcRn-mediated recycling mechanism, increasing the stability of the fusion molecule and extending its in vivo half-life. Furthermore, the Fc fragment can be used to mediate various biological functions, such as ADCC and CDC. However, since such fusion proteins lack the antibody variable region, their 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 different immunoglobulin G subtypes differ. Currently, IgG1 type antibodies are most widely used, but in recent years, with the development of new indications and the emergence of antibody drugs with new mechanisms of action, emphasis has been placed on IgG2 and IgG4 subtypes, which have low cytotoxicity. In the present invention, the "Fc domain" is preferably the Fc domain of an IgG1 type antibody or the Fc domain of an IgG4 type antibody.

[0073] As used herein, the term "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, the term "bispecific" or "multispecific" refers to an antigen-binding protein or antibody having two or more antigen-binding sites, at least two of which bind to different antigens or different epitopes of the same antigen.

[0074] As used herein, the terms "bivalent," "trivalent," and "tetravalent" refer to "valency," i.e., the predetermined number of binding sites present in an antigen-binding protein or antibody molecule. Thus, the terms "bivalent," "trivalent," and "tetravalent" refer to the presence of two, three, or four binding sites, respectively, in an antigen-binding protein or antibody molecule.

[0075] A "VHH domain" (variable domain of heavy chain of heavy-chain antibody), also known as a VHH or VHH antibody fragment, was originally derived from the antigen-binding immunoglobulin variable domain of a "heavy-chain antibody" (hcAb, i.e., an antibody lacking light chains) (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 such variable domains from the heavy-chain variable domains (referred to herein as "VH domains" or "VH domains") present in conventional antibodies consisting of two light chains and two heavy chains (hereinafter abbreviated as "conventional antibodies") and the light-chain variable domains (referred to herein as "VL domains" or "VL domains") present in conventional antibodies. VHH domains specifically bind to an epitope without the need for another antigen-binding domain (this is the opposite of the VH or VL domain in conventional antibodies, which recognize an epitope by the combination of a VL domain and a VH domain in conventional antibodies). A VHH domain is an antigen recognition unit formed from a single immunoglobulin domain.

[0076] As used herein, the terms "heavy chain single domain antibody (single domain antibody, sdAb)", "VHH domain", "VHH", "VHH antibody fragment", "VHH antibody", "nanobody" and "nanobody domain" can be used interchangeably.

[0077] As used herein, the term "homology" refers to the percentage of amino acid residues in a candidate sequence that are identical to those in a specific peptide or polypeptide sequence, after sequence alignment and (if necessary) gap introduction to achieve the maximum percentage of sequence identity, but does not consider conservative substitutions as part of sequence identity. Alignment to determine the percentage of amino acid sequence identity can be performed in a variety of ways within the skill of the art, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or MEGALIGN-(DNASTAR) software. Appropriate parameters for measuring alignment, such as any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared, can be determined by those skilled in the art.

[0078] The term "treatment" as used herein refers to a clinical intervention designed to change the natural process of the individual or cell being treated in the clinical pathological process. The desired therapeutic effect includes reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, it includes alleviating or eliminating one or more symptoms associated with the disease or condition being treated (e.g., cancer, inflammation, autoimmune disease, etc.).

[0079] As used herein, the term "effective amount" refers to an amount of a drug or agent effective to treat a disease or condition in a subject. In the case of cancer, an effective amount of an anti-VEGF single domain antigen-binding fragment, bispecific antibody, multispecific antigen-binding construct, pharmaceutical composition, or immunoconjugate of the present application may reduce the number of cancer cells, reduce tumor size, inhibit (i.e., delay to some extent, and preferably inhibit) cancer cell invasion into surrounding organs, inhibit (i.e., delay to some extent, and preferably prevent) tumor cell metastasis, inhibit tumor growth to some extent, and / or alleviate one or more symptoms associated with cancer to some extent. As understood in a clinical setting, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with other drugs, compounds, or pharmaceutical compositions. Thus, an "effective amount" may be considered when administering one or more therapeutic agents, and when used in combination with one or more other agents, it is believed that providing a single agent in an effective amount will achieve or effect the desired result.

[0080] As used herein, the term "subject" preferably refers to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the individual is a human. DETAILED DESCRIPTION OF THE INVENTION

[0081] Hereinafter, embodiments of the present invention will be described in detail with reference to examples. However, it will be understood by those skilled in the art 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.

[0082] [Example 1] Screening and development of VHHs targeting VEGF Immunogens were prepared by mixing 500 μg of human and mouse VEGF-A proteins with an equal volume of adjuvant. Two-year-old female alpacas were immunized four times at two-week intervals. After immunization, hyperimmune serum was collected, peripheral blood mononuclear cells were isolated, and RNA was extracted. The variable region genes of the nanobodies were amplified by RT-PCR. Subsequently, restriction enzyme digestion, ligation, and electroporation were performed to obtain a VEGF-A immune antibody library.

[0083] The prepared VEGF-A immune antibody library was subjected to two rounds of panning using immunotube solid-phase panning and magnetic bead liquid-phase screening, respectively, and single clones were selected for ELISA detection. Positive clones were sequenced, and 81 distinct antibody genes were finally obtained. VHH-Fc DNA fragments were constructed for all 81 strains obtained in this way, sequenced, and then transfected into 293T cells for eukaryotic expression. The expression supernatants were collected and subjected to ELISA specificity detection. The results showed that strain (#6) 6 was the only antibody that exhibited excellent binding ability to both human and mouse VEGF-A (see Tables 7 and 8 below).

[0084] [Table 14]

[0085] [Table 15]

[0086] Strain 6 (#6) was sequenced and the amino acid sequence was as follows: SEQ ID NO: 8 [CDRs 1-3 are shown in bold and underlined (Kabat numbering)] QVQLQESGGGLVQPGGSLRLSCAASGFTFS TSTMS WYRQAPGKERELVA FITSAGATTNYADSVKD RFTMSRDNDKNTVYLQMNVLKPEDTAMYYCRA LVTLWNVY WGQGTQVTVSS

[0087] [Table 16]

[0088] [Example 2] Humanization and stability optimization of VEGF VHH primary sequence To further improve the thermal stability and degree of humanization of the VHH of strain No. 6, five rounds of sequence optimization and modification were performed in combination with structural biology-based protein structure simulations, and two VHH sequences with good thermal stability and a high degree of humanization were obtained. #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 VTVSS

[0089] [Table 17]

[0090] For the #48 sequence, Q1E+Q5L are located in the FR1 region, A49S is located in the FR2 region, N58Y+D65G are located in the CDR2 region, D74S+V(82B)S+K83R+P84A+M89V are located in the FR3 region, and Q108L is located in the FR4 region.

[0091] In the case of the #49 sequence, Q1E+Q5L are located in the FR1 region, N58Y+D65G are located in the CDR2 region, D74S+V(82B)S+K83R+P84A+M89V are located in the FR3 region, and Q108L is located in the FR4 region.

[0092] Measurements of VEGF binding ability and thermal stability showed that both #48 and #49 exhibited good VEGF binding ability comparable to that of #6 (see Figure 1). Measurement of thermal stability showed that the combined mutant sequences exhibited an improvement of approximately 10°C in thermal stability, with Tm values of 64.1°C and 64.7°C, respectively.

[0093] [Example 3] Screening of candidate bispecific antibody molecular structures and construction of control substances Various bispecific antibody structural formats were evaluated and screened using VHH #48 and antigen-binding fragments of the anti-PD-1 antibodies nivolumab (Opdivo) or pembrolizumab (Keytruda). After comprehensive evaluation of activation, expression level, stability, and other factors, we decided to use the bispecific antibody structure shown in Figure 2. Additionally, bispecific antibody molecules #10 and #16 were constructed and evaluated. Both had bivalent + bivalent (2 + 2) valency and contained functionally silent IgG1 mutations (AEASS) in the Fc. The amino acid sequences of the constructed molecules are shown in Table 18 below.

[0094] [Table 18]

[0095] As a control, a bispecific antibody analogue, AK112, targeting PD-1 and VEGF was used (expressed and prepared in-house from the heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence of SEQ ID NO: 26). SEQ ID NO:25: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVK GRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK SEQ ID NO:26: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0096] [Example 4] Binding of human PD-1 to human VEGF Binding of the antibody to human PD-1 was detected by ELISA. Specifically, 1 μg / mL human PD-1 (Acro, His tag) was coated onto an ELISA plate overnight and blocked. A serial dilution of the bispecific antibody sample and control antibody was added, followed by incubation and washing. Next, an HRP-labeled anti-human IgG Fc secondary antibody was added, incubated, and washed. After adding TMB color development solution, the OD450 absorbance was measured using a microplate reader (see Table 19 below). To detect binding of the antibody to human VEGF using ELISA, an ELISA plate was coated overnight with 50 ng / mL human VEGF (Acro, His tag) and blocked. A serial dilution of the test sample and control antibody was added, incubated, and washed. Next, an HRP-labeled anti-human IgG Fc bispecific antibody was added to the plate, followed by incubation and washing. Finally, TMB color development solution was added, followed by measurement of the OD450 absorbance using a microplate reader (see Table 20 below). As shown by the ELISA results, the binding affinity of #10 and #16 to human PD-1 was comparable to that of Opdivo and Keytruda, but showed a higher Top signal and stronger binding than the control AK112 analog. The binding patterns of #10 and #16 to VEGF were quite unique. Compared to #48, the binding affinity of #10 and #16 to VEGF was slightly lower, with Top absorbances approximately half that of #48. This indicates that structural biology-based optimization altered the binding state of #10 and #16 to VEGF, resulting in greater drug concentration. Meanwhile, a bevacizumab antibody fragment was used in the anti-VEGF portion of the AK112 analog, and its VEGF binding characteristics were similar to those of bevacizumab. (All control substances were expressed and purified in-house.)

[0097] [Table 19]

[0098] [Table 20]

[0099] [Example 5] Dual binding ability of human VEGF and human PD-1 The dual binding ability of #10, #16, and the AK112 analog to both human VEGF and human PD-1 was further verified. A 96-well ELISA plate was coated overnight with 50 ng / mL human VEGF (Sino Biologicals, untagged), washed, and blocked for 1 hour. A serial dilution of the test antibody was then added and incubated for 2 hours. Next, 1 μg / mL human PD-1 (Acro, His-tag) was added and incubated for 2 hours. After washing, an HRP-labeled anti-His antibody was added and incubated for 1 hour. Finally, TMB color development solution was added, and the absorbance at 450 nm was measured using a microplate reader. As shown in Table 21 below, while the single-target antibody failed to simultaneously bind to both targets, #10, #16, and the AK112 analog all exhibited dual binding ability to both human VEGF and human PD-1, with EC50 values of 0.279 nM, 0.326 nM, and 0.254 nM, respectively.

[0100] ELISA dual detection was performed by changing the order of VEGF and PD-1 binding. A 96-well ELISA plate was coated overnight with 1 μg / mL human PD-1 (Acro Biosciences, His tag), washed, and blocked for 1 hour. A serial dilution of specific antibodies was then added and incubated for 2 hours. Next, 50 ng / mL biotin-labeled human VEGF (Acro Biosciences, biotinylated protein) was added and incubated for 2 hours. After washing, HRP-labeled streptavidin was added and incubated for 1 hour. Finally, TMB color development solution was added, and the absorbance at 450 nm was measured using a microplate reader. As shown in Table 21 below, the single-target antibody was unable to simultaneously bind to both targets. However, the #10, #16, and AK112 analogs all exhibited dual binding to both human PD-1 and human VEGF, with EC50 values of 0.114 nM, 0.117 nM, and 0.634 nM, respectively. Comparison of EC50 values revealed that #10 and #16 had 5 to 6 times greater dual binding ability to human PD-1 and human VEGF than the competing AK112 analog.

[0101] [Table 21]

[0102] [Example 6] Binding to foreign antigens Binding of the candidate molecules to competitor antigens from different bacterial species was detected using ELISA. The monkey-derived VEGF sequence was identical to the humanized VEGF sequence, and the binding ability of the candidate antibodies to monkey VEGF was equivalent to that of human VEGF. Binding of the antibodies to monkey PD-1 was detected using ELISA. ELISA plates were coated overnight with 1 μg / mL monkey PD-1 (Acro, His tag) and blocked, followed by incubation with serial dilutions of test samples and control antibodies and washing. Next, an HRP-labeled anti-human IgG Fc bispecific antibody was added, incubated, and washed. Finally, TMB color development was performed, and the OD 450 absorbance was measured using a microplate reader (see Table 22 below). Consistent with the binding results to human PD-1, #10 and #16 demonstrated binding ability to monkey PD-1 comparable to that of Opdivo and Keytruda, but stronger than that of the competitor AK112 analog.

[0103] [Table 22]

[0104] [Example 7] Ability to block binding of PD-1 and PD-L1 The ability of the test antibodies to block the binding of PD-1 to PD-L1 was further verified. An ELISA plate was coated overnight with 1 μg / mL human PD-1 (Acro, Fc tag), washed, and blocked. After this, a mixture of serially diluted antibodies and human PD-L1 (Sino Biological, His tag) was added and incubated for 2 hours. An HRP-conjugated anti-His antibody was then added and incubated for 1 hour. Finally, TMB color development solution was added, and the OD 450 absorbance was measured using a microplate reader (see Table 23 below). As shown in the results, with the exception of #48, which only bound to VEGF, all of the remaining antibodies were able to block the binding of PD-1 to PD-L1, with comparable blocking activity.

[0105] [Table 23]

[0106] [Example 8] Ability to block the binding of VEGF to VEGFR1 or VEGFR2 The ability of the test antibodies to block VEGF binding to VEGFR1 or VEGFR2 was examined. ELISA plates were coated overnight with 2 μg / mL human VEGFR1 and 5 μg / mL human EGFR2 (in-house constructed and expressed, Fc-tagged), washed, blocked, and then incubated for 2 hours with serially diluted antibody and human VEGF (biotinylated protein, Acro Biotech). Next, the plates were incubated for 1 hour with HRP-conjugated streptavidin. Finally, TMB color development was performed, and absorbance at 450 nm was measured using a microplate reader. #10 and #16 demonstrated even stronger blocking abilities against VEGF binding to VEGFR1, with IC50 values of 0.491 nM and 0.507 nM, respectively, superior to those of #48, demonstrating the superiority of the constructed bispecific antibodies due to their spatial conformational changes. The blocking activity of the competitor AK112 analog was comparable to that of bevacizumab, with IC50 values of 4.610 nM and 5.647 nM, respectively. Furthermore, the results of the detection showed that #10 and #16 had even stronger blocking ability against the binding of VEGF to VEGFR2, with IC50 values of 3.643 nM and 3.249 nM, respectively, which was superior to the blocking activity of #48 (IC50 value of 5.841 nM), demonstrating the superiority of the spatial conformational changes of the bispecific antibody. Meanwhile, the blocking activity of the competitor AK112 analog was comparable to that of bevacizumab, with IC50 values of 6.481 nM and 6.637 nM, respectively.

[0107] [Example 9] Verification of T cell responses after PD-1 blockade using mixed lymphocyte reaction (MLR) assay The functions of the test candidate molecule and the competitor AK112 analog were further compared at the cellular level. T cell responses after PD-1 blockade were examined using a mixed lymphocyte reaction (MLR) assay. 2 × 10 cells from one donor were used.4 2 x 10 DC cells from a different donor 5 Equal volumes of CD4+ T cells were mixed, serially diluted test antibodies were added, and the cells were cultured for 72 hours. The cell culture supernatants were collected, and the amounts of IL-2 (Figure 3) and IFNγ (Figure 4) released from the cell supernatants were measured using an ELISA kit. The results showed that the amounts of IL-2 and IFNγ released by CD4+ T cells in the MLR reaction promoted by #10 and #16 were higher than those released by the competitor AK112 analog, indicating that #10 and #16 have a stronger effect on T cells.

[0108] [Example 10] Verification of the ability of the test antibody to inhibit VEGF-induced stimulation of HUVEC cell proliferation using a vascular endothelial cell (HUVEC) proliferation inhibition assay 5 x 10 cells in medium containing 1% FBS on a 96-well cell plate 3 HUVEC cells / well were added and incubated overnight in an incubator at 37°C with 5% carbon dioxide. The next day, the medium was removed, and 70 ng / mL VEGF, serially diluted test antibodies, and the competitor AK112 analog were added. The reaction plate was then placed in an incubator at 37°C with 5% carbon dioxide and incubated for 4 days. After addition of the CTG reagent, the relative fluorescence signal was measured using a microplate reader. The fluorescence value of the cell group treated with 1% FBS-containing ECM medium and VEGF stimulation (no antibody) was defined as corresponding to 0% inhibition, while the fluorescence value of the group treated with PBS alone (no ECM medium containing 1% FBS, no VEGF stimulation, and no antibody) was defined as 100% inhibition. The relative inhibition rates were then fitted as curves. As shown in Figure 5, the maximum inhibition rates of #10 and #16 were approximately 60%, while that of the competitor AK112 analog was approximately 40%, indicating that the inhibition rates of #10 and #16 were higher than those of the competitor AK112 analog.

[0109] [Example 11] Inhibitory effect on HUVEC cell migration A transwell chamber with a pore size of 8 μm was used to detect the ability of antibodies to inhibit HUVEC cell migration. The transwell consists of an upper and a lower chamber. 5 × 10 3 HUVEC cells / well were added to the upper chamber and incubated for 30 minutes at 37°C in a 5% CO2 incubator. Next, 200 ng / mL VEGF and various concentrations of the test antibody and competitor AK112 analog were added to the lower chamber. Three replicate wells per group were incubated for 24 hours at 37°C in a 5% CO2 incubator. Cells were then fixed with 4% paraformaldehyde and stained with crystal violet. After photography, the number of cells that had migrated to the back of the upper chamber was counted. The lower the cell number, the higher the inhibition rate. Figure 6 shows that #10 and #16 exhibited higher inhibition rates than the competitor AK112 analog.

[0110] [Example 12] Antibody concentration increase characteristics Structural biology simulations revealed that the conformation of #10 may alter the binding pattern of anti-VEGF nanoantibodies to VEGF, enabling antibody enrichment using dimeric VEGF proteins. To verify the enhanced antibody enrichment properties of #10, magnetic beads coupled to human VEGF protein (Acro) were diluted to 250 ng / mL, 125 ng / mL, and 50 ng / mL in PBS. 10 nM each of #10, #16, and the competitor AK112 analog were added and incubated for 1 hour. After 2 minutes on a magnet, the supernatant was aspirated, and protein quantification was performed using ELISA. As shown in Figure 7, the residual antibody levels of #10 and #16 were lower than those of the competitor AK112 analog, indicating that greater amounts of bispecific antibodies were enriched using VEGF-containing magnetic beads. To verify the degree of cross-linking between the antibody and VEGF, an ELISA quantification test was performed. First, the antibody was cross-linked to the VEGF magnetic beads, and then the amount of uncross-linked residual antibody was detected using ELISA. The results showed that #10 and #16 cross-linked VEGF to a higher degree than the competitor AK112 analog. This, in agreement with the results of structural biology simulations, suggests that high VEGF expression in tumor tissues can lead to localized tumor enrichment, demonstrating the differentiated characteristics and therapeutic potential of #10 and #16 bispecific antibodies.

[0111] [Example 13] Drug efficacy evaluation in animal models To evaluate the efficacy of the test antibodies in a mouse model, Balb / c mice transfected with human PD-1 were selected and inoculated with wild-type CT26 tumor cells (colon cancer cells). Because neither bevacizumab nor the AK112 analog binds to mouse VEGF, these two groups were excluded. A total of six groups were designed, including human VEGF and VEGF-Trap (a fusion protein consisting of the second domain of VEGFR1, the third domain of VEGFR2, and human IgGFc), which can bind to mouse VEGF. The experimental design, dosage, and route of administration are shown in Table 24.

[0112] [Table 24]

[0113] As shown in Figure 8, the tumor inhibition rates in tumor-bearing mice treated with #10 and #16 were 57% and 62%, respectively. These rates were higher than those observed in the Opdivo monotherapy group (20%) and the VEGF-Trap monotherapy group (45%), and comparable to those observed in the Opdivo and VEGF-Trap combination therapy group (56%). On day 14, the mean tumor volume in the #10 and #16 treatment group was slightly smaller than that in the combination therapy group. Furthermore, changes in mouse weight correlated with the tumor growth pattern. On day 14, weight changes in the #10 and #16 treatment groups were within 10%, demonstrating the favorable efficacy and safety of #10 and #16.

[0114] [Example 14] Synergistic effects of #10 molecules in an immune-reconstituted human melanoma subcutaneous xenograft model To evaluate the synergistic effects of #10 in an immune-reconstituted human melanoma model, immunodeficient mice (B-NDG) were selected and inoculated with human PD-L1-transduced A375 tumor cells (melanoma cells) and human peripheral blood mononuclear cells (huPBMCs) to reconstitute the immune system of tumor-bearing mice. Eight groups were designed, including the anti-PD-1 drug Opdivo, the anti-VEGF drug bevacizumab, and #48 as single-target controls, and the AK112 analog (expressed and prepared in-house) as a control for #10. The experimental design, dosage, and administration route are shown in Table 25 below.

[0115] [Table 25]

[0116] Detection on day 21 revealed that in this experimental system, the Opdivo monotherapy group showed no antitumor effect, and #10 had a tumor inhibition rate (TGI) of 73%, which was significantly higher than the bevacizumab monotherapy group (TGI = 53%), the #48 monotherapy group (TGI = 57%), and the Opdivo and #48 combination therapy group (TGI = 50%). It was also superior to the Opdivo and bevacizumab combination therapy group (based on a tumor inhibition rate of TGI = 68%, the relative tumor inhibition rate of #10 molecule was improved by 7%) and the AK112 analog (based on a tumor inhibition rate of TGI = 64%, the relative tumor inhibition rate of #10 molecule was improved by 14%). These results demonstrate that the bispecific antibodies of the present invention exhibit synergistic effects and are significantly superior to control molecules.

[0117] It should be noted that the above disclosure is merely a few examples of the present invention and does not limit the present invention in any way. Those skilled in the art will understand that the present invention is not limited to each embodiment. Those skilled in the art may make some improvements and modifications without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. (a) a first binding functional region that targets PD-1; (b) a second binding functional region that targets VEGF; the first binding functional domain targeting PD-1 is an anti-PD-1 antibody or an antigen-binding fragment thereof; The second binding functional region targeting VEGF comprises a VHH domain (a heavy chain variable domain of a heavy chain antibody) of CDR1 to CDR3 defined by the following amino acid sequence: the amino acid sequence of the CDR1 is identical to the amino acid sequence set forth in SEQ ID NO: 1; the amino acid sequence of the CDR2 is identical to the amino acid sequence shown in SEQ ID NO: 2 or has one or two amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 2; A bispecific antibody, wherein the amino acid sequence of the CDR3 is identical to the amino acid sequence shown in SEQ ID NO:

3.

2. The bispecific antibody of claim 1, wherein the amino acid mutation site in CDR2 is at positions 58 and / or 65.

3. 3. The bispecific antibody of claim 2, wherein the mutation in CDR2 is an N58Y mutation and / or a D65G mutation.

4. The bispecific antibody of claim 1, wherein the VHH domain is a humanized VHH domain.

5. The second binding functional domain that targets VEGF is 1) a framework region domain FR1 that is identical to the amino acid sequence set forth in SEQ ID NO: 4 or has one or two amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 4, and / or 2) a framework region domain FR2 that is identical to the amino acid sequence set forth in SEQ ID NO: 5 or has one amino acid mutation compared to the amino acid sequence set forth in SEQ ID NO: 5, and / or 3) a framework region domain FR3 that is identical to the amino acid sequence set forth in SEQ ID NO: 6 or has 1 to 5 amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 6, and / or 4) a framework region domain FR4 that is identical to the amino acid sequence set forth in SEQ ID NO: 7 or has one amino acid mutation compared to the amino acid sequence set forth in SEQ ID NO: 7; The bispecific antibody of claim 1 or 3, further comprising:

6. 6. The bispecific antibody of claim 5, wherein the one or two amino acid mutations in the FR1 domain are at positions 1 and / or 5, more preferably Q1E and / or Q5L mutations.

7. 7. The bispecific antibody of claim 6, wherein the single amino acid mutation in the FR2 domain is at position 49, more preferably the A49S mutation.

8. The bispecific antibody of claim 7, wherein the 1 to 5 amino acid mutations in the FR3 domain are 1, 2, 3, 4, or 5 amino acid mutations selected from positions 74, 82B, 83, 84, and 89, more preferably at positions 1, 2, 3, 4, or 5 selected from D74S, V(82B)S, K83R, P84A, and M89V.

9. 9. The bispecific antibody of claim 8, wherein the single amino acid mutation in the FR4 domain is at position 108, more preferably Q108L.

10. The second binding functional domain that targets VEGF is 1) a framework region domain FR1 corresponding to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 9, and / or 2) a framework region domain FR2 corresponding to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 10, and / or 3) a framework region domain FR3 corresponding to the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 11, and / or 4) a framework region domain FR4 corresponding to the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12; The bispecific antibody according to any one of claims 5 to 9, further comprising:

11. The second binding functional region targeting VEGF has a combination of framework regions defined by the following amino acids: 1) FR1 corresponding to the amino acid sequence shown in SEQ ID NO:4, FR2 corresponding to the amino acid sequence shown in SEQ ID NO:5, FR3 corresponding to the amino acid sequence shown in SEQ ID NO:6, and FR4 corresponding to the amino acid sequence shown in SEQ ID NO:7; 2) FR1 corresponding to the amino acid sequence set forth in SEQ ID NO:9, FR2 corresponding to the amino acid sequence set forth in SEQ ID NO:10, FR3 corresponding to the amino acid sequence set forth in SEQ ID NO:11, and FR4 corresponding to the amino acid sequence set forth in SEQ ID NO:12; 3) FR1 corresponding to the amino acid sequence shown in SEQ ID NO:9, FR2 corresponding to the amino acid sequence shown in SEQ ID NO:5, FR3 corresponding to the amino acid sequence shown in SEQ ID NO:11, and FR4 corresponding to the amino acid sequence shown in SEQ ID NO:12; The bispecific antibody of claim 10, further comprising:

12. The bispecific antibody according to claim 1, wherein the amino acid sequence of the second binding functional domain targeting VEGF is identical to the amino acid sequence shown in SEQ ID NO: 8 or has 1 to 11 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO:

8.

13. The bispecific antibody according to claim 12, wherein the amino acid mutation site is preferably selected from positions 1, 5, 49, 58, 65, 74, 82B, 83, 84, 89, and 108.

14. The bispecific antibody according to claim 13, wherein the amino acid mutations are preferably 1 to 11 selected from Q1E, Q5L, A49S, N58Y, D65G, D74S, V(82B)S, K83R, P84A, M89V, and Q108L.

15. The mutation is a combination of the following mutations: 1) Q1E+Q5L+A49S+N58Y+D65G+D74S+V(82B)S+K83R+P84A+M89V+Q108L 2) Q1E+Q5L+N58Y+D65G+D74S+V(82B)S+K83R+P84A+M89V+Q108L 15. The bispecific antibody of claim 14,

16. The bispecific antibody of claim 1, wherein the amino acid sequence of the second binding functional region targeting VEGF corresponds to the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 13 or SEQ ID NO:

14.

17. The bispecific antibody of any one of claims 1 to 16, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is 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.

18. The bispecific antibody of claim 17, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is an IgG antibody.

19. The bispecific antibody of claim 18, wherein the anti-PD-1 antibody or antigen-binding fragment thereof is of the IgG1, IgG2, or IgG4 type.

20. The bispecific antibody of claim 19, wherein the first binding functional domain targeting PD-1 has the same light chain and heavy chain CDR amino acid sequences as those of the nivolumab antibody or the pembrolizumab antibody.

21. The bispecific antibody of claim 20, wherein the first binding functional domain targeting PD-1 has the same amino acid sequence of a heavy chain variable domain as that of a nivolumab antibody or a pembrolizumab antibody.

22. The bispecific antibody of claim 21, wherein the first binding functional domain targeting PD-1 has the same amino acid sequence of a light chain variable domain as that of a nivolumab antibody or a pembrolizumab antibody.

23. The bispecific antibody of claim 22, wherein the heavy chain portion of the first binding functional domain that targets PD-1 further comprises one or more domains selected from a heavy chain constant region CH1 domain, a hinge region, a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain.

24. The bispecific antibody of claim 23, wherein the first binding functional region that targets PD-1 further comprises a light chain constant region domain.

25. The bispecific antibody of claim 24, wherein the anti-PD-1 antibody comprises a heavy chain portion comprising, in order from the N-terminus to the C-terminus, a heavy chain variable region domain, a heavy chain constant region CH1 domain, a hinge region, a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain, and a light chain portion comprising, in order from the N-terminus to the C-terminus, a light chain variable region domain and a light chain constant region domain.

26. 26. The bispecific antibody of claim 25, wherein the Fc domain of the anti-PD-1 antibody is an IgG1 Fc domain or a silent IgG1 mutation thereof.

27. 27. The bispecific antibody of claim 26, wherein the Fc domain is identical to the amino acid sequence set forth in SEQ ID NO: 15 or has 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 set forth in SEQ ID NO:

15.

28. The first binding functional region targeting PD-1 is a combination of a light chain and a heavy chain having the following amino acid sequence: 1) a heavy chain corresponding to the amino acid sequence set forth in SEQ ID NO: 16; and / or a light chain corresponding to the amino acid sequence set forth in SEQ ID NO: 17; or 2) a heavy chain corresponding to the amino acid sequence set forth in SEQ ID NO: 18; and / or a light chain corresponding to the amino acid sequence set forth in SEQ ID NO: 19; 28. The bispecific antibody of claim 27, comprising:

29. The bispecific antibody of claim 28, wherein the second binding functional region is linked to the C-terminus or N-terminus of the first binding functional region.

30. The bispecific antibody of claim 29, wherein the VHH domain is linked directly or via a peptide linker to the C-terminus or N-terminus of the heavy chain portion of the antibody or antigen-binding fragment thereof.

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

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

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

34. The amino acid sequence of the peptide linker is (GGGGS) n 34. The bispecific antibody of claim 33, wherein n is 1, 2, 3, or 4.

35. 31. The bispecific antibody of claim 29 or 30, wherein the bispecific antibody is a bivalent, trivalent or tetravalent bispecific antibody.

36. 2. The bispecific antibody of claim 1, wherein the bispecific antibody comprises a combination of a heavy chain shown in SEQ ID NO: 20 and a light chain shown in SEQ ID NO: 21, or a combination of a heavy chain shown in SEQ ID NO: 22 and a light chain shown in SEQ ID NO:

23.

37. A VHH domain targeting VEGF, comprising CDR1-3 as specified in the amino acid sequence below: the amino acid sequence of the CDR1 is identical to the amino acid sequence set forth in SEQ ID NO: 1; the amino acid sequence of the CDR2 is identical to the amino acid sequence shown in SEQ ID NO: 2 or has one or two amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 2; A VHH domain characterized in that the amino acid sequence of the CDR3 is identical to the amino acid sequence shown in SEQ ID NO:

3.

38. 38. A VHH domain according to claim 37, characterized in that the mutation site in CDR2 is at positions 58 and / or 65.

39. 39. A VHH domain according to claim 38, wherein the mutation in CDR2 is an N58Y mutation and / or a D65G mutation.

40. 40. The VHH domain of claim 39, wherein the VHH domain is a humanized VHH domain.

41. The VHH domain is 1) a framework region domain FR1 that is identical to the amino acid sequence set forth in SEQ ID NO: 4 or has one or two amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 4, and / or 3) a framework region domain FR2 that is identical to the amino acid sequence set forth in SEQ ID NO: 5 or that has one amino acid mutation compared to the amino acid sequence set forth in SEQ ID NO: 5, and / or 3) a framework region domain FR3 that is identical to the amino acid sequence set forth in SEQ ID NO: 6 or has 1 to 5 amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 6, and / or 4) a framework region domain FR4 that is identical to the amino acid sequence set forth in SEQ ID NO: 7 or has one amino acid mutation compared to the amino acid sequence set forth in SEQ ID NO: 7; The VHH domain of claim 40 further comprising:

42. the one or two amino acid mutations in the FR1 domain are at positions 1 and / or 5, more preferably Q1E and / or Q5L mutations; 42. A VHH domain according to claim 41, characterized in that the single amino acid mutation in the FR2 domain is at position 49, more preferably an A49S mutation.

43. The VHH domain of claim 42, wherein the 1 to 5 amino acid mutations in the FR3 domain are 1, 2, 3, 4 or 5 selected from positions 74, 82B, 83, 84 and 89, more preferably 1, 2, 3, 4 or 5 selected from D74S, V(82B)S, K83R, P84A and M89V.

44. 44. A VHH domain according to claim 43, characterized in that the single amino acid mutation in the FR4 domain is at position 108, more preferably Q108L.

45. The VHH domain is 1) a framework region domain FR1 corresponding to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 9, and / or 2) a framework region domain FR2 corresponding to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 10, and / or 3) a framework region domain FR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 11, and / or 4) a framework region domain FR4 comprising the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 12; 45. The VHH domain of claim 44, further comprising:

46. The VHH domain has a combination of framework regions defined by the following amino acids: 1) FR1 corresponding to the amino acid sequence shown in SEQ ID NO:4, FR2 corresponding to the amino acid sequence shown in SEQ ID NO:5, FR3 corresponding to the amino acid sequence shown in SEQ ID NO:6, and FR4 corresponding to the amino acid sequence shown in SEQ ID NO:7; 2) FR1 corresponding to the amino acid sequence set forth in SEQ ID NO:9, FR2 corresponding to the amino acid sequence set forth in SEQ ID NO:10, FR3 corresponding to the amino acid sequence set forth in SEQ ID NO:11, and FR4 corresponding to the amino acid sequence set forth in SEQ ID NO:12; 3) FR1 corresponding to the amino acid sequence shown in SEQ ID NO: 9, FR2 corresponding to the amino acid sequence shown in SEQ ID NO: 5, FR3 corresponding to the amino acid sequence shown in SEQ ID NO: 11, and FR4 corresponding to the amino acid sequence shown in SEQ ID NO: 12; 46. The VHH domain of claim 45, comprising:

47. 38. The VHH domain of claim 37, wherein the amino acid sequence of the VHH domain is identical to the amino acid sequence shown in SEQ ID NO: 8 or has 1 to 11 amino acid mutations compared to the amino acid sequence shown in SEQ ID NO:

8.

48. The VHH domain described in claim 47, characterized in that the amino acid mutation site is preferably selected from positions 1, 5, 49, 58, 65, 74, 82B, 83, 84, 89 and 108.

49. The VHH domain of claim 48, characterized in that the amino acid mutations are preferably 1 to 11 selected from Q1E, Q5L, A49S, N58Y, D65G, D74S, V(82B)S, K83R, P84A, M89V, and Q108L.

50. The mutation is a combination of the following mutations: 1) Q1E+Q5L+A49S+N58Y+D65G+D74S+V(82B) S+K83R+P84A+M89V+Q108L 2) Q1E+Q5L+N58Y+D65G+D74S+V(82B)S+K83 R+P84A+M89V+Q108L 50. The VHH domain of claim 49, wherein:

51. A VHH domain according to claim 50, characterized in that the amino acid sequence of the VHH domain corresponds to the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 13 or SEQ ID NO:

14.

52. A VHH domain according to any one of claims 37 to 51, for use in the construction of a recombinant protein.

53. A recombinant protein comprising a VHH domain according to any one of claims 37 to 51.

54. 54. The recombinant protein of claim 53, which is a bispecific antibody, a multispecific antibody, or an antibody-drug conjugate.

55. 54. A polynucleotide encoding a bispecific antibody according to any one of claims 1 to 36, or a VHH domain according to any one of claims 37 to 51, or a recombinant protein according to claim 53, or a multispecific antibody according to claim 54.

56. 56. An expression vector comprising the polynucleotide of claim 55.

57. A host cell comprising the expression vector of claim 56 or having the polynucleotide of claim 55 integrated into its genome.

58. 54. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 36, or a VHH domain according to any one of claims 37 to 51, or a recombinant protein according to claim 53, or a multispecific antibody according to claim 54, and a pharmaceutically acceptable carrier thereof.

59. 54. Use of a bispecific antibody according to any one of claims 1 to 36, or a VHH domain according to any one of claims 37 to 51, or a recombinant protein according to claim 53, or a multispecific antibody according to claim 54 in the manufacture of a cancer therapeutic.

60. 52. A method for treating cancer, comprising administering to a subject in need thereof an effective amount of a bispecific antibody according to any one of claims 1 to 36, or a VHH domain according to any one of claims 37 to 51, or a recombinant protein according to claim 53, or a multispecific antibody according to claim 54.

61. 61. The use of claim 59 or the method of treatment of claim 60, wherein the cancer is colorectal cancer.

Images