Bispecific antibodies and their use

Abstract

The present application relates to the field of biotechnology, in particular, the present application relates to single domain antibody, bispecific antibody and application thereof, more particularly, the present application relates to single domain antibody, antibody or fusion protein, bispecific antibody, nucleic acid molecule, expression vector, recombinant cell, pharmaceutical composition, pharmaceutical use, detection kit capable of specifically recognizing VEGF and / or ANG-2.The antibody of the present application has higher binding activity with VEGF and / or ANG-2, can effectively block the binding of VEGF, ANG-2 and their respective receptors, has the effect of inhibiting angiogenesis and reducing vascular inflammation, can effectively treat or prevent the related diseases caused by angiogenesis, especially the ophthalmic related diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular, the present application relates to bispecific antibodies and uses thereof, more particularly, the present application relates to single domain antibodies, antibodies or fusion proteins, bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, pharmaceutical compositions, pharmaceutical uses and detection kits capable of specifically recognizing VEGF and / or ANG-2. BACKGROUND

[0002] Vascular endothelial growth factor (VEGF) is a key factor in neovascularization. VEGF can induce the regeneration of existing blood vessels or the growth of new blood vessels (angiogenesis), thus being a key factor in embryonic development and vascular repair. The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factors 1 and 2 (PIGF-1 and PIGF-2, respectively). Members of the VEGF family transduce signals between cells by binding to three tyrosine kinase receptors: VEGF-A and VEGF-B preferentially bind to VEGFR-1; VEGF-A, VEGF-C, VEGF-D, and VEGF-E can bind to VEGFR-2; and VEGF-C and VEGF-D bind to VEGFR-3 (expressed only in hematopoietic cells). Therefore, inhibition of the VEGF signaling pathway can treat ophthalmic diseases related to angiogenesis.

[0003] ANG-2 belongs to the angiopoietin family and is expressed by endothelial cells, usually stored in Weibel-Palade bodies of endothelial cells. When encountering conditions that promote the formation of new blood vessels, such as hypoxia and inflammation, ANG2 is released from Weibel-Palade bodies and competes with ANG1, acting as an antagonist of Tie2 to inhibit the stability of blood vessels, causing pericytes to detach from endothelial cells, increasing the permeability of endothelial cells, and allowing VEGF to better promote the formation of new blood vessels.

[0004] Although CN117545503A and CN116925234A disclose specific antibodies for VEGF and / or ANG-2 in the art, the development of new antibodies with better binding specificity for VEGF and / or ANG-2 is still of great significance for the diagnosis and treatment of angiogenesis-related diseases. SUMMARY

[0005] To solve the above problems, the present application provides a specific antibody capable of specifically binding to VEGF and / or ANG-2. The antibody of the present application has a high binding activity to VEGF and / or ANG-2, can effectively block the binding of VEGF, ANG-2 to their respective receptors, has the effect of inhibiting angiogenesis and reducing vascular inflammation, can effectively treat or prevent diseases related to angiogenesis, especially ophthalmic diseases.

[0006] In a first aspect of the present application, a single-domain antibody capable of specifically recognizing Ang-2 is provided, wherein the heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3, wherein:

[0007] HCDR1 comprises the sequence shown in SEQ ID NO: 18, or an amino acid sequence having at least 80% identity to SEQ ID NO: 18;

[0008] HCDR2 comprises the sequence shown in SEQ ID NO: 19 or 26, or an amino acid sequence having at least 80% identity to SEQ ID NO: 19 or 26;

[0009] HCDR3 comprises the sequence shown in SEQ ID NO: 20, or an amino acid sequence having at least 80% identity to SEQ ID NO: 20.

[0010] According to an embodiment of the present application, the antibody comprises:

[0011] HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO: 18, 19 and 20, respectively, or an amino acid sequence having at least 80% identity to SEQ ID NO: 18, 19 and 20, respectively; or

[0012] HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO: 18, 26 and 20, respectively, or an amino acid sequence having at least 80% identity to SEQ ID NO: 18, 26 and 20, respectively.

[0013] Preferably, the antibody comprises HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NO: 18, 26 and 20, respectively, or an amino acid sequence having at least 80% identity to SEQ ID NO: 18, 26 and 20, respectively.

[0014] According to an embodiment of the present application, the antibody comprises a heavy chain variable region of the amino acid sequence as shown in SEQ ID NO: 21 or 23, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 21 or 23.

[0015] Preferably, the antibody comprises a heavy chain variable region of the amino acid sequence as shown in SEQ ID NO: 23.

[0016] In a second aspect of the present application, the present application provides a fusion protein of an anti-Ang-2 single-domain antibody, comprising the aforementioned anti-Ang-2 single-domain antibody and an Fc fragment of IgG.

[0017] According to an embodiment of the present application, the Fc fragment comprises the amino acid sequence as shown in SEQ ID NO: 13, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence as shown in SEQ ID NO: 13.

[0018] According to an embodiment of the present application, the fusion protein of the anti-Ang-2 antibody comprises the amino acid sequence as shown in SEQ ID NO: 22 or 29. Preferably, the fusion protein of the anti-Ang-2 antibody comprises the amino acid sequence as shown in SEQ ID NO: 29. The inventors have found through experiments that the anti-Ang-2 fusion protein protected by the present application has better blocking efficacy on Tie2 and ang-2.

[0019] In a third aspect of the present application, the present application provides a multispecific antibody, comprising: a first binding region and a second binding region,

[0020] the first binding region comprises a single-domain antibody or an antigen-binding fragment thereof that specifically binds to VEGF, and the second binding region comprises an antibody or an antigen-binding fragment thereof that specifically binds to Ang-2;

[0021] The multispecific antibody further comprises a VEGF receptor or an extracellular domain fragment thereof that specifically binds to VEGF-A.

[0022] According to an embodiment of the present application, the single-domain antibody or the antigen-binding fragment thereof that specifically binds to VEGF is targeted to VEGF-C or VEGF-D.

[0023] According to an embodiment of the present application, the multispecific antibody further comprises at least one connecting peptide.

[0024] According to embodiments of the present application, the multispecific antibody comprises a structure as shown in Formula Ia or Ib from N-terminus to C-terminus:

[0025] P1-L1-P3-L2-P4-L3-P2(Ia);

[0026] P2-L3-P4-L2-P3-L1-P1(Ib);

[0027] wherein,

[0028] P1 is an anti-VEGF single domain antibody,

[0029] P2 is an anti-Ang-2 single domain antibody,

[0030] P3 is an Fc fragment of IgG,

[0031] P4 is a VEGF receptor or an extracellular domain thereof that binds VEGF-A,

[0032] L1, L2 and L3 are each independently a linker or a peptide.

[0033] Preferably, the multispecific antibody has a structure as shown in Formula Ib from N-terminus to C-terminus.

[0034] According to embodiments of the present application, the anti-Ang-2 single domain antibody is any of the foregoing anti-Ang-2 single domain antibodies.

[0035] According to embodiments of the present application, the anti-VEGF single domain antibody heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, wherein:

[0036] HCDR1 comprises a sequence as shown in SEQ ID NO: 1, 4 or 7, or an amino acid sequence having at least 80% identity to SEQ ID NO: 1, 4 or 7;

[0037] HCDR2 comprises a sequence as shown in SEQ ID NO: 2, 5, 8 or 25, or an amino acid sequence having at least 80% identity to SEQ ID NO: 2, 5, 8 or 25;

[0038] HCDR3 comprises a sequence as shown in SEQ ID NO: 3, 6 or 9, or an amino acid sequence having at least 80% identity to SEQ ID NO: 3, 6 or 9.

[0039] According to embodiments of the present application, the anti-VEGF single domain antibody comprises:

[0040] HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences having at least 80% identity to SEQ ID NOs: 1, 2 and 3; or

[0041] HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences having at least 80% identity to SEQ ID NOs: 4, 5 and 6; or

[0042] HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NOs: 7, 8 and 9, respectively, or amino acid sequences having at least 80% identity to SEQ ID NOs: 7, 8 and 9; or

[0043] HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NOs: 7, 25 and 9, respectively, or amino acid sequences having at least 80% identity to SEQ ID NOs: 7, 25 and 9.

[0044] According to embodiments of the present application, the anti-VEGF single-domain antibody comprises a heavy chain variable region as set forth in SEQ ID NOs: 10-12 or SEQ ID NO: 17, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NOs: 10-12 or SEQ ID NO: 17;

[0045] Optionally, the antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 17, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0046] According to embodiments of the present application, the Fc fragment of IgG is an IgG1 Fc or a variant fragment thereof; optionally, the Fc fragment of IgG comprises an amino acid sequence as set forth in SEQ ID NO: 13, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 13. Optionally, the Fc fragment of IgG comprises an amino acid sequence as set forth in SEQ ID NO: 30, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO: 30.

[0047] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 30)

[0048] According to embodiments of the present application, the VEGF receptor or the extracellular domain thereof comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 27.

[0049] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNAT YKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEY PSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHE (SEQ ID NO: 27)

[0050] According to embodiments of the present application, the connecting peptide is a GS connecting peptide, and the sequence of the connecting peptide is (G4S)n, wherein n is 1, 2, 3, 4, 5, or 6, and optionally, n is 3.

[0051] According to embodiments of the present application, the multispecific antibody comprises, from N-terminus to C-terminus, an anti-Ang-2 single-domain antibody, a GS connecting peptide, a VEGF extracellular domain, a Fc fragment of IgG, a GS connecting peptide, and an anti-VEGF single-domain antibody.

[0052] According to embodiments of the present application, the multispecific antibody comprises the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 24.

[0053] QVQLLESGGGLVQPGGSLRLSCAASGFTFSSFAMSWVRQAPGKGLEWVSDINPEGDNTYYADSVKGR

[0054] FTISRDNSKNTVYLQMNSLRAEDTAVYYCTMGPGYWGQGTLVTVSSGGGGSGGGGSGGGGSSDTGRP

[0055] FVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCE

[0056] ATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKK

[0057] LVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK

[0058] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSQVQLLESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGKGREGVAYISSGEGSTYYADSVKGRFTISSDNSKNTVYLQMNSLRAEDTAVYYCAAGLSVVLRKLWAYSFGYDYWGQGTLVTVSS (SEQ ID NO: 24)

[0059] According to an embodiment of the present application, the multispecific antibody is a symmetric structure and comprises two identical peptide chains.

[0060] In a fourth aspect, the present application provides a nucleic acid molecule. According to embodiments of the present application, the nucleic acid molecule comprises the single-domain antibody against Ang-2 according to the first aspect of the present application, the fusion protein of the single-domain antibody against Ang-2 according to the second aspect of the present application, or the multispecific antibody according to the third aspect of the present application.

[0061] According to embodiments of the present application, the nucleic acid molecule can further comprise the following additional technical features:

[0062] According to embodiments of the present application, the nucleic acid molecule is DNA.

[0063] In a fifth aspect, the present application provides an expression vector. According to embodiments of the present application, the expression vector carries the nucleic acid molecule according to the sixth aspect of the present application.

[0064] In a sixth aspect, the present application provides a recombinant cell. According to embodiments of the present application, the recombinant cell carries the nucleic acid molecule according to the sixth aspect of the present application, or the expression vector according to the seventh aspect of the present application.

[0065] In a seventh aspect, the present application provides an immunoconjugate. The immunoconjugate comprises the single-domain antibody against Ang-2 according to the first aspect of the present application, the fusion protein of the single-domain antibody against Ang-2 according to the second aspect of the present application, the multispecific antibody according to the third aspect of the present application, the nucleic acid molecule according to the fourth aspect of the present application, the expression vector according to the fifth aspect of the present application, or the recombinant cell according to the sixth aspect of the present application.

[0066] In an eighth aspect, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition comprises the single-domain antibody against Ang-2 according to the first aspect of the present application, the fusion protein of the single-domain antibody against Ang-2 according to the second aspect of the present application, the multispecific antibody according to the third aspect of the present application, the nucleic acid molecule according to the fourth aspect of the present application, the expression vector according to the fifth aspect of the present application, the recombinant cell according to the sixth aspect of the present application, or the immunoconjugate according to the seventh aspect of the present application.

[0067] In a ninth aspect, the present application provides use of the aforementioned single-domain antibody, fusion protein, multispecific antibody, nucleic acid molecule, expression vector, recombinant cell, immunoconjugate, and / or pharmaceutical composition in the manufacture of a medicament for treating or preventing an ocular disease associated with angiogenesis. According to embodiments of the use of the present application, the medicament is used for treating choroidal neovascularization (CNV).

[0068] In the tenth aspect of the present application, the present application provides a detection kit, which comprises the single-domain antibody, the fusion protein or the multispecific antibody as described above. The kit of the single-domain antibody against VEGF described above can specifically target and bind to VEGF-C / D, so as to specifically detect VEGF. When the antibody is combined with a fluorescent group, the positioning or real-time detection of VEGF can be realized by using a fluorescence detection device. When the antibody is combined with a label such as biotin, the qualitative or quantitative detection of VEGF can be realized by developing color by using a color developing reagent. The antibody can also be combined with an anti-antibody, so as to realize the sandwich or double-sandwich method, and then realize the signal amplification step by step, so as to detect VEGF. The kit of the single-domain antibody against Ang-2 described above can specifically target and bind to Ang-2, so as to specifically detect Ang-2. When the antibody is combined with a fluorescent group, the positioning or real-time detection of Ang-2 can be realized by using a fluorescence detection device. When the antibody is combined with a label such as biotin, the qualitative or quantitative detection of Ang-2 can be realized by developing color by using a color developing reagent. The antibody can also be combined with an anti-antibody, so as to realize the sandwich or double-sandwich method, and then realize the signal amplification step by step, so as to detect Ang-2. The kit of the multispecific antibody against VEGF / Ang-2 described above can specifically target and bind to VEGF-A / C / D and Ang-2, so as to specifically detect VEGF or / and Ang-2. When the antibody is combined with a fluorescent group, the positioning or real-time detection of VEGF or / and Ang-2 can be realized by using a fluorescence detection device. When the antibody is combined with a label such as biotin, the qualitative or quantitative detection of VEGF or / and Ang-2 can be realized by developing color by using a color developing reagent. The antibody can also be combined with an anti-antibody, so as to realize the sandwich or double-sandwich method, and then realize the signal amplification step by step, so as to detect VEGF or / and Ang-2.

[0069] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0070] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the accompanying drawings, wherein:

[0071] Figure 1 For the blocking ELISA function test of VEGF-C and VEGF-R2 of the crude extracts of 39 cloned single-domain antibodies, there are two single-domain antibody crude extract concentrations (1x and 1:3);

[0072] Figure 2 For the blocking ELISA detection results of VEGF-C and VEGF-R2 of the expressed and purified antibodies;

[0073] Figure 3 VEGF-C and VEGF-R2 blocking ELISA results for humanized and sequence engineered antibody VC-Ab3-hz;

[0074] Figure 4 VEGF-C and VEGF-R3 blocking ELISA results for antibody;

[0075] Figure 5 VEGF-D and VEGF-R2 blocking ELISA results for antibody;

[0076] Figure 6 VEGF-D and VEGF-R3 blocking ELISA results for antibody;

[0077] Figure 7 VEGF-C binding ELISA results for antibody;

[0078] Figure 8 VEGF-D binding ELISA results for antibody;

[0079] Figure 9 VEGF-C induced HUVEC cell proliferation inhibition potency results for antibody;

[0080] Figure 10 Ang-2 and Tie2 blocking ELISA results for antibody;

[0081] Figure 11 Amino acid sequence alignment of antibody AN-Ab1-hz variable region and human VH3-23*01 germline gene, CDR regions are boxed, amino acid identical sites between two sequences are indicated by shading;

[0082] Figure 12 Structure of multi-specific antibody fusion protein RYYM002 targeting VEGF-A, VEGF-C / D and Ang-2;

[0083] Figure 13 VEGF-A165-VEGF-R2 mediated intracellular signaling pathway blocking potency results for antibody based on reporter cell assay;

[0084] Figure 14 VEGF-C-VEGF-R2 mediated intracellular signaling pathway blocking potency results for antibody based on reporter cell assay;

[0085] Figure 15The results represent the antibody's blocking efficacy against the VEGF-D-VEGF-R2-mediated intracellular signaling pathway, measured using reporter gene-based cells.

[0086] Figure 16 The results show the inhibitory efficacy of antibodies such as RYYM002 against VEGF-C-induced HUVEC cell proliferation.

[0087] Figure 17 The results show the inhibitory efficacy of the antibody against ang-2-induced intracellular Tie2 phosphorylation.

[0088] Figure 18 In the figure, A represents the statistical results of the spot leakage score of the CNV model of the monkey fundus after antibody treatment, and each data point represents a spot. Figure 18 In the figure, B represents the statistical result of the corresponding light spot leakage area, and each data point represents a light spot. Detailed Implementation

[0089] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0090] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0091] Single-domain antibodies, antibodies

[0092] In this article, the terms "singledomain antibody," "VHH," and "nanobody" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of the antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain. It is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain.

[0093] In the present disclosure, the terms "a single-domain antibody of the present disclosure capable of specifically recognizing VEGF", "a VEGF single-domain antibody of the present disclosure", "an anti-VEGF single-domain antibody", "a VEGF single-domain antibody" have the same meaning and are used interchangeably, and refer to a single-domain antibody that specifically recognizes and binds to VEGF. In some embodiments, a VEGF single-domain antibody of the present disclosure comprises HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NOs: 1, 2 and 3, respectively, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 1, 2 and 3, respectively; or HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NOs: 4, 5 and 6, respectively, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 4, 5 and 6, respectively; or HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NOs: 7, 8 and 9, respectively, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 7, 8 and 9, respectively; or HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NOs: 7, 25 and 9, respectively, or an amino acid sequence having at least 80% identity to SEQ ID NOs: 7, 25 and 9, respectively. In some embodiments, a VEGF single-domain antibody of the present disclosure comprises or has a heavy chain variable region of an amino acid sequence as set forth in SEQ ID NOs: 10-12 or SEQ ID NO: 17, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NOs: 10-12 or SEQ ID NO: 17. In some preferred embodiments, a VEGF single-domain antibody of the present disclosure comprises or has an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 17.

[0094] In the present disclosure, the terms "the Ang-2 binding single domain antibody of the present disclosure", "the Ang-2 binding single domain antibody of the present disclosure", "the anti-Ang-2 single domain antibody", "the Ang-2 binding single domain antibody" have the same meaning and are used interchangeably, and refer to a single domain antibody that specifically recognizes and binds to Ang-2. In some embodiments, the Ang-2 binding single domain antibody of the present disclosure comprises HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NOs: 18, 19 and 20, respectively, or amino acid sequences having at least 80% identity to SEQ ID NOs: 18, 19 and 20, respectively; or HCDR1, HCDR2 and HCDR3 sequences as set forth in SEQ ID NOs: 18, 26 and 20, respectively, or amino acid sequences having at least 80% identity to SEQ ID NOs: 18, 26 and 20, respectively. In some embodiments, the Ang-2 binding single domain antibody of the present disclosure comprises a heavy chain variable region as set forth in SEQ ID NO: 21 or 23, or an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 21 or 23.

[0095] In the present disclosure, the term "antibody" refers to an immunoglobulin molecule capable of binding to a specific antigen. It comprises two light chains with smaller molecular weight and two heavy chains with larger molecular weight, and the heavy chain (H chain) and the light chain (L chain) are connected by a disulfide bond to form a four-chain polypeptide chain molecule. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, which is called the variable region (V region), and the carboxyl-terminal (C-terminal) is relatively stable and varies little, which is called the constant region (C region). The V regions of L chain and H chain are called VL and VH, respectively.

[0096] In the present disclosure, the term "variable" means that some parts of the variable region in the antibody differ in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light chain and heavy chain variable region called the complementarity determining region (CDR) or hypervariable region. The more conserved part of the variable region is called the framework region (FR). The variable region of the natural heavy chain and light chain each contains four FR regions, which are roughly in a b-pleated sheet conformation, connected by three CDRs forming a connecting loop, which in some cases can form a partial b-pleated structure. The CDRs in each chain are closely held together by the FR regions and, together with the CDRs of the other chain, form the antigen binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but it exhibits different effector functions, such as participating in antibody-dependent cellular cytotoxicity.

[0097] In certain regions of the variable region, the amino acid composition and arrangement order have higher degree of variation, which is called hypervariable region (HVR). The HVR is the position of antigen and antibody binding, and is also called complementarity-determining region (CDR). There are three CDR regions in the heavy chain variable region and the light chain variable region. The amino acid sequence of any one of the single domain antibody, antibody or fusion protein, multispecific antibody of the present application can have conservative amino acid substitution. Of course, these conservative amino acid substitutions do not change the biological function of the antibody or antigen binding fragment, and are within the protection scope of the present application. In some specific modes, these conservative amino acid substitutions can occur in the amino acids in the heavy chain variable region and the light chain variable region except the CDR region.

[0098] The naming of the CDR sequence of the present application is determined: the CDR in the single domain antibody, antibody or multispecific antibody of the present application is determined according to AbM rule. The CDR of the variable region of the same antibody may

[0099] Herein, the terms "VEGF antibody", "anti-VEGF antibody", "fusion protein of anti-VEGF single domain antibody" are used interchangeably, and can be understood as comprising or consisting of the Fc fragment of IgG and the anti-VEGF single domain antibody of the present application. In some embodiments of the present application, the term "VEGF antibody", "anti-VEGF antibody", "fusion protein of anti-VEGF single domain antibody" is a VEGF-C / D targeting antibody. In some embodiments, the fusion protein of anti-VEGF single domain antibody of the present application comprises the amino acid sequence as shown in any one of SEQ ID NO: 14-16 or SEQ ID NO: 28.

[0100] Herein, the terms "Ang-2 antibody", "anti-Ang-2 antibody", "fusion protein of anti-Ang-2 antibody" are used interchangeably, and can be understood as comprising or consisting of the Fc fragment of IgG and the anti-Ang-2 single domain antibody of the present application. In some embodiments, the fusion protein of anti-Ang-2 antibody of the present application comprises the amino acid sequence as shown in SEQ ID NO: 22 or 29.

[0101] In the present application, the term "bispecific antibody" "bispecific fusion protein" "multispecific fusion protein" or "multispecific antibody" includes both an antibody or antigen-binding fragment thereof that specifically binds to VEGF and a single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2. In some embodiments of the present application, the antibody that specifically binds to VEGF includes both a single-domain antibody that specifically binds to VEGF and a VEGF receptor or extracellular domain fragment thereof that specifically binds to VEGF-A. The single-domain antibody or antigen-binding fragment thereof that specifically binds to VEGF is targeted to VEGF-C or VEGF-D. The "bispecific antibody" or multispecific antibody includes one or more linker peptides.

[0102] The "VC-Ab1-Fc-containing fusion protein" "VC-Ab2-Fc-containing fusion protein" "VC-Ab3-Fc-containing fusion protein" of the present application refers to an antibody or fusion protein formed by a different single-domain antibody targeting VEGF-C and Fc.

[0103] The "VC-Ab3-hz antibody" of the present application refers to an antibody or fusion protein formed by a humanized single-domain antibody targeting VEGF-C and Fc.

[0104] In the present application, the term "at least 80% identity" means that a sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each of the corresponding reference sequences, which include amino acid sequences, nucleic acid sequences, fusion sequences of DNA and RNA, or fusion sequences of amino acids and nucleic acids.

[0105] In the present application, the term "at least 90% identity" means that a sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity to each of the corresponding reference sequences, which include amino acid sequences, nucleic acid sequences, fusion sequences of DNA and RNA, or fusion sequences of amino acids and nucleic acids.

[0106] Nucleic acid molecules, expression vectors, recombinant cells

[0107] In the process of preparing or obtaining these antibodies or fusion proteins, nucleic acid molecules expressing these single-domain antibodies, antibodies or fusion proteins, multispecific antibodies can be used, which are linked to different vectors and then expressed in different cells to obtain the corresponding antibodies or fusion proteins.

[0108] To this end, the present application also provides an isolated nucleic acid molecule encoding the above-mentioned antibody or fusion protein.

[0109] In some preferred embodiments, the nucleic acid molecule is species-optimized for increased expression in mammalian cells.

[0110] The present application also provides an expression vector comprising the above-mentioned isolated nucleic acid molecule. In linking the above-mentioned isolated polynucleotide to a vector, the polynucleotide can be linked to the control elements on the vector directly or indirectly, as long as the control elements can control the translation and expression of the polynucleotide. Of course, the control elements can be directly from the vector itself, or can be exogenous, i.e. not from the vector itself. Of course, the polynucleotide is operably linked to the control elements. "Operably linked" herein means that the exogenous gene is linked to the vector, so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can exert their expected functions of regulating the transcription and translation of the exogenous gene. Of course, the polynucleotides encoding the heavy and light chains of the antibody can be inserted into different vectors independently, or commonly inserted into the same vector. Commonly used vectors can be plasmids, bacteriophages, etc.

[0111] The present application also provides a recombinant cell comprising the expression vector. The expression vector can be introduced into mammalian cells to construct a recombinant cell, and then the recombinant cell is used to express the antibody or fusion protein provided by the present application. The corresponding antibody can be obtained by culturing the recombinant cell.

[0112] Pharmaceutical compositions, kits and pharmaceutical uses

[0113] The present application also provides a pharmaceutical composition comprising the above-mentioned single-domain antibody, antibody or fusion protein, multispecific antibody of VEGF and / or ANG-2, and a pharmaceutically acceptable carrier, and can further comprise the above-mentioned immune cells, nucleic acid molecules, expression vectors, and recombinant cells.

[0114] The single-domain antibody or fusion protein provided herein can be incorporated into a pharmaceutical composition suitable for administration to a subject. Typically, these pharmaceutical compositions include the antibodies provided herein.

[0115] In some embodiments, these pharmaceutical compositions further include a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, binder, disintegrant, or other liquid excipient, dispersant, flavoring agent or suspending agent, surface active agent, isotonic agent, thickening agent, emulsifying agent, preservative, solid binder, glidant or lubricant, etc., suitable for the particular target dosage form. Except insofar as any conventional excipient is incompatible with the compounds of the present application, such as by producing any adverse biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this application.

[0116] For example, the antibodies of the present application can be incorporated into pharmaceutical compositions suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms. For example, liquid, semi-solid, and solid dosage forms, etc., including but not limited to liquid solutions (e.g., injectable solutions and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injectable solutions or infusible solutions. The antibodies can be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

[0117] Of course, the single-domain antibodies, antibodies and fusion proteins herein can also be made into kits or parts of other diagnostic reagents as needed. According to embodiments of the present application, the present application also provides a kit comprising the above-mentioned ANG-2 antibody or VEGF antibody or multispecific antibody. The kit provided by the present application can be used, for example, in a kit for immunoblotting, immunoprecipitation, etc. involving the use of ANG-2 or VEGF antigen and antibody specific binding performance for detection, etc. These kits can contain any one or more of the following: antagonists, ANG-2 antibodies or VEGF antibodies or multispecific antibodies or pharmaceutical reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. ANG-2 antibodies or VEGF antibodies or multispecific antibodies can be used for different types of diagnostic tests, such as in vitro or in vivo detection of various diseases or the presence of drugs, toxins or other proteins, etc. For example, the presence of the disease can be tested by detecting the serum or blood of the subject. For example, cancer or tumor, which can be any unregulated cell growth.

[0118] In some embodiments, the ANG-2 antibody or VEGF antibody or multispecific antibody can be used in combination with any detection reagent or therapeutic preparation, such as in combination with diagnostic nuclides, nanomaterials, etc. to detect the target site by the radioactivity of the nuclide, and then obtain information about the target site; it can also be used in combination with therapeutic nuclides to specifically kill target cells, tissues, etc. by the radioactivity of the nuclide.

[0119] In the diagnosis or treatment or prevention of the above-mentioned diseases using the ANG-2 antibody or VEGF antibody or multispecific antibody provided by the present application, the ANG-2 antibody or VEGF antibody or multispecific antibody provided by the present application can be provided to the subject. For this purpose, the present application provides a method for treating the above-mentioned diseases, comprising administering the antibody or antigen-binding fragment thereof provided by the present application to a subject in need thereof.

[0120] As used herein, the terms "treatment" and "prevention" and words stemming therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill will recognize as having some potential benefit or therapeutic effect. In this respect, the methods of the present application can provide any amount of any level of treatment or prevention of cancer in a mammal. Moreover, the treatment or prevention provided by the methods of the present application can include the treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Additionally, for purposes herein, "prevention" can encompass delaying the onset of a disease or a symptom or condition thereof.

[0121] In the present context, the term "angiogenesis-related disease" generally refers to a disease caused by angiogenesis (or neovascularization), including but not limited to cancer, ophthalmic disease or inflammatory disease. Exemplarily, the cancer can be solid tumor or blood tumor, including but not limited to hemangioma, hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma and glioma; the ophthalmic disease can be intraocular neovascular syndrome (e.g., proliferative retinopathy, age-related macular degeneration); the inflammatory disease includes but is not limited to rheumatoid arthritis and psoriasis.

[0122] The present application is described in detail below with reference to specific examples, it being understood that these examples are merely illustrative of certain aspects of the application, which is not in any way limited to the specific embodiments presented. Unless otherwise indicated, the techniques or conditions described in the examples are carried out according to the techniques or conditions described in the literature or according to the manufacturer's instructions. Unless otherwise indicated, the reagents or instruments used are conventional products available commercially.

[0123] Example 1 Preparation and screening of single-domain antibody targeting VEGF-C

[0124] 1.1 Immunization of Alpaca

[0125] Four alpacas were immunized subcutaneously with recombinant human VEGF-C protein (purchased from Beijing BPS, item number VEC-H4225). A total of 8 immunizations were performed, with a dose of 0.5 mg per alpaca each time, and the interval between immunizations was 2 weeks. Complete adjuvant was used for the first immunization, and incomplete adjuvant was used for the rest.

[0126] One week after each immunization, 5 mL of peripheral blood was taken from the alpaca, and the serum was gradiently diluted and subjected to ELISA binding detection of VEGF-C coated on the plate to monitor the immunization titer.

[0127] 1.2 Construction of immunization library

[0128] One week after the 8th immunization, 50 mL of peripheral blood of the alpaca was collected, and lymphocytes were obtained by using lymphocyte separation medium (Solarbio, P8610), and then RNA extraction was performed according to the instructions of the RNA extraction reagent (TaKaRa, 9109). Then, the RNA was reversely transcribed using a reverse transcription kit (Thermo Scientific, K1622) to prepare cDNA. Then, the DNA containing the variable region gene of the single-domain antibody was amplified by using a nested PCR method, and was digested by Sfi I enzyme, and then was ligated with the Sfi I enzyme-digested display vector by using T4 DNA ligase. After purification of the ligation product, TG1 competent cells were electrotransformed, and were infected with M13KO7 helper phage to prepare a phage display library. The library capacity was 4.2 x 1011 cfu after the electrotransformed TG1 was counted by using plate gradient dilution. 9 .

[0129] 1.3 Screening of the phage library

[0130] The single-domain antibody targeting VEGF-C was screened by using solid-phase affinity screening method, and two rounds of screening were performed. The VEGF-C protein was coated on a high adsorption 96-well plate at 4°C overnight. Then, 3% bovine serum albumin (BSA) in PBS was added as blocking solution for blocking at 37°C for 1 hour. After removing the blocking solution, the phage (2 x 1011 cfu) of the above-mentioned immune library was added. After incubation at 37°C for 1 hour with shaking, 8 times of washing with PBS containing 0.1% Tween-20 (PBST washing solution) was performed, each time with shaking for 2 min, so as to remove the phage with low or no binding. Finally, 100 μL of glycine-hydrochloride buffer (pH 2.2) was added to dissociate the specific phage, and after slight shaking for 10 min, the eluate containing the specific phage was collected and was neutralized by using Tris solution. 12

[0131] ​Eluent was used to infect E. coli TG1 cells in the logarithmic growth phase (37℃, 20 min). A small amount of infected E. coli TG1 cells was serially diluted, and an appropriate amount of the serial dilution was evenly spread on LB solid medium (plate dilution method). After 12-14 hours, the plates were counted to calculate the eluted phages. The remaining infected TG1 cells were re-infected with M13KO7 helper phage and cultured overnight to amplify an enriched phage library containing specific phages for the next round of screening. 400 clones were randomly picked from each of the plates used in the first and second rounds of elution titer determination and transferred to 96-well plates. 2YT medium containing Carb and M13KO7 helper phages was added and cultured overnight. The next day, the plates were centrifuged at 3000 rpm, and the supernatant was used for phage ELISA identification. The method was as follows: Wells coated with recombinant human VEGF-C protein (coating concentration 2 μg / mL) were used as detection wells, while wells coated with an antigen protein of an unrelated target were used as background control wells. After blocking with PBS containing 3% bovine serum albumin, phage supernatant of the clones to be identified was added in parallel. The mixture was incubated at room temperature with shaking for 1 hour. After washing, anti-M13 antibody conjugated with HRP (horseradish peroxidase) (Sinochem, catalog number 11973-MM05T-H) was added, and the mixture was incubated at room temperature with shaking for 1 hour. After washing, TMB substrate solution was added for color development, and the OD values ​​were read using a microplate reader. 450nm Detection well OD 450 The value is more than 10 times greater than that of the control well, and the OD of the control well is... 450 A reading < 0.1 is considered positive. The clones corresponding to the positive wells are then preserved, plasmids are extracted, and genes are sequenced. The sequenced DNA sequences are translated into amino acid sequences and compared. Repetitive sequences or similar sequences with CDRs differing by no more than 3 amino acids are removed to obtain unique sequences. A total of 39 unique sequences were obtained (clones 1 to 39).

[0132] 1.4 Detection based on single-domain antibody periplasmic extracts

[0133] 1.4.1 Preparation of crude single-domain antibody extract

[0134] Using a bacterial expression system, crude single-domain antibody extracts were prepared from the unique sequence clones obtained in Example 1.3. The steps are as follows:

[0135] (1) Inoculate the TG1 bacterial culture corresponding to the unique sequence into 2 mL of 2×YT medium containing Carb and 0.5% glucose, and incubate at 37℃ and 220 rpm;

[0136] (2) Detect the optical density value of the culture medium. When OD 600When the OD600 reached 0.6, 3 mL of 2xYT medium containing IPTG (final concentration of 0.1 mM) and Carb was added, and the culture was incubated at 28°C, 200 rpm overnight;

[0137] (3) On the second day, the bacterial solution was centrifuged to collect the bacterial cells, which were resuspended in 1 mL of PBS and subjected to multiple freeze-thaw cycles to release the single-domain antibodies expressed in the periplasmic cavity;

[0138] (4) The bacterial cells were centrifuged (12000 rpm, 4°C, 10 minutes), and the supernatant was collected and filtered to prepare the crude extract of the single-domain antibodies in the periplasmic cavity. Meanwhile, a clone expressing single-domain antibodies that bind to an antigen unrelated to the target was inoculated and subjected to the same operation to prepare a negative control crude extract.

[0139] 1.4.2 Detection of blocking activity of single-domain antibodies

[0140] The crude extract prepared in 1.4.1 was used for blocking ELISA detection of human VEGF-R2-VEGFC, and the specific steps were as follows:

[0141] (1) VEGF-R2-Fc (Peprotech, product number VE2-H5255) was coated on a 96-well plate (300 ng per well), and the coating was performed at 4°C overnight;

[0142] (2) 3% BSA (bovine serum albumin) was used for blocking at room temperature for 2 hours. During this period, the single-domain antibody crude extract was co-incubated with Fc-VEGFC-avi (expressed and produced in the laboratory) at a final concentration of 100 ng / mL, and the single-domain antibody was set at a stock solution (1x) and a 1:3 concentration point. OPT-302 (produced in the laboratory, sequence reference patent CN106414487A) at a final concentration of 100 nM was co-incubated with Fc-VEGFC-avi as a positive control. After co-incubation for 30 minutes, 100 μL of the incubation solution was transferred to the blocked well plate;

[0143] (3) After shaking incubation at room temperature for 1 hour, 0.05% Tween-20-containing PBS (PBST washing solution) was used for washing 4 times, and 1:5000 streptavidin-HRP (Peprotech, STN-NH913) was added;

[0144] (4) After shaking incubation at room temperature for 20 minutes, PBST was used for washing 4 times, TMB substrate was added, and color development was performed at room temperature for 1 minute, and color development was terminated using 1M hydrochloric acid solution;

[0145] (5) The absorbance value at 450 nm was read using an enzyme-labeled instrument.

[0146] The results are as follows:Figure 1 As shown, among the 39 unique sequence clones, 3 clones (samples No. 18, 24 and 30) had clear blocking effect on VEGF-R2-VEGFC.

[0147] In addition, the crude extracts of the 39 unique sequence single-domain antibodies were subjected to binding ELISA detection on VEGF-C coated on the plate, and the results showed that 37 of the clones had strong binding to VEGF-C, while not binding to the irrelevant target antigen coated in parallel.

[0148] Example 2 Expression and functional detection of single-domain antibody-Fc fusion protein targeting VEGF-C

[0149] Using the 3 single-domain antibody sequences with preliminary blocking function obtained in 1.4.2, a pCDNA3.1 recombinant vector containing the nucleic acid sequence of the single-domain antibody and human IgG1 Fc (the C-terminus of the VHH is directly connected to the N-terminus of the human Fc hinge region) was synthesized, and the sequence of the human Fc region used and the sequence of the single-domain antibody-Fc fusion protein constructed (including the sequence of the single-domain antibody variable region and the sequence of the CDR region) are shown in Table 1. After amplification of the constructed recombinant vector, it was transfected into Expi293 cells for expression of the single-domain antibody-Fc fusion protein. The specific steps are as follows: 20 μg of plasmid was mixed with PEI (polyethyleneimine) and incubated at room temperature for 30 minutes, then added to a 1 x 10 6 The cells were incubated in a 37°C, 5% CO2 cell incubator on a 125 rpm orbital shaker. On the 7th day after transfection, the supernatant was harvested, purified using Protein A, and the buffer was replaced with PBS. The expressed single-domain antibody-Fc fusion proteins were named VC-Ab1, VC-Ab2 and VC-Ab3.

[0150] Table 1. CDR region sequence and variable region sequence of single-domain antibody targeting VEGF-C and fusion protein sequence with Fc (CDR region is divided by AbM method)

[0151]

[0152]

[0153] Then, the blocking function of the 3 antibodies on VEGF-C and VEGF-R2 was detected using the blocking ELISA system described in Example 1.4.2, and the initial final concentration of the antibodies used was 10 μg / mL, diluted at a gradient of 1:3. The results are shown in Figure 2 As shown, the 3 antibodies all had clear blocking function on the binding of VEGF-C and VEGF-R2, among which the blocking efficacy of VC-Ab3 was the best, slightly better than the control antibody OPT-302.

[0154] Example 3 CDR engineering, humanization and expression-based sequence improvement of the VEGF-C targeting antibody

[0155] 3.1 Engineering of post-translational modification (PTM) sites

[0156] The CDR sequences of VC-Ab3 were analyzed (CDR region division was performed using the AbM method), and it was found that there was an isomerization-prone amino acid pair "DG" in CDR2. The "DG" was mutated to "EG", i.e., the optimized amino acid sequence of VC-Ab3-CDR2 is shown in SEQ ID NO: 25.

[0157] 3.2 Humanization design

[0158] The structure of the single-domain antibody VC-Ab3 was simulated using SWISS-MODEL, and based on the obtained structure, the camel-derived amino acid sites in the framework region were evaluated for humanization mutation. The human germline genes V3-23*01 and J1*01 were used as the target sequences for humanization mutation, and the amino acid sites located on the surface of the antibody structure and not adjacent to the CDR were preferentially mutated, and the sites located in the interior of the antibody structure and adjacent to the CDR were mutated later. According to the above principles, the final humanized antibody sequence was designed.

[0159] 3.3 Expression-based sequence improvement

[0160] The transient transfection expression level of antibody VC-Ab3 in HEK293 was at the level of 100 mg / L, and in order to further improve the expression level, 2 amino acid sites in the framework region that were prone to affect the expression level were empirically engineered in order to improve the expression level and drugability.

[0161] Based on the above design, the final antibody VC-Ab3-hz was obtained, the variable region sequence of which is shown in Table 2, and the Fc region sequence still uses SEQ ID NO: 13. Transient transfection and expression purification in HEK293 were performed, and the yield of protein A one-step purification was measured to be 320 mg / L, and the SEC single peak purity (280 nm) was 98%.

[0162] Table 2. Variable region and antibody sequence of VC-Ab3-hz

[0163]

[0164] Example 4 Functional characterization of the VEGF-C targeting antibody

[0165] 4.1 Blocking ELISA study of VEGF-C binding to VEGF-R2

[0166] The blocking efficacy of VC-Ab3-hz on VEGF-C binding to VEGF-R2 was tested using the blocking ELISA system described in 1.4.2, and the results are shown in Figure 3 VC-Ab3-hz antibody has a clear blocking effect, slightly better than the control antibody OPT-302.

[0167] 4.2 Blocking ELISA study of VEGF-C binding to VEGF-R3

[0168] Human VEGF-R3-Fc (Beijing BPS, FL4-H5251) was coated on the ELISA plate at 300 ng / well, and the gradient-diluted antibody was co-incubated with Fc-VEGFC-avi at a final concentration of 100 ng / mL for 30 min before being transferred to the coated, blocked and washed plate. After 1 h of incubation at room temperature, Streptavidin-HRP secondary antibody was added, and after incubation and washing, color development and reading were performed. As shown in Figure 4 The antibody VC-Ab3-hz has good blocking efficacy on VEGF-C binding to VEGF-R3, better than the control antibody (EC50 values are VC-Ab3-hz: 0.197 μg / mL and OPT-302: 1.239 μg / mL, respectively).

[0169] 4.3 Blocking ELISA study of VEGF-D binding to VEGF-R2

[0170] Human mature VEGF-C and VEGF-D molecules have a closer total sequence length and sequence similarity in the middle of the molecule involved in binding to VEGF receptors compared to VEGF-A, and both VEGF-C and VEGF-D can bind to VEGF-R2 and VEGF-R3, two receptors, with similar binding profiles. During the characterization process, the inventors of the present patent detected the blocking of VEGF-D binding to VEGF-R2 by antibody VC-Ab3-hz, i.e. VEGF-R2-Fc was coated on the ELISA plate at 300 ng / well, and the gradient-diluted antibody was co-incubated with VEGF-D-His (Beijing BPS, VED-H5228) at a final concentration of 5 μg / mL for 30 min before being transferred to the coated, blocked and washed plate. After 1 h of incubation at room temperature, anti-polyhistidine-HRP secondary antibody (Beijing Yiqiao, 105327-MM02T-H) was added, and after incubation and washing, color development and reading were performed. The results are shown in Figure 5As shown, the inventors unexpectedly found that antibody VC-Ab3-hz also has clear blocking effect on VEGF-D, while the other two antibodies of the application, VC-Ab1, VC-Ab2, and another control antibody IBI-333 (sequence reference patent WO2023016516) have no blocking effect on VEGF-D. Studies have shown (Zhou, H.; Zhao, X.; Yuan, M.; Chen, Y. Comparison of cytokine levels in the aqueous humor of polypoidal choroidal vasculopathy and neovascular age-related macular degeneration patients. BMC Ophthalmol. 2020, 20, 15) that in the aqueous humor of patients with nAMD (neovascular age-related macular degeneration) and PCV (polypoidal choroidal vasculopathy), the level of VEGF-D is as high as VEGF-A compared with healthy eyes (P values are all less than 0.0001), and it is noted that OPT-302, which is derived from the extracellular region of human VEGF-R3 protein, has been verified in I, II phase clinicals to show better efficacy than VEGF-A alone targeted therapy, which can also target VEGF-D in addition to targeting VEGF-C, so it is expected that simultaneous targeting of VEGF-D will be beneficial to the treatment of these ophthalmic diseases.

[0171] 4.4 Blocking ELISA study on VEGF-D binding to VEGF-R3

[0172] VEGF-R3-Fc (Beijing BPS, FL4-H5251) was coated on the ELISA plate, 300 ng / well, and gradient-diluted antibodies were co-incubated with VEGF-D-His (Beijing BPS, VED-H5228) at a final concentration of 5 μg / mL for 30 min, then transferred to the coated, blocked and washed plate, and incubated at room temperature for 1 h. Anti-polyhistidine-HRP secondary antibody (Beijing Yiqiaoshenzhou, 105327-MM02T-H) was added after incubation, washing, color development and reading. The results are shown in Figure 6 As shown, antibody VC-Ab3-hz can effectively block the binding of VEGF-D to VEGF-R3, with a blocking effect close to that of OPT-302.

[0173] 4.5 ELISA study of antibody binding to VEGF-C, VEGF-D

[0174] Human VEGF-C-His (Beijing BPS, VEC-H4225) or VEGF-D-His (R&D, 622-VD-025 / CF) was coated on ELISA plates at 200 ng / well, and the antibody was diluted in gradient and added to the blocked and washed plates, incubated at room temperature for 1 h, and then HRP-conjugated anti-human Fc secondary antibody (Beijing Biaoleibo, F030222) was added after washing. Color development and reading were performed after incubation and washing. The results of VEGF-C binding are shown in Figure 7 It can be seen that VC-Ab3-hz has better binding potency to VEGF-C than the two control antibodies. The results of VEGF-D binding are shown in Figure 8 It can be seen that VC-Ab3-hz has clear binding activity to VEGF-D, and the other two control antibodies IBI-333 and Aflibercept (sequence reference patent CN103349781A) do not bind to VEGF-D.

[0175] 4.6 Study of the inhibition potency of VEGF-C-induced HUVEC cell proliferation

[0176] In order to verify the blocking effect of the antibody on VEGF-C at the cellular function level, a VEGF-C-induced HUVEC cell proliferation model was used, and the specific operation was as follows:

[0177] (1) HUVEC cells were cultured in DMEM medium containing 10% fetal bovine serum, and after 2 passages and growth to 80% confluence, the medium was removed, washed once with PBS, then the cells were digested, centrifuged, resuspended with medium, and the cell density was adjusted to 20000 / mL, and then plated in a 96-well white wall cell culture plate at 100 μL / well, and incubated overnight in an incubator to adhere;

[0178] (2) VEGF-C (R&D, 9199-VC-025) was prepared in medium to a final concentration (working concentration) of 40 ng / mL, and co-incubated with the antibody diluted in gradient with medium for 1 h;

[0179] (3) Take the co-incubation solution in (2), add 50 μL / well to the cell culture plate, and incubate in a 37°C, 5% CO2-containing incubator for 72 h;

[0180] (4) Add 100 μL / well of live cell luminescence detection solution (Bi Yun Tian, C0065S) and check the luminescence signal according to the manufacturer's instructions.

[0181] The results are shown in Figure 9As shown, it can be seen that the antibody VC-Ab3-hz has a blocking effect on the HUVEC cell proliferation activity of VEGF-C, which is obviously superior to the control antibody OPT-302.

[0182] Example 5 Screening of single-domain antibodies targeting Ang-2 and functional confirmation

[0183] The llama was immunized with a recombinant human ang-2 protein (Beijing BPS, AN2-H5242), and the rest of the immunization method, construction of phage library, screening, and preparation method of single-domain antibody crude extract were the same as 1.1-1.4 in the present application, except that the target protein used was different.

[0184] The detection method for the blocking activity of the single-domain antibody crude extract is as follows: Tie2-Fc (Beijing BPS, TI2-H5255) is coated in an ELISA plate, 300 ng / well, the single-domain antibody crude extract is co-incubated with ang2-his-avi (Beijing BPS, AN2-H82E9) at a final concentration of 30 ng / mL, then transferred to the blocked and washed plate, incubated at room temperature for 1 h, after washing, Streptavidin-HRP secondary antibody is added, incubated and washed, and then developed and read. After functional screening at the crude extract level, a single-domain antibody showing blocking function for the binding of ang-2 to Tie2 was finally obtained, and the vector construction and transient transfection expression and purification of the single-domain antibody-Fc fusion protein were also carried out, and the obtained antibody was named AN-Ab1, and the related sequences are shown in Table 3 below.

[0185] Table 3. Related sequences of AN-Ab1

[0186]

[0187]

[0188] AN-Ab1 was subjected to PTM site modification (mutation of "DG" in CDR2 to "EG") and humanization design, and transient transfection and expression purification, and the designed antibody was named AN-Ab1-hz, and the variable region sequence thereof is shown in Table 4 below, and the transient transfection expression amount thereof was 329 mg / L, and the SEC single peak purity (280 nm) was 96.5%.

[0189] Table 4. Variable region and antibody sequences of AN-Ab1-hz

[0190]

[0191] The blocking efficiency of AN-Ab1-hz antibody on Tie2 and ang-2 was detected using the above blocking ELISA system, and the results are as follows Figure 10As shown, AN-Ab1-hz has good blocking potency, better than two marketed drugs Nesvacumab and Faricimab.

[0192] In addition, by comparing the sequence of AN-Ab1-hz, it is found that it is similar to human VH 3-23 *01 germline gene (e.g. Figure 11 As shown, in CDR1 (using AbM method to delineate CDR region), it only differs by 1 amino acid from CDR1 of VH3-23*01, and its framework region is similar to VH 3-23 *01 germline gene (e.g.

[0193] Example 6 Construction of multispecific antibody fusion protein targeting VEGF-A, VEGF-C / D and Ang-2

[0194] In order to construct a multispecific antibody / fusion protein that can simultaneously target VEGF-A / C / D and Ang-2, the single-domain antibody of VC-Ab3-hz targeting VEGF-C / D and AN-Ab1-hz targeting Ang-2 are constructed together with VEGF-A trap sequence (containing extracellular second domain derived from human VEGF-R1 and extracellular third domain derived from human VEGF-R2) and human Fc region sequence, and the molecule is named as RYYM002, the structure of which is shown as Figure 12 The sequence is shown in Table 5 below. Transient transfection in HEK293 and expression purification are performed on the molecule, and the yield of protein A one-step purification is measured to be 129 mg / L, and the SEC single peak purity (280 nm) is 94.8%.

[0195] Table 5. Sequence of RYYM002

[0196]

[0197] Example 7 Functional characterization of multispecific antibody fusion protein targeting VEGF-A, VEGF-C / D and Ang-2

[0198] 7.1 Blocking potency determination of antibody on VEGF-A 165 -mediated intracellular signaling pathway

[0199] The luciferase reporter gene cell line (VEGF-R2-NFAT-HEK293, Sanyou Bio, XHA007) downstream of the VEGF-R2 signaling pathway was used to detect the effect of antibodies such as RYYM002 on VEGF-A. 165 The inhibitory effect of the antibody on the intracellular signaling pathway of VEGF-R2 was demonstrated by serially diluting the antibody with a final concentration of 10 ng / mL of VEGF-A. 165 (R&D, 11458-VE-050) was mixed and incubated at room temperature for 30 minutes, then added to reporter cells at a density of 4E+04 cells per well. Cells were incubated at 37°C for 6 hours, followed by the addition of BrightLight (Novizan, DD1204-01), and fluorescence signal intensity was detected. Faricimab and Aflibercept biosimilars were used as positive controls. Results are as follows: Figure 13 As shown, the multispecific molecule RYYM002 has a high blocking effect on VEGF-A activity, which is significantly better than Faricimab and close to that of Aflibercept.

[0200] 7.2 Determination of the antibody's blocking efficacy against the VEGF-C-VEGF-R2-mediated intracellular signaling pathway

[0201] Similarly, the blocking efficacy of the antibody against the VEGF-C-VEGF-R2-mediated intracellular signaling pathway was detected using VEGF-R2 reporter cells: The antibody was serially diluted and mixed with VEGF-C (R&D, 9199-VC-025) to a final concentration of 50 ng / mL, incubated at room temperature for 30 minutes, and then added to reporter cells (2E+04 cells per well). Cells were incubated at 37°C for 6 hours, and then BrightLight (Novazia, DD1204-01) was added, and the fluorescence signal intensity was detected. Results are as follows: Figure 14 As shown, RYYM002 has a blocking efficacy against the VEGF-C-VEGF-R2-mediated intracellular signaling pathway that is close to that of the control antibody OPT-302.

[0202] 7.3 Determination of the antibody's blocking efficacy against the VEGF-D-VEGF-R2-mediated intracellular signaling pathway

[0203] Similarly, the blocking efficacy of the antibody against the VEGF-D-VEGF-R2 mediated intracellular signaling pathway was detected using VEGF-R2 reporter cells: The antibody was serially diluted and mixed with VEGF-D (R&D, 622-VD-025) to a final concentration of 250 ng / mL, incubated at room temperature for 30 minutes, and then added to reporter cells (2E+04 cells per well). Cells were incubated at 37°C for 6 hours, and then BrightLight (Novizan, DD1204-01) was added, and the fluorescence signal intensity was detected. Results are as follows:Figure 15 As shown, it can be seen that RYYM002 has a blocking effect on VEGF-D-VEGF-R2 mediated intracellular signaling pathway close to the control antibody OPT-302.

[0204] 7.4 Study on the inhibitory effect of the antibody on VEGF-C induced HUVEC cell proliferation

[0205] The inhibitory effect of the antibody RYYM002 on VEGF-C induced HUVEC cell proliferation was detected using the experimental system in 4.6, and the results are shown in Figure 16 As shown, it can be seen that in the actual vascular endothelial cell proliferation behavior inhibition experiment, RYYM002 shows a significant inhibition effect better than the control antibody.

[0206] 7.5 Study on the inhibitory effect of the antibody on ang-2 induced intracellular Tie2 phosphorylation

[0207] It is known that after Ang-2 binds to the receptor Tie2 on the cell surface, it promotes the phosphorylation of the tyrosine sites on the intracellular region of Tie2 through the clustering effect, thereby transmitting signals to the downstream, promoting the proliferation, remodeling, increased leakage of blood vessels, and inflammatory response, etc. In order to detect the inhibitory effect of the antibody on the ang-2-tie2 signaling pathway, the following system and operation steps were used:

[0208] (1) 3E+04 / well of huTie2-HEK293 cells were added to a cell culture plate coated with polylysine, and cultured overnight at 37°C;

[0209] (2) After gradient dilution of the antibody, it was co-incubated with ang2-his (R&D, 623-AN-025) at a final concentration of 100 ng / mL;

[0210] (3) The co-incubation solution of the antibody and ang2 was added to the cells, and cultured at 37°C for 90 min;

[0211] (4) After removing the culture solution, the cells were washed with cold PBS, and then lysed and detected for tyrosine phosphorylated Tie2 using the Phospho-tyrosine Tie2 ELISA kit (abcam, ab279956) according to the instructions in the kit;

[0212] (5) After color development and termination according to the instructions in the kit, the absorbance value at 450 nm was read using a microplate reader, and the standard curve was plotted using the Phospho-tyrosine Tie2 reference product in the kit, and the Phospho-tyrosine Tie2 content of each detection well was calculated and plotted.

[0213] The results are shown in Figure 17As shown, the polyclonal antibody RYYM002 has a blocking efficacy against the Ang2-Tie2 signaling pathway that is close to that of Nesvacumab and significantly superior to that of Faricimab.

[0214] Example 8: Therapeutic Experiment of Antibody Against Laser-Induced Choroidal Angiogenesis in Monkey Fundus

[0215] To investigate the therapeutic effects of antibodies on retinal angiogenesis and leakage at the in vivo level, a non-human primate choroidal neovascularization (CNV) model was used. The specific procedures are as follows:

[0216] (1) After anesthetizing and mydriasis of rhesus monkeys, fundus CNV modeling was performed. The fundus was irradiated with laser to rupture Bruch's membrane. When bubbling was observed at the laser irradiation site and a clear penetrating sound was heard, the irradiation was considered successful. A total of 8 monkeys were used, and 6 sites were irradiated in each eye of each monkey;

[0217] (2) Two weeks after irradiation (day 14), FFA (fluorescein fundus angiography) was performed. It was observed that all sites were successfully irradiated and had similar levels of fluorescence leakage. Based on the area of ​​fluorescence leakage, the monkeys were divided into 4 groups. On day 15, 0.5 mg of IgG1 isotype control antibody, 0.5 mg of RYYM002, 0.5 mg of Faricimab, and 0.5 mg of Aflibercept + 0.5 mg of OPT-302 were injected into the vitreous body, respectively.

[0218] (3) Two weeks after administration (day 29), FFA testing was performed again. Professionals scored each spot based on its leakage status, from 1 to 4 points, from low to high, according to the presence and severity of leakage. The scores of each group were then statistically analyzed, and the fluorescence leakage area of ​​each spot was calculated using software.

[0219] The results are as follows Figure 18 As shown, the multispecific molecule RYYM002 has a significantly better score improvement effect than the isotype control group. Figure 18 A, P < 0.0001), and was also significantly better than the Faricimab treatment group (P < 0.01), and showed a better trend of improvement than the Aflibercept + OPT-302 combination therapy group, although no P value was generated; in the statistics of fluorescence leakage area ( Figure 18B), consistent with the leakage rating, RYYM002 showed a significant improvement in the score compared with the control group (P<0.0001), and was significantly better than the Faricimab treatment group (P<0.0001), and showed a more optimal trend than the Aflibercept+OPT-302 combination treatment group, although no significant difference was produced, which can be due to the insufficient number of animals and irradiation points.

[0220] In the description of the present specification, the description referring to the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms are not necessarily directed to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples, without contradiction.

[0221] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A single-domain antibody capable of specifically recognizing Ang-2, characterized in that, The heavy chain variable region of the antibody comprises HCDR1, HCDR2 and HCDR3, wherein: the HCDR1 amino acid sequence is shown as SEQ ID NO: 18; the HCDR2 amino acid sequence is shown as SEQ ID NO: 19 or 26; the HCDR3 amino acid sequence is shown as SEQ ID NO:

20.

2. The single-domain antibody of claim 1, wherein, The amino acid sequence of the antibody heavy chain variable region is shown as SEQ ID NO: 21 or 23.

3. A fusion protein of an anti-Ang-2 antibody, characterized in that, The fusion protein consists of the anti-Ang-2 single-domain antibody of claim 1 or 2 and the Fc fragment of IgG.

4. The fusion protein of claim 3, wherein, The amino acid sequence of the Fc fragment of IgG is shown as SEQ ID NO:

13.

5. The fusion protein of claim 3, wherein, The amino acid sequence of the fusion protein of the anti-Ang-2 antibody is shown as SEQ ID NO: 22 or 29.

6. A multispecific antibody, characterized in that Comprise: a first binding region and a second binding region, the first binding region comprises a single-domain antibody or antigen-binding fragment thereof that specifically binds to VEGF, and the second binding region comprises a single-domain antibody or antigen-binding fragment thereof that specifically binds to Ang-2; the multispecific antibody further comprises a VEGF receptor or extracellular domain fragment thereof that specifically binds to VEGF-A; the single-domain antibody that specifically binds to Ang-2 is the anti-Ang-2 single-domain antibody of claim 1 or 2.

7. The multispecific antibody of claim 6, wherein the single-domain antibody or antigen-binding fragment thereof that specifically binds to VEGF is targeted to VEGF-C and / or VEGF-D.

8. The multispecific antibody of claim 6, wherein The multispecific antibody further comprises at least one linker peptide.

9. The multispecific antibody of claim 8, wherein The linker peptide is a GS linker peptide.

10. The multispecific antibody of claim 6, wherein The multispecific antibody comprises a structure shown as formula Ia or Ib from N-terminus to C-terminus: P1-L1-P3-L2-P4-L3-P2 (Ia); P2-L3-P4-L2-P3-L1-P1 (Ib); wherein, P1 is an anti-VEGF single-domain antibody, P2 is an anti-Ang-2 single-domain antibody, P3 is an Fc fragment of IgG, P4 is a VEGF receptor or extracellular domain thereof that binds to VEGF-A, L1, L2 and L3 are each independently absent or a linker peptide.

11. The multispecific antibody of claim 6, wherein The heavy chain variable region of the anti-VEGF single-domain antibody comprises HCDR1, HCDR2 and HCDR3, wherein: the HCDR1, HCDR2 and HCDR3 amino acid sequences are shown as SEQ ID NO: 1, 2 and 3, respectively; or as SEQ ID NO: 4, 5 and 6, respectively; or as SEQ ID NO: 7, 8 and 9, respectively; or as SEQ ID NO: 7, 25 and 9, respectively.

12. The multispecific antibody of claim 11, wherein The amino acid sequence of the heavy chain variable region of the anti-VEGF single-domain antibody is shown as SEQ ID NO: 10-12 or SEQ ID NO:

17.

13. The multispecific antibody of claim 12, wherein The amino acid sequence of the heavy chain variable region of the anti-VEGF single-domain antibody is shown as SEQ ID NO:

17.

14. The multispecific antibody of claim 10, wherein The Fc fragment of IgG is an IgG1 Fc or a variant fragment thereof.

15. The multispecific antibody of claim 14, wherein The amino acid sequence of the Fc fragment of IgG is shown as SEQ ID NO:

13.

16. The multispecific antibody of claim 14, wherein The Fc fragment amino acid sequence of the IgG is shown as SEQ ID NO:

30.

17. The multispecific antibody of claim 10, wherein The VEGF receptor or the extracellular domain amino acid sequence thereof is shown as SEQ ID NO:

27.

18. The multispecific antibody of claim 10, wherein, The connecting peptide is a GS connecting peptide.

19. The multispecific antibody of claim 18, wherein The sequence of the connecting peptide is (G4S)n, wherein n is 1, 2, 3, 4, 5 or 6.

20. The multispecific antibody of claim 19, wherein, The sequence of the connecting peptide is (G4S)n, wherein n is 3.

21. The multispecific antibody of claim 10, wherein The multispecific antibody sequentially comprises, from N-terminus to C-terminus, an anti-Ang-2 single-domain antibody, a GS connecting peptide, a VEGF extracellular domain, an Fc fragment of IgG, a GS connecting peptide, and an anti-VEGF single-domain antibody.

22. The multispecific antibody of claim 10, wherein The amino acid sequence of the multispecific antibody is shown as SEQ ID NO:

24.

23. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the single-domain antibody of any one of claims 1-2, the fusion protein of any one of claims 3-5, or the multispecific antibody of any one of claims 6-22.

24. An expression vector comprising the nucleic acid of claim 23. The nucleic acid molecule of claim 23.

25. A recombinant cell, comprising: The recombinant cell carries the nucleic acid molecule of claim 23 or the expression vector of claim 24.

26. A pharmaceutical composition comprising, The single-domain antibody of any one of claims 1-2, the fusion protein of any one of claims 3-5, or the multispecific antibody of any one of claims 6-22, the nucleic acid molecule of claim 23, the expression vector of claim 24, or the recombinant cell of claim 25.

27. Use of the single-domain antibody of any one of claims 1-2, the fusion protein of any one of claims 3-5, or the multispecific antibody of any one of claims 6-22, the nucleic acid molecule of claim 23, the expression vector of claim 24, the recombinant cell of claim 25, or the pharmaceutical composition of claim 26 in the preparation of a medicament for treating or preventing an ocular disease associated with angiogenesis; the ocular disease associated with angiogenesis is choroidal neovascularization.

28. A test kit comprising, The single-domain antibody of any one of claims 1-2, the fusion protein of any one of claims 3-5, or the multispecific antibody of any one of claims 6-22, the nucleic acid molecule of claim 23, the expression vector of claim 24, or the recombinant cell of claim 25.

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