Bispecific antibody and application thereof

Abstract

To provide bispecific antibodies targeting vascular endothelial growth factor (VEGF) / VEGF receptor and immune checkpoint inhibitors.SOLUTION: Provided are a VHH targeting VEGF, a bispecific antibody targeting PD-1 and VEGF developed based on the VHH, and an application thereof. The VHH has various good effects such as high stability and easy expression and purification. The constructed bispecific antibody targeting PD-1 and VEGF has high stability, can be targeted and enriched in tumor regions with high expression of VEGF, has good efficacy and safety, and has excellent therapeutic effects.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Programmed death receptor-1 PD-1 is an immunosuppressive receptor that belongs to the immunoglobulin CD28 / B7 superfamily. When PD-1 binds to PD-L1, it binds to phosphatidylinositol-3-carboxylates. phosphorylation of ATP, further activation of protein kinase B, and activation of the stimulatory T cell signaling pathway This is the downstream of T cell activation, as it involves factors such as oxidization, glucose metabolism, and interferon secretion. This inhibits signal transduction, effectively suppressing T cell transcription and ultimately T cell immunity. The immune response is suppressed, and thus it plays an important role in the suppressive control of the immune response. PD-1 antibodies have a broad spectrum of antitumor effects and are currently being investigated in several clinical trials. Blockbuster drugs, such as the one approved in the US on December 22, 2014 Opdivo (trade name: nivolumab) was listed and first approved The indications are malignant melanoma, but also non-small cell lung cancer, renal cell carcinoma, and Hodgkin's disease. Lymphoma, head and neck squamous cell carcinoma, colorectal cancer, hepatocellular carcinoma, urothelial carcinoma, colorectal cancer, liver cell carcinoma, esophageal cancer, esophageal squamous cell carcinoma, pleural mesothelioma, esophageal adenocarcinoma, esophagogastric junction adenocarcinoma, gastric cancer, etc. However, the existing clinical data (160 Analysis of 28,304 patient data from clinical trials (Reference 1: Effic acy of PD-1 / PD-L1 blockade monotherapy in c clinical trials,Bin Zhao,Hong Zhao,et al.,T Her Adv Med Oncol. 2020, Vol. 12, pp. 1-22) Overall, among 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%; 9 5% confidence interval (CI), 18.34-22.15%. Of 19,418 cancer patients, 8 Sixty-two complete responses (CR) were observed. The overall incidence of CR was 3.85% (95% CI, 3.01-3.22). The ORR ranged from 0.06 to 4.73%. Furthermore, ORR varied across different cancer types and PD-L1 expression status. In other words, the therapeutic effect associated with PD-1 / PD-L1 monotherapy was Significant differences were observed among different cancer types and PD-L1 expression levels, indicating unmet clinical needs. There is still a lot of need for flooring.

[0003] vascular endothelial growth factor tor, VEGF) is a vascular permeability factor. It is also known as vascular permeability factor (VPF), which increases vascular permeability, degeneration of the extracellular matrix, and vascular endothelial cells. Highly specific endothelial cell proliferation agent that promotes cell migration, proliferation, and angiogenesis Evidence suggests that VEGF plays an important role in pathological angiogenesis. , tumor growth and metastasis, macular degeneration, diabetic retinal pathology, inflammatory processes (e.g., rheumatoid arthritis) ), ischemic processes (myocardial ischemia), and the onset and progression of diseases such as preeclampsia (Reference 2: Molecular and functional divers ity of vascular endothelial growth factors .Yamazaki Y.,Morita T.,Mol.Divers.Novemb er 2006,Vol.10,No.4,pp.515-527). Vascular endothelial growth factor The high-affinity receptor that specifically binds to vascular endothelial growth factor (VEGF) is the vascular endothelial growth factor receptor (VEGF). ndothelial Growth Factor Receptor (VEGFR) and It is a receptor tyrosine kinase that mediates many signals necessary for angiogenesis and cell migration. It plays an important role in the VEGF signaling pathway and initiates the signal transduction cascade upon binding to VEGF. Therefore, VEGF / VEGFR is mainly expressed in endothelial cells. Because it is expressed only in the endothelial cells of many tumors and is highly upregulated, Currently, anti-VEGF / VEGF inhibitors are being developed for cancer and other pathological processes. It is believed that inhibiting angiogenesis using GFR therapy will be extremely important. Inhibition of endothelial growth factor / vascular endothelial growth factor receptors has been shown to be effective in treating some cancers, such as renal cell carcinoma and hepatocellular carcinoma. It is considered the main treatment for

[0004] Currently, we are investigating the relationship between vascular endothelial growth factor / vascular endothelial growth factor receptor and immune checkpoint inhibitors. Various clinical trials to investigate safety and efficacy of combined cancer treatments Some early studies have shown that single VEGFR inhibitors or immunosuppressants Compared with other drugs, the combination of immune checkpoint inhibitors and VEGFR inhibitors has a better therapeutic effect. may have adverse effects (https: / / www.obroncology.com / a rticle / combining-immune-checkpoint-and-v egf-inhibitors-improves-survival-in-pret However, the risks associated with this favorable outcome are still unknown. (Reference 3: Combining Immune Checkpoint and VE GFR Inhibition in Favorable Risk and Elderly y Patients with Metastatic Renal Cell Carci noma.Varkaris A, Xu W, Davis RB, Healy B, McD ermott DF.Clin Genitourin Cancer.June 2020 , Vol.18, No.3, pp.179-184). Dual inhibition of VEGFR / ICI Several clinical trials (e.g., pembrolizumab ) / Pazopanib combination drug group, nivolumab / Sunitinib combination drug group, nivolumab / pazo In the case of combination drugs such as pazopanib, good therapeutic effects were observed in early clinical studies. (Reference 4: A Phase I / II Study to Assess the e Safety and Efficacy of Pazopanib and Pemb rolizumab Combination Therapy in Patients with Advanced Renal Cell Carcinoma.Chowdhu ry S,Infante JR,et al.Clin Genitourin Canc er.2021 rh October,Vol.19,No.5,pp.434-446 ;Reference 5:Nivolumab(anti-PD-1;BMS-936558,ON O-4538)in combination with sunitinib or pa zopanib in patients (pts) with metastatic r enal cell carcinoma(mRCC).Amin,A.et al.,J Ournal of Clinical Oncology.32,(suppl.),M May 20, 2014, Abstract 5010), and subsequent severe toxicity in the high-dose group. The nivolumab / sunitinib combination group and the nivolumab / pazopanib combination group showed a favorable effect. Grade 3 or higher treatment-related toxicity was observed in 73% and 60% of patients in the EGFR and EGFR groups, respectively. In addition, 23% of patients in the nivolumab / sunitinib combination group experienced an additional adverse event. Treatment was discontinued due to the lack of treatment. Adverse events occurred in 20% of patients in the nivolumab / pazopanib combination group. Similarly, in the pembrolizumab / pazopanib combination group, These combinations were deemed incompatible in phase III clinical trials. In addition, there are other studies regarding dual VEGFR / ICI inhibition. Several clinical trials (e.g., bevacizumab and Phase III clinical trial of combination therapy with atezolizumab (clonal antibody) Study (Reference 6: IMmotion151: a randomized phase I II study of atezolizumab plus bevacizumab v s sunitinib in untreated metastatic renal c ell carcinoma (mRCC), Motzer, RJ et al.Journ al of Clinical Oncology.36,abstr.578(2018 Positive treatment efficacy data were also reported in the study. Although the level increased to some extent, the side effects of the combined therapy also increased, so the MSKCC standard Therefore, only 20% of intent-to-treat patients in this clinical trial are at appropriate risk.

[0005] In addition to investigating combination therapies, several therapeutic options targeting VEGF / VEGF receptors and PD-1 are being investigated. Bispecific antibodies are still being explored. For example, the one developed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. However, until now, there has been no Globally, we are targeting vascular endothelial growth factor / vascular endothelial growth factor receptor and immune checkpoint inhibitors. Bispecific antibodies targeting this target have not yet been approved for sale, and there remains a significant clinical need and There are development challenges. Summary of the Invention

[0006] The inventors have conducted extensive research and performed multiple rounds of screening and optimization to identify V We obtained VHHs that target EGF and combined them with functional domain module components. By combining it with a PD-1 antibody as a target, it can be used to target both PD-1 and VEGF. The bispecific antibody has been developed. (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 an antigen-binding domain. a combined fragment (Ab'), The second binding functional region targeting VEGF is identified by the following amino acid sequence: VHH domains R1 to R3 (variable domain of heavy chain n of heavy-chain antibody).

[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 mutation is the amino acid sequence shown in SEQ ID NO: 24. The amino acid sequence is consistent with that of the nucleotide sequence.

[0012] Preferably, the CDR of the VHH domain of the second binding functional region targeting VEGF The combinations of 1 to 3 are 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) The amino acid sequence shown in SEQ ID NO: 4 is identical to or different from the amino acid sequence shown in SEQ ID NO: 4. A framework region having one or two amino acid mutations compared to the amino acid sequence of the target gene. Main FR1, and / or 2) The amino acid sequence shown in SEQ ID NO: 5 is identical to or different from the amino acid sequence shown in SEQ ID NO: 5. A framework region domain FR having one amino acid mutation compared to the amino acid sequence of the target region. 2, and / or 3) The amino acid sequence shown in SEQ ID NO: 6 is identical to or different from the amino acid sequence shown in SEQ ID NO: 6. A framework region domain having 1 to 5 amino acid variations compared to the amino acid sequence of the target FR3, and / or 4) The amino acid sequence shown in SEQ ID NO: 7 is identical to or different from the amino acid sequence shown in SEQ ID NO: 7. A framework region domain FR having one amino acid mutation compared to the amino acid sequence of the target region. Further includes 4.

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

[0017] Furthermore, the single amino acid mutation in the FR2 domain is at position 49; The most preferred mutation is A49S.

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

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

[0020] Furthermore, the second binding functional domain targeting VEGF may be 1) A framework identical to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9 the FR1 domain, and / or 2) A framework identical to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 the framework region domain FR2, and / or 3) A framework identical to the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 11 the work region domain FR3, and / or 4) A framework identical to the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 12 It further comprises a framework region domain FR4.

[0021] Furthermore, the second binding functional region that targets VEGF is identified by the following amino acids: It includes a combination of framework regions that

[0022] [Table 3]

[0023] Furthermore, the second binding functional domain targeting VEGF is an amino acid sequence represented by SEQ ID NO: 8. or a sequence that is 1 to 10 times larger than the amino acid sequence shown in SEQ ID NO: 8. 1 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) The amino acid mutations are at positions 1, 5, 49, 58, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, Preferably, the number is selected from the following: 5th, 74th, 82Bth, 83rd, 84th, 89th and 108th. Furthermore, the mutations are Q1E, Q5L, A49S, N58Y, D65G, D74S 1 to 11 pieces selected from V(82B)S, K83R, P84A, M89V, Q108L (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11) It is preferable that

[0024] [Table 4]

[0025] More preferably, the amino acid sequence of the second binding functional domain targeting VEGF is: It 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 (Ab') thereof may be a humanized antibody or or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a human antibody or an antigen-binding fragment thereof It is a combined fragment.

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

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

[0032] Furthermore, the first binding functional domain targeting PD-1 is nivolumab (Nivolumab mab, Opdivo) antibody or pembrolizumab (Pembrolizumab, K It has the same light chain and heavy chain CDR amino acid sequences as the eytruda antibody.

[0033] Furthermore, the first binding functional domain targeting PD-1 is nivolumab (Nivolumab mab, Opdivo) antibody or pembrolizumab (Pembrolizumab, K It has the same amino acid sequence of the heavy chain variable region as the eytruda antibody.

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

[0035] Furthermore, the heavy chain portion of the first binding functional domain that targets PD-1 may further comprise a heavy chain constant Constant region CH1 domain (CH1), hinge region (Hinge), heavy chain constant region CH2 domain It contains one or more selected from the heavy chain constant region CH2 domain (CH3).

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

[0037] Furthermore, the anti-PD-1 antibody contains a heavy chain variable region domain (VH) from the N-terminus to the C-terminus. ), heavy chain constant region CH1 domain (CH1), hinge region (Hinge), heavy chain constant region A heavy chain region including a CH2 domain (CH2), a heavy chain constant region CH3 domain (CH3), and From the N-terminus to the C-terminus, there are a light chain variable region domain (VH), a light chain constant region domain ( and a light chain portion comprising, in sequence, a nucleotide sequence consisting of ...

[0038] Furthermore, the Fc domain (i.e., hinge region, heavy chain constant region CH) of the anti-PD-1 antibody 2 domain, heavy chain constant region CH3 domain) is an IgG1 Fc domain or its sibling It is a mutated IgG1.

[0039] Furthermore, the Fc domain corresponds to the amino acid sequence set forth in SEQ ID NO: 15, or Alternatively, a sequence identical to the amino acid sequence shown in SEQ ID NO: 15 by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 1 3%, 94%, 95%, 96%, 97%, 98%, 99% or more homology It is an amino acid sequence.

[0040] [Table 8]

[0041] Furthermore, the first binding functional region targeting PD-1 comprises the following amino acid sequence: It includes a combination of light and heavy chains. 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.

[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 may be a fragment of the antibody (Ab) or antigen-binding fragment (Ab') thereof. It is either directly linked to the C-terminus or N-terminus of the chain or via a peptide linker. are connected.

[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 It is expressed as ( 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 heavy chain shown in SEQ ID NO: 20 and a or a combination of a heavy chain shown in SEQ ID NO: 22 and a light chain shown in SEQ ID NO: 23. Including combinations with the light chains shown.

[0047] [Table 10]

[0048] The present invention further relates to a VHH domain that targets VEGF. contains 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. be. Furthermore, said VHH domain is a humanized VHH domain.

[0051] Furthermore, the VHH domain comprises: 1) The amino acid sequence shown in SEQ ID NO: 4 is identical to or different from the amino acid sequence shown in SEQ ID NO: 4. A framework region having one or two amino acid mutations compared to the amino acid sequence of the target gene. Main FR1, and / or 2) The amino acid sequence shown in SEQ ID NO: 5 is identical to or different from the amino acid sequence shown in SEQ ID NO: 5. A framework region domain FR having one amino acid mutation compared to the amino acid sequence of the target region. 2, and / or 3) The amino acid sequence shown in SEQ ID NO: 6 is identical to or different from the amino acid sequence shown in SEQ ID NO: 6. A framework region domain having 1 to 5 amino acid variations compared to the amino acid sequence of the target FR3, and / or 4) The amino acid sequence shown in SEQ ID NO: 7 is identical to or different from the amino acid sequence shown in SEQ ID NO: 7. A framework region domain FR having one amino acid mutation compared to the amino acid sequence of the target region. Further includes 4.

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

[0053] Furthermore, the 1 to 5 amino acid mutations in the FR3 domain are 1, 2, 3, 4, or 5 selected from the 83rd, 84th, and 89th positions More preferably, D74S, V(82B)S, K83R, P84A and M89V Being selected in 1st or 2nd or 3rd or 4th or 5th place. Furthermore, the single amino acid mutation in the FR4 domain is at position 108 and More preferably, it is 108L.

[0054] Furthermore, the VEGF-targeting VHH domain may comprise: 1) A framework identical to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9 the FR1 domain, and / or 2) A framework identical to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 10 the framework region domain FR2, and / or 3) A framework identical to the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 11 the work region domain FR3, and / or 4) A framework identical to the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 12 It further comprises a framework region domain FR4.

[0055] Furthermore, the VHH domain comprises a combination of framework regions defined by the following amino acids: Includes combinations.

[0056] [Table 12]

[0057] Furthermore, the amino acid sequence of the VHH domain is the same as the amino acid sequence shown in SEQ ID NO: 8. Identical or 1 to 11 (1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) amino acid sites Furthermore, the amino acid mutations are at positions 1, 5, 49, 58, 65, and 70. Preferably, the amino acid sequence is selected from the group consisting of 74, 82B, 83, 84, 89, and 108. Furthermore, the mutations include Q1E, Q5L, A49S, N58Y, D65G, D74S, V (82B)S, K83R, P84A, M89V, Q108L 1 to 11 (1 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) preferable.

[0058] [Table 13]

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

[0060] Furthermore, the recombinant protein may include bispecific antibodies, multispecific antibodies, antibody drug conjugates, and the like. Includes Jugate. The present invention further relates to a bispecific antibody, a multispecific antibody, a recombinant antibody, or a method for producing the same. It relates to a polynucleotide encoding a protein or a VHH domain. The present invention further relates to an expression vector comprising any one of the polynucleotides described above. Regarding.

[0061] The present invention further provides an expression vector according to any one of the above, or a genome thereof. The present invention relates to a host cell into which any one of the polynucleotides described above has been integrated. The present invention further relates to a VHH domain, a bispecific antibody, a multispecific antibody, and a method for producing the same. a pharmaceutical composition comprising an isomeric antibody or recombinant protein and a pharmaceutically acceptable carrier thereof; Regarding.

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

[0063] The beneficial effects of the present invention are as follows: The engineered and optimized VEGF-targeting VHH domain is structurally stable and highly thermostable. It also exhibits high binding activity to both human and mouse VEGF-A, and in animal models This is advantageous for simultaneously verifying the success rate of drug development and for protein The VHH domain has advantageous characteristics for high expression and purification. The bispecific antibodies developed based on this have a unique bispecific antibody structure and are highly human. It is expressed in a manner that does not contain artificially added redundant sequences and is locally concentrated in VEGF-high-expressing tumors. This may result in more selective tumor suppression and more effective tumor suppression. This will solve the problem of insufficient efficacy of PD-1 / PD-L1 inhibitors. This can be done. [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 otherwise specified, the terms of the present invention refer to virology, immunology, and the like, all of which are within the skill of the art. , microbiology, molecular biology, recombinant DNA technology, or other methods commonly known to those skilled in the art. Many of these are listed below for illustrative purposes. Such techniques are well explained in the literature.

[0066] As used herein, the term "antibody" refers to a substantially intact antibody, not an antibody fragment. Specifically, the term "antibody" as used herein preferably refers to an intact antibody. Each of these is composed of two heavy chains (H chains) and two light chains (L chains), and these chains It consists of four polypeptide chains linked together via disulfide bonds and non-covalent bonds. It has a symmetrical structure consisting of four polypeptide chains such that the monomer molecule is formed.

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

[0068] As used herein, the term "humanized antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that is human immunoglobulin G. The HVR residues derived from the receptor are found in mouse, rat, and non-human primates with desired specificity, affinity, and / or capacity, such as herons or non-human primates are replaced by residues from the HVR derived from a human species (donor antibody). Framework ("FR") residues of the immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies or antigen-binding fragments thereof may contain antigens that are not present in the recipient antibody or donor antibody. To further improve antibody properties such as binding affinity, these residues may be included. Generally speaking, a humanized antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment of a humanized antibody. Typically, the antibody comprises substantially all of the two variable domains, including at least one. All or substantially all of the hypervariable loops correspond to those of non-human immunoglobulin sequences. and all or substantially all of the FR domains are those of a human immunoglobulin sequence. However, the FR region has one or more functions that improve antibody performance, such as binding affinity, isomerization, and immunogenicity. can include multiple single FR residue substitutions. Humanized antibodies can optionally include and preferably contains at least a portion of the immunoglobulin constant region (Fc) of a human immunoglobulin. For further details, see, for example, Jones et al., Nature 321:522 -525(1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr.Op.Struct.Biol.2:5 93-596 (1992). See also, for example, Vaswani and Hami lton, Ann. Allergy, Asthma&Immunol.1:105-11 5(1998), Harris,Biochem.Soc.Transactions 23:1035-1038(1995);Hurle and Gross,Curr.Op.B iotech.5:428-433(1994), and U.S. Patent No. 6,982,321 No. 7,087,409.

[0069] As used herein, the term "human antibody" refers to an antibody produced by a human. amino acid sequences and / or using techniques to produce human antibodies This definition of a human antibody includes humanized antibodies that contain non-human antigen-binding residues. Human antibodies are specifically excluded. Human antibodies can be obtained from a wide range of sources, including phage display libraries. These can be created using a variety of techniques known in the art. Winter, J. Mol. Biol., 227:381 (1991); Marks et al. J. Mol. Biol. 222:581 (1991). Furthermore, the human monoclonal antibody For production, see Cole et al., Monoclonal Antibodies and d Cancer Therapy, Alan R. Liss, 77 pages (1985), Boerner et al., J. Immunol., 147(1):86-95(1991), etc. This method is described in the literature by van Dijk and van de Win kel, Curr. Opin. Pharmacol., 5:368-74 (2001) Human antibodies are modified to produce such antibodies in response to antigenic challenge. Transgenic animals in which the endogenous locus is conserved (e.g., immunoxenogeneic mice) The antigen can be prepared by administering the antigen to a mouse (e.g., XENOMOUSE™). (See U.S. Patent Nos. 6,075,181 and 6,150,584 for related technology.) Also, L i et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562( Regarding human antibodies produced by human B cell hybridoma technology disclosed in Please also refer to the contents.

[0070] As used herein, the term "CDR" (i.e., complementarity determining region) refers to a region of a gene that is a complementarity determining region of a gene in the Kabat system. The term is used to refer to the hypervariable regions as defined by the NIH standard. , Sequences of Proteins of Immunological In interest, version 5, Public Health Service, Nati onal Institutes of Health,Bethesda,Md.(19 91). For CDR, see IMGT, Chothia, AbM, C Other definition forms exist, such as contact.

[0071] Unless otherwise stated, the numbering of immunoglobulin residues in the present invention follows the Kabat numbering system. The numbers are based on the stem.

[0072] As used herein, the term "Fc domain" generally refers to a region comprising the hinge region, the heavy chain constant region CH2 domain, including the heavy chain constant region CH3 domain (i.e., hinge-CH2-CH3). The c domain always forms a dimer in the form of a disulfide bond. The fusion partner used is the Fc fragment of immunoglobulin IgG, except that antibody F c is a part of the antibody constant region (including the hinge region - CH2-CH3 and C of the antibody constant region) After fusion with the Fc fragment, the molecular weight increases, and the FcRn-mediated recycling mechanism This mechanism increases the stability of the fusion molecule, prolonging its half-life in vivo and allowing the use of the Fc fragment. These proteins can mediate various biological functions such as ADCC and CDC. The fusion protein does not contain the variable region of the antibody, and its pharmacology and efficacy are mainly determined by the functional moiety fused to Fc. There are four subtypes of human immunoglobulin G, and different immunoglobulin G subtypes The most widely used antibody at present is the IgG1 type, and recently In 2015, with the development of new indications and the emergence of antibody drugs with new mechanisms of action, IgG2 and Ig In the present invention, the "Fc domain" is a subtype of I It is preferably the Fc domain of an IgG1 antibody or the Fc domain of an IgG4 antibody. stomach.

[0073] As used herein, the term "specificity" refers to an antibody that selectively recognizes a specific epitope of an antigen. By "antibody" is meant a native binding protein or antibody. For example, a native antibody is monospecific. The terms "bispecific" or "multispecific" as used herein refer to antigen-binding proteins or antigen-binding proteins. This means that the body has two or more antigen-binding sites, at least two of which are different. They bind to different antigens or different epitopes of the same antigen.

[0074] As used herein, the terms "divalent," "trivalent," and "tetravalent" refer to "atomic valence." 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 binding of an antigen-binding protein or antibody to a This means that there are two, three, and four binding sites in the molecule, respectively. Taste.

[0075] "VHH domain" (variable domain of heavy chain of heavy-chain antibody) is also called VHH or VHH antibody fragment. The first was "heavy-chain antibody" (hcAb, The antigen-binding immunoglobulin variable domains (C .Hamers-Casterman, T.Atarhouch, et al.Natu rally occurring antibodies devoid of ligh t chains.Nature, June 3, 1993, Vol.363, pp.446 The term "VHH domain" refers to such a variable domain consisting of two light chains and Heavy chain variable domains present in conventional antibodies consisting of two heavy chains (hereinafter referred to as "conventional antibodies") domain (herein referred to as "VH domain" or "VH domain") and a conventional The light chain variable domain (referred to herein as a "VL domain" or "VL domain") present in an antibody VHH domains are used to distinguish them from other antigen-binding domains. It specifically binds to the epitope without the VL domain (which is the same as the VL domain in conventional antibodies). The VH domain in conventional antibodies identifies epitopes by combining VH and VH domains. (VHH domains are formed from a single immunoglobulin domain. It is an antigen recognition unit formed by

[0076] As used herein, the term "heavy chain single domain antibody" refers to a antibody, sdAb), "VHH domain", "VHH", "VHH antibody fragment", The terms "VHH antibody," "nanobody," and "nanobody domain" can be used interchangeably. Cut.

[0077] As used herein, the term "homology" refers to achieving the maximum percentage homology of a sequence. After sequence alignment and (if necessary) gap introduction, a specific peptide is identified. A match between an amino acid residue in a peptide or polypeptide sequence and the same amino acid residue in a candidate sequence. Conservative substitutions are not considered as part of the sequence homology. Alignment to determine percent homology of amino acid sequences can be performed using, for example, BLAST, B LAST-2, ALIGN, or MEGALIGN-(DNASTAR) software using commonly available computer software, such as This can be done in a variety of ways within the data, e.g., the number of sequences required to achieve maximum alignment over the entire length of the comparison sequences. Any algorithms etc. can be determined by one skilled in the art.

[0078] As used herein, the term "treatment" refers to an individual or group of individuals who are treated for a clinical pathological process. refers to clinical interventions designed to alter the natural processes of cells or tissues. These include reducing the rate of disease progression, improving or mitigating the disease state, and mitigating or improving the prognosis. For example, the disease or condition being treated (e.g., cancer, inflammation, or autoimmune disease) The reduction or elimination of one or more symptoms associated with a disease, etc.

[0079] As used herein, the term "effective amount" refers to an amount that is effective to treat a disease or condition in a subject. In the case of cancer, the anti-VEGF single domain Antigen-binding fragments, bispecific antibodies, multispecific antigen-binding constructs, pharmaceutical compositions, immunoconjugates According to the effective dose of jugate, it can reduce the number of cancer cells, reduce the size of tumors, and Inhibit (i.e., delay to some extent, preferably inhibit) invasion of surrounding organs and metastasis of tumor cancer cells inhibit (i.e., slow to some extent, preferably prevent) migration, inhibit to some extent tumor growth, and / or may alleviate to some extent one or more symptoms associated with the cancer. As understood in a clinical setting, an effective amount of a drug, compound, or pharmaceutical composition may be administered in combination with other drugs, This may or may not be achieved by using a compound or pharmaceutical composition in combination. Thus, an "effective amount" can be considered when administering one or more therapeutic agents, and When used in combination with one or more other drugs, a single drug may be provided in an effective amount to achieve the desired result. It is believed that the results can be realized or achieved.

[0080] As used herein, the term "subject" is preferably a mammal, and may include humans, bovines, Examples include, but are not limited to, horses, felines, canines, rodents, or primates. In some embodiments, the individual is a human. DETAILED DESCRIPTION OF THE INVENTION

[0081] Hereinafter, the embodiments of the present invention will be described in detail with reference to examples. The examples are used only to illustrate the present invention and are not to be construed as limiting the scope of the present invention. It will be understood by those skilled in the art that this should not be the case.

[0082] [Example 1] Screening and development of VHHs targeting VEGF 500 μg each of human and mouse VEGF-A protein was mixed with an equal volume of adjuvant. The immunogen was prepared by combining the two immunizations, and four rounds of immunization were administered to a two-year-old female alpaca at two-week intervals. After the quarantine, hyperimmune serum was collected, peripheral blood mononuclear cells were isolated, and RNA was extracted. The variable region genes of the nanobody were amplified by the following procedure. After electroporation, a VEGF-A immune antibody library was obtained.

[0083] The prepared VEGF-A immune antibody library was subjected to immunotube solid-phase panel assay. Two rounds of panning were performed using magnetic bead liquid phase screening. A single clone was selected for ELISA detection. Positive clones were sequenced and the final Finally, we obtained 81 different antibody genes. The VHH-Fc DNA fragment was constructed and sequenced, and then transfected into 293T cells. Eukaryotic expression was performed using the ELISA kit, and the expression supernatant was collected for specificity detection. As a result, strain (#6) 6 showed excellent binding ability to both human and mouse VEGF-A. It was found to be the only antibody that

[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 RFTMSRDNDKNT VYLQMNVLKPEDTAMYYCRA 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 VHH of strain No. 6, we conducted a structural biology-based Five rounds of sequence optimization and modification were performed in combination with protein structure simulation. Two VHH sequences with good thermostability and high degree of humanization were obtained. #6 (SEQ ID NO: 8): QVQLQESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYRQAPGKERELVAFITSAGATTNYADSVKDRFTMSRDNDKNT VYLQMNVLKPEDTAMYYCRALVTLWNVYWGQGTQVTVSS #48 (SEQ ID NO: 13): E VQL L ESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYRQAPGKERELV S FITSAGATT Y YADSVK G RFTMSRDN S KNT VYLQMN S L RA EDTA V YYCRALVTLWNVYWGQGT L VTVSS #49 (SEQ ID NO: 14): E VQL L ESGGGLVQPGGSLRLSCAASGFTFSTSTMSWYRQAPGKERELVAFITSAGATT Y YADSVK G RFTMSRDN S KNT VYLQMN S L RA EDTA V YYCRALVTLWNVYWGQGT L VTVSS

[0089] [Table 17]

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

[0091] In the case of sequence #49, Q1E+Q5L is located in the FR1 region, and N58Y+D65G is located in the CDR2 region, and D74S+V(82B)S+K83R+P84A+M89V Q108L is located in the FR4 region, and Q109L is located in the FR3 region.

[0092] Measurement of VEGF binding ability and thermal stability revealed that #48 and #49 were comparable to #6. The combination showed good VEGF binding ability (see Figure 1). The mutant sequences showed an improvement in thermal stability of approximately 10°C, with Tm values ​​of 64.1°C and 64.7°C, respectively. It was.

[0093] [Example 3] Screening of candidate bispecific antibody molecular structures and construction of control substances #48 VHH and anti-PD-1 antibody nivolumab (Opdivo) or pembro Various bispecific antibody structures based on the antigen-binding fragment of lizumab (Keytruda) The activation, expression level, and stability were comprehensively evaluated and screened. After evaluating the results, we decided to use the bispecific antibody structure shown in Figure 2. The bispecific antibody molecule #16 was further constructed and evaluated. +2) and the Fc contained functionally silent IgG1 mutations (AEASS). The amino acid sequence of the molecule is shown in Table 18 below.

[0094] [Table 18]

[0095] As a control, the AK112 analogue, a bispecific antibody targeting PD-1 and VEGF, was administered. (The heavy chain amino acid sequence of SEQ ID NO: 25 and the light chain amino acid sequence of SEQ ID NO: 26 were used in-house. (expressed and prepared in SEQ ID NO:25: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAY LQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEA AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESG GGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRA EDTALYYCANRYGEAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSMSASVGDRVTFTCRASQDIN TYLSWFQQKPGKSPKTLIYRANRLLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELK SEQ ID NO:26: DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQP EDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0096] [Example 4] Binding of human PD-1 to human VEGF The binding of the antibody to human PD-1 was detected by ELISA. L of human PD-1 (Acro, His tag) was coated on an ELISA plate overnight and then blocked. The plate was then incubated with serial dilutions of bispecific antibody samples and control antibodies. Next, HRP-labeled anti-human IgG Fc secondary antibody was added and incubated. After adding TMB coloring solution to develop color, the OD45 was measured using a microplate reader. The absorbance was measured (see Table 19 below). To detect binding, 50 ng / mL human VEGF (Acro, His tag) was used. LISA plates were coated and blocked overnight and incubated with serial dilutions of test samples and control antibodies. The plate was then incubated with HRP-labeled anti-human IgG Fc antibody and washed. Polyspecific antibodies were added, incubated, and washed. Finally, TMB color development solution was added to develop color. After incubation, the OD 450 absorbance was measured using a microplate reader (see Table 20 below). As is clear from the ELISA results, the binding of #10 and #16 to human PD-1 was Performance comparable to Opdivo and Keytruda, but with higher Top signal and control AK The binding patterns of #10 and #16 with VEGF were The binding affinity of #48 to VEGF is very unique, and #10, #16, and #17 are significantly higher than that of #48 to VEGF. The binding ability with GF was slightly reduced, and the Top absorbance was approximately half that of #48. The binding state of #10 and #16 with VEGF was changed by the optimization based on biology. On the other hand, the anti-VEGF portion of the AK112 analogue A bevacizumab antibody fragment was used to bind VEGF, which has a similar binding activity to 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 #10, #16, and AK112 analogs bind to both human VEGF and human PD-1 The dual binding capacity was further verified. A 96-well ELISA plate was filled with 50 ng / mL of human V The cells were coated overnight with EGF (Sino Biological, tag-free), washed, and then blocked for 1 hour. After incubation, serial dilutions of the test antibody were added and incubated for 2 hours. / mL human PD-1 (Acro, His tag) was added and incubated for 2 hours, then washed. Afterwards, HRP-labeled anti-His antibody was added and incubated for 1 hour. After adding the color solution to develop color, measure the OD 450nm absorbance using a microplate reader. As a result, as shown in Table 21 below, the single-target antibody simultaneously bound to two target molecules. However, #10, #16 and the AK112 analog all showed activity against human VEGF and human The EC50 values ​​were 0.279nM and 0.326nM, respectively. M was 0.254 nM.

[0100] ELISA double detection was performed by changing the order of VEGF and PD-1 binding. 96 well The ELISA plate was coated overnight with 1 μg / mL human PD-1 (Acro, His tag). After inversion, washing, and blocking for 1 hour, serial dilutions of specific antibodies were added and incubated for 2 hours. Next, 50 ng / mL of biotin-labeled human VEGF (Acro Biosciences) was added to the cells. After incubation for 2 hours with the addition of chloramphenicol (anticoagulated protein), the cells were washed and then incubated with HRP-labeled streptomycin. Avidin was added and incubated for 1 hour. Finally, TMB color development solution was added to develop the color. The absorbance at 450 nm was measured using a microplate reader. The results are shown in Table 2 below. As shown in #1, the single-target antibody was unable to bind to two target molecules simultaneously, but #10, Both #16 and the AK112 analogue have dual binding ability to human PD-1 and human VEGF. The EC50 values ​​were 0.114 nM, 0.117 nM, and 0.634 nM, respectively. Comparison of EC50 values ​​revealed that #10 and #16 bind to both human PD-1 and human VEGF. Its synthesis ability was found to be 5 to 6 times better than that of the competing AK112 analog.

[0101] [Table 21]

[0102] [Example 6] Binding to foreign antigens ELISA was used to detect binding between the candidate molecules and competitor antigens from different bacterial species. The original VEGF sequence was an exact match with the humanized VEGF sequence, and the candidate antibody was found to be a monkey VEGF The binding ability of the antibody to human VEGF was equivalent to that of human VEGF. Binding to monkey PD-1 was detected. The ELISA plate was filled with 1 μg / mL monkey PD-1 ( After blocking overnight with His tag (Acro Biosciences), test samples and control antibodies were The plate was incubated with serially diluted solutions of HRP-labeled anti-human IgG Fc The bispecific antibody was added, incubated, and washed. Finally, TMB color development solution was added. After coloring, the OD 450 absorbance was measured using a microplate reader (see Table 22 below). Consistent with the results of binding to human PD-1, #10 and #16 did not bind to monkey PD-1. 1 is comparable to Opdivo and Keytruda, and is stronger than the competing AK112 analogue. It was shown to be strong.

[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 examined. ISA plates were coated overnight with 1 μg / mL human PD-1 (Acro, Fc tag). After washing and blocking, serially diluted antibodies and human PD-L1 (Sino Biotechnology) were added. A mixture of HRP-labeled IgG (His tag, manufactured by Caltech) was added and incubated for 2 hours. Anti-His antibody was added and incubated for 1 hour. Finally, TMB coloring solution was added to develop color. After incubation, the OD 450 absorbance was measured using a microplate reader (see Table 23 below). As shown in the results, except for #48, which bound only to VEGF, all the other antibodies bound to P It was found that the binding of D-1 to PD-L1 was blocked and the blocking activity was equivalent. Ta.

[0105] [Table 23]

[0106] [Example 8] Ability to block the binding of VEGF to VEGFR1 or VEGFR2 The ability of the test antibody to block the binding of VEGF to VEGFR1 or VEGFR2 is examined. The ELISA plate was filled with 2 μg / mL human VEGFR1 and 5 μg / mL After coating overnight with human EGFR2 (constructed and expressed in-house, Fc tagged), the cells were washed and After blocking, serially diluted antibodies and human VEGF (Acro Biotin) were incubated with 100µg of VEGF. A mixture of 1000 μg of PEG-1000 (polyacrylamide gel electrophoresis) and 1000 μg of PEG-1000 (polyacrylamide gel electrophoresis) was added to the wells, and the wells were incubated for 2 hours and washed. HRP-labeled streptavidin was added and incubated for 1 hour. Finally, TMB color development was achieved. After adding the solution to develop color, measure the OD 450nm absorbance using a microplate reader. As a result, #10 and #16 were found to have the ability to block the binding of VEGF to VEGFR1. The IC50 values ​​were 0.491nM and 0.507nM, respectively, which was higher than that of blocker #48. The spatial conformational changes of the constructed bispecific antibody are advantageous because they have superior blocking ability. The blocking ability of the competing product AK112 analogue is comparable to that of bevacizumab. The IC50 values ​​were comparable, 4.610 nM and 5.647 nM, respectively. As a result, #10 and #16 showed stronger ability to block the binding of VEGF to VEGFR2. The IC50 values ​​were 3.643nM and 3.249nM, respectively, demonstrating the blocking ability of #48 ( The IC50 value was superior to 5.841nM, indicating that the spatial conformational changes of the bispecific antibody On the other hand, the blocking ability of the competing product AK112 analogue is The blocking potency of the antibody was comparable to that of the antibody with IC50 values ​​of 6.481 nM and 6.637 nM, respectively. It was.

[0107] [Example 9] Verification of T cell responses after PD-1 blockade using mixed lymphocyte reaction (MLR) assay The functions of the test candidate molecules and the competitor AK112 analogue were further compared at the cellular level. The T cell response after D-1 blockade was examined using the mixed lymphocyte reaction (MLR) method. 2 x 10 donor-derived 4 2 x 10 DC cells from a different donor 5 CD4+ T cells The test antibody was added in serial dilutions and incubated for 72 hours. The amount of IL-2 (Figure 3) and IL-2 released from the cell supernatant was measured using an ELISA kit. The contents of IFNγ and IFNγ (Fig. 4) were measured. The amount of IL-2 and IFNγ released by CD4+ T cells in MLR reactions was compared with that of competitor AK1. Since the amount was higher than that of the 12 analogs, #10 and #16 had a stronger effect on T cells. It was shown that

[0108] [Example 10] Using a vascular endothelial cell (HUVEC) proliferation inhibition test, we confirmed that the test antibody inhibits HUVEC proliferation induced by VEGF. Verification of the ability to inhibit stimulation of EC cell proliferation 5 x 10 cells in medium containing 1% FBS on a 96-well cell plate 3 HUVE C cells / well were added and cultured overnight in an incubator at 37°C with 5% carbon dioxide. The medium was removed, and 70 ng / mL of VEGF and serially diluted test antibodies and competitor A were added. The K112 analog was added. The reaction plate was placed in an incubator at 37°C with 5% carbon dioxide. After culturing for 4 days, CTG reagent was added and the relative fluorescence signal was measured using a microplate reader. The values ​​were measured in the ECM medium containing 1% FBS and the cell group stimulated with VEGF (unstimulated). The fluorescence value of the PBS-only group (1% FBS) was defined as corresponding to an inhibition rate of 0. The fluorescence value of the ECM medium containing VEGF and antibodies was 10 The relative inhibition rate was defined as 0%. The relative inhibition rate was then fitted as a curve. As shown in the figure, the maximum inhibition rate of #10 and #16 was approximately 60%, while that of the competitor AK11 The maximum inhibition rate of the two analogs was approximately 40%, so the inhibition rates of #10 and #16 were comparable to those of the competitor AK It was more expensive than the 112 analog.

[0109] [Example 11] Inhibitory effect on HUVEC cell migration Transwell chambers with 8 μm pore size were used to study the effect of antibodies on HUVEC cell migration. The transwell consists of an upper chamber and a lower chamber. 5×10 3 HUVEC cells / well were added to the upper chamber and incubated at 37°C in 5% carbon dioxide. The cells were then cultured in a plain incubator for 30 minutes. Then, 200 ng / mL of VEGF and various concentrations of VEGF were added. Each test antibody and competitor AK112 analog were added to the lower chamber, and three replicates were administered to each group. The wells were then placed in an incubator at 37°C with 5% carbon dioxide and cultured for 24 hours. After fixation with 4% paraformaldehyde, the cells were stained with crystal violet. After the incubation, the number of cells that migrated to the back surface of the upper chamber was counted. The lower the number of cells, the higher the inhibition rate. The results in Figure 6 show that #10 and #16 are higher than the competing AK112 analog. The inhibition rate was high.

[0110] [Example 12] Characteristics of increased antibody concentration Structural biology simulations revealed that the conformation of #10 is consistent with that of the anti-VEGF nanobody and V The binding pattern of EGF may be altered, and the dimeric VEGF protein may induce an anti-VEGF It was revealed that #10 can concentrate the antibody concentration. To verify this, magnetic beads coupled to human VEGF protein (Acro) were used. ) diluted to 250ng / mL, 125ng / mL, and 50ng / mL in PBS, respectively. Then, 10 nM of #10, #16, and the competitor AK112 analog were added and incubated for 1 hour. After incubation, the plate was placed on a magnet and adsorbed for 2 minutes, and the supernatant was aspirated and used for ELISA. As a result, as shown in Figure 7, the residual protein of #10 and #16 was The amount of antibody was less than that of the competitor AK112 analog, which contains VEGF. It was found that the bispecific antibodies were more abundant when enriched with magnetic beads. In addition, an ELISA quantification test was performed to verify the degree of cross-linking between the antibody and VEGF. The antibody was cross-linked to VEGF magnetic beads, and then the remaining uncross-linked antibody was detected by ELISA. The antibody amount was measured. As a result, #10 and #16 showed a V The degree of cross-linking with EGF was higher. These findings support each other, and suggest that high VEGF expression in the tumor localization may contribute to tumor localization. The differentiation characteristics and therapeutic potential of #10 and #16 bispecific antibodies can be demonstrated. was also shown.

[0111] [Example 13] Drug efficacy evaluation in animal models To evaluate the efficacy of the test antibody in a mouse model, we used a mouse model transfected with human PD-1. lb / c mice were selected and inoculated with wild-type CT26 tumor cells (colon cancer cells). Neither mab nor the AK112 analog binds to mouse VEGF, so these two groups were excluded. VEGF-Trap (VEGF-Trap) is a compound that binds to human VEGF and mouse VEGF. a fusion protein consisting of the second domain of VEGFR1, the third domain of VEGFR2, and human IgGFc A total of six groups were designed, with the control group being the IgG1-specific protein. is shown in Table 24 below.

[0112] [Table 24]

[0113] As shown in Figure 8, the tumor inhibition rates of #10 and #16 in tumor-bearing mice were 57% and 57%, respectively. 62%, compared with the Opdivo monotherapy group (20%) and the VEGF-Trap monotherapy group (4%). 5%), and the tumor inhibition rate was higher than that of the Opdivo and VEGF-Trap combination treatment group (56% On day 14, the mean tumor volumes of the #10 and #16 treatment groups were The changes in the mice's weight were slightly smaller than those in the combined treatment group. On the 14th day, the weight change in the #10 and #16 treatment groups was within 10%. It was revealed that #10 and #16 have good pharmacological effects and safety.

[0114] [Example 14] Synergistic effects of #10 molecules in an immune-reconstituted human melanoma subcutaneous xenograft model To evaluate synergy studies of #10 in immune-reconstituted human melanoma models We selected immunodeficient mice B-NDG and transfected A375 tumor cells (malignant tumors) with human PD-L1. melanoma cells) and human peripheral blood mononuclear cells (huPBMCs) were inoculated into tumor-bearing mice. The immune system was reconstructed using the anti-PD-1 drug Opdivo and the anti-VEGF drug A bevacizumab, #48, was used as a single target control, and an AK112 analog (AK112 A total of 8 groups were designed, with #10 as the control drug (an analog, expressed and prepared in-house). The experimental design, dosage and route of administration are shown in Table 25 below.

[0115] [Table 25]

[0116] In this experimental system, the Opdivo monotherapy group showed anti-tumor activity as detected on day 21. #10 showed no tumor suppression effect, and the tumor suppression rate TGI was 73%. bevacizumab monotherapy group (TGI=53%), #48 monotherapy group (TGI=57%), The TGI was significantly higher than that of the Opdivo and #48 combination treatment group (TGI = 50%), and and bevacizumab combination therapy group (based on TGI = 68% tumor inhibition rate, #10 7% improvement in tumor relative inhibition rate by the molecule), and AK112 analog (TGI=64 When the tumor inhibition rate of 10% was used as the standard, the relative tumor inhibition rate of #10 molecule was improved by 14%. As a result, it was revealed that the bispecific antibody of the present invention exhibited synergistic effects. It was found to have a significant advantage over the control molecule.

[0117] It should be noted that what has been disclosed above is merely a few embodiments of the present invention, and it is not intended to limit the scope of the present invention in any way. It is understood by those skilled in the art that the present invention is not limited to the embodiments. It will be appreciated that those skilled in the art will appreciate that certain improvements and modifications may be made without departing from the principles of the present invention. These improvements and modifications are within the scope of the claims of the present invention. do.

Claims

1. 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; the amino acid sequence of the CDR3 is identical to the amino acid sequence shown in SEQ ID NO: 3; Preferably, the site of the mutation in CDR2 is at position 58 and / or 65, Preferably, the mutation in CDR2 is a N58Y mutation and / or a D65G mutation, Preferably, the VHH domain is a humanized VHH domain, Preferably, 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 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) further comprising a framework region domain FR4 that corresponds 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; Preferably, said one or two amino acid mutations in the FR1 domain are at positions 1 and / or 5, more preferably Q1E and / or Q5L mutations; Preferably, said single amino acid mutation in the FR2 domain is at position 49, more preferably an A49S mutation; Preferably, 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 1, 2, 3, 4, or 5 selected from D74S, V(82B)S, K83R, P84A, and M89V; Preferably, the single amino acid mutation in the FR4 domain is at position 108, more preferably Q108L; Preferably, 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) Framework region domain FR4 comprising the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 12 further comprising Preferably, the VHH domain comprises 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 identical to the amino acid sequence shown in SEQ ID NO:9, FR2 identical to the amino acid sequence shown in SEQ ID NO:5, FR3 identical to the amino acid sequence shown in SEQ ID NO:11, and FR4 identical to the amino acid sequence shown in SEQ ID NO:12 Including, A VHH domain characterized by:

2. 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; Preferably, the site of the amino acid mutation is selected from positions 1, 5, 49, 58, 65, 74, 82B, 83, 84, 89, and 108; Preferably, the amino acid mutations are 1 to 11 selected from Q1E, Q5L, A49S, N58Y, D65G, D74S, V(82B)S, K83R, P84A, M89V, and Q108L; Preferably, said mutations are 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 and A VHH domain according to claim 1, characterized in that the amino acid sequence of the VHH domain preferably corresponds to the amino acid sequence shown in SEQ ID NO: 8, SEQ ID NO: 13 or SEQ ID NO:

14.

3. A VHH domain according to claim 1 or 2, used for the construction of a recombinant protein.

4. Comprising a VHH domain according to claim 1 or 2, Preferably, the recombinant protein is a bispecific antibody, a multispecific antibody, or an antibody-drug conjugate.

5. A polynucleotide encoding a VHH domain according to claim 1 or 2, or a recombinant protein according to claim 4.

6. An expression vector comprising the polynucleotide of claim 5.

7. A host cell comprising the expression vector of claim 6 or having the polynucleotide of claim 5 integrated into its genome.

8. A pharmaceutical composition comprising a VHH domain according to claim 1 or 2, or a recombinant protein according to claim 4, and a pharmaceutically acceptable carrier thereof.

9. A drug for cancer treatment comprising the VHH domain of claim 1 or 2 or the recombinant protein of claim 4, Preferably, the cancer is colorectal cancer.

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