WO2021197359A1 - 一种构建多特异性抗体的平台 - Google Patents

一种构建多特异性抗体的平台 Download PDF

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WO2021197359A1
WO2021197359A1 PCT/CN2021/084198 CN2021084198W WO2021197359A1 WO 2021197359 A1 WO2021197359 A1 WO 2021197359A1 CN 2021084198 W CN2021084198 W CN 2021084198W WO 2021197359 A1 WO2021197359 A1 WO 2021197359A1
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antibody
polypeptide
amino acid
seq
serum albumin
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PCT/CN2021/084198
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English (en)
French (fr)
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刘晓林
曾竣玮
孙左宇
缪小牛
王涛
戴爽
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普米斯生物技术(珠海)有限公司
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Priority to EP21782157.8A priority Critical patent/EP4130046A4/en
Priority to JP2022559869A priority patent/JP2023519440A/ja
Priority to BR112022019633A priority patent/BR112022019633A2/pt
Priority to MX2022012238A priority patent/MX2022012238A/es
Priority to CA3178453A priority patent/CA3178453A1/en
Priority to US17/995,287 priority patent/US20230167200A1/en
Priority to AU2021247323A priority patent/AU2021247323A1/en
Priority to KR1020227036232A priority patent/KR20220161362A/ko
Publication of WO2021197359A1 publication Critical patent/WO2021197359A1/zh

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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
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    • A61K47/6879—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C12N2511/00—Cells for large scale production

Definitions

  • the invention belongs to the field of biomedicine or biopharmaceutical technology, and specifically relates to a platform for constructing multispecific antibodies.
  • bispecific antibodies In 1960, Nisonoff and his collaborators at the Roswell Park Memorial Institute in New York first proposed the original concept of bispecific antibodies. Afterwards, with the milestone progress in the fields of antibody engineering and antibody biology, the concept and technology of constructing bispecific antibodies continued to innovate. There are currently more than 100 bispecific antibody structural models, of which about a quarter have been developed into technology platforms and commercialized by biotechnology companies and pharmaceutical companies for new antibody therapies. Up to now, there are more than 20 different commercial technology platforms available for the development of bispecific antibodies, and more than 85 bispecific antibodies are in the clinical development stage.
  • bispecific antibody blinatumomab targeting CD3 and CD19, approved by the FDA in 2014 for the treatment of acute B lymphocytic leukemia
  • T cells Interest and investment Currently, more than 40 T cell redirecting bispecific antibodies are in clinical development for the treatment of hematological tumors and solid tumors.
  • inflammatory diseases have also been the focus of clinical development of bispecific antibodies.
  • Roche's emicizumab targeted coagulation factor X and factor IXa was approved by the FDA in November 2017, and hemophilia became the first non-cancer indication for bispecific antibodies.
  • bispecific antibodies have therapeutic potential in other diseases, such as diabetes, HIV infection, other viral and bacterial infections, Alzheimer's disease, and osteoporosis.
  • the dual-targeting feature of bispecific antibodies (that is, the ability to specifically target two antigens or two different epitopes of one antigen at the same time) makes it a great therapeutic prospect, but it turns the concept of bispecific antibodies into clinical practice The treatment is still challenging.
  • bispecific antibodies are a rapidly expanding and diverse group of molecules. Although compared with monoclonal antibodies, the complexity of the dual-targeting concept has increased, and will bring additional challenges at different stages of discovery and development, but bispecific antibodies provide exciting opportunities for the design and development of new drugs . From the field of disease, the current data show that the industry is more looking forward to the treatment of cancer with bispecific antibodies. The continued development of bispecific antibodies will have a lasting impact on the treatment of diseases such as cancer.
  • the purpose of the present invention is to provide a low-cost and high-efficiency method for constructing multispecific antibodies.
  • a method for constructing a multispecific antibody including the steps:
  • A1, A2, A3, and A4 are each independently an antibody or antigen fragment thereof that targets a target target, and the target antigens targeted by A1, A2, A3, and A4 may be the same or different;
  • L1, L2, L3 and L4 are each independently a non-or joint element
  • B1 and B2 are none, or B1 and B2 are respectively the VL and VH regions of antibodies targeting the same target target;
  • a disulfide bond can be formed between the CL region of the first polypeptide and the CH1 region of the second polypeptide, so that the antibody has a heterodimer form;
  • the CL region of the first polypeptide has the amino acid sequence shown in SEQ ID NO: 9, or has ⁇ 85% (preferably 90%, more preferably 90%, more than the sequence shown in SEQ ID NO: 9). Preferably 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of sequence identity.
  • the CH1 region of the second polypeptide has an amino acid sequence as shown in SEQ ID NO: 3, or has ⁇ 85% (preferably 90%, more preferably 90%) with the sequence shown in SEQ ID NO: 3 Preferably 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of sequence identity.
  • the target target is an antigen, a cell surface receptor, a ligand, or a cytokine.
  • the target target includes but not limited to: PD-1, TIGIT, human serum albumin, VEGF, PD-L1, PD-L2, or 41BB .
  • the target-targeted antibody or antigen fragment thereof is the VHH chain of the Nanobody, the variable region of the antibody heavy chain, and the variable region of the antibody light chain. , Antibody Fc fragment, or a combination thereof.
  • the target target-targeting antibody or antigen fragment thereof includes, but is not limited to: the VHH chain of an anti-TIGIT Nanobody and the VHH chain of an anti-HSA Nanobody , VHH chain of anti-PD-L1 Nanobody, VHH chain of anti-PD-L2 Nanobody, VH chain of anti-VEGF antibody, VL chain of anti-VEGF antibody, VH chain of anti-PD-1 antibody, or anti-PD-1 The VL chain of an antibody.
  • the VHH chain of the anti-TIGIT Nanobody has an amino acid sequence as shown in SEQ ID NO: 6, or has ⁇ 85% (preferably 90%, more preferably 90%) with the sequence shown in SEQ ID NO: 6 Preferably 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of sequence identity.
  • the VHH chain of the anti-HSA Nanobody has the amino acid sequence shown in SEQ ID NO: 5, or has ⁇ 85% (preferably 90%, more preferably 90%, more than the sequence shown in SEQ ID NO: 5). Preferably 95%, 96%, 97%, 98% or 99%) of the amino acid sequence of sequence identity.
  • the VHH chain of the anti-PD-L1 Nanobody has an amino acid sequence as shown in SEQ ID NO: 14 or 28, or ⁇ 85% of the sequence shown in SEQ ID NO: 14 or 28 ( Preferably 90%, more preferably 95%, 96%, 97%, 98% or 99%) sequence identity amino acid sequence.
  • the VHH chain of the anti-PD-L2 Nanobody has the amino acid sequence shown in SEQ ID NO: 19, or has ⁇ 85% (preferably 90%) with the sequence shown in SEQ ID NO: 19 , More preferably 95%, 96%, 97%, 98% or 99%) sequence identity.
  • the antibodies that target the same target include, but are not limited to: anti-PD-1 antibodies and anti-VEGF antibodies.
  • B1 and B2 are the VL region and VH region of the anti-PD-1 antibody, respectively; wherein, the VL region of the anti-PD-1 antibody has the amino acid sequence shown in SEQ ID NO: 8, or An amino acid sequence having ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98% or 99%) sequence identity with the sequence shown in SEQ ID NO: 8, and the anti-PD -1
  • the VH region of the antibody has the amino acid sequence shown in SEQ ID NO: 2, or ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%) with the sequence shown in SEQ ID NO: 2 , 98% or 99%) sequence identity.
  • B1 and B2 are the VL region and VH region of the anti-VEGF antibody, respectively; wherein, the VL region of the anti-VEGF antibody has the amino acid sequence shown in SEQ ID NO: 16, or is the same as SEQ ID NO.
  • the sequence shown in: 16 has an amino acid sequence of ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98% or 99%) sequence identity
  • the VH region of the anti-VEGF antibody Have the amino acid sequence shown in SEQ ID NO: 13, or have ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98% or 99%) with the sequence shown in SEQ ID NO: 13 ) Amino acid sequence of sequence identity.
  • sequence of the linker element is as shown in SEQ ID NO: 4 or 21, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 4 or 21. %, 96%, 97%, 98%, or 99%) sequence identity.
  • the first polypeptide has the amino acid sequence shown in SEQ ID NO:1, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO:1. %, 96%, 97%, 98%, or 99%); and the second polypeptide has an amino acid sequence as shown in SEQ ID NO: 7, or with SEQ ID NO: 7 Shown are amino acid sequences with ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the first polypeptide has the amino acid sequence shown in SEQ ID NO: 10, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 10 %, 96%, 97%, 98%, or 99%); and the second polypeptide has the amino acid sequence shown in SEQ ID NO: 11, or the amino acid sequence shown in SEQ ID NO: 11 Shown are amino acid sequences with ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the first polypeptide has the amino acid sequence shown in SEQ ID NO: 12, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 12 %, 96%, 97%, 98%, or 99%); and the second polypeptide has the amino acid sequence shown in SEQ ID NO: 15, or the amino acid sequence shown in SEQ ID NO: 15 Shown are amino acid sequences with ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the first polypeptide has the amino acid sequence shown in SEQ ID NO: 17, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 17 %, 96%, 97%, 98%, or 99%); and the second polypeptide has the amino acid sequence shown in SEQ ID NO: 18, or the amino acid sequence shown in SEQ ID NO: 18 Shown are amino acid sequences with ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the first polypeptide has the amino acid sequence shown in SEQ ID NO: 20, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 20. %, 96%, 97%, 98%, or 99%); and the second polypeptide has the amino acid sequence shown in SEQ ID NO: 18, or the amino acid sequence shown in SEQ ID NO: 18 Shown are amino acid sequences with ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the first polypeptide has the amino acid sequence shown in SEQ ID NO: 22, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 22. %, 96%, 97%, 98%, or 99%); and the second polypeptide has the amino acid sequence shown in SEQ ID NO: 23, or the amino acid sequence shown in SEQ ID NO: 23 Shown are amino acid sequences with ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the first polypeptide has the amino acid sequence shown in SEQ ID NO: 17, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 17 %, 96%, 97%, 98%, or 99%); and the second polypeptide has the amino acid sequence shown in SEQ ID NO: 24, or the amino acid sequence shown in SEQ ID NO: 24 Shown are amino acid sequences with ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the first polypeptide has the amino acid sequence shown in SEQ ID NO: 17, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 17 %, 96%, 97%, 98%, or 99%); and the second polypeptide has the amino acid sequence shown in SEQ ID NO: 25, or the amino acid sequence shown in SEQ ID NO: 25 Shown are amino acid sequences with ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • a multispecific antibody comprising a first polypeptide as shown in formula I from N-terminus to C-terminus and a first polypeptide as shown in formula II from N-terminus to C-terminus
  • the second polypeptide
  • A1, A2, A3, and A4 are each independently an antibody or antigen fragment thereof that targets a target target, and the target antigens targeted by A1, A2, A3, and A4 may be the same or different;
  • L1, L2, L3 and L4 are each independently a non-or joint element
  • Both B1 and B2 are none, or B1 and B2 are respectively the VL and VH regions of an antibody targeting the same target target;
  • disulfide bonds can be formed between the CL region of the first polypeptide and the CH1 region of the second polypeptide, so that the antibody has a heterodimer form.
  • a fusion protein in the third aspect of the present invention, includes the multispecific antibody as described in the second aspect of the present invention, and the first polypeptide in the multispecific antibody It has a structure as shown in formula III from N end to C end,
  • Fc is the Fc segment of an antibody, including the CH2 domain and the CH3 domain;
  • the fusion protein can form a homodimer through the disulfide bond between the Fc segments.
  • the first polypeptide has the amino acid sequence shown in SEQ ID NO: 27, or has ⁇ 85% (preferably 90%, more preferably 95%) with the sequence shown in SEQ ID NO: 27. %, 96%, 97%, 98%, or 99%); and the second polypeptide has the amino acid sequence shown in SEQ ID NO: 30, or the amino acid sequence shown in SEQ ID NO: 30 Shown are amino acid sequences with ⁇ 85% (preferably 90%, more preferably 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • an isolated polynucleotide combination in the fourth aspect of the present invention, includes a first nucleotide and a second nucleotide, and the first nucleotide encodes the first nucleotide of the present invention.
  • the first polypeptide of the multispecific antibody according to the second aspect or the fusion protein according to the third aspect of the present invention, and the second nucleotide encodes the second polypeptide.
  • a vector which contains the polynucleotide combination according to the fourth aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof; preferably, the expression vectors include viral vectors, such as Lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • a host cell contains the vector according to the fifth aspect of the present invention, or its genome integrates the polynucleotide combination according to the fourth aspect of the present invention;
  • the host cell expresses the multispecific antibody according to the second aspect of the invention or the fusion protein according to the third aspect of the invention.
  • the host cell includes a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.
  • step (b) Purifying and/or separating the culture obtained in step (a) to obtain the antibody.
  • the purification can be purified and separated by affinity chromatography to obtain the target antibody.
  • the purity of the target antibody after purification and separation is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.
  • an immunoconjugate the immunoconjugate containing:
  • a coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combination.
  • the radionuclide includes:
  • Diagnostic isotopes said diagnostic isotopes are selected from the following group: 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
  • Therapeutic isotope said therapeutic isotope is selected from the following group: 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.
  • the coupling moiety is a drug or a toxin.
  • the drug is a cytotoxic drug.
  • the cytotoxic drug is 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 Sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding reagents, DNA alkylating reagents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, auristatins, camptothecin (camptothecins), dokamycin/duocarmycins, etoposides, maytansines and maytansinoids (e.g.
  • DM1 and DM4 taxanes (etoposides) taxanes), benzodiazepines or benzodiazepine containing drugs (such as pyrrolo[1,4] benzodiazepines (PBDs), indoline benzodiazepines (Indolinobenzodiazepines) and oxazolidinobenzodiazepines (oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.
  • PBDs pyrrolo[1,4] benzodiazepines
  • Indolinobenzodiazepines Indolinobenzodiazepines
  • oxazolidinobenzodiazepines oxazolidinobenzodiazepines
  • the toxin is selected from the following group:
  • Otostatin for example, Otostatin E, Otostatin F, MMAE, and MMAF
  • chlortetracycline mettancilol
  • octoxin for example, Otostatin E, Otostatin F, MMAE, and MMAF
  • Lastatin doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, autumn Narcissus, dihydroxy anthracisin diketone, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, acacia toxin, acacia toxin A chain, capsule lotus root toxin A chain, ⁇ -Sarcina, white tree toxin, mitogellin, retstrictocin, phenomycin, enoxomycin
  • the coupling portion is a detectable label.
  • the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computed tomography technology) contrast agents, or can produce detectable Product enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, magnetic nanoparticles, prodrugs Activating enzymes (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutics (such as cisplatin).
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • the immunoconjugate contains: a multivalent (such as bivalent) multispecific antibody as described in the second aspect of the present invention.
  • the multivalent refers to the antibody as described in the second aspect of the present invention or the fusion as described in the third aspect of the present invention comprising multiple repeats in the amino acid sequence of the immunoconjugate protein.
  • the multispecific antibody according to the second aspect of the present invention there is provided the multispecific antibody according to the second aspect of the present invention, the fusion protein according to the third aspect of the present invention, or the immunoconjugate according to the eighth aspect of the present invention.
  • the reagent, detection plate or kit is used to detect whether the target target molecule exists in the sample;
  • the medicament is used to treat or prevent tumors expressing target target molecules.
  • the coupling part of the immunoconjugate is an isotope for diagnosis.
  • the reagent is one or more reagents selected from the following group: isotope tracer, contrast agent, flow detection reagent, cellular immunofluorescence detection reagent, nano magnetic particle and imaging agent .
  • the reagent for detecting the target target molecule in the sample is a contrast agent for detecting the target target molecule (in vivo).
  • the detection is in vivo detection or in vitro detection.
  • the detection includes flow cytometry and cellular immunofluorescence detection.
  • the tumor includes but is not limited to: acute myeloid leukemia, chronic myelogenous leukemia, multiple myelopathy, non-Hodgkin's lymphoma, colorectal cancer, breast cancer, colorectal cancer, gastric cancer , Liver cancer, leukemia, kidney tumors, lung cancer, small bowel cancer, bone cancer, prostate cancer, prostate cancer, cervical cancer, lymphoma, adrenal gland tumors, bladder tumors.
  • a pharmaceutical composition comprising: (i) the multispecific antibody according to the second aspect of the present invention, the fusion protein according to the third aspect of the present invention, or the present invention The immunoconjugate according to the eighth aspect of the invention; and (ii) a pharmaceutically acceptable carrier.
  • 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, levatinib, or pembrolizumab.
  • the pharmaceutical composition is used to treat tumors that express the target target molecule (that is, the target target molecule is positive).
  • the pharmaceutical composition is in the form of injection.
  • the pharmaceutical composition is used to prepare drugs for preventing and treating tumors.
  • a method of treating diseases comprising: administering the multispecific antibody according to the second aspect of the present invention, as described in the third aspect of the present invention, to a subject in need
  • the fusion protein of the present invention, the immunoconjugate according to the eighth aspect of the present invention, or the pharmaceutical composition according to the tenth aspect of the present invention comprising: administering the multispecific antibody according to the second aspect of the present invention, as described in the third aspect of the present invention, to a subject in need.
  • the subject includes mammals, preferably humans.
  • kits which contains the multispecific antibody according to the second aspect of the present invention, the fusion protein according to the third aspect of the present invention, and the third aspect of the present invention.
  • the instructions describe that the kit is used to non-invasively detect the expression of target target molecules in the test subject.
  • the instructions stated that the kit is used for the detection of tumors that express the target target molecule ie, the target target molecule is positive.
  • Figure 1 shows the schematic structure of three trispecific antibodies.
  • Figure 2 shows the results of the antigen co-binding ability of anti-PD-1/TIGIT/human serum albumin trispecific antibodies measured using the Octet system.
  • Figure 3 shows the results of the determination of the binding activity of the anti-PD-1/TIGIT/human serum albumin trispecific antibody to CHO-hPD-1 cells and CHO-hTIGIT cells.
  • Figure 4 shows the binding activity of anti-PD-1/TIGIT/human serum albumin trispecific antibodies to human serum albumin measured by ELISA.
  • Figure 5 shows the blocking effect of the anti-PD-1/TIGIT/human serum albumin trispecific antibody on the binding of human PD-L1 to human PD-1.
  • Figure 6 shows the blocking effect of the anti-PD-1/TIGIT/human serum albumin trispecific antibody on the binding of CD155 to TIGIT.
  • Figure 7 shows the results of the antigen co-binding ability of the anti-VEGF/PD-L1/human serum albumin trispecific antibody measured using the Octet system.
  • Figure 8 shows the results of the determination of the binding activity of anti-VEGF/PD-L1/human serum albumin trispecific antibodies to CHO-hPD-1 cells.
  • Figure 9 shows the binding activity of the anti-VEGF/PD-L1/human serum albumin trispecific antibody to human serum albumin measured by ELISA.
  • Figure 10 shows the binding activity of anti-VEGF/PD-L1/human serum albumin trispecific antibodies to human VEGF protein determined by ELISA.
  • Figure 11 shows a schematic diagram of the structure of three trispecific antibodies and one tetraspecific antibody.
  • Figure 12 shows the results of the determination of the binding activity of three PD-L1/PD-L2/human serum albumin trispecific antibodies to CHO-hPD-L1 cells and CHO-hPD-L2 cells, as well as anti-PD-L1/PD- L2/TIGIT/human serum albumin tetra-specific antibody and CHO-hPD-L1 cell, CHO-hPD-L2 cell and CHO-hTIGIT cell binding activity assay result.
  • Figure 13 shows three anti-PD-L1/PD-L2/human serum albumin trispecific antibodies and anti-PD-L1/PD-L2/TIGIT/human serum albumin tetraspecific antibodies determined by ELISA and human serum Albumin binding activity.
  • Figure 14 shows three anti-PD-L1/PD-L2/human serum albumin trispecific antibodies and anti-PD-L1/PD-L2/TIGIT/human serum albumin tetraspecific antibodies against human PD-L1 or PD -The blocking effect of L2 binding to human PD-1.
  • Figure 15 shows three anti-PD-L1/PD-L2/human serum albumin trispecific antibodies and anti-PD-L1/PD-L2/TIGIT/human serum albumin tetraspecific antibodies simultaneously block PD-L1/PD-L2/TIGIT/human serum albumin in vitro Results of L1/PD-1 and PD-L2/PD-1 signaling pathways.
  • Figure 16 shows a schematic diagram of the structure of a trispecific antibody.
  • Figure 17 shows the results of measuring the binding activity of the PD-L1/41BB/human serum albumin trispecific antibody to CHO-hPD-L1 cells and CHO-41BB cells.
  • Figure 18 shows the binding activity of the PD-L1/41BB/human serum albumin trispecific antibody to human serum albumin measured by ELISA.
  • Figure 19 shows the ability of the anti-PD-L1/41BB/human serum albumin trispecific antibody to bridge cells expressing PD-L1/41BB.
  • Figure 20 shows the blocking effect of the anti-PD-L1/41BB/human serum albumin trispecific antibody on the binding of human PD-L1 to human PD-1.
  • Figure 21 shows a schematic diagram of the structure of the PD-L1/PD-L2 bispecific antibody Fc fusion protein as described in Example 5.
  • Figure 22 shows the results of measuring the binding activity of the anti-PD-L1/PD-L2 bispecific antibody Fc fusion protein to CHO-hPD-L1 cells and CHO-hPD-L2 cells.
  • multispecific antibodies After extensive and in-depth research and extensive screening, the inventors developed a method for constructing multispecific antibodies for the first time. Experiments have proved that by connecting antigen-binding fragments (such as antibody variable regions, single domain antibodies, or Fc, etc.) with natural antibody CL and CH1 fragments, stable heterodimers can be formed.
  • the multispecific antibody constructed by the method of the present application can simultaneously bind to different targets and maintain the binding activity of the original antibody; it is effective when the target is a membrane surface receptor or a target in a solution; it has anti-multiple targets at the same time Biological activity; can be linked to single domain antibodies or normal antibodies or Fc fragments. Therefore, the method and the provided platform of the present invention have huge application prospects. The present invention has been completed on this basis.
  • platform for constructing multispecific antibodies and “construction method of the present invention” are used interchangeably, and refer to the method for constructing multispecific antibodies according to the first aspect of the present invention, wherein CL and CH1
  • the heterodimer formed by the disulfide bond is the core structure, and the antibodies or antigen fragments targeting different target sites are fused.
  • Bispecific/multispecific antibodies are artificial proteins composed of fragments of two or more different monoclonal antibodies, which can bind to two or more different types of antigens.
  • engineered BsAbs simultaneously bind to cytotoxic cells and targets to be killed (such as tumor cells).
  • At least three types of bispecific antibodies have been proposed or tested, including trifunctional antibodies, chemically linked Fabs, and bispecific T cell adapters.
  • trifunctional antibody In order to overcome the manufacturing difficulties, the first-generation BsMAb called trifunctional antibody has been developed. It consists of two heavy chains and two light chains, each from a different antibody. These two Fab regions are directed against two antigens. The Fc region is composed of two heavy chains and forms the third binding site, hence the name.
  • bispecific antibodies have been designed to solve certain problems, such as short half-life, immunogenicity, and side effects caused by cytokine release. They include: chemically linked Fab consisting only of Fab regions, and various types of bivalent and trivalent single-chain variable regions (scFv) (a fusion protein that mimics the variable domains of two antibodies).
  • scFv bivalent and trivalent single-chain variable regions
  • the newly developed form is bispecific T cell adaptor (BiTE) and tetrafunctional antibody.
  • an antibody is a type of immunoglobulin that can specifically bind to an antigen. It consists of four polypeptide chains. The two chains with the larger molecular weight are called heavy chains (H chain), and the two chains with the smaller molecular weight are called heavy chains. It is called the light chain (Light chain, L chain).
  • H chain heavy chains
  • L chain light chain
  • the amino acid composition of the two H chains and the two L chains is exactly the same, while the development of bispecific antibodies is by co-expression of two different H chains and two different L chains.
  • Obtaining functional bispecific antibodies from the 10 possible H2L2 recombination mixtures is one of the initial challenges of bispecific antibody development, which is often referred to as chain-related problems. In the past few decades, researchers have developed many strategies to solve this problem.
  • Fragment-based bispecific antibodies simply combine multiple antibody fragments in one molecule without Fc region, avoiding chain-related problems, and have the advantage of high yield and low cost; the disadvantage is that the half-life is relatively short.
  • fragment-based bispecific antibodies may suffer from stability and polymerization issues.
  • the bispecific antibody of the symmetrical pattern retains the Fc region, which is closer to the natural antibody, but is different in size and structure. These differences may negatively affect the beneficial properties (such as stability and solubility) associated with natural antibodies, which may impair the physicochemical and/or pharmacokinetic properties of these bispecific antibodies.
  • bispecific antibodies with asymmetric patterns are very similar to natural antibodies and are considered to have the lowest immunogenic potential.
  • complex engineering that may be involved in solving chain-related problems may offset this advantage of some asymmetric bispecific antibodies.
  • a class of multispecific antibodies based on the heterodimer form is provided.
  • multispecific antibody of the present invention As used herein, the terms “multispecific antibody of the present invention”, “polyantibody of the present invention”, and “antibody of the present invention” are used interchangeably, and all refer to those constructed using the method for constructing multispecific antibodies provided by the present invention. Multispecific antibodies.
  • the antibody provided by the present invention comprises a first polypeptide as shown in formula I from N-terminus to C-terminus and a second polypeptide as shown in formula II from N-terminus to C-terminus,
  • A1, A2, A3, and A4 are each independently an antibody or antigen fragment thereof that targets a target target, and the target antigens targeted by A1, A2, A3, and A4 may be the same or different;
  • L1, L2, L3 and L4 are each independently a non-or joint element
  • B1 and B2 are none, or B1 and B2 are respectively the VL and VH regions of antibodies targeting the same target target;
  • disulfide bonds can be formed between the CL region of the first polypeptide and the CH1 region of the second polypeptide, so that the antibody has a heterodimer form.
  • the CL region in formula I has an amino acid sequence as shown in SEQ ID NO:9
  • the CH1 region in formula II has an amino acid sequence as shown in SEQ ID NO:3.
  • the multispecific antibody is an anti-PD-1/TIGIT/human serum albumin trispecific antibody, wherein the first polypeptide has an amino acid sequence as shown in SEQ ID NO:1 And the second polypeptide has an amino acid sequence as shown in SEQ ID NO: 7.
  • the multispecific antibody is an anti-PD-1/TIGIT/human serum albumin trispecific antibody, wherein the first polypeptide has an amino acid sequence as shown in SEQ ID NO: 10 And the second polypeptide has an amino acid sequence as shown in SEQ ID NO: 11.
  • the multispecific antibody is an anti-VEGF/PD-L1/human serum albumin trispecific antibody, wherein the first polypeptide has the amino acid sequence shown in SEQ ID NO: 12 And the second polypeptide has an amino acid sequence as shown in SEQ ID NO: 15.
  • the multispecific antibody is an anti-PD-L1/PD-L2/human serum albumin trispecific antibody, wherein, the first polypeptide has the sequence shown in SEQ ID NO: 17 Amino acid sequence, and the second polypeptide has the amino acid sequence shown in SEQ ID NO: 18.
  • the multispecific antibody is an anti-PD-L1/PD-L2/human serum albumin trispecific antibody, wherein, the first polypeptide has the sequence shown in SEQ ID NO: 20 Amino acid sequence, and the second polypeptide has the amino acid sequence shown in SEQ ID NO: 18.
  • the multispecific antibody is an anti-PD-L1/PD-L2/human serum albumin trispecific antibody, wherein, the first polypeptide has the sequence shown in SEQ ID NO: 22 Amino acid sequence, and the second polypeptide has the amino acid sequence shown in SEQ ID NO:23.
  • the multispecific antibody is an anti-PD-L1/PD-L2/TIGIT/human serum albumin tetra-specific antibody, wherein the first polypeptide has a tetraspecific antibody as shown in SEQ ID NO: 17. And the second polypeptide has the amino acid sequence shown in SEQ ID NO: 24.
  • the multispecific antibody is an anti-PD-L1/41BB/human serum albumin trispecific antibody, wherein the first polypeptide has the amino acid sequence shown in SEQ ID NO: 17 And the second polypeptide has an amino acid sequence as shown in SEQ ID NO: 25.
  • the present invention provides a fusion protein.
  • the fusion protein is in the multispecific antibody of the present invention.
  • the Fc segment is fused to the C-terminus of the first polypeptide, so that the multispecific Antibodies can form a more stable homodimer through homodimerization caused by disulfide bonds between Fc segments.
  • the fusion protein is an anti-PD-L1/PD-L2 bispecific antibody, wherein the first polypeptide has an amino acid sequence as shown in SEQ ID NO: 27, and the second polypeptide It has an amino acid sequence as shown in SEQ ID NO: 30.
  • single domain antibody As used herein, the terms “single domain antibody”, “Nanobody VHH”, and “Nanobody” have the same meaning and refer to cloning the variable region of an antibody heavy chain to construct a Nanobody consisting of only one heavy chain variable region ( VHH), it is the smallest antigen-binding fragment with complete functions. Usually, after obtaining an antibody with naturally missing light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.
  • VHH single domain antibody
  • variable means that certain parts of the variable region of an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a -folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partially folded structure.
  • the CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
  • FR framework region
  • the light chain and heavy chain of an immunoglobulin each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • the light chain variable domain can therefore 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 therefore be expressed 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 basically the same as the naturally-occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%. , 97%, 98% or 99%.
  • human framework region is substantially the same (about 85% or more, specifically 90%, 95%, 97%, 99% or 100%) framework region of a naturally occurring human antibody. .
  • affinity is theoretically defined by the balanced association between the intact antibody and the antigen.
  • the affinity of the double antibody of the present invention can be evaluated or determined by the KD value (dissociation constant) (or other measurement methods), such as Bio-layer Interferometry (BLI), which is measured and determined by the FortebioRed96 instrument.
  • KD value dissociation constant
  • BLI Bio-layer Interferometry
  • linker refers to one or more amino acid residues inserted into the antibody of the present invention to provide sufficient mobility for each domain or region.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments of the present invention to form ⁇ conjugate.
  • the present invention also includes cell surface markers or antigens that bind to the multispecific antibody or fragments thereof.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to the multispecific antibody provided by the present invention, which may or may not contain a starting amino acid. Thionine.
  • the invention also provides other proteins or fusion expression products with the antibodies of the invention.
  • the present invention includes any protein or protein conjugate and fusion expression product (ie, immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is compatible with the heavy chain of the antibody of the present invention.
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homology.
  • the present invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to polypeptides that substantially retain the same biological function or activity as the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogues of the present invention may be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide with a substitution group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that prolongs the half-life of the polypeptide, such as Polyethylene glycol) fused to the polypeptide, or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or proprotein sequence, or with Fusion protein formed by 6His tag
  • the antibody of the present invention also includes variant forms of the first polypeptide or the second polypeptide that have the same function as the antibody of the present invention.
  • variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • amino acids with similar or similar properties are substituted
  • the function of the protein is usually not changed.
  • adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • the variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, and DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions.
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • the present invention also provides other polypeptides, such as fusion proteins containing single domain antibodies or fragments thereof.
  • the present invention also includes fragments of single domain antibodies of the present invention.
  • the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.
  • “conservative variants of the antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 compared to the amino acid sequence of the antibody of the present invention. Amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • substitutions 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
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequence) and non-coding sequences of the mature polypeptide .
  • 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 with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • 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; or (2) adding during hybridization There are denaturants, 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, and more Fortunately, hybridization occurs when more than 95%. Moreover, 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 fragments can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • One feasible method is to use artificial synthesis to synthesize relevant sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • the DNA sequence encoding the protein (or fragments, or derivatives thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: 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, and 293 cells.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the steps used are well known in the art.
  • Another method is to use MgCl 2 .
  • the transformation can also be carried out by electroporation.
  • the host is a eukaryote
  • the following DNA transfection methods can be selected: 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 polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. 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 a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the present invention can be used alone, or can be combined or coupled with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of these substances.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or those capable of producing detectable products Enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody 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 Particles; 6. Liposomes; 7. Nano magnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutics ( For example, cisplatin) or any form of nanoparticles.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • a method for constructing a multispecific antibody which includes the steps:
  • A1, A2, A3, and A4 are each independently an antibody or antigenic fragment thereof that targets a target target, and the target antigens targeted by A1, A2, A3, and A4 may be the same or different;
  • L1, L2 , L3 and L4 are each independently a null or linker element;
  • B1 and B2 are both null, or B1 and B2 are respectively the VL region and VH region of an antibody targeting the same target target; and the CL of the first polypeptide A disulfide bond can be formed between the CH1 region and the CH1 region of the second polypeptide, so that the antibody has the form of a heterodimer;
  • the CL region of the first polypeptide has an amino acid sequence as shown in SEQ ID NO: 9
  • the CH1 region of the second polypeptide has an amino acid sequence as shown in SEQ ID NO: 3. Both Disulfide bonds can be formed between them.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the aforementioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH value can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition 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 target target molecules, and thus can be used to treat corresponding diseases.
  • other therapeutic agents can also be used at the same time.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable amount Accepted carrier or excipient.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation 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. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the present invention can also be used together with other therapeutic agents
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases, does not exceed about 50 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • the bi/multispecific antibody structure of the present invention can simultaneously bind to different targets and maintain the binding activity of the original antibody.
  • the bi/multispecific antibody structure of the present invention is effective when the target is a membrane surface receptor or a target in a solution.
  • the bi/multispecific antibody structure of the present invention has biological activity against multiple targets at the same time.
  • the bi/multispecific antibody structure of the present invention can be connected to single domain antibodies or normal antibodies or Fc fragments.
  • the bi/multispecific antibody of the present invention is an antibody or fusion protein constructed with the dimer of CH1-CL as the center.
  • the present invention also provides a multispecific antibody, which includes Fc Fragments can significantly increase the half-life of the protein and simplify the purification process.
  • Example 1 Anti-PD-1/TIGIT/human serum albumin trispecific antibody
  • Bi-70-71 consists of two polypeptide chains, and its structure is shown in Figure 1A.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO:1, which includes the VH amino acid sequence (SEQ ID NO: 2) derived from the anti-PD-1 antibody Pembrolizumab (patent number: US8354509), and the VH amino acid sequence C The end is directly connected to the CH1 amino acid sequence (SEQ ID NO: 3) derived from human IgG1.
  • the C-terminus of the Nanobody ALB8 with SEQ ID NO: 5 anti-human serum albumin (Patent No.: WO2004/041865) was linked to Pembrolizumab's C-terminal through a flexible peptide of 11 amino acid residues (GGGGSGGGGSG) (SEQ ID NO: 4)
  • the N-terminus of the variable region of the heavy chain was linked to the C-terminus of CH1 through a flexible peptide of 11 amino acid residues (GGGGSGGGGSG), thereby obtaining peptide chain #1.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 7, which includes the VL amino acid sequence (SEQ ID NO: 8) derived from the anti-PD-1 antibody Pembrolizumab, the C-terminus of the VL amino acid sequence and the human kappa light chain
  • the constant region (CL) amino acid sequence (SEQ ID NO: 9) is directly connected, thereby obtaining peptide chain #2.
  • Bi-72-73 also consists of two polypeptide chains, and its structure is shown in Figure 1B.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 10, which includes the VH amino acid sequence (SEQ ID NO: 2) derived from the anti-PD-1 antibody Pembrolizumab, the C-terminus of the VH amino acid sequence and the amino acid sequence derived from human IgG1 The CH1 amino acid sequence (SEQ ID NO: 3) is directly connected.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 11, which includes the VL amino acid sequence (SEQ ID NO: 8) derived from the anti-PD-1 antibody Pembrolizumab, the C-terminus of the VL amino acid sequence and the human kappa light chain
  • the constant region (CL) amino acid sequence (SEQ ID NO: 9) is directly connected.
  • the C-terminus of the anti-human serum albumin Nanobody ALB8 (SEQ ID NO: 5) is connected to the N-terminus of the light chain variable region of Pembrolizumab through a flexible peptide of 11 amino acid residues (GGGGSGGGGSG) (SEQ ID NO: 4) ,
  • the N-terminus of the anti-TIGIT Nanobody E-Ye-11 (SEQ ID NO: 6) is connected to the C-terminus of CL through a flexible peptide of 11 amino acid residues GGGGSGGGGSG (SEQ ID NO: 4), thereby obtaining the peptide Chain #2.
  • nucleotide sequence of each two chains of the anti-PD-1/TIGIT/human serum albumin trispecific antibody Bi-70-71 and Bi-72-73 constructed in Example 1.1 will be encoded Linked into the commercially available eukaryotic expression vector pCDNA3.1(+) through multiple cloning sites, and expressed and purified in eukaryotic cells to obtain trispecific antibodies Bi-70-71 and Bi-72-73. Specific operations as follows.
  • the gene sequences encoding the two chains of Bi-70-71 and Bi-72-73 were synthesized by Jin Weizhi Company. Using homologous recombinase (purchased from Vazyme) and EcoR I/Not I double enzyme digestion linearized pCDNA3.1 vector, the process is in accordance with the product specification. The homologous recombination products were transformed into Top10 competent cells, coated with ampicillin resistant plates, incubated overnight at 37°C, and single clones were picked for sequencing.
  • the ExpiCHO TM expression system kit (Thermo) was used to transfer the plasmid into Expi-CHO cells.
  • the transfection method was in accordance with the commercial instructions.
  • the supernatant was collected and purified using a KappaSelect (GE) affinity chromatography column.
  • the specific method is as follows: the sample is filtered through a 0.2um sterile syringe filter PES with a syringe. Equilibrate the column with 5 column volumes of equilibration buffer (20mM PB+0.15M NaCl, pH 7.4) until the conductivity and pH of the effluent do not change. Load the sample at a flow rate of 0.5 ml/min.
  • the purity of the obtained protein was detected by HPLC.
  • the HPLC method is as follows, mobile phase: 150 mM Na 2 HPO 4 ⁇ 12H 2 O, pH 7.0. Chromatographic conditions: detection wavelength: 280nm, column temperature: 25°C, flow rate: 0.35ml/min, detection time: 20min, Zenix-C SEC-300 chromatographic column (SEPAX 4.6 ⁇ 300mm, 3 ⁇ m). SEC results showed that the purity of the bispecific antibody Bi-70-71 was 99.14%, and the purity of Bi-72-73 was 98.27%.
  • the Octet system (manufactured by ForteBio) was used to determine whether the two exemplary anti-PD-1/TIGIT/human serum albumin trispecific antibodies Bi-70-71 and Bi-72-73 of the present invention can be determined by kinetic binding assay. Combines PD-1, TIGIT and human serum albumin at the same time. Half an hour before the start of the experiment, the SA sensor (Pall) was soaked in SD buffer (PBS 1 ⁇ , BSA 0.1%, Tween 20 0.05%) and equilibrated at room temperature.
  • 96-well black polystyrene half-volume microplate To the wells of 96-well black polystyrene half-volume microplate (Greiner), respectively add 100 ⁇ l of SD buffer as a blank control (for background subtraction), 100 ⁇ l and 100nM purified bispecific antibodies Bi-70-71 and Bi-70-71. 72-73, 100 ⁇ l of the biotinylated human PD-1 (100 nM) (Acrobiosystems), human TIGIT (100 nM) (Acrobiosystems) and human serum albumin (Acrobiosystems) solution diluted in SD buffer as an antigen.
  • the SA sensor was immersed in the well containing the biotinylated human PD-1 solution, and the sample was immersed for 60 seconds at room temperature.
  • the anti-PD-1/TIGIT/human serum albumin trispecific antibody Bi-70-71 ( Figure 2A) and Bi-72-73 ( Figure 2B) of the present invention can simultaneously and Human PD-1, human TIGIT and human serum albumin protein binding.
  • CHO cells (CHO-hPD-L1 Cells, CHO-hTIGIT cells). Adjust the cell density of the expanded CHO-hPD-L1/CHO-hTIGIT cells to 2 ⁇ 10 6 cells/ml, add 100 ⁇ l/well to a 96-well flow plate, and centrifuge for use. Dilute the purified trispecific antibody with PBS, start at 400nM and dilute 3 times at 12 points, add 100 ⁇ l/well of the above diluted sample to the above 96-well flow plate with cells, incubate at 4°C for 30 minutes, PBS Wash twice.
  • the anti-PD-1/TIGIT/human serum albumin trispecific antibody of the present invention has binding activity to CHO-hPD-1 cells and CHO-hTIGIT cells. .
  • the human serum albumin (acrobiosystems) was diluted with ELISA coating solution, then added to the ELISA plate, and coated overnight at 4°C. The coating solution was discarded, 250 ⁇ l/well was added with PBST and washed 3 times, and then blocked with 5% BSA for 1 hour at room temperature for later use. The purified antibody and the control antibody were serially diluted and added to the blocked ELISA plate, and incubated at room temperature for 2 hours.
  • the anti-PD-1/TIGIT/human serum albumin trispecific antibody of the present invention binds to human serum albumin at the ELISA level.
  • biotinylated human PD- L1 protein purchased from AcroBiosystems
  • the anti-PD-1/TIGIT/human serum albumin trispecific antibody of the present invention can all block the binding of PD-L1 and PD-1.
  • CHO-hTIGIT cells Adjust the cell density of CHO-hTIGIT cells to 2 ⁇ 10 6 cells/ml, add 100 ⁇ l/well to a 96-well flow plate, and centrifuge for use. Dilute the purified antibody and control antibody samples with PBS, start at 400 nM and start 3-fold dilution for a total of 12 points. Add 60 ⁇ l/well of the above-mentioned diluted sample to a 96-well sample dilution plate, and at the same time add 60 ⁇ l/well to human CD155-mFc protein (purchased from AcroBiosystems), the final concentration is 2 ⁇ g/ml, and the sample is incubated at 4°C for 30 minutes.
  • human CD155-mFc protein purchased from AcroBiosystems
  • the anti-PD-1/TIGIT/human serum albumin trispecific antibody of the present invention can all block the binding of CD155 and TIGIT.
  • the bispecific or multispecific antibody formed by connecting the VH and VL domains above the CH1-CL domain and connecting one or more nanobody domains at the same time can effectively maintain the binding activity of the parent antibody.
  • the VH-VL combination used in this example is derived from Pembrolizumab, which is a domain that binds to the cell surface antigen human PD-1.
  • an anti-VEGF/PD-L1/human serum albumin trispecific antibody was constructed , Named Bi-74-76, and its structure is shown in Figure 1C. It consists of two polypeptide chains.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 12, which contains the anti-VEGF antibody Bevacizumab ( Patent number: WO1998045332) VH amino acid sequence (SEQ ID NO: 13), the C-terminus of the VH amino acid sequence is directly connected to the CH1 amino acid sequence derived from human IgG1 (SEQ ID NO: 3); the anti-human serum albumin
  • the C-terminus of Nanobody ALB8 (SEQ ID NO: 5) is connected to the N-terminus of Bevacizumab heavy chain variable region through a flexible peptide of 11 amino acid residues (GGGGSGGGGSG) (SEQ ID NO: 4).
  • Nanobody C-Ye-8-5 Patent Application No: 2019108631090
  • SEQ ID NO: 14 The N-terminal of Nanobody C-Ye-8-5 (Patent Application No: 2019108631090) (SEQ ID NO: 14) is connected to the C of CH1 through a flexible peptide of 11 amino acid residues (GGGGSGGGGSG) (SEQ ID NO: 4) End, thereby obtaining peptide chain #1.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 15, which includes the VL amino acid sequence (SEQ ID NO: 16) derived from the anti-VEGF antibody Bevacizumab, the C-terminus of the VL amino acid sequence is derived from the human kappa light chain
  • the constant region (CL) amino acid sequence (SEQ ID NO: 9) is directly connected; the N-terminus of the anti-human PD-L1 Nanobody C-Ye-8-5 (SEQ ID NO: 14) is connected through 11 amino acid residues ( The flexible peptide of GGGGSGGGGSG) (SEQ ID NO: 4) is connected to the C terminal of CL, thereby obtaining peptide chain #2.
  • the two nucleotide sequences encoding the anti-VEGF/PD-L1/human serum albumin trispecific antibody Bi-74-76 constructed in Example 2.1 were all connected to the market through a multi-cloning site.
  • the eukaryotic expression vector pCDNA3.1(+) was expressed and purified in eukaryotic cells, and the trispecific antibody Bi-74-76 was obtained.
  • the expression plasmid construction, cell transfection, protein purification and HPLC purity detection methods are the same as in Example 1.2. SEC results showed that the purity of the bispecific antibody Bi-74-76 was 95.89%.
  • the Octet system (manufactured by ForteBio) was used to determine whether the two exemplary anti-VEGF/PD-L1/human serum albumin trispecific antibodies Bi-74-76 of the present invention can simultaneously bind to human PD-L1 by a kinetic binding assay method , VEGF and human serum albumin.
  • the SA sensor (Pall) was soaked in SD buffer (PBS 1 ⁇ , BSA 0.1%, Tween 20 0.05%) and equilibrated at room temperature.
  • the anti-VEGF/PD-L1/human serum albumin trispecific antibody Bi-74-76 of the present invention can simultaneously interact with human PD-L1, human VEGF and Human serum albumin protein binding.
  • the detection method of anti-VEGF/PD-L1/human serum albumin trispecific antibody and CHO-hPD-L1 cell binding activity is the same as in Example 1.4.
  • the anti-VEGF/PD-L1/human serum albumin trispecific antibody of the present invention can bind to CHO-hPD-L1 cells.
  • the human VEGF (acrobiosystems) protein was diluted with ELISA coating solution and added to the ELISA plate, and coated overnight at 4°C. The coating solution was discarded, 250 ⁇ l/well was added with PBST and washed 3 times, and then blocked with 5% BSA for 1 hour at room temperature for later use.
  • the purified antibody Bi-074-076 antibody was serially diluted and added to the blocked ELISA plate, and incubated for 2 hours at room temperature.
  • the purified antibody Bi-74-76 of the present invention can bind to human VEGF protein at the ELISA level ( Figure 10).
  • the bispecific or multispecific antibody formed by connecting the VH and VL domains at the top of the CH1-CL structure and connecting one or more nanobody domains at the same time can effectively maintain the binding activity of the parent antibody.
  • the VH-VL combination used in this example is from Bevacizumab, which is a domain that binds to the free antigen VEGF in the blood.
  • Example 3 Anti-PD-L1/PD-L2/human serum albumin trispecific antibody or anti-PD-L1/PD-L2/TIGIT/human serum albumin tetraspecific antibody
  • the inventors constructed a set of anti-PD-L1/PD-L2/human serum albumin trispecific Antibody or anti-PD-L1/PD-L2/TIGIT/human serum albumin tetra-specific antibody.
  • Bi-78-79 whose structure is shown in Figure 11A, is composed of two polypeptide chains.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 17, and it contains the anti-PD-L1 Nanobody C-Ye -18-5 (SEQ ID NO: 14), the C-terminus of the Nanobody amino acid sequence is directly connected to the CH1 amino acid sequence derived from human IgG1 (SEQ ID NO: 3); the Nanobody ALB8 ( The C-terminus of SEQ ID NO: 5) is connected to the C-terminus of the CH1 region through a flexible peptide of 11 amino acid residues (GGGGSGGGGSG) (SEQ ID NO: 4), thereby obtaining peptide chain #1.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 18, which includes the anti-PD-L2 Nanobody D-Ye-22 amino acid sequence (SEQ ID NO: 19), and the C-terminus of the Nanobody amino acid sequence is directly connected to human ⁇ The light chain constant region (CL) amino acid sequence (SEQ ID NO: 9), thereby obtaining peptide chain #2.
  • peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 20, which includes the anti-PD-L1 nanobody SEQ ID NO :2, the C-terminus of the Nanobody amino acid sequence is directly connected to the CH1 amino acid sequence SEQ ID NO: 6 derived from human IgG1; it includes the anti-PD-L1 Nanobody C-Ye-18-5 (SEQ ID NO: 14 ), the C-terminus of the Nanobody amino acid sequence is directly connected to the CH1 amino acid sequence (SEQ ID NO: 3) derived from human IgG1; the C-terminus of the anti-human serum albumin Nanobody ALB8 (SEQ ID NO: 5) is passed through A flexible peptide of 5 amino acid residues (DKTHT) (SEQ ID NO: 21) is connected to the C-terminus of the CH1 region, thereby obtaining peptide chain #1.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 20, which includes the anti-PD-L1 nanobody SEQ ID NO :2, the
  • Bi-81-82 whose structure is shown in Figure 1A, is composed of two polypeptide chains.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 22, and it contains the anti-PD-L1 Nanobody C-Ye -18-5 (SEQ ID NO: 14), the C-terminus of the Nanobody amino acid sequence is through a flexible peptide chain of 11 amino acids (GGGGSGGGGSG) (SEQ ID NO: 4) and the amino acid sequence of CH1 derived from human IgG1 (SEQ ID NO: 3) connection, connect the C-terminus of the anti-human serum albumin nanobody ALB8 (SEQ ID NO: 5) to the flexible peptide of 11 amino acid residues (GGGGSGGGGSG) (SEQ ID NO: 4) The C-terminus of the CH1 region, thereby obtaining peptide chain #1.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 23, which includes the anti-PD-L2 Nanobody D-Ye-22 amino acid sequence (SEQ ID NO: 19).
  • the C-terminal of the Nanobody amino acid sequence contains 11 A flexible peptide chain of four amino acids (GGGGSGGGGSG) (SEQ ID NO: 4) and a human kappa light chain constant region (CL) amino acid sequence (SEQ ID NO: 9), thereby obtaining peptide chain #2.
  • an anti-PD-L1/PD-L2/TIGIT/human serum albumin tetra-specific antibody was constructed, named Bi-79-83, and its structure diagram is shown in Figure 1A, consisting of two Polypeptide chain composition, peptide chain #1 has the amino acid sequence shown in SEQ ID NO:17.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 24, which includes the amino acid sequence of anti-PD-L2 Nanobody D-Ye-22 (SEQ ID NO: 19), the C-terminus of the Nanobody amino acid sequence and the human kappa light
  • the chain constant region (CL) amino acid sequence (SEQ ID NO: 9) is directly connected; the N-terminus of the anti-TIGIT Nanobody E-Ye-11 passes through the flexibility of 11 amino acid residues (GGGGSGGGGSG) (SEQ ID NO: 4)
  • the peptide is linked to the C-terminus of the CL region, thereby obtaining peptide chain #2.
  • the anti-PD-L1/PD-L2/human serum albumin trispecific antibodies Bi-78-79, Bi-78-80 and Bi-81-82 and the anti-PD-L1/PD-L2/human serum albumin constructed in Example 3.1 will be encoded.
  • the nucleotide sequences of the two chains of the PD-L1/PD-L2/TIGIT/human serum albumin tetra-specific antibody Bi-79-83 are connected to the commercially available eukaryotic expression vector pCDNA3.1 ( +), expression and purification in eukaryotic cells.
  • the expression plasmid construction and protein expression and purification methods are the same as in Example 1.2.
  • Human PD-L1 or human PD-L2 or human TIGIT cDNA purchased from Sino Biological
  • pCHO1.0 vector purchased from Invitrogen
  • CHO-hPD-L1 cells, CHO-hPD-L2 cells, CHO-hTIGIT cells adjusted the cell density of the expanded CHO-hPD-L1/CHO-hPD-L2/CHO-hTIGIT cells to 2 ⁇ 10 6 cells/ml, add 100 ⁇ l/well to a 96-well flow plate, and centrifuge for use.
  • anti-PD-L1/PD-L2/human serum albumin tri-specific antibodies or anti-PD-L1/PD-L2/TIGIT/human serum albumin tetra-specific antibodies and human serum albumin were detected by the ELISA reaction method.
  • the binding ability of the protein, the experimental method is the same as in Example 1.3.
  • PD-L1 and PD-L2 can be co-expressed on tumor cells or immune cells.
  • This example uses CHO cells co-expressing human PD-L1 and human PD-L2 and overexpressing human PD-1 and containing the NFAT-Luciferase reporter gene
  • the method of co-incubation of Jurkat cells detected the effects of purified antibodies Bi-78-79, Bi-78-80, Bi-81-82 and Bi-79-83 on PD-L1/PD-1 pathway and PD-L2/PD-
  • the specific method for simultaneous blocking of 1 pathway is as follows.
  • the functional cells co-expressing human PD-L1 and human PD-L2 (CHO-K1-PD-L1/PD-L2) were adjusted to a density of 5 ⁇ 10 5 cells/ml, and 100 ⁇ l/well was seeded on a 96-well cell culture white bottom plate. Place in 37°C, 5% CO 2 incubator and incubate overnight. After the purified antibody and the control antibody 1640 complete medium were diluted stepwise, they were used for later use.
  • the Jurkat cells Jurkat-PD-1-NFAT
  • overexpressing human PD-1 and containing the NFAT-Luciferase reporter gene were adjusted to a cell density of 2.5 ⁇ 10 5 cells/ml with 1640 complete medium for use.
  • the anti-PD-L1/PD-L2/human serum albumin trispecific antibody or anti-PD-L1/PD-L2/TIGIT/human serum albumin tetraspecific antibody of the present invention can simultaneously block PD-L1/
  • the blocking effect of PD-1 and PD-L2/PD-1 signaling pathways is similar to that of the anti-PD-1 monoclonal antibody Pembrolizumab.
  • Nanobody domains are connected to the N-terminus of the CH1-CL structure, and one Nanobody domain is connected to the C-terminus of CH1 to form three specific antibodies or two different Nanobody domains are connected to the C-terminus of the CH1-CL structure.
  • Four different nanobody domains form 4 specific antibodies, which can effectively maintain the binding activity of the parent antibody.
  • Nanobodies can maintain their antigen binding ability through a flexible peptide chain or directly connected to the N-terminal of CH1 or CL. Nanobodies can maintain their antigen-binding ability through a flexible peptide chain of 11 amino acids (GGGGSGGGGSG) or a shorter peptide chain of 5 amino acids (DKTHT) connected to the C-terminal of CH1.
  • the combination of anti-PD-L1 and anti-PD-L2 nanobodies is used to bind two antigens on the same cell.
  • the results show that all multispecific antibodies can simultaneously bind to PD-L1 and PD-L2 and block PD-L1 and PD-L2.
  • the combination of L1/PD-L2 and PD-1 activates downstream signaling pathways.
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO:17.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 25, which includes the anti-41BB Nanobody amino acid sequence (SEQ ID NO: 26 patent number), and the C-terminus of the Nanobody amino acid sequence is directly connected to the human kappa light chain constant region ( CL) Amino acid sequence SEQ ID NO: 9, thereby obtaining peptide chain #2.
  • nucleotide sequences encoding the two chains of the anti-PD-L1/41BB/human serum albumin trispecific antibody Bi-79-86 constructed in Example 4.1 are all connected by multiple cloning sites
  • the commercially available eukaryotic expression vector pCDNA3.1(+) was used for expression and purification in eukaryotic cells.
  • the expression plasmid construction and protein expression and purification methods are the same as in Example 1.2.
  • the pCHO1.0 vector (purchased from Invitrogen) cloned into MCS human PD-L1 or human 41BB cDNA (purchased from Sino Biological) was used to generate CHO cells (CHO-hPD-L1) overexpressing human PD-L1 or human 41BB. Cells, CHO-41BB cells). Adjust the cell density of the expanded CHO-hPD-L1/CHO-41BB cells to 2 ⁇ 10 6 cells/ml, add 100 ⁇ l/well to a 96-well flow plate, and centrifuge for use.
  • the anti-PD-L1/41BB/human serum albumin trispecific antibody of the present invention can bind to human serum albumin at the ELISA level.
  • Human PD-L1 and human 41BB are expressed on the surface of tumor cells and immune cells, respectively.
  • This example verifies that the trispecific antibody Bi-79-86 can bind to cells overexpressing human PD-L1 (CHO-hPD) through cell bridging experiments. -L1) and cells overexpressing human 41BB (CHO-h41BB), the ability to bring the two cells closer together, the specific method is as follows.
  • the anti-PD-L1/41BB/human serum albumin trispecific antibody Bi-79-86 of the present invention can contain high concentration of human serum albumin in the system.
  • the method for detecting the purified anti-PD-L1/41BB/human serum albumin tri-specific antibody Bi-79-86 to block the binding activity of PD-L1 protein and PD-1 cells is the same as in Example 1.6.
  • the experimental result is shown in Figure 20.
  • the anti-PD-L1/41BB/human serum albumin trispecific antibody Bi-79-86 molecule of the present invention can block PD-L1 protein and PD-1 The combination of cells.
  • connecting two different Nanobody domains at the N-terminus of the CH1-CL structure, and connecting a Nanobody domain at the C-terminus of CH1 to form a 3-specific antibody can effectively maintain the binding activity of the parent antibody.
  • the combination of anti-PD-L1 and anti-41BB Nanobodies is used to bind two antigens on different cells.
  • the results show that the trispecific antibody structure of the present invention can simultaneously bind to PD-L1 and 41BB at the cellular level, and bridge each other. For cells expressing PD-L1 and 41BB, the binding and bridging activities are not affected by human serum albumin in the system.
  • the Fc domain of an antibody can bind to FcRn to extend the half-life of the drug; the Fc domain can bind to other Fc receptors and cause downstream reactions such as ADCC/CDC; the Fc domain and ProteinA have specific binding to facilitate protein purification .
  • Peptide chain #1 has the amino acid sequence shown in SEQ ID NO: 27, which includes the anti-PD-L1 Nanobody SEQ ID NO: 28, the C-terminus of the Nanobody amino acid sequence and SEQ ID NO: 3 derived from human IgG1
  • SEQ ID NO: 27 The shown CH1 amino acid sequence is directly connected; the human IgG1 (LALA mutant) Fc (SEQ ID NO: 29) domain is directly connected to the C-terminus of the CH1 region, thereby obtaining peptide chain #1.
  • Peptide chain #2 has the amino acid sequence shown in SEQ ID NO: 30, which includes the anti-PD-L2 Nanobody HZ-D-NA-96-01 amino acid sequence SEQ ID NO: 31, and the C-terminus of the Nanobody amino acid sequence is directly connected
  • the amino acid sequence of the human kappa light chain constant region (CL) is SEQ ID NO: 9, thereby obtaining peptide chain #2.
  • nucleotide sequences encoding each of the two chains of the anti-PD-L1/PD-L2 bispecific antibody Bi-203-204 constructed in Example 5.1 were connected to the market through multiple cloning sites.
  • the eukaryotic expression vector pCDNA3.1(+) was expressed and purified in eukaryotic cells, and the bispecific antibody Bi-203-204 was obtained.
  • the specific operation is as follows.
  • ExpiCHO TM expression system kit purchased from Thermo
  • the plasmid was transferred 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). Select method to purify the target protein.
  • the magnetic beads were resuspended (1-4 times the volume of the 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, and gently shaken during the period.
  • the sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed 3 times with binding buffer.
  • elution buffer 0.1M sodium citrate, pH 3.2
  • elution buffer 0.1M sodium citrate, pH 3.2
  • the purity of the obtained protein was detected by HPLC.
  • the HPLC method is as follows, mobile phase: 150 mM Na 2 HPO 4 ⁇ 12H 2 O, pH 7.0. Chromatographic conditions: detection wavelength: 280nm, column temperature: 25°C, flow rate: 0.35ml/min, detection time: 20 minutes, Zenix-C SEC-300 chromatographic column (SEPAX 4.6 ⁇ 300mm, 3 ⁇ m).
  • the experimental results show that the anti-PD-L1/PD-L2 bispecific antibody Fc fusion protein Bi-203-204 has good purity (>99%), and according to the size of the purified protein molecule, Bi-203-204 The purified molecule contains 4 peptide chains and they are paired correctly.
  • connecting two different Nanobody domains at the N-terminus of CH1-CL and connecting the human antibody Fc domain at the C-terminus of CH1 to form a bi-specific antibody Fc fusion protein can effectively maintain the binding activity of the parent antibody.
  • the Fc domain can facilitate antibody purification and form a stable 4-mer structure.

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Abstract

本发明提供了一种构建多特异性抗体的方法，包括步骤：(i)分别构建第一多核苷酸和第二多核苷酸，该第一多核苷酸和第二多核苷酸分别编码含有CL区的第一多肽和含有CH1区的第二多肽，并且该第一多肽的CL区和该第二多肽的CH1区之间，可形成二硫键，从而使该抗体具有异二聚体的形式；(ii)表达该第一多核苷酸和第二多核苷酸，从而获得该的第一多肽和第二多肽，使其发生二聚化，从而形成具有异二聚体形式的多特异性抗体。本发明的抗体可以同时结合不同靶点并维持原抗体的结合活性，在靶点为膜表面受体或溶液中的靶点时均有效，并且具有抗多种靶点的生物学活性。

Description

一种构建多特异性抗体的平台 技术领域
本发明属于生物医学或生物制药技术领域，具体涉及一种构建多特异性抗体的平台。
背景技术
1960年，纽约罗斯威尔公园纪念研究所的Nisonoff及其合作者首次提出了双特异性抗体(bispecific antibodys)的原始概念。之后，随着抗体工程和抗体生物学领域的里程碑进展，构建双特异性抗体的概念和技术不断创新。目前有100多种双特异性抗体结构模式，其中，约四分之一已发展成技术平台，并由生物技术公司和制药公司商业化，用于新型抗体疗法。截至目前，已有20多个不同的商业化技术平台可用于双特异性抗体的开发，超过85个双特异性抗体处于临床开发阶段。
一款T细胞参与的双特异性抗体blinatumomab(靶向CD3和CD19，于2014年获FDA批准，用于治疗急性B淋巴细胞白血病)令人印象深刻的临床结果激起了业内对这一概念的兴趣和投资。目前有40多个T细胞重定向双特异性抗体正处于临床开发阶段，用于治疗血液学肿瘤和实体肿瘤。除了癌症，炎症性疾病也一直是双特异性抗体临床开发的重点。罗氏公司的emicizumab(靶向凝血因子X和因子IXa)于2017年11月经FDA批准上市，血友病成了双特异性抗体首个非癌症适应症。目前，很多团队也在探索双特异性抗体在其它疾病领域的治疗潜力，如糖尿病、HIV感染、其它病毒和细菌感染、阿尔茨海默症、骨质疏松症等。双特异性抗体双靶向的特点(即能同时特异性靶向两个抗原或一个抗原的两个不同表位)使其具有巨大的治疗前景，但将双特异性抗体这一概念转化为临床疗法仍具有挑战性。
治疗性双特异性抗体是一个快速扩展的多样化分子群。尽管与单克隆抗体相比，双靶向概念的复杂性增加，在发现和开发的不同阶段会带来额外的挑战，但双特异性抗体为新型药物的设计与开发提供了令人兴奋的机会。从疾病领域来看，目前的数据显示，业界对双特异性抗体治疗癌症更加期待。双特异性抗体的持续发展将对癌症等疾病的治疗带来持久的影响。
因此，本领域迫切需要开发一种低成本、高效率的构建多特异性抗体的方法。
发明内容
本发明的目的就是提供一种低成本、高效率的构建多特异性抗体的方法。
在本发明的第一方面，提供了一种构建多特异性抗体的方法，包括步骤：
(i)分别构建第一多核苷酸和第二多核苷酸，所述第一多核苷酸编码从N端到C端具有如式I所示的结构的第一多肽，并且所述第二多核苷酸编码从N端到C端具有如式II所示的结构的第二多肽，
A1-L1-B1-L2-CL-L3-A2   (式I)
A3-L4-B2-L5-CH1-L6-A4   (式II)
其中，
A1、A2、A3和A4各自独立地为靶向目标靶点的抗体或其抗原片段，并且A1、A2、A3和A4各自靶向的目标抗原可以是相同的或不同的；
L1、L2、L3和L4各自独立地为无或接头元件；
B1和B2均为无，或B1和B2分别为靶向同一目标靶点的抗体的VL区和VH区；
并且所述第一多肽的CL区和所述第二多肽的CH1区之间，可形成二硫键，从而使所述抗体具有异二聚体的形式；
(ii)表达所述第一多核苷酸和第二多核苷酸，从而获得所述的第一多肽和第二多肽，使其发生二聚化，从而形成具有异二聚体形式的多特异性抗体。
在另一优选例中，所述第一多肽的CL区具有如SEQ ID NO:9所示的氨基酸序列，或与SEQ ID NO:9所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述第二多肽的CH1区具有如SEQ ID NO:3所示的氨基酸序列，或与SEQ ID NO:3所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，在A1、A2、A3和A4中，所述目标靶点是抗原、细胞表面受体、配体，或细胞因子。
在另一优选例中，在A1、A2、A3和A4中，所述目标靶点包括但不限于：PD-1、TIGIT、人血清白蛋白、VEGF、PD-L1、PD-L2，或41BB。
在另一优选例中，在A1、A2、A3和A4中，所述靶向目标靶点的抗体或其抗原片段是纳米抗体的VHH链、抗体重链可变区、抗体轻链可变区、抗体Fc片段，或其组合。
在另一优选例中，在A1、A2、A3和A4中，所述靶向目标靶点的抗体或其抗原片段包括但不限于：抗TIGIT纳米抗体的VHH链、抗HSA纳米抗体的VHH链、抗PD-L1纳米抗体的VHH链、抗PD-L2纳米抗体的VHH链、抗VEGF抗体的VH链、抗VEGF抗体的VL链、抗PD-1抗体的的VH链，或抗PD-1抗体的VL链。
在另一优选例中，所述抗TIGIT纳米抗体的VHH链具有如SEQ ID NO:6所示的氨基酸序列，或与SEQ ID NO:6所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述抗HSA纳米抗体的VHH链具有如SEQ ID NO:5所示的氨基酸序列，或与SEQ ID NO:5所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述抗PD-L1纳米抗体的VHH链具有如SEQ ID NO:14或28所示的氨基酸序列，或与SEQ ID NO:14或28所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述抗PD-L2纳米抗体的VHH链具有如SEQ ID NO:19所示的氨基酸序列，或与SEQ ID NO:19所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，在B1和B2中，所述靶向同一目标靶点的抗体包括但不限于：抗PD-1抗体、抗VEGF抗体。
在另一优选例中，B1和B2分别为抗PD-1抗体的VL区和VH区；其中，所述抗PD-1抗体的VL区具有如SEQ ID NO:8所示的氨基酸序列，或与SEQ ID NO:8所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列，并且所述抗PD-1抗体的VH区具有如SEQ ID NO:2所示的氨基酸序列，或与SEQ ID NO:2所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，B1和B2分别为抗VEGF抗体的VL区和VH区；其中，所述抗VEGF抗体的VL区具有如SEQ ID NO:16所示的氨基酸序列，或与SEQ ID NO:16所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列，并且所述抗VEGF抗体的VH区具有如SEQ ID NO:13所示的氨基酸序列，或与SEQ ID NO:13所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述的接头元件的序列为(4GS)n，其中，n为正整数(例如 1、2、3、4、5或6)，优选地，n＝4。
在另一优选例中，所述接头元件的序列如SEQ ID NO：4或21所示，或与SEQ ID NO：4或21所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性。
在另一优选例中，所述第一多肽具有如SEQ ID NO:1所示的氨基酸序列，或与SEQ ID NO:1所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列；并且所述第二多肽具有如SEQ ID NO:7所示的氨基酸序列，或与SEQ ID NO:7所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述第一多肽具有如SEQ ID NO:10所示的氨基酸序列，或与SEQ ID NO:10所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列；并且所述第二多肽具有如SEQ ID NO:11所示的氨基酸序列，或与SEQ ID NO:11所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述第一多肽具有如SEQ ID NO:12所示的氨基酸序列，或与SEQ ID NO:12所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列；并且所述第二多肽具有如SEQ ID NO:15所示的氨基酸序列，或与SEQ ID NO:15所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述第一多肽具有如SEQ ID NO:17所示的氨基酸序列，或与SEQ ID NO:17所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列；并且所述第二多肽具有如SEQ ID NO:18所示的氨基酸序列，或与SEQ ID NO:18所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述第一多肽具有如SEQ ID NO:20所示的氨基酸序列，或与SEQ ID NO:20所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列；并且所述第二多肽具有如SEQ ID NO:18所示的氨基酸序列，或与SEQ ID NO:18所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述第一多肽具有如SEQ ID NO:22所示的氨基酸序列，或与SEQ ID NO:22所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、 98％或99％)的序列同一性的氨基酸序列；并且所述第二多肽具有如SEQ ID NO:23所示的氨基酸序列，或与SEQ ID NO:23所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述第一多肽具有如SEQ ID NO:17所示的氨基酸序列，或与SEQ ID NO:17所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列；并且所述第二多肽具有如SEQ ID NO:24所示的氨基酸序列，或与SEQ ID NO:24所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在另一优选例中，所述第一多肽具有如SEQ ID NO:17所示的氨基酸序列，或与SEQ ID NO:17所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列；并且所述第二多肽具有如SEQ ID NO:25所示的氨基酸序列，或与SEQ ID NO:25所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在本发明的第二方面，提供了一种多特异性抗体，所述抗体包含从N端到C端如式I所示的第一多肽和从N端到C端如式II所示的第二多肽，
A1-L1-B1-L2-CL-L3-A2   (式I)
A3-L4-B2-L5-CH1-L6-A4   (式II)
其中，
A1、A2、A3和A4各自独立地为靶向目标靶点的抗体或其抗原片段，并且A1、A2、A3和A4各自靶向的目标抗原可以是相同的或不同的；
L1、L2、L3和L4各自独立地为无或接头元件；
B1和B2均为无，或B1和B2分别为靶向同一目标靶点的抗体的VL区和VH区；
并且所述第一多肽的CL区和所述第二多肽的CH1区之间，可形成二硫键，从而使所述抗体具有异二聚体的形式。
在本发明的第三方面，提供了一种融合蛋白，所述融合蛋白中包括如本发明第二方面所述的多特性抗体，并且，所述多特异性抗体中的所述第一多肽从N端到C端具有如式III所示的结构，
A1-L1-CL-L3-Fc   (式III)
其中，Fc是为抗体的Fc段，包含CH2结构域和CH3结构域；
并且，所述的融合蛋白可通过Fc段之间的二硫键作用形成同源二聚体。
在另一优选例中，所述第一多肽具有如SEQ ID NO:27所示的氨基酸序列，或与SEQ ID NO:27所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列；并且所述第二多肽具有如SEQ ID NO:30所示的氨基酸序列，或与SEQ ID NO:30所示序列具有≥85％(优选地90％，更优选地95％、96％、97％、98％或99％)的序列同一性的氨基酸序列。
在本发明的第四方面，提供了一种分离的多核苷酸组合，所述多核苷酸组合包括第一核苷酸和第二核苷酸，所述第一核苷酸编码如本发明第二方面所述的多特异性抗体或如本发明第三方面所述的融合蛋白的第一多肽，并且所述第二核苷酸编码第二多肽。
在本发明的第五方面，提供了一种载体，所述载体含有如本发明第四方面所述的多核苷酸组合。
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。
在本发明的第六方面，提供了一种宿主细胞，所述宿主细胞含有如本发明第五方面所述的载体，或其基因组中整合有如本发明第四方面所述的多核苷酸组合；
或者，所述的宿主细胞表达如本发明第二方面所述的多特异性抗体或如本发明第三方面所述的融合蛋白。
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。
在本发明的第七方面，提供了一种产生抗体的方法，包括步骤：
(a)在合适的条件下，培养如本发明第六方面所述的宿主细胞，从而获得含如本发明第二方面所述的多特异性抗体或如本发明第三方面所述的融合蛋白的培养物；和
(b)对步骤(a)中得到的培养物进行纯化和/或分离，获得所述的抗体。
在另一优选例中，所述纯化可以通过亲和层析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％。
在本发明的第八方面，提供了一种免疫偶联物，所述免疫偶联物含有：
(a)如本发明第二方面所述的多特异性抗体或如本发明第三方面所述的融合蛋白；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)的如本发明第二方面所述的多特异性抗体。
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二方面所述的抗体或如本发明第三方面所述的融合蛋白。
在本发明的第九方面，提供了如本发明第二方面所述的多特异性抗体、如本发明第三方面所述的融合蛋白或如本发明第八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：检测样品中是否存在所述的目标靶点分子；
并且，所述药剂用于治疗或预防表达目标靶点分子的肿瘤。
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，所述检测样品中目标靶点分子的试剂为(体内)检测目标靶点分子的造影剂。
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。
在本发明的第十方面，提供了一种药物组合物，含有：(i)如本发明第二方面所述的多特异性抗体、如本发明第三方面所述的融合蛋白，或如本发明第八方面所述的免疫偶联物；以及(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，或派姆单抗。
在另一优选例中，所述的药物组合物用于治疗表达目标靶点分子(即目标靶点分子阳性)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。
在本发明的第十一方面，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二方面所述的多特异性抗体、如本发明第三方面所述的融合蛋白、如本发明第八方面所述的免疫偶联物，或如本发明第十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。
在本发明的第十二方面，提供了一种试剂盒，所述试剂盒含有如本发明第二方面所述的多特异性抗体、如本发明第三方面所述的融合蛋白、本发明第八方面所 述的免疫偶联物或如本发明第十方面所述的药物组合物，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目标靶点分子的表达。
在另一优选例中，所述的说明书记载，所述的试剂盒用于表达目标靶点分子(即目标靶点分子阳性)的肿瘤的检测。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了三种三特异性抗体的结构示意图。
图2显示了使用Octet系统测定的抗PD-1/TIGIT/人血清白蛋白三特异性抗体的抗原共结合能力的结果。
图3显示了抗PD-1/TIGIT/人血清白蛋白三特异性抗体与CHO-hPD-1细胞及CHO-hTIGIT细胞结合活性的测定结果。
图4显示了通过ELISA测定的抗PD-1/TIGIT/人血清白蛋白三特异性抗体与人血清白蛋白的结合活性。
图5显示了抗PD-1/TIGIT/人血清白蛋白三特异性抗体对人PD-L1与人PD-1结合的阻断效果。
图6显示了抗PD-1/TIGIT/人血清白蛋白三特异性抗体对CD155与TIGIT结合的阻断效果。
图7显示了使用Octet系统测定的抗VEGF/PD-L1/人血清白蛋白三特异性抗体的抗原共结合能力的结果。
图8显示了抗VEGF/PD-L1/人血清白蛋白三特异性抗体与CHO-hPD-1细胞结合活性的测定结果。
图9显示了通过ELISA测定的抗VEGF/PD-L1/人血清白蛋白三特异性抗体与人血清白蛋白的结合活性。
图10显示了通过ELISA测定的抗VEGF/PD-L1/人血清白蛋白三特异性抗体与人VEGF蛋白的结合活性。
图11显示了三种三特异性抗体和一种四特异性抗体的结构示意图。
图12显示了三种PD-L1/PD-L2/人血清白蛋白三特异性抗体与CHO-hPD-L1细胞及CHO-hPD-L2细胞结合活性的测定结果，以及抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体与CHO-hPD-L1细胞、CHO-hPD-L2细胞及CHO-hTIGIT细胞结合活性的测定结果。
图13显示了通过ELISA测定的三种抗PD-L1/PD-L2/人血清白蛋白三特异性抗体以及抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体与人血清白蛋白的结合活性。
图14显示了三种抗PD-L1/PD-L2/人血清白蛋白三特异性抗体以及抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体对人PD-L1或PD-L2与人PD-1结合的阻断效果。
图15显示了三种抗PD-L1/PD-L2/人血清白蛋白三特异性抗体以及抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体在体外同时阻断PD-L1/PD-1和PD-L2/PD-1信号通路的结果。
图16显示了一种三特异性抗体的结构示意图。
图17显示了PD-L1/41BB/人血清白蛋白三特异性抗体与CHO-hPD-L1细胞及CHO-41BB细胞的结合活性的测定结果。
图18显示了通过ELISA测定的PD-L1/41BB/人血清白蛋白三特异性抗体与人血清白蛋白的结合活性。
图19显示了抗PD-L1/41BB/人血清白蛋白三特异性抗体桥接表达PD-L1/41BB细胞的能力。
图20显示了抗PD-L1/41BB/人血清白蛋白三特异性抗体对人PD-L1与人PD-1结合的阻断效果。
图21显示了如实施例5所述的PD-L1/PD-L2双特异性抗体Fc融合蛋白的结构示意图。
图22显示了抗PD-L1/PD-L2双特异性抗体Fc融合蛋白与CHO-hPD-L1细胞及CHO-hPD-L2细胞的结合活性的测定结果。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种构建多特异性抗体的方法。实验证明，将抗原结合片段(如抗体可变区、单域抗体或Fc等)和天然抗体CL和CH1片段连接，可以形成稳定的异源二聚体。本申 请的方法所构建的多特异性抗体能够同时结合不同靶点并维持原抗体的结合活性；在靶点为膜表面受体或溶液中的靶点时均有效；同时具有抗多种靶点的生物学活性；可以连接单域抗体或者正常抗体或者Fc片段。因此本发明方法和所提供的平台具有巨大的应用前景。在此基础上完成了本发明。
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。
如本文所用，术语“构建多特异性抗体的平台”、“本发明构建方法”可互换使用，是指本发明第一方面所述的构建多特异性抗体的方法，其中，以CL和CH1之间通过二硫键作用所形成的异源二聚体为核心结构，并融合靶向不同目标靶点的抗体或其抗原片段。
多特异性抗体
双特异/多特异性抗体(BsAb,MsAb)是由两种或多种不同单克隆抗体的片段组成的人造蛋白，因而可结合到两种或多种不同类型抗原。例如，在癌症免疫治疗中，经过工程化的BsAbs同时结合至细胞毒性细胞及要被杀灭的靶标(如肿瘤细胞)。至少三种类型的双特异抗体己经被提出或测试，包括三官能抗体、化学连接的Fab和双特异T细胞衔接体。为了克服制造上的难点，己经开发了被称作三官能抗体的第一代BsMAb。它由两个重链和两个轻链组成，每个来自不同的抗体。这两个Fab区针对两种抗原。Fc区由两个重链组成并形成第三个结合位点，由此得名。
己设计其他类型的双特异抗体来解决某些问题，例如，半衰期短、免疫原性和细胞因子释放引起的副作用。它们包括：仅由Fab区组成的化学连接的Fab，以及各种类型二价及三价的单链可变区(scFv)(模拟两种抗体可变结构域的融合蛋白)。最新开发的形式是双特异的T细胞衔接体(BiTE)及四官能的抗体。
众所周知，抗体是一类能与抗原特异性结合的免疫球蛋白，由四条多肽链组成，分子量较大的两条链称为重链(heavy chain，H链)，而分子量较小的两条链称为轻链(Light chain，L链)。在单抗中，两条H链和两条L链的氨基酸组成完全相同，而双特异性抗体的开发是通过共表达两条不同的H链和两条不同的L链。从10种可能的H2L2重组混合物中获得功能性双特异性抗体是双特异性抗体开发的 最初挑战之一，这通常称为链相关问题。在过去几十年里，研究者们已经开发了许多策略，以解决这一问题。
基于片段的模式(Fragment-based formats)
基于片段的双特异性抗体简单地将多个抗体片段结合在一个分子中，不含Fc区域，避免了链相关问题，优势是产量高、成本低；缺点是半衰期相对较短。此外，基于片段的双特异性抗体可能会出现稳定性和聚合问题。
对称模式(Symmetric formats)
对称模式的双特异性抗体保留了Fc区域，更接近于天然抗体，但在大小和结构上有所不同。这些差异可能对与天然抗体相关的有利特性(如稳定性和溶解度)产生负面影响，从而可能损害这些双特异性抗体的理化和/或药代动力学特性。
不对称模式(Asymmetric formats)
大多数不对称模式的双特异性抗体与天然抗体非常相似，被认为具有最低免疫原性的潜力。不过，解决链相关问题可能涉及到的复杂工程可能会抵消一些不对称模式双特异性抗体的这一优势。
而在本发明中，提供了一类基于异源二聚体形式的多特异性抗体。
如本文所用，术语“本发明的多特异性抗体”、“本发明的多抗”、“本发明抗体”可互换使用，均指使用本发明所提供的构建多特异性抗体的方法所构建出的多特异性抗体。
优选地，本发明提供的抗体包含从N端到C端如式I所示的第一多肽和从N端到C端如式II所示的第二多肽，
A1-L1-B1-L2-CL-L3-A2  (式I)
A3-L4-B2-L5-CH1-L6-A4  (式II)
其中，
A1、A2、A3和A4各自独立地为靶向目标靶点的抗体或其抗原片段，并且A1、A2、A3和A4各自靶向的目标抗原可以是相同的或不同的；
L1、L2、L3和L4各自独立地为无或接头元件；
B1和B2均为无，或B1和B2分别为靶向同一目标靶点的抗体的VL区和VH区；
并且所述第一多肽的CL区和所述第二多肽的CH1区之间，可形成二硫键，从而使所述抗体具有异二聚体的形式。
在一个优选的实施方式中，式I中的CL区具有如SEQ ID NO:9所示的氨基酸序列，并且式II中的CH1区具有如SEQ ID NO:3所示的氨基酸序列。
在一个实施方式中，所述的多特异性抗体为抗PD-1/TIGIT/人血清白蛋白三特异性抗体，其中，所述第一多肽具有如SEQ ID NO:1所示的氨基酸序列，并且所述第二多肽具有如SEQ ID NO:7所示的氨基酸序列。
在一个实施方式中，所述的多特异性抗体为抗PD-1/TIGIT/人血清白蛋白三特异性抗体，其中，所述第一多肽具有如SEQ ID NO:10所示的氨基酸序列，并且所述第二多肽具有如SEQ ID NO:11所示的氨基酸序列。
在一个实施方式中，所述的多特异性抗体为抗VEGF/PD-L1/人血清白蛋白三特异性抗体，其中，所述第一多肽具有如SEQ ID NO:12所示的氨基酸序列，并且所述第二多肽具有如SEQ ID NO:15所示的氨基酸序列。
在一个实施方式中，所述的多特异性抗体为抗PD-L1/PD-L2/人血清白蛋白三特异性抗体，其中，所述第一多肽具有如SEQ ID NO:17所示的氨基酸序列，并且所述第二多肽具有如SEQ ID NO:18所示的氨基酸序列。
在一个实施方式中，所述的多特异性抗体为抗PD-L1/PD-L2/人血清白蛋白三特异性抗体，其中，所述第一多肽具有如SEQ ID NO:20所示的氨基酸序列，并且所述第二多肽具有如SEQ ID NO:18所示的氨基酸序列。
在一个实施方式中，所述的多特异性抗体为抗PD-L1/PD-L2/人血清白蛋白三特异性抗体，其中，所述第一多肽具有如SEQ ID NO:22所示的氨基酸序列，并且所述第二多肽具有如SEQ ID NO:23所示的氨基酸序列。
在一个实施方式中，所述的多特异性抗体为抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体，其中，所述第一多肽具有如SEQ ID NO:17所示的氨基酸序列，并且所述第二多肽具有如SEQ ID NO:24所示的氨基酸序列。
在一个实施方式中，所述的多特异性抗体为抗PD-L1/41BB/人血清白蛋白三特异性抗体，其中，所述第一多肽具有如SEQ ID NO:17所示的氨基酸序列，并且所述第二多肽具有如SEQ ID NO:25所示的氨基酸序列。
在另一个实施方式中，本发明提供了一种融合蛋白，所述融合蛋白是在本发明的多特异性抗体中，在第一多肽的C端融合Fc段，使得所述的多特异性抗体能够通过Fc段之间二硫键作用引起的同源二聚化，而形成一个更加稳定的同源二聚体。
优选地，所述的融合蛋白为抗PD-L1/PD-L2双特异性抗体，其中，所述第一 多肽具有如SEQ ID NO:27所示的氨基酸序列，并且所述第二多肽具有如SEQ ID NO:30所示的氨基酸序列。
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KD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”是指插入本发明抗体中为各结构域或区域提供足够的可动性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多特异性抗体或其片段结合的细胞表面标记物或抗原。
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，均指本发明所提供的多特异性抗体，其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。
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。
本发明抗体还包括具有与本发明抗体相同功能的、其中所述第一多肽或第二多肽的变异形式。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。
本发明的构建方法
在本发明中，提供了一种构建多特异性抗体的方法，包括步骤：
(i)分别构建第一多核苷酸和第二多核苷酸，所述第一多核苷酸编码从N端到C端具有如式I所示的结构的第一多肽，并且所述第二多核苷酸编码从N端到C端具有如式II所示的结构的第二多肽，
A1-L1-B1-L2-CL-L3-A2   (式I)
A3-L4-B2-L5-CH1-L6-A4   (式II)
其中，A1、A2、A3和A4各自独立地为靶向目标靶点的抗体或其抗原片段，并且A1、A2、A3和A4各自靶向的目标抗原可以是相同的或不同的；L1、L2、L3和L4各自独立地为无或接头元件；B1和B2均为无，或B1和B2分别为靶向同一目标靶点的抗体的VL区和VH区；并且所述第一多肽的CL区和所述第二多肽的CH1区之间，可形成二硫键，从而使所述抗体具有异二聚体的形式；
(ii)表达所述第一多核苷酸和第二多核苷酸，从而获得所述的第一多肽和第二多肽，使其发生二聚化，从而形成具有异二聚体形式的多特异性抗体。
优选地，所述第一多肽的CL区具有如SEQ ID NO:9所示的氨基酸序列，并且所述第二多肽的CH1区具有如SEQ ID NO:3所示的氨基酸序列，两者之间可以形成二硫键。
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目标靶点分子，因而可用于治疗相应的疾病。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。
本发明的主要优点包括：
1)本发明的双/多特异性抗体结构可以同时结合不同靶点并维持原抗体的结合活性。
2)本发明的双/多特异性抗体结构在靶点为膜表面受体或溶液中的靶点时均有效。
3)本发明的双/多特异性抗体结构同时具有抗多种靶点的生物学活性。
4)本发明的双/多特异性抗体结构可以连接单域抗体或者正常抗体或者Fc片段。
5)本发明的双/多特异性抗体是以CH1-CL这个二聚体为中心所构建的抗体或者融合蛋白，在此基础上，本发明还提供了一种多特异性抗体，其包括Fc片段，可以显著提高蛋白半衰期及简化纯化过程。
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。
表B本发明的序列总结
Figure PCTCN2021084198-appb-000001
Figure PCTCN2021084198-appb-000002
实施例1：抗PD-1/TIGIT/人血清白蛋白三特异性抗体
1.1抗PD-1/TIGIT/人血清白蛋白三特异性抗体的构建
为了证实M-Body技术，在本实施例中，构建了2种抗PD-1/TIGIT/人血清白蛋白三特异性抗体：
Bi-70-71由2条多肽链组成，其结构如图1A所示。肽链#1具有SEQ ID NO:1所示的氨基酸序列，其包含衍生自抗PD-1抗体Pembrolizumab(专利号：US8354509)的VH氨基酸序列(SEQ ID NO:2)，所述VH氨基酸序列C端和衍生自人IgG1的CH1氨基酸序列(SEQ ID NO:3)直接相连。将具有SEQ ID NO:5抗人血清白蛋白的纳米抗体ALB8(专利号：WO2004/041865)的C端通过11个氨基酸残基(GGGGSGGGGSG)(SEQ ID NO：4)的柔性肽连接于Pembrolizumab的重链可变区N端。将抗TIGIT的纳米抗体E-Ye-11(SEQ ID NO:6)的N端通过11个氨基酸残基(GGGGSGGGGSG)的柔性肽连接于CH1的C端，由此获得肽链#1。肽链#2具有SEQ ID NO:7所示的氨基酸序列，其包含衍生自抗PD-1抗体Pembrolizumab的VL氨基酸序列(SEQ ID NO：8)，所述VL氨基酸序列C端和人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:9)直接相连，由此获得肽链#2。
Bi-72-73也由2条多肽链组成，其结构如图1B所示。肽链#1具有SEQ IDNO:10所示的氨基酸序列，其包含衍生自抗PD-1抗体Pembrolizumab的VH氨基酸序列(SEQ ID NO:2)，所述VH氨基酸序列C端和衍生自人IgG1的CH1氨基酸序列(SEQ ID NO:3)直接相连。肽链#2具有SEQ ID NO:11所示的氨基酸序列，其包含衍生自抗PD-1抗体Pembrolizumab的VL氨基酸序列(SEQ ID NO：8)，所述VL氨基酸序列C端和人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:9)直接相连。将抗人血清白蛋白的纳米抗体ALB8(SEQ ID NO:5)的C端通过11个氨基酸残基(GGGGSGGGGSG)(SEQ  ID NO:4)的柔性肽连接于Pembrolizumab的轻链可变区N端，将抗TIGIT的纳米抗体E-Ye-11(SEQ ID NO:6)的N端通过11个氨基酸残基GGGGSGGGGSG(SEQ ID NO:4)的柔性肽连接于CL的C端，由此获得肽链#2。
1.2抗PD-1/TIGIT/人血清白蛋白三特异性抗体的表达和纯化
在本实施例中，将编码实施例1.1中构建的抗PD-1/TIGIT/人血清白蛋白三特异性抗体Bi-70-71和Bi-72-73的各2条链的核苷酸序列通过多克隆位点连接入市售的真核表达载体pCDNA3.1(+)，在真核细胞中进行表达和纯化，获得了三特异性抗体Bi-70-71和Bi-72-73，具体操作如下。
抗体基因构建入pCDNA3.1表达载体
编码Bi-70-71和Bi-72-73的各2条链的基因序列有金维智公司合成。利用同源重组酶(购自Vazyme)和EcoR I/Not I双酶切线性化的pCDNA3.1载体中，流程按照商品说明书。同源重组产物化转入Top10感受态细胞，涂布氨苄抗性平板，37℃培养过夜，挑取单克隆测序。
蛋白表达及纯化
采用ExpiCHO TM表达系统试剂盒(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KappaSelect(GE)亲和层析柱进行纯化。具体方法如下：样品用注射器通过0.2um无菌针头式过滤器PES进行样品过滤。用5倍柱体积的平衡缓冲液(20mM PB+0.15M NaCl，pH 7.4)平衡层析柱，至流出液电导和pH不变。流速0.5ml/分钟上样。上样完毕后继续用平衡缓冲液冲洗层析柱，冲洗至穿透完全，UV值不再下降。用洗脱缓冲液(0.1M glycine-HCl，pH 3.0)洗脱，收集流出液。洗脱后，应立刻用碱性缓冲液(如1M Tris/HCl，pH 8.0)将收集到的抗体溶液中和到抗体稳定的pH。
利用HPLC检测获得蛋白的纯度。HPLC方法如下，流动相：150mM Na 2HPO 4·12H 2O，pH7.0。色谱条件：检测波长：280nm，柱温：25℃，流速：0.35ml/min，检测时间：20min，Zenix-C SEC-300色谱柱(SEPAX 4.6×300mm，3μm)。SEC结果显示，双特异性抗体Bi-70-71纯度为99.14％，Bi-72-73纯度为98.27％。
1.3测定抗PD-1/TIGIT/人血清白蛋白三特异性抗体的抗原共结合能力
使用Octet系统(ForteBio公司生产)通过动力学结合测定法确定本发明上述两种示例性抗PD-1/TIGIT/人血清白蛋白三特异性抗体Bi-70-71和Bi-72-73能否 同时结合PD-1、TIGIT及人血清白蛋白。在实验开始前半个小时，将SA传感器(Pall)浸泡于SD缓冲液(PBS 1×，BSA 0.1％，吐温20 0.05％)中于室温平衡。向96孔黑色聚苯乙烯半量微孔板(Greiner)的孔中分别加入100μl的SD缓冲液作为空白对照(用于扣除背景)、100μl 100nM纯化的双特异性抗体Bi-70-71和Bi-72-73、100μl稀释于SD缓冲液中作为抗原的生物素化标记的人PD-1(100nM)(Acrobiosystems)、人TIGIT(100nM)(Acrobiosystems)和人血清白蛋白(Acrobiosystems)的溶液。将SA传感器浸没于含生物素化标记的人PD-1溶液的孔中，在室温浸没60秒上样。随后将传感器在SD缓冲液中洗涤至达到基线，然后浸没于含100μl抗体溶液的孔中，监测抗体与抗原的缔合，随后将传感器转移至含有100μl SD缓冲液的孔，监测抗体解离；然后再将传感器转移至含有100nM人TIGIT溶液的孔中，检测抗体与人TIGIT的结合，随后将传感器转移至含有100μl SD缓冲液的孔，监测抗原解离；然后将传感器转移至含有100nM人血清白蛋白溶液的孔中，检测抗体与人血清白蛋白的结合，随后将传感器转移至含有100μl SD缓冲液的孔，监测抗原解离。转速为1000转/分钟，温度为30℃。
在如上方法的测定实验中，本发明的抗PD-1/TIGIT/人血清白蛋白三特异性抗体Bi-70-71(如图2A)和Bi-72-73(如图2B)能够同时和人PD-1、人TIGIT及人血清白蛋白蛋白结合。
1.4测定抗PD-1/TIGIT/人血清白蛋白三特异性抗体的抗原的结合能力
通过转染克隆到MCS的人PD-1或者人TIGIT cDNA(购自Sino Biological)的pCHO1.0载体(购自Invitrogen)产生过表达人PD-L1或人TIGIT的CHO细胞(CHO-hPD-L1细胞、CHO-hTIGIT细胞)。将扩大培养的CHO-hPD-L1/CHO-hTIGIT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。将纯化的三特异性抗体用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30分钟，PBS清洗两次。纯化抗体样品孔100μl/孔加入用PBS稀释的鼠抗人IgG-Fab(PE)(购自Abcam)，4℃孵育30分钟，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3所示，本发明的抗PD-1/TIGIT/人血清白蛋白三特异性抗体和CHO-hPD-1细胞及CHO-hTIGIT细胞均有结合活性。
1.5抗PD-1/TIGIT/人血清白蛋白三特异性抗体与人血清白蛋白在ELISA水平的结合
用ELISA包被液将人血清白蛋白(acrobiosystems)稀释后加入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。将纯化抗体和对照抗体梯度稀释后加入封闭好的ELISA板，室温孵育2小时。加入PBST洗3次，向纯化抗体样品孔中加入羊抗人Fab-HRP(abcam)，室温孵育1小时,加入PBST洗3次后加入ELISA显色液，室温放置3分钟后加入ELISA终止液，读取450nm处吸光度数值。
在如上方法的测定实验中，实验结果如图4所示，本发明抗PD-1/TIGIT/人血清白蛋白三特异性抗体在ELISA水平与人血清白蛋白均有结合。
1.6抗PD-1/TIGIT/人血清白蛋白三特异性抗体阻断人PD-L1与人PD-1结合
将CHO-hPD-1细胞调整细胞密度至2×10 6cells/ml,100μl/孔加入96孔流式板，离心备用。将纯化抗体和对照抗体样品用PBS稀释，400nM开始3倍稀释共12个点，将上述稀释好的样品60μl/孔加入96孔样品稀释板，同时60μl/孔加入生物素化标记的人PD-L1蛋白(购自AcroBiosystems)，终浓度为500ng/ml，与样品4℃孵育30分钟。将共孵育样品100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育30分钟，PBS清洗两次。100μl/孔加入用PBS稀释100倍的Streptavidin,R-Phycoerythrin Conjugate(购自Thermo fisher)，4℃孵育30分钟,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5所示，本发明的抗PD-1/TIGIT/人血清白蛋白三特异性抗体均可以阻断PD-L1与PD-1的结合。
1.7抗PD-1/TIGIT/人血清白蛋白三特异性抗体阻断人CD155与人TIGIT结合
将CHO-hTIGIT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。将纯化抗体和对照抗体样品用PBS稀释，400nM开始3倍稀释共12个点，将上述稀释好的样品60μl/孔加入96孔样品稀释板，同时60μl/孔加入人CD155-mFc蛋白(购自AcroBiosystems)，终浓度为2μg/ml，与样品4℃孵育30分钟。将共孵育样品100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育30分钟,PBS清洗两次。100μl/孔加入用PBS稀释100倍的Goat  anti-mouse IgG Fc-APC(购自Biolegend)，4℃孵育30分钟,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6所示，本发明的抗PD-1/TIGIT/人血清白蛋白三特异性抗体均可以阻断CD155与TIGIT的结合。
综上，在CH1-CL的结构域上方连接VH和VL结构域，同时连接1个或多个纳米抗体结构域形成的双特异性或多特异性抗体可以有效维持亲本抗体的结合活性。本实施例所使用的的VH-VL组合来自Pembrolizumab，是结合细胞表面抗原人PD-1的结构域。
实施例2：抗VEGF/PD-L1/人血清白蛋白三特异性抗体
2.1.抗VEGF/PD-L1/人血清白蛋白三特异性抗体的构建
为了证实M-body在连接的VH-VL结构域是靶向血液中游离的抗原时是否适用，在本实施例中，构建了1种抗VEGF/PD-L1/人血清白蛋白三特异性抗体，命名为Bi-74-76，其结构示意图如图1C所示，由2条多肽链组成，肽链#1具有SEQ ID NO:12所示的氨基酸序列，其包含衍生自抗VEGF抗体Bevacizumab(专利号：WO1998045332)的VH氨基酸序列(SEQ ID NO:13)，所述VH氨基酸序列C端和衍生自人IgG1的CH1氨基酸序列(SEQ ID NO:3)直接相连；将抗人血清白蛋白的纳米抗体ALB8(SEQ ID NO:5)的C端通过11个氨基酸残基(GGGGSGGGGSG)(SEQ ID NO:4)的柔性肽连接于Bevacizumab重链可变区N端，将抗人PD-L1的纳米抗体C-Ye-8-5(专利申请号：2019108631090)(SEQ ID NO:14)的N端通过11个氨基酸残基(GGGGSGGGGSG)(SEQ ID NO:4)的柔性肽连接于CH1的C端，由此获得肽链#1。肽链#2具有SEQ ID NO:15所示的氨基酸序列，其包含衍生自抗VEGF抗体Bevacizumab的VL氨基酸序列(SEQ ID NO:16)，所述VL氨基酸序列C端和衍生自人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:9)直接相连；将抗人PD-L1的纳米抗体C-Ye-8-5(SEQ ID NO:14)的N端通过11个氨基酸残基(GGGGSGGGGSG)(SEQ ID NO:4)的柔性肽连接于CL的C端，由此获得肽链#2。
2.2抗VEGF/PD-L1/人血清白蛋白三特异性抗体的表达和纯化
在本实施例中，将编码实施例2.1中构建的抗VEGF/PD-L1/人血清白蛋白三 特异性抗体Bi-74-76的2条核苷酸序列均通过多克隆位点连接入市售的真核表达载体pCDNA3.1(+)，在真核细胞中进行表达和纯化，获得了三特异性抗体Bi-74-76。表达质粒构建、细胞转染、蛋白纯化及HPLC纯度检测方法同实施例1.2。SEC结果显示，双特异性抗体Bi-74-76纯度为95.89％。
2.3测定抗VEGF/PD-L1/人血清白蛋白三特异性抗体的抗原共结合能力
使用Octet系统(ForteBio公司生产)通过动力学结合测定法确定本发明上述两种示例性抗VEGF/PD-L1/人血清白蛋白三特异性抗体Bi-74-76能否同时结合人PD-L1、VEGF及人血清白蛋白。在实验开始前半个小时，将SA传感器(Pall)浸泡于SD缓冲液(PBS 1×，BSA 0.1％，吐温20 0.05％)中于室温平衡。向96孔黑色聚苯乙烯半量微孔板(Greiner)的孔中分别加入100μl的SD缓冲液作为空白对照(用于扣除背景)、100μl 100nM纯化的三特异性抗体Bi-74-76、100μl稀释于SD缓冲液中作为抗原的生物素化标记的人VEGF(100nM)(Acrobiosystems)、人PD-L1(100nM)(Acrobiosystems)和人血清白蛋白(Acrobiosystems)的溶液。将SA传感器浸没于含生物素化标记的VEGF溶液的孔中，在室温浸没60秒上样。随后将传感器在SD缓冲液中洗涤至达到基线，然后浸没于含100μl抗体溶液的孔中，监测抗体与抗原的缔合，随后将传感器转移至含有100μl SD缓冲液的孔，监测抗体解离；然后再将传感器转移至含有100nM人PD-L1溶液的孔中，检测抗体与人PD-L1的结合，随后将传感器转移至含有100μl SD缓冲液的孔，监测抗原解离；然后将传感器转移至含有100nM人血清白蛋白溶液的孔中，检测抗体与人血清白蛋白的结合，随后将传感器转移至含有100μl SD缓冲液的孔，监测抗原解离。转速为1000转/分钟，温度为30℃。
在如上方法的测定实验中，实验结果如图7所示，本发明的抗VEGF/PD-L1/人血清白蛋白三特异性抗体Bi-74-76能够同时和人PD-L1、人VEGF及人血清白蛋白蛋白结合。
2.4测定抗VEGF/PD-L1/人血清白蛋白三特异性抗体的抗原结合能力
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、CHO-hTIGIT细胞)。抗VEGF/PD-L1/人血清白蛋白三特异性抗体和CHO-hPD-L1细胞结合活性的检测方法同实施例1.4。
在如上方法的测定实验中，实验结果如图8所示，本发明抗VEGF/PD-L1/人血清白蛋白三特异性抗体能够和CHO-hPD-L1细胞有结合。
2.5测定抗VEGF/PD-L1/人血清白蛋白三特异性抗体和人血清白蛋白在ELISA水平的结合
本实验通过ELISA反应的方法检测了抗VEGF/PD-L1/人血清白蛋白三特异性抗体和人血清白蛋白的结合能力，实验方法同实施例1.5。在如上方法的测定实验中，本发明纯化抗体在能够在ELISA水平与人血清白蛋白结合(如图9)。
2.6抗VEGF/PD-L1/人血清白蛋白三特异性抗体与人VEGF在ELISA水平的结合
用ELISA包被液将人VEGF(acrobiosystems)蛋白稀释后加入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。将纯化的抗体Bi-074-076抗体梯度稀释后加入封闭好的ELISA板，室温孵育2小时。加入PBST洗3次，向纯化抗体样品孔中加入羊抗人Fab-HRP(abcam),向对照抗体样品孔中加入羊抗人Fc-HRP(abcam)，室温孵育1小时,加入PBST洗3次后加入ELISA显色液，室温放置3min后加入ELISA终止液，读取450nm处吸光度数值。
在如上方法的测定实验中，本发明中纯化抗体Bi-74-76能够在ELISA水平与人VEGF蛋白结合(如图10)。
综上，在CH1-CL的结构上方连接VH和VL结构域，同时连接1个或多个纳米抗体结构域形成的双特异性或多特异性抗体可以有效维持亲本抗体的结合活性。本实施例所使用的的VH-VL组合来自Bevacizumab，是结合血液中游离抗原VEGF的结构域。
实施例3：抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体
3.1抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体的构建
为了证实M-body在连接的是两个靶向位于同一细胞的不同靶点的纳米抗体时 是否适用，本发明人构建了一组抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体。
在本实施例中，构建了3种抗抗PD-L1/PD-L2/人血清白蛋白三特异性抗体：
Bi-78-79，其结构示意图如图11A所示，由2条多肽链组成，肽链#1具有SEQ ID NO:17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:14)，所述纳米抗体氨基酸序列C端和衍生自人IgG1的CH1氨基酸序列(SEQ ID NO:3)直接连接；将抗人血清白蛋白的纳米抗体ALB8(SEQ ID NO：5)的C端通过11个氨基酸残基(GGGGSGGGGSG)(SEQ ID NO：4)的柔性肽连接于CH1区C端，由此获得肽链#1。肽链#2具有SEQ ID NO:18所示的氨基酸序列，其包含抗PD-L2纳米抗体D-Ye-22氨基酸序列(SEQ IDNO:19)，所述纳米抗体氨基酸序列C端直接连接人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:9)，由此获得肽链#2。
Bi-78-80，其结构示意图如图1A所示，由2条多肽链组成，肽链#1具有SEQ ID NO:20所示的氨基酸序列，其包含抗PD-L1的纳米抗体SEQ ID NO:2，所述纳米抗体氨基酸序列C端和衍生自人IgG1的CH1氨基酸序列SEQ ID NO:6直接连接；其包含抗PD-L1的纳米抗体C-Ye-18-5(SEQ ID NO:14)，所述纳米抗体氨基酸序列C端和衍生自人IgG1的CH1氨基酸序列(SEQ ID NO:3)直接连接；将抗人血清白蛋白的纳米抗体ALB8(SEQ ID NO：5)的C端通过5个氨基酸残基(DKTHT)(SEQ ID NO：21)的柔性肽连接于CH1区C端，由此获得肽链#1。肽链#2具有SEQ ID NO:18所示的氨基酸序列。
Bi-81-82，其结构示意图如图1A所示，由2条多肽链组成，肽链#1具有SEQ ID NO:22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:14)，所述纳米抗体氨基酸序列C端通过一段11个氨基酸(GGGGSGGGGSG)(SEQ ID NO：4)的柔性肽链和衍生自人IgG1的所示CH1氨基酸序列(SEQ ID NO:3)连接,将抗人血清白蛋白的纳米抗体ALB8(SEQ ID NO：5)的C端通过11个氨基酸残基(GGGGSGGGGSG)(SEQ ID NO：4)的柔性肽连接于CH1区C端，由此获得肽链#1。肽链#2具有SEQ ID NO:23所示的氨基酸序列，其包含抗PD-L2纳米抗体D-Ye-22氨基酸序列(SEQ IDNO:19)，所述纳米抗体氨基酸序列C端通过一段含有11个氨基酸(GGGGSGGGGSG)(SEQ ID NO：4)的柔性肽链和人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:9)，由此获得肽链#2。
在本实施例中，构建了1种抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体，命名为Bi-79-83，其结构示意图如图1A所示，由2条多肽链组成，肽链#1 具有SEQ ID NO:17所示的氨基酸序列。肽链#2具有SEQ ID NO:24所示的氨基酸序列，其包含抗PD-L2纳米抗体D-Ye-22氨基酸序列(SEQ IDNO:19)，所述纳米抗体氨基酸序列C端和人κ轻链恒定区(CL)氨基酸序列(SEQ ID NO:9)直接连接；将抗TIGIT的纳米抗体E-Ye-11的N端通过11个氨基酸残基(GGGGSGGGGSG)(SEQ ID NO：4)的柔性肽连接于CL区C端，由此获得肽链#2。
3.2 PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体的表达和纯化
在本实施例中，将编码实施例3.1中构建的抗PD-L1/PD-L2/人血清白蛋白三特异性抗体Bi-78-79、Bi-78-80和Bi-81-82及抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体Bi-79-83的2条链的核苷酸序列均通过多克隆位点连接入市售的真核表达载体pCDNA3.1(+)，在真核细胞中进行表达和纯化。表达质粒构建及蛋白表达和纯化方法同实施例1.2。
本研究利用SEC法对纯化产物纯度进行了检测，检测方法同实施例1.2。实验结果表明，4种多特异性抗体均就有较高的纯度(Bi-78-79：98.07％；Bi-78-80：98.62％；Bi-81-82：96.31；Bi-79-83：99.14％)。
3.3测定抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体的抗原结合能力
通过转染克隆到MCS的人PD-L1或者人PD-L2或者人TIGIT cDNA(购自Sino Biological)的pCHO1.0载体(购自Invitrogen)产生过表达人PD-L1或者人PD-L2或者人TIGIT的CHO细胞(CHO-hPD-L1细胞、CHO-hPD-L2细胞、CHO-hTIGIT细胞)。将扩大培养的CHO-hPD-L1/CHO-hPD-L2/CHO-hTIGIT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。将纯化的三特异性抗体用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30分钟，PBS清洗两次。纯化抗体样品孔100μl/孔加入用PBS稀释的鼠抗人IgG-Fab(PE)(购自Abcam)，对照抗体样品孔加入用PBS稀释的羊F(ab’)2抗人IgG-Fc(PE)(购自Abcam)，4℃孵育30分钟，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12所示，本发明的纯化样品 Bi-78-79、Bi-78-80和Bi-81-82和CHO-hPD-L1细胞及CHO-hPD-L2细胞均有结合活性；本发明的纯化样品Bi-79-83和CHO-hPD-L1细胞、CHO-hPD-L2细胞及CHO-hTIGIT细胞都有结合活性。
3.4测定抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体和人血清白蛋白在ELISA水平的结合
本实验通过ELISA反应的方法检测了抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体和人血清白蛋白的结合能力，实验方法同实施例1.3。
在如上方法的测定实验中，实验结果如图13所示。本发明所有抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体均能够在ELISA水平与人血清白蛋白结合。
3.5测定抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体阻断人PD-L1/PD-L2与PD-1结合活性
将CHO-hPD-1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。将纯化抗体Bi-78-79、Bi-78-80、Bi-81-82及Bi-79-83和对照抗体样品用PBS稀释，400nM开始3倍稀释共12个点，将上述稀释好的样品60μl/孔加入96孔样品稀释板，同时60μl/孔加入生物素化标记的人PD-L1蛋白或生物素化标记的人PD-L2蛋白(购自AcroBiosystems)，终浓度为500ng/ml，与样品4℃孵育30分钟。将共孵育样品100μl/孔加入上述带有细胞的96孔流式板中，4℃孵育30分钟,PBS清洗两次。100μl/孔加入用PBS稀释100倍的Streptavidin,R-Phycoerythrin Conjugate(购自Thermo fisher)，4℃孵育30分钟,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14所示。本发明所有抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体均可以阻断人PD-L1及人PD-L2与细胞表面人PD-1的结合。
3.6抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体阻断PDL1/PDL2/PD1/luc信号通路实验
PD-L1和PD-L2能够共表达于肿瘤细胞或免疫细胞上，本实施例利用共表达人PD-L1和人PD-L2的CHO细胞和过表达人PD-1且含有NFAT-Luciferase报告基因的Jurkat细胞共孵育的方法检测了纯化抗体Bi-78-79、Bi-78-80、Bi-81-82及Bi-79-83对PD-L1/PD-1通路和PD-L2/PD-1通路的同时阻断作用，具体方法如下。
共表达人PD-L1和人PD-L2的功能细胞(CHO-K1-PD-L1/PD-L2)调整密度为5×10 5细胞/ml，100μl/孔接种于96孔细胞培养白底板，置于37℃，5％CO 2培养箱培养过夜。将纯化抗体和对照抗体1640完全培养基梯度稀释后，备用。将过表达人PD-1且含有NFAT-Luciferase报告基因的Jurkat细胞(Jurkat-PD-1-NFAT)用1640完全培养基调整细胞密度至2.5×10 5细胞/ml，备用。取出白底板，吸去培养上清，将上述稀释好的样品40μl/孔加入白底板，同时40μl/孔加入Jurkat-PD-1-NFAT效应细胞悬液，置于37℃，5％CO 2培养箱贴壁培养6小时。向每孔中加入Bio-Glo TM试剂(Promega)，使用多功能酶标仪读取荧光信号值。
在如上方法的测定实验中，实验结果如图15所示。本发明的抗PD-L1/PD-L2/人血清白蛋白三特异性抗体或抗PD-L1/PD-L2/TIGIT/人血清白蛋白四特异性抗体可以在体外同时阻断PD-L1/PD-1和PD-L2/PD-1信号通路，其阻断效果和抗PD-1单克隆抗体Pembrolizumab相似。
综上，在CH1-CL的结构N端连接两个不同的纳米抗体结构域，在CH1的C端连接1个纳米抗体结构域形成3特异性抗体或在CH1-CL的结构C端分别连接两个不同的纳米抗体结构域形成4特异性抗体均能有效维持亲本抗体的结合活性。纳米抗体通过一段柔性肽链或者直接和CH1或CL的N端连接均可保持抗原结合能力。纳米抗体通过一段11个氨基酸的柔性肽链(GGGGSGGGGSG)或者通过一段5个氨基酸的较短肽链(DKTHT)和CH1的C端连接均可保持抗原结合能力。
本实施例使用抗PD-L1和抗PD-L2纳米抗体组合是结合同一细胞上的两种抗原，结果表明所有多特异性抗体均可同时和PD-L1和PD-L2结合并阻断PD-L1/PD-L2和PD-1的结合，激活下游信号通路。
实施例4：抗PD-L1/41BB/人血清白蛋白三特异性抗体
4.1抗PD-L1/41BB/人血清白蛋白三特异性抗体的构建
为了证实M-body在连接的是两个靶向位于不同细胞的不同靶点的纳米抗体时是否适用，本发明人构建了抗PD-L1/41BB/人血清白蛋白三特异性抗体命名为Bi-79-86，该三特异性抗体含有2种不同多肽，其结构示意图如图16所示。肽链#1具有SEQ ID NO:17所示的氨基酸序列。肽链#2具有SEQ ID NO:25所示的氨基酸序列，其包含抗41BB纳米抗体氨基酸序列(SEQ IDNO:26专利号)，所述纳米抗体氨基酸序列C端直接连接人κ轻链恒定区(CL)氨基酸序列SEQ ID NO:9，由此获得肽链#2。
4.2抗PD-L1/41BB/人血清白蛋白三特异性抗体的表达和纯化
在本实施例中，将编码实施例4.1中构建的抗PD-L1/41BB/人血清白蛋白三特异性抗体Bi-79-86的2条链的核苷酸序列均通过多克隆位点连接入市售的真核表达载体pCDNA3.1(+)，在真核细胞中进行表达和纯化。表达质粒构建及蛋白表达和纯化方法同实施例1.2。
本研究利用SEC法对纯化产物纯度进行了检测，检测方法同实施例1.2。实验结果表明，本研究获得的抗PD-L1/41BB/人血清白蛋白三特异性抗体纯度为95.08％。
4.3测定抗PD-L1/41BB/人血清白蛋白三特异性抗体的抗原结合能力
通过转染克隆到MCS的人PD-L1或者人41BB cDNA(购自Sino Biological)的pCHO1.0载体(购自Invitrogen)产生过表达人PD-L1或者人41BB的CHO细胞(CHO-hPD-L1细胞、CHO-41BB细胞)。将扩大培养的CHO-hPD-L1/CHO-41BB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。将纯化的三特异性抗体用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30分钟，PBS清洗两次。纯化抗体样品孔100μl/孔加入用PBS稀释的鼠抗人IgG-Fab(PE)(购自Abcam)，对照抗体样品孔加入用PBS稀释的羊F(ab’)2抗人IgG-Fc(PE)(购自Abcam)，4℃孵育30分钟，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17所示，本发明的抗PD-L1/41BB/人血清白蛋白三特异性抗体Bi-79-86和CHO-hPD-L1细胞及CHO-41BB细胞均有结合活性。
4.4测定抗PD-L1/41BB/人血清白蛋白三特异性抗体三特异性抗体和人血清白蛋白在ELISA水平的结合
本实验通过ELISA反应的方法检测了抗PD-L1/41BB/人血清白蛋白三特异性抗体Bi-79-86和人血清白蛋白的结合能力，实验方法同实施例1.3。
在如上方法的测定实验中，实验结果如图18所示，本发明抗PD-L1/41BB/人血清白蛋白三特异性抗体能够在ELISA水平与人血清白蛋白结合。
4.5测定抗PD-L1/41BB/人血清白蛋白三特异性抗体桥接表达PD-L1/41BB细胞的能力
人PD-L1和人41BB分别表达在肿瘤细胞和免疫细胞表面，本实施例通过细胞桥接实验验证了三特异性抗体Bi-79-86通过同时结合过表达人PD-L1的细胞(CHO-hPD-L1)和过表达人41BB的细胞(CHO-h41BB)，将两种细胞拉近的能力，具体方法如下。
各取2×10 7个CHO-hPDL1和CHO-h41BB细胞离心后用CellTrace TM CFSE、CellTracker TM Violet BMQC Dye两种染料分别重悬，37℃避光孵育12分钟；将Bi-079-086、A-Na-19、αHSA(Ablynx benchmark)用PBS稀释，400nM开始2倍稀释共12个点。将CHO-hPDL1和CHO-h41BB细胞离心后，加入PBS重悬，细胞比1:1混合后，100μl/孔加入96孔板中，离心弃去上清。用PBS稀释人血清白蛋白至2μg/ml，50μl/孔加入细胞板，同时加入50μl/孔加入上述配制完成的抗体。混匀，37℃避光孵育2小时，上CytoFlex(Bechman)流式细胞仪进行检测。
在如上方法的测定实验中，实验结果如图19所示，本发明抗PD-L1/41BB/人血清白蛋白三特异性抗体Bi-79-86能够在体系中含有高浓度人血清白蛋白的情况下拉近CHO-hPDL1和CHO-h41BB细胞，证明Bi-79-86可以同时结合表达于不同细胞表面的不同抗原，同时结合活性不受体系中的人血清白蛋白影响。
4.6抗PD-L1/41BB/人血清白蛋白三特异性抗体阻断人PD-L1与人PD-1结合
检测纯化的抗PD-L1/41BB/人血清白蛋白三特异性抗体Bi-79-86阻断PD-L1蛋白和PD-1细胞结合活性的方法同实施例1.6。在如上方法的测定实验中，实验结果如图20所示，本发明抗PD-L1/41BB/人血清白蛋白三特异性抗体Bi-79-86分 子可以阻断PD-L1蛋白与PD-1细胞的结合。
综上，在CH1-CL的结构N端连接两个不同的纳米抗体结构域，在CH1的C端连接1个纳米抗体结构域形成3特异性抗体能有效维持亲本抗体的结合活性。本实施例使用抗PD-L1和抗41BB纳米抗体组合是结合不同细胞上的两种抗原，结果表明本发明的三特异性抗体结构可以在细胞水平同时和PD-L1及41BB结合，并桥接分别表达PD-L1和41BB的细胞，该结合和桥接活性不受体系中的人血清白蛋白的影响。
实施例5：抗PD-L1/PD-L2双特异性抗体Fc融合蛋白
5.1抗PD-L1/PD-L2双特异性抗体Fc融合蛋白的构建
抗体的Fc结构域可以和FcRn结合，延长药物的体能半衰期；Fc结构域可以和其他Fc受体结合，引起下游的反应如ADCC/CDC；Fc结构域和ProteinA有特异性结合，可以便于蛋白纯化。
为了证实M-Body技术在和Fc融合的情况下是否适用，本发明人构建了抗PD-L1/PD-L2双特异性抗体命名为Bi-203-204，该三特异性抗体含有2种不同多肽，其结构示意图如图21所示。肽链#1具有SEQ ID NO:27所示的氨基酸序列，其包含抗PD-L1的纳米抗体SEQ ID NO:28，所述纳米抗体氨基酸序列C端和衍生自人IgG1的SEQ ID NO:3所示CH1氨基酸序列直接连接；将人IgG1(LALA突变型)Fc(SEQ ID NO：29)结构域直接和CH1区C端连接，由此获得肽链#1。肽链#2具有SEQ ID NO:30所示的氨基酸序列，其包含抗PD-L2纳米抗体HZ-D-NA-96-01氨基酸序列SEQ IDNO:31，所述纳米抗体氨基酸序列C端直接连接人κ轻链恒定区(CL)氨基酸序列SEQ ID NO:9，由此获得肽链#2。
5.2抗PD-L1/PD-L2双特异性抗体Fc融合蛋白的表达和纯化
在本实施例中，将编码实施例5.1中构建的抗PD-L1/PD-L2双特异性抗体Bi-203-204的各2条链的核苷酸序列均通过多克隆位点连接入市售的真核表达载体pCDNA3.1(+)，在真核细胞中进行表达和纯化，获得了双特异性抗体Bi-203-204。具体操作如下。
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，pH 7.4)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柠檬酸钠,pH 3.2)室温振荡5-10分钟，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，pH 8.54)的收集管中混匀。
利用HPLC检测获得蛋白的纯度。HPLC方法如下，流动相：150mM Na 2HPO 4·12H 2O，pH7.0。色谱条件：检测波长：280nm，柱温：25℃，流速：0.35ml/min，检测时间：20分钟，Zenix-C SEC-300色谱柱(SEPAX 4.6×300mm，3μm)。
实验结果显示，抗PD-L1/PD-L2双特异性抗体Fc融合蛋白Bi-203-204具有较好的纯度(>99％)，且根据纯化获得的蛋白分子大小可知，Bi-203-204的纯化分子含有4条肽链且正确配对。
5.3测定抗PD-L1/PD-L2双特异性抗体Fc融合蛋白的抗原结合能力
将扩大培养的CHO-hPD-L1/CHO-hPD-L2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。将纯化的双特异性抗体用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30分钟，PBS清洗两次。纯化抗体样品孔100μl/孔加入用PBS稀释羊F(ab’)2抗人IgG-Fc(PE)(购自Abcam)，4℃孵育30分钟，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22所示，本发明的抗PD-L1/PD-L2双特异性抗体Fc融合蛋白Bi-203-204和CHO-hPD-L1细胞及CHO-hPD-L2细胞均有结合活性。
综上，在CH1-CL的结构N端连接两个不同的纳米抗体结构域，在CH1的C端连接人抗体Fc结构域形成双特意特异性抗体Fc融合蛋白能有效维持亲本抗体的结合活性。同时Fc结构域可以有利于抗体纯化，并形成稳定的4聚体结构。
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 (10)

  1. 一种构建多特异性抗体的方法，其特征在于，包括步骤：
    (i)分别构建第一多核苷酸和第二多核苷酸，所述第一多核苷酸编码从N端到C端具有如式I所示的结构的第一多肽，并且所述第二多核苷酸编码从N端到C端具有如式II所示的结构的第二多肽，
    A1-L1-B1-L2-CL-L3-A2  (式I)
    A3-L4-B2-L5-CH1-L6-A4  (式II)
    其中，
    A1、A2、A3和A4各自独立地为靶向目标靶点的抗体或其抗原片段，并且A1、A2、A3和A4各自靶向的目标抗原可以是相同的或不同的；
    L1、L2、L3和L4各自独立地为无或接头元件；
    B1和B2均为无，或B1和B2分别为靶向同一目标靶点的抗体的VL区和VH区；
    并且所述第一多肽的CL区和所述第二多肽的CH1区之间，可形成二硫键，从而使所述抗体具有异二聚体的形式；
    (ii)表达所述第一多核苷酸和第二多核苷酸，从而获得所述的第一多肽和第二多肽，使其发生二聚化，从而形成具有异二聚体形式的多特异性抗体。
  2. 一种多特异性抗体，其特征在于，所述抗体包含从N端到C端如式I所示的第一多肽和从N端到C端如式II所示的第二多肽，
    A1-L1-B1-L2-CL-L3-A2  (式I)
    A3-L4-B2-L5-CH1-L6-A4  (式II)
    其中，
    A1、A2、A3和A4各自独立地为靶向目标靶点的抗体或其抗原片段，并且A1、A2、A3和A4各自靶向的目标抗原可以是相同的或不同的；
    L1、L2、L3和L4各自独立地为无或接头元件；
    B1和B2均为无，或B1和B2分别为靶向同一目标靶点的抗体的VL区和VH区；
    并且所述第一多肽的CL区和所述第二多肽的CH1区之间，可形成二硫键，从而使所述抗体具有异二聚体的形式。
  3. 一种融合蛋白，其特征在于，所述融合蛋白中包括如权利要求2所述的多 特性抗体，并且，所述多特异性抗体中的所述第一多肽从N端到C端具有如式III所示的结构，
    A1-L1-CL-L3-Fc  (式III)
    其中，Fc是为抗体的Fc段，包含CH2结构域和CH3结构域；
    并且，所述的融合蛋白可通过Fc段之间的二硫键作用形成同源二聚体。
  4. 一种分离的多核苷酸组合，其特征在于，所述多核苷酸组合包括第一核苷酸和第二核苷酸，所述第一核苷酸编码如权利要求2所述的多特异性抗体或如权利要求3所述的融合蛋白的第一多肽，并且所述第二核苷酸编码第二多肽。
  5. 一种载体，其特征在于，所述载体含有如权利要求4所述的多核苷酸组合。
  6. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求5所述的载体，或其基因组中整合有如权利要求4所述的多核苷酸组合；
    或者，所述的宿主细胞表达如权利要求2所述的多特异性抗体或如权利要求3所述的融合蛋白。
  7. 一种产生抗体的方法，其特征在于，包括步骤：
    (a)在合适的条件下，培养如权利要求6所述的宿主细胞，从而获得含如权利要求2所述的多特异性抗体或如权利要求3所述的融合蛋白的培养物；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)如权利要求2所述的多特异性抗体或如权利要求3所述的融合蛋白；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. 如权利要求2所述的多特异性抗体、如权利要求3所述的融合蛋白或如权利要求8所述的免疫偶联物的用途，其特征在于，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：检测样品中是否存在所述的目标靶点分子；
    并且，所述药剂用于治疗或预防表达目标靶点分子的肿瘤。
  10. 一种药物组合物，其特征在于，含有：(i)如权利要求2所述的多特异性抗体、如权利要求3所述的融合蛋白，或如权利要求8所述的免疫偶联物；以及(ii)药学上可接受的载体。
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