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WO2022042719A1 - 抗vegf-抗pd-l1双特异性抗体、其药物组合物及用途 - Google Patents

抗vegf-抗pd-l1双特异性抗体、其药物组合物及用途 Download PDF

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WO2022042719A1
WO2022042719A1 PCT/CN2021/115308 CN2021115308W WO2022042719A1 WO 2022042719 A1 WO2022042719 A1 WO 2022042719A1 CN 2021115308 W CN2021115308 W CN 2021115308W WO 2022042719 A1 WO2022042719 A1 WO 2022042719A1
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antibody
vegf
bispecific antibody
seq
amino acid
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PCT/CN2021/115308
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English (en)
French (fr)
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缪小牛
陈乘
袁志军
黄威峰
曾竣玮
孙左宇
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普米斯生物技术(珠海)有限公司
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Priority to KR1020237010695A priority Critical patent/KR20230061433A/ko
Priority to EP21860551.7A priority patent/EP4209513A4/en
Priority to AU2021334042A priority patent/AU2021334042A1/en
Priority to CA3192812A priority patent/CA3192812A1/en
Priority to US18/023,701 priority patent/US20230340158A1/en
Priority to JP2023513787A priority patent/JP2023539501A/ja
Priority to MX2023002331A priority patent/MX2023002331A/es
Priority to CN202180052341.1A priority patent/CN116261595A/zh
Publication of WO2022042719A1 publication Critical patent/WO2022042719A1/zh

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Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to an anti-VEGF-anti-PD-L1 bispecific antibody, a pharmaceutical composition and uses thereof.
  • VEGF Vascular endothelial growth factor
  • VPF vascular permeability factor
  • vasculotropin is a highly specific homodimer that promotes the growth of vascular endothelial cells. body protein.
  • VEGF family proteins include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placental growth factor (PIGF), among which VEGF-A is involved in the early formation of blood vessels Play an important role.
  • VEGF-A vascular permeability factor
  • PIGF placental growth factor
  • VEGF vascular endothelial growth factor
  • VEGF is involved in the pathogenesis and progression of many angiogenesis-dependent diseases, including cancer, certain inflammatory diseases, and diabetic retinopathy. Therefore, VEGF is an important target in antitumor drug research.
  • the main receptors of VEGF proteins are VEGFR1, VEGFR2, VEGFR3, NRP1, NRP2 and NRP3.
  • VEGFA can bind to VEGFR1 and VEGFR2, activate endogenous kinase activation, and promote new blood vessels. generate. Blocking the binding of VEGF to the receptor can be applied to the treatment of various cancers, such as breast cancer, colon cancer, lung cancer, ovarian cancer, endometrial cancer, mesothelioma, cervical cancer, kidney cancer (Rakesh R. Ramjiawan, Arjan W. Griffioen, and Dan G. Duda, Angiogenesis. 2017 20(2):185–204.).
  • Bevacizumab is approved in the United States, the European Union and other places for the treatment of colorectal cancer, non-small cell lung cancer, breast cancer, glioblastoma and renal cell carcinoma.
  • Ranibizumab is a second-generation humanized anti-VEGF recombinant mouse monoclonal antibody fragment Fab part, which is obtained from the same parental mouse antibody as Bevacizumab. It was approved by the US FDA on June 30, 2006 for the treatment of patients with age-related macular degeneration (AMD). Compared with bevacizumab, ranibizumab maintains a better affinity for VEGFA and can better inhibit angiogenesis. It has been developed for gastric cancer, rectal cancer and other indications.
  • Apacept is a human IgG-Fc recombinant protein composed of VEGFR1 and VEGFR2 extracellular fragments, which can simultaneously block the binding of VEGFR1 to VEGFR2 and VEGFA, thereby blocking the angiogenesis of vascular epithelial cells.
  • Apacept is primarily indicated for the treatment of patients with neovascular (wet) age-related macular degeneration (AMD).
  • AMD age-related macular degeneration
  • the clinical treatment of advanced colorectal cancer is carried out (Caemen Stancan, etc, Rom J Morphol Embryol. 2018 59(2):455–467). Since apatacept possesses functional fragments of VEGFR1 and VEGFR2, it has the function of blocking VEGF and receptor binding similar to antibodies.
  • PD-L1 Programmed death 1 ligand 1
  • CD274 is a member of the B7 family and is a ligand of PD-1.
  • PD-L1 is a type I transmembrane protein with a total of 290 amino acids, including an IgV-like domain, an IgC-like domain, a transmembrane hydrophobic domain and an intracellular domain consisting of 30 amino acids.
  • PD-L1 negatively regulates immune responses. Studies have found that PD-L1 is mainly expressed in activated T cells, B cells, macrophages and dendritic cells, etc.
  • PD-L1 In addition to lymphocytes, PD-L1 is also expressed in other tissues such as thymus, heart, placenta, etc. Endothelial cells, and various non-lymphoid lineages such as melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophagus cancer, head and neck cancer, etc. (Akintunde Akinleye&Zoaib Rasool, Journal of Hematology&Oncology volume 12, Article number: 92 (2019)). PD-L1 is broad in regulating autoreactive T, B cells, and immune tolerance, and plays a role in peripheral tissue T and B cell responses. High expression of PD-L1 on tumor cells is associated with poor prognosis in cancer patients.
  • bifunctional antibodies are a direction of antibody drug development, they face many challenges, such as preclinical evaluation models, low expression levels, poor stability, complex processes, and large differences in quality control. Therefore, the development of bifunctional antibodies has always been difficult. Heavy.
  • anti-VEGF-anti-PD-L1 bispecific antibody an anti-VEGF-anti-PD-L1 bispecific antibody (hereinafter also referred to as anti-PD-L1/VEGF bispecific antibody) through intensive research and creative work.
  • anti-PD-L1/VEGF bispecific antibody of the present invention has high affinity for the dual targets of PD-L1 and VEGF, has the common biological activities of the two targets, and has a small molecular weight at the same time. It can penetrate the tumor area flexibly and has good safety.
  • One aspect of the present invention pertains to a bispecific antibody comprising:
  • the first protein functional domain is an anti-VEGF antibody or an antigen-binding fragment thereof, or, the first protein functional domain comprises a VEGF receptor or a fragment having a VEGF receptor function;
  • the second protein functional region is an anti-PD-L1 single domain antibody.
  • the bispecific antibody consists of the first protein functional domain and the second protein functional domain and an optional linker.
  • the bispecific antibody wherein,
  • the heavy chain variable region of the anti-VEGF antibody comprises HCDR1 whose amino acid sequence is shown in SEQ ID NO:21, HCDR2 whose amino acid sequence is shown in SEQ ID NO:22, and HCDR2 whose amino acid sequence is shown in SEQ ID NO:23 HCDR3;
  • the heavy chain variable region of the anti-VEGF antibody comprises HCDR1 whose amino acid sequence is shown in SEQ ID NO: 27, HCDR2 whose amino acid sequence is shown in SEQ ID NO: 22, and whose amino acid sequence is shown in SEQ ID NO: 28 HCDR3;
  • the light chain variable region of the antibody against VEGF comprises LCDR1 with amino acid sequence as shown in SEQ ID NO: 24, LCDR2 with amino acid sequence as shown in SEQ ID NO: 25, and amino acid sequence as shown in SEQ ID NO: 26 LCDR3.
  • variable regions of the light and heavy chains determine the binding of antigens; the variable regions of each chain contain three hypervariable regions, called complementarity determining regions (CDRs), of which the CDRs of the heavy chain (H) include HCDR1, HCDR2, HCDR3, the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3.
  • CDRs are defined by the IMGT numbering system, please refer to Ehrenmann F, Kaas Q, Lefranc M P.
  • IMGT/3Dstructure-DB and IMGT/DomainGapAlign a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF[J].Nucleic acids research,2009;38(suppl_1):D301-D307.
  • the bispecific antibody wherein,
  • the anti-PD-L1 single domain antibody comprises a heavy chain variable region, and the heavy chain variable region comprises HCDR1 whose amino acid sequence is shown in SEQ ID NO: 29, and HCDR2 whose amino acid sequence is shown in SEQ ID NO: 30 And the HCDR3 whose amino acid sequence is as shown in SEQ ID NO:31;
  • amino acid sequence of the anti-PD-L1 single domain antibody is shown in SEQ ID NO:5.
  • the bispecific antibody wherein,
  • amino acid sequence of the heavy chain variable region of the anti-VEGF antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO:9; or
  • amino acid sequence of the heavy chain variable region of the anti-VEGF antibody is shown in SEQ ID NO: 13
  • amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 15.
  • the bispecific antibody wherein,
  • amino acid sequence of the heavy chain variable region of the anti-VEGF antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO:9; or
  • amino acid sequence of the heavy chain variable region of the anti-VEGF antibody is shown in SEQ ID NO: 13
  • amino acid sequence of the light chain variable region thereof is shown in SEQ ID NO: 15;
  • amino acid sequence of the anti-PD-L1 single domain antibody is shown in SEQ ID NO:5.
  • the bispecific antibody wherein,
  • the anti-VEGF antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single chain antibody, humanized antibody, chimeric antibody or diabody antibody.
  • the bispecific antibody wherein,
  • the constant region of the anti-VEGF antibody is derived from a human antibody
  • the constant region is selected from the constant region of human IgGl, IgG2, IgG3 or IgG4.
  • the bispecific antibody wherein,
  • the heavy chain constant region of the anti-VEGF antibody is a human Ig gamma-1 chain C region or a human Ig gamma-4 chain C region, and the light chain constant region thereof is a human Ig kappa chain C region;
  • amino acid sequence of the light chain constant region of the anti-VEGF antibody is shown in SEQ ID NO: 10;
  • amino acid sequence of the light chain of the anti-VEGF antibody is shown in SEQ ID NO:8 or SEQ ID NO:14.
  • the bispecific antibody wherein,
  • the heavy chain constant region of the anti-VEGF antibody further comprises the L234A mutation and the L235A mutation according to the EU numbering system; optionally, the G237A mutation;
  • amino acid sequence of the heavy chain constant region of the anti-VEGF antibody is shown in SEQ ID NO:4.
  • the bispecific antibody wherein:
  • the VEGF is VEGF-A;
  • the VEGF receptor is VEGFR1 and/or VEGFR2.
  • the bispecific antibody wherein the single-domain antibody is linked to the C-terminus or N-terminus of the first protein functional region, for example, the single-domain antibody is two, each One end of the single domain antibody is respectively connected to the C-terminus or N-terminus of the two heavy chains of the anti-VEGF antibody, or is connected to the C-terminus or N-terminus of a VEGF receptor or a fragment with VEGF receptor function;
  • the single domain antibody is directly connected with the first protein functional region or connected by a connecting fragment
  • amino acid sequence of the linker fragment is independently selected from SEQ ID NO:6 and SEQ ID NO:7;
  • amino acid sequence of the peptide chain obtained by linking the single domain antibody to the first protein functional region is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11 or SEQ ID NO: 12.
  • the present invention relates to a bispecific antibody comprising:
  • the first protein functional region is 1, and the second protein functional region is 2;
  • the first protein functional region is an anti-VEGF antibody or an antigen-binding fragment thereof, and the second protein functional region is an anti-PD-L1 single-domain antibody;
  • the single-domain antibody is connected to the C-terminus of the heavy chain of the anti-VEGF antibody through a connecting fragment;
  • the amino acid sequence of the peptide chain obtained after connecting is such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 11 or SEQ ID NO:12 shown;
  • amino acid sequence of the light chain of the anti-VEGF antibody is shown in SEQ ID NO:8 or SEQ ID NO:14.
  • the bispecific antibody wherein,
  • the first protein functional region comprises: VEGF receptor or a fragment with VEGF receptor function, and the Fc segment of IgG1;
  • amino acid sequence of the fragment with VEGF receptor function is shown in SEQ ID NO: 17;
  • the Fc segment of the IgG1 comprises the L234A mutation and the L235A mutation according to the EU numbering system;
  • amino acid sequence of the Fc segment of IgG1 is shown in SEQ ID NO: 18.
  • the bispecific antibody wherein,
  • the bispecific antibody is a dimer; preferably, an amino acid sequence such as SEQ ID NO: 16 or SEQ ID NO: A dimer of the polypeptide shown in 19.
  • the bispecific antibody according to any one of the present invention is used for the treatment and/or prevention of malignant tumor; preferably, the malignant tumor is selected from the group consisting of melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer cancer, breast cancer, esophageal cancer, and head and neck cancer.
  • Another aspect of the present invention pertains to isolated nucleic acid molecules encoding the bispecific antibodies of any one of the present invention.
  • Yet another aspect of the present invention relates to a vector comprising the isolated nucleic acid molecule of the present invention.
  • Yet another aspect of the present invention relates to a host cell comprising an isolated nucleic acid molecule of the present invention, or a vector of the present invention.
  • a further aspect of the present invention pertains to a method of making a bispecific antibody of any one of the present invention, comprising culturing a host cell of the present invention under suitable conditions, and recovering the bispecific from the cell culture Antibody steps.
  • Yet another aspect of the present invention relates to a conjugate comprising a bispecific antibody and a coupling moiety, wherein the bispecific antibody is the bispecific antibody of any one of the present invention, and the coupling moiety is a detectable label; preferably, the coupling moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme.
  • the conjugate of the present invention is used for the treatment and/or prevention of malignant tumors; preferably, the malignant tumors are selected from melanoma, liver cancer, gastric cancer, renal cell cancer, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer.
  • a further aspect of the present invention relates to a kit comprising the bispecific antibody of any one of the present invention, or a conjugate of the present invention;
  • the kit further comprises a secondary antibody capable of specifically binding to the bispecific antibody; optionally, the secondary antibody further comprises a detectable label such as a radioisotope, fluorescent substance, luminescent substance , colored substances or enzymes.
  • a detectable label such as a radioisotope, fluorescent substance, luminescent substance , colored substances or enzymes.
  • a further aspect of the present invention relates to the use of the bispecific antibody of any one of the present invention in the preparation of a kit for detecting the presence or level of VEGF and/or PD-L1 in a sample .
  • Yet another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody of any one of the present invention or the conjugate of the present invention; optionally, it further comprises pharmaceutically acceptable excipients .
  • Another aspect of the present invention relates to the use of the bispecific antibody or the conjugate of the present invention in the preparation of a medicament for preventing and/or treating malignant tumors; preferably, the malignant tumor is selected from From melanoma, liver, stomach, renal cell, ovarian, colon, breast, esophagus, and head and neck cancers.
  • Yet another aspect of the present invention relates to a method of treating and/or preventing malignant tumors, comprising administering to a subject in need thereof an effective amount of the bispecific antibody of any one of the present invention or the conjugate of the present invention
  • the malignant tumor is selected from the group consisting of melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer and head and neck cancer.
  • the method wherein the step of administering to a subject in need thereof an effective amount of the bispecific antibody of any one of the present invention is before or after surgical treatment, and/or before or after radiation therapy.
  • the single administration dose of the bispecific antibody of the present invention is 0.1-100 mg per kilogram of body weight, preferably 4.8-24 mg or 1-10 mg; or, the single administration dose of the bispecific antibody of the present invention is each subject or 10-1000mg, preferably 50-500mg, 100-400mg, 150-300mg, 150-250mg or 200mg;
  • it is administered every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks or 3 weeks;
  • the mode of administration is intravenous drip or intravenous injection.
  • EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that elicits 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having one "light” (L) chain and one "heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino terminus to carboxy terminus.
  • the assignment of amino acids to regions or domains follows Bethesda Md, Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al.
  • antibody is not limited by any particular method of producing an antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, that is, excluding natural mutations that may arise spontaneously, A population of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on an antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which generally comprise at least two or more different antibodies that generally recognize different epitopes on an antigen.
  • Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. nature, 1975; 256(5517): 495), but can also be obtained by recombinant DNA technology (eg, see US Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (acceptor antibody) with the CDR regions of a non-human antibody (donor antibody)
  • the antibody or antibody fragment of which the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, to further improve or optimize the performance of the antibody.
  • antigen-binding fragments of antibodies are diabodies, in which the VH and VL domains are expressed on a single polypeptide chain, but linkers that are too short are used to allow for both domains on the same chain Pairing between the domains forces the domains to pair with the complementary domains of the other chain and create two antigen-binding sites (see, eg, Holliger P. et al., Proc. Natl. Acad. Sci. USA 1993;90:6444 -6448 and Poljak RJet al., Structure 1994;2:1121-1123).
  • a fusion protein as described herein is a protein product of co-expression of two genes obtained by DNA recombination.
  • Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art (eg, Cold Spring Harbor's Technical Guide to Antibody Assays, Chapters 5-8 and 15).
  • isolated refers to artificially obtained from the natural state. If an "isolated” substance or component occurs in nature, it may be due to a change in its natural environment, or separation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolated of.
  • isolated or isolated
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1 derived artificial chromosomes (PACs) ; Phage such as ⁇ phage or M13 phage and animal viruses.
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PACs P1 derived artificial chromosomes
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses eg, adeno-associated viruses
  • herpesviruses eg, herpes simplex virus
  • poxviruses baculoviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg SV40
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc., Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus, etc.
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • the term "pharmaceutically acceptable excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which are well known in the art (See, eg, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed . Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include but are not limited to phosphate buffers; surfactants include but are not limited to cationic, anionic or nonionic surfactants such as Tween-80; ionic strength enhancers include but are not limited to sodium chloride.
  • the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect.
  • a disease-prophylactically effective amount refers to an amount sufficient to prevent, arrest, or delay the onset of a disease (eg, a tumor);
  • a therapeutically-effective amount refers to an amount sufficient to cure or at least partially prevent the development of a disease in a patient already suffering from the disease. The amount of disease and its complications. Determining such effective amounts is well within the ability of those skilled in the art.
  • an effective amount for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently etc.
  • first and second are for the purpose of distinction in reference or clarity of expression, Does not have a typical sequential meaning.
  • the present invention also relates to any one of the following items 1 to 10:
  • bispecific antibody is characterized in that, described bispecific antibody comprises:
  • bispecific antibody according to item 1, characterized in that, the bispecific antibody contains the polypeptides having the structures shown in formula I and formula II,
  • A1 is the heavy chain variable region VH of the anti-VEGF antibody
  • A2 is the light chain variable region VL of the anti-VEGF antibody
  • B is an anti-PD-L1 single domain antibody
  • L1, L2 and L3 are each independently a no or connecting element
  • CH is a human IgG heavy chain constant region CH (preferably LALA mutant);
  • CL is the human kappa light chain constant region CL
  • polypeptide represented by formula I and the polypeptide represented by formula II form a heterodimer through disulfide bond interaction.
  • bispecific antibody according to item 1 wherein the bispecific antibody is a polypeptide having the structure shown in formula III or formula IV,
  • A3 is the domain that can bind VEGF and block its activity
  • B is an anti-PD-L1 single domain antibody
  • L4, L5, L6 and L7 are each independently a no or connecting element
  • Fc is the Fc segment of human IgG (preferably LALA mutant).
  • a vector comprising the polynucleotide according to item 4.
  • a host cell characterized in that the host cell contains the vector described in item 5, or the polynucleotide described in item 4 is integrated into its genome;
  • the host cell expresses the bispecific antibody described in item 1.
  • a method for producing a bispecific antibody as described in item 1, comprising the steps of:
  • step (b) purifying and/or separating the culture obtained in step (a) to obtain the bispecific antibody.
  • immunoconjugate characterized in that the immunoconjugate comprises:
  • conjugation moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or their combination.
  • a pharmaceutical composition characterized in that the pharmaceutical composition contains:
  • the present invention also relates to any one of the following first to fourteenth aspects:
  • a bispecific antibody comprising:
  • the bispecific antibody includes 1-3 anti-PD-L1 single-domain antibodies, preferably, includes 1 or 2 anti-PD-L1 single-domain antibodies.
  • the PD-L1 single domain antibody can block the interaction between PD-1 and PD-L1.
  • the bispecific antibody further comprises an Fc segment.
  • the Fc segment of the bispecific antibody is selected from the group consisting of a human IgG domain, a CH1+CL1 domain, or a combination thereof.
  • the human IgG domain is an engineered mutant IgG domain, preferably a LALA mutant IgG domain.
  • the bispecific antibody contains polypeptides with structures as shown in formula I and formula II,
  • A1 is the heavy chain variable region VH of the anti-VEGF antibody
  • A2 is the light chain variable region VL of the anti-VEGF antibody
  • B is an anti-PD-L1 single domain antibody
  • L1, L2 and L3 are each independently a no or connecting element
  • CH is a human IgG heavy chain constant region CH (preferably LALA mutant);
  • CL is the human kappa light chain constant region CL
  • polypeptide represented by formula I and the polypeptide represented by formula II form a heterodimer through disulfide bond interaction.
  • the bispecific antibody is a polypeptide having the structure shown in formula III or formula IV,
  • A3 is the domain that can bind VEGF and block its activity
  • B is an anti-PD-L1 single domain antibody
  • L4, L5, L6 and L7 are each independently a no or connecting element
  • Fc is the Fc segment of human IgG (preferably LALA mutant).
  • the anti-VEGF heavy chain variable region VH has the heavy chain variable region amino acid sequence derived from Bevacizumab, the amino acid sequence of which is shown in SEQ ID NO: 3, or Has ⁇ 85% (preferably 90%, more preferably 95%) sequence identity with the sequence shown in SEQ ID NO:3.
  • the light chain variable region VL of the anti-VEGF has the amino acid sequence of the light chain variable region derived from Bevacizumab, the amino acid sequence of which is shown in SEQ ID NO: 9, or Has ⁇ 85% (preferably 90%, more preferably 95%) sequence identity with the sequence shown in SEQ ID NO:9.
  • the heavy chain variable region VH of the anti-VEGF has the heavy chain variable region amino acid sequence derived from Ranibizumab, the amino acid sequence of which is shown in SEQ ID NO: 13, or Has ⁇ 85% (preferably 90%, more preferably 95%) sequence identity with the sequence shown in SEQ ID NO: 13.
  • the light chain variable region VL of the anti-VEGF has the amino acid sequence of the light chain variable region derived from Ranibizumab, the amino acid sequence of which is shown in SEQ ID NO: 15, or Has a sequence identity of > 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 15.
  • amino acid sequence of the domain that can bind to VEGF and block its activity is shown in SEQ ID NO: 17, or has ⁇ 85% (preferably 90%) of the sequence shown in SEQ ID NO: 17 %, more preferably 95%) sequence identity.
  • amino acid sequence of the anti-PD-L1 single domain antibody is as shown in SEQ ID NO:5, or has ⁇ 85% (preferably 90%, more preferably ⁇ 85% of the sequence shown in SEQ ID NO:5) 95%) sequence identity.
  • the human IgG heavy chain constant region CH has a LALA mutation, and its amino acid sequence is shown in SEQ ID NO: 4, or has ⁇ 85% of the sequence shown in SEQ ID NO: 4 (preferably 90%, more preferably 95%) sequence identity.
  • amino acid sequence of the human kappa light chain constant region CL is as shown in SEQ ID NO: 10, or has ⁇ 85% (preferably 90%, more preferably ⁇ 85% with the sequence shown in SEQ ID NO: 10) 95%) sequence identity.
  • the Fc segment of the human IgG has a LALA mutation, and its amino acid sequence is as shown in SEQ ID NO: 18, or ⁇ 85% (preferably 90%) of the sequence shown in SEQ ID NO: 18 , more preferably 95%) sequence identity.
  • the sequence of the linker element is (G 4 S) n , wherein n is a positive integer (eg 1, 2, 3, 4, 5 or 6), preferably, n is 2 or 4 .
  • the amino acid sequence of the linker element is as shown in SEQ ID NO: 6 or 7, or has ⁇ 85% (preferably 90%, more preferably ⁇ 85% of the sequence shown in SEQ ID NO: 6 or 7) 95%) sequence identity.
  • the bispecific antibody contains polypeptides with structures shown in formula I and formula II, wherein the amino acid sequence of the polypeptides with structures shown in formula I is shown in SEQ ID NO: 1, and the The amino acid sequence of the polypeptide with the structure shown in the formula II is shown in SEQ ID NO: 8, namely Ava-2GS-NSD.
  • the bispecific antibody contains polypeptides with structures shown in formula I and formula II, wherein the amino acid sequence of the polypeptides with structures shown in formula I is shown in SEQ ID NO: 2, and the The amino acid sequence of the polypeptide of the structure shown in the formula II is shown in SEQ ID NO: 8, namely Ava-4GS-NSD).
  • the bispecific antibody contains polypeptides with structures shown in formula I and formula II, wherein the amino acid sequence of the polypeptides with structures shown in formula I is shown in SEQ ID NO: 11, and the The amino acid sequence of the polypeptide with the structure shown in the formula II is shown in SEQ ID NO: 14, namely Luc-2GS-NSD).
  • the bispecific antibody contains polypeptides with structures shown in formula I and formula II, wherein the amino acid sequence of the polypeptides with structures shown in formula I is shown in SEQ ID NO: 12, and the The amino acid sequence of the polypeptide of the structure shown in the formula II is shown in SEQ ID NO: 14, namely Luc-4GS-NSD).
  • the bispecific antibody is a polypeptide having the structure shown in formula III, and its amino acid sequence is shown in SEQ ID NO: 16, namely NSD-Elyea).
  • the bispecific antibody is a polypeptide having the structure shown in formula III, and its amino acid sequence is shown in SEQ ID NO: 19, namely Elyea-NSD).
  • the bispecific antibody contains both the polypeptides shown in formula I and formula II, and the polypeptide shown in formula I and the polypeptide shown in formula II form a heterozygous complex through disulfide bond interaction.
  • the homodimer ii is formed through the interaction of disulfide bonds between the CH domains.
  • the bispecific antibody is a polypeptide having the structure shown in formula III or formula IV, and the polypeptides with the structure shown in formula III or formula IV pass through the Fc segment disulfide bonds between them to form homodimers.
  • an isolated polynucleotide encoding the bispecific antibody according to the first aspect of the present invention is provided.
  • the bispecific antibody contains the polypeptides with the structures shown in Formula I and Formula II
  • the polynucleotide sequence encoding the structure polypeptide shown in Formula I is the same as the The ratio of the polynucleotide sequences encoding the structural polypeptide represented by the formula II is 1:1.
  • a vector comprising the polynucleotide according to the second aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof; preferably, the expression vector includes a viral vector, such as Lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • a host cell contains the vector according to the third aspect of the present invention, or the polynucleotide according to the second aspect of the present invention is integrated into its genome;
  • the host cell expresses the bispecific antibody according to the first aspect of the invention.
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cell is selected from the group consisting of: Escherichia coli, yeast cells, and mammalian cells.
  • a method for producing a bispecific antibody as described in the first aspect of the present invention comprising the steps of:
  • step (b) purifying and/or separating the culture obtained in step (a) to obtain the bispecific antibody.
  • the purification can be carried out by protein A affinity column purification and separation to obtain the target antibody.
  • the purity of the purified and separated target antibody is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, preferably 100%.
  • an immunoconjugate is provided, the immunoconjugate contains:
  • conjugation moiety selected from the group consisting of detectable labels, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or their combination.
  • the radionuclide includes:
  • a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or
  • a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd- 103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133Yb-169, Yb-177, or a combination thereof.
  • the coupling moiety is a drug or a toxin.
  • the drug is a cytotoxic drug.
  • the cytotoxic drugs are selected from the group consisting of anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating reagents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy A sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecins camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (eg DM1 and DM4), taxanes ( taxanes), benzodiazepines, or benzodiazepine-containing drugs (eg, pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines) indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • PPDs pyrrolo[1,4]benzodiazepines
  • indoline benzodiazepines indolinobenzodiazepines and oxazolidinobenzodiazepines
  • vinca alkaloids or combinations thereof.
  • the toxin is selected from the following group:
  • Auristatins eg, auristatin E, auristatin F, MMAE, and MMAF
  • chlortetracycline maytansoid, gamatoxin, gamatoxin A-chain, combretastatin, docarmicin, Lastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, autumn Narcissin, Dihydroxyanthraxdione, Actinomycin, Diphtheria Toxin, Pseudomonas Exotoxin (PE) A, PE40, Acacia toxin, Acacia A chain, Capsule root toxin A chain, ⁇ - Sarcinus, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin,
  • the coupling moiety is a detectable label.
  • the conjugate is selected from: fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or capable of producing detectable Enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles, prodrugs Activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin).
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate contains: a multivalent (eg, bivalent) bispecific antibody according to the first aspect of the present invention.
  • the multivalent refers to the bispecific antibody according to the first aspect of the present invention comprising multiple repetitions in the amino acid sequence of the immunoconjugate.
  • the coupling part of the immunoconjugate is a diagnostic isotope.
  • the reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agents .
  • the reagent for detecting PD-L1 and/or VEGF in the sample is a contrast agent for (in vivo) detecting PD-L1 and/or VEGF molecules.
  • the detection is in vivo detection or in vitro detection.
  • the detection includes flow detection and cellular immunofluorescence detection.
  • the agent is used to block the interaction between PD-1 and PD-L1, and at the same time block the interaction between VEGF and VEGFR.
  • the tumors include but are not limited to: acute myeloid leukemia, chronic myeloid leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer , liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor.
  • a pharmaceutical composition contains:
  • the coupling part of the immunoconjugate is a drug, a toxin, and/or a therapeutic isotope.
  • the pharmaceutical composition also contains other drugs for treating tumors, such as cytotoxic drugs.
  • the other drugs for treating tumors include paclitaxel, doxorubicin, cyclophosphamide, axitinib, lenvatinib, and pembrolizumab.
  • the drug is used to block the interaction between PD-1 and PD-L1, and at the same time block the interaction between VEGF and VEGFR.
  • the pharmaceutical composition is used to block PD-1/PD-L1 and/or VEGF/VEGFR signaling pathway.
  • the pharmaceutical composition is used to treat tumors expressing PD-L1 protein (ie PD-L1 positive) and/or expressing VEGF protein (ie VEGF positive).
  • the pharmaceutical composition is in the form of injection.
  • the pharmaceutical composition is used to prepare a medicine for preventing and treating tumors.
  • the tumor is a tumor expressing PD-L1 protein (ie PD-L1 positive) and/or expressing VEGF protein (ie VEGF positive).
  • the use is non-diagnostic and non-therapeutic.
  • a recombinant protein having: (i) the bispecific antibody of the first aspect of the present invention; and (ii) optionally assisted expression and/or or purified tag sequences.
  • the tag sequence includes 6His tag, HA tag and Fc tag.
  • the recombinant protein specifically binds to PD-L1 and/or VEGF.
  • a method for detecting PD-L1 and/or VEGF in a sample comprising the steps of: (1) combining the sample with the bispecific according to the first aspect of the present invention antibody contact; (2) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PD-L1 and/or VEGF in the sample.
  • a method for treating a disease comprising: administering to a subject in need the bispecific antibody according to the first aspect of the present invention, as described in the sixth aspect of the present invention
  • the immunoconjugate, or the pharmaceutical composition according to the eighth aspect of the present invention comprising: administering to a subject in need the bispecific antibody according to the first aspect of the present invention, as described in the sixth aspect of the present invention
  • the immunoconjugate, or the pharmaceutical composition according to the eighth aspect of the present invention comprising: administering to a subject in need the bispecific antibody according to the first aspect of the present invention, as described in the sixth aspect of the present invention
  • the immunoconjugate, or the pharmaceutical composition according to the eighth aspect of the present invention comprising: administering to a subject in need the bispecific antibody according to the first aspect of the present invention, as described in the sixth aspect of the present invention
  • the immunoconjugate, or the pharmaceutical composition according to the eighth aspect of the present invention comprising: administering to a subject in need the bispecific antibody according to the first aspect of the
  • the subject includes mammals, preferably humans.
  • the detection reagent comprises the immunoconjugate according to the sixth aspect of the present invention and a detection acceptable carrier .
  • the coupling part of the immunoconjugate is a diagnostic isotope.
  • the detectably acceptable carrier is a non-toxic, inert aqueous carrier medium.
  • the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow detection reagents, cellular immunofluorescence detection reagents, magnetic nanoparticles and imaging agent.
  • the detection reagent is used for in vivo detection.
  • the dosage form of the detection reagent is liquid or powder (eg, water preparation, injection, freeze-dried powder, tablet, buccal preparation, aerosol preparation).
  • kits for detecting PD-L1 and/or VEGF contains the immunoconjugate according to the sixth aspect of the present invention or the thirteenth aspect of the present invention The detection reagents described in the aspect, and the instructions.
  • the description describes that the kit is used to non-invasively detect the expression of PD-L1 and/or VEGF in the subject to be tested.
  • the kit is used for the detection of tumors expressing PD-L1 protein (ie PD-L1 positive) and/or expressing VEGF protein (ie VEGF positive).
  • bispecific antibody of the present invention As used herein, the terms "bispecific antibody of the present invention”, “diabody of the present invention”, “anti-PD-L1/VEGF bispecific antibody” have the same meaning and refer to specific recognition and binding of PD-L1 and VEGF bispecific antibodies.
  • a VEGF receptor eg VEGFR1 and/or VEGFR2
  • Proteins are also broadly referred to in the present invention as bispecific antibodies.
  • the present invention provides an anti-PD-L1/VEGF bispecific antibody, comprising: an anti-PD-L1 single domain antibody and an anti-VEGF antibody or element.
  • the bispecific antibody contains polypeptides with structures shown in formula I and formula II,
  • A1 is the heavy chain variable region VH of the anti-VEGF antibody
  • A2 is the light chain variable region VL of the anti-VEGF antibody
  • B is an anti-PD-L1 single domain antibody
  • L1, L2 and L3 are each independently a no or connecting element
  • CH is a human IgG heavy chain constant region CH (preferably LALA mutant);
  • CL is the human kappa light chain constant region CL
  • polypeptide represented by formula I and the polypeptide represented by formula II form a heterodimer through disulfide bond interaction.
  • the bispecific antibody is a polypeptide having a structure as shown in formula III or IV,
  • A3 is the domain that can bind VEGF and block its activity
  • B is an anti-PD-L1 single domain antibody
  • L4, L5, L6 and L7 are each independently a no or connecting element
  • Fc is the Fc segment of human IgG (preferably LALA mutant).
  • the bispecific antibody contains the polypeptides of formula I and formula II at the same time, and the polypeptide of formula I and the polypeptide of formula II form heterologous through disulfide bond interaction dimer i;
  • the homodimer ii is formed through the interaction of disulfide bonds between the CH domains.
  • the bispecific antibody is a polypeptide having the structure shown in formula III or formula IV, and the polypeptides with the structure shown in formula III or formula IV pass through the Fc segment disulfide bonds between them to form homodimers.
  • single domain antibody As used herein, the terms “single domain antibody”, “Nanobody VHH”, “Nanobody” have the same meaning and refer to cloning the variable region of an antibody heavy chain, constructing a Nanobody consisting of only one heavy chain variable region ( VHH), which is the smallest fully functional antigen-binding fragment.
  • VHH single domain antibody
  • an antibody that naturally lacks light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a Nanobody (VHH) consisting of only one heavy chain variable region.
  • variable means that certain portions of the variable regions of an antibody differ in sequence that contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable regions are called the framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • the variable domains of native heavy and light chains each contain four FR regions, which are generally in a -folded configuration, connected by three CDRs that form a linking loop, and in some cases may form a partially folded structure.
  • the CDRs in each chain are tightly packed together by the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as involvement in antibody-dependent cytotoxicity of the antibody.
  • FR framework region
  • the light and heavy chains of immunoglobulins each have four FRs, designated FR1-L, FR2-L, FR3-L, FR4-L, and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • a light chain variable domain may thus be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can thus be represented as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is substantially identical to a naturally occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96% , 97%, 98% or 99%.
  • human framework region is a framework region that is substantially identical (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) to that of a naturally occurring human antibody .
  • affinity is theoretically defined by an equilibrium association between intact antibody and antigen.
  • the affinity of the double antibody of the present invention can be evaluated or determined by K D value (dissociation constant) (or other measurement methods), such as Bio-layer interferometry (BLI), measured using FortebioRed96 instrument.
  • K D value dissociation constant
  • BLI Bio-layer interferometry
  • linker refers to insertion into an immunoglobulin domain to provide sufficient mobility for the domains of the light and heavy chains to fold into an exchange of one or more amino acid residues of a dual variable region immunoglobulin base.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments thereof of the present invention to form the conjugate.
  • the present invention also includes cell surface markers or antigens that bind to the PD-L1/VEGF bispecific antibody or fragment thereof.
  • variable region is used interchangeably with “complementarity determining region (CDR)”.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to a polypeptide that specifically binds to PD-L1 and/or VEGF protein, such as having a heavy chain variable region protein or polypeptide. They may or may not contain the starting methionine.
  • the present invention also provides other protein or fusion expression products with the antibodies of the present invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a variable region-containing heavy chain, as long as the variable region is associated with the heavy chain of an antibody of the invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable regions which are separated into four framework regions (FRs), four FR amino acids
  • FRs framework regions
  • FRs framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a circular structure, and the ⁇ -sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody.
  • Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Accordingly, the present invention includes those molecules having CDR-bearing antibody heavy chain variable regions, as long as their CDRs have greater than 90% (preferably greater than 95%, optimally greater than 98%) homology to the CDRs identified herein sex.
  • the present invention includes not only intact antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Accordingly, the present invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to polypeptides that retain substantially the same biological function or activity of an antibody of the invention.
  • a polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide with another compound (such as a compound that prolongs the half-life of a polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or a secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or with 6His-tagged fusion protein.
  • the antibody of the present invention refers to a double antibody with PD-L1 and/or VEGF protein binding activity.
  • the term also includes variant forms of polypeptides comprising the same CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitution with amino acids of similar or similar properties generally does not alter the function of the protein. As another example, the addition of one or more amino acids to the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNAs capable of hybridizing with the DNA encoding the antibody of the present invention under conditions of high or low stringency
  • the encoded protein, and the polypeptide or protein obtained using the antiserum against the antibody of the present invention are included in the polypeptide.
  • the invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof.
  • the present invention also includes fragments of the single domain antibodies of the present invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “conservative variants of the antibody of the present invention” means that compared with the amino acid sequence of the antibody of the present invention, there are at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3
  • the amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • These conservatively variant polypeptides are best produced by amino acid substitutions according to Table A.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be the coding or non-coding strand.
  • Polynucleotides encoding mature polypeptides of the invention include: coding sequences encoding only the mature polypeptide; coding sequences and various additional coding sequences for the mature polypeptide; coding sequences (and optional additional coding sequences) for the mature polypeptide and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • a feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • Biomolecules nucleic acids, proteins, etc.
  • Biomolecules include biomolecules in isolated form.
  • DNA sequences encoding the proteins of the present invention can be obtained entirely by chemical synthesis.
  • This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present invention.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Antibodies of the invention may be used alone, or may be conjugated or conjugated to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or those capable of producing detectable products. enzymes.
  • Therapeutic agents that can be combined or conjugated with the antibodies of the present invention include but are not limited to: 1. Radionuclides; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents ( For example, cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the This will vary depending on the nature of the formulation material and the condition to be treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind PD-L1 and/or VEGF protein molecules, and thus can be used to treat tumors.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (eg, 0.001-99 wt %, preferably 0.01-90 wt %, more preferably 0.1-80 wt %) of the above-mentioned single domain antibody (or its conjugate) of the present invention and a pharmaceutical an acceptable carrier or excipient.
  • a pharmaceutical an acceptable carrier or excipient include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the present invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is generally at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the antibody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels or fluorescent labels.
  • colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the PD-L1/VEGF bispecific antibody can be labeled with colloidal gold to obtain a colloidal gold-labeled antibody.
  • the present invention also relates to methods of detecting PD-L1 and/or VEGF proteins.
  • the method steps are roughly as follows: obtaining a cell and/or tissue sample; lysing the sample in a medium; detecting the level of PD-L1 and/or VEGF protein in the lysed sample.
  • the sample to be used is not particularly limited, and a representative example is a cell-containing sample existing in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention.
  • the kit further includes a container, an instruction manual, a buffer, and the like.
  • the present invention also provides a detection kit for detecting the level of PD-L1 and/or VEGF
  • the kit includes an antibody that recognizes PD-L1 and/or VEGF protein, a lysis medium for dissolving the sample, and a general purpose required for the detection Reagents and buffers, such as various buffers, detection labels, detection substrates, etc.
  • the detection kit may be an in vitro diagnostic device.
  • the bispecific antibody of the present invention can be highly specific to human PD-L1 protein, can inhibit the PD-1/PD-L1 pathway on the basis of targeting VEGF that neutralizes the tumor microenvironment, and can make T cells. Restore activity and enhance immune response, thereby more effectively improving the effect of inhibiting the occurrence and development of tumors.
  • the single-domain antibody of the present invention has a wide range of biological application value and clinical application value, and its application involves diagnosis and treatment of diseases related to PD-L1 and/or VEGF, basic medical research, biological research, etc. field.
  • a preferred application is for clinical diagnosis and targeted therapy against PD-L1 and/or VEGF, such as tumor therapy.
  • the bispecific antibody of the present invention has a strong affinity.
  • Inhibiting the PD-1/PD-L1 pathway on the basis of targeting VEGF to neutralize the tumor microenvironment can restore the activity of T cells, enhance the immune response, and more effectively improve the effect of inhibiting the occurrence and development of tumors. potential for the preparation of antitumor drugs.
  • the bispecific antibody of the present invention there is likely to be a synergistic effect between the first protein functional domain and the second protein functional domain of the bispecific antibody of the present invention, for example, its affinity with VEGF is often better than that of anti-VEGF monoclonal antibody, and its affinity with PD-L1 Its affinity is often better than that of anti-PD-L1 monoclonal antibody; it is also better than anti-VEGF monoclonal antibody or anti-PD-L1 monoclonal antibody in inducing mixed lymphocytes to secrete IL2 or INF, which shows that the bispecific antibody of the present invention can better activate T cells.
  • Figure 1A Schematic diagram of the structure of Ava-2GS-NSD or Ava-4GS-NSD.
  • Figure 1B Schematic representation of the structure of Luc-2GS-NSD or Luc-4GS-NSD.
  • Figure 1C Schematic diagram of the structure of NSD-Elyea.
  • Figure 1D Schematic diagram of the structure of Elyea-NSD.
  • Figure 2 Graph of bispecific antibody and CHO-PDL1 cell binding.
  • Figure 3A Graph of bispecific antibody blocking VEGF and VEGFR2 binding.
  • Figure 3B Graph of bispecific antibody blocking VEGF and VEGFR2 binding.
  • FIG. 4A Bispecific antibodies block secretion of INF-gamma concentrations in mixed cultures of lymphocytes. For each bispecific antibody sample, the concentrations were 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM from left to right.
  • FIG. 4B Bispecific antibodies block secretion of IL-2 concentrations in mixed cultures of lymphocytes.
  • the concentration was 100 nM, 10 nM, 1 nM, 0.1 nM and 0.01 nM from left to right.
  • Figure 5A Graph of the binding of bispecific antibodies to CHO-PDL1 cells for different days of storage.
  • Figure 5B Graph of the binding of bispecific antibodies to CHO-PDL1 cells for different days of storage.
  • Figure 5C Graph of the binding of bispecific antibodies to CHO-PDL1 cells for different days of storage.
  • Figure 5D Graph of the binding of bispecific antibodies to CHO-PDL1 cells for different days of storage.
  • Figure 5E Graph of bispecific antibody and VEGF binding profiles for different days of storage.
  • Figure 5F Graph of bispecific antibody and VEGF binding profiles for different days of storage.
  • Figure 5G Graph of bispecific antibody and VEGF binding profiles for different days of storage.
  • Figure 5H Graph of bispecific antibody and VEGF binding profiles for different days of storage.
  • Figure 6A Graph of the effect of bispecific antibodies on tumor volume in mice.
  • Figure 6B Graph of the effect of bispecific antibodies on body weight in mice.
  • Figure 7A Schematic representation of the structure of Ava-2GS-NSD or Ava-4GS-NSD.
  • Figure 7B Schematic diagram of the structure of Luc-2GS-NSD or Luc-4GS-NSD.
  • Figure 7C Schematic diagram of the structure of NSD-Elyea.
  • Figure 7D Schematic diagram of the structure of Elyea-NSD.
  • anti-VEGF/PD-L1 bispecific antibodies were constructed, which are:
  • Ava-2GS-NSD/Ava-4GS-NSD It consists of 4 polypeptide chains (two heavy chains connected to C-Ye-18-5, two light chains respectively), the schematic diagram of its structure is shown in Figure 1A and Figure 7A shown that both peptide chains #1 have the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, which comprise VH amino acids derived from the anti-VEGF antibody Bevacizumab (Patent No.: WO1998045332) Sequence (SEQ ID NO: 3), directly linked at the C-terminus of the VH amino acid sequence derived from the human IgG1 CH amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 4), the anti-PD-L1 Nanobody C - The N-terminus of Ye-18-5 (patent application number: 2019108631090) (SEQ ID NO: 5) is passed through 11 amino acid residues (G 4 S) 2 G (SEQ ID NO: 6) (Ava-2GS-NSD) or a
  • Both peptide chains #2 have the amino acid sequence shown in SEQ ID NO: 8, which comprises the VL amino acid sequence (SEQ ID NO 9) derived from the anti-VEGF antibody Bevacizumab, and the VL amino acid sequence in said VL amino acid sequence.
  • SEQ ID NO: 10 C-terminal human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 10).
  • Luc-2GS-NSD/Luc-4GS-NSD It consists of 4 polypeptide chains (two heavy chains connected to C-Ye-18-5, two light chains respectively), the schematic diagrams of which are shown in Figure 1B and Figure 7B
  • Both peptide chains #1 have the amino acid sequences shown in SEQ ID NO: 11 or SEQ ID NO: 12, which comprise VH amino acids derived from the anti-VEGF antibody Ranibizumab (patent number: WO2018175752) Sequence (SEQ ID NO: 13), directly linked at the C-terminus of the VH amino acid sequence derived from the human IgG1 CH amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO: 4), the anti-PD-L1 Nanobody C -
  • the N-terminus of Ye-18-5 (SEQ ID NO: 5) is defined by 11 amino acid residues (G 4 S) 2 G (SEQ ID NO: 6) (Luc-2GS-NSD) or 21 amino acid residues ( The flexible peptide of G4S)
  • Both peptide chains #2 have the amino acid sequence shown in SEQ ID NO: 14, which comprises the VL amino acid sequence (SEQ ID NO: 15) derived from the anti-VEGF antibody Ranibizumab, and the amino acid sequence in the VL Human kappa light chain constant region (CL) amino acid sequence C-terminal to the sequence (SEQ ID NO: 10).
  • NSD-Elyea consists of 2 identical polypeptide chains (dimer), its structural schematic diagram is shown in Figure 1C and Figure 7C, the peptide chain has the amino acid sequence shown in SEQ ID NO: 16, which contains derived from anti-VEGF
  • the N-terminus of the anti-PD-L1 Nanobody C-Ye-18-5 is connected to the C of the heavy chain through a flexible peptide of 21 amino acid residues (G 4 S) 4 G (SEQ ID NO: 7). end.
  • Elyea-NSD consists of 2 identical polypeptide chains (dimer), its structural schematic diagram is shown in Figure 1D and Figure 7D, the peptide chain has the amino acid sequence shown in SEQ ID NO: 19, which contains anti-PD-L1
  • the Nanobody C-Ye-18-5 (SEQ ID NO:5), directly linked at the C-terminus of the Nanobody sequence derived from a human IgG1 Fc amino acid sequence (LALA mutation was introduced to reduce Fc function, SEQ ID NO:18),
  • the N-terminus of the VEGF-binding region (Elyea, SEQ ID NO: 17) derived from the anti-VEGF fusion protein Aflibercept is attached to the heavy chain by a flexible peptide of 21 amino acid residues (G4S)4G (SEQ ID NO: 7 ) the C terminal.
  • the ExpiCHO TM expression system kit (purchased from Thermo) was used to transfer the plasmid into Expi-CHO cells.
  • the transfection method was in accordance with the commercial instructions. After 5 days of cell culture, the supernatant was collected using protein A magnetic beads (purchased from GenScript). Purification of target protein by selective method. The magnetic beads were resuspended (1-4 times the volume of magnetic beads) with an appropriate volume of binding buffer (PBS+0.1% Tween 20, pH 7.4) and added to the sample to be purified, incubated at room temperature for 1 hour, with gentle shaking during the period. . The samples were placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer.
  • the results show that all the bispecific antibody samples of the present invention have binding activity to PD-L1 and VEGF proteins.
  • the bispecific antibody of the present invention Compared with the control antibody C-Ye-18-5 (PD-L1 single domain antibody), the bispecific antibody of the present invention has similar or even better binding activity to PD-L1; and/or, compared with the control antibody ranibizumab Compared with Ranibizumab and Bevacizumab, the bispecific antibody of the present invention has similar or even better binding activity to VEGF.
  • CHO cells overexpressing human PD-L1 (CHO-hPD-L1 cells) were generated by transfection of the pCHO1.0 vector (purchased from Invitrogen) of human PD-L1 cDNA (purchased from Sino Biological) cloned into MCS.
  • the expanded CHO-hPD-L1 cells were adjusted to a cell density of 2 ⁇ 10 6 cells/ml, 100 ⁇ l/well was added to a 96-well flow plate, and centrifuged for use.
  • the human VEGFR protein was diluted to an appropriate concentration with ELISA coating solution, added to the ELISA plate, and coated overnight at 4°C. Block with 5% BSA for 1 hour at room temperature.
  • the test samples were serially diluted and incubated with biotinylated human VEGF protein for 1 hour at room temperature. Add the incubated samples to the blocked ELISA plate and react at room temperature for 2 hours. Wash 3 times with PBS washing solution, add diluted streptavidin (HRP) to react at room temperature for 1 hour, wash 3 times with PBS washing solution, add ELISA color developing solution, leave at room temperature for 3 min, add ELISA stop solution, read at 450nm absorbance value.
  • HRP diluted streptavidin
  • the activity of the bispecific antibody sample to activate T cells was detected by the mixed lymphocyte reaction (MLR) assay.
  • MLR mixed lymphocyte reaction
  • PBMC cells purchased from SAILY BIO, SLB-HPB
  • PBMCs Resuscitate PBMC cells (purchased from SAILY BIO, SLB-HPB), centrifuge, resuspend PBMCs with 10 ml of X-VIVO-15 medium (purchased from LONZA), and culture in a cell incubator at 37 °C for 2 h, remove the unattached cells parietal cells.
  • DC medium X-VIVO-15 medium with 10ng/ml GM-CSF (purchased from R&D), 20ng/ml IL-4
  • DC maturation medium X-VIVO-15 medium with 1000U/ml TNF- ⁇ (purchased from R&D), 10ng/ml IL-6 (purchased from R&D), 5ng/ml IL-1 ⁇ (purchased from R&D) ), 1 ⁇ M PGE2 (purchased from Tocris)
  • DC maturation medium X-VIVO-15 medium with 1000U/ml TNF- ⁇ (purchased from R&D), 10ng/ml IL-6 (purchased from R&D), 5ng/ml IL-1 ⁇ (purchased from R&D)
  • 1 ⁇ M PGE2 purchasedd from Tocris
  • PBMC cells from another donor (purchased from SAILY BIO, SLB-HPB) were thawed, centrifuged, and PBMCs were resuspended in 10 ml of X-VIVO-15 medium.
  • CD4 + T cells were enriched with CD4 + T cell sorting kit (purchased from Stemcell), CD4 + T cells were resuspended in X-VIVO-15, and the cell density was adjusted to 2 ⁇ 10 6 cells/ml.
  • the CD4 + T cells were mixed with the mature DC cells collected above at a ratio of 1:1, and 100 ⁇ l/well was added to a 96-well U bottom plate.
  • the long-term thermal stability of the bispecific antibody was evaluated by detecting the changes in the purity and biological activity of the bispecific antibody after being placed at 40°C for 30 days.
  • the purity of the target antibody after being placed at 40°C for 0, 14 and 30 days was determined by the method of SEC.
  • the FACS method was used to detect the binding of the accelerated stability test sample and CHO-PDL1 cells, and the method was the same as that of Example 3.
  • the binding of accelerated stability samples to VEGF was detected by ELISA.
  • the method was as follows: VEGFA recombinant protein was diluted to 1 ⁇ g/ml with ELISA coating solution, 100 ⁇ l/well was added to ELISA plate, and coated overnight at 4°C. The coating solution was discarded, 250 ⁇ l/well was added with PBST for 3 washes, and the cells were blocked with 5% BSA for 1 hour at room temperature. The purified samples were serially diluted with 1% BSA.
  • Blood collection time points 3 min, 4 hours, 10 hours, 24 hours, 48 hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours, and 672 hours after administration, blood was collected from the jugular vein of rats.
  • Whole blood samples were placed at 2-8°C for 30min, centrifuged at 12000rpm for 5min to collect serum, the obtained serum was centrifuged at 2-8°C, 12000rpm for 5min, and stored at -80°C.
  • the molecular weight of free Ava-2GS-NSD or Elyea-NSD in serum was detected by ELISA.
  • human colon cancer LOVO cells/NOG mice were injected with human PBMC model to determine the anti-tumor effect of bispecific antibodies.
  • Sufficient LOVO cells purchased from Addexbio
  • cells were collected after trypsinization, washed with PBS for 3 times, counted, and inoculated into 8-week-old female NOG severely immunized mice at 2 ⁇ 10 6 cells/mouse Deficient mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were subcutaneously placed on the right abdomen.
  • the subcutaneous tumor formation of NOG mice was observed every day, and 6 ⁇ 10 6 PBMCs were injected into the tail vein of each mouse 8 days after inoculation.
  • the maximum wide axis W and the maximum long axis L of the subcutaneous tumor in the right abdomen of each animal were measured with a vernier caliper, and the weight of each mouse was weighed with an electronic balance.
  • the mice with too large and too small tumor volume were removed, and the NOG mice were divided into 4 groups according to the average tumor volume, with 6 mice in each group.
  • the groups were grouped and injected with corresponding doses of Ava-2GS-NSD according to the group dosing schedule shown in Table 5 below.
  • Group 1 PBS 6 times 2 times a week
  • Group 2 Ava-2GS-NSD 24mg/kg 6 times 2 times a week
  • Group 3 Ava-2GS-NSD 4.8mg/kg 6 times 2 times a week
  • Group 4 Ava-2GS-NSD 1mg/kg 6 times 2 times a week
  • Mouse tumor volume and mouse body weight were measured twice a week. The mouse body weight and tumor volume were last measured 31 days after tumor cell inoculation, and the mice were euthanized.
  • mice in each group The state of the mice in each group was observed daily and no abnormality was found; the weight of the mice was weighed twice a week, and the weight of the mice in each group did not decrease significantly. Body weight, the change did not exceed 13%, and the body weight of the mice in the PBS group also decreased by 6%, which is more likely to be due to the GvHD caused by the reconstitution of PBMC in the mice. ); at the end of the test, no obvious lesions were found in the liver, kidney, lung and other major organs of the mice in each group by anatomical observation, indicating that the drugs in each group had no obvious toxicity to the mice at the doses used in this test.

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Abstract

本发明属于生物医药领域，具体涉及一种抗VEGF-抗PD-L1双特异性抗体、其药物组合物及用途。具体地，本发明涉及一种双特异性抗体，其包括: 靶向VEGF的第一蛋白功能区，和靶向PD-L1的第二蛋白功能区; 其中: 所述第一蛋白功能区为抗VEGF的抗体或其抗原结合片段，或者，所述第一蛋白功能区包含VEGF受体或具有VEGF受体功能的片段; 所述第二蛋白功能区为抗PD-L1的单域抗体。本发明的双特异性抗体能够通过同时激活免疫系统并阻断肿瘤血管生成，具有良好的抗肿瘤前景。

Description

抗VEGF-抗PD-L1双特异性抗体、其药物组合物及用途 技术领域
本发明属于生物医药领域，具体涉及一种抗VEGF-抗PD-L1双特异性抗体、其药物组合物及用途。
背景技术
血管内皮生长因子(Vascular endothelial growth factor,VEGF)又称血管通透因子(vascular permeability factor,VPF)或血管调理素(vasculotropin)，是一种具有高度特异性的促血管内皮细胞生长的同型二聚体的蛋白。VEGF家族蛋白包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E、VEGF-F和胎盘生长因子(Placental growth factor,PIGF)等，其中VEGF-A在血管早期的形成中发挥重要作用。1983年，senger等最先从豚鼠的肝癌细胞分离出来，具有增加微静脉、小静脉通透性，促进血管内皮细胞分裂、增殖以及诱导血管形成等作用。同时，VEGF参与许多血管生成依赖性疾病的发病及其进展，包括癌症、某些炎性疾病以及糖尿病视网膜病变等。因此，在抗肿瘤药物研究中，VEGF是一个重要的靶点。
VEGF蛋白主要受体有VEGFR1，VEGFR2，VEGFR3，NRP1，NRP2NRP3，然而，VEGF家族蛋白成员与VEGF受体的结合具有选择性，其中VEGFA可以VEGFR1，VEGFR2结合，激活内源性激酶活化，促进新生血管生成。阻断VEGF与受体的结合能够应用于多种癌症的治疗，例如乳腺癌、结肠癌、肺癌、卵巢癌、子宫内膜癌、间皮瘤、宫颈癌、肾癌(Rakesh R.Ramjiawan,Arjan W.Griffioen,and Dan G.Duda,Angiogenesis.2017 20(2):185–204.)。
目前在全球市场中，上市或通过审批的VEGF药物共有23种，并覆盖了45个适应症，其中贝伐珠单抗获批的适应症数量最多。贝伐珠单抗在美国、欧盟等地批准用于结直肠癌、非小细胞肺癌、乳腺癌、恶性胶质瘤和肾细胞癌等疾病的治疗。雷珠单抗，是第二代人源化的抗VEGF重组鼠单克隆抗体片段Fab部分，其与贝伐单抗(Bevacizumab)是从相同亲本鼠抗体获得。美国FDA于2006年6月30日批准其用于治疗年龄相关性黄斑变性(AMD)的患者。雷珠单抗相对贝伐珠单抗保持更好的VEGFA的亲和力，能更好的抑制血管生成，已开展胃癌，直肠癌等适应症。
阿帕西普是VEGFR1与VEGFR2胞外部分片段组成的人IgG-Fc重组蛋白，可以 同时阻断VEGFR1与VEGFR2和VEGFA结合，从而阻断血管上皮细胞的血管生成。阿帕西普主要适用于治疗有新生血管(湿)年龄相关黄斑变性(AMD)患者。同时在临床上开展晚期肠癌治疗(Caemen Stancan,etc,Rom J Morphol Embryol.2018 59(2):455–467)。由于阿帕西普拥有VEGFR1和VEGFR2的功能片段，具有与抗体类似的阻断VEGF和受体结合的功能。
程序性死亡因子1配体1(programmed death 1ligand 1，PD-L1)又称CD274，为B7家族成员，是PD-1的配体。PD-L1属于I型跨膜蛋白，共290个氨基酸，包含1个IgV样区、1个IgC样区、1个跨膜疏水区和1个由30个氨基酸组成的胞内区。与其他B7家族分子不同的是，PD-L1具有负向调节免疫应答的作用。研究发现，PD-L1主要表达于活化的T细胞、B细胞、巨噬细胞和树突状细胞等，除淋巴细胞外，PD-L1也表达于其它多种组织如胸腺、心脏、胎盘等的内皮细胞，以及各类非淋巴系如黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌、头颈癌等(Akintunde Akinleye&Zoaib Rasool，Journal of Hematology&Oncology volume 12,Article number:92(2019))。PD-L1在调节自身反应性T、B细胞和免疫耐受方面具有一定广泛性，并且在外周组织T和B细胞应答起作用。PD-L1在肿瘤细胞上的高表达与癌症患者的不良预后相关。
双功能抗体虽然是抗体药物研发的一个方向，但面临诸多挑战，比如临床前评价模型、表达量低、稳定性差、工艺复杂、质控差异性大等问题，因此一直以来双功能抗体的研发困难重重。
因此，需要开发一种特异性佳、疗效好且易于制备的针对PD-L1和VEGF两个靶点的双特异性抗体。
发明内容
本发明人深入的研究和创造性的劳动，开发了一种抗VEGF-抗PD-L1双特异性抗体(后文中也表述为抗PD-L1/VEGF双特异性抗体)。本发明人惊奇地发现，本发明的抗PD-L1/VEGF双特异性抗体具有针对PD-L1和VEGF双靶点的高亲和力，兼具两个靶点的共同生物活性，同时分子量较小，可以灵活的渗透肿瘤区域，并且安全性良好。由此提供了下述发明：
本发明的一个方面涉及一种双特异性抗体，其包括：
靶向VEGF的第一蛋白功能区，和
靶向PD-L1的第二蛋白功能区；
其中：
所述第一蛋白功能区为抗VEGF的抗体或其抗原结合片段，或者，所述第一蛋白功能区包含VEGF受体或具有VEGF受体功能的片段；
所述第二蛋白功能区为抗PD-L1的单域抗体。
在本发明的一些实施方式中，所述的双特异性抗体，其由所述第一蛋白功能区和第二蛋白功能区以及可选的连接片段(linker)组成。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗VEGF的抗体的重链可变区包含氨基酸序列如SEQ ID NO:21所示的HCDR1、氨基酸序列如SEQ ID NO:22所示的HCDR2以及氨基酸序列如SEQ ID NO:23所示的HCDR3；
或者所述抗VEGF的抗体的重链可变区包含氨基酸序列如SEQ ID NO:27所示的HCDR1、氨基酸序列如SEQ ID NO:22所示的HCDR2以及氨基酸序列如SEQ ID NO:28所示的HCDR3；
并且所述抗VEGF的抗体的轻链可变区包含氨基酸序列如SEQ ID NO:24所示的LCDR1、氨基酸序列如SEQ ID NO:25所示的LCDR2以及氨基酸序列如SEQ ID NO:26所示的LCDR3。
轻链和重链的可变区决定抗原的结合；每条链的可变区均含有三个高变区，称互补决定区(CDR)，其中重链(H)的CDR包含HCDR1、HCDR2、HCDR3，轻链(L)的CDR包含LCDR1、LCDR2、LCDR3。本发明中，CDR由IMGT编号系统定义，请参见Ehrenmann F,Kaas Q,Lefranc M P.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF[J].Nucleic acids research,2009；38(suppl_1):D301-D307。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗PD-L1单域抗体包含一个重链可变区，所述重链可变区包含氨基酸序列如SEQ ID NO:29所示的HCDR1、氨基酸序列如SEQ ID NO:30所示的HCDR2以及氨基酸序列如SEQ ID NO:31所示的HCDR3；
优选地，所述抗PD-L1单域抗体的氨基酸序列如SEQ ID NO:5所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗VEGF的抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，其轻链可变区的氨基酸序列如SEQ ID NO:9所示；或者
所述抗VEGF的抗体的重链可变区的氨基酸序列如SEQ ID NO:13所示，其轻链可变区的氨基酸序列如SEQ ID NO:15所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗VEGF的抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，其轻链可变区的氨基酸序列如SEQ ID NO:9所示；或者
所述抗VEGF的抗体的重链可变区的氨基酸序列如SEQ ID NO:13所示，其轻链可变区的氨基酸序列如SEQ ID NO:15所示；
并且所述抗PD-L1单域抗体的氨基酸序列如SEQ ID NO:5所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗VEGF的抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗VEGF的抗体的恒定区来自人抗体；
优选地，所述恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗VEGF的抗体的重链恒定区为人Ig gamma-1 chain C region或人Ig gamma-4 chain C region，并且其轻链恒定区为人Ig kappa chain C region；
优选地，所述抗VEGF的抗体的轻链恒定区的氨基酸序列如SEQ ID NO:10所示；
优选地，所述抗VEGF的抗体的轻链的氨基酸序列如SEQ ID NO:8或SEQ ID NO:14所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述抗VEGF的抗体的重链恒定区还包含按照EU编号系统的L234A突变和L235A突变；可选地，还包含G237A突变；
优选地，所述抗VEGF的抗体的重链恒定区的氨基酸序列如SEQ ID NO:4所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中：
所述VEGF为VEGF-A；
所述VEGF受体为VEGFR1和/或VEGFR2。
在本发明的一些实施方式中，所述的双特异性抗体，其中，所述单域抗体连接在第一蛋白功能区的C末端或N末端，例如，所述单域抗体为两条，每条单域抗体的一端分别连接在抗VEGF的抗体的两条重链的C末端或N末端，或者连接在VEGF受体或具有VEGF受体功能的片段的C末端或N末端；
并且所述单域抗体与第一蛋白功能区直接连接或者通过连接片段连接；
优选地，所述连接片段的氨基酸序列独立地选自SEQ ID NO:6和SEQ ID NO:7；
优选地，单域抗体连接在第一蛋白功能区得到的肽链的氨基酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:11或SEQ ID NO:12所示。
本发明涉及一种双特异性抗体，其包含：
靶向VEGF的第一蛋白功能区，和
靶向PD-L1的第二蛋白功能区；
其中，
所述第一蛋白功能区为1个，所述第二蛋白功能区为2个；
所述第一蛋白功能区为抗VEGF抗体或其抗原结合片段，所述第二蛋白功能区为抗PD-L1的单域抗体；
所述单域抗体通过连接片段连接在抗VEGF抗体的重链的C末端；连接后得到的肽链的氨基酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:11或SEQ ID NO:12所示；
所述抗VEGF抗体的轻链的氨基酸序列如SEQ ID NO:8或SEQ ID NO:14所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
所述第一蛋白功能区包含：VEGF受体或具有VEGF受体功能的片段，以及IgG1的Fc段；
优选地，所述具有VEGF受体功能的片段的氨基酸序列如SEQ ID NO:17所示；
优选地，所述IgG1的Fc段包含按照EU编号系统的L234A突变和L235A突变；
优选地，所述IgG1的Fc段的氨基酸序列如SEQ ID NO:18所示。
在本发明的一些实施方式中，所述的双特异性抗体，其中，
当所述第一蛋白功能区为VEGF受体或具有VEGF受体功能的片段时，所述双特异性抗体为二聚体；优选地，为氨基酸序列如SEQ ID NO:16或SEQ ID NO:19所示多肽的二聚体。
根据本发明中任一项所述的双特异性抗体，其用于治疗和/或预防恶性肿瘤；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
本发明的另一方面涉及分离的核酸分子，其编码本发明中任一项所述的双特异性抗体。
本发明的再一方面涉及一种载体，其包含本发明的分离的核酸分子。
本发明的再一方面涉及一种宿主细胞，其包含本发明的分离的核酸分子，或者本发明的载体。
本发明的再一方面涉及制备本发明中任一项所述的双特异性抗体的方法，其包括在合适的条件下培养本发明的宿主细胞，以及从细胞培养物中回收所述双特异性抗体的步骤。
本发明的再一方面涉及偶联物，其包括双特异性抗体以及偶联部分，其中，所述双特异性抗体为本发明中任一项所述的双特异性抗体，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
根据本发明的偶联物，其用于治疗和/或预防恶性肿瘤；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
本发明的再一方面涉及试剂盒，其包含本发明中任一项所述的双特异性抗体，或者包含本发明的偶联物；
优选地，所述试剂盒还包含第二抗体，其能够特异性结合所述双特异性抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
本发明的再一方面涉及本发明中任一项所述的双特异性抗体在制备试剂盒中的用途，所述试剂盒用于检测VEGF和/或PD-L1在样品中的存在或其水平。
本发明的再一方面涉及一种药物组合物，其包含本发明中任一项所述的双特异性抗体或者包含本发明的偶联物；可选地，其还包括药学上可接受的辅料。
本发明的再一方面涉及本发明中任一项所述的双特异性抗体或者本发明的偶联物在制备预防和/或治疗恶性肿瘤的药物中的用途；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
本发明的再一方面涉及一种治疗和/或预防恶性肿瘤的方法，包括给予有需求的受试者以有效量的本发明中任一项所述的双特异性抗体或者本发明的偶联物的步骤；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
在本发明的一些实施方式中，所述的方法，其中，给予有需求的受试者以有效量的本发明中任一项所述的双特异性抗体的步骤为在手术治疗之前或之后，和/或在放射治疗之前或之后。
在本发明的一些实施方式中，所述的方法，其中，
本发明的双特异性抗体的单次给药剂量为每千克体重0.1-100mg，优选4.8-24mg或1-10mg；或者，本发明的双特异性抗体的单次给药剂量为每位受试者10-1000mg，优选50-500mg、100-400mg、150-300mg、150-250mg或200mg；
优选地，每3天、4天、5天、6天、10天、1周、2周或3周给药一次；
优选地，给药方式为静脉滴注或静脉注射。
如本文中所使用的，术语EC 50是指半最大效应浓度(concentration for 50％of maximal effect)，是指能引起50％最大效应的浓度。
如本文中所使用的，术语“抗体”是指通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Bethesda M.d.,Kabat Sequences  of Proteins of Immunological Interest(National Institutes of Health,(1987and 1991))，或Chothia&Lesk J.Mol.Biol.1987；196:901-917；Chothia等人Nature1989；342:878-883，或者IMGT编号系统定义，见Ehrenmann F,Kaas Q,Lefranc M P.IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF[J].Nucleic acids research,2009；38(suppl_1):D301-D307的定义。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(
Figure PCTCN2021115308-appb-000001
G,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975；256(5517):495)，但也可采用重组DNA技术获得(如参见U.S.Patent 4,816,567)。
如本文中所使用的，术语“人源化抗体”是指，人源免疫球蛋白(受体抗体)的全部或部分CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或反应性的非人源(例如，小鼠、大鼠或兔)抗体。此外，受体抗体的构架区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或被其他抗体的氨基酸残基替换，以进一步完善或优化抗体的性能。关于人源化抗体的更多详细内容，可参见例如，Jones et al.,Nature 1986；321:522 525；Reichmann et al.,Nature,1988；332:323329；Presta,Curr.Op.Struct.Biol.1992；2:593-596；和Clark,Immunol.Today 2000；21:397 402。在一些情况下，抗体的抗原结合片段是双抗体(Diabodies)，其中V H和V L结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.et al.,Proc.Natl.Acad.Sci.USA 1993；90:6444-6448和Poljak R.J.et al.,Structure 1994；2:1121-1123)。
如本文中所述的融合蛋白是一种通过DNA重组得到的两个基因共表达的蛋白产物。现有技术中熟知生产和纯化抗体和抗原结合片段的方法(如冷泉港的抗体实验技术指南，5-8章和15章)。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草杆菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，GS细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“药学上可接受的辅料”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19 th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂。例如，pH调节剂包括但不限于磷酸盐缓冲液；表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80；离子强度增强剂包 括但不限于氯化钠。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如肿瘤)有效量是指，足以预防，阻止，或延迟疾病(例如肿瘤)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途的有效量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
在本发明中，如果没有特别说明，所述“第一”(例如第一蛋白功能区)和“第二”(例如第二蛋白功能区)是为了指代上的区分或表述上的清楚，并不具有典型的次序上的含义。
本发明还涉及如下1至10项中的任意一项：
1.一种双特异性抗体，其特征在于，所述双特异性抗体包括：
(a)抗PD-L1单域抗体；和
(b)抗VEGF的抗体或元件。
2、如第1项所述的双特异性抗体，其特征在于，所述的双特异性抗体含有如式I和式II所示结构的多肽，
A1-L1-CH-L2-B      (式I)
A2-L3-CL           (式II)
其中，
A1是抗VEGF抗体的重链可变区VH；
A2是抗VEGF抗体的轻链可变区VL；
B是抗PD-L1单域抗体；
L1、L2和L3各自独立地为无或连接元件；
CH是人IgG重链恒定区CH(优选地为LALA突变型)；
CL是人κ轻链恒定区CL；和
“-”为肽键；
并且其中，式I所示的多肽与式II所示的多肽通过二硫键作用而形成异源二聚体。
3、如第1项所述的双特异性抗体，其特征在于，所述双特异性抗体是具有如式III或式IV所示结构的多肽，
A3-L4-Fc-L5-B    (式III)
B-L6-Fc-L7-A3    (式IV)
其中，
A3是可以结合VEGF并阻断其活性的结构域；
B是抗PD-L1单域抗体；
L4、L5、L6和L7各自独立地为无或连接元件；
Fc是人IgG的Fc区段(优选地为LALA突变型)；和
“-”为肽键。
4、一种分离的多核苷酸，其特征在于，所述多核苷酸编码如第1项所述的双特异性抗体。
5、一种载体，其特征在于，所述载体含有如第4项所述的多核苷酸。
6、一种宿主细胞，其特征在于，所述宿主细胞含有如第5项所述的载体，或其基因组中整合有如第4项所述的多核苷酸；
或者，所述的宿主细胞表达如第1项所述的双特异性抗体。
7、一种产生如第1项所述双特异性抗体的方法，其特征在于，包括步骤：
(a)在合适的条件下，培养如第6项所述的宿主细胞，从而获得含所述双特异性抗体的培养物；和
(b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的双特异性抗体。
8、一种免疫偶联物，其特征在于，所述免疫偶联物含有：
(a)如第1项所述的双特异性抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
9、如第1项所述的双特异性抗体，或如第8项所述的免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；其中，所述试剂、检测板或试剂盒用于：检测样品中PD-L1和/或VEGF；其中，所述药剂用于治疗或预防表达PD-L1(即PD-L1阳性)的肿瘤或是表达VEGF的肿瘤。
10、一种药物组合物，其特征在于，所述药物组合物含有：
(i)如第1项所述的双特异性抗体，或如第8项所述的免疫偶联物；和
(ii)药学上可接受的载体。
本发明还涉及如下的第一方面至第十四方面中的任一方面：
在本发明的第一方面，提供了一种双特异性抗体，所述双特异性抗体包括：
(a)抗PD-L1单域抗体；和
(b)抗VEGF的抗体或元件。
在另一优选例中，所述的双特异性抗体包括1-3个抗PD-L1单域抗体，较佳地，包括1或2个抗PD-L1单域抗体。
在另一优选例中，所述的PD-L1单域抗体可以阻断PD-1和PD-L1的相互作用。
在另一优选例中，所述的双特异性抗体还包含Fc段。
在另一优选例中，所述双特异性抗体的Fc段选自下组：人IgG结构域、CH1+CL1结构域，或其组合。
在另一优选例中，所述人IgG结构域是经过改造的突变型IgG结构域，优选地为LALA突变型的IgG结构域。
在另一优选例中，所述的双特异性抗体含有如式I和式II所示结构的多肽，
A1-L1-CH-L2-B      (式I)
A2-L3-CL           (式II)
其中，
A1是抗VEGF抗体的重链可变区VH；
A2是抗VEGF抗体的轻链可变区VL；
B是抗PD-L1单域抗体；
L1、L2和L3各自独立地为无或连接元件；
CH是人IgG重链恒定区CH(优选地为LALA突变型)；
CL是人κ轻链恒定区CL；和
“-”为肽键；
并且其中，式I所示的多肽与式II所示的多肽通过二硫键作用而形成异源二聚体。
在另一优选例中，所述双特异性抗体是具有如式III或式IV所示结构的多肽，
A3-L4-Fc-L5-B     (式III)
B-L6-Fc-L7-A3     (式IV)
其中，
A3是可以结合VEGF并阻断其活性的结构域；
B是抗PD-L1单域抗体；
L4、L5、L6和L7各自独立地为无或连接元件；
Fc是人IgG的Fc区段(优选地为LALA突变型)；和
“-”为肽键。
在另一优选例中，所述抗VEGF的重链可变区VH具有衍生自贝伐单抗(Bevacizumab)的重链可变区氨基酸序列，其氨基酸序列如SEQ ID NO:3所示，或与SEQ ID NO:3所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述抗VEGF的轻链可变区VL具有衍生自贝伐单抗(Bevacizumab)的轻链可变区氨基酸序列，其氨基酸序列如SEQ ID NO:9所示，或与SEQ ID NO:9所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述抗VEGF的重链可变区VH具有衍生自雷尼单抗(Ranibizumab)的重链可变区氨基酸序列，其氨基酸序列如SEQ ID NO:13所示，或与SEQ ID NO:13所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述抗VEGF的轻链可变区VL具有衍生自雷尼单抗(Ranibizumab)的轻链可变区氨基酸序列，其氨基酸序列如SEQ ID NO:15所示，或与SEQ ID NO:15所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述可以结合VEGF并阻断其活性的结构域的氨基酸序列如SEQ ID NO:17所示，或与SEQ ID NO:17所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述抗PD-L1单域抗体的氨基酸序列如SEQ ID NO:5所示，或与SEQ ID NO:5所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述人IgG重链恒定区CH中具有LALA突变，其氨基酸序列如SEQ ID NO:4所示，或与SEQ ID NO:4所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述人κ轻链恒定区CL的氨基酸序列如SEQ ID NO:10所示，或与SEQ ID NO:10所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述人IgG的Fc区段具有LALA突变，其氨基酸序列如SEQ ID NO:18所示，或与SEQ ID NO:18所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述接头元件的序列为(G 4S) n，其中，n为正整数(例如1、2、3、4、5或6)，优选地，n为2或4。
在另一优选例中，所述接头元件的氨基酸序列如SEQ ID NO:6或7所示，或与SEQ ID NO:6或7所示序列具有≥85％(优选地90％，更优选地95％)的序列同一性。
在另一优选例中，所述双特异性抗体含有如式I和式II所示结构的多肽，其中所述式I所示结构的多肽的氨基酸序列如SEQ ID NO:1所示，并且所述式II所示结构的多肽的氨基酸序列如SEQ ID NO:8所示，即Ava-2GS-NSD。
在另一优选例中，所述双特异性抗体含有如式I和式II所示结构的多肽，其中所述式I所示结构的多肽的氨基酸序列如SEQ ID NO:2所示，并且所述式II所示结构的多肽的氨基酸序列如SEQ ID NO:8所示，即Ava-4GS-NSD)。
在另一优选例中，所述双特异性抗体含有如式I和式II所示结构的多肽，其中所述式I所示结构的多肽的氨基酸序列如SEQ ID NO:11所示，并且所述式II所示结构的多肽的氨基酸序列如SEQ ID NO:14所示，即Luc-2GS-NSD)。
在另一优选例中，所述双特异性抗体含有如式I和式II所示结构的多肽，其中所述式I所示结构的多肽的氨基酸序列如SEQ ID NO:12所示，并且所述式II所示结构的多肽的氨基酸序列如SEQ ID NO:14所示，即Luc-4GS-NSD)。
在另一优选例中，所述双特异性抗体是具有如式III所示结构的多肽，其氨基酸序列如SEQ ID NO:16所示，即NSD-Elyea)。
在另一优选例中，所述双特异性抗体是具有如式III所示结构的多肽，其氨基酸序列如SEQ ID NO:19所示，即Elyea-NSD)。
在另一优选例中，所述的双特异性抗体同时含有如式I和式II所示结构的多肽，并且式I所示的多肽与式II所示的多肽通过二硫键作用而形成异源二聚体i；
并且，所述异源二聚体i之间，通过CH结构域之间的二硫键作用而形成同源二聚体ii。
在另一优选例中，所述的双特异性抗体是具有如式III或式IV所示结构的多肽，并且所述的如式III或式IV所示结构的多肽之间，通过Fc区段之间的二硫键作用而形成同源二聚体。
在本发明的第二方面，提供了一种分离的多核苷酸，所述多核苷酸编码如本发明第一方面所述的双特异性抗体。
在另一优选例中，当所述双特异性抗体含有如式I和式II所示结构的多肽时，所 述多核苷酸中，编码所述式I所示结构多肽的多核苷酸序列与编码所述式II所示结构多肽的多核苷酸序列的比例为1:1。
在本发明的第三方面，提供了一种载体，所述载体含有如本发明第二方面所述的多核苷酸。
在另一优选例中，所述的载体选自下组：DNA、RNA、病毒载体、质粒、转座子、其他基因转移系统、或其组合；优选地，所述表达载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。
在本发明的第四方面，提供了一种宿主细胞，所述宿主细胞含有如本发明第三方面所述的载体，或其基因组中整合有如本发明第二方面所述的多核苷酸；
或者，所述的宿主细胞表达如本发明第一方面所述的双特异性抗体。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、哺乳动物细胞。
在本发明的第五方面，提供了一种产生如本发明第一方面所述双特异性抗体的方法，包括步骤：
(a)在合适的条件下，培养如本发明第四方面所述的宿主细胞，从而获得含所述双特异性抗体的培养物；和
(b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的双特异性抗体。
在另一优选例中，所述纯化可以通过蛋白A亲和柱纯化分离获得目标抗体。
在另一优选例中，所述经过纯化分离后的目标抗体纯度大于95％，大于96％、大于97％、大于98％、大于99％，优选为100％。
在本发明的第六方面，提供了一种免疫偶联物，所述免疫偶联物含有：
(a)如本发明第一方面所述的双特异性抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
在另一优选例中，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188、或其组合；和/或
(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、 Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133Yb-169、Yb-177、或其组合。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述的药物为细胞毒性药物。
在另一优选例中，所述的细胞毒性药物选自下组：抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱、或其组合。
特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(auristatins)、喜树碱(camptothecins)、多卡霉素/倍癌霉素(duocarmycins)、依托泊甙(etoposides)、美登木素(maytansines)和美登素类化合物(maytansinoids)(例如DM1和DM4)、紫杉烷(taxanes)、苯二氮卓类(benzodiazepines)或者含有苯二氮卓的药物(benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(oxazolidinobenzodiazepines))、长春花生物碱(vinca alkaloids)、或其组合。
在另一优选例中，所述的毒素选自下组：
耳他汀类(例如，耳他汀E、耳他汀F、MMAE和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(mitogellin)、局限曲菌素(retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂、糖皮质激素、或其组合。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联物选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2)、抗体、抗体Fc片段、抗体scFv片 段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(如DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(如顺铂)。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明第一方面所述的双特异性抗体。
在另一优选例中，所述多价是指在所述免疫偶联物的氨基酸序列中包含多个重复的如本发明第一方面所述的双特异性抗体。
在本发明的第七方面，提供了如本发明第一方面所述的双特异性抗体，或本发明第六方面所述的免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；其中，所述试剂、检测板或试剂盒用于：检测样品中PD-L1和/或VEGF；其中，所述药剂用于治疗或预防表达PD-L1(即PD-L1阳性)的肿瘤或是表达VEGF的肿瘤。
在另一优选例中，所述的免疫偶联物的偶联部分为诊断用同位素。
在另一优选例中，所述的试剂为选自下组的一种或多种试剂：同位素示踪剂、造影剂、流式检测试剂、细胞免疫荧光检测试剂、纳米磁粒和显像剂。
在另一优选例中，所述检测样品中PD-L1和/或VEGF的试剂为(体内)检测PD-L1和/或VEGF分子的造影剂。
在另一优选例中，所述的检测为体内检测或体外检测。
在另一优选例中，所述的检测包括流式检测、细胞免疫荧光检测。
在另一优选例中，所述的药剂用于阻断PD-1和PD-L1的相互作用，同时阻断VEGF和VEGFR的相互作用。
在另一优选例中，所述的肿瘤包括但不限于：急性髓细胞白血病、慢性粒细胞性白血病、多发性骨髓病、非霍奇金淋巴瘤、结直肠癌、乳腺癌、大肠癌、胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、前列腺癌、宫颈癌、淋巴癌、肾上腺肿瘤、膀胱肿瘤。
在本发明的第八方面，提供了一种药物组合物，所述药物组合物含有：
(i)如本发明第一方面所述的双特异性抗体，或如本发明第六方面所述的免疫偶联物；和
(ii)药学上可接受的载体。
在另一优选例中，所述的免疫偶联物的偶联部分为药物、毒素、和/或治疗用同位素。
在另一优选例中，所述的药物组合物中还含有治疗肿瘤的其他药物，如细胞毒性 药物。
在另一优选例中，所述的治疗肿瘤的其他药物包括紫杉醇、多柔比星、环磷酰胺、阿西替尼、乐伐替尼、派姆单抗。
在另一优选例中，所述的药物用于阻断PD-1和PD-L1的相互作用，同时阻断VEGF和VEGFR的相互作用。
在另一优选例中，所述的药物组合物用于阻断PD-1/PD-L1和/或VEGF/VEGFR信号通路。
在另一优选例中，所述的药物组合物用于治疗表达PD-L1蛋白(即PD-L1阳性)和/或表达VEGF蛋白(即VEGF阳性)的肿瘤。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备防治肿瘤的药物。
在本发明的第九方面，提供了如本发明第一方面所述的双特异性抗体的一种或多种选自下组的用途，包括：
(i)用于检测人PD-L1分子和/或VEGF分子；(ii)用于流式检测；(iii)用于细胞免疫荧光检测；(iv)用于治疗肿瘤；(v)用于肿瘤诊断；(vi)用于阻断PD-1和PD-L1的相互作用；和(vii)用于阻断VEGF和VEGFR的相互作用。
在另一优选例中，所述的肿瘤为表达PD-L1蛋白(即PD-L1阳性)和/或表达VEGF蛋白(即VEGF阳性)的肿瘤。
在另一优选例中，所述用途为非诊断的和非治疗的。
在本发明的第十方面，提供了一种重组蛋白，所述的重组蛋白具有：(i)如本发明第一方面所述的双特异性抗体；以及(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签、HA标签和Fc标签。
在另一优选例中，所述的重组蛋白特异性结合于PD-L1和/或VEGF。
在本发明的第十一方面，提供了一种检测样品中PD-L1和/或VEGF的方法，所述方法包括步骤：(1)将样品与如本发明第一方面所述的双特异性抗体接触；(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在PD-L1和/或VEGF。
在本发明的第十二方面，提供了一种治疗疾病的方法，所述方法包括：给需要的对象施用如本发明第一方面所述的双特异性抗体、如本发明第六方面所述的免疫偶联物，或如本发明第八方面所述的药物组合物。
在另一优选例中，所述的对象包括哺乳动物，优选地是人。
在本发明的第十三方面，提供了一种PD-L1和/或VEGF检测试剂，所述的检测试剂包含如本发明第六方面所述的免疫偶联物和检测学上可接受的载体。
在另一优选例中，所述的免疫偶联物的偶联部分为诊断用同位素。
在另一优选例中，所述的检测学上可接受的载体为无毒的、惰性的水性载体介质。
在另一优选例中，所述的检测试剂为选自下组的一种或多种试剂：同位素示踪剂、造影剂、流式检测试剂、细胞免疫荧光检测试剂、纳米磁粒和显像剂。
在另一优选例中，所述的检测试剂用于体内检测。
在另一优选例中，所述的检测试剂的剂型为液态或粉状(如水剂、针剂、冻干粉、片剂、含服剂、吸雾剂)。
在本发明的第十四方面，提供了一种检测PD-L1和/或VEGF的试剂盒，所述试剂盒含有如本发明第六方面所述的免疫偶联物或如本发明第十三方面所述的检测试剂，以及说明书。
在另一优选例中，所述的说明书记载，所述的试剂盒用于非侵入性地检测待测对象的PD-L1和/或VEGF表达。
在另一优选例中，所述的试剂盒用于表达PD-L1蛋白(即PD-L1阳性)和/或表达VEGF蛋白(即VEGF阳性)的肿瘤的检测。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
双特异性抗体
如本文所用，术语“本发明的双特异性抗体”、“本发明的双抗”、“抗PD-L1/VEGF双特异性抗体”具有相同的含义，均指特异性识别和结合PD-L1和VEGF的双特异性抗体。对于具有VEGF结合活性的蛋白功能区，当其为VEGF受体(例如VEGFR1和/或VEGFR2)或其功能片段时，由于具有与抗体类似的阻断VEGF和受体结合的功能，此时的融合蛋白在本发明中亦广义上地称为双特异性抗体。
本发明提供了一种抗PD-L1/VEGF双特异性抗体，其包括：抗PD-L1单域抗体和抗VEGF的抗体或元件。
优选地，所述的双特异性抗体含有如式I和式II所示结构的多肽，
A1-L1-CH-L2-B      (式I)
A2-L3-CL           (式II)
其中，
A1是抗VEGF抗体的重链可变区VH；
A2是抗VEGF抗体的轻链可变区VL；
B是抗PD-L1单域抗体；
L1、L2和L3各自独立地为无或连接元件；
CH是人IgG重链恒定区CH(优选地为LALA突变型)；
CL是人κ轻链恒定区CL；和
“-”为肽键；
并且其中，式I所示的多肽与式II所示的多肽通过二硫键作用而形成异源二聚体。
或者，所述双特异性抗体是具有如式III或式IV所示结构的多肽，
A3-L4-Fc-L5-B     (式III)
B-L6-Fc-L7-A3     (式IV)
其中，
A3是可以结合VEGF并阻断其活性的结构域；
B是抗PD-L1单域抗体；
L4、L5、L6和L7各自独立地为无或连接元件；
Fc是人IgG的Fc区段(优选地为LALA突变型)；和
“-”为肽键。
在一个实施方式中，所述的双特异性抗体同时含有如式I和式II所示结构的多肽，并且式I所示的多肽与式II所示的多肽通过二硫键作用而形成异源二聚体i；
并且，所述异源二聚体i之间，通过CH结构域之间的二硫键作用而形成同源二聚体ii。
在另一个实施方式中，所述的双特异性抗体是具有如式III或式IV所示结构的多肽，并且所述的如式III或式IV所示结构的多肽之间，通过Fc区段之间的二硫键作用而形成同源二聚体。
如本文所用，术语“单域抗体”、“纳米抗体VHH”、“纳米抗体”具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)，它是具 有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为构架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本文所用，术语“框架区”(FR)指插入CDR间的氨基酸序列，即指在单一物种中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。
如本文所用，术语″人框架区″是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％)框架区。
如本文所用，术语“亲和力”理论上通过完整抗体和抗原间的平衡缔合来定义。本发明双抗的亲和力可以通过K D值(解离常数)(或其它测定方式)进行评估或测定，例如生物膜层干涉技术(Bio-layer interferometry BLI)，使用FortebioRed96仪器测量确定。
如本文所用，术语“接头”是指插入免疫球蛋白结构域中为轻链和重链的结构域提供足够的可动性以折叠成交换双重可变区免疫球蛋白的一个或多个氨基酸残基。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的PD-L1/VEGF双特异性抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“可变区”与“互补决定区(complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合PD-L1和/或VEGF蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或 多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有PD-L1和/或VEGF蛋白结合活性的双抗。该术语还包括具有与本发明抗体相同功能的、包含相同CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含单域抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明单域抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度 和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。 如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合PD-L1和/或VEGF蛋白分子，因而可用于治疗肿瘤。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单域抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10 微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
标记的抗体
在本发明的一个优选例中，所述抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，PD-L1/VEGF双特异性抗体可以用胶体金标记，得到胶体金标记的抗体。
检测方法
本发明还涉及检测PD-L1和/或VEGF蛋白的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中PD-L1和/或VEGF蛋白的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测PD-L1和/或VEGF水平的检测试剂盒，该试剂盒包括识别PD-L1和/或VEGF蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
应用
实验结果表明，本发明的双特异性抗体能够高特异性针对人的PD-L1蛋白，能够在靶向中和肿瘤微环境的VEGF基础上抑制PD-1/PD-L1通路，可以使T细胞恢复活 性，增强免疫应答，从而更有效地提高抑制肿瘤发生和发展的效果。
如上所述，本发明的单域抗体有广泛生物应用价值和临床应用价值，其应用涉及到与PD-L1和/或VEGF相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对PD-L1和/或VEGF的临床诊断和靶向治疗，如肿瘤治疗。
发明的有益效果
本发明取得了如下技术效果中的一项或多项：
(1)本发明双特异性抗体的亲和力强。
(2)在靶向中和肿瘤微环境的VEGF基础上抑制PD-1/PD-L1通路，可以使T细胞恢复活性，增强免疫应答，更有效地提高抑制肿瘤发生和发展的效果，具有良好的制备抗肿瘤药物的潜力。
(3)本发明双特异性抗体的生产简便。
(4)本发明的双特异性抗体的第一蛋白功能区和第二蛋白功能区之间很可能存在协同效应，例如，其与VEGF的亲和力往往优于抗VEGF单抗，其与PD-L1的亲和力往往优于抗PD-L1单抗；在诱导混合淋巴细胞分泌IL2或者INF方面，也优于抗VEGF单抗或抗PD-L1单抗，显示出本发明的双特异性抗体能够更好地激活T细胞。
附图说明
图1A：Ava-2GS-NSD或Ava-4GS-NSD的结构示意图。
图1B：Luc-2GS-NSD或Luc-4GS-NSD的结构示意图。
图1C：NSD-Elyea的结构示意图。
图1D：Elyea-NSD的结构示意图。
图2：双特异性抗体和CHO-PDL1细胞结合曲线图。
图3A：双特异性抗体阻断VEGF和VEGFR2结合曲线图。
图3B：双特异性抗体阻断VEGF和VEGFR2结合曲线图。
图4A：双特异性抗体阻断在淋巴细胞混合培养中分泌INF-gamma浓度。对于每个双特异性抗体样品，其浓度从左往右依次为100nM、10nM、1nM、0.1nM和0.01nM。
图4B：双特异性抗体阻断在淋巴细胞混合培养中分泌IL-2浓度。对于每个双特 异性抗体样品，其浓度从左往右依次为100nM、10nM、1nM、0.1nM和0.01nM。
图5A：存储不同天数双特异性抗体和CHO-PDL1细胞结合曲线图。
图5B：存储不同天数双特异性抗体和CHO-PDL1细胞结合曲线图。
图5C：存储不同天数双特异性抗体和CHO-PDL1细胞结合曲线图。
图5D：存储不同天数双特异性抗体和CHO-PDL1细胞结合曲线图。
图5E：存储不同天数双特异性抗体和VEGF结合曲线图。
图5F：存储不同天数双特异性抗体和VEGF结合曲线图。
图5G：存储不同天数双特异性抗体和VEGF结合曲线图。
图5H：存储不同天数双特异性抗体和VEGF结合曲线图。
图6A：双特异性抗体对小鼠的肿瘤体积影响图。
图6B：双特异性抗体对小鼠的体重影响图。
图7A：Ava-2GS-NSD或Ava-4GS-NSD的结构示意图。
图7B：Luc-2GS-NSD或Luc-4GS-NSD的结构示意图。
图7C：NSD-Elyea的结构示意图。
图7D：Elyea-NSD的结构示意图。
本发明涉及的部分序列如下面的表B所示
表B
Figure PCTCN2021115308-appb-000002
Figure PCTCN2021115308-appb-000003
Figure PCTCN2021115308-appb-000004
Figure PCTCN2021115308-appb-000005
Figure PCTCN2021115308-appb-000006
Figure PCTCN2021115308-appb-000007
Figure PCTCN2021115308-appb-000008
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非 另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1：抗VEGF/PD-L1双特异性抗体的克隆和表达
1.1抗体结构设计
在本实施例中，构建了6种抗VEGF/PD-L1双特异性抗体，分别为：
Ava-2GS-NSD/Ava-4GS-NSD：由4条多肽链组成(两条分别连接C-Ye-18-5的重链，两条轻链)，其结构示意图如图1A和图7A所示，两条肽链#1均具有SEQ ID NO:1或SEQ ID NO:2所示的氨基酸序列，其包含衍生自抗VEGF抗体贝伐单抗(Bevacizumab)(专利号：WO1998045332)的VH氨基酸序列(SEQ ID NO:3)，在所述VH氨基酸序列C端直接连接衍生自人IgG1CH氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:4),将抗PD-L1的纳米抗体C-Ye-18-5(专利申请号：2019108631090)(SEQ ID NO:5)的N端通过11个氨基酸残基(G 4S) 2G(SEQ ID NO:6)(Ava-2GS-NSD)或21个氨基酸残基(G 4S) 4G(SEQ ID NO:7)(Ava-4GS-NSD)的柔性肽连接于重链的C端。两条肽链#2均具有SEQ ID NO:8所示的氨基酸序列，其包含衍生自抗VEGF抗体贝伐单抗(Bevacizumab)的VL氨基酸序列(SEQ IDNO 9)，以及在所述VL氨基酸序列C端的人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:10)。
Luc-2GS-NSD/Luc-4GS-NSD：由4条多肽链组成(两条分别连接C-Ye-18-5的重链，两条轻链)，其结构示意图如图1B和图7B所示，两条肽链#1均具有SEQ ID NO:11或SEQ ID NO:12所示的氨基酸序列，其包含衍生自抗VEGF抗体雷尼单抗(Ranibizumab)(专利号：WO2018175752)的VH氨基酸序列(SEQ ID NO:13)，在所述VH氨基酸序列C端直接连接衍生自人IgG1CH氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:4),将抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:5)的N端通过11个氨基酸残基(G 4S) 2G(SEQ ID NO:6)(Luc-2GS-NSD)或21个氨基酸残基(G 4S) 4G(SEQ ID NO:7)(Luc-4GS-NSD)的柔性肽连接于重链的C端。两条肽链#2均具有SEQ ID NO:14所示的氨基酸序列，其包含衍生自抗VEGF抗体雷尼单抗(Ranibizumab)的VL氨基酸序列(SEQ IDNO:15)，以及在所述VL氨基酸序列C端的人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:10)。
NSD-Elyea:由2条相同的多肽链组成(二聚体)，其结构示意图如图1C和图7C所示，肽链具有SEQ ID NO:16所示的氨基酸序列，其包含衍生自抗VEGF融合蛋白Aflibercept(专利号：US7070959)的VEGF结合区域(Elyea，SEQ ID NO:17)，在所述 氨基酸序列C端直接连接衍生自人IgG1Fc氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID:18),将抗PD-L1的纳米抗体C-Ye-18-5的N端通过21个氨基酸残基(G 4S) 4G(SEQ ID NO:7)的柔性肽连接于重链的C端。
Elyea-NSD：由2条相同的多肽链组成(二聚体)，其结构示意图如图1D和图7D所示，肽链具有SEQ ID NO:19所示的氨基酸序列，其包含抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:5)，在所述纳米抗体序列C端直接连接衍生自人IgG1Fc氨基酸序列(引入LALA突变以降低Fc功能，SEQ ID NO:18)，衍生自抗VEGF融合蛋白Aflibercept的VEGF结合区域(Elyea，SEQ ID NO:17)的N端通过21个氨基酸残基(G 4S) 4G(SEQ ID NO:7)的柔性肽连接于重链的C端。
1.2基因克隆及蛋白制备
参照表B中的序列，将编码相应氨基酸序列的基因片段构建入pCDNA3.1载体中。对于Ava-2GS-NSD中的肽链#1和肽链#2，这两条序列在瞬时转染的时候是分别表达在两个不同质粒中的，在细胞表达的过程中会自动形成二硫键。
采用ExpiCHO TM表达系统试剂盒(购自Thermo)，将质粒转入Expi-CHO细胞中，转染方法按照商品说明书，细胞培养5天后收集上清利用蛋白A磁珠(购自金斯瑞)分选法纯化目的蛋白。将磁珠用适当体积的结合缓冲液(PBS+0.1％吐温20，pH7.4)重悬(1-4倍磁珠体积)后加入至待纯化样品中，室温孵育1小时，期间温柔振荡。样品置于磁力架上(购自海狸)，弃去上清，磁珠用结合缓冲液清洗3遍。按照磁珠体积的3-5倍体积加入洗脱缓冲液(0.1M sodium citrate,pH3.2)室温振荡5-10min，置回磁力架上，收集洗脱缓冲液，转移至已加入中和缓冲液(1M Tris，pH8.54)的收集管中混匀。用于后面的实验。
实施例2：双特异性抗体抗体亲和力测定
ForteBio亲和力测定按照现有的方法(Estep,P等人，基于溶液的高通量抗体-抗原亲和力和表位分级的测量，MAbs，2013.5(2):p.270-8)进行。简言之，传感器在分析缓冲液中线下平衡30min，然后线上检测60s建立基线，在线加载如上所述获得的经纯化的抗体至AHC传感器上。再将传感器放入100nM的PD-L1或VEGF抗原中作用5min，之后将传感器转移至PBS中解离5min。使用1:1结合模型进行动力学的分析。
结果如下面的表1和表2所示。
表1：候选分子和PD-L1结合亲和力
编号 K D(M) Kon(1/Ms) Koff(1/s)
Ava-2GS-NSD 8.48E-09 3.19E+05 2.70E-03
Luc-2GS-NSD 9.52E-09 2.81E+05 2.67E-03
Ava-4GS-NSD 7.12E-09 3.54E+05 2.52E-03
Luc-4GS-NSD 8.08E-09 3.21E+05 2.60E-03
NSD-Elyea 5.33E-09 3.22E+05 1.72E-03
Elyea-NSD 8.34E-09 3.39E+05 2.83E-03
C-Ye-18-5 8.75E-09 1.98E+05 1.73E-03
表2：候选分子和VEGF结合亲和力
编号 K D(M) Kon(1/Ms) Koff(1/s)
Ava-2GS-NSD 1.38E-09 2.59E+05 3.58E-04
Luc-2GS-NSD 1.33E-09 1.77E+05 2.36E-04
Ava-4GS-NSD 1.51E-09 2.49E+05 3.75E-04
Luc-4GS-NSD 1.35E-09 1.82E+05 2.45E-04
NSD-Elyea 1.10E-09 6.48E+05 7.12E-04
Elyea-NSD 4.01E-10 9.14E+05 3.66E-04
Ranibizumab 2.29E-09 1.75E+05 4.00E-04
Bevacizumab 1.79E-09 1.71E+05 3.06E-04
结果显示，本发明所有的双特异性抗体样品均与PD-L1及VEGF蛋白有结合活性。与对照抗体C-Ye-18-5(PD-L1单域抗体)相比，本发明的双特异性抗体和PD-L1的结合活性相似甚至更优；和/或，与对照抗体雷尼单抗(Ranibizumab)及贝伐单抗(Bevacizumab)相比，本发明的双特异性抗体与VEGF的结合活性相似甚至更优。
实施例3：纯化的双特异性抗体与PD-L1结合
通过转染克隆到MCS的人PD-L1cDNA(购自Sino Biological)的pCHO1.0载体(购自Invitrogen)产生过表达人PD-L1的CHO细胞(CHO-hPD-L1细胞)。将扩大培养的CHO-hPD-L1细胞调整细胞密度至2×10 6细胞/ml，100μl/孔加入96孔流式板，离 心备用。将纯化的PD-L1抗体用PBS稀释，400nM开始3倍稀释共12个点，将上述稀释好的样品100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育30min，PBS清洗两次。100μl/孔加入用PBS稀释100倍的羊F(ab’)2抗人IgG-Fc(PE)(购自Abcam)，4℃孵育30min，PBS清洗两次。100μl/孔加入PBS重悬细胞，在CytoFlex(Bechman)流式细胞仪上进行检测并计算对应的MFI。
在如上方法的测定实验中，实验结果如图2所示，本发明所有的纯化样品和CHO-hPD-L1细胞均有结合活性，且纯化样品的结合活性与对照抗体C-Ye-18-5相似甚至更优。
实施例4：ELISA检测VEGF/VEGFR蛋白水平结合阻断实验
用ELISA包被液将人VEGFR蛋白稀释至合适浓度，加入ELISA板，4℃包被过夜。5％BSA室温封闭1小时。将待测样品梯度稀释并与生物素化标记的人VEGF蛋白室温共孵育1小时。将孵育好的样品加入封闭好的ELISA板，室温反应2小时。PBS洗液洗涤3次，加入稀释好的链霉亲和素(HRP)室温反应1小时,PBS洗液洗涤3次，加入ELISA显色液，室温放置3min，加入ELISA终止液，读取450nm处吸光度数值。
结果如图3A-图3B所示。
结果显示，Ava-2GS-NSD、Luc-2GS-NSD、Ava-4GS-NSD、Luc-4GS-NSD(图3A)及NSD-Elyea、Elyea-NSD(图3B)均可以完全阻断VEGF和VEGFR蛋白的相互作用。
实施例5：混合淋巴细胞反应实验
本实施例通过混合淋巴细胞反应实验(MLR)检测双特异性抗体样品激活T细胞的活性。具体实验方法如下。
复苏PBMC细胞(购自SAILY BIO,SLB-HPB)，离心，用10ml X-VIVO-15培养基(购自LONZA)重悬PBMC，于细胞培养箱内37℃贴壁培养2h，吸去未贴壁细胞。加入10ml DC培养基(X-VIVO-15培养基加入10ng/ml GM-CSF(购自R&D)，20ng/ml IL-4)，培养3天，补加5ml DC培养基，继续培养至第6天，加入DC成熟培养基(X-VIVO-15培养基加入1000U/ml TNF-α(购自R&D)，10ng/ml IL-6(购自R&D)，5ng/ml IL-1β(购自R&D)，1μM PGE2(购自Tocris)),培养2天，收集成熟的DC细胞，用X-VIVO-15培养基调整细胞密度为2×10 5细胞/ml。
复苏另一位捐献者的PBMC细胞(购自SAILY BIO,SLB-HPB)，离心，用10ml X-VIVO-15培养基重悬PBMC。用CD4 +T细胞分选试剂盒(购自Stemcell)富集CD4 +T细胞，X-VIVO-15重悬CD4 +T细胞，调整细胞密度为2×10 6细胞/ml。将CD4 +T细胞与上述收集的成熟DC细胞按1:1比例混合，100μl/孔加入96孔U底板。
用X-VIVO-15培养基稀释纯化的双特异性抗体样品，200nM开始3倍稀释共9个点，100μl/孔加入上述混合细胞孔，培养5天，收集上清，ELISA(购自eBioscience)方法检测IFN-γ(图4A)和IL2(图4B)表达量。
结果如图4A和图4B所示，Ava-2GS-NSD、Luc-2GS-NSD、Ava-4GS-NSD、Luc-4GS-NSD、NSD-Elyea、Elyea-NSD均在MLR实验中显示较好的生物学活性，且激活水平与对照抗体C-Ye-18-5相似或优于对照抗体。例如，在一些浓度上，双特异性抗体样品诱导IL2或者INF的分泌比对照抗体更高，显示出本发明的双特异性抗体能够更好地激活T细胞。
实施例6：加速稳定性实验测定
6.1加速稳定性样品纯度测定
本实验通过检测双特异性抗体在40℃放置30天之后的纯度及生物学活性的变化，从而评价该抗体的长期热稳定性。使用SEC的方法测定了目的抗体在40℃放置0、14及30天后的纯度。
实验结果如表3所示。
表3：加速稳定性样品单体比例
样品名 0天 14天 30天
Ava-2GS-NSD 98.9％ 98.1％ 97.0％
Luc-2GS-NSD 97.4％ 97.3％ 96.3％
NSD-Elyea 93.0％ 97.1％ 94.1％
Elyea-NSD 99.7％ 100.0％ 98.4％
结果显示，双特异性抗体Ava-2GS-NSD、Luc-2GS-NSD、NSD-Elyea、Elyea-NSD的纯度没有明显变化。
6.2加速稳定性样品活性测定
本实验利用FACS方法检测了加速稳定性实验样品和CHO-PDL1细胞的结合情况，方法同实施例3。用ELISA方法检测了加速稳定性样品和VEGF的结合情况，方 法如下：用ELISA包被液将VEGFA重组蛋白蛋白稀释至1μg/ml,100μl/孔加入ELISA板，4℃包被过夜。弃去包被液，250μl/孔加入PBST洗3次，用5％BSA室温封闭1小时备用。将纯化的样品用1％BSA梯度稀释。将ELISA板弃去封闭液，将上述稀释好的样品100μl/孔加入封闭好的ELISA板，室温孵育2小时。250μl/孔加入PBST洗3次，100μl/孔加入用1％BSA稀释的羊抗人Fc-HRP,室温孵育1小时,250μl/孔加入PBST洗3次，100μl/孔加入ELISA显色液，室温放置3min,50μl/孔加入ELISA终止液，读取450nm处吸光度数值。
实验结果如图5A-图5H所示，双特异性抗体和CHO-PDL1细胞的结合(图5A-图5D)及和VEGF蛋白结合(图5E-图5H)的EC 50没有明显变化。结果表明，本发明的双特异性抗体具有较好的热稳定性。
实施例7：大鼠药代动力学评价
本实验检测了抗PD-L1/VEGF双特异性抗体在大鼠体内的药代动力学性质，使用的SD大鼠6只(雌雄各半，12/12小时光/暗调节，温度24±2℃，湿度40-70％,自由进水饮食)购自浙江维通利华实验技术有限公司。实验当天对SD大鼠单次尾静脉注射Ava-2GS-NSD或Elyea-NSD分子，注射剂量为10mg/kg。
取血时间点：给药后3min、4小时、10小时、24小时、48小时、72小时、120小时、168小时、240小时、336小时、504小时、672小时于大鼠颈静脉采血。全血样品2-8℃放置30min，12000rpm离心5min收集血清，所得血清再于2-8℃，12000rpm离心5min，-80℃保存，ELISA检测血清中游离Ava-2GS-NSD或Elyea-NSD分子量。
实验结果如表4所示。
表4：双特异性抗体在SD大鼠中的T1/2
受试药物 给药方式 T1/2
Ava-2GS-NSD IV 320.6小时
Elyea-NSD IV 38.1小时
结果显示，本发明Ava-2GS-NSD游离状态分子在SD大鼠体内半衰期约为320.6小时，Elyea-NSD游离状态分子在SD大鼠体内半衰期约为38.1小时。
实施例8：双特异性抗体的肿瘤抑制活性研究
本实验采用人结肠癌LOVO细胞/NOG小鼠注射人PBMC模型测定双特异性抗 体的抗肿瘤作用。体外培养扩增足够的LOVO细胞(购自Addexbio)，胰酶消化后收集细胞，用PBS清洗3遍后计数，按2×10 6细胞/小鼠的量接种到雌性8周龄的NOG重度免疫缺陷小鼠(购自北京维通利华实验动物技术有限公司)右侧腹部皮下。每日观察细胞在NOG小鼠皮下成瘤情况，接种8天后每只小鼠尾静脉注射6×10 6PBMC。注射完PBMC过3天后，使用游标卡尺测量每只动物右侧腹部皮下肿瘤的最大宽轴W和最大长轴L，使用电子天平称量每只小鼠的体重。按肿瘤体积T＝1/2×W×W×L计算每只小鼠右侧腹部皮下肿瘤体积。剔除瘤体积过大和过小的小鼠，按平均瘤体积将NOG小鼠平均分为4组，每组6只。按照下面的表5所示的分组给药方案进行分组并注射相应剂量的Ava-2GS-NSD。
表5：抗PD-L1/VEGF双特异性抗体肿瘤抑制活性实验方案
组别 给药类别 给药剂量 给药频率
Group 1 PBS — 每周2次共6次
Group 2 Ava-2GS-NSD 24mg/kg 每周2次共6次
Group 3 Ava-2GS-NSD 4.8mg/kg 每周2次共6次
Group 4 Ava-2GS-NSD 1mg/kg 每周2次共6次
每周2次测量小鼠肿瘤体积与小鼠体重。于接种肿瘤细胞31天后最后一次测量小鼠体重与肿瘤体积，对小鼠执行安乐死。
实验结果如图6A、图6B以及表6所示。
表6：各处理组对NOG小鼠皮下接种的LOVO肿瘤体积和TGI的影响
Figure PCTCN2021115308-appb-000009
Figure PCTCN2021115308-appb-000010
注：与PBS组相比，“***”P<0.001；“**”P<0.01。
结果显示，与PBS组相比，Ava-2GS-NSD能剂量依赖性地抑制肿瘤的生长，1mg/kg、4.8mg/kg和24mg/kg剂量下的TGI分别为16.7％、44.9％和55.6％；其中，4.8mg/kg和24mg/kg组肿瘤体积与PBS组相比具有显著性差异。肿瘤平均重量的结果，以及肿瘤生长抑制率与肿瘤体积的趋势基本一致(图6A与表6)。
每日观察各组小鼠状态未发现异常；每周两次称量小鼠体重，各组小鼠体重未发生明显降低，实验结束时各剂量组小鼠体重相比刚开始给药治疗时的体重，变化未超过13％，而且PBS组小鼠体重亦下降6％，考虑更可能是因为PBMC在小鼠体内重建导致的GvHD造成每组小鼠体重在实验后期均有所下降(见图6B)；试验结束时各组小鼠解剖观察肝脏、肾脏、肺脏等主要脏器亦未见明显病变，表明各组药物在本试验所采用的给药剂量下对小鼠未见明显毒性。
实验结果表明，Ava-2GS-NSD可剂量依赖性地抑制LOVO皮下移植瘤的生长，起效剂量为4.8mg/kg；3个给药剂量下(1mg/kg、4.8mg/kg和24mg/kg)对小鼠未见明显毒性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (33)

  1. 一种双特异性抗体，其包括：
    靶向VEGF的第一蛋白功能区，和
    靶向PD-L1的第二蛋白功能区；
    其中：
    所述第一蛋白功能区为抗VEGF的抗体或其抗原结合片段，或者，所述第一蛋白功能区包含VEGF受体或具有VEGF受体功能的片段；
    所述第二蛋白功能区为抗PD-L1的单域抗体。
  2. 根据权利要求1所述的双特异性抗体，其中，
    所述抗VEGF的抗体的重链可变区包含氨基酸序列如SEQ ID NO:21所示的HCDR1、氨基酸序列如SEQ ID NO:22所示的HCDR2以及氨基酸序列如SEQ ID NO:23所示的HCDR3；
    或者所述抗VEGF的抗体的重链可变区包含氨基酸序列如SEQ ID NO:27所示的HCDR1、氨基酸序列如SEQ ID NO:22所示的HCDR2以及氨基酸序列如SEQ ID NO:28所示的HCDR3；
    并且所述抗VEGF的抗体的轻链可变区包含氨基酸序列如SEQ ID NO:24所示的LCDR1、氨基酸序列如SEQ ID NO:25所示的LCDR2以及氨基酸序列如SEQ ID NO:26所示的LCDR3。
  3. 根据权利要求1至2中任一权利要求所述的双特异性抗体，其中，
    所述抗PD-L1单域抗体包含一个重链可变区，所述重链可变区包含氨基酸序列如SEQ ID NO:29所示的HCDR1、氨基酸序列如SEQ ID NO:30所示的HCDR2以及氨基酸序列如SEQ ID NO:31所示的HCDR3；
    优选地，所述抗PD-L1单域抗体的氨基酸序列如SEQ ID NO:5所示。
  4. 根据权利要求1至3中任一权利要求所述的双特异性抗体，其中，
    所述抗VEGF的抗体的重链可变区的氨基酸序列如SEQ ID NO:3所示，其轻链可变区的氨基酸序列如SEQ ID NO:9所示；或者
    所述抗VEGF的抗体的重链可变区的氨基酸序列如SEQ ID NO:13所示，其轻链可变区的氨基酸序列如SEQ ID NO:15所示。
  5. 根据权利要求1至4中任一权利要求所述的双特异性抗体，其中，
    所述抗VEGF的抗体或其抗原结合片段选自Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、单链抗体、人源化抗体、嵌合抗体或双抗体。
  6. 根据权利要求1至5中任一权利要求所述的双特异性抗体，其中，
    所述抗VEGF的抗体的恒定区来自人抗体；
    优选地，所述恒定区选自人IgG1、IgG2、IgG3或IgG4的恒定区。
  7. 根据权利要求1至6中任一权利要求所述的双特异性抗体，其中，
    所述抗VEGF的抗体的重链恒定区为人Ig gamma-1 chain C region或人Ig gamma-4chain C region，并且其轻链恒定区为人Ig kappa chain C region；
    优选地，所述抗VEGF的抗体的轻链恒定区的氨基酸序列如SEQ ID NO:10所示；
    优选地，所述抗VEGF的抗体的轻链的氨基酸序列如SEQ ID NO:8或SEQ ID NO:14所示。
  8. 根据权利要求1至7中任一权利要求所述的双特异性抗体，其中，
    所述抗VEGF的抗体的重链恒定区还包含按照EU编号系统的L234A突变和L235A突变；可选地，还包含G237A突变；
    优选地，所述抗VEGF的抗体的重链恒定区的氨基酸序列如SEQ ID NO:4所示。
  9. 根据权利要求1至8中任一权利要求所述的双特异性抗体，其中：
    所述VEGF为VEGF-A；
    所述VEGF受体为VEGFR1和/或VEGFR2。
  10. 根据权利要求1至9中任一权利要求所述的双特异性抗体，其中，所述单域抗体连接在第一蛋白功能区的C末端或N末端，例如，所述单域抗体为两条，每条单域抗体的一端分别连接在抗VEGF的抗体的两条重链的C末端或N末端，或者连接在VEGF 受体或具有VEGF受体功能的片段的C末端或N末端；
    并且所述单域抗体与第一蛋白功能区直接连接或者通过连接片段连接；
    优选地，所述连接片段的氨基酸序列独立地选自SEQ ID NO:6和SEQ ID NO:7；
    优选地，单域抗体连接在第一蛋白功能区得到的肽链的氨基酸序列如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:11或SEQ ID NO:12所示。
  11. 根据权利要求1至10中任一权利要求所述的双特异性抗体，其中，
    所述第一蛋白功能区包含：VEGF受体或具有VEGF受体功能的片段，以及IgG1的Fc段；
    优选地，所述具有VEGF受体功能的片段的氨基酸序列如SEQ ID NO:17所示；
    优选地，所述IgG1的Fc段包含按照EU编号系统的L234A突变和L235A突变；
    优选地，所述IgG1的Fc段的氨基酸序列如SEQ ID NO:18所示。
  12. 根据权利要求1至11中任一权利要求所述的双特异性抗体，其中，
    当所述第一蛋白功能区为VEGF受体或具有VEGF受体功能的片段时，所述双特异性抗体为二聚体；优选地，为氨基酸序列如SEQ ID NO:16或SEQ ID NO:19所示多肽的二聚体。
  13. 分离的核酸分子，其编码权利要求1至12中任一权利要求所述的双特异性抗体。
  14. 一种载体，其包含权利要求13所述的分离的核酸分子。
  15. 一种宿主细胞，其包含权利要求13所述的分离的核酸分子，或者权利要求14所述的载体。
  16. 制备权利要求1至12中任一权利要求所述的双特异性抗体的方法，其包括在合适的条件下培养权利要求15的宿主细胞，以及从细胞培养物中回收所述双特异性抗体的步骤。
  17. 偶联物，其包括双特异性抗体以及偶联部分，其中，所述双特异性抗体为权利 要求1至12中任一权利要求所述的双特异性抗体，所述偶联部分为可检测的标记；优选地，所述偶联部分为放射性同位素、荧光物质、发光物质、有色物质或酶。
  18. 试剂盒，其包含权利要求1至12中任一权利要求所述的双特异性抗体，或者包含权利要求17所述的偶联物；
    优选地，所述试剂盒还包含第二抗体，其能够特异性结合所述双特异性抗体；任选地，所述第二抗体还包括可检测的标记，例如放射性同位素、荧光物质、发光物质、有色物质或酶。
  19. 权利要求1至12中任一权利要求所述的双特异性抗体在制备试剂盒中的用途，所述试剂盒用于检测VEGF和/或PD-L1在样品中的存在或其水平。
  20. 一种药物组合物，其包含权利要求1至12中任一权利要求所述的双特异性抗体或者包含权利要求17所述的偶联物；可选地，其还包括药学上可接受的辅料。
  21. 权利要求1至12中任一权利要求所述的双特异性抗体或者权利要求17所述的偶联物在制备预防和/或治疗恶性肿瘤的药物中的用途；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
  22. 一种治疗和/或预防恶性肿瘤的方法，包括给予有需求的受试者以有效量的权利要求1至12中任一权利要求所述的双特异性抗体或者权利要求17所述的偶联物的步骤；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
  23. 根据权利要求1至12中任一权利要求所述的双特异性抗体或者权利要求17所述的偶联物，其用于治疗和/或预防恶性肿瘤；优选地，所述恶性肿瘤选自黑色素瘤、肝癌、胃癌、肾细胞癌、卵巢癌、结肠癌、乳腺癌、食道癌和头颈癌。
  24. 一种双特异性抗体，其特征在于，所述双特异性抗体包括：
    (a)抗PD-L1单域抗体；和
    (b)抗VEGF的抗体或元件。
  25. 如权利要求24所述的双特异性抗体，其特征在于，所述的双特异性抗体含有如式I和式II所示结构的多肽，
    A1-L1-CH-L2-B  (式I)
    A2-L3-CL       (式II)
    其中，
    A1是抗VEGF抗体的重链可变区VH；
    A2是抗VEGF抗体的轻链可变区VL；
    B是抗PD-L1单域抗体；
    L1、L2和L3各自独立地为无或连接元件；
    CH是人IgG重链恒定区CH(优选地为LALA突变型)；
    CL是人κ轻链恒定区CL；和
    “-”为肽键；
    并且其中，式I所示的多肽与式II所示的多肽通过二硫键作用而形成异源二聚体。
  26. 如权利要求24所述的双特异性抗体，其特征在于，所述双特异性抗体是具有如式III或式IV所示结构的多肽，
    A3-L4-Fc-L5-B  (式III)
    B-L6-Fc-L7-A3  (式IV)
    其中，
    A3是可以结合VEGF并阻断其活性的结构域；
    B是抗PD-L1单域抗体；
    L4、L5、L6和L7各自独立地为无或连接元件；
    Fc是人IgG的Fc区段(优选地为LALA突变型)；和
    “-”为肽键。
  27. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码如权利要求24所述的双特异性抗体。
  28. 一种载体，其特征在于，所述载体含有如权利要求27所述的多核苷酸。
  29. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求28所述的载体，或其基因组中整合有如权利要求27所述的多核苷酸；
    或者，所述的宿主细胞表达如权利要求24所述的双特异性抗体。
  30. 一种产生如权利要求24所述双特异性抗体的方法，其特征在于，包括步骤：
    (a)在合适的条件下，培养如权利要求29所述的宿主细胞，从而获得含所述双特异性抗体的培养物；和
    (b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的双特异性抗体。
  31. 一种免疫偶联物，其特征在于，所述免疫偶联物含有：
    (a)如权利要求24所述的双特异性抗体；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
  32. 如权利要求24所述的双特异性抗体，或如权利要求31所述的免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；其中，所述试剂、检测板或试剂盒用于：检测样品中PD-L1和/或VEGF；其中，所述药剂用于治疗或预防表达PD-L1(即PD-L1阳性)的肿瘤或是表达VEGF的肿瘤。
  33. 一种药物组合物，其特征在于，所述药物组合物含有：
    (i)如权利要求24所述的双特异性抗体，或如权利要求31所述的免疫偶联物；和
    (ii)药学上可接受的载体。
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