WO2023109888A1 - 抗ang2-vegf双特异性抗体及其用途 - Google Patents

抗ang2-vegf双特异性抗体及其用途 Download PDF

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WO2023109888A1
WO2023109888A1 PCT/CN2022/139203 CN2022139203W WO2023109888A1 WO 2023109888 A1 WO2023109888 A1 WO 2023109888A1 CN 2022139203 W CN2022139203 W CN 2022139203W WO 2023109888 A1 WO2023109888 A1 WO 2023109888A1
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amino acid
acid sequence
polypeptide
sequence
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郎国竣
刘婵娟
王立燕
曹静丽
司远青
李文
苏飞
闫闰
张震
张文海
胡宇豪
孙兴鲁
陈克让
江茹兰
林紫绮
戴珊珊
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三优生物医药(上海)有限公司
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Definitions

  • the invention belongs to the field of biomedicine. Specifically, the present invention provides an anti-ANG2-VEGF bispecific antibody, a pharmaceutical composition comprising the bispecific antibody, and uses thereof. The present invention further provides polynucleotides and expression vectors encoding the bispecific antibody and methods for producing the bispecific antibody.
  • VEGF-A Vascular endothelial growth factor A
  • VEGFR1 and VEGFR2 which are expressed in vascular endothelial cells.
  • VEGFR2 is the main receptor tyrosine kinase receptor that mediates angiogenesis.
  • VEGF activates VEGFR2 to promote mitosis of vascular endothelial cells and increase vascular permeability, thereby promoting cardiovascular sprouting. Therefore, targeting VEGF or VEGFR2 can effectively inhibit abnormal vascular proliferation (Ferrara, N. (2010). Mol Biol Cell 21 (5): 687-690.).
  • Angiopoietin is part of the angiogenesis factor family.
  • ANG2 also known as ANGPT2 belongs to the angiopoietin family, expressed by endothelial cells, and is usually stored in Weibel–Palade bodies of endothelial cells. When hypoxia, inflammation, etc. promote the formation of new blood vessels, ANG2 It is released from Weibo body and competes with ANG1. As an antagonist of Tie2, it inhibits the stability of blood vessels, detaches pericytes from endothelial cells, increases the permeability of endothelial cells, and enables VEGF to better play the role of promoting neovascularization. Effect (Saharinen, P., et al. (2017).
  • VEGF and ANG2 The complementary coordination and joint effect of VEGF and ANG2 in abnormal blood vessel formation have been confirmed in vivo functional experiments.
  • Vanucizumab see, for example, WO2010040508A1
  • Faricimab see, for example, WO2014009465A1
  • BI836880 see, for example, US9527925B2
  • the invention provides a bispecific antibody comprising a first antigen-binding portion that binds ANG2 and a second antigen-binding portion that binds VEGF, the first antigen-binding portion comprising a heavy chain variable region and a light chain A variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences are selected from ( Any one of 1)-(3): (1) HCDR1 sequence shown in SEQ ID NO:25; HCDR2 sequence shown in SEQ ID NO:26; HCDR3 sequence shown in SEQ ID NO:27; SEQ ID NO:28 LCDR1 sequence; LCDR2 sequence shown in SEQ ID NO:29; and LCDR3 sequence shown in SEQ ID NO:30; (2) HCDR1 sequence shown in SEQ ID NO:33; HCDR2 sequence shown in SEQ ID NO:33;
  • variable region of the heavy chain and the variable region of the light chain are selected from any one of (1)-(3): (1) the variable region of the heavy chain, which comprises SEQ ID NO:31 Amino acid sequence; light chain variable region, which comprises the amino acid sequence of SEQ ID NO:32; (2) heavy chain variable region, which comprises the amino acid sequence of SEQ ID NO:39; light chain variable region, which comprises SEQ ID The amino acid sequence of NO:40; (3) heavy chain variable region, it comprises the aminoacid sequence of SEQ ID NO:47; Light chain variable region, it comprises the aminoacid sequence of SEQ ID NO:48.
  • said second antigen binding moiety comprises an immunoglobulin single variable domain (VHH) that binds VEGF.
  • VHH immunoglobulin single variable domain
  • the immunoglobulin single variable domain comprises: the CDR1 sequence shown in SEQ ID NO:49, the CDR2 sequence shown in SEQ ID NO:50 and the CDR3 sequence shown in SEQ ID NO:51.
  • said first antigen binding moiety and said second antigen binding moiety are linked by a linker.
  • the bispecific antibody of the present invention further comprises a human IgG1 heavy chain constant region and a human kappa light chain constant region.
  • said human IgG1 heavy chain constant region comprises a leucine to alanine mutation at positions 234 and 235 according to EU numbering.
  • the present invention also provides a polynucleotide encoding the bispecific antibody of the present invention.
  • the present invention also provides an expression vector comprising the polynucleotide of the present invention.
  • the present invention also provides a host cell comprising the polynucleotide or expression vector of the present invention.
  • the present invention further provides a method for producing the bispecific antibody of the present invention, which comprises:
  • the present invention also provides a pharmaceutical composition, which comprises the bispecific antibody of the present invention and a pharmaceutically acceptable carrier.
  • the present invention also provides the use of the bispecific antibody or the pharmaceutical composition of the present invention in the preparation of a medicament for treating the following diseases: (1) eye diseases related to angiogenesis; or (2) cancer.
  • Figure 1A-1E shows the schematic structure of anti-ANG2-VEGF bispecific antibody: Figure 1A, BsAb1, BsAb13 and BsAb21; Figure 1B, BsAb2, BsAb14 and BsAb22; Figure 1C, BsAb3, BsAb15 and BsAb23; BsAb16 and BsAb24; Figure IE, BsAb29, BsAb30 and BsAb31.
  • 2A-2E show the binding activity of anti-ANG2-VEGF bispecific antibody to recombinant protein hVEGF-His.
  • 3A-3E show the binding activity of anti-ANG2-VEGF bispecific antibody to recombinant protein hANG2-His.
  • Figures 4A-4C show the activity of anti-ANG2-VEGF bispecific antibodies to neutralize VEGF.
  • Figures 5A-5B show the blocking activity of anti-ANG2-VEGF bispecific antibodies on ANG2/Tie2.
  • Figures 6A-6C show the activity of anti-ANG2-VEGF bispecific antibodies in inhibiting the proliferation of HUVEC cells.
  • Figures 7A-7C show the effect of anti-ANG2-VEGF bispecific antibodies on inhibiting the growth of human colon cancer cell subcutaneous xenografts:
  • Figure 7A shows the changes in the average tumor volume of mice in each group over time;
  • Figure 7B shows the changes in the average tumor volume of mice in each group The change of average body weight over time;
  • Figure 7C shows the average tumor weight of mice in each group at the end of the experiment.
  • the expressions “comprising”, “comprising”, “containing” and “having” are open-ended, meaning that listed elements, steps or components are included but other unlisted elements, steps or components are not excluded.
  • the expression “consisting of” does not include any element, step or component not specified.
  • the expression “consisting essentially of” means that the scope is limited to the elements, steps or components specified, plus optional elements, steps or components that do not materially affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions “consisting essentially of” and “consisting of” are encompassed within the meaning of the expression “comprising”.
  • bispecific antibody refers to an antibody that specifically binds two different antigens.
  • the bispecific antibody of the present invention comprises an antigen-binding portion that specifically binds to two different antigens, ANG2 and VEGF.
  • antibody refers to an immunoglobulin or fragment thereof that specifically binds an antigenic epitope through at least one antigen binding site.
  • the term “antibody” includes multispecific antibodies (eg, bispecific antibodies), fully human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, and antigen-binding fragments.
  • Antibodies can be synthetic (eg, produced by chemical or biological conjugation), enzymatically processed, or recombinantly produced.
  • Antibodies include any immunoglobulin class (eg, IgG, IgM, IgD, IgE, IgA, and IgY), any class (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass (eg, IgG2a and IgG2b).
  • Antibodies can be multivalent, ie, contain multiple antigen combining sites. For example, antibodies can be bivalent, trivalent, tetravalent, pentavalent, and hexavalent, comprising two, three, four, five, and six antigen-binding sites, respectively.
  • antigen-binding fragment refers to a portion of a full-length antibody that is less than full-length, but comprises at least part of the variable region of a full-length antibody (e.g., comprising one or more CDRs and/or one or more antigen-binding site), and thus retain at least part of the full-length antibody's ability to specifically bind the antigen.
  • antigen binding fragments include, but are not limited to, sdAb (e.g., the variable domain of a heavy chain antibody), Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', F(ab') 2 , diabody, Fd, and Fd ' fragments as well as other fragments (such as those containing modifications).
  • sdAb e.g., the variable domain of a heavy chain antibody
  • a “full-length antibody” typically comprises four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region of a full-length antibody may comprise CH1-hinge region-CH2-CH3 from N-terminus to C-terminus.
  • the heavy chain constant region may comprise, from N-terminus to C-terminus, CH1-hinge-CH2-CH3-CH4.
  • the light and heavy chain variable regions can each comprise three highly variable "complementarity determining regions (CDRs)" and four relatively conserved “framework regions (FRs)” Sequential linkage of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDRs of the light chain variable region CDRL or LCDR
  • LCDR1, LCDR2 and LCDR3 the CDRs of the heavy chain variable region
  • the CDRs of antibodies can be defined by various methods in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (see for example Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), Kabat based on antibody sequence variability (see e.g. Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest , Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No.91-3242), AbM (Martin, A.C.R. and J.
  • CDR complementarity determining region
  • variable region e.g., variable region
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • a different protocol e.g. Different assignment system rules or combinations
  • framework region and “framework region” are used interchangeably.
  • framework region refers to those amino acid residues in an antibody variable region other than the CDR sequences as defined above.
  • the "Fv" fragment composed of one VH and one VL through non-covalent interaction is the smallest antigen-binding fragment containing an antigen-binding site.
  • single variable domains single variable domain antibodies
  • a "single-chain Fv (scFv)" can be obtained by linking VH and VL via a peptide linker.
  • dsFv disulfide bond-stabilized Fv
  • scdsFv or dsscFv can be obtained by introducing a disulfide bond into Fv or scFv, respectively.
  • Fab comprises a complete antibody light chain (VL-CL) and an antibody heavy chain variable region and a heavy chain constant region (VH-CH1, also known as Fd).
  • a single-chain “Fab (scFab)” can be obtained by linking CL and CH1 in a “Fab” with a peptide linker.
  • F(ab') 2 essentially comprises two Fab fragments linked by a disulfide bond in the hinge region.
  • Fab' is the half of F(ab') 2 , which can be obtained by reducing the disulfide bonds in the hinge region of F(ab') 2 .
  • single domain antibody refers to an antibody comprising a single immunoglobulin variable domain (single variable domain) as a functional antigen-binding fragment.
  • single variable domains Like the variable regions of full-length antibodies, single variable domains generally comprise CDR1, CDR2, and CDR3 that form the antigen-binding site and supporting framework regions.
  • the single variable domain can be, for example, the variable domain of a heavy-chain antibody (variable domain of heavy-chain antibody, VHH), the IgNAR variable domain of a shark, the variable domain of a human light-chain antibody, and the variable domain of a heavy-chain antibody. domain.
  • percentage (%) sequence identity and “sequence identity” of amino acid sequences have definitions recognized in the art, which refer to two sequences determined by sequence alignment (for example, by manual inspection or known algorithms). The percentage of identity between polypeptide sequences. It can be determined using methods known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, Clustal Omega and FASTA software.
  • Polypeptides can be modified, eg, by one or more amino acid substitutions, additions and/or deletions, without altering the function of the polypeptide. Substitutions are preferably conservative substitutions of amino acids. Suitable conservative substitutions are well known to those skilled in the art.
  • antibodies can be modified using methods known in the art to alter their properties, such as altering the type of glycosylation modification of the antibody, altering the ability to form interchain disulfide bonds, altering the effector function of the antibody (e.g., enhancing or reducing antibody-dependent Cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), or provide active groups for the preparation of antibody conjugates.
  • ADCC antibody-dependent Cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • Affinity or "binding affinity” is a measure of the strength of non-covalent binding between an antibody and an antigen.
  • the magnitude of "affinity” can usually be reported as the equilibrium dissociation constant KD or EC50 .
  • Affinity can be determined by conventional techniques known in the art, such as biomembrane interferometry (for example, ForteBio Octet or Gator detection systems can be used), surface plasmon resonance, enzyme-linked immunoassay (ELISA) or flow cytometry (FACS), etc. .
  • isolated means that a substance (such as a polynucleotide or polypeptide) is separated from its source or environment in which it exists, ie does not substantially contain any other components.
  • the bispecific antibodies of the invention, or polynucleotides encoding them, may be isolated.
  • polynucleotide and “nucleic acid” are used interchangeably to denote an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives.
  • Polynucleotides may include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • a "vector” is a medium for introducing exogenous polynucleotides into host cells, and when the vector is transformed into an appropriate host cell, the exogenous polynucleotides are amplified or expressed.
  • the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (eg, yeast artificial chromosomes and mammalian artificial chromosomes), and the like.
  • Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpesviral vectors, poxviral vectors, baculoviral vectors, and the like.
  • expression vector refers to a vector capable of expressing a polypeptide of interest.
  • An expression vector may generally comprise a polynucleotide sequence encoding a polypeptide of interest and regulatory sequences (such as a promoter and ribosome binding site) operably linked thereto.
  • a "host cell” is a cell used to receive, maintain, replicate or amplify a vector. Host cells can also be used to express polynucleotides or polypeptides encoded by vectors. Host cells can be eukaryotic or prokaryotic. Prokaryotic cells such as Escherichia coli (E.coli) or Bacillus subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus, insect cells (such as S2 Drosophila cells or Sf9), and animal cells (such as fibroblasts, CHO cells , COS cells, HeLa cells, NSO cells or HEK293 cells).
  • E.coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells , COS cells, HeLa cells, NSO cells or
  • treatment refers to the improvement of the disease/symptom, such as reducing or disappearing the disease/symptom, preventing or slowing down the occurrence, progression and/or deterioration of the disease/symptom.
  • an “effective amount” means the amount of an active substance (such as an antibody or a pharmaceutical composition of the present invention) that induces a biological or medical response or a desired therapeutic effect on a tissue, system, animal, mammal or human.
  • an “effective amount” may be that amount required to prevent, cure, ameliorate, arrest or partially arrest a disease or condition (eg, cancer).
  • a disease or condition eg, cancer
  • Those skilled in the art can determine the effective amount according to factors such as the subject's age, physical condition, sex, severity of symptoms, specific composition or route of administration.
  • the term "pharmaceutically acceptable carrier” refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • mammals include, but are not limited to, humans, non-human primates, rats, mice, cows, horses, pigs, sheep, alpacas, dogs, cats, and the like.
  • subject refers to a mammal, such as a human.
  • the subject is a patient, such as a cancer patient.
  • the invention provides a bispecific antibody comprising a first antigen-binding portion that binds ANG2 and a second antigen-binding portion that binds VEGF, the first antigen-binding portion comprising a heavy chain variable region and a light chain A variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequences are selected from ( Any one of 1)-(3): (1) HCDR1 sequence shown in SEQ ID NO:25; HCDR2 sequence shown in SEQ ID NO:26; HCDR3 sequence shown in SEQ ID NO:27; SEQ ID NO:28 LCDR1 sequence; LCDR2 sequence shown in SEQ ID NO:29; and LCDR3 sequence shown in SEQ ID NO:30; (2) HCDR1 sequence shown in SEQ ID NO:33; HCDR2 sequence shown in SEQ ID NO:33;
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:31, SEQ ID NO:39, or SEQ ID NO:47. In some embodiments, the heavy chain variable region comprises at least 80%, at least 85%, at least 90%, at least 91%, at least Amino acid sequences having 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity. In some embodiments, the heavy chain variable region polypeptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid substitutions, additions and/or deletions of the amino acid sequence. Preferably, said amino acid substitutions, additions and/or deletions do not occur in CDR regions.
  • the light chain variable region comprises the amino acid sequence of SEQ ID NO:32, SEQ ID NO:40 or SEQ ID NO:48. In some embodiments, the light chain variable region comprises at least 80%, at least 85%, at least 90%, at least 91%, at least Amino acid sequences having 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity. In some embodiments, the light chain variable region comprises one or more (e.g., 1, 2, 3, 4, 5 , 6, 7, 8, 9 or 10) amino acid substitutions, additions and/or deletions of amino acid sequences. Preferably, said amino acid substitutions, additions and/or deletions do not occur in CDR regions.
  • the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:31, SEQ ID NO:39 or SEQ ID NO:47; the light chain variable region comprises SEQ ID NO:32, SEQ ID NO:32, SEQ ID NO:39 or SEQ ID NO:47; The amino acid sequence of ID NO:40 or SEQ ID NO:48.
  • variable region of the heavy chain and the variable region of the light chain are selected from any one of (1)-(3): (1) the variable region of the heavy chain, which comprises SEQ ID NO:31 The amino acid sequence of; Light chain variable region, it comprises the aminoacid sequence of SEQ ID NO:32; (2) heavy chain variable region, it comprises the aminoacid sequence of SEQ ID NO:39; Light chain variable region, it comprises SEQ The amino acid sequence of ID NO:40; (3) heavy chain variable region, it comprises the aminoacid sequence of SEQ ID NO:47; Light chain variable region, it comprises the aminoacid sequence of SEQ ID NO:48.
  • the first antigen binding portion and the second antigen binding portion may comprise any form of antigen binding fragment.
  • the first antigen binding moiety is a scFv, Fab, Fab', F(ab') 2 , Fv fragment or disulfide stabilized Fv (dsFv).
  • the first antigen binding portion is a fully human antibody or an antigen binding portion thereof.
  • the second antigen binding moiety comprises an immunoglobulin single variable domain that binds VEGF.
  • said single variable domain is VHH.
  • said single variable domain is a humanized VHH.
  • the immunoglobulin single variable domain comprises: the CDR1 sequence shown in SEQ ID NO:49, the CDR2 sequence shown in SEQ ID NO:50 and the CDR3 sequence shown in SEQ ID NO:51.
  • said immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 11.
  • said immunoglobulin single variable domain comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least Amino acid sequences having 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the first and second antigen binding moieties are linked by a linker.
  • the linker may be a peptide linker or a chemical bond, preferably a peptide linker (also referred to as a "linker peptide").
  • a peptide linker may contain no more than 30 amino acids.
  • Exemplary peptide linkers can include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S), or combinations thereof, such as GGAS, GGGS, GGGSG, or (G 4 S) n , where n is 1 An integer of -20. Preferably, n is an integer of 1-5.
  • the peptide linker comprises the amino acid sequence of SEQ ID NO:12.
  • the bispecific antibodies of the invention further comprise an immunoglobulin constant region.
  • the immunoglobulin constant regions can be derived from the constant heavy (CH) and light (CL) chain regions of an immunoglobulin of any species.
  • the heavy chain constant region can be derived from the immune response of any subtype (e.g., IgA, IgD, IgE, IgG, and IgM), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass (e.g., IgG2a and IgG2b).
  • the heavy chain constant region comprises at least an Fc region.
  • the heavy chain constant region is that of human IgG1, an exemplary amino acid sequence of which is shown in SEQ ID NO:1.
  • the bispecific antibody of the present invention further comprises the Fc region of human IgG1, an exemplary amino acid sequence of which is shown in SEQ ID NO:3.
  • the bispecific antibodies of the invention have reduced ADCC and CDC effector functions, for example by including amino acid substitutions (mutations) in the heavy chain constant region.
  • the bispecific antibodies of the invention may comprise the heavy chain constant region of human IgG1.
  • the heavy chain constant region of human IgG1 may comprise a mutation selected from the group consisting of mutation of leucine to alanine at position 234 according to EU numbering (corresponding to position 117 of SEQ ID NO: 1) Acid (L234A), Leucine at position 235 (corresponding to position 118 of SEQ ID NO:1) is mutated to alanine (L235A), position 265 (corresponding to position 148 of SEQ ID NO:1) Mutation of aspartate to alanine (D265A), asparagine at position 297 (corresponding to position 180 of SEQ ID NO:1) to alanine (N297A) or glycine (N297G) or glutamine Amide (N297Q), lysine at position 322 (corresponding to position 205 of SEQ ID NO:1) is mutated to alanine (K322A), position 329 (corresponding to position 212 of SEQ ID NO:1) Proline to alanine (P329A) and combinations
  • the bispecific antibodies of the invention may comprise the heavy chain constant region of human IgG4.
  • the heavy chain constant region of human IgG4 may comprise a mutation selected from the group consisting of mutation of serine at position 228 to proline (S228P), mutation of phenylalanine at position 234 to Alanine (F234A), leucine at position 235 to alanine (L235A) and combinations thereof.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • cytotoxic cells e.g., natural killer (NK) cells, Fc receptors (FcRs) on the surface of neutrophils and macrophages
  • ADCC activity of an antibody can be assessed by methods known in the art (see e.g. US5821337A).
  • complement-dependent cytotoxicity refers to the activation of the complement system by antibodies that bind to an antigen on a target cell, thereby causing lysis of the target cell.
  • the CDC activity of an antibody can be assessed by methods known in the art (see, eg, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996)).
  • the light chain constant region may be derived from a lambda (Lambda) light chain or a kappa (Kappa) light chain constant region.
  • the light chain constant region is a human kappa light chain constant region.
  • the light chain constant region comprises the amino acid sequence of SEQ ID NO:2.
  • the bispecific antibody of the invention further comprises a heavy chain constant region (CH) and a light chain constant region (CL) of an immunoglobulin.
  • the heavy chain constant region is a human IgGl or human IgG4 heavy chain constant region
  • the light chain constant region is a human kappa light chain constant region.
  • the heavy chain constant region of the human IgGl comprises a leucine to alanine mutation (L234A/L235A) at positions 234 and 235 according to EU numbering.
  • the heavy chain constant region of the human IgG4 comprises serine to proline, phenylalanine to alanine and leucine to Mutation of alanine (S228P/F234A/L235A).
  • the VH and VL of the first antigen-binding moiety are fused to the N-terminus of CH and CL, respectively, and the single variable domain (e.g., VHH) of the second antigen-binding moiety is fused, optionally via a linker, to the N-terminus of CH and CL, respectively.
  • a bispecific antibody of the invention comprises a first polypeptide and a second polypeptide.
  • the first polypeptide comprises the heavy chain variable region (VH) and the heavy chain constant region (CH) of the first antigen-binding portion
  • the second polypeptide comprises the light chain region of the first antigen-binding portion.
  • VL Chain variable region
  • CL light chain constant region
  • VHH single variable domain of the second antigen-binding portion
  • the single variable domain of the second antigen-binding portion is optionally fused to: (1) the N-terminus of said VH by a linker; ( 2) the N-terminal of the VL; (3) the N-terminal of the VH and VL; (4) the C-terminal of the CH; (5) the C-terminal of the CL; (6) the C-terminal of the CH and CL C-terminal; (8) the N-terminal of the VH and the C-terminal of the CH; or (9) the N-terminal of the VL and the C-terminal of the CH.
  • first polypeptide and the second polypeptide are selected from any one of (1)-(5):
  • the first polypeptide has the structure of formula (I), and the second polypeptide has the structure of formula (II)
  • the first polypeptide has the structure of formula (III), and the second polypeptide has the structure of formula (II)
  • the first polypeptide has the structure of formula (III), and the second polypeptide has the structure of formula (IV)
  • the first polypeptide has the structure of formula (V), and the second polypeptide has the structure of formula (IV)
  • the first polypeptide has a structure of formula (V), and the second polypeptide has a structure of formula (VI)
  • VH and VL are the heavy chain variable region and the light chain variable region, respectively, of the first antigen-binding portion
  • VHH is the single variable domain of the second antigen binding portion
  • CH and CL are the heavy chain constant region and light chain constant region of immunoglobulin, respectively;
  • Linker is a connector
  • first polypeptide and the second polypeptide are selected from any one of (1)-(15):
  • said first polypeptide comprises a leucine to alanine mutation at the following amino acid position: positions 238 and 239 of SEQ ID NO: 5 or SEQ ID NO: 13 (L238A/L239A); 242 and 243 of SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:17 or SEQ ID NO:21 (L242A/L243A); 382 and 383 of SEQ ID NO:14 (L382A/L383A ); or the 386th and 387th positions (L386A/L387A) of SEQ ID NO:18 or SEQ ID NO:22.
  • first polypeptide and the second polypeptide are selected from any one of (1)-(15):
  • a first polypeptide comprising an amino acid sequence having L238A and L239A mutations compared with SEQ ID NO:13; a second polypeptide comprising an amino acid sequence of SEQ ID NO:6;
  • a first polypeptide comprising an amino acid sequence having L242A and L243A mutations compared to SEQ ID NO:7; a second polypeptide comprising an amino acid sequence of SEQ ID NO:19;
  • a first polypeptide comprising an amino acid sequence having L386A and L387A mutations compared to SEQ ID NO:22; a second polypeptide comprising an amino acid sequence of SEQ ID NO:23.
  • the bispecific antibodies of the invention may be multivalent antibodies, such as bivalent, trivalent, tetravalent, pentavalent and hexavalent.
  • the bispecific antibodies of the invention are tetravalent antibodies comprising two first antigen binding portions and two second antigen binding portions.
  • the bispecific antibody of the invention is a hexavalent antibody comprising two first antigen binding moieties and four second antigen binding moieties.
  • the bispecific antibody of the present invention comprises two first polypeptides and two second polypeptides, wherein the first polypeptides are the same or different, and the second polypeptides are the same or different.
  • the bispecific antibody of the invention comprises two identical first polypeptides and two identical second polypeptides. The first and second polypeptides are as described above.
  • the bispecific antibodies of the invention specifically bind ANG2 and VEGF. In some embodiments, the bispecific antibodies of the invention block the binding of ANG2 and VEGF to their respective receptors. In some embodiments, the bispecific antibody of the invention specifically binds ANG2, but does not bind or substantially does not bind ANG1.
  • the expression "does not bind” or “substantially does not bind” as used herein means that the bispecific antibody of the present invention has significantly lower binding ability to ANG1 relative to the binding ability to ANG2, for example as described in Example 4.
  • Antibodies or antigen-binding fragments thereof can be prepared and produced using methods known in the art. Such methods can include, for example, preparation and isolation of nucleic acids encoding antibodies or antigen-binding fragments from phage display libraries, yeast display libraries, immortalized B cells (e.g., mouse B cell hybridoma cells or EBV immortalized B cells) . It is also possible to immunize animals, for example, immunize animals (such as humanized mice) with antigens or DNA encoding antigens, and then isolate antibody-expressing B cells from the immunized animals.
  • immunize animals for example, immunize animals (such as humanized mice) with antigens or DNA encoding antigens, and then isolate antibody-expressing B cells from the immunized animals.
  • Polynucleotides encoding antibodies or antigen-binding fragments thereof can also be isolated from immunized animals or humans or prepared by chemical synthesis, and then the polynucleotides can be used to construct expression vectors expressing antibodies or antigen-binding fragments.
  • the invention provides a polynucleotide encoding a bispecific antibody of the invention.
  • polynucleotides of the present invention can be obtained using methods known in the art.
  • polynucleotides of the invention can be isolated from humans, phage display libraries, yeast display libraries, immunized animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemically synthesis.
  • Polynucleotides can be codon-optimized for the host cell for expression.
  • the present invention also provides an expression vector comprising a polynucleotide of the present invention.
  • Expression vectors may further comprise additional polynucleotide sequences, such as transcriptional regulatory sequences and antibiotic resistance genes.
  • the present invention also provides a host cell comprising the polynucleotide or expression vector of the present invention.
  • the polynucleotide or expression vector of the present invention can be introduced into suitable host cells by various methods known in the art. Such methods include, but are not limited to, viral transduction, lipofection, electroporation, calcium phosphate transfection, and the like.
  • host cells are used to express the bispecific antibodies of the invention. Examples of host cells include, but are not limited to, prokaryotic cells (eg bacteria such as E. coli) and eukaryotic cells (eg yeast, insect cells, mammalian cells).
  • Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK293 cells, Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for expressing antibodies.
  • the present invention also provides a method for producing the bispecific antibody of the present invention, which comprises:
  • the present invention also provides a pharmaceutical composition, which comprises the bispecific antibody of the present invention, and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include, but are not limited to: diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients Excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, Propyl gallate, ⁇ -tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.), solubilizers, gelling agents, softeners, solvents (for example, water, alcohol, acetic acid and syrup), buffers (e.g., phosphate buffer, histidine buffer, and a
  • suitable carriers may be selected from buffers (e.g. citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine buffer, salt buffer), isotonic agents (such as trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (such as polysorbate 80, polysorbate 20, poloxamer) or its combination.
  • buffers e.g. citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine buffer, salt buffer
  • isotonic agents such as trehalose, sucrose, mannitol, sorbitol, lactose, glucose
  • nonionic surfactants such as polysorbate 80, polysorbate 20, poloxamer
  • compositions provided herein can be in a variety of dosage forms including, but not limited to, solid, semi-solid, liquid, powder, or lyophilized forms.
  • the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or infusion).
  • compositions provided herein can be administered by a variety of routes.
  • Routes of administration include, but are not limited to, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, or intracavity), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual or topical).
  • parenteral e.g., intravenous, subcutaneous, intradermal, intramuscular, or intracavity
  • topical e.g., intratumoral
  • epidural e.g., epidural
  • mucosal e.g., intranasal, oral, vaginal, rectal, sublingual or topical
  • the method of administration may be, for example, injection or infusion.
  • the bispecific antibodies of the invention may be administered in a dosage range of about 0.0001 to 100 mg/kg, more typically 0.01 to 20 mg/kg of the subject's body weight.
  • the dose administered can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight or 20 mg/kg body weight, or within the range of 1-20 mg/kg.
  • Exemplary treatment regimens entail weekly dosing, once every two weeks, once every three weeks, once every four weeks, once monthly, once every 3 months, once every 3-6 months, or an initial dosing interval The interval between dosing is slightly shorter and the later period is longer.
  • the bispecific antibodies of the invention block ANG2 and VEGF signaling pathways by binding to ANG2 and VEGF, including blocking angiogenesis associated with ANG2 and VEGF signaling pathways.
  • angiogenesis refers to the formation of new blood vessels. Studies have shown that angiogenesis is associated with various diseases, such as cancer and angiogenesis-related eye diseases.
  • a bispecific antibody or pharmaceutical composition of the invention for use in cancer and angiogenesis-related eye diseases.
  • the present invention also provides the use of the bispecific antibody or pharmaceutical composition of the present invention in the preparation of medicines for treating cancer and angiogenesis-related eye diseases.
  • the present invention also provides a method of treating cancer and angiogenesis-related eye diseases in a subject, comprising administering to the subject an effective amount of the bispecific antibody or pharmaceutical composition of the present invention.
  • Angiogenesis-associated ocular diseases may be ocular diseases associated with choroidal and retinal vascular diseases, including but not limited to choroidal neovascular diseases, retinal neovascular diseases, and diseases associated with vascular leakage.
  • the angiogenesis-related eye disease is macular degeneration (eg, dry or wet age-related macular degeneration (AMD)), retinal vein occlusion, retinopathy, retinopathy of prematurity (ROP), diabetic retinopathy, neonatal Vascular glaucoma, pathological myopia, macular edema, retinal edema, diabetic macular edema (DME), or choroidal neovascular disease.
  • macular degeneration eg, dry or wet age-related macular degeneration (AMD)
  • AMD age-related macular degeneration
  • ROP retinopathy of prematurity
  • diabetic retinopathy neonatal Vascular glaucoma
  • pathological myopia ma
  • cancer refers to or describes the physiological condition in an individual that is often characterized by unregulated cell growth.
  • the cancer is a solid tumor associated with angiogenesis.
  • Cancer can include primary cancer and metastatic cancer.
  • Non-limiting examples of cancers include lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, skin cancer, head and neck cancer, melanoma , ovarian cancer, colorectal cancer, gastric cancer, breast cancer, prostate cancer, uterine cancer, Hodgkin's lymphoma, esophageal cancer, anal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, kidney cancer (such as renal cell carcinoma , renal pelvis, and adrenal gland), soft tissue sarcomas, bladder cancer, central nervous system (CNS) tumors, mesothelioma, glioma, meningioma, and pituitary adenoma.
  • the cancer is colorectal cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer or hepatocellular
  • the present invention also provides a kit comprising the anti-ANG2-VEGF bispecific antibody or pharmaceutical composition of the present invention.
  • the kit further comprises instructions for the use of the bispecific antibody or pharmaceutical composition of the invention.
  • the kit may also comprise suitable containers, such as ampoules.
  • the kit also includes a device for administration.
  • the kit may also include a label indicating the intended use and/or method of use of the kit contents.
  • label includes any written or recorded material provided on or with the kit or otherwise provided with the kit.
  • the anti-ANG2-VEGF bispecific antibody of the present invention achieves at least one of the following beneficial effects:
  • Embodiment 1 Raw material preparation
  • antigen VEGF hVEGF, UniProt ID:P15692-4, SEQ ID NO:52
  • antigen Ang2 hANG2, Uniprot ID:O15123-1, SEQ ID NO:53
  • PCR Provided by General Biotechnology Co., Ltd.
  • pcDNA3.4 eukaryotic expression vector pcDNA3.4
  • the recombinant protein expression vectors were transformed into Escherichia coli DH5 ⁇ , cultured overnight at 37°C, and then the plasmids were extracted using the endotoxin-free plasmid extraction kit (OMEGA, D6950-01), and then extracted by the Expi293 TM expression system kit (ThermoFisher, A14635).
  • OEGA endotoxin-free plasmid extraction kit
  • Expi293 TM expression system kit ThermoFisher, A14635.
  • the cell expression supernatant was centrifuged at high speed for 10 min, and the expression supernatant of hANG2-Fc and hVEGF-Fc was affinity purified with COLUMN XK16/20 (purchased from Cytiva), and then purified with 100 mM sodium acetate (pH3.0)
  • the target protein was eluted, then neutralized with 1M Tris-HCl; hANG2-His or hVEGF-His expression supernatant was affinity-purified with Ni Smart Beads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd., SA036050), and then used with gradient concentration
  • the imidazole elutes the target protein.
  • the eluted proteins were respectively exchanged into PBS buffer through ultrafiltration concentrator tubes (Millipore, UFC901096). After SDS-PAGE identification and activity identification, it was frozen at -80°C until use.
  • the anti-VEGF positive control antibody used was Bevacizumab (synthesized according to the antibody sequence information provided in the patent US9527925B2) and P30-10-26 (VHH VEGF -huFc, amino acid sequence shown in SEQ ID NO: 4) independently developed by the applicant;
  • the anti-ANG2 positive control antibody used was Nesvacumab (synthesized according to the sequence disclosed in the patent application US20170355762A1) and the fully human antibody 17 of IgG1 type independently developed by the applicant (the heavy chain amino acid sequence is shown in SEQ ID NO: 5, the light chain amino acid sequence as shown in SEQ ID NO:6), 78A14 (heavy chain amino acid sequence as shown in SEQ ID NO:7, light chain amino acid sequence as shown in SEQ ID NO:8) and 78A74-7 (heavy chain amino acid sequence As shown in SEQ ID NO: 9, the amino acid sequence of the light chain is shown in SEQ ID NO: 10); the anti-ANG2-VEGF positive control antibody used is Farici
  • HEK293 cell line overexpressing human Tie2 (hereinafter referred to as huTie2-HEK293 cell line) and a HEK293 cell line overexpressing VEGFR2 and NFAT luciferase reporter gene (hereinafter referred to as VEGFR2/NFAT-HEK293 cell line) were constructed.
  • Cell line construction method They are as follows:
  • a DNA fragment (NCBI Gene ID: 7010) encoding human Tie2 protein was synthesized by gene synthesis technology, and cloned into the expression vector pLVX-Puro (Clontech, 632164) to obtain the Tie2 expression plasmid.
  • Use Invitrogen's electroporation kit (Cat. No.: MPK10096) and electroporation machine (Cat.
  • NFAT-HEK293 cell line the pGL4.30 plasmid (promega, #E8481) containing the luciferase gene (its transcription and expression are driven by the NFAT response element (NFAT-RE)) was passed through an electroporator (Invitrogen, Neon TM Transfection System, MP922947) into HEK293 cells Then, the monoclonal cell lines were screened with Hygromycin B (Yuanpei, S160J7) at a final concentration of 500 ⁇ g/mL for identification.
  • Hygromycin B Hygromycin B
  • the plasmid expressing VEGFR2 (see NCBI Gene ID: 3791, synthesized by General Biosynthesis) was electrotransferred into the NFAT-HEK293 cell line by the method of Example 1.3.1, and obtained by FACS identification VEGFR2/NFAT-HEK293 cell line.
  • This example describes the construction of an anti-ANG2-VEGF bispecific antibody (BsAb) in which the first antigen-binding portion uses the heavy chain variable region (VH) and light Chain variable region (VL) (HCDR1-3 and LCDR1-3 defined according to AbM and the amino acid sequences (SEQ ID NO:) of VH and VL are shown in Table 1A), the second antigen-binding portion was humanized anti-VEGF
  • the VHH of the single domain antibody P30-10-26 the amino acid sequences of CDR1, CDR2 and CDR defined according to AbM are shown in SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51 respectively; the amino acid sequences of VHH are shown in SEQ ID NO: 11).
  • the bispecific antibody of the present invention also comprises a human IgG1 heavy chain constant region (SEQ ID NO: 1) or a human IgG1 heavy chain constant region mutant (abbreviated as LALA; the 117th and 118th leucines of SEQ ID NO: 1) mutated to alanine) and the human Kappa light chain constant region (SEQ ID NO: 2), fused to the C-termini of the VH and VL of the first antigen-binding portion, respectively; and a linker peptide (SEQ ID NO: 12).
  • Figures 1A-1E show schematic structures of candidate bispecific antibodies.
  • Candidate bispecific antibodies contain two identical first polypeptides and two identical second polypeptides, and the amino acid sequences are shown in Table 1B.
  • the specific method for preparing the expression vectors of the first polypeptide and the second polypeptide is as follows: the target fragments are amplified by the PCR method, the fragments are connected by the overlap extension PCR method, and then respectively constructed into the transformed true fragments by the homologous recombination method.
  • the expression vectors for the first polypeptide and the second polypeptide were obtained on the nuclear expression vector plasmid pcDNA3.4 (Invitrogen).
  • the expression vectors of the first polypeptide and the second polypeptide were respectively transformed into Escherichia coli DH5 ⁇ , and then the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain the first polypeptide without endotoxin
  • OEGA endotoxin-free plasmid extraction kit
  • the anti-ANG2-VEGF bispecific antibody in Example 2 was expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133), and the specific method was as follows: on the day of transfection, confirm that the cell density was 7 ⁇ 10 6 to 1 ⁇ 10 7 Viable cells/mL is about, and the cell survival rate is >98%. At this time, adjust the cells to a final concentration of 6 ⁇ 10 6 cells/mL with fresh ExpiCHO expression medium pre-warmed at 37°C.
  • OptiPRO TM SFM Dilute the target plasmid with 4°C pre-cooled OptiPRO TM SFM (add 1 ⁇ g of plasmid to 1 mL of the medium), and at the same time, dilute ExpiFectamine TM CHO with OptiPRO TM SFM, then mix the two in equal volumes and gently blow and mix to prepare Form ExpiFectamine TM CHO/plasmid DNA mixture, incubate at room temperature for 1-5min, slowly add to the prepared cell suspension and shake gently at the same time, and finally place in a cell culture shaker at 37°C, 8% CO 2 conditions under cultivation.
  • the target protein was eluted, then neutralized with 1M Tris-HCl, and finally the resulting protein was replaced into PBS buffer through an ultrafiltration concentration tube (Millipore, UFC901096).
  • the purified antibody was identified by SDS-PAGE and SEC-HPLC with a purity greater than 90%, and it was subpackaged and stored at -80°C.
  • the recombinant protein hANG2-His was coated on a 96-well ELISA plate overnight at 4°C. On the next day, the well plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 hours. After the plate was washed 3 times with PBST, different concentrations of antibodies to be tested were added and incubated for 1 hour. Afterwards, after washing with PBST for 3 times, the secondary antibody Anti-human-IgG-Fc-HRP (abcam, ab79225) was added and incubated for 1 h. After incubation, the plate was washed six times with PBST, and TMB (SurModics, TMBS-1000-01) was added to develop the color.
  • TMB PurModics, TMBS-1000-01
  • hANG1-Fc 2 ⁇ g/mL hANG1-Fc (hANG1 (Uniprot ID: Q15389-1) and human IgG1 Fc fragment (SEQ ID NO: 3) fusion protein coated on a 96-well ELISA plate; see Example 1.1 for the preparation method ), overnight at 4°C.
  • the plate was washed 3 times with PBST and blocked with 5% skimmed milk for 2 hours. After the plate was washed 3 times with PBST, the antibody to be tested was added in gradient dilution and incubated for 1 hour.
  • VEGFR2/NFAT-HEK293 cell line constructed in Example 1 was used to screen candidate bispecific antibodies capable of neutralizing VEGF activity.
  • VEGF protein activates the transcriptional activity of the intracellular transcription factor NFAT through the VEGF-VEGFR2 signaling axis, thereby initiating the transcription and expression of the luciferase reporter gene; when the candidate bispecific antibody is added, the candidate bispecific antibody The antibody neutralizes VEGF and blocks its binding to VEGFR2, thereby blocking luciferase reporter gene expression.
  • the specific method is as follows: resuscitate the VEGFR2/NFAT-HEK293 cell line, use the cells that have been passaged for 2-4 times and grow well in the experiment, resuspend the cells after trypsinization, centrifuge at room temperature and 300g to remove the supernatant, and then use the Resuspend in DMEM medium, count and adjust the cell density to 4 ⁇ 105 cells/mL, add 100 ⁇ L per well to a new 96-well plate, and place in a 37°C cell culture incubator. At the same time, use DMEM medium to dilute the antibody to be tested, add 60ng/mL recombinant protein hVEGF-Fc, mix and incubate at room temperature for 30min.
  • the co-incubated antibody/hVEGF-Fc mixture was added to a 96-well plate, and incubated in a 37° C. incubator for 18 hours. After the incubation, 30 ⁇ L of luciferase substrate Bright-Lite (Vazyme, DD1204-03) was added to each well, and after shaking for 2 minutes, the fluorescence value of the 96-well plate was detected and the IC 50 was calculated.
  • Antibody name Neutralize VEGF activity IC 50 (nM) BsAb3 0.194 BsAb14 0.213 BsAb22 0.418 P30-10-26 0.292
  • Antibody name Neutralize VEGF activity IC 50 (nM) BsAb14-LALA 0.469 BsAb22-LALA 0.401 Faricimab 0.582 P30-10-26 0.482
  • Example 6 Based on the FACS method to detect the activity of anti-ANG2-VEGF bispecific antibody in blocking the binding of ANG2 and Tie2
  • the binding activity of candidate antibodies to block ANG2 and cell surface expressed receptor Tie2 was evaluated based on FACS method.
  • the specific method is as follows: 100 ⁇ L of the antibody to be tested that is serially diluted in FACS buffer (1 ⁇ PBS+2% FBS) is added to a 96-well round bottom plate. Then 2 ⁇ g/mL biotin-labeled hANG2-Fc protein (biotin-hANG2-Fc) in 100 ⁇ L FACS buffer was added to the 96-well plate, and then incubated at 4° C. for 1 h.
  • the huTie2-HEK293 cells that had been passaged 2-4 times and grown well were used in the experiment. Add 1 ⁇ 10 6 cells/mL cells in FACS buffer to a new 96-well round bottom plate at 100 ⁇ L per well, centrifuge at 4°C/300g and remove the supernatant; then add pre-incubated antibody/biotin- hANG2-Fc mixture, 180 ⁇ L per well, and then incubated at 4°C for 30 min; then the cells were centrifuged at 4°C/300g and the supernatant was removed, then 200 ⁇ L/well of FACS buffer was added to resuspend the cells, and centrifuged at 4°C/300g to remove supernatant; repeat this step twice.
  • PE-labeled Streptavidin (Invitrogen, 12-4317-87; 1:200 diluted in FACS buffer) was added to the wells at 200 ⁇ L/well and the cells were gently pipetted to resuspend the cells, and then the cells were incubated at 4°C in the dark for 30 min . After the incubation, the cells were centrifuged at 4°C and 300g to remove the supernatant, and FACS buffer was added to resuspend the cells, and this step was repeated twice. Finally, the amount of biotin-hANG2-Fc bound to the cells (expressed as mean fluorescence intensity (MFI) of PE) was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102).
  • MFI mean fluorescence intensity
  • Figures 5A-5B and Tables 3A-3B show that the blocking effects of candidate bispecific antibodies BsAb14, BsAb30, BsAb31 and BsAb22 are equivalent to those of antibodies 78A14 and 78A74-7;
  • Figure 5B and Table 3B show that candidate bispecific antibodies BsAb14-LALA and BsAb22-
  • the blocking effect of LALA was comparable to that of antibodies 78A14 and 78A74-7, and significantly better than that of Faricimab.
  • the inhibitory effect of anti-ANG2-VEGF bispecific antibodies BsAb14 and BsAb22 on VEGF-induced endothelial cell proliferation was verified.
  • the specific implementation method is as follows: resuscitate human umbilical vein endothelial cell line (HUVEC) (purchased from Beina Biotechnology), use the cells that have been passed down for 2-4 times and grow well in the experiment, resuspend the cells after trypsinization, and store at room temperature, Centrifuge at 300g to remove the supernatant, then resuspend the cells with 0.5% FBS EBM-2 medium, count and adjust the cell density to 5 ⁇ 104 cells/mL, add 50 ⁇ L per well to a new 96-well cell culture Plates were incubated overnight in a 37°C cell culture incubator.
  • HUVEC human umbilical vein endothelial cell line
  • the tumor inhibitory effects of anti-ANG2-VEGF bispecific antibodies BsAb3, BsAb14 and BsAb22 in human colon cancer cell Colo205 xenograft model were verified, and compared with antibodies Nesvacumab, Bevacizumab and P30-10-26.
  • the specific method is as follows: 5 ⁇ 10 6 Colo205 cells (purchased from Beina Biotechnology Co., Ltd.) were subcutaneously inoculated into 6-8 week-old male nude mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). Experimental mice were maintained under standard conditions. When the tumor-bearing volume reached about 130 mm 3 , the operations of dividing into groups and administering drugs were carried out.
  • tumor-bearing nude mice in each group 7 groups in total, including 3 candidate bispecific antibody groups (BsAb3 3.4952 mg/kg, BsAb14 3.5224 mg/kg and BsAb22 3.528 mg/kg; equimolar dose), 3 positive controls Antibody group (Nesvacumab 3mg/kg and Bevacizumab 3mg/kg, equimolar dose to bispecific antibody; P30-10-26 3mg/kg, molar dose 2.3 times that of bispecific antibody) and PBS negative control group.
  • BsAb3 3.4952 mg/kg, BsAb14 3.5224 mg/kg and BsAb22 3.528 mg/kg equimolar dose
  • 3 positive controls Antibody group Nesvacumab 3mg/kg and Bevacizumab 3mg/kg, equimolar dose to bispecific antibody
  • P30-10-26 3mg/kg molar dose 2.3 times that of bispecific antibody

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Abstract

本发明属于生物医药领域。具体地，本发明提供抗ANG2-VEGF双特异性抗体、包含所述双特异性抗体的药物组合物及其用途。本发明进一步提供编码所述双特异性抗体的多核苷酸和表达载体以及产生所述双特异性抗体的方法。

Description

抗ANG2-VEGF双特异性抗体及其用途
相关申请的交叉引用
本申请要求2021年12月16日提交的中国申请202111541547.9的优先权，其内容整体援引加入本文。
技术领域
本发明属于生物医药领域。具体地，本发明提供抗ANG2-VEGF双特异性抗体、包含所述双特异性抗体的药物组合物及其用途。本发明进一步提供编码所述双特异性抗体的多核苷酸和表达载体以及产生所述双特异性抗体的方法。
背景技术
血管内皮生长因子A(VEGF-A，也简称为VEGF)为促进血管生成的关键细胞因子。VEGF的受体有VEGFR1和VEGFR2，表达于血管内皮细胞。VEGFR2为介导血管生成主要的受体酪氨酸激酶受体，VEGF激活VEGFR2促进血管内皮细胞有丝分裂和血管渗透性增加，从而促进心血管出芽。因此，靶向VEGF或者VEGFR2能够有效抑制异常的血管增生(Ferrara,N.(2010).Mol Biol Cell 21(5):687-690.)。
血管生成素(Angiopoietin)是血管生长因子家族的一部分。ANG2(又称为ANGPT2)属于血管生成素家族，由内皮细胞表达，通常储存于内皮细胞韦博小体(Weibel–Palade bodies)，当遇到缺氧、炎症等促进新生血管形成的情况，ANG2从韦博小体释放出来和ANG1竞争，作为Tie2的拮抗剂抑制血管的稳定性，使周细胞从内皮细胞上脱离下来，内皮细胞通透性增加，使VEGF更好的发挥促进新生血管形成的作用(Saharinen,P.,et al.(2017).Nat Rev Drug Discov 16(9):635-661.)。与此同时，诸多研究表明ANG2的过表达在促进肿瘤的淋巴结转移起关键作用，因此ANG2是病理性血管生成相关疾病以及肿瘤治疗的重要靶点(Holash,J.,et al.(1999).Science 284(5422):1994-1998；Gengenbacher,N.,et al.(2021).Cancer Discov 11(2):424-445.)。2004年，首次通过中和ANG2与其受体Tie2相互作用的抗体，证实了阻断ANG2活性能够有效抑制肿瘤新生血管形成及肿瘤生长(Oliner,J.,et al.(2004).Cancer Cell 6(5):507-516.)。
VEGF与ANG2在异常血管形成中的互补协调以及共同作用在体内功能实验中得到了证实，在靶向ANG2和靶向VEGF的抗体联合用药、以及双特异性抗体在动物模型中表现出显著的抑瘤效果，且优于任意靶点单克隆抗体的药效(Hashizume,H.,et al.(2010).Cancer Res 70(6):2213-2223.Kienast,Y.,et al.(2013).Clin Cancer Res 19(24):6730-6740.)。因此，同时阻断VEGF和ANG2可以更有效抑制异常血管生成，达到治疗与血管生成相关的疾病的目的。
目前本领域内报道了几种处于临床阶段的同时阻断VEGF和ANG2信号通路的双特异性抗体及其用途，包括Vanucizumab(参见例如WO2010040508A1)、Faricimab(参见例如WO2014009465A1)和BI836880(参见例如US9527925B2)，但仍亟待于研发新的高效的抗ANG2-VEGF双特异性抗体。
发明内容
在一方面，本发明提供一种双特异性抗体，其包含结合ANG2的第一抗原结合部分以及结合VEGF的第二抗原结合部分，所述第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3序列选自(1)-(3)中任一项：(1)SEQ ID NO:25所示HCDR1序列；SEQ ID NO:26所示HCDR2序列；SEQ ID NO:27所示HCDR3序列；SEQ ID NO:28所示LCDR1序列；SEQ ID NO:29所示LCDR2序列；和SEQ ID NO:30所示LCDR3序列；(2)SEQ ID NO:33所示HCDR1序列；SEQ ID NO:34所示HCDR2序列；SEQ ID NO:35所示HCDR3序列；SEQ ID NO:36所示LCDR1序列；SEQ ID NO:37所示LCDR2序列；和SEQ ID NO:38所示LCDR3序列；(3)SEQ ID NO:41所示HCDR1序列；SEQ ID NO:42所示HCDR2序列；SEQ ID NO:43所示HCDR3序列；SEQ ID NO:44所示LCDR1序列；SEQ ID NO:45所示LCDR2序列；和SEQ ID NO:46所示LCDR3序列。
在一实施方案中，所述重链可变区和轻链可变区选自(1)-(3)中任一项：(1)重链可变区，其包含SEQ ID NO:31的氨基酸序列；轻链可变区，其包含SEQ ID NO:32的氨基酸序列；(2)重链可变区，其包含SEQ ID NO:39的氨基酸序列；轻链可变区，其包含SEQ ID NO:40的氨基酸序列；(3)重链可变区，其包含SEQ ID NO:47的氨基酸序列；轻链可变区，其包含SEQ ID NO:48的氨基酸序列。
在一实施方案中，所述第二抗原结合部分包含结合VEGF的免疫球蛋白单可变结构域(VHH)。在优选的实施方案中，所述免疫球蛋白单可变结构域包含：SEQ ID NO:49所示CDR1序列、SEQ ID NO:50所示CDR2序列以及SEQ ID NO:51所示CDR3序列。
在一实施方案中，所述第一抗原结合部分和所述第二抗原结合部分通过接头连接。
在一实施方案中，本发明的双特异性抗体进一步包含人IgG1的重链恒定区和人κ轻链恒定区。在优选的实施方案中，所述人IgG1的重链恒定区在根据EU编号的第234和235位包含亮氨酸至丙氨酸的突变。
本发明还提供一种多核苷酸，其编码本发明的双特异性抗体。
本发明还提供一种表达载体，其包含本发明的多核苷酸。
本发明还提供一种宿主细胞，其包含本发明的多核苷酸或表达载体。
本发明进一步提供一种产生本发明的双特异性抗体的方法，其包括：
(Ⅰ)在合适条件下培养本发明的宿主细胞以表达所述双特异性抗体，以及
(Ⅱ)从宿主细胞或其培养物分离所述双特异性抗体。
本发明还提供一种药物组合物，其包含本发明的双特异性抗体，以及药学上可接受的载剂。
本发明还提供本发明的双特异性抗体或药物组合物在制备用于治疗以下疾病的药物中的用途：(1)血管生成相关的眼病；或(2)癌症。
附图说明
图1A-1E显示了抗ANG2-VEGF双特异性抗体的结构示意图：图1A，BsAb1、BsAb13和BsAb21；图1B，BsAb2、BsAb14和BsAb22；图1C，BsAb3、BsAb15和BsAb23；图1D，BsAb4、BsAb16和BsAb24；图1E，BsAb29、BsAb30和BsAb31。
图2A-2E显示了抗ANG2-VEGF双特异性抗体对重组蛋白hVEGF-His的结合活性。
图3A-3E显示了抗ANG2-VEGF双特异性抗体对重组蛋白hANG2-His的结合活性。
图4A-4C显示了抗ANG2-VEGF双特异性抗体中和VEGF的活性。
图5A-5B显示了抗ANG2-VEGF双特异性抗体对ANG2/Tie2的阻断活性。
图6A-6C显示了抗ANG2-VEGF双特异性抗体抑制HUVEC细胞增殖的活性。
图7A-7C显示了抗ANG2-VEGF双特异性抗体抑制人结肠癌细胞皮下移植瘤生长效果：图7A显示了各组小鼠的平均肿瘤体积随时间变化情况；图7B显示了各组小鼠的平均体重随时间变化情况；图7C显示了实验结束时各组小鼠的平均瘤重。
具体实施方式
定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文所用，表述“包括”、“包含”、“含有”和“具有”是开放式的，表示包括所列举的元素、步骤或组分但不排除其他未列举的元素、步骤或组分。表述“由……组成”不包括未指定的任何元素、步骤或组分。表述“基本上由……组成”是指范围限于指定的元素、步骤或组分，加上不显著影响要求保护的主题的基本和新颖性质的任选存在的元素、步骤或组分。应当理解，表述“基本上由……组成”和“由……组成”涵盖在表述“包括”的含义之内。
如本文所用，“双特异性抗体”是指特异性结合两种不同抗原的抗体。本发明的双特异性抗体包含特异性结合ANG2和VEGF两种不同抗原的抗原结合部分。
如本文所用，“抗体”指免疫球蛋白或其片段，其通过至少一个抗原结合位点特异性结合抗原表位。术语“抗体”包括多特异性抗体(例如双特异性抗体)、全人源抗体、非人抗体、人源化抗体、嵌合抗体、单域抗体以及抗原结合片段。抗体可以是合成的(例如通过化学偶联或生物偶联产生的)、酶促处理得到的或重组产生的。抗体包括任何免疫球蛋白类型(例如，IgG、IgM、IgD、IgE、IgA和IgY)、任何类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类(例如，IgG2a和IgG2b)。抗体可以是多价的，即包含多个抗原结合位点。例如，抗体可以是二价、三价、四价、五价和六价，其分别包含两个、三个、四个、五个和六个抗原结合位点。
如本文所用，“抗原结合片段”指全长抗体的部分，其少于全长，但是至少包含全长抗体的部分可变区(例如包含一个或多个CDR和/或一个或多个抗原结合位点)，并因此保留全长抗体的至少部分特异性结合抗原的能力。抗原结合片段的实例包括但不限于sdAb(例如重链抗体的可变结构域)、Fv、scFv、dsFv、scdsFv、Fab、scFab、Fab'、F(ab') 2、双抗体、Fd和Fd'片段以及其他片段(例如包含修饰的片段)。
如本文所用，“全长抗体”通常包含四条多肽：两条重链(HC)和两条轻链(LC)。每条轻链包含轻链可变区(VL)和轻链恒定区(CL)。每条重链包含重链可变区(VH)以及重链恒定区(CH)。一般而言，全长抗体的重链恒定区从N端至C端可以包含CH1-铰链区-CH2-CH3。在某些免疫球蛋白类型(例如IgM和IgE)中，重链恒定区从N端至C端可以包含CH1-铰链区-CH2-CH3-CH4。
轻链可变区和重链可变区各自可以包含三个高度可变的“互补决定区(CDR)”和四个相对保守的“框架区(FR)”，并且从N端至C端以FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的次序连接。在本文中，轻链可变区的CDR(CDRL或LCDR)可以称为LCDR1、LCDR2和LCDR3，重链可变区的CDR(CDRH或HCDR)可以称为HCDR1、HCDR2和HCDR3。
在本领域中可以通过多种方法来定义抗体的CDR，例如基于抗体的三维结构和CDR环的拓扑学的Chothia(参见例如Chothia,C.et al.,Nature,342,877-883(1989)；和Al-Lazikani,B.et al.,J.Mol.Biol.,273,927-948(1997))、基于抗体序列可变性的Kabat(参见例如Kabat,E.A.et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242)、AbM(Martin,A.C.R.and J.Allen(2007)“Bioinformatics tools for antibody engineering,”in S.Dübel(ed.),Handbook of Therapeutic Antibodies.Weinheim:Wiley-VCH Verlag,pp.95–118)、Contact(MacCallum,R.M.et al.,(1996)J.Mol.Biol.262:732-745)、IMGT(Lefranc,M.-P.,2011(6),IMGT,the International ImMunoGeneTics Information System Cold Spring Harb Protoc.；和Lefranc,M.-P.et al.,Dev.Comp.Immunol.,27,55-77(2003))，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应理解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明的权利要求中请求保护的范围是基于AbM定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当落在本发明的保护范围中。
因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
如本文所用，术语“框架区”和“构架区”可以互换使用。如本文中所使用的，术语“框架区”、“构架区”或“FR”残基是指抗体可变区中除了如上定义的CDR序列以外的那些氨基酸残基。
通常认为，由一个VH和一个VL通过非共价作用构成的“Fv”片段为包含抗原结合位点的最小的抗原结合片段。但是单可变结构域(单域抗体)也具有抗原结合能力。可以通过肽接头将VH和VL连接来获得“单链Fv(scFv)”。通过向Fv或scFv中引入二硫键可以分别获得“二硫键稳定的Fv(dsFv)”或“单链二硫键稳定的Fv(scdsFv或dsscFv)”。
如本文所用，“Fab”包含一个完整的抗体轻链(VL-CL)和抗体重链可变区和一个重链恒定区(VH-CH1，也称为Fd)。用肽接头将“Fab”中的CL和CH1连接可以获得单链“Fab(scFab)”。“F(ab') 2”基本上包含通过铰链区的二硫键连接的两个Fab片段。“Fab'”为F(ab') 2的一半，其可以通过还原F(ab') 2铰链区的二硫键获得。
如本文所用，“单域抗体(sdAb)”或“纳米抗体”是指包含单个免疫球蛋白可变结构域(单可变结构域)作为功能性抗原结合片段的抗体。与全长抗体的可变区类似，单可变结构域通常包含形成抗原结合位点的CDR1、CDR2和CDR3以及起支持作用的框架区。单可变结构域可以例如是重链抗体的可变结构域(variable domain of heavy-chain antibody，VHH)、鲨鱼的IgNAR可变结构域、人轻链抗体可变结构域和重链抗体可变结构域。
如本文所用，氨基酸序列的“百分比(％)序列相同性”、“序列相同性”具有本领域公认的定义，其指通过序列比对(例如通过人工检视或可公知的算法)确定的两个多肽序列之间相同的百分比。可以 使用本领域技术人员已知的方法确定，例如使用可公开获得的计算机软件如BLAST、BLAST-2、Clustal Omega和FASTA软件。
可以修饰多肽(例如抗体的CDR区、框架区和恒定区)，例如进行一个或多个氨基酸的取代、添加和/或缺失，而不改变多肽的功能。取代优选为氨基酸的保守取代。合适的保守取代是本领域技术人员熟知的。此外，可以使用本领域已知的方法修饰抗体来改变其性能，例如改变抗体糖基化修饰的类型，改变形成链间二硫键的能力，改变抗体的效应功能(例如增强或降低抗体依赖性细胞介导的细胞毒性(ADCC)和/或补体依赖性细胞毒性(CDC)，或者为抗体缀合物的制备提供活性基团。这类修饰的抗体也涵盖在本发明的抗体的范围之内。
“亲和力”或“结合亲和力”用来衡量抗体和抗原之间通过非共价作用相互结合的强度。“亲和力”的大小通常可以报告为平衡解离常数K D或EC 50。K D可以通过测量平衡缔合常数(ka)和平衡解离常数(kd)来计算：K D＝kd/ka。可以用本领域已知的常规技术测定亲和力，例如生物膜干涉技术(可以采用例如ForteBio Octet或Gator检测系统)、表面等离子共振法、酶联免疫测定(ELISA)或流式细胞术(FACS)等。
如本文所用，表述“分离的”是指物质(例如多核苷酸或多肽)与其存在的来源或环境是分离的，即基本上不包含其他任何成分。本发明的双特异性抗体或编码其的多核苷酸可以是分离的。
在本文中，术语“多核苷酸”和“核酸”可以互换用于表示包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物。多核苷酸可以包括脱氧核糖核酸(DNA)和核糖核酸(RNA)。
在本文中，“载体”是用于将外源多核苷酸导入宿主细胞的媒介，当载体转化入适当的宿主细胞时，外源多核苷酸得以扩增或表达。如本文所用，载体的定义涵盖质粒、线性化质粒、病毒载体、粘粒、噬菌体载体、噬菌粒、人工染色体(例如，酵母人工染色体和哺乳动物人工染色体)等。病毒载体包括但不限于逆转录病毒载体(包括慢病毒载体)、腺病毒载体、腺相关病毒载体、疱疹病毒载体、痘病毒载体和杆状病毒载体等。如本文所用，“表达载体”指能够表达感兴趣多肽的载体。表达载体通常可以包含编码感兴趣多肽的多核苷酸序列和与其可操作地连接的调控序列(如启动子和核糖体结合位点)。
如本文所用，“宿主细胞”是用于接受、保持、复制或扩增载体的细胞。宿主细胞还可以用来表达多核苷酸或载体所编码的多肽。宿主细胞可以是真核细胞或原核细胞。原核细胞例如大肠杆菌(E.coli)或枯草芽孢杆菌(Bacillus subtilis)，真菌细胞例如酵母细胞或曲霉属、昆虫细胞(如S2果蝇细胞或Sf9)以及动物细胞(如成纤维细胞、CHO细胞、COS细胞、HeLa细胞、NSO细胞或HEK293细胞)。
如本文所用，术语“治疗”指对疾病/症状的改善，例如使疾病/症状减轻或消失、防止或减缓疾病/症状的发生、进展和/或恶化。
如本文所用，“有效量”意指活性物质(例如本发明抗体或药物组合物)针对组织、系统、动物、哺乳动物或人诱发的生物或医学反应或期望治疗效应的量。因此，“有效量”可以是防止、治愈、改善、阻滞或部分阻滞疾病或症状(例如癌症)所需的量。本领域技术人员可以根据例如受试者的年龄、身体状况、性别、症状的严重程度、特定组合物或给药途径等因素来确定有效量。
如本文所用，术语“药学上可接受的载剂”是指在药理学和/或生理学上与受试者和活性成分相容的载剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)。
如本文所用，哺乳动物的实例包括但不限于人、非人灵长类动物、大鼠、小鼠、牛、马、猪、羊、羊驼、狗、猫等。如本文所用，术语“受试者”是指哺乳动物，例如人。在一些实施方案中，受试者是患者，例如癌症患者。
抗ANG2-VEGF双特异性抗体
在一方面，本发明提供一种双特异性抗体，其包含结合ANG2的第一抗原结合部分以及结合VEGF的第二抗原结合部分，所述第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3序列选自(1)-(3)中任一项：(1)SEQ ID NO:25所示HCDR1序列；SEQ ID NO:26所示HCDR2序列；SEQ ID NO:27所示HCDR3序列；SEQ ID NO:28所示LCDR1序列；SEQ ID NO:29所示LCDR2序列；和SEQ ID NO:30所示LCDR3序列；(2)SEQ ID NO:33所示HCDR1序列；SEQ ID NO:34所示HCDR2序列；SEQ ID NO:35所示HCDR3序列；SEQ ID NO:36所示LCDR1序列；SEQ ID NO:37所示LCDR2序列；和SEQ ID NO:38所示LCDR3序列；(3)SEQ ID NO:41所示HCDR1序列；SEQ ID NO:42所示HCDR2序列；SEQ ID NO:43所示HCDR3序列；SEQ ID NO:44所示LCDR1序列；SEQ ID NO:45所示LCDR2序列；和SEQ ID NO:46所示LCDR3序列。
在一些实施方案中，所述重链可变区包含SEQ ID NO:31、SEQ ID NO:39或SEQ ID NO:47的氨基酸序列。在一些实施方案中，所述重链可变区包含与SEQ ID NO:31、SEQ ID NO:39或SEQ ID NO:47具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。在一些实施方案中，所述重链可变区多肽包含与SEQ ID NO:31、SEQ ID NO:39或SEQ ID NO:47相比具有一个或多个(例如1、2、3、4、5、6、7、8、9或10个)氨基酸的取代、添加和/或缺失的氨基酸序列。优选地，所述氨基酸的取代、添加和/或缺失不发生在CDR区。
在一些实施方案中，所述轻链可变区包含SEQ ID NO:32、SEQ ID NO:40或SEQ ID NO:48的氨基酸序列。在一些实施方案中，所述轻链可变区包含与SEQ ID NO:32、SEQ ID NO:40或SEQ ID NO:48具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。在一些实施方案中，所述轻链可变区包含与SEQ ID NO:32、SEQ ID NO:40或SEQ ID NO:48相比具有一个或多个(例如1、2、3、4、5、6、7、8、9或10个)氨基酸的取代、添加和/或缺失的氨基酸序列。优选地，所述氨基酸的取代、添加和/或缺失不发生在CDR区。
在一些实施方案中，所述重链可变区包含SEQ ID NO:31、SEQ ID NO:39或SEQ ID NO:47的氨基酸序列；所述轻链可变区包含SEQ ID NO:32、SEQ ID NO:40或SEQ ID NO:48的氨基酸序列。
在一具体实施方案中，所述重链可变区和轻链可变区选自(1)-(3)中任一项：(1)重链可变区，其包含SEQ ID NO:31的氨基酸序列；轻链可变区，其包含SEQ ID NO:32的氨基酸序列；(2)重链可变区，其包含SEQ ID NO:39的氨基酸序列；轻链可变区，其包含SEQ ID NO:40的氨基酸序列；(3)重链可变区，其包含SEQ ID NO:47的氨基酸序列；轻链可变区，其包含SEQ ID NO:48的氨基酸序列。
第一抗原结合部分和第二抗原结合部分可以包含任何形式的抗原结合片段。在一些实施方案中，第一抗原结合部分为scFv、Fab、Fab'、F(ab') 2、Fv片段或二硫键稳定的Fv(dsFv)。在一实施方案中，第一抗原结合部分为全人源抗体或其抗原结合部分。
在一些实施方案中，第二抗原结合部分包含结合VEGF的免疫球蛋白单可变结构域。在一实施方案中，所述单可变结构域为VHH。在一实施方案中，所述单可变结构域为人源化的VHH。在一实施方案中，所述免疫球蛋白单可变结构域包含：SEQ ID NO:49所示CDR1序列、SEQ ID NO:50所示CDR2序列以及SEQ ID NO:51所示CDR3序列。在一具体实施方案中，所述免疫球蛋白单可变结构域包含SEQ ID NO:11的氨基酸序列。在又一具体实施方案中，所述免疫球蛋白单可变结构域包含与SEQ ID NO:11的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列相同性的氨基酸序列。
在一些实施方案中，所述第一抗原结合部分和第二抗原结合部分通过接头连接。接头可以是肽接头或化学键，优选肽接头(又可以称为“连接肽”)。肽接头可以包含不超过30个氨基酸。示例性的肽接头可以包括但不限于聚甘氨酸(G)、聚丙氨酸(A)、聚丝氨酸(S)或其组合，例如GGAS、GGGS、GGGSG或者(G 4S) n，其中n为1-20的整数。优选地，n为1-5的整数。在一具体实施方案中，肽接头包含SEQ ID NO:12的氨基酸序列。
在一些实施方案中，本发明的双特异性抗体进一步包含免疫球蛋白恒定区。免疫球蛋白恒定区可以衍生自任何物种的免疫球蛋白的重链恒定区(CH)和轻链恒定区(CL)。
重链恒定区可以衍生自任何亚型(例如IgA、IgD、IgE、IgG和IgM)、类别(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或亚类(例如，IgG2a和IgG2b)的免疫球蛋白的重链恒定区或其组合。在优选的实施方案中，重链恒定区至少包含Fc区。在一优选实施方案中，重链恒定区为人IgG1的重链恒定区，其示例性氨基酸序列示于SEQ ID NO:1。在一实施方案中，本发明的双特异性抗体进一步包含人IgG1的Fc区，其示例性氨基酸序列示于SEQ ID NO:3。
在一些实施方案中，本发明的双特异性抗体具有降低的ADCC和CDC效应功能，例如通过在重链恒定区中包含氨基酸取代(突变)。在一些实施方案中，本发明的双特异性抗体可以包含人IgG1的重链恒定区。在一示例性实施方案中，人IgG1的重链恒定区可以包含选自以下的突变：根据EU编号第234位(对应于SEQ ID NO:1的第117位)的亮氨酸突变为丙氨酸(L234A)、第235位(对应于SEQ ID NO:1的第118位)的亮氨酸突变为丙氨酸(L235A)、第265位(对应于SEQ ID NO:1的第148位)的天冬氨酸突变为丙氨酸(D265A)、第297位(对应于SEQ ID NO:1的第180位)的天冬酰胺突变为丙氨酸(N297A)或甘氨酸(N297G)或谷氨酰胺(N297Q)、第322位(对应于SEQ ID NO:1的第205位)的赖氨酸突变为丙氨酸(K322A)、第329位(对应于SEQ ID NO:1的第212位)的脯氨酸突变为丙氨酸(P329A)及其组合。在一些实施方案中，本发明的双特异性抗体可以包含人IgG4的重链恒定区。在一示例性实施方案中，人IgG4的重链恒定区可以包含选自以下的突变：根据EU编号第228位的丝氨酸突变为脯氨酸(S228P)、第234位的苯丙氨酸突变为丙氨酸(F234A)、第235位的亮氨酸突变为丙氨酸(L235A)及其组合。
如本文所用，“抗体依赖性细胞介导的细胞毒性(ADCC)”是指与靶细胞上的抗原结合的抗体(通过其Fc区)结合到细胞毒性细胞(例如，自然杀伤(NK)细胞、中性粒细胞和巨噬细胞)表面的Fc受体(FcR)上，从而将这些细胞毒性细胞靶向至靶细胞，并且随后利用细胞毒素杀死靶细胞。可以通过本 领域已知的方法评估抗体的ADCC活性(参见例如US5821337A)。
如本文所用，“补体依赖的细胞毒性(CDC)”是指与靶细胞上的抗原结合的抗体激活补体系统，从而引起对靶细胞的裂解。可以通过本领域已知的方法评估抗体的CDC活性(参见例如Gazzano-Santoro et al.,J.Immunol.Methods 202:163(1996))。
轻链恒定区可以衍生自λ(Lambda)轻链或κ(Kappa)轻链恒定区。在一优选实施方案中，轻链恒定区为人κ轻链恒定区。在一实施方案中，轻链恒定区包含SEQ ID NO:2的氨基酸序列。
在一些实施方案中，本发明的双特异性抗体进一步包含免疫球蛋白的重链恒定区(CH)和轻链恒定区(CL)。在一实施方案中，所述重链恒定区为人IgG1或人IgG4的重链恒定区，并且所述轻链恒定区为人κ轻链恒定区。在一实施方案中，所述人IgG1的重链恒定区在根据EU编号的第234和235位包含亮氨酸至丙氨酸的突变(L234A/L235A)。在一实施方案中，所述人IgG4的重链恒定区在根据EU编号第228位、第234位和235位分别包含丝氨酸至脯氨酸、苯丙氨酸至丙氨酸和亮氨酸至丙氨酸的突变(S228P/F234A/L235A)。
在一实施方案中，第一抗原结合部分的VH和VL分别融合至CH和CL的N端，并且第二抗原结合部分的单可变结构域(例如VHH)任选地通过接头融合至所述VH的N端、所述VL的N端、所述CH的C端和/或所述CL的C端。
在一些实施方案中，本发明的双特异性抗体包含第一多肽和第二多肽。在一实施方案中，所述第一多肽包含第一抗原结合部分的重链可变区(VH)和重链恒定区(CH)，所述第二多肽包含第一抗原结合部分的轻链可变区(VL)和轻链恒定区(CL)，第二抗原结合部分的单可变结构域(例如VHH)任选地通过接头融合至：(1)所述VH的N端；(2)所述VL的N端；(3)所述VH和VL的N端；(4)所述CH的C端；(5)所述CL的C端；(6)所述CH和CL的C端；(8)所述VH的N端和所述CH的C端；或者(9)所述VL的N端和所述CH的C端。
在一实施方案中，所述第一多肽和第二多肽选自(1)-(5)中任一项：
(1)所述第一多肽具有式(Ⅰ)的结构，所述第二多肽具有式(Ⅱ)的结构
VH-CH-Linker-VHH   式(Ⅰ)，
VL-CL   式(Ⅱ)；
(2)所述第一多肽具有式(Ⅲ)的结构，所述第二多肽具有式(Ⅱ)的结构
VHH-Linker-VH-CH   式(Ⅲ)；
(3)所述第一多肽具有式(Ⅲ)的结构，所述第二多肽具有式(Ⅳ)的结构
VHH-Linker-VL-CL   式(Ⅳ)；
(4)所述第一多肽具有式(Ⅴ)的结构，所述第二多肽具有式(Ⅳ)的结构
VH-CH   式(Ⅴ)；
(5)所述第一多肽具有式(Ⅴ)的结构，所述第二多肽具有式(Ⅵ)的结构
VL-CL-Linker-VHH   式(Ⅵ)；
其中
VH和VL分别为第一抗原结合部分的重链可变区和轻链可变区；
VHH为第二抗原结合部分的单可变结构域；
CH和CL分别为免疫球蛋白的重链恒定区和轻链恒定区；
Linker为接头。
在一具体实施方案中，所述第一多肽和第二多肽选自(1)-(15)中任一项：
(1)第一多肽，其包含SEQ ID NO:13的氨基酸序列；第二多肽，其包含SEQ ID NO:6的氨基酸序列；
(2)第一多肽，其包含SEQ ID NO:14的氨基酸序列；第二多肽，其包含SEQ ID NO:6的氨基酸序列；
(3)第一多肽，其包含SEQ ID NO:5的氨基酸序列；第二多肽，其包含SEQ ID NO:15的氨基酸序列；
(4)第一多肽，其包含SEQ ID NO:5的氨基酸序列；第二多肽，其包含SEQ ID NO:16的氨基酸序列；
(5)第一多肽，其包含SEQ ID NO:17的氨基酸序列；第二多肽，其包含SEQ ID NO:8的氨基酸序列；
(6)第一多肽，其包含SEQ ID NO:18的氨基酸序列；第二多肽，其包含SEQ ID NO:8的氨基酸序列；
(7)第一多肽，其包含SEQ ID NO:7的氨基酸序列；第二多肽，其包含SEQ ID NO:19的氨基酸序列；
(8)第一多肽，其包含SEQ ID NO:7的氨基酸序列；第二多肽，其包含SEQ ID NO:20的氨基酸序列；
(9)第一多肽，其包含SEQ ID NO:21的氨基酸序列；第二多肽，其包含SEQ ID NO:10的氨基酸序列；
(10)第一多肽，其包含SEQ ID NO:22的氨基酸序列；第二多肽，其包含SEQ ID NO:10的氨基酸序列；
(11)第一多肽，其包含SEQ ID NO:9的氨基酸序列；第二多肽，其包含SEQ ID NO:23的氨基酸序列；
(12)第一多肽，其包含SEQ ID NO:9的氨基酸序列；第二多肽，其包含SEQ ID NO:24的氨基酸序列；
(13)第一多肽，其包含SEQ ID NO:14的氨基酸序列；第二多肽，其包含SEQ ID NO:15的氨基酸序列；
(14)第一多肽，其包含SEQ ID NO:18的氨基酸序列；第二多肽，其包含SEQ ID NO:19的氨基酸序列；
(15)第一多肽，其包含SEQ ID NO:22的氨基酸序列；第二多肽，其包含SEQ ID NO:23的氨基酸序列。
在一实施方案中，所述第一多肽在以下氨基酸位置包含亮氨酸至丙氨酸的突变：SEQ ID NO:5或SEQ ID NO:13的第238和239位(L238A/L239A)；SEQ ID NO:7、SEQ ID NO:9、SEQ ID NO:17或SEQ ID NO:21的第242和243位(L242A/L243A)；SEQ ID NO:14的第382和383位(L382A/L383A)；或者SEQ ID NO:18或SEQ ID NO:22的第386和387位(L386A/L387A)。
在一具体实施方案中，所述第一多肽和第二多肽选自(1)-(15)中任一项：
(1)第一多肽，其包含与SEQ ID NO:13相比具有L238A和L239A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:6的氨基酸序列；
(2)第一多肽，其包含与SEQ ID NO:14相比具有L382A和L383A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:6的氨基酸序列；
(3)第一多肽，其包含与SEQ ID NO:5相比具有L238A和L239A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:15的氨基酸序列；
(4)第一多肽，其包含与SEQ ID NO:5相比具有L238A和L239A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:16的氨基酸序列；
(5)第一多肽，其包含与SEQ ID NO:17相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:8的氨基酸序列；
(6)第一多肽，其包含与SEQ ID NO:18相比具有L386A和L387A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:8的氨基酸序列；
(7)第一多肽，其包含与SEQ ID NO:7相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:19的氨基酸序列；
(8)第一多肽，其包含与SEQ ID NO:7相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:20的氨基酸序列；
(9)第一多肽，其包含与SEQ ID NO:21相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:10的氨基酸序列；
(10)第一多肽，其包含与SEQ ID NO:22相比具有L386A和L387A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:10的氨基酸序列；
(11)第一多肽，其包含与SEQ ID NO:9相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:23的氨基酸序列；
(12)第一多肽，其包含与SEQ ID NO:9相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:24的氨基酸序列；
(13)第一多肽，其包含与SEQ ID NO:14相比具有L382A和L383A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:15的氨基酸序列；
(14)第一多肽，其包含与SEQ ID NO:18相比具有L386A和L387A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:19的氨基酸序列；
(15)第一多肽，其包含与SEQ ID NO:22相比具有L386A和L387A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:23的氨基酸序列。
本发明的双特异性抗体可以是多价抗体，例如二价、三价、四价、五价和六价。在一些实施方案中，本发明的双特异性抗体是四价抗体，其包含两个第一抗原结合部分和两个第二抗原结合部分。在一实施方案中，本发明的双特异性抗体是六价抗体，其包含两个第一抗原结合部分和四个第二抗原结合部分。
在一实施方案中，本发明的双特异性抗体包含两条第一多肽和两条第二多肽，其中所述第一多肽相同或不同，所述第二多肽相同或不同。在一具体实施方案中，本发明的双特异性抗体包含两条相同的第一多肽和两条相同的第二多肽。所述第一多肽和第二多肽如上所述。
在一些实施方案中，本发明的双特异性抗体特异性结合ANG2和VEGF。在一些实施方案中，本发明的双特异性抗体阻断ANG2和VEGF与其各自受体的结合。在一些实施方案中，本发明的双特异性抗体特异性结合ANG2，但不结合或基本不结合ANG1。本文所用的表述“不结合”或“基本不 结合”指，相对于对ANG2的结合能力而言，本发明的双特异性抗体针对ANG1的结合能力明显低，例如如实施例4所述。
可以使用本领域已知的方法制备和生产抗体或其抗原结合片段。这类方法可以包括例如，从噬菌体展示文库、酵母展示文库、永生化的B细胞(例如，小鼠B细胞杂交瘤细胞或EBV永生化的B细胞)制备和分离抗体或抗原结合片段的编码核酸。还可以用免疫动物的方法，例如用抗原或编码抗原的DNA免疫动物(例如人源化小鼠)，然后从免疫后的动物分离表达抗体的B细胞。还可以从免疫动物或人体中分离或者采用化学合成的方法制备编码抗体或其抗原结合片段的多核苷酸，然后利用多核苷酸构建表达抗体或抗原结合片段的表达载体。
多核苷酸、载体和宿主细胞
在另一方面，本发明提供一种多核苷酸，其编码本发明的双特异性抗体。
本发明的多核苷酸可以利用本领域已知的方法获得。例如，本发明的多核苷酸可以分离自人体、噬菌体展示文库、酵母展示文库、免疫动物、永生化的细胞(例如，小鼠B细胞杂交瘤细胞、EBV介导的永生化B细胞)或者化学合成。多核苷酸可以针对用于表达的宿主细胞进行密码子优化。
在又一方面，本发明还提供了包含本发明的多核苷酸的表达载体。表达载体可以进一步包含额外的多核苷酸序列，例如转录调控序列和抗生素抗性基因。
本发明还提供一种宿主细胞，其包含本发明的多核苷酸或表达载体。可以采用本领域已知的各种方法将本发明的多核苷酸或表达载体导入合适的宿主细胞中。这类方法包括但不限于病毒转导、脂质体转染、电穿孔和磷酸钙转染等。在优选的实施方案中，宿主细胞用于表达本发明的双特异性抗体。宿主细胞的实例包括但不限于原核细胞(例如细菌，例如大肠杆菌)和真核细胞(例如酵母、昆虫细胞、哺乳动物细胞)。适合于抗体表达的哺乳动物宿主细胞包括但不限于骨髓瘤细胞、HeLa细胞、HEK293细胞、中国仓鼠卵巢(CHO)细胞和其他适于表达抗体的哺乳动物细胞。
本发明还提供了一种产生本发明的双特异性抗体的方法，其包括：
(Ⅰ)在合适条件下培养本发明宿主细胞以表达本发明的双特异性抗体，以及
(Ⅱ)从宿主细胞或其培养物分离所述双特异性抗体。
药物组合物
本发明还提供药物组合物，其包含本发明的双特异性抗体，以及药学上可接受的载剂。
药学上可接受的载剂可以包括但不限于：稀释剂、粘合剂和胶粘剂、润滑剂、崩解剂、防腐剂、媒介物、分散剂、助流剂、甜味剂、包衣、赋形剂、防腐剂、抗氧化剂(如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠、抗坏血酸棕榈酸酯、丁羟茴醚(BHA)、丁羟甲苯(BHT)、卵磷脂、没食子酸丙酯、α-生育酚、柠檬酸、乙二胺四乙酸(EDTA)、山梨糖醇、酒石酸、磷酸等)、增溶剂、胶凝剂、软化剂、溶剂(例如，水、酒精、乙酸和糖浆)、缓冲剂(例如，磷酸盐缓冲剂、组氨酸缓冲剂和乙酸盐缓冲剂)、表面活性剂(例如非离子表面活性剂，例如聚山梨酯80、聚山梨酯20、泊洛沙姆或聚乙二醇)、抗细菌剂、抗真菌剂、等渗剂(例如海藻糖、蔗糖、甘露醇、山梨醇、乳糖、葡萄糖)、吸收延迟剂、螯合剂和乳化剂。对于包含抗体或者抗体缀合物的组合物而言，合适的载剂可以选自缓冲剂(例如柠檬酸盐缓冲液、乙酸盐缓冲液、磷酸盐缓冲液、组氨酸缓冲液、组氨酸盐缓冲液)、等渗剂(例如海藻糖、蔗糖、甘露醇、山梨醇、乳糖、葡萄糖)、非离子表面活 性剂(例如聚山梨酯80、聚山梨酯20、泊洛沙姆)或其组合。
本文提供的药物组合物可以为多种剂型，包括但不限于固体、半固体、液体、粉末或冻干形式。优选地，药物组合物适合于静脉内、肌内、皮下、肠胃外、脊柱或表皮施用(如通过注射或输注)。
本文提供的药物组合物可以通过多种途径给药。给药途径包括但不限于肠胃外(例如，静脉内、皮下、皮内、肌肉内或腔内)、局部(例如瘤内)、硬膜外或粘膜(例如鼻内、口服、阴道、直肠、舌下或局部)。给药方法可以为例如注射或输注。
作为一般性指导，本发明的双特异性抗体的给药剂量范围可以为约0.0001至100mg/kg，更通常为0.01至20mg/kg受试者体重。例如，给药剂量可以是0.3mg/kg体重、1mg/kg体重、3mg/kg体重、5mg/kg体重，10mg/kg体重或20mg/kg体重，或在1-20mg/kg范围内。示例性的治疗方案需要每周给药一次、每两周一次、每三周一次、每四周一次、每月一次、每3个月一次、每3-6个月一次、或起始给药间隔略短后期给药间隔加长。
治疗
不希望受任何理论束缚，本发明的双特异性抗体通过结合至ANG2和VEGF来阻断ANG2和VEGF信号通路，包括阻断与ANG2和VEGF信号通路相关的血管生成。如本文所用，“血管生成”是指新血管的形成。研究表明，血管生成与多种疾病相关，例如癌症和血管生成相关的眼病。
在一总的方面，提供本发明的双特异性抗体或药物组合物用于癌症和血管生成相关的眼病。本发明还提供本发明的双特异性抗体或药物组合物在制备用于治疗癌症和血管生成相关的眼病的药物中的用途。本发明还提供在受试者中治疗癌症和血管生成相关的眼病的方法，其包括向所述受试者给药有效量的本发明的双特异性抗体或药物组合物。
血管生成相关的眼病可以是与脉络膜和视网膜血管性疾病相关的眼部疾病，包括但不限于脉络膜新生血管性疾病、视网膜新生血管性疾病和与血管渗漏相关的疾病。在一实施方案中，血管生成相关的眼病为黄斑变性(例如干性或湿性年龄相关性黄斑变性(AMD))、视网膜静脉阻塞、视网膜病变、早产儿视网膜病变(ROP)、糖尿病视网膜病变、新生血管性青光眼、病理性近视、黄斑水肿、视网膜水肿、糖尿病性黄斑水肿(DME)或脉络膜新生血管性疾病。
如本文所用，术语“癌症”或“肿瘤”是指或描述个体中通常以不受调节的细胞生长为特征的生理状况。优选地，癌症为与血管生成相关的实体瘤。癌症可以包括原发癌和转移癌。癌症的非限制性实例包括肺癌(例如非小细胞肺癌、小细胞肺癌、肺腺癌和肺鳞状细胞癌)、肝癌(例如肝细胞癌)、胰腺癌、皮肤癌、头颈癌、黑素瘤、卵巢癌、结肠直肠癌、胃癌、乳腺癌、前列腺癌、子宫癌、霍奇金淋巴瘤、食管癌、肛门癌、内分泌系统癌症、甲状腺癌、甲状旁腺癌、肾癌(例如肾细胞癌、肾盂癌和肾上腺癌)、软组织肉瘤、膀胱癌、中枢神经系统(CNS)肿瘤、间皮瘤、胶质瘤、脑脊膜瘤和垂体腺瘤。在一优选实施方案中，所述癌症为结肠直肠癌、肺癌、乳腺癌、卵巢癌、胃癌或肝细胞癌。
试剂盒
本发明还提供试剂盒，其包含本发明的抗ANG2-VEGF双特异性抗体或药物组合物。在一实施方案中，试剂盒还包含本发明的双特异性抗体或药物组合物的使用说明。试剂盒还可以包含合适的容器，例如安瓿瓶。在一些实施方案中，试剂盒还包括给药的装置。试剂盒还可以包含标签，其用 于表明试剂盒内容物的预期用途和/或使用方法。术语“标签”包括在试剂盒上或与试剂盒一起提供的或以其他方式随试剂盒提供的任何书面的或记录的材料。
有益效果
本发明的抗ANG2-VEGF双特异性抗体实现以下有益效果中的至少一个：
(1)特异性结合ANG2，但不结合或基本不结合ANG1；
(2)特异性结合VEGF并中和VEGF；
(3)阻断ANG2与Tie2的结合；
(4)抑制ANG2和VEGF活性相关的血管生成；和
(5)抑制肿瘤生长。
实施例
通过参考以下实施例将更容易地理解本文一般地描述的本发明，这些实施例是以举例说明的方式提供的，并且不旨在限制本发明。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书进行。
实施例1 原材料制备
1.1抗原制备
通过PCR扩增在抗原VEGF(hVEGF，UniProt ID:P15692-4，SEQ ID NO:52)和抗原Ang2(hANG2，Uniprot ID:O15123-1，SEQ ID NO:53)的编码序列(由通用生物科技股份有限公司合成)的3’端连接人IgG1 Fc段(SEQ ID NO:3)或His标签的编码序列，然后通过同源重组的方法构建至真核表达载体pcDNA3.4(Invitrogen)来制备重组蛋白hVEGF-Fc、hANG2-Fc、hANG2-His和hVEGF-His的表达载体。将重组蛋白表达载体分别转化到大肠杆菌DH5α中，37℃过夜培养，然后利用无内毒素质粒提取试剂盒(OMEGA，D6950-01)进行质粒提取，通过Expi293 TM表达系统试剂盒(ThermoFisher，A14635)表达，瞬转方法参见该试剂盒说明书。
在转染7天后，将细胞表达上清高速离心10min，hANG2-Fc和hVEGF-Fc表达上清用COLUMN XK16/20(购自Cytiva)进行亲和纯化，然后用100mM乙酸钠(pH3.0)洗脱目的蛋白，接着用1M Tris-HCl中和；hANG2-His或hVEGF-His表达上清用Ni Smart Beads 6FF(常州天地人和生物科技有限公司，SA036050)进行亲和纯化，然后用梯度浓度的咪唑洗脱目的蛋白。洗脱下来的各蛋白分别通过超滤浓缩管(Millipore，UFC901096)换液至PBS缓冲液中。经SDS-PAGE鉴定和活性鉴定合格后于-80℃冻存待用。
1.2阳性对照抗体的制备
使用的抗VEGF阳性对照抗体为Bevacizumab(根据专利US9527925B2中提供的抗体序列信息合成)以及本申请人自主研发的P30-10-26(VHH VEGF-huFc，氨基酸序列SEQ ID NO:4所示)；使用的抗ANG2阳性对照抗体为Nesvacumab(根据专利申请US20170355762A1所披露的序列合成)以及本申请人自主研发的IgG1型式的全人源抗体17(重链氨基酸序列如SEQ ID NO:5所示，轻链氨基酸序列如SEQ ID NO:6所示)、78A14(重链氨基酸序列如SEQ ID NO:7所示，轻链氨基酸序列如SEQ ID NO:8所示)和78A74-7(重链氨基酸序列如SEQ ID NO:9所示，轻链氨基酸序列如SEQ ID NO:10 所示)；使用的抗ANG2-VEGF阳性对照抗体为Faricimab(根据专利WO2016073157A1中提供的抗体序列信息合成，购自泰州市百英生物科技有限公司)。
除Faricimab外上述其余对照抗体都采用ExpiCHO瞬转表达系统(Gibco，A29133)进行表达，所得上清经0.22μm滤膜过滤后，采用Protein A/G亲和层析柱亲和法进行纯化，然后用100mM乙酸钠(pH3.0)洗脱目的蛋白，接着用1M Tris-HCl中和，最后通过超滤浓缩管(Millipore，UFC901096)将所得蛋白置换至PBS缓冲液中。纯化的抗体经SDS-PAGE鉴定和活性鉴定后分装保存于-80℃。
1.3细胞株构建
构建了过表达人Tie2的HEK293细胞株(以下简称huTie2-HEK293细胞株)和过表达VEGFR2和NFAT荧光素酶报告基因的HEK293细胞株(以下简称VEGFR2/NFAT-HEK293细胞株)，细胞株构建方法分别如下：
1.3.1 huTie2-HEK293细胞株的构建
通过基因合成技术合成含有编码人Tie2蛋白的DNA片段(NCBI Gene ID:7010)，并将其克隆至表达载体pLVX-Puro(Clontech，632164)获得Tie2表达质粒。使用Gibco的DMEM无血清培养基(货号：12634010)培养HEK293细胞
Figure PCTCN2022139203-appb-000001
使用Invitrogen的电转试剂盒(货号：MPK10096)和电转仪(货号：MP922947)将Tie2表达质粒导入HEK293细胞中，通过终浓度2μg/mL的puromycin的培养基筛选单克隆细胞株，通过FACS鉴定获得huTie2-HEK293细胞株。
1.3.2 VEGFR2/NFAT-HEK293细胞株的构建
首先构建NFAT-HEK293细胞株：将含有荧光素酶基因(其转录和表达处于NFAT应答元件(NFAT-RE)驱动下)的pGL4.30质粒(promega，#E8481)通过电转仪(Invitrogen，Neon TM Transfection System，MP922947)电转化至HEK293细胞
Figure PCTCN2022139203-appb-000002
然后通过终浓度为500μg/mL的Hygromycin B(源培，S160J7)筛选单克隆细胞株进行鉴定。NFAT-HEK293细胞株鉴定合格后，通过实施例1.3.1的方法将表达VEGFR2(基因序列参见NCBI Gene ID:3791，由通用生物合成)的质粒电转至NFAT-HEK293细胞株中，通过FACS鉴定获得VEGFR2/NFAT-HEK293细胞株。
实施例2 抗ANG2-VEGF双特异性抗体的构建
本实施例描述了抗ANG2-VEGF双特异性抗体(BsAb)的构建，其中第一抗原结合部分使用抗ANG2全人源抗体17、78A14或78A74-7的重链可变区(VH)和轻链可变区(VL)(根据AbM定义的HCDR1-3和LCDR1-3以及VH和VL的氨基酸序列(SEQ ID NO:)示于表1A)，第二抗原结合部分使用人源化的抗VEGF单域抗体P30-10-26的VHH(根据AbM定义的CDR1、CDR2和CDR的氨基酸序列分别示于SEQ ID NO:49、SEQ ID NO:50和SEQ ID NO:51；VHH的氨基酸序列示于SEQ ID NO:11)。本发明的双特异性抗体还包含人IgG1重链恒定区(SEQ ID NO:1)或人IgG1重链恒定区突变体(简称为LALA；SEQ ID NO:1的第117和118位亮氨酸突变成丙氨酸)和人Kappa轻链恒定区(SEQ ID NO:2)，分别融合至第一抗原结合部分的VH和VL的C端；以及连接肽(SEQ ID NO:12)。图1A-1E示出候选双特异性抗体的结构示意图。候选双特异性抗体含有两条相同的第一多肽和两条相同的第二多肽，氨基酸序列示于表1B。
制备第一多肽和第二多肽的表达载体，具体方法如下：通过PCR方法扩增目的片段，重叠延伸PCR法将各个片段连接起来，再通过同源重组方法，分别构建至经过改造的真核表达载体质粒pcDNA3.4(Invitrogen)上以获得第一多肽和第二多肽的表达载体。将第一多肽和第二多肽的表达载体 分别转化到大肠杆菌DH5α中，随后利用无内毒素质粒提取试剂盒(OMEGA，D6950-01)进行质粒提取，得到无内毒素的第一多肽链和第二多肽链的表达质粒以供真核表达使用。
表1A
抗体名称 HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 VH VL
17 25 26 27 28 29 30 31 32
78A14 33 34 35 36 37 38 39 40
78A74-7 41 42 43 44 45 46 47 48
表1B
Figure PCTCN2022139203-appb-000003
实施例3 抗ANG2-VEGF双特异性抗体的表达和纯化
实施例2的抗ANG2-VEGF双特异性抗体是通过ExpiCHO瞬转表达系统(Thermo Fisher，A29133)表达的，具体方法如下：转染当天，确认细胞密度为7×10 6至1×10 7个活细胞/mL左右，细胞存活率>98％，此时，用37℃预温的新鲜ExpiCHO表达培养基将细胞调整到终浓度为6×10 6个细胞/mL。用4℃预冷的OptiPRO TM SFM稀释目的质粒(向1mL所述培养基中加入1μg质粒)，同时，用OptiPRO TMSFM稀释ExpiFectamine TMCHO，再将两者等体积混合并轻轻吹打混匀制备成ExpiFectamine TMCHO/质粒DNA混合液，室温孵育1-5min，缓慢加入到准备好的细胞悬液中并同时轻轻摇晃，最后置于细胞培养摇床中，在37℃、8％CO 2条件下培养。在转染后18-22h，向培养液中添加ExpiCHO TMEnhancer和ExpiCHO TMFeed，摇瓶放置于32℃摇床和5％CO 2条件下继续培养。在转染后的第5天，添加相同体积的ExpiCHO TMFeed，缓慢加入的同时轻轻混匀细胞混悬液。在转染7-15天后，将表达有目的蛋白的细胞培养上清于15000g高速离心10min，所得上清用COLUMN XK16/20(购自Cytiva)进行亲和纯化，然后用100mM乙酸钠(pH3.0)洗脱目的蛋白，接着用1M Tris-HCl中和，最后通过超滤浓缩管(Millipore，UFC901096)将所得蛋白置换至PBS缓冲液中。纯化的抗体通过SDS-PAGE和SEC-HPLC鉴定后纯度大于90％，将其分装保存于-80℃。
实施例4 抗ANG2-VEGF双特异性抗体的抗原结合活性分析
4.1 ELISA方法检测抗ANG2-VEGF双特异性抗体对重组蛋白hVEGF-His的结合活性
在96孔ELISA板上包被重组蛋白hVEGF-His，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h，用PBST洗板3次后，加入不同浓度的待测抗体孵育1h。之后，用PBST清洗3次后加入二抗Anti-human-IgG-Fc-HRP(abcam，ab79225)并孵育1h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色。根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)读取OD450值。利用PRISM TM(GraphPad Software，San Diego，CA)分析数据，并且计算EC 50值。
ELISA结合测定结果如图2A-2E所示，候选双特异性抗体表现出与抗体P30-10-26和Faricimab相当的对重组蛋白hVEGF-His的结合活性。
4.2 ELISA方法检测候选双特异性抗体对重组蛋白hANG2-His的结合活性
在96孔ELISA板上包被重组蛋白hANG2-His，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h，用PBST洗板3次后，加入不同浓度的待测抗体孵育1h。之后，用PBST清洗3次后加入二抗Anti-human-IgG-Fc-HRP(abcam，ab79225)并孵育1h。孵育完成后，PBST洗板六次，加TMB(SurModics，TMBS-1000-01)显色。根据显色结果，加入2M HCl终止反应，通过酶标仪(Molecular Devices，SpecterMax 190)读取OD450值。利用PRISM TM(GraphPad Software，San Diego，CA)分析数据，并且计算EC 50值。
ELISA结合测定结果如图3A-3E所示，候选双特异性抗体表现出与抗体17、78A14、78A74-7相当的对重组蛋白hANG2-His的结合活性，且显著优于阳性双特异性抗体Faricimab。
4.3基于ELISA检测抗ANG2全人源抗体对hANG1-Fc的结合活性
在96孔ELISA板上包被30μL/孔的2μg/mL hANG1-Fc(hANG1(Uniprot ID：Q15389-1)与人IgG1 Fc段(SEQ ID NO:3)的融合蛋白；制备方法参见实施例1.1)，4℃过夜。次日，将孔板用PBST洗3次后用5％脱脂牛奶封闭2h，用PBST洗板3次后，加入梯度稀释的待测抗体并孵育1h。之后，用PBST清洗3次后加入anti-human Kappa HRP(Millipore，AP502P)并孵育1h。孵育完成后PBST洗板六次，加TMB显色，根据显色结果加入2M HCl终止反应，通过酶标仪读取OD450值。
结果显示，抗体17、78A14和78A74-7均不与hANG1结合。
实施例5 抗ANG2-VEGF双特异性抗体中和VEGF活性检测
采用实施例1中构建的VEGFR2/NFAT-HEK293细胞株筛选能够中和VEGF活性的候选双特异性抗体。在此细胞株培养体系下，VEGF蛋白通过VEGF-VEGFR2信号轴激活胞内转录因子NFAT的转录活性，从而启动荧光素酶报告基因的转录和表达；当加入候选双特异性抗体，候选双特异性抗体中和VEGF并阻断其与VEGFR2的结合，从而阻断荧光素酶报告基因表达。
具体方法如下：将VEGFR2/NFAT-HEK293细胞株复苏，将传代2-4次且生长状态良好的细胞用于实验，胰酶消化后重悬细胞，常温、300g离心去除上清，随后将细胞用DMEM培养基重悬，计数后将细胞密度调整为4×10 5个细胞/mL，以每孔100μL加至新的96孔板中，置于37℃细胞培养箱中。同时，使用DMEM培养基梯度稀释待测抗体，加入60ng/mL重组蛋白hVEGF-Fc，混合后室温孵育30min。随后，将共孵育后的抗体/hVEGF-Fc混合液加入至96孔板，在37℃培养箱中培养18h。培养结束后，每孔加入30μL荧光素酶底物Bright-Lite(Vazyme，DD1204-03)，震荡2min 后检测96孔板的荧光值并计算IC 50。
结果如图4A-4C和表2A-2C所示，候选双特异性抗体BsAb3、BsAb14、BsAb30、BsAb31、BsAb22和BsAb29中和VEGF的活性与P30-10-26相当；候选双特异性抗体BsAb14-LALA和BsAb22-LALA中和VEGF的效果与P30-10-26和Faricimab相当。
表2A
抗体名称 中和VEGF活性IC 50(nM)
BsAb3 0.194
BsAb14 0.213
BsAb22 0.418
P30-10-26 0.292
表2B
抗体名称 中和VEGF活性IC 50(nM)
BsAb29 1.130
BsAb30 0.608
BsAb31 0.754
P30-10-26 0.878
表2C
抗体名称 中和VEGF活性IC 50(nM)
BsAb14-LALA 0.469
BsAb22-LALA 0.401
Faricimab 0.582
P30-10-26 0.482
实施例6 基于FACS方法检测抗ANG2-VEGF双特异性抗体的阻断ANG2与Tie2结合的活性
基于FACS方法评价候选抗体阻断ANG2和细胞表面表达受体Tie2的结合活性。具体方法如下：将在FACS缓冲液(1×PBS+2％FBS)中梯度稀释的待测抗体以100μL每孔加入96孔圆底板中。然后向该96孔板加入100μL FACS缓冲液中的2μg/mL生物素标记的hANG2-Fc蛋白(biotin-hANG2-Fc)，然后于4℃孵育1h。
将传代2-4次且生长状态良好的huTie2-HEK293细胞用于实验。将1×10 6个/mL的FACS缓冲液中的细胞以每孔100μL加至新的96孔圆底板中，4℃/300g离心并去除上清；随后向细胞加入预先孵育的抗体/biotin-hANG2-Fc混合液，每孔180μL，然后于4℃孵育30min；然后将细胞于4℃/300g离心并去除上清，随后加入200μL/孔的FACS缓冲液并重悬细胞，4℃/300g离心去上清；重复该步骤两次。将PE标记的Streptavidin(Invitrogen，12-4317-87；1:200稀释在FACS缓冲液中)以200μL/孔加入孔中并轻轻吹打重悬细胞，随后将细胞放置于4℃避光孵育30min。孵育结束后将细胞于4℃、300g离心去除上清，加入FACS缓冲液重悬细胞，重复该步骤两次。最后通过流式细胞仪(Beckman，CytoFLEX AOO-1-1102)检测结合至细胞上的biotin-hANG2-Fc的量(表示为PE的平均荧光强度(MFI))。
结果如图5A-5B和表3A-3B所示。其中，图5A和表3A显示候选双特异性抗体BsAb14、BsAb30、 BsAb31和BsAb22的阻断效果与抗体78A14和78A74-7相当；图5B和表3B显示候选双特异性抗体BsAb14-LALA和BsAb22-LALA的阻断效果与抗体78A14和78A74-7相当，显著优于Faricimab。
表3A
抗体名称 阻断IC 50值(nM)
BsAb14 0.415
BsAb22 0.350
BsAb30 0.362
BsAb31 0.299
78A14 0.390
78A74-7 0.226
表3B
抗体名称 阻断IC 50值(nM)
BsAb14-LALA 0.411
BsAb22-LALA 0.320
78A14 0.464
78A74-7 0.440
Faricimab 17.76
实施例7 抗ANG2-VEGF双特异性抗体抑制HUVEC增殖药效检测
本实施例中，验证了抗ANG2-VEGF双特异性抗体BsAb14和BsAb22对VEGF诱导的内皮细胞增殖的抑制效果。具体实施方式如下：将人脐静脉内皮细胞株(HUVEC)(购自北纳生物)复苏，将传代2-4次且生长状态良好的细胞用于实验，胰酶消化后重悬细胞，常温、300g离心去除上清，随后将细胞用0.5％FBS的EBM-2培养基重悬，计数后将细胞密度调整为5×10 4个细胞/mL，以每孔50μL加至新的96孔细胞培养板中，置于37℃细胞培养箱中培养过夜。翌日，使用EBM-2培养基梯度稀释待测抗体，加入400ng/mL重组蛋白hVEGF-Fc，混合后室温孵育30min。随后，将共孵育后的抗体/hVEGF-Fc混合液加入至96孔细胞板，在37℃培养箱中培养72h。培养结束后，每孔加入20μL MTS(Progema，G3581)，震荡2min后检测96孔板OD492值。通过各自的OD492计算增殖抑制率，增殖抑制率(％)＝[1-(OD492 样品-OD492 仅细胞)/(OD492 VEGF-OD492 仅细胞)]×100％。
结果如图6A-6C所示，候选双特异性抗体BsAb14和BsAb22显著抑制VEGF诱导的HUVEC增殖，并且活性与P30-10-26相当；在抗体浓度较高的情况下，例如，抗体浓度达到100nM时，P30-10-26抑制VEGF诱导的HUVEC增殖的活性比Bevacizumab更优。
实施例8 抗ANG2-VEGF双特异性抗体抑制Colo205肿瘤细胞动物模型药效检测
验证了抗ANG2-VEGF双特异性抗体BsAb3、BsAb14和BsAb22在人结肠癌细胞Colo205移植瘤模型中的抑瘤效果，并与抗体Nesvacumab、Bevacizumab和P30-10-26进行比较。具体方法如下：将5×10 6的Colo205细胞(购自北纳生物)皮下注射接种于6-8周龄的雄性裸鼠(北京维通利华实验动物技术有限公司)。实验小鼠在标准条件下饲养。待荷瘤体积达130mm 3左右时，进行分组分笼和给药操作。每组8只荷瘤裸鼠，共7组，包括3个候选双特异性抗体组(BsAb3 3.4952mg/kg、BsAb143.5224mg/kg和BsAb22 3.528mg/kg；等摩尔剂量)、3个阳性对照抗体组(Nesvacumab 3mg/kg和 Bevacizumab 3mg/kg，与双特异性抗体等摩尔剂量；P30-10-26 3mg/kg，摩尔剂量是双特异性抗体的2.3倍)和PBS阴性对照组。给药方式为腹腔注射给药，剂量如图7A-7C所示，每3-4天给药一次，每周给药2次并测量2次肿瘤体积(V＝(L×W 2)/2，其中L是肿瘤直径中最长的，W是肿瘤直径中最短的(mm))，共给药9次。给药结束后10天取肿瘤并测量瘤重。分析各组小鼠的平均肿瘤体积、瘤重和体重的变化，计算抑瘤率，结果示于图7A-7C和表4中。
如图7B所示，各组小鼠体重之间无明显差异，表明小鼠对抗体的耐受性良好。如图7A、7C和表4所示，PBS组小鼠肿瘤增长最快；和PBS组比较，所有抗体都具有显著的抑瘤效果；在等摩尔剂量下，候选双特异性抗体BsAb3、BsAb14和BsAb22的抑瘤效果优于Nesvacumab和Bevacizumab；出人意料地，BsAb14和BsAb22与摩尔数高达其2.3倍的抗VEGF抗体P30-10-26的抑瘤效果相当，表现出优异的抗肿瘤活性。
表4
Figure PCTCN2022139203-appb-000004
本领域技术人员将进一步认识到，在不脱离其精神或中心特征的情况下，本发明可以以其他具体形式来实施。由于本公开的前述描述仅公开了其示例性实施方案，应该理解的是，其他变化被认为是在本发明的范围内。因此，本发明不限于在此详细描述的特定实施方案。相反，应当参考所附权利要求来指示本发明的范围和内容。
序列表
Figure PCTCN2022139203-appb-000005
Figure PCTCN2022139203-appb-000006
Figure PCTCN2022139203-appb-000007
Figure PCTCN2022139203-appb-000008
Figure PCTCN2022139203-appb-000009
Figure PCTCN2022139203-appb-000010
Figure PCTCN2022139203-appb-000011
Figure PCTCN2022139203-appb-000012
Figure PCTCN2022139203-appb-000013

Claims (16)

  1. 一种双特异性抗体，其包含结合ANG2的第一抗原结合部分以及结合VEGF的第二抗原结合部分，所述第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含HCDR1、HCDR2和HCDR3，所述轻链可变区包含LCDR1、LCDR2和LCDR3，其中所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3序列选自(1)-(3)中任一项：
    (1)SEQ ID NO:25所示HCDR1序列；SEQ ID NO:26所示HCDR2序列；SEQ ID NO:27所示HCDR3序列；SEQ ID NO:28所示LCDR1序列；SEQ ID NO:29所示LCDR2序列；和SEQ ID NO:30所示LCDR3序列；
    (2)SEQ ID NO:33所示HCDR1序列；SEQ ID NO:34所示HCDR2序列；SEQ ID NO:35所示HCDR3序列；SEQ ID NO:36所示LCDR1序列；SEQ ID NO:37所示LCDR2序列；和SEQ ID NO:38所示LCDR3序列；
    (3)SEQ ID NO:41所示HCDR1序列；SEQ ID NO:42所示HCDR2序列；SEQ ID NO:43所示HCDR3序列；SEQ ID NO:44所示LCDR1序列；SEQ ID NO:45所示LCDR2序列；和SEQ ID NO:46所示LCDR3序列。
  2. 权利要求1的双特异性抗体，其中所述重链可变区包含：
    (1)SEQ ID NO:31、SEQ ID NO:39或SEQ ID NO:47的氨基酸序列；或者
    (2)与SEQ ID NO:31、SEQ ID NO:39或SEQ ID NO:47具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。
  3. 权利要求1或2的双特异性抗体，其中所述轻链可变区包含：
    (1)SEQ ID NO:32、SEQ ID NO:40或SEQ ID NO:48的氨基酸序列；或者
    (2)与SEQ ID NO:32、SEQ ID NO:40或SEQ ID NO:48具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列相同性的氨基酸序列。
  4. 权利要求1-3中任一项的双特异性抗体，其中所述重链可变区和轻链可变区选自(1)-(3)中任一项
    (1)重链可变区，其包含SEQ ID NO:31的氨基酸序列；轻链可变区，其包含SEQ ID NO:32的氨基酸序列；
    (2)重链可变区，其包含SEQ ID NO:39的氨基酸序列；轻链可变区，其包含SEQ ID NO:40的氨基酸序列；
    (3)重链可变区，其包含SEQ ID NO:47的氨基酸序列；轻链可变区，其包含SEQ ID NO:48的氨基酸序列。
  5. 权利要求1-4中任一项的双特异性抗体，其中所述第二抗原结合部分包含结合VEGF的免疫球蛋白单可变结构域(VHH)；
    优选地，所述免疫球蛋白单可变结构域包含：SEQ ID NO:49所示CDR1序列、SEQ ID NO:50所示CDR2序列以及SEQ ID NO:51所示CDR3序列；
    更优选地，所述免疫球蛋白单可变结构域包含：1)SEQ ID NO:11的氨基酸序列；或者2)与SEQ ID NO:11的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％序列相同性的氨基酸序列。
  6. 权利要求1-5中任一项的双特异性抗体，其中所述第一抗原结合部分和所述第二抗原结合部分通过接头连接。
  7. 权利要求1-6中任一项的双特异性抗体，其进一步包含免疫球蛋白的重链恒定区(CH)和轻链恒定区(CL)；
    优选地，所述重链恒定区为人IgG1的重链恒定区，并且所述轻链恒定区为人κ轻链恒定区；
    更优选地，所述人IgG1的重链恒定区在根据EU编号的第234和235位包含亮氨酸至丙氨酸的突变。
  8. 权利要求7的双特异性抗体，其包含第一多肽和第二多肽，其中所述第一多肽包含所述重链可变区(VH)和所述重链恒定区(CH)，所述第二多肽包含所述轻链可变区(VL)和所述轻链恒定区(CL)，第二抗原结合部分的单可变结构域任选地通过接头融合至：(1)所述VH的N端；(2)所述VL的N端；(3)所述VH和VL的N端；(4)所述CH的C端；(5)所述CL的C端；(6)所述CH和CL的C端；(8)所述VH的N端和所述CH的C端；或者(9)所述VL的N端和所述CH的C端。
  9. 权利要求8的双特异性抗体，其中所述第一多肽和第二多肽选自(1)-(15)中任一项：
    (1)第一多肽，其包含SEQ ID NO:13的氨基酸序列或者与SEQ ID NO:13相比具有L238A和L239A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:6的氨基酸序列；
    (2)第一多肽，其包含SEQ ID NO:14的氨基酸序列或者与SEQ ID NO:14相比具有L382A和L383A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:6的氨基酸序列；
    (3)第一多肽，其包含SEQ ID NO:5的氨基酸序列或者与SEQ ID NO:5相比具有L238A和L239A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:15的氨基酸序列；
    (4)第一多肽，其包含SEQ ID NO:5的氨基酸序列或者与SEQ ID NO:5相比具有L238A和L239A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:16的氨基酸序列；
    (5)第一多肽，其包含SEQ ID NO:17的氨基酸序列或者与SEQ ID NO:17相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:8的氨基酸序列；
    (6)第一多肽，其包含SEQ ID NO:18的氨基酸序列或者与SEQ ID NO:18相比具有L386A和L387A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:8的氨基酸序列；
    (7)第一多肽，其包含SEQ ID NO:7的氨基酸序列或者与SEQ ID NO:7相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:19的氨基酸序列；
    (8)第一多肽，其包含SEQ ID NO:7的氨基酸序列或者与SEQ ID NO:7相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:20的氨基酸序列；
    (9)第一多肽，其包含SEQ ID NO:21的氨基酸序列或者与SEQ ID NO:21相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:10的氨基酸序列；
    (10)第一多肽，其包含SEQ ID NO:22的氨基酸序列或者与SEQ ID NO:22相比具有L386A和L387A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:10的氨基酸序列；
    (11)第一多肽，其包含SEQ ID NO:9的氨基酸序列或者与SEQ ID NO:9相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:23的氨基酸序列；
    (12)第一多肽，其包含SEQ ID NO:9的氨基酸序列或者与SEQ ID NO:9相比具有L242A和L243A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:24的氨基酸序列；
    (13)第一多肽，其包含SEQ ID NO:14的氨基酸序列或者与SEQ ID NO:14相比具有L382A和L383A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:15的氨基酸序列；
    (14)第一多肽，其包含SEQ ID NO:18的氨基酸序列或者与SEQ ID NO:18相比具有L386A和L387A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:19的氨基酸序列；
    (15)第一多肽，其包含SEQ ID NO:22的氨基酸序列或者与SEQ ID NO:22相比具有L386A和L387A突变的氨基酸序列；第二多肽，其包含SEQ ID NO:23的氨基酸序列。
  10. 权利要求8或9的双特异性抗体，其包含两条所述第一多肽和两条所述第二多肽，其中所述第一多肽相同或不同，所述第二多肽相同或不同。
  11. 一种多核苷酸，其编码权利要求1-10中任一项的双特异性抗体。
  12. 一种表达载体，其包含权利要求11的多核苷酸。
  13. 一种宿主细胞，其包含权利要求11的多核苷酸或权利要求12的表达载体。
  14. 一种产生权利要求1-10中任一项的双特异性抗体的方法，其包括：
    (Ⅰ)在合适条件下培养权利要求13的宿主细胞以表达所述双特异性抗体，以及
    (Ⅱ)从宿主细胞或其培养物分离所述双特异性抗体。
  15. 一种药物组合物，其包含权利要求1-10中任一项的双特异性抗体，以及药学上可接受的载剂。
  16. 权利要求1-10中任一项的双特异性抗体或权利要求15的药物组合物在制备用于治疗以下疾病的药物中的用途：(1)血管生成相关的眼病；或(2)癌症；
    优选地，
    所述血管生成相关的眼病为黄斑变性、视网膜静脉阻塞、视网膜病变、早产儿视网膜病变、糖尿病视网膜病变、新生血管性青光眼、病理性近视、黄斑水肿、视网膜水肿、糖尿病性黄斑水肿或脉络膜新生血管性疾病；
    所述癌症为肺癌、肝癌、胰腺癌、皮肤癌、头颈癌、黑素瘤、卵巢癌、结肠直肠癌、胃癌、乳腺癌、前列腺癌、子宫癌、霍奇金淋巴瘤、食管癌、肛门癌、内分泌系统癌症、甲状腺癌、甲状旁腺癌、肾癌、软组织肉瘤、膀胱癌、中枢神经系统(CNS)肿瘤、间皮瘤、胶质瘤、脑脊膜瘤或垂体腺瘤。
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