WO2022171109A1 - 抗vegf抗体及其用途 - Google Patents

抗vegf抗体及其用途 Download PDF

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WO2022171109A1
WO2022171109A1 PCT/CN2022/075600 CN2022075600W WO2022171109A1 WO 2022171109 A1 WO2022171109 A1 WO 2022171109A1 CN 2022075600 W CN2022075600 W CN 2022075600W WO 2022171109 A1 WO2022171109 A1 WO 2022171109A1
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seq
amino acid
binding protein
acid sequence
isolated antigen
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PCT/CN2022/075600
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English (en)
French (fr)
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王宗达
顾春银
曹晓丹
刘小五
邓俗俊
潘忠宗
王学萍
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上海济煜医药科技有限公司
江西济民可信集团有限公司
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Application filed by 上海济煜医药科技有限公司, 江西济民可信集团有限公司 filed Critical 上海济煜医药科技有限公司
Priority to BR112023016095A priority Critical patent/BR112023016095A2/pt
Priority to CN202280014211.3A priority patent/CN116897164A/zh
Priority to JP2023548780A priority patent/JP2024506664A/ja
Priority to IL305092A priority patent/IL305092A/en
Priority to US18/276,630 priority patent/US20240287168A1/en
Priority to KR1020237030457A priority patent/KR20230142838A/ko
Priority to CA3207763A priority patent/CA3207763A1/en
Priority to EP22752269.5A priority patent/EP4292661A1/en
Priority to AU2022220965A priority patent/AU2022220965A1/en
Priority to MX2023009392A priority patent/MX2023009392A/es
Publication of WO2022171109A1 publication Critical patent/WO2022171109A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the application relates to the field of biomedicine, in particular to an anti-VEGF antibody and its use in preparing medicines
  • Malignant tumor is a disease that seriously threatens human health on a global scale, and is the main type of disease that causes human death.
  • effective therapeutic drugs need to be developed urgently.
  • the development of anti-angiogenesis drugs has become a research hotspot in recent years.
  • the growth and metastasis of tumors require abundant blood vessels to provide them with sufficient oxygen and nutrients, and tumor tissue can secrete a variety of pro-angiogenic substances.
  • the epidermal growth factor VEGF can specifically stimulate the proliferation of endothelial cells during tumor growth and plays a key role in various types of tumor angiogenesis.
  • the combination of VEGF and epidermal growth factor receptor VEGFR can mediate the transcription and expression of intracellular related protein genes through downstream signaling pathways, and promote the proliferation of vascular endothelial cells.
  • VEGF epidermal growth factor receptor VEGFR
  • a number of humanized monoclonal antibodies targeting human VEGF, such as Bevacizumab have been developed, but there are still phenomena such as low response rate and easy generation of drug resistance during the treatment process. Therefore, there is an urgent need for anti-tumor VEGF antibodies with stable structure, good efficacy and suitable for large-scale industrial production.
  • the present application provides an isolated antigen-binding protein having one or more of the following properties: (1) capable of binding to VEGF protein with a KD value of 1 x 10-7 M or less; and (2) Can block the binding between VEGF protein and VEGFR protein.
  • the VEGF protein comprises VEGF165.
  • the VEGFR protein comprises VEGFR2.
  • the isolated antigen-binding protein comprises an antibody or antigen-binding fragment thereof.
  • the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and/or dAb.
  • the antigen-binding fragment is a VHH.
  • the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.
  • the isolated antigen binding protein is capable of competing with a reference antibody for binding to the VEGF protein, wherein the reference antibody comprises a heavy chain variable region VH, the VH of the reference antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 of the reference antibody comprises the amino acid sequence shown in SEQ ID NO: 9, the HCDR2 of the reference antibody comprises the amino acid sequence shown in SEQ ID NO: 23, and the reference The HCDR3 of the antibody comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the HCDR1 of the reference antibody comprises the amino acid sequence set forth in SEQ ID NO:9.
  • the HCDR2 of the reference antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs: 12-14.
  • the HCDR3 of the reference antibody comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17-19.
  • the isolated antigen binding protein comprises at least one CDR in a VH comprising the amino acid sequence set forth in SEQ ID NO:29.
  • the isolated antigen binding protein comprises at least one CDR in a VH comprising the amino acid sequence set forth in any of SEQ ID NOs: 1-6.
  • the isolated antigen binding protein comprises HCDR3, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:24.
  • the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17-19.
  • the isolated antigen binding protein comprises HCDR2, and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:23.
  • the HCDR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 12-14.
  • the isolated antigen binding protein comprises HCDR1, and the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:9.
  • the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:9 and the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:23 sequence, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.
  • the isolated antigen binding protein comprises HCDR1, HCDR2 and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:9 and the HCDR2 comprises any of SEQ ID NOs:12-14 an amino acid sequence set forth in one, and the HCDR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17-19.
  • the isolated antigen binding protein comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 are selected from any of the following group of amino acid sequences:
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 12
  • HCDR3 SEQ ID NO: 17;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 13
  • HCDR3 SEQ ID NO: 17;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 14
  • HCDR3 SEQ ID NO: 17;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 13
  • HCDR3 SEQ ID NO: 18
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 13
  • HCDR3 SEQ ID NO: 19.
  • the isolated antigen binding protein comprises a heavy chain variable region VH, wherein the VH comprises a framework region H-FR1, the C-terminus of the H-FR1 being directly or the N-terminus of the HCDR1 Indirectly linked, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:25.
  • the H-FR1 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 7-8.
  • the VH comprises a framework region H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence set forth in SEQ ID NO: 26 .
  • the H-FR2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 10-11.
  • the VH comprises a framework region H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence set forth in SEQ ID NO:27 .
  • the H-FR3 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 15-16.
  • the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence set forth in SEQ ID NO:28.
  • the H-FR4 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 20-22.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 comprises the amino acid sequence shown in SEQ ID NO: 25, the The H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 26, the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 27 and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO: 28.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, wherein the H-FR1 comprises SEQ ID NO:7 or SEQ ID NO:8
  • the H-FR2 comprises the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11
  • the H-FR3 comprises the amino acid sequence shown in SEQ ID NO: 15 or SEQ ID NO: 16
  • the H-FR4 comprises the amino acid sequence shown in any one of 20-22.
  • the isolated antigen binding protein comprises H-FR1, H-FR2, H-FR3 and H-FR4, and the H-FR1, H-FR2, H-FR3 and H-FR4 are selected from from any of the following sets of amino acid sequences:
  • F-FR1 SEQ ID NO: 7
  • H-FR2 SEQ ID NO: 10
  • H-FR3 SEQ ID NO: 15
  • H-FR4 SEQ ID NO: 20;
  • H-FR1 SEQ ID NO: 8
  • H-FR2 SEQ ID NO: 11
  • H-FR3 SEQ ID NO: 16
  • H-FR4 SEQ ID NO: 21;
  • H-FR1 SEQ ID NO: 8
  • H-FR2 SEQ ID NO: 11
  • H-FR3 SEQ ID NO: 16
  • H-FR4 SEQ ID NO: 22.
  • the isolated antigen binding protein comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence set forth in SEQ ID NO:29.
  • the VH comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-6.
  • the application provides isolated one or more nucleic acid molecules encoding the isolated antigen binding proteins.
  • the application provides a vector comprising the nucleic acid molecule.
  • the application provides a cell comprising the nucleic acid molecule or the vector.
  • the present application provides a method of preparing the isolated antigen binding protein, the method comprising culturing the cell under conditions such that the isolated antigen binding protein is expressed.
  • the application provides a pharmaceutical composition comprising the isolated antigen binding protein, the nucleic acid molecule, the carrier and/or the cell, and optionally a pharmaceutically acceptable vector.
  • the application provides polypeptides comprising the isolated antigen binding protein.
  • the application provides immunoconjugates comprising the isolated antigen binding protein or the polypeptide.
  • the application provides the isolated antigen binding protein, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition, the polypeptide and/or the immunoconjugate Use of the compound in the preparation of a medicament for preventing, relieving and/or treating tumors.
  • the tumor comprises a VEGF-overexpressing tumor.
  • the tumor comprises a solid tumor and/or a non-solid tumor.
  • the tumor comprises lung cancer, colorectal cancer, breast cancer, kidney cancer, gastric cancer, liver cancer, blastoma, cervical cancer, and/or ovarian cancer.
  • the present application provides a method of preventing, ameliorating or treating a tumor, the method comprising administering the isolated antigen-binding protein, the nucleic acid molecule, the carrier, the the cell, the pharmaceutical composition, the polypeptide and/or the immunoconjugate.
  • the tumor comprises a VEGF-overexpressing tumor.
  • the tumor comprises a solid tumor and/or a non-solid tumor.
  • the tumor comprises lung cancer, colorectal cancer, breast cancer, kidney cancer, gastric cancer, liver cancer, blastoma, cervical cancer, and/or ovarian cancer.
  • the present application provides the isolated antigen binding protein, the nucleic acid molecule, the carrier, the cell, the pharmaceutical composition, the polypeptide and/or the immunoconjugate, which For the prevention, alleviation or treatment of tumors.
  • the tumor comprises a VEGF-overexpressing tumor.
  • the tumor comprises a solid tumor and/or a non-solid tumor.
  • the tumor comprises lung cancer, colorectal cancer, breast cancer, kidney cancer, gastric cancer, liver cancer, blastoma, cervical cancer, and/or ovarian cancer.
  • the present application provides a method for inhibiting the binding of a VEGF protein to a VEGFR protein, comprising administering the isolated antigen-binding protein, the nucleic acid molecule, the carrier, the cell, and the drug The composition, the polypeptide and/or the immunoconjugate.
  • Figure 1 shows a schematic diagram of the yeast display library construction method.
  • Figure 2 shows that the antigen binding proteins Ab1910VE8, Ab1910VE9 and Ab1910VE6 described herein block the binding of human VEGF165 and human VEGFR2.
  • Figure 3 shows that the antigen binding proteins Ab1910VE18, Ab1910VE21 and Ab1910VE24 described herein block the binding of human VEGF165 and human VEGFR2.
  • VEGF generally refers to vascular endothelial cell growth factor, including its naturally occurring allelic and processed forms.
  • the VEGF may include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and/or PlGF.
  • the VEGF may be VEGF-A, which may also be referred to as VEGF165.
  • VEGF-A can bind to receptor tyrosine kinases, namely VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), VEGFR3 and Neuropilin-1 (NRP-1).
  • VEGF165 After VEGF165 binds to VEGFR2, it mediates the transcription and expression of intracellular related protein genes through the downstream PLC- ⁇ -PKC-Raf-MEK-MAPK signaling pathway, and promotes the proliferation of vascular endothelial cells (Takahashi T et al., Oncogene, 18(13):2221- 2230. (1999)).
  • the term "VEGF” may also refer to VEGF from non-human species such as mice, rats or primates. In this application, VEGF from a specific species can be expressed as follows, hVEGF refers to human VEGF, and mVEGF refers to murine VEGF. Generally, VEGF may refer to human VEGF.
  • VEGF is also used to refer to truncated forms or fragments of intact VEGF, and also includes functional variants, isoforms, species homologues, derivatives, analogs of VEGF, and those having at least one epitope in common with VEGF analogs of .
  • VEGF sequences are known in the art.
  • exemplary full-length human VEGFA protein sequences can be found under NCBI Accession Nos. NP_001020537, NP_001020538, NP_001020539, NP_001020540, or NP_001020541.
  • VEGFR generally refers to the vascular endothelial cell growth factor receptor, including its naturally occurring allelic and processed forms.
  • VEGFRs can include VEGFRl, CEGFR2, and VEGFR3, as well as other alternatively spliced variants.
  • VEGFR can be membrane bound or soluble.
  • the term "VEGFR” can also refer to VEGFRs from non-human species such as mice, rats or primates. In the present application, VEGFR from a specific species can be expressed as follows, hVEGFR refers to human VEGFR, and mVEGFR refers to murine VEGFR. Generally, VEGFR may refer to human VEGFR.
  • VEGFR is also used to refer to truncated forms or fragments of the intact VEGFR, and also includes functional variants, isoforms, species homologues, derivatives, analogs of VEGFR, as well as having at least one epitope in common with VEGFR analogs of .
  • VEGFR sequences are known in the art. For example, an exemplary full-length human VEGFR2 protein sequence can be found under NCBI Accession No. NP_002244.
  • homologue generally refers to an amino acid sequence or nucleotide sequence that has some homology to a wild-type amino acid sequence and a wild-type nucleotide sequence.
  • the term “homology” may be equivalent to sequence "identity”.
  • homologous sequences can include amino acid sequences that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • a homologue will contain the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity.
  • a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of.
  • antigen-binding protein generally refers to a protein comprising an antigen-binding moiety, and optionally a scaffold or backbone moiety that allows the antigen-binding moiety to adopt a conformation that facilitates the binding of the antigen-binding protein to the antigen.
  • Antigen binding proteins can typically comprise antibody light chain variable regions (VL), antibody heavy chain variable regions (VH), or both, and functional fragments thereof. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments, immunoconjugates, multispecific antibodies (eg, bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, etc., so long as they show The desired antigen-binding activity can be obtained.
  • antibody generally refers to an immunoglobulin reactive against a specified protein or peptide or fragment thereof.
  • Antibodies can be antibodies from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (eg, IgGl, IgG2, IgG3, and IgG4).
  • the antibody may have a heavy chain constant region selected from, eg, IgGl, IgG2, IgG3, or IgG4.
  • the antibody may also have a light chain selected from, for example, kappa ( ⁇ ) or lambda ( ⁇ ).
  • the antibodies of the present application can be derived from any species.
  • antigen-binding fragment generally refers to a portion of an antibody molecule comprising amino acid residues that interact with and confer specificity and affinity for the antigen to the antibody.
  • antigen-binding fragments may include, but are not limited to, Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAbs.
  • Fab generally refers to a fragment containing the variable domain of the heavy chain and the variable domain of the light chain, and also containing the constant domain of the light chain and the first constant domain (CH1) of the heavy chain
  • Fab' generally refers to a fragment that differs from Fab by adding a small number of residues (including one or more cysteines from the antibody hinge region) to the carboxy terminus of the heavy chain CH1 domain
  • F(ab"') 2 generally refers to a dimer of Fab', an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region.
  • Fv generally refers to the smallest antibody fragment containing the entire antigen recognition and binding site.
  • the fragment may consist of a heavy chain variable region and a light chain variable region in a tightly non-covalently bound dimer;
  • dsFv generally refers to disulfide-stabilized Fv fragments, The bond between its single light chain variable region and single heavy chain variable region is a disulfide bond.
  • dAb fragment generally refers to antibody fragments consisting of VH domains.
  • the term "scFv” generally refers to a monovalent molecule formed by covalently linking and pairing one heavy chain variable domain and one light chain variable domain of an antibody through a flexible peptide linker; such scFv molecules may have a general Structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • VHH generally refers to an antibody comprising the variable antigen binding domain of a heavy chain antibody (see Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHHs may also be referred to as Nanobodies (Nb).
  • variable region or “variable domain” generally refers to the domain of an antibody heavy or light chain that is involved in the binding of an antibody to an antigen.
  • variable generally refers to certain portions of the sequence of the variable domains of antibodies that vary strongly, resulting in the binding and specificity of each particular antibody for its particular antigen. Variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments in the light and heavy chain variable regions, called complementarity determining regions (CDRs) or hypervariable regions (HVRs), LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. The more highly conserved portions of the variable domains are referred to as framework regions (FRs).
  • CDRs complementarity determining regions
  • HVRs hypervariable regions
  • FRs framework regions
  • variable domains of native heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4) , mostly adopting a ⁇ -sheet configuration, connected by three loop regions of the CDR structure.
  • the CDRs in each chain are brought together in close proximity by the FR regions, and together with the CDRs from the other chain form the antigen-binding site of the antibody.
  • variable regions of an antibody or the CDRs of an antibody can be encoded by a variety of methods, such as the Kabat numbering scheme and definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, 5.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous antibodies, ie the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or In addition to post-translational modifications (eg, isomerization, amidation). Monoclonal antibodies are highly specific, directed against a single antigenic site.
  • chimeric antibody generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species.
  • the variable regions are derived from antibodies from experimental animals such as rodents ("parental antibodies”), and the constant regions are derived from human antibodies, such that the resulting chimeric antibody is more robust in human subjects than the parental (eg, mouse-derived) antibody Reduced likelihood of triggering an adverse immune response.
  • humanized antibody generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (eg, a mouse antibody) have been replaced by corresponding amino acids derived from human immunoglobulins. In the CDR regions, additions, deletions, insertions, substitutions or modifications of amino acids are also permissible as long as they still retain the ability of the antibody to bind to a particular antigen.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region.
  • a "humanized antibody” retains antigenic specificity similar to the original antibody.
  • “Humanized” forms of non-human (eg, murine) antibodies may minimally comprise chimeric antibodies that contain sequences derived from non-human immunoglobulins.
  • CDR region residues in a human immunoglobulin can be substituted with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or ability , rabbit or non-human primate) CDR region residue replacement.
  • donor antibody such as mouse, rat
  • FR region residues of the human immunoglobulin can be replaced with corresponding non-human residues.
  • humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody.
  • the term "fully human antibody” generally refers to the antibody expressed by the human antibody gene-encoding gene transferred into a genetically engineered antibody gene-deficient animal. All parts of an antibody, including the variable and constant regions of the antibody, are encoded by genes of human origin. Methods for obtaining fully human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology and RNA-polypeptide technology.
  • binding generally refer to a measurable and reproducible interaction, such as binding between an antigen and an antibody, which can be determined in the presence of a molecule
  • a target in the context of a heterogeneous population (including biological molecules).
  • an antibody binds to an epitope through its antigen binding domain, and this binding requires some complementarity between the antigen binding domain and the epitope.
  • an antibody that specifically binds a target is an antibody that binds to that target with greater affinity, avidity, easier, and/or for a greater duration than it binds to other targets.
  • An antibody is said to "specifically bind" to an antigen when it binds to an epitope more readily through its antigen-binding domain than it would bind to a random, unrelated epitope.
  • KD KD
  • KD KD
  • KD the equilibrium dissociation constant
  • kdis the dissociation rate constant
  • koff the dissociation rate constant
  • kon the association rate constant
  • KD equilibrium dissociation constant
  • association and dissociation rate constants are well known in the art and include, but are not limited to, Biofilm Interferometry (BLI), Radioimmunoassay (RIA), Equilibrium Dialysis, Surface Plasmon Resonance (SPR), Fluorescence Resonance Energy Transfer (FRET) , co-immunoprecipitation (Co-IP) and protein chip technology.
  • BBI Biofilm Interferometry
  • RIA Radioimmunoassay
  • SPR Surface Plasmon Resonance
  • FRET Fluorescence Resonance Energy Transfer
  • Co-IP co-immunoprecipitation
  • the measured affinity for a particular protein-protein interaction may vary if measured under different conditions (eg, salt concentration, pH).
  • the term "primate” generally refers to monkey and ape species, and includes monkey species such as those from the genus Macaque (eg, Macaca fascicularis and or rhesus monkey (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (from tamarinds ( Saguinus), and ape species, such as chimpanzees (Pan troglodytes), and also including Homo sapiens.
  • monkey species such as those from the genus Macaque (eg, Macaca fascicularis and or rhesus monkey (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri
  • polypeptide or “protein” are used interchangeably and generally refer to a polymer of amino acid residues.
  • the term also applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers.
  • the term may also include modified amino acid polymers, eg, by addition of sugar residues to form glycoproteins or modified by phosphorylation.
  • Polypeptides and proteins may be produced by naturally occurring and non-recombinant cells or by genetically engineered or recombinant cells, and may comprise molecules having the amino acid sequence of the native protein, or deletions, additions, or deletions of one or more amino acids of the native sequence and/or substituted molecules.
  • polypeptide and “protein” specifically include deleted, added and/or substituted sequences of one or more amino acids of the antigen binding proteins described herein.
  • isolated generally refers to biological material (eg, viruses, nucleic acids, or proteins) that is substantially free of components that normally accompany or interact with its naturally occurring environment.
  • the isolated biological material optionally contains additional material that the biological material is not found to have in its natural environment (eg, nucleic acids or proteins).
  • isolated when referring to a protein, “isolated” generally refers to the separation and separation of the molecule in question from the entire organism in which the molecule is found to occur naturally, or the substantial absence of other biological macromolecules of the same type.
  • nucleic acid molecule it is completely or partially separated from the sequence with which it is naturally associated, or the nucleic acid has a heterologous sequence associated with it, or the nucleic acid is separated from the chromosome.
  • immunoconjugate generally refers to a substance formed by linking an antigen-binding protein with other active agents, which can be small molecule active agents, such as chemotherapeutic agents, toxins, immunotherapeutic agents, imaging probes or spectral probes.
  • nucleic acid generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, isolated from their natural environment or artificially synthesized, or analogs thereof.
  • the term "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by culturing a suitable host cell containing the vector.
  • the term "cell” generally refers to a plasmid or vector that may contain or already contains a nucleic acid molecule described herein, or an individual cell, cell line or cell culture capable of expressing an antigen binding protein described herein thing.
  • the cells may include progeny of a single host cell. Due to natural, accidental or intentional mutations, the progeny cells may not necessarily be morphologically or genomically identical to the original parental cells, but are capable of expressing the antibodies or antigen-binding fragments thereof described herein.
  • the cells can be obtained by transfecting cells in vitro using the vectors described herein.
  • the cells may be prokaryotic cells (eg E.
  • the cells can be mammalian cells.
  • the mammalian cells can be CHO-K1 cells.
  • the term "pharmaceutical composition” generally refers to a formulation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain substances that are unacceptably toxic to the subject to whom the composition is to be administered. additional ingredients.
  • treatment generally refers to the desire to alter the natural course of the disease in the individual being treated, and may be a clinical intervention to achieve prevention or during the course of a clinical disease.
  • Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or ameliorating the disease state, and alleviating or improving prognosis.
  • antigen binding proteins eg, anti-VEGF antibodies
  • administration generally refers to the administration of a dose of a compound (eg, an anticancer therapeutic agent) or a pharmaceutical composition (eg, a pharmaceutical composition comprising an anticancer therapeutic agent) to a subject (eg, a patient).
  • a pharmaceutical composition eg, a pharmaceutical composition comprising an anticancer therapeutic agent
  • Administration can be by any suitable means, including parenteral, intrapulmonary and intranasal, and, if desired for topical treatment, intralesional administration.
  • Parenteral infusions include, for example, intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • tumor generally refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
  • the tumor may be a tumor with high expression of VEGF or VEGFR in cells and tissues.
  • Tumors can include solid tumors and/or non-solid tumors (eg, hematological tumors, lymphomas).
  • the term "between” generally means that the C-terminus of a certain amino acid fragment is directly or indirectly connected to the N-terminus of the first amino acid fragment, and its N-terminus is directly or indirectly connected to the C-terminus of the second amino acid fragment.
  • indirect connection In the light chain, for example, the N-terminus of the L-FR2 is directly or indirectly linked to the C-terminus of the LCDR1, and the C-terminus of the L-FR2 is directly or indirectly linked to the N-terminus of the LCDR2.
  • the N-terminus of the L-FR3 is directly or indirectly linked to the C-terminus of the LCDR2, and the C-terminus of the L-FR3 is directly or indirectly linked to the N-terminus of the LCDR3.
  • the N-terminus of the H-FR2 is directly or indirectly linked to the C-terminus of the HCDR1
  • the C-terminus of the H-FR2 is directly or indirectly linked to the N-terminus of the HCDR2.
  • the N-terminus of the H-FR3 is directly or indirectly linked to the C-terminus of the HCDR2
  • the C-terminus of the H-FR3 is directly or indirectly linked to the N-terminus of the HCDR3.
  • first amino acid fragment" and "second amino acid fragment” can be any amino acid fragment that is the same or different.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the application provides an isolated antigen-binding protein capable of binding primate (eg, human)-derived VEGF with a KD value of 1 x 10-8 M or less (eg, VEGF165).
  • the binding affinity of the VEGF antigen binding protein to VEGF can be determined by any method known in the art. In certain instances, binding affinity can be determined by surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), bound antigen precipitation, equilibrium dialysis, biofilm interference (BLI). In certain instances, the binding affinity and KD value of the VEGF antigen binding protein for VEGF can be determined by biofilm interference (BLI). For example, the ForteBio Octet Molecular Interaction Analyzer can be used to analyze the binding kinetics between antigen and antibody.
  • the isolated antigen binding protein is capable of binding VEGF (eg, VEGF165) with a KD value of 1 ⁇ 10 ⁇ 7 M or less.
  • the KD can be at a value of about 5 ⁇ 10 ⁇ 8 M or less, about 4 ⁇ 10 ⁇ 8 M or less, about 3 ⁇ 10 ⁇ 8 M or less, about 2 ⁇ 10 ⁇ 8 M or less, about 1 ⁇ 10 -8 M or less, about 9 ⁇ 10 -9 M or less, about 8 ⁇ 10 -9 M or less, about 7 ⁇ 10 -9 M or less, about 6 ⁇ 10 -9 M or less, about 5 x 10 -9 M or less, about 4 x 10 -9 M or less, about 3 x 10 -9 M or less, about 2 x 10 -9 M or less, about 1 x 10 -9 M or less, about Values of 9 x 10 -10 M or less, about 8 x 10 -10 M or less, about 7 x 10 -10 M or less bind human-derived VEGF, eg, as
  • the antigen binding proteins described herein are capable of blocking the binding of VEGF (eg, VEGF165) to VEGFR (eg, VEGFR2).
  • VEGF eg, VEGF165
  • VEGFR eg, VEGFR2
  • the blocking of the binding of VEGF to VEGFR by the antigen binding protein can be determined by flow cytometry (FACS), enzyme-linked immunosorbent assay (ELISA).
  • human VEGFR eg, VEGFR2
  • VEGFR vascular endothelial growth factor receptor 2
  • VEGF vascular endothelial growth factor
  • the IC50 for blocking activity is between about 0.001 ⁇ g/mL and about 10 ⁇ g/mL, between about 0.001 ⁇ g/mL and about 5 ⁇ g/mL, between about 0.01 ⁇ g/mL and about 1 ⁇ g/mL, about 0.02 ⁇ g /mL to about 0.5 ⁇ g/mL, between about 0.2 ⁇ g/mL to about 15 ⁇ g/mL, between about 0.2 ⁇ g/mL to about 12 ⁇ g/mL, between about 0.2 ⁇ g/mL to about 10 ⁇ g/mL, Between about 0.3 ⁇ g/mL and about 8 ⁇ g/mL, between about 0.3 ⁇ g/mL and about 6 ⁇ g/mL, between about 0.5 ⁇ g/mL and about 5 ⁇ g/mL, between about 0.1 ⁇ g/mL and about 2 ⁇ g/mL time, or between about 0.5 ⁇ g/mL to about 1.5 ⁇ g/mL
  • the isolated antigen binding protein is capable of competing with a reference antibody for binding to a VEGF protein
  • the reference antibody may comprise a heavy chain variable region VH
  • the VH of the reference antibody may comprise HCDR1, HCDR2 and HCDR3
  • the HCDR1 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:9
  • the HCDR2 of the reference antibody may comprise the amino acid sequence shown in SEQ ID NO:23
  • the reference The HCDR3 of the antibody can comprise the amino acid sequence set forth in SEQ ID NO:24.
  • the isolated antigen binding protein is capable of competing with a reference antibody for binding to a VEGF protein
  • the reference antibody may comprise HCDR1, HCDR2 and HCDR3
  • the HCDR1 of the reference antibody may comprise SEQ ID NO: 9
  • the amino acid sequence shown the HCDR2 of the reference antibody may comprise the amino acid sequence shown in any one of SEQ ID NOs: 12-14
  • the HCDR3 of the reference antibody may comprise any one of SEQ ID NOs: 17-19 An amino acid sequence shown.
  • the isolated antigen binding protein is capable of competing with a reference antibody for binding to a VEGF protein
  • the reference antibody may comprise HCDR1, HCDR2 and HCDR3
  • the HCDR1, HCDR2 and HCDR3 of the reference antibody may comprise selected from any of the following sets of amino acid sequences:
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 12
  • HCDR3 SEQ ID NO: 17;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 13
  • HCDR3 SEQ ID NO: 17;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 14
  • HCDR3 SEQ ID NO: 17;
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 13
  • HCDR3 SEQ ID NO: 18
  • HCDR1 SEQ ID NO: 9
  • HCDR2 SEQ ID NO: 13
  • HCDR3 SEQ ID NO: 19.
  • the isolated antigen binding protein may comprise at least one CDR from a VH, wherein the VH may comprise the amino acid sequence set forth in SEQ ID NO:29.
  • the isolated antigen binding protein may comprise at least one CDR from a VH, wherein the VH may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1-6.
  • the isolated antigen binding protein may be a VHH.
  • the isolated antigen binding protein may comprise HCDR3, and the HCDR3 may comprise the CDR3 of VH whose amino acid sequence is as shown in SEQ ID NO:29.
  • the isolated antigen binding protein may comprise a HCDR3, and the HCDR3 may comprise a CDR3 of a VH having an amino acid sequence as shown in any one of SEQ ID NOs: 1-6.
  • the isolated antigen binding protein can comprise HCDR3, which can comprise the amino acid sequence set forth in SEQ ID NO: 24 : RLRIX 5X6X7X8X9X10ERLDY ( SEQ ID NO : 24 ) , wherein X5 is P or T, X6 is D or H, X7 is E, Q or W, X8 is R, W or Y, X9 is R or S, and X10 is R or T.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the HCDR3 may comprise at least an amino acid substitution at a position selected from the group consisting of: X 5 , X 6 , X 7 , X 8 , compared to the amino acid sequence shown in SEQ ID NO: 17, Amino acid substitutions at X9 and/or X10 .
  • the HCDR3 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 17-19.
  • the isolated antigen binding protein may comprise HCDR2, and the HCDR2 may comprise the CDR2 of VH whose amino acid sequence is shown in SEQ ID NO:29.
  • the isolated antigen binding protein may comprise a HCDR2, and the HCDR2 may comprise a CDR2 of a VH having an amino acid sequence as shown in any one of SEQ ID NOs: 1-6.
  • the isolated antigen binding protein can comprise HCDR2, which can comprise the amino acid sequence set forth in SEQ ID NO: 23: AVX 3 AX 5 X 6 WX 8 YVEDSVX 15 G (SEQ ID NO: 23), wherein , X3 is F or L, X5 is E or P, X6 is D or G, X8 is R or S, and X15 is K or R.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the HCDR2 may comprise at least amino acid substitutions at positions selected from the group consisting of: at X3 , X5 , X6 , X8 , and /or amino acid substitution at X15 .
  • the HCDR2 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 12-14.
  • the isolated antigen binding protein may comprise HCDR1, and the HCDR1 may comprise the CDR1 of VH whose amino acid sequence is as shown in SEQ ID NO:29.
  • the isolated antigen binding protein may comprise HCDR1, and the HCDR1 may comprise the CDR1 of the VH whose amino acid sequence is set forth in any one of SEQ ID NOs: 1-6.
  • the isolated antigen binding protein can comprise HCDR1, and the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the CDR1 of the VH with the amino acid sequence as shown in SEQ ID NO: 29, and the HCDR2 may comprise the amino acid sequence as shown in SEQ ID NO: 29 The CDR2 of the VH shown in NO:29, the HCDR3 may comprise the CDR3 of the VH whose amino acid sequence is shown in SEQ ID NO:29.
  • the isolated antigen binding protein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the CDR1 of the VH whose amino acid sequence is shown in any one of SEQ ID NOs: 1-6, and the HCDR2 may be Comprising the CDR2 of the VH having the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, the HCDR3 may comprise the CDR3 of the VH having the amino acid sequence set forth in any one of SEQ ID NOs: 1-6.
  • the isolated antigen-binding protein described herein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:9, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:23 The amino acid sequence shown, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:24.
  • the isolated antigen binding proteins described herein may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:9, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:12 - the amino acid sequence shown in any one of 14, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NOs: 17-19.
  • the isolated antigen binding proteins described herein can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:12 amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 17.
  • the isolated antigen binding proteins described herein can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:13 amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 17.
  • the isolated antigen binding proteins described herein can comprise HCDR1, HCDR2 and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:14 amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 17.
  • the isolated antigen binding proteins described herein can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:13 amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the isolated antigen binding proteins described herein can comprise HCDR1, HCDR2, and HCDR3, the HCDR1 can comprise the amino acid sequence set forth in SEQ ID NO:9, and the HCDR2 can comprise the amino acid sequence set forth in SEQ ID NO:13 amino acid sequence, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO: 19.
  • the isolated antigen binding protein package may comprise H-FR1, and the H-FR1 may comprise the amino acid sequence shown in SEQ ID NO: 25 : X1VQLVESGGGLVQX14GGSX18RLSCAASGSTSD ( SEQ ID NO: 25), wherein X 1 is A or E, X 14 is A or P, and X 18 is A or L.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the H-FR1 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 1 , X 14 and/or X 18 compared to the amino acid sequence set forth in SEQ ID NO:7 amino acid substitutions.
  • the H-FR1 may comprise the amino acid sequence set forth in SEQ ID NO: 7 or 8.
  • the isolated antigen-binding protein may comprise H-FR2, and the H-FR2 may comprise the amino acid sequence shown in SEQ ID NO: 26: WYRQAPGKERXQQLVX 14 (SEQ ID NO: 26), wherein, X 11 is D or E, and X 14 is A or S.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the H-FR2 may comprise at least amino acid substitutions at positions selected from the group consisting of amino acids at X 11 and/or X 14 compared to the amino acid sequence shown in SEQ ID NO: 10 replace.
  • the H-FR2 can comprise the amino acid sequence set forth in SEQ ID NO: 10 or 11.
  • the isolated antigen-binding protein may comprise H-FR3, and the H-FR3 may comprise the amino acid sequence shown in SEQ ID NO: 27:
  • RFTISRDNX 9 KNTVX 14 LQMNX 19 LX 21 X 22 EDTAX 27 YYCNV (SEQ ID NO: 27), wherein X 9 is S or T, X 14 is D or Y, X 19 is N or S, and X 21 is K or R , X 22 is A or P, and X 27 is I or V.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the H-FR3 may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 9 , X 14 , X 19 , X compared to the amino acid sequence shown in SEQ ID NO: 15 Amino acid substitutions at 21 , X22 and/or X27 .
  • the H-FR3 may comprise the amino acid sequence set forth in SEQ ID NO: 15 or 16.
  • the isolated antigen-binding protein may comprise H-FR4, and the H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:28: WGX3GTX6VTVSS (SEQ ID NO:28), wherein X 3 is K or Q and X6 is L, Q or T.
  • the sequence may be a sequence determined according to the rules defined by Chothia.
  • the H-FR4 may comprise at least an amino acid substitution at a position selected from the group consisting of amino acids at X3 and/or X6 compared to the amino acid sequence shown in SEQ ID NO: 20 replace.
  • the H-FR4 can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 20-22.
  • the isolated antigen binding protein may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO: 29:
  • the VH may comprise at least an amino acid substitution at a position selected from the group consisting of: at X 1 , X 14 , X 18 , X 46 , compared to the amino acid sequence shown in SEQ ID NO: 1 , X49 , X52 , X54 , X55 , X57 , X64 , X74 , X79 , X84 , X86 , X87 , X92 , X102 , X103 , X104 , X105 , X106 Amino acid substitutions at , X 107 , X 115 and/or X 118 .
  • the VH may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1-6.
  • the isolated antigen binding protein may be a VHH.
  • the VHH may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 1-6.
  • each heavy or light chain amino acid sequence of the antigen binding protein is homologous to the corresponding amino acid sequence in an antibody from a particular species, or belongs to a particular class.
  • the variable and constant portions of the light and heavy chains are derived from the variable and constant regions of antibodies of one animal species (eg, human).
  • the homologue may be at least about 85% (eg, having at least about 85%) the amino acid sequence of the protein and/or the polypeptide (eg, an antibody or fragment thereof that specifically binds the VEGF protein). about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence identity derived protein or polypeptide.
  • the homology generally refers to the similarity, similarity or relatedness between two or more sequences. Alignment to determine percent sequence homology can be accomplished in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W.R. Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Comparison", Proc. Natl.
  • the present application also provides isolated one or more nucleic acid molecules that encode the antigen binding proteins described herein.
  • each of the one or more nucleic acid molecules can encode the entire antigen binding protein or a portion thereof (eg, HCDR1-3, LCDR1-3, VL, VH, light chain or one or more of the heavy chains).
  • the nucleic acid molecules described herein can be isolated. For example, it may be produced or synthesized by: (i) amplified in vitro, for example by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified either (iv) synthetic, eg by chemical synthesis.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • nucleic acids encoding the antibodies, antigen-binding fragments thereof can be prepared by a variety of methods known in the art, including, but not limited to, manipulation using restriction fragments or using synthetic oligonucleotides. Overlap extension PCR.
  • the application provides one or more vectors comprising one or more nucleic acid molecules described herein.
  • One or more of the nucleic acid molecules may be included in each vector.
  • other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions.
  • the vector may also contain expression control elements that allow the correct expression of the coding region in an appropriate host.
  • the vector is an expression vector.
  • the application provides host cells that may comprise one or more nucleic acid molecules described herein and/or one or more vectors described herein.
  • each or each host cell may comprise one or one nucleic acid molecule or vector described herein.
  • each or each host cell may contain a plurality (e.g., 2 or more) or more (e.g., 2 or more) of the nucleic acid molecules or vectors described herein
  • the present application provides methods of making said antibodies or antigen-binding fragments thereof.
  • the method may comprise culturing the host cell described herein under conditions such that the antibody or antigen-binding fragment thereof is expressed.
  • these methods can be understood by those of ordinary skill in the art by using an appropriate medium, appropriate temperature and incubation time, and the like.
  • the application provides a pharmaceutical composition, which can comprise the antigen binding protein described in the application, the polypeptide, the nucleic acid molecule, the vector, the host cell, and any A pharmaceutically acceptable carrier is selected.
  • the pharmaceutically acceptable adjuvants are non-toxic to recipients at the doses and concentrations employed, and the pharmaceutical compositions herein may also contain more than one active compound, usually one that does not adversely affect each other's properties. those active compounds with complementary activities.
  • the type and effective amount of such drugs depends, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.
  • the pharmaceutical composition can be used to inhibit tumor growth.
  • the pharmaceutical compositions of the present application can inhibit or delay the development or progression of a disease, can reduce tumor size (or even substantially eliminate a tumor), and/or can alleviate and/or stabilize a disease state.
  • compositions described herein may comprise a prophylactically and/or therapeutically effective amount of the antibody, antigen-binding fragment thereof.
  • the prophylactically and/or therapeutically effective amount is that amount required to prevent and/or treat (at least in part) a disease or disorder and/or any complications thereof in a subject having or at risk of developing it.
  • the present application provides the use of the antigen binding protein and/or the fusion protein in the preparation of medicine.
  • the medicament is used to treat cancer, inhibit tumor growth and/or inhibit tumor cell proliferation.
  • the tumor comprises a VEGF-overexpressing tumor.
  • the tumor comprises a VEGFR-overexpressing tumor.
  • the tumor comprises a solid tumor and/or a non-solid tumor.
  • the tumor comprises lung cancer, colorectal cancer, breast cancer, kidney cancer, gastric cancer, liver cancer, blastoma, cervical cancer, and/or ovarian cancer.
  • the present application provides a method of inhibiting the binding of a VEGF protein to a VEGFR protein, comprising administering the antigen binding protein and/or the polypeptide described herein.
  • the method can be an ex vivo or in vitro method.
  • the method may be a non-therapeutic method.
  • the method can include contacting a biological sample with an antigen binding protein and/or VEGFR described herein under conditions that allow the antigen binding protein and/or VEGFR to bind VEGF, and detecting the presence of the antigen at the antigen Whether a complex is formed between the binding protein and VEGF, and whether a complex is formed between VEGF and VEGFR is detected.
  • the present application provides a method for detecting the presence and/or content of a VEGF protein, comprising administering the isolated antigen binding protein and/or the polypeptide.
  • the method can be an ex vivo or in vitro method.
  • the method may be a non-therapeutic method.
  • the present application also provides the use of an antigen binding protein in a method of diagnosing a subject with a tumor or cancer, the method comprising: determining by contacting a sample with the antigen binding protein of the present application and detecting the presence of bound antibody The presence or level of expression of VEGF in a sample obtained from a subject.
  • an antibody drug conjugate which may comprise a cytotoxic agent, and an antigen-binding fragment as described herein.
  • Antibody drug conjugates usually refer to the use of specific linkers to connect antibodies and small molecule cytotoxic drugs, and its main components can include antibodies, linkers and small molecule cytotoxic drugs.
  • the present application provides a kit, which may comprise the antigen binding protein described in the present application, the chimeric antigen receptor, the genetically modified cell, the antibody drug conjugate, and/or the antigen binding protein described in the present application.
  • pharmaceutical composition may include the antigen binding proteins, chimeric antigen receptors, genetically modified cells, and/or antibody drug conjugates described herein, optionally together with one or more therapeutic agents, in a single conventional container. Combinations, optionally formulated together in pharmaceutical compositions.
  • the present application provides a drug delivery device, which can be used to administer the antigen binding protein or the pharmaceutical composition thereof described herein.
  • RNA was extracted from lymphocytes isolated from blood samples, and a phage-displayed nanobody library was constructed. The size of the library was 3 ⁇ 10 9 cfu. Take 6 ml of transformed antibody library bacteria to prepare phage for specific panning, and the total amount of bacteria is 50 times greater than the library capacity.
  • Antigen-specifically bound phage was eluted with 450ul of 100mM hydrochloric acid, 50ul of 1M Tris-HCl pH11 was added to neutralize and infect E. coli SS320 in logarithmic growth phase, and phage was produced and purified for the next round of screening.
  • the screening method was the same as the first round, only the antigen dosage was reduced to 4ug.
  • the phage enriched after two rounds of screening were taken to identify the enrichment situation by enzyme-linked immunosorbent assay (ELISA).
  • the yeast display library was sorted for two rounds using a flow sorter.
  • the yeast obtained by sorting was coated with auxotrophic plate medium, and 46 single clones were selected for sequencing, and finally 5 single domains with unique sequences were obtained. heavy chain antibodies.
  • the identification scheme is shown in Table 2.
  • the corresponding yeast monoclonal colonies were analyzed by flow staining, and 1 ⁇ 10 6 cells were taken and stained according to the protocols in Table 2.
  • the strength of the combination of protocol 1 and the human VEGF165-Bio cell population was determined by the PE mean fluorescence signal intensity (MFI). Reflect, in the same way scheme 2 and scheme 3 can evaluate Mouse VEGF-Bio and non-specific binding level, scheme 4 competition signal is reflected by APC mean fluorescence signal intensity (MFI), the results are shown in Table 3.
  • MFI PE mean fluorescence signal intensity
  • IMGT/Domain Gap Align was performed on the sequence of monoclonal antibody Y20A6, and the human germline with the highest homology was found to be IGHV3-23*04.
  • the antibody sequence was numbered according to the Chothia rule, and the sites with different site sequences such as H1, H14, and H18 were mutated as shown in Table 4.
  • the protein number Ab1910VE6 was the original antibody before humanization.
  • Antibody number Theoretical isoelectric point Yield (mg) Expression amount (mg/L) Concentration (mg/mL) Ab1910VE6 7 0.89 89 4.6 Ab1910VE8 7.4 0.48 48 4.8 Ab1910VE9 7 0.75 75 3.8
  • Example 4 Determination of the binding activity of the antigen-binding protein of the present application to human VEGF165
  • Each cycle consists of the following steps: 1) immersion in buffer for 60 seconds; 2) detection of non-specific binding of antigen to the sensor; 3) regeneration with 10 mM glycine solution pH 1.7; 4) immersion in buffer for 60 seconds; 5) antibody immobilization On the sensor, the time was 20 seconds; 6) the sensor was immersed in the buffer for 180 seconds; 7) the antigen-antibody binding time was 180 seconds; 8) the dissociation of the antigen-antibody, the time was 10 minutes; 9) the sensor regeneration.
  • Example 5 Determination of the activity of the antigen-binding protein of the present application to block the binding of human VEGF165 to human VEGFR2
  • a competitive ELISA method was used to determine the single-domain antibody blocking the binding of VEGF165 and human VEGFR2.
  • the specific implementation steps are as follows: Human VEGF R2 (Acro, Cat. No.: KDR-H5227) was diluted with PBS (Gibco, Cat. No.: 10010-023) to 1 ⁇ g/ mL, added to a 96-well plate, 100 ⁇ L per well, affixed with a sealing film, and incubated overnight at 4°C.
  • the plate was washed 3 times with washing solution PBST (0.05% TWEEN-20:sigma-alorich, P1379), and then the blocking solution PBST+2% BSA (BSA: VWR, 0332-1KG) was prepared with washing solution, and added to each well. 300 ⁇ L of blocking solution, 37°C for 1 hour.
  • Anti-VEGF and Biotin-hVEGF (Acro, product number: VE5-H82Q0) were prepared with blocking solution, the initial concentration of Anti-VEGF was 200 ⁇ g/mL, and 5-fold gradient dilution (7 concentration points + 1 0 concentration point) , the preparation concentration of Biotin-hVEGF is 90ng/mL.
  • the sealed ELISA plate wash the plate three times with washing solution, add 50 ⁇ L of diluted Anti-VEGF to the 96-well plate, and add 50 ⁇ L of Biotin-hVEGF to the 96-well plate, and incubate at 37°C. 1 hour.
  • the plate was washed three times with washing solution, the secondary antibody SA-HRP (sigma, S2438) was diluted 1:5000 in blocking solution, 100 ⁇ L/well was added to the ELISA plate, and incubated at 37°C for 1 hour.
  • the Ab1910VE9 sequence was selected for affinity maturation, and the CDR region of Ab1910VE9 was defined by Chothia.
  • CDR1, CDR2, and CDR3, NNK mutation primers were designed to carry out polymerase chain reaction (PCR) amplification of each CDR mutation library gene fragment.
  • PCR polymerase chain reaction
  • each CDR mutation library gene fragment and yeast display plasmid were respectively transformed into Saccharomyces cerevisiae strain EBY100 (purchased from ATCC), so that each CDR mutation library was displayed on the yeast surface.
  • the parental sequence of Ab1910VE9 was displayed on the surface of yeast as a control.
  • Biotin-Human VEGF165 was used for three rounds of sorting, with an initial concentration of 3 nM and a 10-fold serial dilution.
  • the cell population with high display level and strong antigen-binding ability was collected; after sorting, the cells were spread on SD-Trp solid medium and cultured at 30°C for 3 days.
  • the single domain antibodies in Table 7 were expressed and purified. SEC-HPLC purity analysis of the expressed antibody was performed as follows:
  • Example 7 Determination of kinetic parameters of antigen-binding protein of the present application
  • Example 8 Experiment on the binding function of the antigen-binding protein of the present application to block the binding of human VEGF165 and human VEGFR2
  • the binding function experiment of the affinity matured VEGF antibody blocking human VEGF165 and human VEGFR2 was determined, wherein the preparation concentration of Biotin-hVEGF165 was 700ng/mL.
  • the experimental results are shown in Figure 3.
  • the blocking effect of Ab1910VE18, Ab1910VE21, and Ab1910VE24 antibodies is comparable to that of Bevacizumab. This shows that the antigen-binding protein of the present application has a good effect of blocking the binding of VEGF and VEGFR.

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Abstract

提供一种分离的抗原结合蛋白，其能够结合VEGF蛋白，和/或能够阻断VEGF蛋白与VEGFR蛋白之间结合。还提供了所述分离的抗原结合蛋白在制备药物中的用途。

Description

抗VEGF抗体及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种抗VEGF抗体及其在制备药物中的用途
背景技术
恶性肿瘤是目前全球范围内严重威胁人类健康的疾病，是疾病导致人类死亡的主要类型。随着国内人口老龄化的日渐加剧，肿瘤发生率不断提高，有效的治疗药物亟待开发，其中抗血管生成药物的开发为近年来研究热点。肿瘤的生长、转移需要丰富的血管为其提供足够的氧气和营养物质，肿瘤组织可分泌多种促血管生成物质。
在肿瘤的生长过程中表皮生长因子VEGF能特异性刺激内皮细胞的增殖，在多种类型的肿瘤血管生成中起关键作用。VEGF与表皮生长因子受体VEGFR结合后能够通过下游信号通路介导胞内相关蛋白基因转录表达，促进血管内皮细胞增殖。目前已开发出诸如贝伐单抗(Bevacizumab)等多款靶向人VEGF人源化单克隆抗体，但在治疗过程中仍存在响应率低，易产生耐药性等现象。因此，亟需结构稳定、疗效好且适合大规模工业化生产的抗肿瘤VEGF抗体。
发明内容
本申请提供了一种分离的抗原结合蛋白，其具有下述性质中的一种或多种：(1)能够以1×10 -7M或更低的KD值结合VEGF蛋白；和(2)能够阻断VEGF蛋白与VEGFR蛋白之间结合。
在某些实施方式中，所述VEGF蛋白包括VEGF165。
在某些实施方式中，所述VEGFR蛋白包括VEGFR2。
在某些实施方式中，所述分离的抗原结合蛋白包括抗体或其抗原结合片段。
在某些实施方式中，所述抗原结合片段包括Fab，Fab’，F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv、VHH和/或dAb。
在某些实施方式中，所述抗原结合片段为VHH。
在某些实施方式中，所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
在某些实施方式中，所述分离的抗原结合蛋白能够与参比抗体竞争结合所述VEGF蛋白，其中所述参比抗体包含重链可变区VH，所述参比抗体的VH包含HCDR1、HCDR2和HCDR3， 且所述参比抗体的HCDR1包含如SEQ ID NO:9所示的氨基酸序列，所述参比抗体的HCDR2包含如SEQ ID NO:23所示的氨基酸序列，和，所述参比抗体的HCDR3包含如SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，所述参比抗体的HCDR1包含SEQ ID NO：9所示的氨基酸序列。
在某些实施方式中，所述参比抗体的HCDR2包含SEQ ID NO：12-14中任一项所示的氨基酸序列。
在某些实施方式中，所述参比抗体的HCDR3包含SEQ ID NO：17-19中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含VH中的至少一个CDR，所述VH包含如SEQ ID NO:29所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含VH中的至少一个CDR，所述VH包含如SEQ ID NO:1-6中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR3，且所述HCDR3包含SEQ ID NO：24所示的氨基酸序列。
在某些实施方式中，所述HCDR3包含SEQ ID NO：17-19中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR2，且所述HCDR2包含SEQ ID NO：23所示的氨基酸序列。
在某些实施方式中，所述HCDR2包含SEQ ID NO：12-14中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1，且所述HCDR1包含SEQ ID NO：9所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO:23所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO:12-14中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:17-19中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3选自以下任一组氨基酸序列：
(1)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:12和HCDR3：SEQ ID NO:17；
(2)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:13和HCDR3：SEQ ID NO:17；
(3)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:14和HCDR3：SEQ ID NO:17；
(4)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:13和HCDR3：SEQ ID NO:18；和
(5)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:13和HCDR3：SEQ ID NO:19。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，其中所述VH包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:25所示的氨基酸序列。
在某些实施方式中，所述H-FR1包含SEQ ID NO:7-8中任一项所示的氨基酸序列。
在某些实施方式中，所述VH包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:26所示的氨基酸序列。
在某些实施方式中，所述H-FR2包含SEQ ID NO:10-11中任一项所示的氨基酸序列。
在某些实施方式中，所述VH包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:27所示的氨基酸序列。
在某些实施方式中，所述H-FR3包含SEQ ID NO:15-16中任一项所示的氨基酸序列。
在某些实施方式中，所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:28所示的氨基酸序列。
在某些实施方式中，所述H-FR4包含SEQ ID NO:20-22中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1包含SEQ ID NO:25所示的氨基酸序列，所述H-FR2包含SEQ ID NO:26所示的氨基酸序列，所述H-FR3包含SEQ ID NO:27所示的氨基酸序列且所述H-FR4包含SEQ ID NO:28所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1包含SEQ ID NO:7或SEQ ID NO:8所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10或SEQ ID NO:11所示的氨基酸序列，所述H-FR3包含SEQ ID NO:15或SEQ ID NO:16所示的氨基酸序列，且所述H-FR4包含20-22中任一项所示的氨基酸序列。
在某些实施方式中，所述分离的抗原结合蛋白包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1、H-FR2、H-FR3和H-FR4选自以下任一组氨基酸序列：
(1)F-FR1：SEQ ID NO:7，H-FR2：SEQ ID NO:10，H-FR3：SEQ ID NO:15和H-FR4：SEQ ID NO:20；
(2)H-FR1：SEQ ID NO:8，H-FR2：SEQ ID NO:11，H-FR3：SEQ ID NO:16和H- FR4：SEQ ID NO:21；和
(3)H-FR1：SEQ ID NO:8，H-FR2：SEQ ID NO:11，H-FR3：SEQ ID NO:16和H-FR4：SEQ ID NO:22。
在某些实施方式中，所述分离的抗原结合蛋白包含重链可变区VH，且所述VH包含SEQ ID NO:29所示的氨基酸序列。
在某些实施方式中，所述VH包含SEQ ID NO:1-6中任一项所示的氨基酸序列。
另一方面，本申请提供了分离的一种或多种核酸分子，其编码所述分离的抗原结合蛋白。
另一方面，本申请提供了载体，其包含所述的核酸分子。
另一方面，本申请提供了细胞，其包含所述的核酸分子或所述的载体。
另一方面，本申请提供了制备所述的分离的抗原结合蛋白的方法，所述方法包括在使得所述的分离的抗原结合蛋白表达的条件下，培养所述的细胞。
另一方面，本申请提供了药物组合物，其包含所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体和/或所述的细胞，以及任选地药学上可接受的载体。
另一方面，本申请提供了多肽，其包含所述的分离的抗原结合蛋白。
另一方面，本申请提供了免疫缀合物，其包含所述的分离的抗原结合蛋白或所述的多肽。
另一方面，本申请提供了所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的药物组合物、所述的多肽和/或免疫缀合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗肿瘤。
在某些实施方式中，所述肿瘤包括VEGF过表达的肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤包括肺癌、结直肠癌、乳腺癌、肾癌、胃癌、肝癌、母细胞瘤、宫颈癌和/或卵巢癌。
另一方面，本申请提供了预防、缓解或治疗肿瘤的方法，所述方法包括向有需要的受试者施用所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的药物组合物、所述多肽和/或所述免疫缀合物。
在某些实施方式中，所述肿瘤包括VEGF过表达的肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤包括肺癌、结直肠癌、乳腺癌、肾癌、胃癌、肝癌、母细胞瘤、宫颈癌和/或卵巢癌。
另一方面，本申请提供了所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、 所述的细胞、所述的药物组合物、多肽和/或免疫缀合物，其用于预防、缓解或治疗肿瘤。
在某些实施方式中，所述肿瘤包括VEGF过表达的肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。
在某些实施方式中，所述肿瘤包括肺癌、结直肠癌、乳腺癌、肾癌、胃癌、肝癌、母细胞瘤、宫颈癌和/或卵巢癌。
另一方面，本申请提供了抑制VEGF蛋白与VEGFR蛋白结合的方法，其包括施用所述的分离的抗原结合蛋白、所述的核酸分子、所述的载体、所述的细胞、所述的药物组合物、所述多肽和/或所述免疫缀合物。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是酵母展示文库构建方法示意图。
图2显示的是本申请所述抗原结合蛋白Ab1910VE8、Ab1910VE9和Ab1910VE6阻断人VEGF165和人VEGFR2的结合。
图3显示的是本申请所述抗原结合蛋白Ab1910VE18、Ab1910VE21和Ab1910VE24阻断人VEGF165和人VEGFR2的结合。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“VEGF”通常是指血管内皮细胞生长因子，包括其天然存在的等位基因形式和加工形式。所述VEGF可包括VEGF-A，VEGF-B，VEGF-C，VEGF-D，VEGF-E， VEGF-F和/或PlGF。在本申请中，所述VEGF可以为VEGF-A，也可称为VEGF165。VEGF-A可结合受体酪氨酸激酶，即VEGFR-1(Flt-1)、VEGFR-2(Flk-1/KDR)、VEGFR3和Neuropilin-1(NRP-1)。VEGF165与VEGFR2结合后通过下游PLC-γ-PKC-Raf-MEK-MAPK信号通路介导胞内相关蛋白基因转录表达，促进血管内皮细胞增殖(Takahashi T等人，Oncogene,18(13):2221-2230.(1999))。术语“VEGF”还可以指来自非人物种诸如小鼠，大鼠或灵长类动物的VEGF。在本申请中，来自特定物种的VEGF可以表示如下，hVEGF表示人VEGF，mVEGF表示鼠VEGF。通常，VEGF可以指人VEGF。术语“VEGF”还用于指完整VEGF的截短形式或片段，还包括VEGF的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与VEGF共同表位的类似物。VEGF序列是本领域已知的。例如，示例性的全长人VEGFA蛋白序列可在NCBI登录号NP_001020537、NP_001020538、NP_001020539、NP_001020540或NP_001020541下找到。
在本申请中，术语“VEGFR”通常是指血管内皮细胞生长因子受体，包括其天然存在的等位基因形式和加工形式。VEGFR可包括VEGFR1、CEGFR2和VEGFR3，以及其他选择性剪接变体。VEGFR可以是膜结合的或可溶的。术语“VEGFR”还可以指来自非人物种诸如小鼠，大鼠或灵长类动物的VEGFR。在本申请中，来自特定物种的VEGFR可以表示如下，hVEGFR表示人VEGFR，mVEGFR表示鼠VEGFR。通常，VEGFR可以指人VEGFR。术语“VEGFR”还用于指完整VEGFR的截短形式或片段，还包括VEGFR的功能性变体、同工型、物种同源物、衍生物、类似物，以及具有至少一个与VEGFR共同表位的类似物。VEGFR序列是本领域已知的。例如，示例性的全长人VEGFR2蛋白序列可在NCBI登录号NP_002244下找到。
在本申请中，术语“同源物”通常是指与野生型氨基酸序列和野生型核苷酸序列具有一定同源性的氨基酸序列或核苷酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以包括可以与主题序列是至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。在本申请中，提及的氨基酸序列或核苷酸序列的SEQ ID NO中的任一项具有百分比同一性的序列是指在所提及的SEQ ID NO的整个长度上具有所述百分比同一性的序列。
在本申请中，术语“抗原结合蛋白”通常是指包含结合抗原部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。抗原结合 蛋白可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者，及其功能性片段。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗原结合蛋白的实例包括但不限于抗体、抗原结合片段、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，术语“抗体”通常是指对指定蛋白质或肽或其片段有反应性的免疫球蛋白。抗体可以是来自任何类的抗体，包括但不限于IgG、IgA、IgM、IgD和IgE，及来自任何亚类(例如IgG1、IgG2、IgG3、和IgG4)的抗体。抗体可具有选自例如IgG1、IgG2、IgG3、或IgG4的重链恒定区。抗体还可具有选自例如kappa(κ)或lambda(λ)的轻链。本申请的抗体可衍生自任何物种。
在本申请中，术语“抗原结合片段”通常是指抗体分子的某部分，该部分包含氨基酸残基，该氨基酸残基与抗原相互作用并赋予抗体对于抗原的特异性和亲和力。抗原结合片段的实例可包括但不限于Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb。在本申请中，术语“Fab”通常是指含有重链可变结构域和轻链可变结构域的片段，并且还含有轻链的恒定结构域和重链的第一恒定结构域(CH1)；术语“Fab’”通常是指在重链CH1结构域的羧基端添加少量残基(包括一个或多个来自抗体铰链区的半胱氨酸)而不同于Fab的片段；术语“F(ab') 2”通常是指Fab’的二聚体，包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。术语“Fv”通常是指含有完整抗原识别与结合位点的最小抗体片段。在某些情形中，该片段可以由一个重链可变区和一个轻链可变区以紧密非共价结合的二聚体组成；术语“dsFv”通常是指二硫键稳定的Fv片段，其单个轻链可变区与单个重链可变区之间的键是二硫键。术语“dAb片段”通常是指由VH结构域组成的抗体片段。在本申请中，术语“scFv”通常是指抗体的一个重链可变结构域和一个轻链可变结构域通过柔性肽连接子共价连接配对形成的单价分子；此类scFv分子可具有一般结构：NH 2-VL-连接子-VH-COOH或NH 2-VH-连接子-VL-COOH。在本申请中，术语“VHH”通常是指包含重链抗体的可变抗原结合结构域的抗体(参见Vanlandschoot P.等人，2011，Antiviral Research 92，389-407)。VHH也可称为纳米抗体(Nanobody)(Nb)。
在本申请中，术语“可变区”或“可变结构域”通常是指参与抗体与抗原的结合的抗体重链或轻链的结构域。在本申请中，术语“可变”通常是指，抗体的可变结构域的序列的某些部分变化强烈，形成各种特定抗体对其特定抗原的结合和特异性。变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链可变区和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)，分别为LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和 HCDR3。可变域中更高度保守的部分被称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区(H-FR1，H-FR2，H-FR3，H-FR4，L-FR1，L-FR2，L-FR3，L-FR4)，大部分采用β-折叠构型，通过三个CDR结构环区连接。每条链中的CDR通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。
在本领域中，可以通过多种方法来编码抗体的可变区或划分抗体的CDR，例如基于序列可变性的Kabat编号方案和定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))，基于结构环区域位置的Chothia编号方案和定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)，efranc等人的基于种系V基因的氨基酸序列比对的IMGT编号方案和定义规则，还有Honneger’s编号方案(AHo’s)，Martin编号方案，Gelfand编号方案等，可参见Mathieu Dondelinger等人，Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition,Front.Immunol.,16 October 2018.
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的抗体获得的抗体，即构成群体的各个抗体相同，除了可能以极小量存在的可能的天然存在突变和/或翻译后修饰(例如异构化、酰胺化)外。单克隆抗体是高度特异性的，针对单一抗原性位点。
在本申请中，术语“嵌合抗体”通常是指其中可变区源自一个物种，而恒定区源自另一个物种的抗体。通常，可变区源自实验动物诸如啮齿动物的抗体(“亲本抗体”)，且恒定区源自人类抗体，使得所得嵌合抗体与亲本(例如小鼠来源)抗体相比，在人类个体中引发不良免疫反应的可能性降低。
在本申请中，术语“人源化抗体”通常是指非人抗体(例如小鼠抗体)的CDR区以外的部分或全部有的氨基酸被源自人免疫球蛋白的相应的氨基酸置换的抗体。在CDR区中，氨基酸的添加、缺失、插入、置换或修饰也可以是允许的，只要它们仍保留抗体结合特定抗原的能力。人源化抗体可任选地包含人类免疫球蛋白恒定区的至少一部分。“人源化抗体”保留类似于原始抗体的抗原特异性。非人(例如鼠)抗体的“人源化”形式可以最低限度地包含衍生自非人免疫球蛋白的序列的嵌合抗体。在某些情形中，可以将人免疫球蛋白(受体抗体)中的CDR区残基用具有所期望性质、亲和力和/或能力的非人物种(供体抗体)(诸如小鼠，大鼠，家兔或非人灵长类动物)的CDR区残基替换。在某些情形中，可以将人免疫球蛋白的FR区残基用相应的非人残基替换。此外，人源化抗体可包含在受体抗体中或在供体抗体中没有的氨基酸修饰。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因转移至基因工程改造 的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变区和恒定区)均由人类来源的基因所编码。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术、核糖体展示技术和RNA-多肽技术等。
在本申请中，术语“结合”、“特异性结合”或“对…特异性的”通常是指可测量且可再现的相互作用，诸如抗原和抗体之间的结合，其可以确定在存在分子(包括生物学分子)的异质群体的情况中靶物的存在。例如，抗体通过其抗原结合域与表位结合，并且该结合需要抗原结合域和表位之间的一些互补性。例如，特异性结合靶物(其可以是表位)的抗体是以比其结合其它靶物更大的亲和力、亲合力、更容易和/或以更大的持续时间结合此靶物的抗体。当抗体相比于其将结合随机的、不相关的表位而言更容易通过其抗原结合域与表位结合时，抗体被称为“特异性结合”该抗原。
在本申请中，术语“KD”、“K D”可互换地使用，通常是指平衡解离常数，“KD”是解离速率常数(kdis，也称为“解离率(off-rate)(koff)”或“kd”)与结合速率常数(kon，也称为“结合率(kon)”或“ka”)的比值。可使用结合速率常数(kon)、解离速率常数(kdis)和平衡解离常数(KD)表示抗原结合蛋白(例如抗体)对抗原的结合亲和力。确定结合和解离速率常数的方法为本领域熟知，包括但不限于生物膜干涉技术(BLI)、放射免疫法(RIA)、平衡透析法、表面等离子共振(SPR)、荧光共振能量迁移(FRET)、免疫共沉淀(Co-IP)以及蛋白质芯片技术。如果在不同的条件(例如盐浓度、pH)下测量，则所测得的某种特定蛋白-蛋白相互作用的亲和力可不同。
在本申请中，术语“灵长类动物”通常是指猴和猿物种，并包括猴物种，诸如来自弥猴属(例如，食蟹猴(Macaca fascicularis)和或恒河猴(Macaca mulatta))和狒狒(豚尾狒狒(Papio ursinus))的猴，以及狨猴(来自狨(Callithrix)属的物种)，松鼠猴(来自松鼠猴(Saimiri)属的物种)和绢毛猴(来自柽柳猴(Saguinus)属的物种)，以及猿物种，诸如黑猩猩(Pan troglodytes)，并且还包括智人(Homo sapiens)。
在本申请中，术语“多肽”或“蛋白质”可互换地使用，通常是指氨基酸残基的聚合物。该术语也适用于其中一个或多个氨基酸残基是相应的天然存在的氨基酸的类似物或模拟物的氨基酸聚合物、以及天然存在的氨基酸聚合物。该术语也可包括修饰的氨基酸聚合物，例如，通过添加糖残基以形成糖蛋白或被磷酸化修饰。多肽和蛋白质可由天然存在的和非重组的细胞或由遗传工程改造的或重组的细胞产生，并且可包含具有天然蛋白质的氨基酸序列的分子、或具有天然序列的一个或多个氨基酸的缺失、添加和/或取代的分子。术语“多 肽”和“蛋白质”特别包括本申请所述的抗原结合蛋白的一个或多个氨基酸的缺失、添加和/或取代的序列。
在本申请中，术语“分离的”通常是指大体上不含其天然存在的环境中通常伴随或与之相互作用的组分的生物材料(例如病毒、核酸或蛋白质)。所述分离的生物材料任选地包含在其天然环境(例如，核酸或蛋白质)中所述生物材料未发现具有的另外的材料。在本申请中，当涉及蛋白质时，“分离”通常是指所述的分子从发现该分子天然存在的整个生物体中分离和分开，或基本不存在其它相同类型的生物大分子。当涉及核酸分子时，它与天然与其结合的序列完全或部分分离，或该核酸具有与其结合的异源序列，或该核算从染色体分离。
在本申请中，术语“免疫缀合物”通常是指抗原结合蛋白与其它活性剂连接形成的物质，其他活性剂可以是小分子活性剂，例如化疗剂、毒素、免疫治疗剂、成像探针或光谱探针。
在本申请中，术语“核酸”分子通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。
在本申请中，术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子，其将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。
在本申请中，术语“细胞”通常是指可以包含或已经含有包括本申请所述的核酸分子的质粒或载体，或者能够表达本申请所述的抗原结合蛋白的个体细胞、细胞系或细胞培养物。所述细胞可以包括单个宿主细胞的子代。由于天然的、意外的或故意的突变，子代细胞与原始亲本细胞在形态上或在基因组上可能不一定完全相同，但能够表达本申请所述的抗体或其抗原结合片段即可。所述细胞可以通过使用本申请所述的载体体外转染细胞而得到。所述细胞可以是原核细胞(例如大肠杆菌)，也可以是真核细胞(例如酵母细胞，例如COS细胞，中国仓鼠卵巢(CHO)细胞，HeLa细胞，HEK293细胞，COS-1细胞，NS0细胞或骨髓瘤细胞)。在某些情形中，所述细胞可以是哺乳动物细胞。例如，所述哺乳动物细胞可以是CHO-K1细胞。
在本申请中，术语“药物组合物”通常是指这样的制剂，其以允许活性成分的生物学活性有效的形式存在，并且不包含对将施用所述组合物的对象具有不可接受的毒性的另外的成分。
在本申请中，术语“治疗”通常是指期望改变所治疗个体的天然病程，且可为实现防治或在临床病变过程中进行的临床介入。合乎需要的治疗效果包括但不限于防止疾病发生或复发性、减轻症状、减弱疾病的任何直接或间接病理学后果、防止转移、降低疾病进展速率、改善或缓解疾病状态以及缓和或改善预后。在一些情形中，抗原结合蛋白(例如，抗VEGF抗体)可用来延迟疾病发展或减缓疾病进展。
在本申请中，术语“施用”通常是指向受试者(例如，患者)给予一定剂量的化合物(例如，抗癌治疗剂)或药物组合物(例如，包含抗癌治疗剂的药物组合物)的方法。施用可通过任何合适的方式进行，包括肠胃外、肺内和鼻内，以及(如果局部治疗需要)损伤内施用。胃肠外输注包括例如肌肉内、静脉内、动脉内、腹膜内或皮下施用。
在本申请中，术语“肿瘤”通常是指所有赘生性细胞生长和增殖(无论恶性还是良性)以及所有癌前和癌性细胞和组织。在本申请中，所述肿瘤可以为细胞和组织的VEGF或VEGFR高表达的肿瘤。肿瘤可包括实体瘤和/或非实体瘤(例如，血液瘤、淋巴瘤)。
在本申请中，术语“在……之间”通常是指某种氨基酸片段的C端与第一氨基酸片段的N端直接或间接连接，并且其N端与第二氨基酸片段的C端直接或间接连接。在轻链中，例如，所述L-FR2的N末端与所述LCDR1的C末端直接或间接相连，且所述L-FR2的C末端与所述LCDR2的N末端直接或间接相连。又例如，所述L-FR3的N末端与所述LCDR2的C末端直接或间接相连，且所述L-FR3的C末端与所述LCDR3的N末端直接或间接相连。在重链中，例如，所述H-FR2的N末端与所述HCDR1的C末端直接或间接相连，且所述H-FR2的C末端与所述HCDR2的N末端直接或间接相连。又例如，所述H-FR3的N末端与所述HCDR2的C末端直接或间接相连，且所述H-FR3的C末端与所述HCDR3的N末端直接或间接相连。在本申请中，“第一氨基酸片段”和“第二氨基酸片段”可以为相同或不同的任意一段氨基酸片段。
在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
抗原结合蛋白
一方面，本申请提供一种分离的抗原结合蛋白，所述的分离的抗原结合蛋白能够以1×10 - 8M或更低的KD值结合源自灵长类动物(例如，人)的VEGF(例如，VEGF165)。所述VEGF抗原结合蛋白对VEGF的结合亲和力可通过本领域已知的任何方法测定。在某些情形中，结合亲和力可通过表面等离子共振法(SPR)、酶联免疫法(ELISA)、结合抗原沉淀法、平衡透析法、生物膜干涉(BLI)测定。在某些情形中，VEGF抗原结合蛋白对VEGF的结合亲和力和KD值可通过生物膜干涉(BLI)测定。例如，可使用ForteBio Octet分子相互作用分析仪，来进行抗原抗体之间的结合动力学分析。
本申请中，所述分离的抗原结合蛋白能够以1×10 -7M或更低的KD值结合VEGF(例如，VEGF165)。例如，所述KD的值可以以约5×10 -8M或以下、约4×10 -8M或以下、约3×10 -8M或以下、约2×10 -8M或以下、约1×10 -8M或以下、约9×10 -9M或以下、约8×10 -9M或以下、约7×10 -9M或以下、约6×10 -9M或以下、约5×10 -9M或以下、约4×10 -9M或以下、约3×10 -9M或以下、约2×10 -9M或以下、约1×10 -9M或以下、约9×10 -10M或以下、约8×10 -10M或以下、约7×10 -10M或以下的值结合源自人的VEGF，例如，使用FortieBio Octet分子相互作用分析仪所检测的。
本申请所述的抗原结合蛋白能够阻断VEGF(例如，VEGF165)与VEGFR(例如VEGFR2)的结合。在某些情形中，所述的抗原结合蛋白阻断VEGF与VEGFR的结合可通过流式细胞技术FACS、酶联免疫法ELISA测定。
例如，首先将人VEGFR(例如VEGFR2)与递减量的未标记的所述抗原结合蛋白孵育，随后用被标记的VEGF蛋白孵育。然后，检测OD450，以证实所述抗原结合蛋白阻断VEGF(例如，VEGF165)与VEGFR(例如VEGFR2)结合。例如，阻断活性的IC50在约0.001μg/mL至约10μg/mL之间，约0.001μg/mL至约5μg/mL之间，约0.01μg/mL至约1μg/mL之间，约0.02μg/mL至约0.5μg/mL之间，约0.2μg/mL至约15μg/mL之间，约0.2μg/mL至约12μg/mL之间，约0.2μg/mL至约10μg/mL之间，约0.3μg/mL至约8μg/mL之间，约0.3μg/mL至约6μg/mL之间，约0.5μg/mL至约5μg/mL之间，约0.1μg/mL至约2μg/mL之间，或约0.5μg/mL至约1.5μg/mL之间。
在本申请中，所述分离的抗原结合蛋白能够与参比抗体竞争结合VEGF蛋白，其中所述参比抗体可包含重链可变区VH，所述参比抗体的VH可包含HCDR1、HCDR2和HCDR3，且所述参比抗体的HCDR1可包含如SEQ ID NO:9所示的氨基酸序列，所述参比抗体的HCDR2可包含如SEQ ID NO:23所示的氨基酸序列，和，所述参比抗体的HCDR3可包含如SEQ ID NO:24所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白能够与参比抗体竞争结合VEGF蛋白，所述参比 抗体可包含HCDR1、HCDR2和HCDR3，且所述参比抗体的HCDR1可包含SEQ ID NO：9所示的氨基酸序列，所述参比抗体的HCDR2可包含SEQ ID NO：12-14中任一项所示的氨基酸序列，所述参比抗体的HCDR3可包含SEQ ID NO：17-19中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白能够与参比抗体竞争结合VEGF蛋白，所述参比抗体可包含HCDR1、HCDR2和HCDR3，且所述参比抗体的HCDR1、HCDR2和HCDR3可以包含选自以下任一组氨基酸序列：
(1)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:12和HCDR3：SEQ ID NO:17；
(2)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:13和HCDR3：SEQ ID NO:17；
(3)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:14和HCDR3：SEQ ID NO:17；
(4)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:13和HCDR3：SEQ ID NO:18；和
(5)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:13和HCDR3：SEQ ID NO:19。
在本申请中，所述分离的抗原结合蛋白可包含至少一个来自VH的CDR，其中所述VH可包含SEQ ID NO：29所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含至少一个来自VH的CDR，其中所述VH可包含SEQ ID NO:1-6中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以是VHH。
在本申请中，所述分离的抗原结合蛋白可包含HCDR3，所述HCDR3可包含氨基酸序列如SEQ ID NO:29所示的VH的CDR3。在本申请中，所述分离的抗原结合蛋白可包含HCDR3，所述HCDR3可包含氨基酸序列如SEQ ID NO:1-6中任一项所示的VH的CDR3。
例如，所述分离的抗原结合蛋白可包含HCDR3，所述HCDR3可包含如SEQ ID NO:24所示的氨基酸序列：RLRIX 5X 6X 7X 8X 9X 10ERLDY(SEQ ID NO:24)，其中，X 5为P或T，X 6为D或H，X 7为E，Q或W，X 8为R，W或Y，X 9为R或S，且X 10为R或T。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:17所示的氨基酸序列相比，所述HCDR3可至少包含在选自下组位置处的氨基酸取代：在X 5、X 6、X 7、X 8、X 9和/或X 10处的氨基酸取代。
例如，所述HCDR3可包含如SEQ ID NO:17-19中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含氨基酸序列如SEQ ID NO:29所示的VH的CDR2。在本申请中，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含氨基酸序列如SEQ ID NO:1-6中任一项所示的VH的CDR2。
例如，所述分离的抗原结合蛋白可包含HCDR2，所述HCDR2可包含如SEQ ID NO:23所示的氨基酸序列：AVX 3AX 5X 6WX 8YVEDSVX 15G(SEQ ID NO:23)，其中，X 3为F或L，X 5为E或P，X 6为D或G，X 8为R或S，且X 15为K或R。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:12所示的氨基酸序列相比，所述HCDR2可至少包含在选自下组位置处的氨基酸取代：在X 3、X 5、X 6、X 8和/或X 15处的氨基酸取代。
例如，所述HCDR2可包含如SEQ ID NO:12-14中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1，所述HCDR1可包含氨基酸序列如SEQ ID NO:29所示的VH的CDR1。在本申请中，所述分离的抗原结合蛋白可包含HCDR1，所述HCDR1可包含氨基酸序列如SEQ ID NO:1-6中任一项所示的VH的CDR1。
例如，所述分离的抗原结合蛋白可包含HCDR1，所述HCDR1可包含如SEQ ID NO:9所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含氨基酸序列如SEQ ID NO:29所示的VH的CDR1，所述HCDR2可包含氨基酸序列如SEQ ID NO:29所示的VH的CDR2，所述HCDR3可包含氨基酸序列如SEQ ID NO:29所示的VH的CDR3。
在本申请中，所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含氨基酸序列如SEQ ID NO:1-6中任一项所示的VH的CDR1，所述HCDR2可包含氨基酸序列如SEQ ID NO:1-6中任一项所示的VH的CDR2，所述HCDR3可包含氨基酸序列如SEQ ID NO:1-6中任一项所示的VH的CDR3。
在本申请中，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含如SEQ ID NO:9所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:23所示的氨基酸序列，且所述HCDR3可包含如SEQ ID NO:24所示的氨基酸序列。
在本申请中，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含如SEQ ID NO:9所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:12-14中任一项所示的氨基酸序列，且所述HCDR3可包含如SEQ ID NO:17-19中任一项所示的氨基酸序列。
例如，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含如SEQ ID NO:9所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:12所示的氨基酸序列，且所述HCDR3可包含如SEQ ID NO:17所示的氨基酸序列。
例如，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含如SEQ ID NO:9所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:13所示的氨基酸序列，且所述HCDR3可包含如SEQ ID NO:17所示的氨基酸序列。
例如，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含如SEQ ID NO:9所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:14所示的氨基酸序列，且所述HCDR3可包含如SEQ ID NO:17所示的氨基酸序列。
例如，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含如SEQ ID NO:9所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:13所示的氨基酸序列，且所述HCDR3可包含如SEQ ID NO:18所示的氨基酸序列。
例如，本申请所述分离的抗原结合蛋白可包含HCDR1、HCDR2和HCDR3，所述HCDR1可包含如SEQ ID NO:9所示的氨基酸序列，所述HCDR2可包含如SEQ ID NO:13所示的氨基酸序列，且所述HCDR3可包含如SEQ ID NO:19所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR1，所述H-FR1可包含如SEQ ID NO:25所示的氨基酸序列：X 1VQLVESGGGLVQX 14GGSX 18RLSCAASGSTSD(SEQ ID NO:25)，其中，X 1为A或E，X 14为A或P，且X 18为A或L。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:7所示的氨基酸序列相比，所述H-FR1可至少包含在选自下组位置处的氨基酸取代：在X 1、X 14和/或X 18处的氨基酸取代。
例如，所述H-FR1可包含如SEQ ID NO:7或8所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR2，所述H-FR2可包含如SEQ ID NO:26所示的氨基酸序列：WYRQAPGKERXQQLVX 14(SEQ ID NO:26)，其中，X 11为D或E，且X 14为A或S。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:10所示的氨基酸序列相比，所述H-FR2可至少包含在选自下组位置处的氨基酸取代：在X 11和/或X 14处的氨基酸取代。
例如，所述H-FR2可包含如SEQ ID NO:10或11所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR3，所述H-FR3可包含如SEQ ID NO:27所示的氨基酸序列：
RFTISRDNX 9KNTVX 14LQMNX 19LX 21X 22EDTAX 27YYCNV(SEQ ID NO:27)，其中，X 9为S或T，X 14为D或Y，X 19为N或S，X 21为K或R，X 22为A或P，且X 27为I或V。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:15所示的氨基酸序列相比，所述H-FR3可至少包含在选自下组位置处的氨基酸取代：在X 9、X 14、X 19、X 21、X 22和/或X 27处的氨基酸取代。
例如，所述H-FR3可包含如SEQ ID NO:15或16所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白包可含H-FR4，所述H-FR4可包含如SEQ ID NO:28所示的氨基酸序列：WGX3GTX6VTVSS(SEQ ID NO:28)，其中，X 3为K或Q，且X 6为L、Q或T。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:20所示的氨基酸序列相比，所述H-FR4可至少包含在选自下组位置处的氨基酸取代：在X 3和/或X 6处的氨基酸取代。
例如，所述H-FR4可包含如SEQ ID NO:20-22中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可包含重链可变区VH，所述VH可包含SEQ ID NO:29所示的氨基酸序列：
X 1VQLVESGGGLVQX 14GGSX 18RLSCAASGSTSDIVSMAWYRQAPGKERX 46LVX 49AVX 5 2AX 54X 55WX 57YVEDSVX 64GRFTISRDNX 74KNTVX 79LQMNX 84LX 86X 87EDTAX 92YYCNVRLRIX 102X 103X 104X 105X 106X 107ERLDYWGX 115GTX 118VTVSS(SEQ ID NO:29)，其中，X 1为A或E，X 14为A或P，X 18为A或L，X 46为D或E，X 49为A或S，X 52为F或L，X 54为E或P，X 55为D或G，X 57为R或S，X 64为K或R，X 74为S或T，X 79为D或Y，X 84为N或S，X 86为K或R，X 87为A或P，X 92为I或V，X 102为P或T，X 103为D或H，X 104为E，Q或W，X 105为R，W或Y，X 106为R或S，X 107为R或T，X 115为K或Q，且X 118为L、Q或T。例如，该序列可以是根据Chothia定义规则确定的序列。
在某些情形中，与SEQ ID NO:1所示的氨基酸序列相比，所述VH可至少包含在选自下组位置处的氨基酸取代：在X 1、X 14、X 18、X 46、X 49、X 52、X 54、X 55、X 57、X 64、X 74、X 79、X 84、X 86、X 87、X 92、X 102、X 103、X 104、X 105、X 106、X 107、X 115和/或X 118处的氨基酸取代。
例如，所述VH可包含SEQ ID NO:1-6中任一项所示的氨基酸序列。
在本申请中，所述分离的抗原结合蛋白可以是VHH。例如，所述VHH可包含SEQ ID NO:1-6中任一项所示的氨基酸序列。
在本申请中，所述抗原结合蛋白每个重链或轻链氨基酸序列的一部分与来自特定物种的抗体中相应氨基酸序列同源，或者属于特定的类别。例如，轻链和重链的可变区及恒定部分均来自一个动物物种(如人)的抗体的可变区及恒定区。在本申请中，所述同源物可以为，与所述蛋白质和/或所述多肽(例如，特异性结合VEGF蛋白的抗体或其片段)的氨基酸序列具有至少约85％(例如，具有至少约85％、约90％、约91％、约92％、约93％、约94％、约 95％、约96％、约97％、约98％、约99％或更高的)序列同源性的蛋白质或多肽。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
核酸、载体、宿主细胞和制备方法
在另一个方面，本申请还提供了分离的一种或多种核酸分子，所述一种或多种核酸分子可编码本申请所述的抗原结合蛋白。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述抗原结合蛋白，也可以编码其中的一部分(例如，HCDR1-3、LCDR1-3、VL、VH、轻链或重链中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
在本申请中，可以通过本领域已知的多种方法来制备编码所述抗体、其抗原结合片段的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR。
在另一个方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。例如，所述载体为表达载体。
在另一个方面，本申请提供了宿主细胞，所述宿主细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。在某些实施方式中，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包 含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体
在另一个方面，本申请提供了制备所述的抗体或其抗原结合片段的方法。所述方法可包括，在使得所述的抗体或其抗原结合片段表达的条件下，培养所述本申请所述的宿主细胞。例如，可通过使用适当的培养基、适当的温度和培养时间等，这些方法是本领域普通技术人员所了解的。
药物组合物、方法、用途
在另一个方面，本申请提供了一种药物组合物，其可包含本申请所述的抗原结合蛋白，所述多肽，所述的核酸分子，所述的载体，所述的宿主细胞，以及任选地药学上可接受的载体。所述药学上可接受的佐剂在所采用的剂量和浓度下对接受者无毒性，本申请中的药物组合物还可含有多于一种活性化合物，通常为不会不利地影响彼此的具有互补活性的那些活性化合物。此类药物的类型和有效量取决于例如制剂中存在的拮抗剂的量和类型，以及受试者的临床参数。
所述药物组合物可以用于抑制肿瘤生长。例如，本申请的药物组合物可以抑制或延缓疾病的发展或进展，可以减小肿瘤大小(甚至基本消除肿瘤)，和/或可以减轻和/或稳定疾病状态。
本申请所述的药物组合物可以包含预防和/或治疗有效量的所述抗体、其抗原结合片段。所述预防和/或治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的疾病或病症和/或其任何并发症而所需的剂量。
另一方面，本申请提供了所述抗原结合蛋白和/或所述融合蛋白在制备药物中的用途。所述药物用于治疗癌症，抑制肿瘤生长和/或抑制肿瘤细胞增殖。在某些实施方式中，所述肿瘤包括VEGF过表达的肿瘤。在某些实施方式中，所述肿瘤包括VEGFR过表达的肿瘤。在某些实施方式中，所述肿瘤包括实体瘤和/或非实体瘤。在某些实施方式中，所述肿瘤包括肺癌、结直肠癌、乳腺癌、肾癌、胃癌、肝癌、母细胞瘤、宫颈癌和/或卵巢癌。
另一方面，本申请提供了抑制VEGF蛋白与VEGFR蛋白结合的方法，包括施用本申请所述的抗原结合蛋白和/或所述多肽。例如，所述方法可以是离体或体外方法。例如，所述方法可以是非治疗目的的方法。在某些情形中，所述方法可包括使生物样品与本申请所述的抗原结合蛋白和/或VEGFR在容许所述抗原结合蛋白和/或VEGFR结合VEGF的条件下接触，检测在所述抗原结合蛋白与VEGF之间是否形成复合物，和检测VEGF与VEGFR之间是否形成复合物。
另一方面，本申请提供了一种用于检测VEGF蛋白的存在和/或含量的方法，其包括施用 所述分离的抗原结合蛋白和/或所述的多肽。例如，所述方法可以是离体或体外方法。例如，所述方法可以是非治疗目的的方法。
本申请还提供了抗原结合蛋白在诊断患有肿瘤或癌症的受试者的方法中的用途，所述方法包括：通过使样品与本申请的抗原结合蛋白接触并检测结合的抗体的存在来确定获自受试者的样品中VEGF的存在或表达水平。
另一方面，本申请提供了一种抗体药物偶联物，其可以包含细胞毒性剂，以及本申请所述的抗原结合片段。抗体药物偶联物通常是指采用特定的连接子将抗体和小分子细胞毒药物连接起来，其主要组成成分可以包括抗体、连接子和小分子细胞毒药物。
另一方面，本申请提供了一种试剂盒，其可以包含本申请所述的抗原结合蛋白，嵌合抗原受体，基因修饰的细胞，抗体药物偶联物，和/或本申请所述的药物组合物。其可在单一常用容器中包括本申请所述的抗原结合蛋白，嵌合抗原受体，基因修饰的细胞，和/或抗体药物偶联物，也可任选地与一种或多种治疗剂组合，任选地一起配制于药物组合物中。
另一方面，本申请提供了一种给药装置，它可以用来施用本申请所述的抗原结合蛋白或其药物组合物。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
实施例1抗VEGF单域抗体制备与检测
采取三只未免疫目的抗原的羊驼的血样，每只取150ml。从血样中分离淋巴细胞提取总RNA，构建噬菌体展示纳米抗体文库，库容大小为3×10 9cfu。取6ml转化的抗体文库菌制备噬菌体用于特异性淘选，菌体总量大于库容50倍。
将50ml Streptavidin Magnetic Beads(Thermo fisher,货号：88817)预结合1ml噬菌体室温孵育30min，去除非特异性结合。取除背景后的纳米抗体文库噬菌体加入10ug Biotinylated Human VEGF165(百普赛斯，货号：VE5-H82Q0),150ul Streptavidin Magnetic Beads，室温孵育15min,PBST(PBS中含有0.05％Tween-20)洗14遍，洗去不结合的噬菌体。用450ul 100mM盐酸洗脱抗原特异性结合的噬菌体，加入50ul pH11的1M Tris-HCl中和并感染处于对数生长期的大肠杆菌SS320，产生并纯化噬菌体用于下一轮的筛选。筛选方法与第一轮相同，仅将抗原用量减为4ug。取两轮筛选后富集的噬菌体用酶联免疫(ELISA)鉴定富集情况，结果如下表1所示，结果表明，经过两轮淘选后噬菌体富集明显。
表1第二轮富集后的Phage Elisa鉴定
包被抗原 样品孔 对照孔
OD450nm吸收值 1.0713 0.1799
实施例2单域抗体筛选及体外活性鉴定
以实施例1中的单域抗体库两轮淘选后质粒为模板，设计引物进行聚合酶链式反应(PCR)扩增纳米抗体基因(V HH)；PCR扩增的V HH基因片段回收后与酵母展示质粒共转入酿酒酵母菌株EBY100(购自ATCC)，通过酿酒酵母的同源重组使VHH基因插入至酵母展示质粒中，进而实现在酵母细胞壁表面展示单域抗体，构建方案如图1所示。
使用流式分选仪对酵母展示文库进行两轮分选，将分选获得的酵母菌涂布营养缺陷型平板培养基，挑46个单克隆进行测序，最终获得5个独一序列的单域重链抗体。鉴定方案如表2所示。对相应的酵母单克隆菌落进行流式染色分析，取1×10 6个细胞按表2各方案进行染色，方案1与human VEGF165-Bio结合细胞群的强弱由PE平均荧光信号强度(MFI)反映，同理方案2和方案3可以评估Mouse VEGF-Bio以及非特异性的结合水平，方案4竞争信号由APC平均荧光信号强度(MFI)反映，结果如表3。由表3可知，排除与人VEGF、鼠VEGF不结合的克隆，最终获得抗人VEGF165单域重链抗体的单克隆Y20A6，Y20A6的结合及竞争的能力均理想。
表2单克隆酵母菌落流式染色鉴定方案
Figure PCTCN2022075600-appb-000001
表3酵母展示文库二轮筛选后酵母单克隆菌落流式染色分析结果
克隆编号 人VEGF结合信号 鼠VEGF结合信号 无关抗原结合信号 竞争信号
Y20A6 1477 1441 21.8 25.4
实施例3单域抗体人源化
3.1单域抗体人源化设计与表达
对单克隆抗体Y20A6的序列进行IMGT/Domain Gap Align，查找与其同源性最高的人源的germline为IGHV3-23*04。抗体序列按Chothia规则编号，对H1，H14，H18等位点序列不同的位点进行如表4突变，蛋白编号Ab1910VE6为人源化前的初始抗体。
表4单域抗体序列人源化设计
Figure PCTCN2022075600-appb-000002
将表4中各全长序列与IgG1 Fc N297A(采购自泰州市百英生物科技有限公司)融合，进行密码子优化，基因合成后装入表达载体pcDNA3.4(Life Technologies)。表达质粒扩增和质粒抽提后质粒转入ExpiCHO细胞(ThermoFisher Scientific，A29133)，根据供应商ExpiCHO表达系统方法进行抗体瞬转表达，得到纯化的抗体见表5。
表5单域抗体表达、纯化数据
抗体编号 理论等电点 产量(mg) 表达量(mg/L) 浓度(mg/mL)
Ab1910VE6 7 0.89 89 4.6
Ab1910VE8 7.4 0.48 48 4.8
Ab1910VE9 7 0.75 75 3.8
实施例4本申请抗原结合蛋白与人VEGF165的结合活性测定
(1)采用Octet RED96e(Fortebio)测定Ab1910VE6、AB1910VE8及Ab1910VE9与人VEGF165(Acro,货号：VE5-H82Q0)的亲和力，抗原及抗体均用1xPBST(1xPBS：生工，B548117-0500；0.02％吐温20：sigma-alorich，P1379)稀释，抗原使用浓度为100nM，抗体使用浓度为50nM。
(2)样品上机检测(Octet Data Acquisition 11.1.0.11)：首先，将样品加入96孔板(Greiner bio-one，655209)，体系为200μL/well。然后设置软件参数，板温设定为30℃，收集标准动 力学信号的频率为5.0HZ。接着，用1xPBST预湿AHC传感器(Fortébio,货号：18-0015)10分钟，然后上机检测。每个循环包含以下步骤：1)浸入缓冲液60秒；2)检测抗原是否与传感器有非特异性结合；3)10mM pH1.7的甘氨酸溶液再生；4)浸入缓冲液60秒；5)抗体固化在传感器上，时间为20秒；6)传感器浸入缓冲液180秒；7)抗原与抗体结合，时间180秒；8)抗原抗体的解离，时间10分钟；9)传感器再生。
(3)数据分析
采用Fortebio的Data Analysis 11.0软件，对抗原-抗体以1:1的结合方式，测定结合速率(Ka)和解离速率(Kd)，以此计算抗体的平衡解离常数(KD)。结果如表6。Ab1910VE6、AB1910VE8和Ab1910VE9与人VEGF亲和力均较强。说明本申请抗原结合蛋白与VEGF的结合亲和力高。
表6单域抗体与人VEGF165的亲和力
候选抗体 响应值 K D(M) kon(1/Ms) kdis(1/s)
AB1910VE6 0.4416 6.304E-09 1.41E+06 8.88E-03
AB1910VE8 0.4095 1.052E-08 1.75E+06 1.84E-02
AB1910VE9 0.4062 9.913E-09 1.82E+06 1.81E-02
Bevacizumab 0.3414 <1.0E-12 6.75E+05 <1.0E-07
实施例5本申请抗原结合蛋白阻断人VEGF165与人VEGFR2结合的活性测定
采用竞争ELISA方法测定单域抗体阻断VEGF165和人VEGFR2的结合，具体实施步骤如下：将人VEGF R2(Acro,货号：KDR-H5227)用PBS(Gibco,货号：10010-023)稀释到1μg/mL，加入到96孔板中，每孔100μL，贴封板膜，置于4度孵育过夜。第二天用洗涤液PBST(0.05％TWEEN-20:sigma-alorich，P1379)洗板3次，继而用洗涤液配制封闭液PBST+2％BSA(BSA：VWR,0332-1KG)，每孔加入300μL封闭液，37℃封闭1小时。用封闭液配制Anti-VEGF和Biotin-hVEGF(Acro,货号：VE5-H82Q0)，Anti-VEGF的配制起始浓度为200μg/mL，5倍梯度稀释(7个浓度点+1个0浓度点)，Biotin-hVEGF的配制浓度为90ng/mL。继而取出封闭好的酶标板，用洗涤液洗板3次，取50μL稀释后的Anti-VEGF加入到96孔板中，再取50μL的Biotin-hVEGF加入到96孔板中，37℃共孵育1小时。用洗涤液洗板3次，封闭液1：5000稀释二抗SA-HRP(sigma，S2438)，以100μL/孔加至酶标板内，37℃孵育1小时。用洗涤液洗板3次，每孔加入100μL TMB显色液(Biopanda，货号：TMB-S-003)，室温避光显色，然后每孔加入50μL终止液(Solarbio，货号：C1058)终止反应，在450nm波长处测定吸光值。结果显示(图2)，Ab1910VE8、Ab1910VE9和Ab1910VE6 的阻断能力均较强。说明本申请抗原结合蛋白具有良好的阻断VEGF和VEGFR结合的效果。
实施例6本申请抗原结合蛋白亲和力成熟及体外鉴定
6.1单域抗体亲和力成熟文库构建
选择Ab1910VE9序列进行亲和力成熟，将Ab1910VE9的CDR区按Chothia定义。对可变区CDR1、CDR2、CDR3，设计NNK突变引物进行聚合酶链式反应(PCR)扩增各CDR突变文库基因片段。根据文献所述方法，将各CDR突变文库基因片段与酵母展示质粒分别转入酿酒酵母菌株EBY100(购自ATCC)，使各CDR突变文库展示于酵母表面。同时将Ab1910VE9的亲本序列展示于酵母表面，作为对照使用。
文库经过培养、诱导后，使用Biotin-Human VEGF165进行三轮分选，起始浓度3nM，10倍梯度稀释。收集展示水平高且与抗原结合能力强的细胞群；分选后细胞涂布于SD-Trp固体培养基，30℃静置培养3天。
6.2单克隆鉴定
挑取单克隆送测序，对获得的单克隆进行流式染色鉴定，与1nM Biotin-Human VEGF165孵育染色，比较不同克隆的展示平均荧光信号强度与抗原结合平均荧光信号强度的比值，反映了单个分子与抗原的结合能力。根据结合力数值，选取各CDR区不同突变序列共3个分子进行后续体外评估，具体见表7。
表7单克隆鉴定结果与抗体表达编号
Figure PCTCN2022075600-appb-000003
6.3亲和力成熟单域抗体表达及SEC-HPLC纯度分析
将表7中各单域抗体进行表达、纯化。对表达抗体进行SEC-HPLC纯度分析，方法如下：
(1)将样品稀释至1mg/mL，混匀，12000rpm离心5min，取上清转至样品瓶，放入HPLC样品盘。设置色谱条件如下：
Figure PCTCN2022075600-appb-000004
Figure PCTCN2022075600-appb-000005
(2)色谱柱采用流动相(200mM磷酸盐缓冲液，pH6.8)平衡后，进样分析，用色谱软件进行数据分析，峰面积归一化法计算各个峰的峰面积百分比。
具体结果见表8。
表8亲和力成熟单域抗体表达、纯化数据
抗体编号 理论等电点 产量(mg) 表达(mg/L) 浓度(mg/ml) 单体率(％)
Ab1910VE18 7.5 3.26 130 2.17 96.4
Ab1910VE21 7.6 3.54 142 2.36 96.9
Ab1910VE23 7.5 2.54 101 1.69 96.2
Ab1910VE24 7.3 2.79 112 1.86 96.5
实施例7本申请抗原结合蛋白动力学参数测定
参照实施例4的方法，测定亲和力成熟前后抗体与人VEGF165的亲和力。
表9亲和力成熟抗体与人VEGF165的亲和力
本申请抗体 响应值 KD(M) kon(1/Ms) kdis(1/s)
Ab1910VE18 0.1421 2.227E-09 1.81E+06 4.03E-03
Ab1910VE21 0.1536 4.126E-10 1.24E+06 5.10E-04
Ab1910VE23 0.1164 7.276E-10 1.43E+06 1.04E-03
Ab1910VE24 0.1532 6.257E-10 1.37E+06 8.57E-04
Bevacizumab 0.2211 <1.0E-12 3.50E+05 <1.0E-07
结果如表9，Ab1910VE18为亲和力成熟前的分子，成熟后的抗体分子亲和力提高约2-5倍。说明本申请抗原结合蛋白与VEGF的结合亲和力高。
实施例8本申请抗原结合蛋白阻断人VEGF165和人VEGFR2的结合功能实验
参照实施例5的方法，测定亲和力成熟VEGF抗体阻断人VEGF165和人VEGFR2的结合功能实验，其中Biotin-hVEGF165的配制浓度为700ng/mL。实验结果如图3，Ab1910VE18，Ab1910VE21,Ab1910VE24抗体的阻断效果和Bevacizumab相当。说明本申请抗原结合蛋白具有良好的阻断VEGF和VEGFR结合的效果。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (54)

  1. 分离的抗原结合蛋白，其具有下述性质中的一种或多种：
    (1)能够以1×10 -7M或更低的K D值结合VEGF蛋白；和
    (2)能够阻断VEGF蛋白与VEGFR蛋白之间结合。
  2. 根据权利要求1所述的分离的抗原结合蛋白，其中所述VEGF蛋白包括VEGF165。
  3. 根据权利要求1-2中任一项所述的分离的抗原结合蛋白，其中所述VEGFR蛋白包括VEGFR2。
  4. 根据权利要求1-3中任一项所述的分离的抗原结合蛋白，其包括抗体或其抗原结合片段。
  5. 根据权利要求4所述的分离的抗原结合蛋白，其中所述抗原结合片段包括Fab，Fab’，F(ab) 2、Fv片段、F(ab’) 2、scFv、di-scFv、VHH和/或dAb。
  6. 根据权利要求4或5所述的分离的抗原结合蛋白，其中所述抗原结合片段为VHH。
  7. 根据权利要求4-6中任一项所述的分离的抗原结合蛋白，其中所述抗体选自下组：单克隆抗体、嵌合抗体、人源化抗体和全人源抗体。
  8. 根据权利要求1-7中任一项所述的分离的抗原结合蛋白，其能够与参比抗体竞争结合所述VEGF蛋白，其中所述参比抗体包含重链可变区VH，所述参比抗体的VH包含HCDR1、HCDR2和HCDR3，且所述参比抗体的HCDR1包含如SEQ ID NO:9所示的氨基酸序列，所述参比抗体的HCDR2包含如SEQ ID NO:23所示的氨基酸序列，和，所述参比抗体的HCDR3包含如SEQ ID NO:24所示的氨基酸序列。
  9. 根据权利要求8所述的分离的抗原结合蛋白，其中所述参比抗体的HCDR1包含SEQ ID NO：9所示的氨基酸序列。
  10. 根据权利要求8-9中任一项所述的分离的抗原结合蛋白，其中所述参比抗体的HCDR2包含SEQ ID NO：12-14中任一项所示的氨基酸序列。
  11. 根据权利要求8-10中任一项所述的分离的抗原结合蛋白，其中所述参比抗体的HCDR3包含SEQ ID NO：17-19中任一项所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的分离的抗原结合蛋白，其包含VH中的至少一个CDR，所述VH包含如SEQ ID NO:29所示的氨基酸序列。
  13. 根据权利要求1-12中任一项所述的分离的抗原结合蛋白，其包含VH中的至少一个CDR，所述VH包含如SEQ ID NO:1-6中任一项所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的分离的抗原结合蛋白，其包含HCDR3，且所述HCDR3包含SEQ ID NO：24所示的氨基酸序列。
  15. 根据权利要求14所述的分离的抗原结合蛋白，其中所述HCDR3包含SEQ ID NO：17-19中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的分离的抗原结合蛋白，其包含HCDR2，且所述HCDR2包含SEQ ID NO：23所示的氨基酸序列。
  17. 根据权利要求16所述的分离的抗原结合蛋白，其中所述HCDR2包含SEQ ID NO：12-14中任一项所示的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的分离的抗原结合蛋白，其包含HCDR1，且所述HCDR1包含SEQ ID NO：9所示的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的分离的抗原结合蛋白，其包含HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO:23所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:24所示的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的分离的抗原结合蛋白，其包含HCDR1、HCDR2和HCDR3，其中所述HCDR1包含SEQ ID NO:9所示的氨基酸序列，所述HCDR2包含SEQ ID NO:12-14中任一项所示的氨基酸序列，且所述HCDR3包含SEQ ID NO:17-19中任一项所示的氨基酸序列。
  21. 根据权利要求1-20中任一项所述的分离的抗原结合蛋白，其包含HCDR1、HCDR2和HCDR3，且所述HCDR1、HCDR2和HCDR3选自以下任一组氨基酸序列：
    (1)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:12和HCDR3：SEQ ID NO:17；
    (2)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:13和HCDR3：SEQ ID NO:17；
    (3)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:14和HCDR3：SEQ ID NO:17；
    (4)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:13和HCDR3：SEQ ID NO:18；和
    (5)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:13和HCDR3：SEQ ID NO:19。
  22. 根据权利要求1-21中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，其中所述VH包括框架区H-FR1，所述H-FR1的C末端与所述HCDR1的N末端直接或间接相连，且所述H-FR1包含SEQ ID NO:25所示的氨基酸序列。
  23. 根据权利要求22所述的分离的抗原结合蛋白，其中所述H-FR1包含SEQ ID NO:7-8中任一项所示的氨基酸序列。
  24. 根据权利要求22-23中任一项所述的分离的抗原结合蛋白，其中所述VH包括框架区H-FR2，所述H-FR2位于所述HCDR1与所述HCDR2之间，且所述H-FR2包含SEQ ID NO:26所示的氨基酸序列。
  25. 根据权利要求24所述的分离的抗原结合蛋白，其中所述H-FR2包含SEQ ID NO:10-11 中任一项所示的氨基酸序列。
  26. 根据权利要求22-25中任一项所述的分离的抗原结合蛋白，其中所述VH包括框架区H-FR3，所述H-FR3位于所述HCDR2与所述HCDR3之间，且所述H-FR3包含SEQ ID NO:27所示的氨基酸序列。
  27. 根据权利要求26所述的分离的抗原结合蛋白，其中所述H-FR3包含SEQ ID NO:15-16中任一项所示的氨基酸序列。
  28. 根据权利要求22-27中任一项所述的分离的抗原结合蛋白，其中所述H-FR4的N末端与所述HCDR3的C末端相连，且所述H-FR4包含SEQ ID NO:28所示的氨基酸序列。
  29. 根据权利要求28所述的分离的抗原结合蛋白，其中所述H-FR4包含SEQ ID NO:20-22中任一项所示的氨基酸序列。
  30. 根据权利要求1-29中任一项所述的分离的抗原结合蛋白，其包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1包含SEQ ID NO:25所示的氨基酸序列，所述H-FR2包含SEQ ID NO:26所示的氨基酸序列，所述H-FR3包含SEQ ID NO:27所示的氨基酸序列且所述H-FR4包含SEQ ID NO:28所示的氨基酸序列。
  31. 根据权利要求1-30中任一项所述的分离的抗原结合蛋白，其包含H-FR1、H-FR2、H-FR3和H-FR4，其中所述H-FR1包含SEQ ID NO:7或SEQ ID NO:8所示的氨基酸序列，所述H-FR2包含SEQ ID NO:10或SEQ ID NO:11所示的氨基酸序列，所述H-FR3包含SEQ ID NO:15或SEQ ID NO:16所示的氨基酸序列，且所述H-FR4包含20-22中任一项所示的氨基酸序列。
  32. 根据权利要求1-31中任一项所述的分离的抗原结合蛋白，其包含H-FR1、H-FR2、H-FR3和H-FR4，且所述H-FR1、H-FR2、H-FR3和H-FR4选自以下任一组氨基酸序列：
    (1)F-FR1：SEQ ID NO:7，H-FR2：SEQ ID NO:10，H-FR3：SEQ ID NO:15和H-FR4：SEQ ID NO:20；
    (2)H-FR1：SEQ ID NO:8，H-FR2：SEQ ID NO:11，H-FR3：SEQ ID NO:16和H-FR4：SEQ ID NO:21；和
    (3)H-FR1：SEQ ID NO:8，H-FR2：SEQ ID NO:11，H-FR3：SEQ ID NO:16和H-FR4：SEQ ID NO:22。
  33. 根据权利要求1-32中任一项所述的分离的抗原结合蛋白，其包含重链可变区VH，且所述VH包含SEQ ID NO:29所示的氨基酸序列。
  34. 根据权利要求33所述的分离的抗原结合蛋白，其中所述VH包含SEQ ID NO:1-6中任 一项所示的氨基酸序列。
  35. 分离的一种或多种核酸分子，其编码权利要求1-34中任一项所述的分离的抗原结合蛋白。
  36. 载体，其包含根据权利要求35所述的核酸分子。
  37. 细胞，其包含根据权利要求35所述的核酸分子或根据权利要求36所述的载体。
  38. 制备权利要求1-34中任一项所述的分离的抗原结合蛋白的方法，所述方法包括在使得权利要求1-34中任一项所述的分离的抗原结合蛋白表达的条件下，培养根据权利要求37所述的细胞。
  39. 药物组合物，其包含权利要求1-34中任一项所述的分离的抗原结合蛋白、权利要求35所述的核酸分子、权利要求36所述的载体和/或权利要求37所述的细胞，以及任选地药学上可接受的载体。
  40. 多肽，其包含权利要求1-34中任一项所述的分离的抗原结合蛋白。
  41. 免疫缀合物，其包含权利要求1-34中任一项所述的分离的抗原结合蛋白或权利要求40所述的多肽。
  42. 权利要求1-34中任一项所述的分离的抗原结合蛋白、权利要求35所述的核酸分子、权利要求36所述的载体、权利要求37所述的细胞、权利要求39所述的药物组合物、权利要求40所述的多肽和/或权利要求41所述的免疫缀合物在制备药物中的用途，所述药物用于预防、缓解和/或治疗肿瘤。
  43. 根据权利要求42所述的用途，其中所述肿瘤包括VEGF过表达的肿瘤。
  44. 根据权利要求42或43所述的用途，其中所述肿瘤包括实体瘤和/或非实体瘤。
  45. 根据权利要求42-44中任一项所述的用途，其中所述肿瘤包括肺癌、结直肠癌、乳腺癌、肾癌、胃癌、肝癌、母细胞瘤、宫颈癌和/或卵巢癌。
  46. 预防、缓解或治疗肿瘤的方法，所述方法包括向有需要的受试者施用权利要求1-34中任一项所述的分离的抗原结合蛋白、权利要求35所述的核酸分子、权利要求36所述的载体、权利要求37所述的细胞、权利要求39所述的药物组合物、权利要求40所述的多肽和/或权利要求41所述的免疫缀合物。
  47. 根据权利要求46所述的方法，其中所述肿瘤包括VEGF过表达的肿瘤。
  48. 根据权利要求46或47所述的方法，其中所述肿瘤包括实体瘤和/或非实体瘤。
  49. 根据权利要求46-48中任一项所述的方法，其中所述肿瘤包括肺癌、结直肠癌、乳腺癌、肾癌、胃癌、肝癌、母细胞瘤、宫颈癌和/或卵巢癌。
  50. 权利要求1-34中任一项所述的分离的抗原结合蛋白、权利要求35所述的核酸分子、权 利要求36所述的载体、权利要求37所述的细胞、权利要求39所述的药物组合物、权利要求40所述的多肽和/或权利要求41所述的免疫缀合物，其用于预防、缓解或治疗肿瘤。
  51. 根据权利要求50所述的分离的抗原结合蛋白、核酸分子、载体、细胞、药物组合物、多肽和/或免疫缀合物，其中所述肿瘤包括VEGF过表达的肿瘤。
  52. 根据权利要求50或51所述的分离的抗原结合蛋白、核酸分子、载体、细胞、药物组合物、多肽和/或免疫缀合物，其中所述肿瘤包括实体瘤和/或非实体瘤。
  53. 根据权利要求50-52中任一项所述的分离的抗原结合蛋白、核酸分子、载体、细胞、药物组合物、多肽和/或免疫缀合物，其中所述肿瘤包括肺癌、结直肠癌、乳腺癌、肾癌、胃癌、肝癌、母细胞瘤、宫颈癌和/或卵巢癌。
  54. 抑制VEGF蛋白与VEGFR蛋白结合的方法，其包括施用权利要求1-34中任一项所述的分离的抗原结合蛋白、权利要求35所述的核酸分子、权利要求36所述的载体、权利要求37所述的细胞、权利要求39所述的药物组合物、权利要求40所述的多肽和/或权利要求41所述的免疫缀合物。
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