WO2018205755A1 - 多特异性蛋白药物及其文库、以及制备方法和应用 - Google Patents

多特异性蛋白药物及其文库、以及制备方法和应用 Download PDF

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WO2018205755A1
WO2018205755A1 PCT/CN2018/080058 CN2018080058W WO2018205755A1 WO 2018205755 A1 WO2018205755 A1 WO 2018205755A1 CN 2018080058 W CN2018080058 W CN 2018080058W WO 2018205755 A1 WO2018205755 A1 WO 2018205755A1
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protein drug
nucleic acid
antibody
protein
dna
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French (fr)
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周界文
潘利强
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Assembly Medicine LLC
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Assembly Medicine LLC
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Priority to AU2018266091A priority Critical patent/AU2018266091B2/en
Priority to EP18797570.1A priority patent/EP3623503A4/en
Priority to KR1020197036149A priority patent/KR102384538B1/ko
Priority to CA3062962A priority patent/CA3062962C/en
Priority to JP2020513386A priority patent/JP6927618B2/ja
Priority to CN201880029385.0A priority patent/CN110621814B/zh
Priority to US16/612,004 priority patent/US11331365B2/en
Publication of WO2018205755A1 publication Critical patent/WO2018205755A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/54—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 organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/56—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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
    • C07K16/2809—Immunoglobulins [IG], 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 the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • a conventional monoclonal antibody specifically binds to an antigenic site, and its Fc end binds to an Fc receptor on the surface of NK cells, thereby further eliciting immune cell activity.
  • it is unable to recruit T cells with great lethality, so it is impossible to maximize the mobilization of immune system activity.
  • traditional monospecific antibodies are difficult to fully utilize or block the signaling pathway based on the antigen or its associated compensatory pathway by binding to one antigenic site, resulting in unsatisfactory therapeutic effects or prone to drug resistance, such as for CD20.
  • Antibodies recognize different sites on the surface of CD20, so the activity of each antibody is significantly different; antibody therapy targeting VEGF on the surface of glioblastoma (GBM) cells can lead to up-regulation of angiopoietin-2 (Ang-2) expression, leading to anti-VEGF Antibody resistance.
  • GBM glioblastoma
  • the present invention provides a platform technology for linking a plurality of antibody drugs to form a dual or multispecific drug using a L-nucleic acid chain framework, which can conveniently and efficiently couple a plurality of antibody drugs together to form an individual for disease.
  • a protein drug library comprising C different protein drug monomers, wherein the protein drug monomer comprises a protein drug element portion and the protein a portion of the nucleic acid element to which the drug element is partially linked, and a portion of the nucleic acid element of the protein drug monomer and the nucleic acid element portion of the at least one different protein drug monomer may form a double-stranded pair structure by complementation, thereby constituting the multimer Body protein drug;
  • the multimeric protein drug is a multispecific protein drug.
  • the "depolymerization” refers to the dissociation of a multimeric protein drug to form a protein drug monomer.
  • the ratio Q (ie, E2/E1) (Q is a molar ratio) of the nucleic acid element portion E2 to the protein drug element portion E1 is 10-1.
  • Q is a molar ratio
  • the ratio Q (ie, E2/E1) (Q is a molar ratio) of the nucleic acid element portion E2 to the protein drug element portion E1 is 10-1.
  • 4-1 more preferably 2-1, or about 1-1.
  • P is a protein drug molecule (ie, a protein drug component part);
  • A is a nucleic acid sequence selected from the group consisting of a left-handed nucleic acid, a peptide nucleic acid, a locked nucleic acid, a thio-modified nucleic acid, a 2'-fluoro-modified nucleic acid, a 5-hydroxymethylcytosine nucleic acid, or a combination thereof;
  • the proteinaceous drug molecule P is selected from the group consisting of an antibody, a ligand that activates or inhibits a receptor or other protein, a biologically active enzyme, or a combination thereof.
  • the antibody is selected from the group consisting of a single chain antibody, a Nanobody, a Fab, a monoclonal antibody, or a combination thereof.
  • the antibody is selected from the group consisting of an anti-PD-1 single chain antibody, an anti-PD-L1 single chain antibody, an anti-CTLA-4 single chain antibody, an anti-CD-3 single chain antibody, or a combination thereof .
  • the antibody is selected from the group consisting of an antibody for treating a cancer, an autoimmune disease, an immune checkpoint, an organ transplant rejection, rheumatoid arthritis, diabetes, hemophilia.
  • the target to which the antibody is directed is selected from the group consisting of CD3, HER2, PD-1.
  • the mutation does not affect drug function.
  • the mutation comprises introducing one or more cysteine residues at the carboxy terminus (C-terminus) of the antibody.
  • X is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.
  • the haloacetyl group is selected from the group consisting of iodoacetyl and bromoacetyl.
  • the Y is a left-handed nucleic acid.
  • the Y is AAAA, AAA or AA.
  • Z is 0-30 nucleotides.
  • Z is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
  • the Z is AAAA, AAA or AA.
  • the position of the modifying group on the nucleic acid A and/or Y is selected from the group consisting of: the 5' end, the 3' end, and any intermediate position.
  • any two complementary pairing regions in the nucleic acid A have a transition region between 0 and 10 nt in length.
  • the complementary pairing region has a length of from 5 to 100 nt; preferably from 8 to 50 nt; more preferably from 10 to 30 nt; still more preferably from 12 to 20 nt; most preferably from 10 to 15 nt. .
  • helper nucleic acid molecule is in a single-stranded form.
  • the nucleic acid T is a nucleic acid of an unconjugated protein drug.
  • the length of the nucleic acid T is 1-1.5 times the sum of the number of pairs of monomeric nucleic acids in all (b).
  • the pharmaceutical information is protein drug information required for treating a disease of a subject to be treated, including a type, a combination (eg, antibody combination), and a ratio of the protein drug (the ratio of any two protein drugs P is 1: 1-1:20).
  • the assembly conditions are: 5-50 degrees (preferably 25-40 degrees), and the reaction is 1-15 minutes (preferably 5-10 minutes).
  • a multimeric protein drug which is a polymer formed by a D protein drug monomer which forms a double-stranded pair structure by nucleic acid complementation, wherein D is a positive integer ⁇ 2;
  • the protein drug monomer comprises a protein drug element portion and a nucleic acid element portion linked to the protein drug element portion, and the nucleic acid element portion of one of the protein drug monomers is different from another protein drug
  • the nucleic acid element portion of the monomer can form a double-stranded paired structure by complementation.
  • the nucleic acid element portion is nuclease resistant.
  • the nucleic acid element portion is selected from the group consisting of a left-handed nucleic acid, a peptide nucleic acid, a locked nucleic acid, a thio-modified nucleic acid, a 2'-fluoro-modified nucleic acid, a 5-hydroxymethylcytosine nucleic acid, or combination.
  • D is a positive integer from 2 to 20; preferably D is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 Or 20.
  • the multimeric protein drug is a multispecific protein drug.
  • the multimeric protein drug is an anticancer drug.
  • the half-life H1 of the multimeric protein drug that depolymerizes in vivo is greater than the in vivo half-life H2 of the protein drug component alone.
  • the ratio of H1/H2 is from 1 to 100, preferably from 10 to 50, more preferably from 10 to 20.
  • a pharmaceutical composition comprising
  • Figure 1 is a schematic representation of a multispecific antibody based on self-assembly of L-nucleic acid. It consists of a plurality of antibodies or antibody fragments, a plurality of self-assemblable L-nucleic acids, and a linker.
  • Figure 2 is a schematic diagram showing the shape and pairing pattern of the L-DNA framework of the tetraspecific antibody.
  • Figure 3 is a graph showing the self-assembly results of four SMCC-L-DNAs. 3% agarose gel electrophoresis.
  • the first to fourth lanes are SMCC-L-DNA single strands, wherein the first lane is strand 1, the second lane is strand 2, the third lane is strand 3, and the fourth lane is strand 4.
  • Lanes 5-8 are assembled bands in which the magnesium ion concentration in lane 5 is 0 mM and the magnesium ion concentrations in lanes 6, 7, and 8 are 1 mM, 2 mM, and 4 mM, respectively.
  • Figure 4 is a graph showing the results of self-assembly of tetraspecific antibodies. SDS-PAGE gel was stained with ethidium bromide (EB) and Coomassie blue, respectively, to visualize the DNA and protein fractions.
  • Lane 1 is an unconjugated anti-PD-1 single chain antibody
  • lane 2 is an anti-PD-L1 single chain antibody conjugated to strand 1 (L-DNA)
  • lane 3 is a coupled strand 2 (L-DNA)
  • Anti-PD-L1 single-chain antibody lane 4 is an anti-PD-1 single-chain antibody conjugated to chain 3 (L-DNA)
  • lane 5 is an anti-CD3 single-chain antibody conjugated to chain 4 (L-DNA) .
  • Lane 6 is a mixture of four single-chain antibody-L-DNA reaction solutions.
  • FIG. 5 is a schematic diagram showing the expression results of MBP-fused single-chain antibody mutants.
  • Lane 1 in the left is a control experiment without IPTG induction.
  • Lanes 2, 3, and 4 are MBP-anti-CD3 single-chain antibodies, MBP-anti-CEA. Single-chain antibody, protein expression of MBP-anti-PDL1 single-chain antibody.
  • the right panel is a schematic representation of the solubility of MBP-fused single-chain antibody mutants.
  • Lane 1 is the whole bacterial lysate
  • lane 2 is the soluble fraction
  • lane 3 is the inclusion body component.
  • Figure 8M is a DNA standard with a minimum band of 25 bp and other bands increasing by 25 bp.
  • Lanes 1-4 were four L-DNA (20 uM), respectively, and the loading was 5 ul.
  • Lanes 5 and 6 are assembled at a room temperature and 37 degrees, respectively, with the addition of a trimer followed by a fourth L-DNA.
  • Lane 7 is an assembly method in which four L-DNAs are directly mixed under conditions of 37 degrees.
  • Figure 10M is a broad molecular weight protein marker; 1-4 is a 1 uM L-DNA-fusion protein monomer, 5 is a 1-4 assembled product; and 6-9 is a 2 uM L-DNA-fusion protein monomer, 10 is a product assembled from 1 to 4.
  • Figure 12 Results of in vitro activity of CEA/PD-L1/CD3 tetraspecific antibodies prepared based on the L-DNA framework.
  • the colorectal cancer cell line LS174T was positive for CEA as a cell model for this in vitro activity assay.
  • FIG. 13 Experimental results of anti-CEA/PD-L1/CD3 tetraspecific antibody-activated T cells.
  • IFN- ⁇ secreted by CD3 positive cells was used as a test subject.
  • the positive control was Dynabeads (fine beads conjugated with anti-CD28/CD3 antibody on the surface to efficiently activate T cells) and the negative control was the buffer used for the antibody.
  • a protein drug library comprising ⁇ 2 different protein drug monomers, the protein drug monomers including protein drug component parts and a nucleic acid element portion partially linked to a protein drug element, the nucleic acid element portion being a nucleic acid resistant to degradation by a nuclease in vivo (eg, a left-handed nucleic acid), and the nucleic acid element portion of one of the protein drug monomers is different from at least one
  • the nucleic acid element portion of the protein drug monomer can form a double-stranded pairing structure by complementation, and the corresponding protein drug monomer can be selected from the protein drug library according to needs (such as the condition and diagnosis result of an individual), rapid (1)
  • the present invention has been completed on the basis of high-efficiency, low-cost, high-yield assembly of multi-target, in vivo stable multispecific protein drugs (for example, multispecific antibodies).
  • protein drug library As used herein, the terms “protein drug library”, “protein drug library of the invention”, “pharmaceutical library of the invention” are used interchangeably.
  • multimeric protein drug of the invention As used herein, the terms “multimeric protein drug of the invention”, “multimeric protein drug”, “multispecific protein drug”, “multimeric drug protein of the invention”, “multimeric drug protein”, The “multimeric proteins of the invention” are used interchangeably.
  • the present invention provides a protein drug library comprising C different protein drug monomers, wherein the protein drug monomer comprises a protein drug element portion and a nucleic acid linked to the protein drug element portion a component portion, and wherein the nucleic acid element portion of one of the protein drug monomers and the nucleic acid element portion of at least one different protein drug monomer can form a double-stranded pairing structure by complementation, thereby constituting a multimeric protein drug; , C is a positive integer ⁇ 2.
  • the library of the present invention further contains at least two or more protein drug monomers, and the preferred protein drug monomer has the structure of the above formula I.
  • the protein drug monomer of the present invention has a specific structure, not only can the drug protein in a multimeric form be rapidly assembled, but also the multimeric protein drug formed is multi-specific, and can simultaneously target a plurality of different proteins. Targets to meet the needs of multiple targets simultaneously or sequentially during disease treatment.
  • the multimeric protein drugs of the present invention also have unexpected stability in vivo and can be present in the body for a long time and remain active without being rapidly degraded.
  • the antibody element in the protein drug monomer is not particularly limited, and a representative example (selected from the group below): a single chain antibody, a Nanobody, a Fab, a monoclonal antibody, or a combination thereof.
  • antibodies of various origins can be used to prepare protein drug monomers.
  • An outstanding feature of the library of the present invention is that antibody fragments expressed by prokaryotic systems such as E. coli or eukaryotic systems such as yeast, CHO cells can be used, thereby greatly reducing production costs.
  • the corresponding protein drug monomers which can be coupled to each other are selected from the library, and after mixing in the desired antibody ratio, the assembly process can be completed in 1 minute.
  • the nucleic acid element of the protein drug monomer can be designed into a polymer framework such as a dimer, a trimer or a tetramer by a sequence, thereby completing a trispecific or even a tetra which cannot be easily achieved by conventional antibody engineering. Preparation of multispecific antibodies such as specific antibodies.
  • Multimeric protein drug of the invention and preparation thereof are Multimeric protein drug of the invention and preparation thereof
  • the nucleic acid element portion is nuclease resistant.
  • the nucleic acid element portion is selected from the group consisting of a left-handed nucleic acid, a peptide nucleic acid, a locked nucleic acid, a thio-modified nucleic acid, a 2'-fluoro-modified nucleic acid, a 5-hydroxymethylcytosine nucleic acid, or combination.
  • the multimeric protein drug of the present invention can be formed by, for example, assembly of a protein drug monomer of Formula I.
  • a multimeric protein drug refers to a multimeric antibody (multispecific antibody), such as a two, three, four, five or six specific antibody.
  • multispecific antibody such as a two, three, four, five or six specific antibody.
  • the multimeric antibody of the present invention contains the specificity of two or more antibodies, and can target an epitope that binds to multiple antigens, or multiple epitopes of one antigen, thereby sufficiently blocking the downstream of the antigen itself.
  • the protein drug of the present invention is a multispecific antibody using a L-nucleic acid.
  • a nucleic acid is a double-stranded molecule which can be rapidly and specifically paired. Therefore, if an antibody fragment (such as a single-chain antibody, a Nanobody, a Fab, etc.) is coupled to a nucleic acid single strand, it can be designed.
  • the nucleic acid sequence allows two or more nucleic acid single strands to be rapidly paired to form a multimer, thereby guiding the antibody fragment to form a multimer, thereby completing the preparation of the multispecific antibody.
  • the pharmaceutical composition of the present invention can be directly used for treatment (e.g., anti-tumor treatment), and thus can be used to prolong the half-life of the drug, and further, other therapeutic agents can be used at the same time.
  • treatment e.g., anti-tumor treatment
  • 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 per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
  • specific doses should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the multi-specific antibody of the invention is simple and rapid to prepare, and the assembly of various antibodies can be completed by using a L-nucleic acid chain in one minute;
  • the multispecific antibody of the present invention has a wide transformation space, and any type of antibody (such as single chain antibody, Nanobody, Fab) can be assembled into a multispecific antibody;
  • each part of the antibody of the multispecific antibody can be separately prepared, and then subjected to simple in vitro assembly;
  • an antibody drug library can be constructed, and an antibody drug suitable for individualized precise treatment can be quickly and easily prepared according to the provided disease and/or pharmaceutical information, and the cost is low and versatile. it is good.
  • the L-nucleic acid strand framework is formed by base pairing of two or more L-nucleic acid single strands.
  • the 5' or 3' end of each L-nucleic acid single strand is activated to a group for subsequent modification (such as NH 2 or the like), and then one end of a linker (such as SMCC, SM (PEG), SPDP, etc.) is used.
  • the activating group on the L-nucleic acid single strand is coupled.
  • L-nucleic acids with a linker can be assembled into the desired L-nucleic acid strand framework. After determining that the L-nucleic acid with the linker can be successfully self-assembled into a framework, the L-nucleic acid single strand with the linker can be coupled to the antibody for subsequent assembly.
  • a N is an antibody or an antibody fragment, such as a single-chain antibody, a Nanobody, a Fab, etc.
  • the chain nucleic acid is composed, and one end of the single-stranded nucleic acid has a reactive group modification, such as NH 2 or the like; the number of single-stranded nucleic acids can be adjusted according to the type of the multi-specific antibody, for example, the tetra-specific antibody requires a minimum number of single-stranded nucleic acids.
  • a linker is used to link a reactive group of a single-stranded nucleic acid to a specific ligation site on the antibody (eg, an SH group on a mutant cysteine residue).
  • the L-nucleic acid framework of the present invention can be basically prepared by the following procedure.
  • Determining the type of multispecific antibody to be prepared eg, a trispecific antibody
  • determining the desired number of L-nucleic single strands M based on the number of antibodies in the multispecific antibody designing the corresponding number of L-nucleic acid single stranded sequences
  • the stability of the nucleic acid frame of interest is regulated, and the possibility of non-specific pairing between nucleic acid strands is reduced.
  • non-specific pairing is less than -10 kcal/mole, meaning that the tetramer is assembled more easily than the non-specific pairwise pair, the form of the tetramer It is the most stable in the reaction system.
  • Activation of the L-nucleic acid includes reactive group modification at its 5' or 3' end and subsequent ligation of the linker.
  • the reactive group modification can be custom made by the nucleic acid synthesis company; the linker generally has a bifunctional group, that is, a reactive group at one end that can couple the nucleic acid, and the other end can be linked to a specific site on the antibody (such as SH).
  • all of the L-nucleic acids constituting the framework are added with an NH 2 modification at the 5' end, and then a linker, a bi-functional cross-linking agent, SMCC (4-(N-maleimidomethyl), is used.
  • SMCC bi-functional cross-linking agent
  • Cyclohexane-1-carboxylic acid succinimide ester sodium salt The NH 2 on the nucleic acid is coupled via an amide bond.
  • the maleimide group at the other end of the linker is in a free state, and can be used for subsequent coupling of the thiol group (SH) on the antibody, thereby completing the activation of the L-nucleic acid.
  • the degree of nucleic acid frame polymerization can be verified by, for example, agarose gel electrophoresis.
  • 3% agarose gel electrophoresis is selected to analyze the degree of nucleic acid frame polymerization; comparing the single-strand size of the L-nucleic acid, a frame formed by mixing a plurality of L-nucleic acids can be easily derived. Size, and thus the degree of polymerization.
  • L-nucleic acid frameworks contemplated by the present invention can be similarly prepared in accordance with the methods described above and in the preferred embodiments, without limitation.
  • a specific site eg, a mutation site, Cys
  • a specific site is introduced into the antibody for conjugation of the linker.
  • a single-chain antibody against PD-L1/PD-1/CD3 is selected for preparation of a trispecific antibody, wherein PD-1 and CD3 are sites located on the surface of T cells, the main effects are respectively To suppress the inhibition of anti-tumor activity and activate CD8-positive T cells.
  • PD-L1 is located on the surface of some tumor cells and prevents T cells from further killing by acting with PD-1.
  • each single-chain antibody for conjugated SMCC-activated L-DNA single strands, each ligated to a different L-DNA single strand, and Two anti-PD-L1 single-chain antibodies are at one end of the multispecific antibody, while single-chain antibodies against PD-1 and CD3 are at the other end, facilitating the recruitment of T cells to tumor cells.
  • the 5' or 3' end of the L-nucleic acid is modified with NH 2 and then according to the difference of the linker, the following main preparation methods can be used, wherein one end functional group of the linker is NHS (N-hydroxysuccinimide) or Sulfo. -NHS (N-hydroxysuccinimide sulfonate sodium salt) for rapid coupling of NH 2 groups at one end of the L-nucleic acid.
  • the linker comprising the diisomeric group first reacts with the NH 2 of the L-nucleic acid, and secondly, after the sulfhydryl group on the reduced antibody, the other end group reacts with the thiol group to form a stable chemical bond.
  • SMCC (N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester
  • PEG polyethylene glycol modified 4-(N) - Maleimide methyl) cyclohexane-1-carboxylic acid succinimide ester
  • Haloacetyl (Haloacetyl).
  • the group of the linker used to couple the sulfhydryl group on the antibody is a haloacetyl group such as iodine or bromoacetyl.
  • Halogen ions may be substituted by nucleophilic groups to form stable thioether bonds.
  • Common linkers are SBAP (N-maleimidomethyl[4-bromoacetyl]aminobenzoate), SIAB (N-maleimidomethyl[4-iodoacetyl]aminobenzene Formate) and so on.
  • Pyridyldithiol The group of the linker used to couple the thiol group on the antibody is thiopyridine.
  • the thiopyridine can react with the free sulfhydryl group to form a disulfide bond.
  • Common linkers are SPDP (3-(2-pyridinedithio)propionic acid N-hydroxysuccinimide ester) and the like.
  • any one L-nucleic acid single strand can be specifically complementary paired with the other two L-nucleic acid single strands, but not paired with the fourth strand.
  • the Gibbs change ⁇ G of the specific complementary pair is much smaller than the non-specific pairing, and the Gibbs change ⁇ G of the specific complementary pair is about -34 kcal/mole, not the special The heterosexual pairing is less than -10 kcal/mole, which means that the tetramer is assembled more easily than the non-specific pairwise pair, and the tetramer form is the most stable in the reaction system.
  • Chain 4 (L-DNA4): SEQ ID NO: 4
  • the 5' end has an NH 2 group modification for coupling the NHS of the SMCC.
  • the base sequences following AAAA and AAA are paired with the other two strands, respectively, and the paired Gibbs energy change ⁇ G of each fraction is about -34 kcal/mole.
  • the L-DNA single strand was dissolved in phosphate buffer (50 mM NaH 2 PO 4 , 150 mM NaCl, pH 7.0) to prepare a mother liquor having a final concentration of 200 uM.
  • the SMCC powder was dissolved in dimethyl sulfoxide (DMSO) and a 250 mM SMCC mother liquor was freshly prepared. 10 to 50 times the molar amount of the SMCC mother liquid is added to the L-DNA single-strand mother liquor, and the mixture is rapidly mixed and reacted at room temperature for 30 minutes to 2 hours.
  • DMSO dimethyl sulfoxide
  • the concentration of each SMCC-L-DNA single strand was determined.
  • Four appropriate SMCC-L-DNA single strands to be reacted were preheated at 40 degrees for 5 min, and then four SMCC-L-DNA single strands were mixed in an equimolar amount at 40 °C and incubated for 1 min.
  • the reaction system was set to different magnesium ion concentrations to explore the effect of magnesium ion concentration on the formation of the framework. 0.25 ul of SMCC-L-DNA single strand and reaction product were analyzed by 3% agarose gel electrophoresis. As shown in Fig.
  • the SMCC-L-DNA single strand has a size of about 25 bp, and the main band formed after mixing is about 100 bp, indicating that four different SMCC-L-DNA single strands form a tetramer.
  • the body frame, and different magnesium ion concentrations do not affect its self-assembly, showing its extremely high stability.
  • the gene sequence of the anti-PD-1/PD-L1/CD3 single-chain antibody was optimized to the codon of E. coli preference, and NcoI and XhoI restriction sites were added to both ends of the gene, but subcloned into the pET22b plasmid. Between the NcoI/XhoI sites.
  • the amino acid sequences of the anti-PD-1/PD-L1/CD3 single chain antibodies are SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.
  • the pET22b plasmid can direct the secretion of single-chain antibodies into the periplasmic space due to the pelB signal peptide sequence.
  • the cells after expression were collected by centrifugation, resuspended in phosphate buffer (50 mM NaH 2 PO 4 , 150 mM NaCl, pH 7.0), protease inhibitor cocktail (Sigma), and disrupted by sonication.
  • phosphate buffer 50 mM NaH 2 PO 4 , 150 mM NaCl, pH 7.0
  • protease inhibitor cocktail Sigma
  • the DNase I hydrolase was added and incubated on ice for 1 hour. After the incubation, the bacterial solution was centrifuged at 17,000 rpm for 20 minutes to collect the supernatant.
  • the single-chain antibody in the supernatant was purified using a Hitrap Protein L affinity column, and the supernatant was passed through the column at a rate of 0.25 ml/min, and then washed with a large amount of phosphate buffer (50 mM NaH 2 PO 4 , 150 mM NaCl, pH 7.0) at 1 ml/min. The flow rate washes the column until the heteroprotein no longer flows out (according to UV absorption on the AKTA protein chromatography system), gradient elution from 0 to 100% elution buffer (50 mM NaH 2 PO 4 , 150 mM NaCl, pH 2.3) is combined on the column.
  • Single-chain antibody The single chain antibody fraction was collected and the pH was adjusted to 7.0.
  • the purified single-chain antibody is incubated with a 10-50 molar excess of a reducing agent (such as TCEP, DTT, mercaptoethanol, etc.) for 30 min at room temperature. After the completion of the incubation, the reducing agent in the reaction system was quickly removed using a PD-10 desalting column while the buffer was replaced with a phosphate buffer (50 mM NaH 2 PO 4 , 150 mM NaCl, pH 7.0). After measuring the concentration of the single-chain antibody, an SMCC-L-DNA single chain (prepared in Example 2) in an excess of 1 to 4 times the molar ratio was immediately added, mixed uniformly, and reacted at room temperature for 1 hour.
  • a reducing agent such as TCEP, DTT, mercaptoethanol, etc.
  • the single-chain antibody-L-DNA is separated and purified by an anion exchange column (HiTrap Q HP column) to remove unreacted single-chain antibody and excess SMCC-L-DNA single strand.
  • the separation process was carried out by gradient elution with a loading buffer of 50 mM NaH 2 PO 4 , pH 7.0, elution buffer of 50 mM NaH 2 PO 4 , 1 M NaCl pH 7.0, 0-100% elution buffer for gradient washing.
  • the unreacted single-chain antibody, the single-chain antibody-L-DNA, and the excess SMCC-L-DNA single strand were sequentially peaked.
  • the single-chain antibody-L-DNA was collected, and after concentration, the buffer was replaced with 50 mM NaH 2 PO 4 , 150 mM NaCl, pH 7.0 using a PD-10 desalting column.
  • the effluent was a single-chain antibody after excision of the MBP fusion protein, and the effluent was collected. Since the nucleic acid such as DNA is negatively charged, the single-chain antibody-L-DNA is separated and purified by an anion exchange column (HiTrap Q HP column) to remove the TEV enzyme. The separation process was carried out by gradient elution.
  • L-DNA Compared to D-DNA, L-DNA has the advantage of being unable to be degraded by DNase in nature. There are a variety of DNases in the human body. To verify whether the L-DNA tetramer framework can be degraded or depolymerized by DNase, DNAse I, T7 endonuclease, S1 nuclease, exonuclease are selected. I (Exonuclease I) treats the D-DNA and L-DNA tetramer framework. The four monomer sequences of D-DNA and L-DNA correspond one-to-one, and the assembly method is assembled by the improved two-step method of Example 9. After various enzymes were added to the D-DNA or L-DNA tetramer framework, they were sampled in a 37 degree water bath for 17 hours, and analyzed by 2% agarose electrophoresis.
  • the L-DNA tetramer framework can withstand the treatment of four DNases without any degradation.
  • D-DNA tetramers are almost completely degraded by DNAse I and S1 nucleases, and can also be disrupted by exonuclease 1 and T7 DNA endonucleases. Therefore, the L-DNA tetramer framework cannot be degraded by common DNases.
  • the L-DNA tetramer framework of Example 1 was used to assemble an MBP (maltose binding protein)-anti-PDL1 single-chain antibody fusion protein (hereinafter referred to as a fusion protein, A tetramer having a molecular weight of 69 kDa).
  • MBP maltose binding protein
  • a tetramer having a molecular weight of 69 kDa The MBP-anti-PDL1 single-chain antibody fusion protein was prepared as in Example 6.
  • Four DNAs of the L-DNA tetramer framework were coupled to the fusion protein and purified according to the method described in Example 7 to obtain four L-DNA-fusion proteins.
  • Example 12 Evaluation of in vitro activity of tetraspecific antibodies prepared based on L-DNA framework
  • Example 13 Ability of anti-CEA/PD-L1/CD3 tetraspecific antibodies to activate T cells

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Abstract

提供了多特异性蛋白药物及其文库、以及制备方法和应用。具体地，提供了一种蛋白药物文库，所述蛋白药物文库包括C种不同的蛋白药物单体，其中，所述的蛋白药物单体包括蛋白药物元件部分以及与所述蛋白药物元件部分相连的核酸元件部分，并且一个所述蛋白药物单体的所述的核酸元件部分与至少一个不同的蛋白药物单体的核酸元件部分可通过互补形成双链配对结构，从而构成多聚体的蛋白药物；其中，C为≥2的正整数。

Description

多特异性蛋白药物及其文库、以及制备方法和应用 技术领域
本发明涉及药物领域。具体地涉及一种蛋白药物文库、及其构建多特异性蛋白药物(抗体)的方法和应用。
背景技术
传统的单克隆抗体可特异性结合一个抗原位点，且其Fc端可结合在NK细胞表面的Fc受体，因而进一步激发免疫细胞活性。然而，它无法募集具有极大杀伤力的T细胞，所以无法将免疫系统活性的调动最大化。此外，传统单特异性抗体通过结合一个抗原位点很难充分利用或阻断基于该抗原的信号通路或与其相关代偿通路，导致治疗效果不理想或容易产生耐药性，如针对CD20的多个抗体识别CD20表面不同的位点，因此每个抗体的活性差异显著；靶向glioblastoma(GBM)细胞表面VEGF的抗体治疗可导致angiopoietin-2(Ang-2)表达水平的上调，从而导致对抗VEGF抗体的耐药。
多特异性抗体含有两种及以上抗体的特异性，能靶向结合多个抗原的表位，或一个抗原的多个表位，因此可充分阻断抗原本身下游通路或其与其他蛋白的相互作用，从而提高抗体治疗效果、减少耐药性。以双特异性抗体为例，全球范围内目前已有60余家双特性抗体研发企业、上百个在研双特异性抗体药物，它们多为肿瘤细胞靶点-T细胞募集位点的形式(比如通过CD3募集并激活杀伤性T细胞、通过CD16募集自然杀伤细胞(NK细胞)，从而通过局部富集的免疫细胞靶向杀死肿瘤细胞)和双靶点位点的形式(如VEGF-PDGF、VEGF-Ang2、Her2-Her3，通过抑制两条相关的信号通路，减少潜在的耐药性)。还有部分双特异抗体针对一个抗原的多个位点，如MedImmune公司的MEDI4276,是一种同时靶向Her2第二和第四结构域的双特异性抗体偶联药物(Bispecific ADC)。因此，相比单特异性抗体，多特异性抗体提供了更多的组合可能性、协同效应，且直接增加了T细胞的参与度，在降低给药量的同时极大增强了抗体的免疫治疗效果(如抗肿瘤效果)。
现阶段最具发展潜力的多特异性(双特异性)抗体技术平台主要为BiTE、DART、tandAB、Bi-nanobody、CrossMAB、Triomab等。这些平台的主要通过不同的抗体工程技术实现将不同的抗体识别区域组装在一个蛋白质分子内，实现 多特异性的目的。比如BiTE和Bi-nanobody技术，均将两个单链(scFv)或纳米抗体(nanobody)通过一段柔性肽连接肽串在一起，同时保留两个抗体单元的亲和特性；Crossmab则是通过在两个抗体的Fc重链区域引入不同的突变，使得同个抗体的重链由于空间位阻无法组装，而不同抗体的重链则是空间互补，可以通过二硫键顺利组装成完整的抗体分子，从而成功制备出双特异性抗体。但通过蛋白质工程将两个抗体或片段组装成一个分子，很容易造成抗体的亲和力下降或丢失，比如BiTE，不同的单链抗体组合需要尝试不同的轻重链排列顺序才能得到最优的双特异抗体分子；Crossmab等全长抗体则面临轻链错配的问题，虽然这可以通过通用轻链技术解决，却增加了更多的设计、筛选步骤，且无法作为通用技术直接应用于其他双特异性抗体组合；Triomab、Crossmab、DVD-Ig、Ortho-Fab-IgG等全长抗体形式的多特异性抗体仅能在哺乳动物细胞表达体系(如CHO、HEK293)中才可大规模生产，工艺相比抗体片段(scFv、Fab)更为复杂、制备成本更高。
因此本领域亟待开发一种通用、成本低、得率高的多特异性抗体制备技术，以构建适于个体化精准治疗的蛋白(如抗体)药物文库。
发明内容
本发明的目的是提供一种适于个体化精准治疗的蛋白药物文库。
具体地，本发明提供了用左旋核酸链框架将多个抗体药物连接形成双或多特异性药物的平台技术，可便捷而有效的将多个抗体药物偶联在一起，形成可用于疾病个体化精准治疗的抗体药物文库。
在本发明的第一方面，提供了一种蛋白药物文库，所述蛋白药物文库包括C种不同的蛋白药物单体，其中，所述的蛋白药物单体包括蛋白药物元件部分以及与所述蛋白药物元件部分相连的核酸元件部分，并且一个所述蛋白药物单体的所述的核酸元件部分与至少一个不同的蛋白药物单体的核酸元件部分可通过互补形成双链配对结构，从而构成多聚体的蛋白药物；
其中，C为≥2的正整数。
在另一优选例中，所述的多聚体的蛋白药物为多特异性蛋白药物。
在另一优选例中，C为3-100000的任一正整数；较佳地，C为3-10000；更佳地C为5-5000；最佳地C为10-5000。
在另一优选例中，所述的多聚体的蛋白药物是耐核酸酶降解的。
在另一优选例中，所述的核酸元件部分是耐核酸酶降解的。
在另一优选例中，所述的蛋白药物单体是耐核酸酶降解的。
在另一优选例中，所述的多聚体的蛋白药物在体内发生解聚的半衰期H1大于单独的蛋白药物元件的体内半衰期H2。
在另一优选例中，H1/H2的比值为1-100，较佳地10-50，更佳地10-20。
在另一优选例中，所述的“发生解聚”指多聚体的蛋白药物发生解离，从而形成蛋白药物单体。
在另一优选例中，所述的体内指在人或非人哺乳动物体内。
在另一优选例中，所述的蛋白药物元件部分与所述核酸元件部分直接相连，或间接相连。
在另一优选例中，对于一种蛋白药物单体而言，其所含的核酸元件部分E2与蛋白药物元件部分E1之比Q(即E2/E1)(Q为molar ratio)为10-1，较佳地，4-1，更佳地2-1，或约1-1。
在另一优选例中，优选地，Q为2、1.5、1.2、1.1或1.05。
在另一优选例中，所述的蛋白药物为静脉内给药的蛋白药物。
在另一优选例中，所述的蛋白药物单体具有式I所示的结构：
P─X─L─Y─A─Z    (I)
式中，
P为蛋白类药物分子(即蛋白药物元件部分)；
X为无或冗余肽；
L为连接物分子；
Y和Z为无或冗余核酸；
A为一核酸序列，所述核酸序列选自下组：左旋核酸、肽核酸、锁核酸、硫代修饰核酸、2’-氟修饰核酸、5-羟甲基胞嘧啶核酸、或其组合；
“─”为共价键；
其中任一种蛋白药物单体的核酸A具有至少一个互补配对区，所述互补配对区与所述蛋白药物文库中的至少一种蛋白药物单体的核酸A的互补配对区部分或完全互补。
在另一优选例中，所述蛋白类药物分子P选自下组：抗体、激活或抑制受体或 其他蛋白的配体、生物活性酶、或其组合。
在另一优选例中，所述抗体选自下组：单链抗体、纳米抗体、Fab、单克隆抗体、或其组合。
在另一优选例中，所述抗体选自下组：抗PD-1单链抗体、抗PD-L1单链抗体、抗CTLA-4单链抗体、抗CD-3单链抗体、或其组合。
在另一优选例中，所述抗体选自用于治疗下述疾病的抗体：癌症、自身免疫疾病、免疫检查点、器官移植排斥、类风湿性关节炎、糖尿病、血友病。
在另一优选例中，所述抗体针对的靶点选自下组：CD20、CD19、CD30、HER2、VEGFR、EGFR、RANK、VEGFR2、SLAMF7、GD2、CD33、TNF-a、IL12、IL23、IL6R、IL17、BlyS、CD11a、PD-1、CTLA-4、TIM-3、OX40、CD47、CD3、IL-2R、PCSK9、GPCR。
在另一优选例中，所述抗体针对的靶点选自下组：TNF-a、IL17。
在另一优选例中，所述抗体针对的靶点选自下组：CD3、HER2、PD-1。
在另一优选例中，所述蛋白类药物分子P为野生型或突变型。
在另一优选例中，所述突变不影响药物功能。
在另一优选例中，所述突变包括在抗体的羧基末端(C-末端)引入一个或多个半胱氨酸残基。
在另一优选例中，X为0-30个氨基酸。
在另一优选例中，X为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个氨基酸。
在另一优选例中，所述连接物分子L具有双功能接头，可偶联核酸A或Y的具有修饰基团的修饰端和抗体P或X的特异性连接位点。
在另一优选例中，所述连接物分子L的活性基团选自：马来酰亚胺、卤乙酰基、硫代吡啶。
在另一优选例中，所述卤乙酰基选自：碘乙酰基、溴乙酰基。
在另一优选例中，Y为0-30个核苷酸。
在另一优选例中，所述Y为左旋核酸。
在另一优选例中，Y为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸。
在另一优选例中，所述Y为AAAA、AAA或AA。
在另一优选例中，Z为0-30个核苷酸。
在另一优选例中，所述Z为左旋核酸。
在另一优选例中，Z为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸。
在另一优选例中，所述Z为AAAA、AAA或AA。
在另一优选例中，所述核酸A为左旋核酸。
在另一优选例中，所述核酸A选自：DNA、RNA。
在另一优选例中，所述修饰基团选自下组：NH 2、炔基、巯基(SH)、羧基(COOH)、或其组合。
在另一优选例中，所述修饰基团为NH 2。
在另一优选例中，所述修饰基团在所述核酸A和/或Y上的位置选自：5’端、3’端、中间任意位点。
在另一优选例中，所述核酸A中的任意2个互补配对区之间具有长度为0-10nt的过渡区。
在另一优选例中，所述过渡区为AAAA、AAA或AA。
在另一优选例中，所述互补配对区的长度为5-100nt；较佳地为8-50nt；更佳地为10-30nt；又佳地为12-20nt；最佳地为10-15nt。
在本发明的第二方面，提供了一种组装用于个性化治疗的蛋白药物的方法，包括：
(a)基于制药信息，从本发明的第一方面所述的蛋白药物文库中选取至少二种蛋白药物单体；和
(b)将所述的至少两种蛋白药物单体进行混合，从而组装形成多聚体形式的多特异性蛋白药物。
在另一优选例中，所述的组装是通过所述核酸元件部分的互补形成双链配对结构。
在另一优选例中，在所述的多聚体形式的多特异性蛋白药物中，每一个蛋白药物单体的核酸元件部分与一种或两种或三种不同的蛋白药物单体的核酸元件部 分形成双链配对结构。
在另一优选例中，所述的组装是通过所述核酸元件部分与辅助核酸分子(即核酸T)的单链互补序列发生互补形成双链配对结构。
在另一优选例中，所述的辅助核酸分子为单链形式。
在另一优选例中，所述核酸T为未偶联蛋白药物的核酸。
在另一优选例中，所述核酸T为左旋核酸，或经修饰基团修饰的核酸。
在另一优选例中，所述核酸T的长度为所有(b)中单体核酸配对数目总和的1-1.5倍。
在另一优选例中，所述制药信息为治疗待治疗对象的疾病所需的蛋白药物信息，包括蛋白药物的种类、组合(例如抗体组合)、比例(任意2个蛋白药物P比例为1:1-1:20)。
在另一优选例中，所述组装条件为：5-50度(优选25-40度)，反应1-15分钟(优选5-10分钟)。
在另一优选例中，所述组装条件为：pH 6-9。
在本发明的第三方面，提供了一种多聚体蛋白药物，所述多聚体蛋白药物为D种蛋白药物单体通过核酸互补形成双链配对结构而形成的多聚体，其中D为≥2的正整数；
其中，所述的蛋白药物单体包括蛋白药物元件部分以及与所述蛋白药物元件部分相连的核酸元件部分，并且一个所述蛋白药物单体的所述的核酸元件部分与另一不同的蛋白药物单体的核酸元件部分可通过互补形成双链配对结构。
在另一优选例中，所述的核酸元件部分是耐核酸酶降解的。
在另一优选例中，所述的核酸元件部分选自下组：左旋核酸、肽核酸、锁核酸、硫代修饰核酸、2’-氟修饰核酸、5-羟甲基胞嘧啶核酸、或其组合。
在另一优选例中，所述的蛋白药物单体为来自本发明的第一方面所述的蛋白药物文库中的蛋白药物单体。
其中D为2-20的正整数；较佳地D为2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20。
在另一优选例中，所述的多聚体的蛋白药物为多特异性蛋白药物。
在另一优选例中，所述的多聚体的蛋白药物为抗癌药物。
在另一优选例中，所述的多聚体的蛋白药物在体内发生解聚的半衰期H1大于单独的蛋白药物元件的体内半衰期H2。
在另一优选例中，H1/H2的比值为1-100，较佳地10-50，更佳地10-20。
在本发明的第四方面，提供了一种药物组合物，所述的药物组合物含有
(i)本发明的第三方面所述的多聚体蛋白药物作为活性成分；和
(ii)药学上可接受的载体。
在另一优选例中，所述药物组合物的剂型选自下组：注射剂、冻干剂。
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为基于左旋核酸自组装的多特异性抗体示意图。它由多个抗体或抗体片段、可自组装的多条L-核酸、连接物(linker)所构成。
图2为四特异性抗体的L-DNA框架形状及配对方式示意图。
图3为四条SMCC-L-DNA的自组装结果图。3％琼脂糖凝胶电泳。第1-第4泳道为SMCC-L-DNA单链，其中第1泳道为链1,第2泳道为链2,第3泳道为链3,第4泳道为链4。第5-第8泳道为组装后的条带，其中第5泳道中的镁离子浓度为0mM，第6、7、8泳道的镁离子浓度分别为1mM、2mM和4mM。
图4为四特异性抗体的自组装结果图。SDS-PAGE胶先后用溴化乙锭(EB)、考马斯亮蓝法染色，分别显色DNA和蛋白部分。泳道1为未偶联的抗PD-1单链抗体，泳道2为偶联了链1(L-DNA)的抗PD-L1单链抗体，泳道3为偶联了链2(L-DNA)的抗PD-L1单链抗体，泳道4为偶联了链3(L-DNA)的抗PD-1单链抗体，泳道5为偶联了链4(L-DNA)的抗CD3单链抗体。泳道6为四种单链抗体-L-DNA反应液的混合物。
图5左图为MBP融合单链抗体突变体的表达结果示意图，左图泳道1为未加IPTG诱导的对照实验，泳道2、3、4分别为MBP-抗CD3单链抗体，MBP-抗CEA单链抗体，MBP-抗PDL1单链抗体的蛋白表达情况。右图为MBP融合单链抗体 突变体的可溶情况示意图，右图泳道1为全菌裂解液，泳道2为可溶组分，泳道3为包涵体组分。
图6为抗CD3-L-DNA2纯化结果示意图。左图为Superdex 200 10/300GL层析柱纯化结果。右图为聚丙烯酰氨凝胶电泳技术检验蛋白样品纯度结果。
图7为多特异性抗体自组装结果示意图。左图为聚丙烯酰氨凝胶电泳技术监测多特异性抗体三聚体组装结果示意图，泳道1、2、3分别为抗CEA-L-DNA1、抗PDL1-L-DNA2、抗CEA-L-DNA3蛋白条带，泳道4为三种特异性抗体自组装后的蛋白条带。右图为聚丙烯酰氨凝胶电泳技术监测多特异性抗体四聚体组装结果示意图，泳道1为与抗CD3-L-DNA4反应前三聚体，泳道2为与抗CD3-L-DNA4反应后四聚体。
图8M为DNA标准品，最小的条带为25bp，其它条带以25bp递增。泳道1-4分别为四条L-DNA(20uM)，上样量为5ul。泳道5和6分别为室温和37度条件下，先组装三聚体再加第四条L-DNA的组装方式。泳道7为37度条件下直接混合四条L-DNA的组装方式。
图9泳道1为未经任何核酸酶处理的L-DNA四聚体(上图)和D-DNA四聚体(下图)；泳道2-5分别为DNAse I、外切酶I(Exonuclease I)、T7DNA内切酶(endonuclease)、S1核酸酶(nuclease)处理后的L-DNA四聚体样品或D-DNA四聚体样品。核酸标准(Marker)的最小条带为25bp，且每个条带相差25bp。
图10M为宽分子量蛋白标准(marker)；1-4为1uM的L-DNA-融合蛋白单体，5为1-4组装的产物；6-9为2uM的L-DNA-融合蛋白单体，10为1-4组装的产物。
图11L-DNA四聚体框架介导组装的融合蛋白四聚体分子筛分析结果。所用的层析柱为Superose 6 10/300分子排阻色谱柱(GE公司)。
图12基于L-DNA框架制备的CEA/PD-L1/CD3四特异性抗体体外活性结果。大肠癌细胞系LS174T为CEA阳性，作为该体外活性实验的细胞模型。
图13抗CEA/PD-L1/CD3四特异性抗体激活T细胞的实验结果。采用CD3阳性细胞分泌的IFN-γ作为检测对象。阳性对照为Dynabeads(表面偶联了抗CD28/CD3抗体的细珠，可高效激活T细胞)，阴性对照为抗体所用的缓冲液。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一种蛋白药物文库，所述蛋白药物文库包括≥2种不同的蛋白药物单体，所述的蛋白药物单体包括蛋白药物元 件部分以及与所述蛋白药物元件部分相连的核酸元件部分，所述核酸元件部分为耐体内核酸酶降解的核酸(例如左旋核酸)，并且一个所述蛋白药物单体的所述的核酸元件部分与至少一个不同的蛋白药物单体的核酸元件部分可通过互补形成双链配对结构，可根据需要(如某一个体的病情和诊断结果)，从所述蛋白药物文库中选择相应的蛋白药物单体，快速(1分钟内)、高效、低成本、高得率地组装针对多靶点、体内稳定的多特异性蛋白药物(例如多特异性抗体)，在此基础上完成本发明。
术语
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
如本文所用，术语“蛋白药物单体”、“本发明蛋白药物单体”、“本发明药物单体”可互换使用。
如本文所用，术语“蛋白药物文库”、“本发明蛋白药物文库”、“本发明药物文库”可互换使用。
如本文所用，术语“本发明的多聚体蛋白药物”、“多聚体蛋白药物”、“多特异性蛋白药物”、“本发明多聚体药物蛋白”、“多聚体药物蛋白”、“本发明多聚体蛋白”可互换使用。
蛋白药物文库
本发明提供了一种蛋白药物文库，所述蛋白药物文库包括C种不同的蛋白药物单体，其中，所述的蛋白药物单体包括蛋白药物元件部分以及与所述蛋白药物元件部分相连的核酸元件部分，并且一个所述蛋白药物单体的所述的核酸元件部分与至少一个不同的蛋白药物单体的核酸元件部分可通过互补形成双链配对结构，从而构成多聚体的蛋白药物；其中，C为≥2的正整数。
本发明文库还含有至少2种以上的蛋白药物单体，优选的蛋白药物单体具有上述式I结构。
由于本发明的蛋白药物单体具有特定的结构，因此不仅可以快速组装成多聚体形式的药物蛋白，而且所形成的多聚体的蛋白药物为多特异性，可同时靶向多个不同的靶点，从而满足在疾病治疗过程中同时或先后针对多个靶点的需要。此外，本发明的多聚体蛋白药物还具有出乎意料的体内的稳定性，可以长时间在体内存在并保持活性，而不会被很快降解。
例如，在癌症治疗中，往往涉及多个靶点和多个通路，而且每个病人不仅病因不同，而且在一个病人中还存在肿瘤的异质性，因此往往需要采用针对多靶点的药物。随着个性化治疗或精准治疗技术的发展，本领域迫切需要开发能够快速制备，成本低、靶向性好、稳定性的蛋白质药物(如多特异性抗体)。本发明的文库满足了这类需求。
应理解，虽然本发明文库含有或主要含有或全部含有本发明上述蛋白药物单体，但是该文库还含有其他的治疗剂，尤其是其他的蛋白药物，代表性的例子包括(但并不限于)：抗体、化合物、融合蛋白。例如，本发明文库可额外地含有一种或多种具有治疗作用的常规抗体。
应理解，本发明文库中的蛋白药物单体的数量没有限制，可以为≥2的任一正整数C。例如，C为3-100000的任一正整数；C为3-10000；更佳地C为5-5000；最佳地C为10-5000。
此外，在本发明中，蛋白药物单体中的抗体元件没有特别限制，代表性的例子(选自下组)：单链抗体、纳米抗体、Fab、单克隆抗体、或其组合。
对于本发明文库而言，可以使用各种来源的抗体来制备蛋白药物单体。本发明文库的一个突出特点是，可使用通过原核系统(如大肠杆菌)或真核系统(如酵母、CHO细胞)表达的抗体片段，从而极大地降低了生产成本。
典型地，在使用时，可根据需要(如某一个体的病情和诊断结果)，选择相应的蛋白药物单体，简便地通过核酸互补框架就完成多特异抗体。例如，在应用时，根据病人疾病的靶点情况，相应确定单体种类、数量或比例(例如2种、3种、4种、或4种以上)，然后进行组装。
在制备多聚体蛋白药物时，从文库中选出相应的可相互配对偶联的蛋白药物单体，并按所需的抗体比例混合后，就可在1分钟内完成组装过程。
在本发明文库中，蛋白药物单体的核酸元件可通过序列设计成为二聚体、三聚体、四聚体等高聚体框架，由此完成通过传统抗体工程无法轻易达到的三特异甚至四特异性抗体等多特异性抗体制备。
一旦组装形成多特异性的多聚体蛋白药物，就可根据治疗目的，用于相应的个体。
左旋核酸(L-核酸)
左旋核酸是指相对自然界存在的右旋核酸(D-核酸)而言，成镜像存在，可分为左旋DNA(L-DNA)和左旋RNA(L-RNA)。左旋(手性中心)主要存在于核酸的脱氧核糖或核糖部分，呈镜像翻转。因此，左旋核酸无法被血浆中无处不在的核酸酶(如核酸外切酶、核酸内切酶)所降解。
本发明多聚体的蛋白药物及其制备
本发明多聚体的蛋白药物是指D种蛋白药物单体通过核酸互补形成双链配对结构而形成的多聚体，其中D为≥2的正整数；其中，所述的蛋白药物单体包括蛋白药物元件部分以及与所述蛋白药物元件部分相连的核酸元件部分，并且一个所述蛋白药物单体的所述的核酸元件部分与另一不同的蛋白药物单体的核酸元件部分可通过互补形成双链配对结构。
在另一优选例中，所述的核酸元件部分是耐核酸酶降解的。
在另一优选例中，所述的核酸元件部分选自下组：左旋核酸、肽核酸、锁核酸、硫代修饰核酸、2’-氟修饰核酸、5-羟甲基胞嘧啶核酸、或其组合。
本发明的多聚体的蛋白药物可通过例如式I所示的蛋白药物单体的组装而形成。
典型地，多聚体的蛋白药物是指多聚体抗体(多特异性抗体)，例如二、三、四、五或六特异性抗体。
在本发明中，本发明的多聚体抗体含有两种及以上抗体的特异性，能靶向结合多个抗原的表位，或一个抗原的多个表位,因此可充分阻断抗原本身下游通路或其与其他蛋白的相互作用，从而提高抗体治疗效果、减少耐药性。
在一个优选例中，本发明的蛋白药物为采用左旋核酸的多特异性抗体。本发明的研究表明，核酸是一种可以快速进行特异性配对的双链分子，因此，若 是将抗体片段(如单链抗体、纳米抗体、Fab等)与核酸单链偶联，则可通过设计核酸序列，让两个及以上的核酸单链快速配对形成多聚体，从而引导抗体片段也形成多聚体，完成多特异性抗体的制备。
在本发明中，为了提高治疗效果，需要采用特定结构的蛋白药物单体。优选例中，通过使用左旋核酸(如L-DNA、L-RNA等)替代右旋核酸(如D-DNA、D-RNA)的方式，可以显著提高治疗效果。一个原因是L-核酸无法被人体内存在的外切核酸酶、内切核酸酶等降解，所以由L-核酸(左旋核酸)自组装所介导的多特异性抗体组合在体内将极为稳定。
药物组合物
本发明还提供了一种组合物。在优选例中，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)：口服、呼吸道、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治疗(例如抗肿瘤治疗)，因而可用于延长药物的半衰期，此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(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1微克/千克体重-约10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8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1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。
本发明的主要优点在于：
(1)本发明的多特异性抗体制备简便、快捷，在一分钟内即可利用左旋核酸链介导完成多种抗体的组装；
(2)本发明的多特异性抗体改造空间广泛，可将任意形式的抗体(如单链抗体、纳米抗体、Fab)组装成多特异性抗体；
(3)本发明的制备多特异性抗体的平台技术，可单独制备多特异性抗体的各部分抗体，然后进行简单的体外组装；
(4)本发明的制备多特异性抗体的平台技术，可储备本发明中多特异性抗体的中间产物(L-核酸-抗体偶联物)，可根据需要灵活制备任意靶向不同抗原或位点的抗体组合，以及调整多特异性抗体中某个抗体的比例；
(5)基于本发明的制备多特异性抗体的平台技术构建抗体药物文库，可根据提供的疾病和/或制药信息，快速、简便制备适于个体化精准治疗的抗体药物，成本低、通用性好。
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，否则百分比和份数按重量计算。本发明中所涉及的实验材料如无特殊说明均可从市售渠道获得。
通用方法
1.L-核酸链框架的设计与制备
根据本发明，L-核酸链框架由两条及以上的L-核酸单链通过碱基配对形成。每条L-核酸单链的5’或3’端均活化成可供后续修饰的基团(如NH 2等)，然后用连接物(如SMCC、SM(PEG)、SPDP等)的一端与L-核酸单链上的活化基团偶联。 带连接物的L-核酸可组装成所需的L-核酸链框架。在确定带连接物的L-核酸可以成功自组装成框架后，则可将带连接物的L-核酸单链分别偶联抗体进行后续的组装。
图1为基于左旋核酸自组装制备而成的多特异性抗体结构示意图，其中，A N为抗体或抗体片段，如单链抗体、纳米抗体、Fab等；L-核酸框架由数目不等的单链核酸组成，且单链核酸的一端有活性基团修饰，比如NH 2等；单链核酸的数目可根据多特异性抗体的类型调整，比如四特异性抗体则需要单链核酸的数目最低为4；连接物(linker)用于连接单链核酸的活性基团和抗体上的特异性连接位点(如突变半胱氨酸残基上的SH基团)。
本发明L-核酸框架基本上可通过如下步骤制备。
1.1设计可快速自组装的L-核酸单链
确定需要制备的多特异性抗体的类型(例如三特异性抗体)；根据多特异性抗体中的抗体数目N确定所需的L-核酸单链数目M；设计相应数目的L-核酸单链序列，通过增减碱基配对数目调节目的核酸框架稳定性，减少核酸链间非特异性配对的可能性。
根据本发明一个优选的实施方式，为设计一个四聚体L-核酸框架(M＝4)，设计四条可按照一定规则配对的L-核酸(如图2所示)。其中，任意一条L-核酸单链可与另两条L-核酸单链进行特异性互补配对，而不于第四条配对。且特异性互补配对的吉布斯能变化量△G要远小于非特异性配对，比如该优选的实施方式中，特异性互补配对的吉布斯能变化量△G约为-34千卡每摩尔(kcal/mole)，而非特异性配对则均小于-10千卡每摩尔(kcal/mole)，意味着四聚体的组装方式要比非特异性的两两配对更容易发生，四聚体的形式在反应体系中最为稳定。
1.2 L-DNA或L-RNA的活化
L-核酸的活化包括其5’端或3’端的活性基团修饰和后续的连接物偶联。活性基团修饰可由核酸合成公司订制完成；连接物一般拥有双功能基团，即一端可偶联核酸的活性基团，另一端可与抗体上的特异性位点(如SH)连接。
根据本发明一个优选的实施方式，组成框架的所有L-核酸在5’端添加NH 2 修饰，然后利用连接物即双异官能团交联试剂SMCC(4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯钠盐)通过酰胺键偶联核酸上的NH 2。此时连接物的另外一端马来酰亚胺基团为游离状态，可用于后续偶联抗体上的巯基(SH)，至此完成L-核酸的活化。
1.3核酸框架聚合度的验证
核酸框架聚合度可以通过例如琼脂糖凝胶电泳来验证。
根据本发明一个优选的实施方式，选择3％的琼脂糖凝胶电泳来分析核酸框架聚合度；对比L-核酸的单链大小，可轻易推算出多条L-核酸单链混合后形成的框架大小，从而得到聚合度。
本领域技术人员可以理解，按照上述反应路线以及优选的实施方式中所描述的方法，可以类似地制备本发明里提到的其它L-核酸框架，并不具有局限性。
2.抗体的选择与制备方法
本发明的抗体根据多特异性抗体的用途和目的进行选择。若是用于实体瘤治疗，需要穿透性高的多特异性抗体，则选择较小的抗体片段(如单链抗体、纳米抗体等)；若是用于血液瘤，则选择抗体或抗体片段均可。具体的选择需要根据治疗的用途和机理而定。对于抗体片段的制备，选择大肠杆菌或酵母等低成本表达系统；而全长抗体则需哺乳动物细胞表达系统。
为便于与活化的L-核酸偶联，抗体上会引入一个特异性的位点(如突变位点，Cys)，用于连接物的偶联。
根据本发明一个优选的实施方式，选择抗PD-L1/PD-1/CD3的单链抗体用于制备三特异性抗体，其中PD-1和CD3为位于T细胞表面的位点，主要作用分别为解除抗肿瘤活性的抑制作用以及激活CD8阳性T细胞。而PD-L1则位于部分肿瘤细胞表面，通过与PD-1作用阻止T细胞对其进一步杀伤。因此，使用两个抗PD-L1单链抗体，一个抗PD-1单链抗体以及一个抗CD3单链抗体用于制备三特异性抗体，使用于靶向肿瘤和免疫细胞的单链抗体数目达到平衡，因此所需的L-核酸框架为四聚体(M＝4)。在每个单链抗体的羧基末端(C-末端)引入一个半胱氨酸突变，用于偶联SMCC活化的L-DNA单链，每个单链抗体连接不同的L-DNA单链，且两个抗PD-L1单链抗体在多特异性抗体的一端，而抗PD-1和CD3的单链抗体则 在另一端，便于募集T细胞至肿瘤细胞。
3.抗体-L-核酸复合物的制备方法
首先L-核酸的5’或3’端用NH 2修饰，然后根据连接物的不同可为以下几种主要制备方法，其中连接物的一端官能团为NHS(N-羟基琥珀酰亚胺)或Sulfo-NHS(N-羟基琥珀酰亚胺磺酸钠盐)，用于快速偶联L-核酸一端的NH 2基团。包含双异官能团的连接物均首先与L-核酸的NH 2反应，其次在还原抗体上的巯基后，另一端的基团再与巯基反应形成稳定的化学键。
3.1马来酰亚胺(Maleimide)。连接物用于偶联抗体上巯基的基团为马来酰亚胺。马来酰亚胺可快速与抗体上游离的巯基反应形成硫醚键。常见的连接物有SMCC(4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯)、SM(PEG)(聚乙二醇修饰的4-(N-马来酰亚胺甲基)环己烷-1-羧酸琥珀酰亚胺酯)等。
3.2卤乙酰基(Haloacetyl)。连接物用于偶联抗体上巯基的基团为卤乙酰基，比如碘、溴乙酰基。卤素离子可能抗体上的巯基通过亲核作用替换形成稳定的硫醚键。常见的连接物有SBAP(N-马来酰亚胺甲基[4-溴乙酰基]氨基苯甲酸酯),SIAB(N-马来酰亚胺甲基[4-碘乙酰基]氨基苯甲酸酯)等。
3.3硫代吡啶(Pyridyldithiol)。连接物用于偶联抗体上巯基的基团为硫代吡啶。硫代吡啶可与游离的巯基反应形成二硫键。常见的连接物有SPDP(3-(2-吡啶二硫代)丙酸N-羟基琥珀酰亚胺酯)等。
实施例1：四聚体DNA结构框架的设计
设计四条可按照四边形形状配对的L-核酸(如图2所示)。其中，任意一条L-核酸单链可与另两条L-核酸单链进行特异性互补配对，而不与第四条配对。且特异性互补配对的吉布斯能变化量△G要远小于非特异性配对，特异性互补配对的吉布斯能变化量△G约为-34千卡每摩尔(kcal/mole)，而非特异性配对则均小于-10千卡每摩尔(kcal/mole)，意味着四聚体的组装方式要比非特异性的两两配对更容易发生，四聚体的形式在反应体系中最为稳定。
按照以上原则设计的四条L-DNA单链序列如下(从5’到3’)：
链1(L-DNA1)：SEQ ID NO:1
5’AAAA CGACAGTCCGATGTGCC AAA CGGCTGGAAGTTGAGC AA 3’
链2(L-DNA2)：SEQ ID NO:2
5’AAAA GGCACATCGGACTGTCG AAA GGCGTAGCCTAGTGCC AA 3’
链3(L-DNA3)：SEQ ID NO:3
5’AAAA CGCTGATATGCGACCTG AAA GCTCAACTTCCAGCCG AA 3’
链4(L-DNA4)：SEQ ID NO:4
5’AAAA CAGGTCGCATATCAGCG AAA GGCACTAGGCTACGCC AA 3’
5’端有NH 2基团修饰，用于偶联SMCC的NHS。AAAA和AAA后面的碱基序列分别与另外两条链互补配对，每一部分的配对吉布斯能变化量△G约为-34千卡每摩尔(kcal/mole)。
实施例2：四聚体DNA框架的合成与验证
由生物技术服务公司合成5’端NH 2修饰的L-DNA单链，四条单链的序列如实施例1中所示。
用磷酸缓冲液(50mM NaH 2PO 4，150mM NaCl，pH 7.0)将L-DNA单链溶解，配制成终浓度为200uM的母液。用二甲亚砜(DMSO)溶解SMCC粉末，新鲜配制250mM的SMCC母液。在L-DNA单链母液中加入10～50倍摩尔量的SMCC母液，迅速混合后在室温下反应30min～2h。反应完成后加入反应液10％体积的1M Tris-HCl(pH 7.0),混合后室温孵育20分钟，用于终止过量的SMCC继续反应。孵育结束后，加入反应液2倍体积的100％无水乙醇，混合均匀后在-20度冰箱中放置25分钟，充分沉淀L-DNA。离心(12000rpm，10min)取沉淀，用1mL 70％乙醇洗涤，12000rpm离心1min去除上清，重复洗涤5次，充分去除过量的SMCC。剩下的白色沉淀在空气中自然干燥5～10min，然后用磷酸缓冲液重悬、溶解，即得到SMCC-L-DNA复合物(即SMCC-L-DNA单链)。
测定各条SMCC-L-DNA单链的浓度。取适量四条待反应的SMCC-L-DNA单链在40度预热5min，然后在40度条件下混合等摩尔量的四条SMCC-L-DNA单链，孵育1min。反应体系设置不同的镁离子浓度，探索镁离子浓度对框架形成的影响。取0.25ul的SMCC-L-DNA单链和反应产物用3％琼脂糖凝胶电泳分析。结果如附图3所示，SMCC-L-DNA单链的大小约为25bp左右，而混合后形成的主条带则在 100bp左右，表明四条不同的SMCC-L-DNA单链形成了四聚体框架，且不同镁离子浓度均不影响其自组装，表现其具有极高的稳定性。
实施例3：单链抗体突变体的制备
单链抗体的羧基末端引入半胱氨酸突变。由于单链抗体中存在二硫键，而大肠杆菌细胞质中环境不利于二硫键的形成，因此需要将单链抗体分泌至大肠杆菌的周质空间进行折叠、形成二硫键。
将抗PD-1/PD-L1/CD3单链抗体的基因序列优化为大肠杆菌偏爱的密码子，并在基因两端分别添加NcoI和XhoI限制性酶切位点，然而亚克隆至pET22b质粒中的NcoI/XhoI位点之间。抗PD-1/PD-L1/CD3单链抗体的氨基酸序列分别为SEQ ID NO:5、SEQ ID NO:6和SEQ ID NO:7。
SEQ ID NO:5，抗PD1单链抗体突变体(Anti-PD1single-chain antibody mutants)氨基酸序列：
Figure PCTCN2018080058-appb-000001
SEQ ID NO:6，抗PD-L1单链抗体突变体(Anti-PD-L1single-chain antibody mutants)氨基酸序列：
Figure PCTCN2018080058-appb-000002
SEQ ID NO:7，抗CD3单链抗体突变体(Anti-CD3single-chain antibody mutants)氨基酸序列：
Figure PCTCN2018080058-appb-000003
Figure PCTCN2018080058-appb-000004
pET22b质粒由于带有pelB信号肽序列，可引导单链抗体分泌至周质空间。取1ul构建好的表达载体转化大肠杆菌BL21(DE3)，挑取转化后的BL21(DE3)单菌落至LB培养基中(含100ug/mL氨苄青霉素)，37度培养至OD600＝0.7，加入终浓度为1mM的IPTG诱导表达，37度继续培养3到4小时。离心收集表达完成后的菌体，重悬于磷酸缓冲液(50mM NaH 2PO 4，150mM NaCl，pH 7.0)，蛋白酶抑制剂cocktail(Sigma)，利用超声破碎。加入DNaseI水解酶在冰上孵育1小时。孵育结束后，菌液在17000rpm离心20分钟收集上清。用Hitrap Protein L亲和柱纯化上清中的单链抗体，上清以0.25ml/min速度过柱后，用大量磷酸缓冲液(50mM NaH 2PO 4，150mM NaCl，pH 7.0)以1ml/min的流速洗涤柱子直至杂蛋白不再流出(根据AKTA蛋白层析系统上的紫外吸收)，0～100％洗脱缓冲液(50mM NaH 2PO 4，150mM NaCl，pH 2.3)梯度洗脱结合在柱子上的单链抗体。收集单链抗体组分，调节pH至7.0。
实施例4：单链抗体-L-DNA的偶联与纯化
将纯化后单链抗体用10-50倍摩尔比过量的还原剂(如TCEP、DTT、巯基乙醇等)在室温下孵育30min。孵育结束后用PD-10脱盐柱快速除去反应体系中的还原剂，同时将缓冲液置换为磷酸缓冲液(50mM NaH 2PO 4，150mM NaCl，pH 7.0)。测定单链抗体的浓度后，马上加入1～4倍摩尔比过量的SMCC-L-DNA单链(实施例2中制备)，混合均匀后在室温下反应1h。
由于DNA等核酸带负电，利用阴离子交换柱(HiTrap Q HP柱)分离纯化单链抗体-L-DNA，除去未反应的单链抗体、过量的SMCC-L-DNA单链。分离过程通过梯度洗脱实现，上样缓冲液为50mM NaH 2PO 4，pH 7.0，洗脱缓冲液为50mM NaH 2PO 4，1M NaCl pH 7.0，0-100％的洗脱缓冲液进行梯度洗脱，未反应的单链抗体、单链抗体-L-DNA、过量的SMCC-L-DNA单链先后出峰。收集单链抗体-L-DNA，浓缩后用PD-10脱盐柱将缓冲液置换为50mM NaH 2PO 4，150mM NaCl，pH 7.0。
实施例5：多特异性抗体的自组装
为排除单链抗体自己形成多聚体的可能性，利用实施例4中刚完成偶联反应的单链抗体-L-DNA反应液进行自组装实验。偶联反应中的单链抗体/SMCC-L-DNA反应比例为1:0.5，确保反应结束后有未偶联的单链抗体，但是需要除去未反应的SMCC-L-DNA单链。因此反应完成后，加入适量Protein L填料，孵育10min，12000rpm离心1min去除上清，加入1mL磷酸缓冲液(50mM NaH 2PO 4，150mM NaCl，pH 7.0)洗涤填料，离心去除上清，如此重复四次。加入洗脱缓冲液(50mM NaH 2PO 4，150mM NaCl，pH 2.3)，孵育10min，用于洗脱吸附在填料表面的单链抗体以及单链抗体-L-DNA，调节pH至7.0。实施例1中的链1(L-DNA1)与抗PD-L1单链抗体偶联，链2(L-DNA2)与抗PD-L1单链抗体偶联，链3(L-DNA3)与抗PD-1单链抗体偶联，链4(L-DNA4)则与抗CD3单链抗体偶联。
取100ul上述经Protein L纯化的单链抗体-L-DNA反应液在40度预热5min，然后在40度条件下混合四种等体积的单链抗体-L-DNA反应液，孵育1min。反应后取30ul用SDS-PAGE分析。电泳结束后，SDS-PAGE胶首先用2ug/ml的溴化乙锭溶液染色20min，用超纯水洗涤三次后在紫外下显色，可观察到包含DNA的条带(如单链抗体-L-DNA的单体、多特异性抗体)。然后将同一块胶用考马斯亮蓝法染色，可观察所有的蛋白样品电泳情况。
结果如图4所示，偶联了L-DNA的单链抗体相比未偶联的单链抗体发生了明显的偏移，且仅有偶联了L-DNA的单链抗体可在紫外光下成像。在溴化乙锭(EB)染色后，可在紫外灯下观察到多聚物的存在，根据分子量大小可判断为四聚体和二聚体。二聚体的存在是由于抗CD3单链抗体-L-DNA的量要显著少于另外三种单链抗体-L-DNA，因此无法全部自组装成四聚体，导致其他三种单链抗体-L-DNA倾向于非特异性地两两组合形成二聚体。考马斯亮蓝染色后，可以看到几种单链抗体-L-DNA混合后，单体的条带消失，而在150kDa左右出现了模糊的条带，根据EB染色结果可以判断为四聚体。未反应的单链抗体条带未发生任何变化，表明四聚体的形成是因为L-DNA的相互配对形成的。因此，该L-DNA四聚体框架可以用于快速制备四特异性抗体等多特异性抗体。
实施例6：MBP融合单链抗体突变体的表达与制备
单链抗体在大肠杆菌中单独表达往往形成不具生物活性的包涵体，为了提高单链抗体的可溶性及生物活性，构建麦芽糖结合蛋白MBP和三种单链抗体(抗PD-L1/CD3/CEA单链抗体，Seq.No 1,2和3)融合表达的载体，形成MBP-ScFv融合蛋白。MBP与单链抗体中间引入TEV酶切位点用于MBP标签切除，并在基因两端分别添加NcoI和XhoI限制性酶切位点，然后亚克隆至pET21b质粒中的NcoI/XhoI位点之间。
取1ul构建好的表达载体转化大肠杆菌BL21(DE3)，挑取转化后的BL21(DE3)单菌落至LB培养基中(含100ug/mL氨苄青霉素)，37度培养至OD600＝0.7，加入终浓度为0.1mM的IPTG诱导表达，16度继续培养12到16小时。取等量菌液少许，利用聚丙烯酰氨凝胶电泳技术对蛋白表达情况进行监测，如图5左图所示，其中泳道1为未加IPTG诱导的对照实验，泳道2、3、4分别为MBP-抗CD3单链抗体，MBP-抗CEA单链抗体，MBP-抗PDL1单链抗体的蛋白表达情况，表明MBP-ScFv融合蛋白在大肠杆菌表达体系中表达情况稳定，表达量较高，蛋白分子量约为69kDa。
以MBP-抗CEA单链抗体为例，离心收集表达完成后的菌体，重悬于HEPES缓冲液(20mM HEPES+150mM NaCl,pH＝7.4)，加入蛋白酶抑制剂cocktail(sigma)，还原剂巯基乙醇，DNaseI水解酶，超声破碎，39000g离心40分钟收集上清为可溶组分，重悬沉淀于相同体积HEPES缓冲液为包涵体组分，利用聚丙烯酰氨凝胶电泳技术对蛋白可溶情况进行监测，如图5右图所示，其中泳道1为全菌裂解液，泳道2为可溶组分，泳道3为包涵体组分，表明MBP-ScFv融合蛋白在大肠杆菌表达体系中可溶性良好。
用镍柱亲和层析的方法对MBP融合单链抗体突变体进行初步纯化。将MBP-ScFv融合蛋白加入镍柱，吸附30分钟后除去流出液，分别用20mM及40mM咪唑洗脱杂蛋白，400mM咪唑洗脱收集目的蛋白，等待单链抗体-L-DNA的后续偶联与纯化。
实施例7：单链抗体-L-DNA的偶联与纯化
用PD-10脱盐柱快速除去初步纯化的MBP融合单链抗体中的过量还原剂巯 基乙醇，马上加入1～4倍摩尔比过量的SMCC-L-DNA单链(实施例2中制备)，混合均匀后在室温下反应1h。通过直链淀粉树脂亲和层析的方法除去未反应DNA，目的蛋白与直链淀粉柱结合，加入10CV HEPES缓冲液(20mM HEPES+150mM NaCl,pH＝7.4)进行冲洗，除去未反应DNA。然后加入TEV酶孵育3小时，在直链淀粉柱上切除融合蛋白，流出液为切除MBP融合蛋白后的单链抗体，将流出液收集。由于DNA等核酸带负电，利用阴离子交换柱(HiTrap Q HP柱)分离纯化单链抗体-L-DNA，除去TEV酶。分离过程通过梯度洗脱实现，上样缓冲液为20mM Tris-Cl+15mM NaCl,pH＝8.5，洗脱缓冲液为20mM Tris-Cl+1M NaCl,pH＝8.5，0-100％的洗脱缓冲液进行梯度洗脱，TEV酶及单链抗体-L-DNA先后出峰，收集单链抗体-L-DNA。将单链抗体-L-DNA进行快速蛋白质液相层析纯化，样品通过HEPES缓冲液(20mM HEPES+150mM NaCl,pH＝7.4)平衡后的Superdex 200 10/300GL column(GE Healthcare)纯化分离后，根据紫外吸收A280取样，利用聚丙烯酰氨凝胶电泳技术检验出峰位置以及样品纯度。以抗CD3-L-DNA2为例，如图6所示，最终可得到生物物理性质均一，纯度较高的单链抗体-L-DNA偶联样品，其分子量大小约为40kDa。
实施例8：多特异性抗体的自组装
实施例1中的链1(L-DNA1)与抗CEA单链抗体偶联，链2(L-DNA2)与抗PD-L1单链抗体偶联，链3(L-DNA3)与抗CEA单链抗体偶联，链4(L-DNA4)则与抗CD3单链抗体偶联。各取300μl抗CEA-L-DNA1、抗PDL1-L-DNA2、抗CEA-L-DNA3在37度预热5min，然后37度条件下等体积混合三种单链抗体-L-DNA，孵育5min。反应后取30ul反应液利用聚丙烯酰氨凝胶电泳技术对抗体组装情况进行监测，如图7左图所示，泳道1、2、3分别为抗CEA-L-DNA1、抗PDL1-L-DNA2、抗CEA-L-DNA3蛋白条带，泳道4为三种特异性抗体自组装后的蛋白条带，其蛋白分子量表明抗CEA-L-DNA1、抗PDL1-L-DNA2、抗CEA-L-DNA3可自组装形成三聚体。将三聚体进行快速蛋白质液相层析纯化去除未反应单链抗体-L-DNA单体。将纯化后的三聚体稀释至终浓度为0.1μM，37度条件下等体积等浓度加入抗CD3-L-DNA4，孵育5min。反应后取30μl反应液利用聚丙烯酰氨凝胶电泳技术对抗体组装情况进行监测，如图7右图所示，泳道1为与抗CD3-L-DNA4反应前三聚体，泳道2为 与抗CD3-L-DNA4反应后四聚体，其四聚体蛋白质分子量约为168kDa。因此，以上实验证明该L-DNA四聚体框架可以用于快速制备四特异性抗体等多特异性抗体。
实施例9：四聚体DNA框架的组装优化
用磷酸缓冲液(50mM NaH 2PO 4，150mM NaCl，pH 7.0)将四条L-DNA单链溶解，配制成终浓度为20uM的储存液。为优化四聚体DNA框架的组装，将组装过程分为两种方式进行比较：1，先混合三条L-DNA单链，室温或37度混合反应5分钟，等待30分钟后，再加入第四条L-DNA单链；2，同时混合四条L-DNA单链，37度混合反应5分钟。反应结束后，各组样品取5ul用2％琼脂糖凝胶进行分析。
结果如图8所示，四条L-DNA同时混合反应时，主要产物为四聚体，但同时存在很多非特异的高聚合度组装产物(泳道7)。而当三条L-DNA先混合，再加入第四条L-DNA时，室温和37度的反应结果均为单一的四聚体产物，没有任何非特异条带(泳道5和泳道6)。表明对于该DNA框架的组装，先组装三聚体再加入第四条L-DNA的组装方式要远优于四条直接混合的方式。
实施例10：D-DNA和L-DNA四聚体框架的耐降解实验
相比D-DNA，L-DNA的优势在于无法被自然界中的DNA酶所降解。人体内存在多种DNA酶，为验证L-DNA四聚体框架是否可被DNA酶所降解或解聚，选取DNAse I、T7DNA内切酶(endonuclease)、S1核酸酶(nuclease)、外切酶I(Exonuclease I)对D-DNA和L-DNA四聚体框架进行处理。D-DNA和L-DNA的四条单体序列一一对应，且组装方式采用实施例9中改进的两步法组装。在将各种酶加入D-DNA或L-DNA四聚体框架中后，在37度水浴17小时，取样后用2％琼脂糖电泳分析。
结果如图9所示，L-DNA四聚体框架可以耐受四种DNA酶的处理，没有任何的降解存在。而D-DNA四聚体则被DNAse I和S1核酸酶几乎完全降解，还可被外切酶1和T7DNA内切酶破坏双螺旋结构。因此，L-DNA四聚体框架无法被常见的各种DNA酶所降解。
实施例11：将L-DNA框架用于组装大分子量蛋白质的四聚体
为表明L-DNA框架也可用于大分子量蛋白质的组装，将实施例1中L-DNA四聚体框架用于组装MBP(麦芽糖结合蛋白)-抗PDL1单链抗体融合蛋白(以下简称融合蛋白，分子量为69kDa)的四聚体。MBP-抗PDL1单链抗体融合蛋白按照实施例6进行制备。L-DNA四聚体框架的四条DNA按照实施例7所述的方法偶联融合蛋白并加以纯化，得到四种L-DNA-融合蛋白。将上述四种L-DNA-融合蛋白的缓冲液置换为磷酸缓冲液(50mM NaH 2PO 4，150mM NaCl，pH 7.0)，并以终浓度为1uM和2uM在37度进行四聚体组装，反应产物用10％SDS-PAGE和分子筛进行分析。
蛋白电泳结果如图10所示，融合蛋白在偶联了L-DNA之后，分子量变大，因此在SDS-PAGE上的条带上移。在两种组装浓度下(1uM和2uM)，四种L-DNA-融合蛋白均可特异性的组装为融合蛋白四聚体，而未反应的融合蛋白单体(即无L-DNA偶联的融合蛋白)则没有参与组装。表明L-DNA四聚体框架介导了融合蛋白四聚体的组装，且融合蛋白的大分子量并未影响该L-DNA框架的组装效率。
分子筛结果如图11所示，融合蛋白四聚体呈现单一的洗脱峰，且峰型对称，表明融合蛋白四聚体非常均一，只存在一种组装方式。
实施例12：基于L-DNA框架制备的四特异性抗体体外活性评价
为分析抗CEA/PD-L1/CD3四特异性抗体的体外活性，用大肠癌细胞系LS174T(CEA阳性细胞)作为细胞模型。将2万个LS174T细胞铺48孔板，24小时后，用3-十八烷基-2-[3-(3-十八烷基-2(3H)-苯并恶唑-2-亚基)-1-丙烯-1-基]苯并恶唑高氯酸盐(DiOC18，DIO细胞膜绿色荧光探针)染色，然后加入40万个PBMC(外周血单核细胞)进一步孵育，同时加入浓度梯度稀释的抗CEA/PD-L1/CD3四特异性抗体(0.001nM-20nM)，共孵育96小时。用碘化丙啶(PI)标记死亡细胞后，流式细胞仪检测绿色荧光探针和碘化丙啶荧光双信号的细胞数量。阳性对照为Triton-X100处理，以及Dynabeads(表面偶联了抗CD28/CD3抗体的细珠，可高效激活T细胞)，阴性对照为抗体所用的缓冲液。以Triton-X100处理组的细胞死亡量作为100％杀伤，缓冲液组作为0％杀伤。
结果如图12所示，抗CEA/PD-L1/CD3四特异性抗体高效介导了T细胞对LS174T细胞的杀伤，且该杀伤活性呈剂量依赖。抗CEA/PD-L1/CD3四特异性抗 体的EC50约为0.7nM。
实施例13：抗CEA/PD-L1/CD3四特异性抗体激活T细胞的能力
为分析抗CEA/PD-L1/CD3四特异性抗体激活T细胞的能力，选择干扰素γ(IFN-γ)作为检测对象。具体过程如下：用大肠癌细胞系LS174T作为细胞模型。将2万个LS174T细胞铺48孔板，24小时后，然后加入40万个PBMC(外周血单核细胞)进一步孵育，同时加入浓度梯度稀释的抗CEA/PD-L1/CD3四特异性抗体(0.001nM-20nM)，共孵育96小时。用布雷非德菌素A(Brefeldin A)将IFN-γ固定在T细胞表面，再用荧光标记抗CD3抗体标记T细胞，然后用流式细胞仪检测IFN-γ/CD3双阳性的细胞数量。阳性对照为Dynabeads(表面偶联了抗CD28/CD3抗体的细珠，可高效激活T细胞)，阴性对照为抗体所用的缓冲液。
结果如图13所示，各个浓度的抗CEA/PD-L1/CD3四特异性抗体均可以激活T细胞，使其释放IFN-γ，这与实施例12中的体外活性实验结果相一致。抗CEA/PD-L1/CD3四特异性抗体对T细胞的激活能力与阳性对照(Dynabeads)相当，而阴性对照(缓冲液)则没有明显的IFN-γ释放。
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. 一种蛋白药物文库，其特征在于，所述蛋白药物文库包括C种不同的蛋白药物单体，其中，所述的蛋白药物单体包括蛋白药物元件部分以及与所述蛋白药物元件部分相连的核酸元件部分，并且一个所述蛋白药物单体的所述的核酸元件部分与至少一个不同的蛋白药物单体的核酸元件部分可通过互补形成双链配对结构，从而构成多聚体的蛋白药物；
    其中，C为≥2的正整数。
  2. 如权利要求1所述的蛋白药物文库，其特征在于，所述的蛋白药物元件部分与所述核酸元件部分直接相连，或间接相连。
  3. 如权利要求1所述的蛋白药物文库，其特征在于，所述的蛋白药物单体具有式I所示的结构：
    P─X─L─Y─A─Z  (I)
    式中，
    P为蛋白类药物分子(即蛋白药物元件部分)；
    X为无或冗余肽；
    L为连接物分子；
    Y和Z为无或冗余核酸；
    A为一核酸序列，所述核酸序列选自下组：左旋核酸、肽核酸、锁核酸、硫代修饰核酸、2’-氟修饰核酸、5-羟甲基胞嘧啶核酸、或其组合；
    “─”为共价键；
    其中任一种蛋白药物单体的核酸A具有至少一个互补配对区，所述互补配对区与所述蛋白药物文库中的至少一种蛋白药物单体的核酸A的互补配对区部分或完全互补。
  4. 如权利要求1所述的蛋白药物文库，其特征在于，所述蛋白类药物分子P选自下组：抗体、激活或抑制受体或其他蛋白的配体、生物活性酶、或其组合。
  5. 如权利要求1所述的蛋白药物文库，其特征在于，所述抗体选自用于治疗下述疾病的抗体：癌症、自身免疫疾病、免疫检查点、器官移植排斥、类风湿性关节炎、糖尿病、血友病。
  6. 如权利要求1所述的蛋白药物文库，其特征在于，所述连接物分子L具有双 功能接头，可偶联核酸A或Y的具有修饰基团的修饰端和抗体P或X的特异性连接位点。
  7. 一种组装用于个性化治疗的蛋白药物的方法，其特征在于，包括：
    (a)基于制药信息，从权利要求1所述的蛋白药物文库中选取至少二种蛋白药物单体；和
    (b)将所述的至少两种蛋白药物单体进行混合，从而组装形成多聚体形式的多特异性蛋白药物。
  8. 一种多聚体蛋白药物，其特征在于，所述多聚体蛋白药物为D种蛋白药物单体通过核酸互补形成双链配对结构而形成的多聚体，其中D为≥2的正整数；
    其中，所述的蛋白药物单体包括蛋白药物元件部分以及与所述蛋白药物元件部分相连的核酸元件部分，并且一个所述蛋白药物单体的所述的核酸元件部分与另一不同的蛋白药物单体的核酸元件部分可通过互补形成双链配对结构。
  9. 如权利要求8所述的多聚体蛋白药物，其特征在于，所述的核酸元件部分选自下组：左旋核酸、肽核酸、锁核酸、硫代修饰核酸、2’-氟修饰核酸、5-羟甲基胞嘧啶核酸、或其组合。
  10. 一种药物组合物，其特征在于，所述的药物组合物含有
    (i)权利要求8所述的多聚体蛋白药物作为活性成分；和
    (ii)药学上可接受的载体。
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