WO2021115497A2 - 蛋白-药物偶联物和定点偶联方法 - Google Patents

蛋白-药物偶联物和定点偶联方法 Download PDF

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WO2021115497A2
WO2021115497A2 PCT/CN2021/083024 CN2021083024W WO2021115497A2 WO 2021115497 A2 WO2021115497 A2 WO 2021115497A2 CN 2021083024 W CN2021083024 W CN 2021083024W WO 2021115497 A2 WO2021115497 A2 WO 2021115497A2
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protein
antigen
drug conjugate
connecting peptide
cys1
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PCT/CN2021/083024
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French (fr)
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WO2021115497A3 (zh
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何云
石磊
曹旸
王明
张云
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和铂医药(上海)有限责任公司
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Priority to US18/256,422 priority Critical patent/US20240050583A1/en
Priority to EP21731365.9A priority patent/EP4241789A2/en
Priority to CN202180000978.6A priority patent/CN113164621B/zh
Publication of WO2021115497A2 publication Critical patent/WO2021115497A2/zh
Publication of WO2021115497A3 publication Critical patent/WO2021115497A3/zh
Priority to TW110145684A priority patent/TW202222349A/zh

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    • 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
    • A61K47/68—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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • A61K47/68—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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • 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
    • A61K47/68—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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61K47/68—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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • 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
    • A61K47/68—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
    • 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
    • A61K47/6851—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 targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention generally relates to the field of biomedicine, and specifically relates to a protein-drug conjugate and a method for preparing the protein-drug conjugate.
  • Antibody-Drug Conjugate is produced by coupling an antibody targeting tumor antigen to a highly effective cytotoxic small molecule chemical drug through a linker. It uses the characteristics of specific binding of the antibody and the target antigen to Targeted delivery of molecular drugs to tumor cells in order to play a role in killing tumors.
  • ADC Antibody-Drug Conjugate
  • ADC technology has also been applied to non-tumor fields; for example, AbbVie Co. conjugates steroids to anti-TNF ⁇ antibodies for the treatment of various TNF ⁇ -mediated autoimmune diseases.
  • Cysteine coupling technology is a representative of early chemical coupling methods; it uses the four natural interchain disulfide bonds of IgG1 antibody as the active sulfhydryl group for coupling. Compared with lysine amide coupling technology, this The technology has a controllable DAR value (drug/antibody ratio), but still produces a mixture of distributions with a DAR value of 0 to 8. Due to the uneven composition of the coupling product produced by this kind of technology, its stability and pharmaceutical properties are quite insufficient.
  • the coupling technology represented by the third generation ADC mainly includes ThioMab, ThioBridge, introduction of unnatural amino acids, transpeptidation reaction, N-sugar chain coupling, etc.
  • ThioMab technology was first developed by Genentech. Based on the cysteine coupling technology, it introduced two or more cysteines as coupling sites at specific sites other than natural disulfide bonds in antibodies.
  • the ThioBridge technology is to reduce the disulfide bond of the monoclonal antibody itself, and use reagents such as dibromomaleimide or dibromopyridinediphenyl ether to react with the reduced interchain cysteine to re-bridge the bridge to obtain the main group.
  • the score is a product with a DAR of 4, but this technology still has the risk of disulfide bond mismatch.
  • Unnatural amino acid coupling technology uses a special tRNA synthetase that encodes unnatural amino acids, allowing cells to synthesize various antibodies with p-acetylphenylalanine residues, using p-acetylphenylalanine
  • the carbonyl group of the acid residue and the linker with the alkoxyamine group undergo a coupling reaction to obtain a product with a DAR of 2 as the main component; because this technology requires the use of modified host cells to produce unnatural amino acids,
  • the obvious disadvantage is that the yield of antibody production is lower and the cost is higher.
  • the coupling technology using transpeptidation reaction is to introduce a sequence of LPETG into the C'end of the heavy or light chain of the antibody.
  • transpeptidase Sortase A specifically recognizes the sequence and hydrolyzes the peptide bond between threonine and glycine, and then Conjugates containing glycine are linked to threonine; however, additional peptide sequences are introduced into the coupling products produced by this technology, and therefore also face the risk of potential immunogenicity.
  • N-sugar chain coupling technology such as GlycoConnect technology uses the endoglycosidase Endo S2 to trim the sugar chain of Asn297 (Eu number) located in the CH2 region of the antibody, and then uses the mutant galactosyltransferase GalT (Y289L) and N-azido Acetylgalactosamine (GalNAz) introduces an azide group, and click Chemistry (Click Chemistry) is used to couple the azide group with a small molecule drug to produce a stable and uniform antibody-conjugated drug with a DAR of 2; this technology requires the use of Special mutant enzymes have higher process development costs.
  • GlycoConnect technology uses the endoglycosidase Endo S2 to trim the sugar chain of Asn297 (Eu number) located in the CH2 region of the antibody, and then uses the mutant galactosyltransferase GalT (Y289L) and N-azido Acetylgalact
  • This application provides a method for site-directed coupling of protein-drug conjugates and protein-drug conjugates prepared by the method.
  • the present application provides a protein-drug conjugate, which comprises an antigen-binding protein portion and a drug conjugate portion;
  • the antigen-binding protein portion includes one or more antigen-binding fragments, and binds to the antigen At least two connecting peptides directly or indirectly connected to the fragment;
  • the at least two connecting peptides include a first connecting peptide and a second connecting peptide; wherein, the first connecting peptide includes a first cysteine Cys1 and a second half Cys2, the drug conjugate part is coupled to the first connecting peptide through the Cys1; and the second connecting peptide comprises a first cysteine Cys1 and a second cysteine Cys2, The drug conjugate part is coupled to the second connecting peptide through the Cys1.
  • the Cys2 of the first connecting peptide and the Cys2 of the second connecting peptide are connected by a disulfide bond.
  • the one or more antigen-binding fragments do not include a functional group that can affect the coupling of the drug conjugate with the first connecting peptide and/or the second connecting peptide at Cys1, or
  • the effect is manifested in significantly reducing the coupling probability of the drug conjugate at the Cys1 site.
  • the one or more antigen-binding fragments do not include a functional group capable of forming a disulfide bond with the Cys1 of the first connecting peptide and/or the Cys1 of the second connecting peptide Or a conjugate formed by the aforementioned functional group and other drug conjugates.
  • the one or more antigen-binding fragments do not include a half capable of forming a disulfide bond with the Cys1 of the first connecting peptide and/or the Cys1 of the second connecting peptide. Cystine or a conjugate formed by the aforementioned cysteine and other drug conjugates.
  • the amino acid sequence of the first connecting peptide and the second connecting peptide may be the same or different.
  • the first connecting peptide and/or the second connecting peptide comprise the amino acid sequence shown in Cys1-(X)n-Cys2 from N-terminus to C-terminus; wherein, n is greater than or equal to An integer of 3, X is any amino acid other than Cys.
  • the first connecting peptide and/or the second connecting peptide are derived from an antibody hinge region sequence or a sequence derived therefrom.
  • the derivative sequence is obtained by modifying the sequence of the hinge region of the antibody, and the modification does not involve the change of Cys1 and Cys2 in the hinge region of the antibody.
  • the modification is to adjust the type and number of amino acids between Cys1 and Cys2 in the sequence of the antibody hinge region and/or the addition of flexible linking amino acids before Cys1.
  • the connecting peptide is derived from a human IgG hinge region, for example, the antibody hinge region is a human IgG1 hinge region.
  • the connecting peptide is the hinge region of human IgG1, the sequences of the first connecting peptide and the second connecting peptide are each independently shown in SEQ ID NO: 73, and the Cys1 in the connecting peptide
  • the hinge region of human IgG1 is Cys-220 coded according to EU
  • the Cys2 is Cys-226 of human IgG1 hinge region coded according to EU.
  • the first connecting peptide and the second connecting peptide may each independently be derived from a derived sequence of the hinge region of human IgG1, the amino acid sequence of which is shown in SEQ ID NOs: 74-105, 145-146 As shown in any one of the above; the Cys1 in the first connecting peptide is Cys corresponding to the human IgG1 hinge region Cys-220, and the Cys2 in the first connecting peptide is corresponding to the human IgG1 hinge region Cys-226 The Cys; in the second connecting peptide, the Cys1 is the Cys corresponding to the human IgG1 hinge region Cys-220, and the Cys2 in the second connecting peptide is the Cys corresponding to the human IgG1 hinge region Cys-226.
  • the connecting peptide is the hinge region of mouse IgG2c, and the sequences of the first connecting peptide and the second connecting peptide are each independently shown in SEQ ID NO: 147, and the connecting peptide
  • the Cys1 in is the first cysteine of the hinge region sequence of mouse IgG2c, and the Cys2 is the second cysteine of the sequence.
  • multiple antigen-binding fragments included in the antigen-binding protein portion of the protein-drug conjugate are connected to the same connecting peptide or to different connecting peptides.
  • the plurality of antigen-binding fragments may be partly or all the same or different from each other.
  • the plurality of antigen-binding fragments includes a first antigen-binding fragment and a second antigen-binding fragment.
  • the first antigen-binding fragment and the second antigen-binding fragment may be the same or different.
  • the first antigen-binding fragment and the second antigen-binding fragment bind to the same target and have the same amino acid sequence; for example, the first antigen-binding fragment and the second antigen-binding fragment bind to the same target but The amino acid sequence is not the same; for example, the first antigen-binding fragment and the second antigen-binding fragment bind different targets.
  • the plurality of antigen-binding fragments comprise a first antigen-binding fragment, a second antigen-binding fragment, a third antigen-binding fragment, and a fourth antigen-binding fragment.
  • the first antigen-binding fragment, the second antigen-binding fragment, the third antigen-binding fragment, and the fourth antigen-binding fragment may be partly or all the same or different from each other.
  • the first antigen-binding fragment and the second antigen-binding fragment bind to the same target and have the same amino acid sequence; for example, the first antigen-binding fragment and the second antigen-binding fragment bind to the same target but The amino acid sequence is not the same; for example, the first antigen-binding fragment and the second antigen-binding fragment bind different targets.
  • the third antigen-binding fragment and the fourth antigen-binding fragment bind to the same target and have the same amino acid sequence; for example, the third antigen-binding fragment and the fourth antigen-binding fragment bind to the same target but The amino acid sequence is not the same; for example, the third antigen-binding fragment and the fourth antigen-binding fragment bind different targets.
  • the first antigen-binding fragment and the second antigen-binding fragment and the third antigen-binding fragment and the fourth antigen-binding fragment bind to different targets.
  • the one or more antigen-binding fragments may each independently be a VHH domain, VH, VL, Fab, ScFv, soluble extracellular domain of receptor protein, ligand protein, lipocalin. , Neural cell adhesion molecule NCAM, fibronectin fibronectin and/or ankyrin repeat fragment protein DARPins and other derivative protein structures.
  • the one or more antigen-binding fragments can specifically bind to tumor antigens or non-tumor antigens.
  • the tumor antigens include CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, CD38, CD123, CD44v6, B7H3, B7H4, KIT, IL- 13Ra2, IL-11Ra, PSCA, PSMA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, fucosyl GM1 (fucosyl GM1), sLe, GM3, TGS5, HMWMAA, GD2, FOLR1, FOLR2, TEM1/CD248, TEM7R, CLDN6, CLDN18.2, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR -1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51
  • the first antigen-binding fragment and/or the second antigen-binding fragment each independently comprise HCDR1, HCDR2, and HCDR3, and the antigen-binding fragment comprises any one selected from the following A set of amino acid sequences: (1) HCDR1: SEQ ID NO: 7, HCDR2: SEQ ID NO: 22, HCDR3: SEQ ID NO: 38; (2) HCDR1: SEQ ID NO: 10, HCDR2: SEQ ID NO: 25 , HCDR3: SEQ ID NO: 41; (3) HCDR1: SEQ ID NO: 11, HCDR2: SEQ ID NO: 26, HCDR3: SEQ ID NO: 42; (4) HCDR1: SEQ ID NO: 13, HCDR2: SEQ ID NO: 28, HCDR3: SEQ ID NO: 44; (5) HCDR1: SEQ ID NO: 14, HCDR2: SEQ ID NO: 29, HCDR3: SEQ ID NO: 42; (6) HCDR1: SEQ ID NO: 9
  • the antigen-binding fragment includes VH, and the VH includes the amino acid sequence shown in any one of SEQ ID NOs: 55-59, 61, and 62.
  • the first antigen-binding fragment and/or the second antigen-binding fragment each independently comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, and said HCDR1, HCDR2, and HCDR3, LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 12, SEQ ID NO: 27, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 53 in sequence. .
  • the first antigen-binding fragment and/or the second antigen-binding fragment each independently comprise VL and VH, and the VL comprises the amino acid sequence shown in SEQ ID NO: 63, the VH includes the amino acid sequence shown in SEQ ID NO:60.
  • the first antigen-binding fragment and/or the second antigen-binding fragment each independently comprise a pair of VH and VL and/or another VH, and the pair of VH and VL respectively It includes the amino acid sequence shown in SEQ ID NO: 60 and SEQ ID NO: 63, and the other VH includes the amino acid sequence shown in SEQ ID NO: 62.
  • the pair of VH and VL may form a scFv.
  • the first antigen-binding fragment and/or the second antigen-binding fragment each independently comprise VH, and the VH comprises the amino acid sequence shown in SEQ ID NO: 62; and, the The third antigen-binding fragment and/or the fourth antigen-binding fragment each independently comprise a pair of VH and VL, and the pair of VH and VL respectively comprise the amino acids shown in SEQ ID NO: 130 and SEQ ID NO: 131 sequence.
  • the third antigen-binding fragment and/or the fourth antigen-binding fragment may comprise Fab.
  • the antigen-binding protein portion of the protein-drug conjugate may also include a pairing unit portion capable of pairing the first connecting peptide and the second connecting peptide, and the pairing unit may include at least a first connecting peptide.
  • a pairing subunit and a second pairing subunit, and the first pairing subunit can interact with the second pairing subunit through a covalent bond or a non-covalent bond.
  • the pairing unit is an Fc fragment or a mutated Fc fragment.
  • the N-terminus of the first pairing subunit is connected to the C-terminus of the first connecting peptide
  • the N-terminus of the second pairing subunit is connected to the C-terminus of the second connecting peptide.
  • the N-terminus of the first pairing subunit is connected to the C-terminus of the second connecting peptide
  • the N-terminus of the second pairing subunit is connected to the C-terminus of the first connecting peptide.
  • the protein-drug conjugate comprises the amino acid sequence shown in any one of SEQ ID NOs: 64-71, 106-129, and 148-149.
  • the drug conjugate portion of the protein-drug conjugate includes two drug conjugates respectively connected to a first connecting peptide and a second connecting peptide, the drug conjugate Contains the drug load and optionally a linker.
  • the drug conjugate in the protein-drug conjugate includes at least one loaded drug.
  • the loaded drug may include, but is not limited to, small molecule compounds, toxin molecules, antibiotics, oligonucleotides, protein degradation targeted chimeras (PROTAC), affinity ligands, fluorescent or radionuclide labeling groups, polypeptides, immunology Regulators, etc.
  • the linker includes a cleavable linker or a non-cleavable linker; wherein the cleavable linker includes a protease cleavable linker.
  • the drug conjugate includes mc-vc-PAB-MMAE (as shown in Figure 6).
  • the drug conjugate includes PROTAC, such as a BRD4 protein degrading agent (as shown in Figure 64).
  • the protein-drug conjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the present application provides a method for preparing the protein-drug conjugate described in the present application, which comprises making the drug conjugate and the first connecting peptide and/or the second connecting peptide The reactive sulfhydryl group of Cys1 on the above is covalently bonded.
  • the protein-drug conjugate includes an antigen-binding protein portion and a drug conjugate portion; the antigen-binding protein portion includes one or more antigen-binding fragments, and at least two that are directly or indirectly connected to the antigen-binding fragments Connecting peptide; the two connecting peptides include a first connecting peptide and a second connecting peptide, wherein the first connecting peptide includes a first cysteine Cys1 and a second cysteine Cys2, and the second connecting peptide
  • the peptide comprises a first cysteine Cys1 and a second cysteine Cys2; the method includes linking the drug conjugate portion to the first connecting peptide through the Cys1 of the first connecting peptide, And/or the drug conjugate portion is connected to the second connecting peptide through the Cys1 of the second connecting peptide.
  • the one or more antigen-binding fragments do not include a functional group that can affect the coupling of the drug conjugate with the first connecting peptide and/or the second connecting peptide at Cys1, or
  • the effect is manifested in significantly reducing the coupling probability of the drug conjugate at the Cys1 site.
  • the one or more antigen-binding fragments in the method do not include the ability to form a second connecting peptide with the Cys1 of the first connecting peptide and/or the Cys1 of the second connecting peptide.
  • the one or more antigen-binding fragments do not include a cysteine capable of forming a disulfide bond with the Cys1 of the first connecting peptide and/or the Cys1 of the second connecting peptide.
  • the amino acid sequence of the first connecting peptide and the second connecting peptide in the method may be the same or different.
  • the first connecting peptide and/or the second connecting peptide in the method comprises the amino acid sequence shown in Cys1-(X)n-Cys2 from N-terminus to C-terminus; wherein, n is an integer greater than or equal to 3, and X refers to any amino acid that is not Cys.
  • the first connecting peptide and/or the second connecting peptide are derived from an antibody hinge region sequence or a sequence derived therefrom.
  • the derivative sequence in the method is obtained by modifying the sequence of the hinge region of the antibody, and the modification does not involve changes in Cys1 and Cys2 in the hinge region of the antibody.
  • the modification is to adjust the type and number of amino acids between Cys1 and Cys2 in the sequence of the antibody hinge region and/or the addition of flexible linking amino acids before Cys1.
  • the connecting peptide in the method is a human IgG hinge region, preferably a human IgG1 hinge region.
  • the first connecting peptide in the method is the hinge region of human IgG1, the sequence of the first connecting peptide is shown in SEQ ID NO: 73, and the Cys1 in the first connecting peptide
  • the hinge region of human IgG1 is Cys-220 coded according to EU
  • the Cys2 is Cys-226 of human IgG1 hinge region coded according to EU.
  • the second connecting peptide in the method is the hinge region of human IgG1, the sequence of the second connecting peptide is shown in SEQ ID NO: 73, and the Cys1 in the second connecting peptide
  • the hinge region of human IgG1 is Cys-220 coded according to EU
  • the Cys2 is Cys-226 of human IgG1 hinge region coded according to EU.
  • the first connecting peptide in the method may be derived from a derived sequence of the hinge region of human IgG1, and its amino acid sequence is as shown in any one of SEQ ID NOs: 74-105, 145-146.
  • the Cys1 in the first connecting peptide is Cys corresponding to Cys-220 in the hinge region of human IgG1
  • the Cys2 in the connecting peptide is Cys corresponding to Cys-226 in the hinge region of human IgG1.
  • the second connecting peptide in the method may be derived from a derived sequence of the hinge region of human IgG1, and its amino acid sequence is as shown in any one of SEQ ID NOs: 74-105, 145-146.
  • the Cys1 in the second connecting peptide is Cys corresponding to Cys-220 in the hinge region of human IgG1
  • the Cys2 in the connecting peptide is Cys corresponding to Cys-226 in the hinge region of human IgG1.
  • the first connecting peptide in the method is the hinge region of mouse IgG2c, the amino acid sequence of which is shown in SEQ ID NO: 147, and the Cys1 in the first connecting peptide is The first cysteine of the sequence, and the Cys2 is the second cysteine of the sequence.
  • the second connecting peptide in the method is the hinge region of mouse IgG2c, the amino acid sequence of which is shown in SEQ ID NO: 147, and the Cys1 in the second connecting peptide is The first cysteine of the sequence, and the Cys2 is the second cysteine of the sequence.
  • the method includes reduction, coupling, and optionally purification steps.
  • the reduction includes reducing the disulfide bond between the Cys1 of the first connecting peptide and the Cys1 of the second connecting peptide under the condition of a reducing agent.
  • the reduction includes the use of reducing agents DTT and/or TCEP; in certain embodiments, the reducing agent is TCEP.
  • the amount of the reducing agent is 1 to 50 mole multiples; in some embodiments, the amount of the reducing agent is 1 to 6 mole multiples; in some embodiments, the amount of reducing agent is It is 1.5 to 3 mole multiples.
  • the reaction time of the reduction is 1 to 17 hours; in some embodiments, the reaction time of the reduction is 1 to 3 hours; in some embodiments, the reaction time of the reduction is 1.5 to 2 hours.
  • the reaction temperature of the reduction is 0-40°C; in some embodiments, the reaction temperature of the reduction is 0°C or room temperature (25-30°C) or 37°C.
  • the pH of the reduction reaction buffer is 4.0 to 9.0; in some embodiments, the pH of the reduction reaction buffer is 5.0 to 8.0; in some embodiments, the pH is 5.0 to 7.0; in some embodiments, the pH is 5.0 to 6.0.
  • the coupling includes covalently binding the drug conjugate to the reactive sulfhydryl group of Cys1 on the first connecting peptide and/or the second connecting peptide.
  • the drug conjugate includes mc-vc-PAB-MMAE or PROTAC or CpG oligonucleotides.
  • the amount of the drug conjugate is 1 to 50 mole multiples; in some embodiments, the amount of the drug conjugate is 1 to 10 mole multiples; in some embodiments , The dosage of the drug conjugate is 3 to 7 mole multiples.
  • the drug conjugate is mc-vc-PAB-MMAE, and the amount of mc-vc-PAB-MMAE is 1 to 50 molar multiples; in some embodiments, mc-vc-PAB The amount of MMAE is 3 to 18 mole multiples; in some embodiments, the amount of mc-vc-PAB-MMAE is 3 to 7 mole multiples.
  • the amount of the drug conjugate PROTAC is 10 to 15 molar multiples.
  • the coupling reaction time is 0.5 to 10 hours; in some embodiments, the coupling reaction time is 0.5 to 3 hours; in some embodiments, the coupling reaction time is 0.5 to 3 hours. For 0.5 to 1 hour.
  • reaction temperature of the coupling is 0-40°C; in some embodiments, the reaction temperature of the coupling is room temperature (25-30°C).
  • the pH of the coupled reaction buffer is 4.0 to 9.0; in some embodiments, the pH of the coupled reaction buffer is 5.0 to 8.0; in some embodiments, the pH of the coupled reaction buffer is 5.0 to 8.0; The pH is 5.0 to 7.0; in some embodiments, the pH is 7.0 to 8.0; in some embodiments, the pH is 5.0 to 6.0.
  • the purity of the protein-drug conjugate is greater than 80%.
  • the purity of the protein-drug conjugate is greater than 82%, greater than 85%, greater than 88%, or greater than 90%.
  • the preparation method can provide a combination of reaction steps and reaction condition parameters including "reduction” and "coupling”; the combination of the reaction steps and reaction condition parameters can further improve the coupling
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the protein-drug conjugate and a pharmaceutically acceptable carrier.
  • the present application provides a method for preventing and/or treating diseases, which comprises administering the protein-drug conjugate and/or the pharmaceutical composition, optionally, the protein-drug conjugate
  • the drug and/or the pharmaceutical composition are used in combination with other therapies or drugs.
  • the present application provides the use of the protein-drug conjugate and/or the pharmaceutical composition in the preparation of medicines for the treatment of tumors or other diseases.
  • the application provides the use of the protein-drug conjugate in combination with other therapies or drugs in the preparation of medicines for the treatment of tumors or other diseases.
  • the other therapies or drugs are selected from the group consisting of chemotherapy, radiotherapy, miRNA and oligonucleotides.
  • the tumor is selected from any one or more of the following group: lymphoma, multiple myeloma, breast cancer, ovarian cancer, kidney cancer, endometrial cancer, melanoma, pancreatic cancer , Lung cancer, gastric cancer, liver cancer, mesothelioma, esophageal cancer, head and neck cancer, cholangiocarcinoma, gallbladder cancer, bladder cancer, thymic cancer and colorectal cancer.
  • the present application provides a method for diagnosing specific diseases, which includes using the protein-drug conjugate and/or the pharmaceutical composition.
  • the present application provides a method for detecting a specific target, which includes using the protein-drug conjugate and/or the pharmaceutical composition, preferably, the detection is for non-disease diagnosis purposes.
  • the present application provides a kit for detection, which includes the protein-drug conjugate and/or the pharmaceutical composition, and optionally instructions for use.
  • the present application provides a drug delivery device, which includes the protein-drug conjugate and/or the pharmaceutical composition, and administration of the protein-drug conjugate and/or the drug combination ⁇ Device.
  • Cys-220 (Eu code) in the hinge region of the human IgG1 heavy chain has the opportunity to form a disulfide bond between the heavy chains, but this disulfide bond has nothing else
  • the disulfide bond is stable, and only a weaker reducing agent is needed to distinguish the disulfide bond of Cys-220 from other disulfide bonds, so that the disulfide bond of Cys-220 can be effectively opened while the other disulfide bonds are maintained. Intact, thus making Cys-220 almost the only free cysteine residue as a site-directed coupling site for covalent binding to the drug conjugate.
  • the hinge region-derived sequence can also be used as a connecting peptide to achieve the corresponding Site-directed coupling at Cys-220 in the natural hinge region.
  • Figure 1 shows the disulfide bond structure of human IgG1 antibody.
  • Figure 2 shows the structure of a heavy chain antibody HCAb with the sequence of the hinge region of a human IgG1 antibody, and the difference between the disulfide bond structure of the hinge region between human IgG1 and HCAb.
  • Figure 3 shows the two possible states of Cys-220 at position 220 (Eu numbering) of the hinge region in the HCAb structure: Cys-220 has formed or has not formed a disulfide bond between the heavy chains.
  • Figure 4 shows the mass spectrometry deconvolution processing map of the molecular weight analysis of the non-reduced sample of HCAb PR000020.
  • Figure 5 shows the results of papain digestion analysis of HCAb PR000020: (A) Hypothesis of whether Cys-220 forms disulfide bonds between heavy chains and the corresponding digestion product composition; (B) digestion product Non-reduced SDS-PAGE results.
  • Figure 6 shows (A) a schematic diagram of the ADC containing the drug conjugate mc-vc-PAB-MMAE and the structure of mc-vc-PAB-MMAE (also known as VcMMAE).
  • mc-vc-PAB-MMAE consists of the linker mc -vc-PAB and drug-loaded MMAE, ("antibody” and “ADCs” in the figure are only general illustrations, and do not refer to the specific structure of the "antigen binding protein” or “protein-drug conjugate” of the present invention ); and (B) the characteristic fragment structure of mc-vc-PAB-MMAE in mass spectrometry analysis.
  • Figure 7 shows the binding activity of HCAb PR000020 and its conjugate product PR000020-ADC to cells that highly express CTLA4.
  • Figure 8 shows the HIC-HPLC analysis results of HCAb PR000020 and its coupling product PR000020-ADC: (A) before HCAb coupling; (B) after coupling and before purification; (C) after coupling and one-step HIC purification.
  • Figure 9 shows the RP-HPLC analysis results of HCAb PR000020 and its coupling product PR000020-ADC: (A) before HCAb coupling; (B) after coupling and before purification; (C) after coupling and one-step HIC purification.
  • Figure 10 shows the mass spectrometry deconvolution processing pattern of the molecular weight analysis of the coupling product PR000020-ADC of HCAb PR000020: (A) non-reduced sample; (B) reduced sample.
  • Figure 11 shows the analysis of the coupling site of PR000020-ADC using LC-MS peptide spectrogram: (A) the amino acid sequence of PR000020-ADC, the peptide fragments containing Cys-IAM are marked with a gray background; (B) PR000020-ADC The sample contains Cys-IAM polypeptide fragments; (C) PR000020 sample contains Cys-IAM polypeptide fragments; (B) and (C) for the definition of each column name in the table below (C).
  • Figure 12 shows the SEC-HPLC analysis results of the sample after HCAb PR000759 protein expression and purification.
  • Figure 13 shows the HIC-HPLC analysis results of a sample of the coupling product PR000759-ADC of HCAb PR000759 after one-step HIC purification.
  • Figure 14 shows the analysis of the coupling sites of PR000759-ADC using LC-MS peptide spectroscopy and the selected peptides with coupling sites; the interpretation of each column name in the table is the same as Figure 11(C).
  • Figure 15 shows the mass spectrometry deconvolution processing map of the molecular weight analysis of HCAb PR000759 and its coupling product PR000759-ADC: (A) PR000759 non-reduced sample; (B) PR000759-ADC non-reduced sample; (C) PR000759- A reduced sample of ADC.
  • Figure 16 shows the SEC-HPLC analysis results of the sample after HCAb PR001046 protein expression and purification.
  • Figure 17 shows the HIC-HPLC analysis results of a sample of the coupling product PR001046-ADC of HCAb PR001046 after one-step HIC purification.
  • Figure 18 shows the LC-MS peptide spectrogram analysis of PR001046-ADC compound coupling sites and the screened peptides with coupling sites; the interpretation of each column name in the table is the same as Figure 11(C).
  • Figure 19 shows the mass spectrometry deconvolution processing pattern of molecular weight analysis of HCAb PR001046 and its coupling product PR001046-ADC: (A) PR001046 non-reduced sample; (B) PR001046-ADC non-reduced sample; (C) PR001046- A reduced sample of ADC.
  • Figure 20 shows the binding activity of HCAb PR001046 and its conjugate product PR001046-ADC to cells that highly express BCMA.
  • Figure 21 shows the specific cytotoxicity of PR001046-ADC: (A) specifically kills BCMA+ cells NCI-H929 and mixed cells; (B) the effect of ADCs of different purity (D2 components) on cytotoxicity.
  • Figure 22 shows the SEC-HPLC analysis results of the sample after HCAb PR004432 protein expression and purification.
  • Figure 23 shows the RP-HPLC analysis results of a sample of the coupling product PR004432-ADC of HCAb PR004432 after one-step HIC purification.
  • Figure 24 shows the use of LC-MS peptide spectrogram to analyze the coupling sites of PR004432-ADC compounds, including the selected peptides with coupling sites and the corresponding coupling site coverage.
  • Figure 25 shows the mass spectrometry deconvolution processing pattern of the molecular weight analysis of the coupling product PR004432-ADC of HCAb PR004432: (A) non-reduced sample; (B) reduced sample.
  • Figure 26 shows the binding activity of HCAb PR004432 and its conjugate product PR004432-ADC to cells that highly express 5T4.
  • Figure 27 shows the specific cytotoxicity of PR004432-ADC to cells that highly express 5T4.
  • Figure 28 shows the SEC-HPLC analysis results of the sample after HCAb PR004433 protein expression and purification.
  • Figure 29 shows the HIC-HPLC analysis results of a sample of the coupling product PR004433-ADC of HCAb PR004433 after one-step HIC purification.
  • Figure 30 shows the HIC-HPLC analysis results of HCAb PR004433 and its coupling product PR004433-ADC: (A) before HCAb coupling; (B) after coupling and before purification; (C) after coupling and one-step HIC purification.
  • Figure 31 shows the mass spectrometry deconvolution processing pattern of the molecular weight analysis of the coupling product PR004433-ADC of HCAb PR004433: (A) non-reduced sample; (B) reduced sample.
  • Figure 32 shows the binding activity of HCAb PR004433 and its conjugate product PR004433-ADC in binding to cells expressing BCMA: (A) binding to HEK293T-hBCMA cells expressing human BCMA; (B) binding to cells NCI-H929 expressing human BCMA ; (C) Does not bind to BCMA-negative cells SNU-16.
  • Figure 33 shows the specific cytotoxicity of PR004433-ADC: (A) Specific cytotoxicity to HEK293T-hBCMA; (B) No cytotoxicity to HEK293T; (C) Specific cytotoxicity to NCI-H929 ; (D) No cytotoxicity to SNU-16.
  • Figure 34 shows the use of LC-MS peptide spectrogram to analyze the coupling sites of PR004433-ADC compounds, including the selected peptides with coupling sites and the corresponding coupling site coverage.
  • Figure 35 shows the secondary spectrum corresponding to the peptides with coupling sites selected when the LC-MS peptide spectrogram is used to analyze the coupling sites of PR000020-ADC compounds.
  • Figure 36 shows the use of LC-MS peptide spectrogram to analyze the compound coupling site of PR000020-ADC, the enzymatically hydrolyzed peptide PHGSDIWGQGTMVTVSSEPKSC#DK (# indicates coupling site) in ADC sample (top) and monoclonal antibody sample (bottom) ) XIC diagram.
  • Figure 37 shows the analysis of the disulfide bond of Cys-220 in PR002129 by LC-MS: (A) shows the two possible states of Cys-220 in the structure of PR002129; (B) the molecular weight analysis of non-reduced samples of PR002129 Deconvolution of the mass spectrum to process the spectrum.
  • Figure 38 shows the DSC thermodynamic analysis curve of HCAb PR000184 and its C220S derivative variants: (A) PR000184; (B) PR000184 (C220S).
  • Figure 39 shows the DSC thermodynamic analysis curve of HCAb PR000453 and its C220S derivative variants: (A) PR000453; (B) PR000453 (C220S).
  • Figure 40 shows the melting temperature Tm of HCAb PR004432, its C220S derivative variants and its coupling products were determined using the Uncle analysis platform: (A) PR004432; (B) PR004432 (C220S); (C) PR004432-ADC.
  • Figure 41 illustrates the components and related terms of the "protein-drug conjugate" in the specific embodiment of the present invention.
  • Figure 42 shows exemplary antigen binding proteins: (A) an antigen binding protein with two antigen binding fragments and two connecting peptides; (B) an antigen binding protein with four antigen binding fragments and two connecting peptides.
  • Figure 43 shows an exemplary protein-drug conjugate produced by coupling the antigen-binding protein shown in Figure 42: (A) a protein-drug conjugate with two antigen-binding fragments and two connecting peptides; (B) A protein-drug conjugate with four antigen-binding fragments and two connecting peptides.
  • Figure 44 shows the hinge region sequence and disulfide bond structure of different species: (A) the hinge region sequence of different IgG isotypes of human, mouse and alpaca, Cys of cysteine is identified; (B) human IgG1 hinge Region sequence and disulfide bond structure; (C) human IgG4 hinge region sequence and disulfide bond structure; (D) mouse IgG1 hinge region sequence and disulfide bond structure; (E) mouse IgG2a hinge region sequence and disulfide bond structure.
  • Figure 45 shows the total ion chromatogram (TIC) of the mass spectrum obtained by LC-MS analysis of the complete molecular weight of the non-reduced sample of the coupling products prepared by HCAb PR004433 under different reduction and coupling reaction conditions, respectively corresponding to The experiment numbers in Table 10-1 and Table 10-2 of Example 10.1: (A) Experiment #8; (B) Experiment #9; (C) Experiment #11; (D) Experiment #12.
  • TIC total ion chromatogram
  • Figure 46 shows the binding activity of HCAb PR004433 and its variant molecules to NCI-H929 cells.
  • Figure 47 shows the mass spectrometry deconvolution processing pattern of the intact molecular weight analysis of the non-reduced sample of the coupling product PR006468-ADC of HCAb PR006468.
  • Figure 48 shows the use of LC-MS peptide spectroscopy to analyze the coupling sites of PR006468-ADC compounds, including the selected peptides with coupling sites and the corresponding coupling site coverage.
  • Figure 49 shows the binding activity of BCMA binding protein (HCAb PR004433, HCAb PR006468) and its coupling products (PR004433-ADC, PR006468-ADC) to NCI-H929 cells.
  • Figure 50 shows the cytotoxicity of PR006468-ADC and PR004433-ADC to NCI-H929 cells.
  • Figure 51 shows the HIC-HPLC analysis results of the PR002129 coupling product PR002129-ADC: (A) after coupling and before purification; (B) after coupling and one-step HIC purification.
  • Figure 52 shows the mass spectrometry deconvolution processing pattern of the molecular weight analysis of the PR002129 coupling product PR002129-ADC: (A) the non-reduced molecular weight of the sample before HIC purification; (B) the reduced molecular weight of the sample after HIC purification; (C) HIC The non-reduced molecular weight of the purified sample.
  • Figure 53 shows the use of LC-MS peptide spectroscopy to analyze the coupling sites of PR002129-ADC compounds, including the selected peptides with coupling sites and the corresponding coupling site coverage.
  • Figure 54 shows the binding activity of PR002129 and its conjugate product PR002129-ADC to cells with high ROR1 expression.
  • Figure 55 shows the HIC-HPLC analysis results of the pre-purification sample of the coupling product PR006345-ADC of the SIRPa-Fc fusion protein PR006345.
  • Figure 56 shows the non-reducing molecular weight analysis of the pre-purification sample of the coupling product PR006345-ADC of the SIRPa-Fc fusion protein PR006345 and the mass spectrometric deconvolution processing pattern.
  • Figure 57 shows the analysis of PR006345-ADC compound coupling sites using LC-MS peptide spectra, including the selected peptides with coupling sites and the corresponding coupling site coverage.
  • Figure 58 shows the binding activity of PR006345 and its conjugate product PR006345-ADC to cells with high CD47 expression.
  • Figure 59 shows the structure of the tetravalent bispecific antigen binding protein PR005744.
  • Figure 60 shows the internalization of PR005744 on NCI-H929 cells.
  • Figure 61 shows the use of LC-MS peptide spectroscopy to analyze the coupling sites of PR005744-ADC compounds, including the selected peptides with coupling sites and the corresponding coupling site coverage.
  • Figure 62 shows the binding activity of PR005744 and its conjugate product PR005744-ADC to NCI-H929 cells.
  • Figure 63 shows the cytotoxicity of PR005744-ADC to NCI-H929 cells.
  • FIG 64 shows the molecular structure of BRD4 protein degradation agent (PROTAC).
  • Figure 65 shows the non-reduced molecular weight analysis of the mass spectrometry deconvolution processing spectrum of the pre-purification sample of the product PR004433-PROTAC, a product of HCAb PR004433 and PROTAC coupling.
  • Figure 66 shows the binding activity of HCAb PR004433 and its conjugate product PR004433-PROTAC to NCI-H929 cells.
  • Figure 67 shows the structure of the bivalent bispecific antigen binding protein 2129/4433.
  • Figure 68 shows the HIC-HPLC analysis results of the pre-purification sample of the 2129/4433 coupling product 2129/4433-ADC.
  • Figure 69 shows the coupling site analysis of 2129/4433-ADC using LC-MS peptide spectroscopy, including the selected peptides with coupling sites and the corresponding coupling site coverage.
  • Figure 70 shows the secondary spectra corresponding to the peptides with coupling sites selected when the LC-MS peptide spectra are used to analyze the coupling sites of PR001046-ADC compounds.
  • Figure 71 shows the secondary spectra corresponding to the peptides with coupling sites selected when the LC-MS peptide spectra are used to analyze the coupling sites of PR006468-ADC compounds.
  • Figure 72 shows the secondary spectra corresponding to the peptides with coupling sites selected when the LC-MS peptide spectra are used to analyze the coupling sites of the 2129/4433-ADC compounds.
  • the basic four-chain antibody unit is a heterotetrameric protein, which is composed of two identical light chains (L) and two identical heavy chains (H).
  • L light chains
  • H heavy chains
  • each L chain is connected to the H chain by a covalent disulfide bond
  • two H chains are connected to each other by one or more disulfide bonds.
  • the number of disulfide bonds depends on the same species of the H chain. type.
  • Each H and L chain also has regularly spaced intrachain disulfide bonds.
  • Each H chain has a variable domain (VH) at the amino terminus, followed by three (for each alpha and gamma chain) or four (for mu and epsilon isotypes) constant domains (CH).
  • Each L chain has a variable domain (VL) at the amino terminus, followed by a constant domain (CL).
  • Human IgG has four subtypes: IgG1, IgG2, IgG3 and IgG4.
  • Human or murine antibody L chains are further divided into ⁇ chain and ⁇ chain.
  • its variable domain is divided into V ⁇ and V ⁇
  • its constant domain is divided into C ⁇ and C ⁇ .
  • binding protein or "antigen-binding protein” generally refers to a protein containing a portion that binds to an antigen, and optionally a scaffold or framework that allows the portion that binds to the antigen to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen. section.
  • the "binding protein” or “antigen binding protein” may comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both.
  • VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR) and relatively conserved framework regions (FR). Each VH and VL can be composed of three CDRs and four FR regions.
  • VH and VL contain binding sites that interact with antigens.
  • the three CDRs of VH are represented as HCDR1, HCDR2, and HCDR3, respectively, and can also be represented as VH CDR1, VH CDR2, and VH CDR3;
  • the three CDRs of VL are represented as LCDR1, LCDR2 and LCDR3, respectively, and can also be represented as VL CDR1, VL CDR2 And VL CDR3.
  • antigen-binding proteins may include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab) 2 , Fv fragments, F(ab') 2 , scFv, di-scFv and/or dAb), immunoconjugates Compounds, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, soluble extracellular domains of receptor proteins, ligand proteins or other forms of fusion proteins, etc., as long as they show all The required antigen binding activity is sufficient.
  • Fab antigen-binding fragments
  • Fv fragments F(ab') 2
  • scFv di-scFv and/or dAb
  • immunoconjugates Compounds, multispecific antibodies (such as bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, soluble extracellular domains of receptor proteins, ligand proteins or other forms of fusion proteins, etc., as long as they show all The required antigen
  • the amino acid sequences of the CDRs are shown in accordance with Chothia's definition rules.
  • the CDR of an antibody can be defined in a variety of ways in the art, such as the Kabat definition rule based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, U.S. National Institute of Health, Bethesda, Maryland (1991)) and Chothia definition rules based on the location of structural loop regions (see JMol Biol 273:927-48, 1997).
  • the combined definition rule including the Kabat definition and Chothia definition can also be used to determine the amino acid residues in the variable domain sequence.
  • the Combined definition rule is to combine the Kabat definition and Chothia definition range, based on this, a larger range is taken, as shown in the following table for details.
  • CDR complementarity determining region
  • Complementary decision area defined by any one of the known schemes.
  • Laa-Lbb can refer to the amino acid sequence starting from the N-terminus of the antibody light chain, from position aa (Chothia coding rules) to position bb (Chothia coding rules);
  • Haa-Hbb can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain , The amino acid sequence from position aa (Chothia coding rule) to position bb (Chothia coding rule).
  • L24-L34 can refer to the amino acid sequence from the 24th to the 34th starting from the N-terminus of the antibody light chain according to the Chothia coding rules
  • H26-H32 can refer to the amino acid sequence starting from the N-terminus of the antibody heavy chain and according to the Chothia coding rules The amino acid sequence from position 26 to position 32.
  • the term "monoclonal antibody” generally refers to an antibody obtained from a population of substantially homogeneous cells, that is, the antibodies in the population are the same, except for a small number of natural mutations that may exist.
  • Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen.
  • the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture without being contaminated by other immunoglobulins.
  • the modifier "monoclonal” refers to the characteristics of an antibody obtained from a substantially homogeneous antibody population, but is not interpreted as requiring the production of the antibody by any specific method.
  • the monoclonal antibody used according to the present invention can be produced in hybridoma cells, or can be produced by recombinant DNA methods.
  • the term "fully human antibody” generally refers to an antibody expressed by the animal by transferring all the genes encoding the human antibody to a genetically engineered animal with a gene deletion of the antibody. All parts of an antibody (including the variable and constant regions of the antibody) are composed of amino acid sequences of human origin. Fully human antibodies can greatly reduce the immune side effects caused by heterologous antibodies on the human body. Methods for obtaining fully human antibodies in this field can include phage display technology, transgenic mouse technology, and the like.
  • the term "specific binding” generally refers to the binding of an antigen binding protein to an epitope, and this binding requires some complementarity between the antigen binding protein and the epitope. According to this definition, when an antibody binds to an epitope through its antigen binding protein more easily than it will bind to a random, unrelated epitope, the antibody is said to “specifically bind” the antigen.
  • “Epitope” refers to a specific atomic group (for example, sugar side chain, phosphoryl, sulfonyl) or amino acid that binds to an antigen-binding protein (such as an antibody) on an antigen.
  • Fab generally refers to the part of a conventional antibody (such as IgG) that binds to the antigen, including the heavy chain variable region VH, light chain variable region VL, and heavy chain constant region CH1 of the antibody, as well as light Chain constant region CL.
  • the C-terminus of VH is connected to the N-terminus of CH1 to form a heavy chain Fd fragment
  • the C-terminus of VL is connected to the N-terminus of CL to form a light chain
  • the C-terminus of CH1 is further connected to the hinge region of the heavy chain and other constants.
  • the domains are joined to form a heavy chain.
  • “Fab” also refers to a variant structure of Fab.
  • the C-terminus of VH is connected to the N-terminus of CL to form a polypeptide chain
  • the C-terminus of VL is connected to the N-terminus of CH1 to form another polypeptide chain, forming a Fab (cross VH/VL) structure.
  • the CH1 of the Fab is not connected to the hinge region, but the C-terminus of the CL is connected to the hinge region of the heavy chain to form a Fab (cross Fd/LC) structure.
  • VH generally refers to the VH domain of the heavy chain variable region of an antibody.
  • VH may be the heavy chain variable region VH of a conventional antibody (H2L2 structure) of humans or other animals.
  • VH may also be the heavy chain variable region VHH of a heavy chain antibody (HCAb structure) of animals such as camelid.
  • VH can also be the VH of the heavy chain variable region of a fully human heavy chain antibody (HCAb structure) produced using Harbour HCAb transgenic mice.
  • the term "antigen-binding fragment” generally refers to any functional region of a protein that can specifically bind to an antigen.
  • the "antigen-binding fragment” may be “Fab”.
  • the "antigen-binding fragment” can also be "VH”.
  • the "antigen-binding fragment” can also be a single-chain antibody scFv.
  • the "antigen-binding fragment” can also be other antigen-binding forms (such as the soluble extracellular domain of receptor proteins, ligand proteins, lipocalins, neural cell adhesion molecules (NCAM), Derivative protein structures such as fibronectin and DARPins).
  • the term "pairing unit” generally refers to a group of structures containing at least two subunits that can be paired with each other.
  • the subunits that are paired with each other can be combined with each other, and the combination requires complementarity between the subunits; the subunits that are paired with each other can interact with each other by covalent bonding or non-covalent bonding;
  • Valence bond interactions can include van der Waals forces, hydrogen bonds, hydrophobic interactions, electrostatic interactions, dipole interactions, and the like.
  • the "pairing unit” may be a collection of naturally-occurring or modified polypeptide chains in the form of a homologous or heteromultimer; for example, the “pairing unit” may be a dimer form of an antibody Fc fragment or a variant thereof;
  • the "pairing unit” can be the ⁇ chain and the ⁇ chain of CD79; the “pairing unit” can be the ⁇ chain and the ⁇ chain of CD8; the “pairing unit” can be the ⁇ chain and the ⁇ chain of the T cell receptor (TCR).
  • Fc fragment generally refers to a fragment crystallizable (Fc) of an antibody, that is, a polypeptide chain composed of heavy chain constant domains CH2 and CH3.
  • Fc fragment may be in the form of a natural dimer; in other embodiments, the Fc fragment may be in the form of a modified monomer.
  • CTLA4 generally refers to cytotoxic T lymphocyte-associated protein-4 (also known as CD152), its functional variants and/or its functional fragments.
  • the CTLA4 sequence is known in the art.
  • an exemplary full-length human CTLA4 sequence can be found in Uniprot accession number P16410; an exemplary full-length cynomolgus monkey CTLA4 sequence can be found in Uniprot accession number G7PL88.
  • Drugs targeting CTLA4 may be used to treat tumors such as melanoma, lung cancer, colon cancer, liver cancer, head and neck cancer, kidney cancer, breast cancer, pancreatic cancer, and bladder cancer.
  • BCMA tumor necrosis factor receptor superfamily member 17
  • CD269 B cell maturation protein
  • the BCMA sequence is known in the art.
  • an exemplary full-length human BCMA sequence can be found in Uniprot accession number Q02223; an exemplary full-length cyno BCMA sequence can be found in NCBI accession number XP_005591343.
  • Drugs for BCMA may be used to treat multiple myeloma and other hematological malignancies.
  • MSLN generally refers to a mesothelin molecule (also known as MPF), its functional variants and/or its functional fragments.
  • the MSLN sequence is known in the art.
  • an exemplary full-length human MSLN sequence can be found in Uniprot accession number Q13421; an exemplary full-length cynomolgus monkey MSLN sequence can be found in NCBI accession number XP_005590873.
  • Drugs targeting MSLN may be used to treat tumors such as mesothelioma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, and endometrial cancer.
  • the term "5T4" generally refers to trophoblast glycoprotein (also known as TPBG), its functional variants and/or its functional fragments.
  • the 5T4 sequence is known in the art.
  • an exemplary full-length human 5T4 sequence can be found in Uniprot accession number Q13641; an exemplary full-length cynomolgus monkey 5T4 sequence can be found in Uniprot accession number Q4R8Y9.
  • Drugs targeting 5T4 may be used to treat thymic cancer, lung cancer, esophageal cancer, gastric cancer, small bowel cancer, pancreatic cancer, liver cancer, gallbladder cancer, kidney cancer, bladder cancer, ovarian cancer, endometrial cancer, cervical cancer and other tumors.
  • ROR1 generally refers to the inactivated tyrosine protein kinase transmembrane receptor ROR1 (also referred to as NTRKR1), its functional variants and/or its functional fragments.
  • the ROR1 sequence is known in the art.
  • an exemplary full-length human ROR1 sequence can be found in Uniprot accession number Q01973; an exemplary full-length cynomolgus monkey ROR1 sequence can be found in NCBI accession number XP_015290264.
  • Drugs targeting ROR1 may be used to treat leukemia, non-Hodgkin’s lymphoma, mantle cell lymphoma, breast cancer, lung cancer, ovarian cancer and other tumors.
  • CD47 generally refers to the leukocyte surface antigen CD47, its functional variants and/or its functional fragments.
  • the CD47 sequence is known in the art.
  • an exemplary full-length human CD47 sequence can be found in Uniprot accession number Q08722.
  • Drugs targeting CD47 may be used to treat tumors such as leukemia, non-Hodgkin's lymphoma, multiple myeloma, melanoma, and head and neck cancer.
  • SIRP ⁇ generally refers to signal regulatory protein ⁇ (also known as BIT, MFR, MYD1, PTPNS1, SHPS1 or SIRP), its functional variants and/or its functional fragments.
  • SIRP ⁇ is a receptor for CD47.
  • the SIRP ⁇ sequence is known in the art.
  • an exemplary full-length human SIRP ⁇ sequence can be found in Uniprot accession number P78324.
  • protein-drug conjugate generally refers to a type of drug molecule formed by covalently bonding a drug conjugate to an antigen binding protein.
  • the drug conjugate contains at least one loaded drug.
  • the protein-drug conjugate may be an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • exemplary protein-drug conjugates may include, but are not limited to, trastuzumab, ocrelizumab, pertuzumab, rituximab )
  • other antibodies can covalently attach the drug-loading module to the antibody via a linker unit to form an antibody-drug conjugate to achieve a targeted therapeutic effect.
  • protein-drug conjugates may include antigen-binding fragments, linking peptides, and drug conjugates.
  • the drug conjugate of the "protein-drug conjugate” may be mc-vc-PAB-MMAE (also known as VcMMAE).
  • drug conjugate generally refers to a collection of atoms and groups with a specific structure that can be covalently bonded to other molecules through specific functional groups; it contains at least one drug loaded, And optionally a linker.
  • Various drug loads, various linkers or linker components are known in the art.
  • the drug conjugate may be mc-vc-PAB-MMAE (also known as VcMMAE).
  • drug loaded generally refers to a class of small molecule compounds with pharmacological activity or toxins or other drug molecular forms, which can be but not limited to small molecule compounds, toxin molecules, antibiotics, oligonucleotides, Protein degradation targeting chimera (PROTAC), affinity ligand, fluorescent group, nuclide group, polypeptide, immunomodulatory molecule, etc.
  • Various drug loadings are known in the art, such as monomethyl auristatin E ("MMAE”, monomethyl auristatin E), maytansine (“DM1", Mertansine), and Toll-like receptor agonist molecules.
  • connecting peptide generally refers to a part of the polypeptide chain in the "protein-drug conjugate” described in this application, and the connecting peptide contains at least one cysteine to As a site for covalent binding to the drug conjugate.
  • the connecting peptide may include a first connecting peptide and/or a second connecting peptide, and the amino acid sequence of the first connecting peptide and the second connecting peptide may be the same or different.
  • linker generally refer to those that can be used to connect one or more loaded drugs to an antigen-binding protein to form a "protein-drug coupling
  • the chemical functional module of "thing" The linker may include one or more linker members, and a variety of linker members are known in the art, for example, maleimidocaproyl ("MC”, maleimidocaproyl), valine-citrulline ("val -cit” or “vc”, valine-citrulline), p-aminobenzyloxycarbonyl (“PAB”, p-aminobenzyloxycarbonyl).
  • the “linker” may include a polypeptide linker composed of amino acids, but the "linker” is different from the aforementioned "connecting peptide", which is a part of the antigen binding protein.
  • conjugate generally refers to a collection of atoms and groups formed by covalent bonding of the "drug conjugate” described in this application with a specific functional group.
  • the conjugate may be formed by the reaction of a drug conjugate with a sulfhydryl group.
  • the conjugate may be a stable atom/group assembly formed by the addition reaction of the maleimide reactive group of mc-vc-PAB-MMAE and the sulfhydryl group. .
  • DAR Drug-Antibody Ratio
  • drug-antibody ratio that is, the average value of the number of drug-loaded antibodies connected to the antibody.
  • the drug loading capacity of current protein-drug conjugates is usually 0-8 drug-loaded molecules (D0-D8)/antibody.
  • DAR protein- Drug conjugate components
  • D0 is also referred to as "naked antibody”.
  • the HIC-HPLC analysis method is a common analysis method to determine the DAR and drug loading distribution of protein-drug conjugates.
  • CAR Conjugate/Antigen binding protein Ratio
  • a drug conjugate contains at least one loaded drug; in some embodiments, the prior art can make a drug conjugate part contain only one loaded drug, that is, in this case, the values of "CAR” and “DAR” can be Equivalent; in other embodiments, the prior art can make a drug conjugate part to connect multiple loaded drugs through a linker (see, Kumara et al., Bioorganic&Medicinal Chemistry Letters (2016), 28, 3617-3621) In this case, the value of "DAR” can be several times the value of "CAR”; for example, when a drug conjugate contains two loaded drugs, the DAR value can be twice the CAR value.
  • the cysteine Cys-220 (Eu numbering) in the natural sequence (SEQ ID NO: 73) of the human IgG1 antibody heavy chain hinge region is used as the coupling site to link the application
  • the drug conjugate can produce a protein-drug conjugate with a uniform CAR value.
  • derived from generally refers to a derived amino acid or nucleotide sequence derived from a parent amino acid or nucleotide sequence. It usually refers to the structural similarity between the parent sequence and the derived sequence, and does not imply or include the reference to the source.
  • the process of deriving a sequence from a parent sequence or source is limited, so when using "derived” to discuss a protein or polynucleotide, regardless of its physical origin.
  • "derived from” can mean that the derived sequence is an unmodified, part of the parental sequence.
  • Cys1 and Cys2 may be part of the unmodified natural antibody hinge region sequence.
  • a protein-drug conjugate which basically consists of a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprising a first cysteine (Cys1) and a second cysteine (Cys2), the second polypeptide chain includes a first cysteine (Cys1) and a second cysteine (Cys2), and the Cys1 of the first polypeptide chain is coupled with the structure -L1-P1 The Cys1 of the second polypeptide chain is coupled with the structure -L2-P2.
  • the drug loaded includes small molecule compounds, toxin molecules, oligonucleotides, protein degradation targeted chimeras (PROTAC), Affinity ligands, labeling groups, antibiotics, polypeptides and/or immunomodulatory molecules.
  • small molecule compounds toxin molecules, oligonucleotides, protein degradation targeted chimeras (PROTAC), Affinity ligands, labeling groups, antibiotics, polypeptides and/or immunomodulatory molecules.
  • antigen-binding fragment comprises VH, VL, scFv, Fab, dAb, soluble extracellular region of receptor protein and/or derivative Protein structure, etc.
  • the protein-drug conjugate according to any one of embodiments 35-41, wherein the fourth polypeptide chain, from N-terminus to C-terminus, comprises the VH and the VH of the fourth antigen-binding fragment in sequence.
  • the CH 1 of the fourth antigen-binding fragment comprises the VH and the VH of the fourth antigen-binding fragment in sequence.
  • the extracellular region, the first connecting peptide for example, the hinge region of IgG), CH 2 and CH 3 .
  • the application provides an antigen binding protein.
  • the antigen binding protein includes at least two connecting peptides and one or more antigen binding fragments.
  • the two connecting peptides are the first connecting peptide and the second connecting peptide; the amino acid sequences of the first connecting peptide and the second connecting peptide may be the same or different.
  • the first connecting peptide may include at least a first cysteine Cys1 and a second cysteine Cys2, and the second connecting peptide may include at least a first cysteine. Cys1 and the second cysteine Cys2.
  • a disulfide bond can be formed between Cys1 of the first connecting peptide and Cys1 of the second connecting peptide, which is called “disulfide bond Cys1-Cys1".
  • a disulfide bond can be formed between Cys2 of the first connecting peptide and Cys2 of the second connecting peptide, which is called “disulfide bond Cys2-Cys2".
  • the "disulfide bond Cys1-Cys1" is weaker than the "disulfide bond Cys2-Cys2".
  • the disulfide bond Cys1-Cys1 is easier to be opened by the reducing agent than the disulfide bond Cys2-Cys2.
  • the Cys1 and the Cys2 of the first connecting peptide in the antigen binding protein may be separated by at least 3 (for example, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, or more) amino acids that are not cysteine.
  • the Cys1 and the Cys2 of the second connecting peptide in the antigen binding protein may be separated by at least 3 (for example, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, or more) amino acids that are not cysteine.
  • the first connecting peptide may comprise the amino acid sequence shown in Cys1-(X)n-Cys2 from the N-terminus to the C-terminus, wherein n may be an integer greater than or equal to 3 (for example, n may be 3, n may Is 4, n can be 5, n can be 6, n can be 7, n can be 8, n can be 9, n can be 10, n can be 11, n can be 12, n can be 13, and n can be Is 14, n can be 15, or n can be an integer greater than 15); the X refers to any amino acid that is not Cys.
  • the second connecting peptide may include the amino acid sequence shown in Cys1-(X)n-Cys2 from the N-terminus to the C-terminus, where n may be an integer greater than or equal to 3 (for example, n may be 3, n may be Is 4, n can be 5, n can be 6, n can be 7, n can be 8, n can be 9, n can be 10, n can be 11, n can be 12, n can be 13, and n can be Is 14, n can be 15, or n can be an integer greater than 15); the X refers to any amino acid that is not Cys.
  • the connecting peptide in the antigen binding protein may be a naturally occurring amino acid sequence containing at least two cysteines (Cys1 and Cys2), or it may be a sequence of amino acids containing at least two cysteines.
  • the naturally-occurring amino acid sequence of an acid is a derivative sequence obtained by modification.
  • the "engineering" does not involve the alteration of Cys1 and Cys2 in the naturally-occurring amino acid sequence; for example, the modification is to adjust the type and number of amino acids between Cys1 and Cys2 in the naturally-occurring amino acid sequence and/or the flexibility before Cys1 Addition of connecting peptides (such as GSGS, GGGGS).
  • the first connecting peptide or the second connecting peptide may be the hinge region of human IgG (SEQ ID NO: 73), and the Cys1 is Cys-220 encoded by the human IgG hinge region according to EU.
  • the first connecting peptide may be the hinge region of human IgG1, the sequence of the connecting peptide is shown in SEQ ID NO: 73, the Cys1 in the connecting peptide is Cys-220 encoded by the EU in the hinge region of natural IgG1, and the Cys2 is The natural IgG1 hinge region is based on Cys-226 encoded by EU.
  • the first connecting peptide may be a derived sequence derived from the hinge region of human IgG1, and its amino acid sequence is as shown in any one of SEQ ID NOs: 74-105 and 145-146.
  • the Cys1 is Cys corresponding to Cys-220 of the human IgG1 hinge region
  • the Cys2 in the connecting peptide is the Cys corresponding to Cys-226 of the human IgG1 hinge region.
  • the second connecting peptide may be the hinge region of human IgG1, the sequence of the connecting peptide is shown in SEQ ID NO: 73, the Cys1 in the connecting peptide is Cys-220 encoded by the EU in the hinge region of natural IgG1, and the Cys2 is The natural IgG1 hinge region is based on Cys-226 encoded by EU.
  • the second connecting peptide may be a derived sequence derived from the hinge region of human IgG1, and its amino acid sequence is as shown in any one of SEQ ID NOs: 74-105 and 145-146.
  • the Cys1 is Cys corresponding to Cys-220 of the human IgG1 hinge region
  • the Cys2 in the connecting peptide is the Cys corresponding to Cys-226 of the human IgG1 hinge region.
  • the first connecting peptide or the second connecting peptide may be the hinge region of mouse IgG2c (SEQ ID NO: 147), the Cys1 is the first cysteine of the sequence, and the Cys2 is the sequence The second cysteine.
  • the "multiple antigen-binding fragments" in the “one or more antigen-binding fragments” may be partly or all the same or different; the “same” refers to the combination of two or more antigen-binding fragments.
  • the amino acid sequence is the same, and the “different” means that the amino acid sequences of two or more antigen-binding fragments are different.
  • Two or more antigen-binding fragments can recognize different antigens or epitopes, or two or more antigens.
  • the binding protein recognizes the same antigen or the same epitope, but the sequence and structure are different.
  • the antigen binding protein may include at least two of the antigen binding fragments.
  • the antigen-binding protein may comprise a first antigen-binding fragment and a second antigen-binding fragment (as shown in Figure 42(A)), and the amino acid sequence of the first antigen-binding fragment and the second antigen-binding fragment may be The same or different, the targets of the first antigen-binding fragment and the second antigen-binding fragment may be the same or different.
  • the antigen-binding protein may further include a third antigen-binding fragment and/or a fourth antigen-binding fragment (as shown in Figure 42(B)); the first, second, third, and fourth antigen-binding fragments It can be partly or all the same or different.
  • one or more antigen-binding fragments can be connected to the same connecting peptide or different connecting peptides.
  • the antigen-binding fragment may be located at the N-terminus or C-terminus of the connecting peptide.
  • the antigen-binding protein includes a first connecting peptide and the second connecting peptide, and includes a first antigen-binding fragment and a second antigen-binding fragment
  • the antigen-binding protein may include the following structure:
  • the C-terminus of the first antigen-binding fragment may be directly or indirectly connected to the N-terminus of the first connecting peptide, and the C-terminus of the second antigen-binding fragment may be connected to the N-terminus of the second connecting peptide.
  • the terminals are directly or indirectly connected, with the structure shown in Figure 42(A);
  • the N-terminus of the first antigen-binding fragment may be directly or indirectly connected to the C-terminus of the first connecting peptide, and the C-terminus of the second antigen-binding fragment may be connected to the N-terminus of the second connecting peptide. Connect directly or indirectly at the end;
  • the N-terminus of the first antigen-binding fragment may be directly or indirectly connected to the C-terminus of the first connecting peptide, and the N-terminus of the second antigen-binding fragment may be connected to the C-terminus of the second connecting peptide. Connect directly or indirectly at the end;
  • the C-terminus of the first antigen-binding fragment may be directly or indirectly connected to the N-terminus of the first connecting peptide, and the N-terminus of the second antigen-binding fragment may be connected to the C-terminus of the second connecting peptide.
  • the terminals are directly or indirectly connected.
  • the first antigen-binding fragment and/or the second antigen-binding fragment may refer to any functional protein domain that can specifically bind to an antigen, and it may be a VHH domain derived from a camel or an HCAb transgenic mouse, It can also be a single-chain antibody scFv; or other antigen-binding forms (such as soluble extracellular domain of receptor protein, ligand protein, lipocalin lipocalins, neural cell adhesion molecule NCAM, fibronectin fibronectin, ankyrin repeat fragments) DARPins and other derived protein structures).
  • the first antigen-binding fragment and/or the second antigen-binding fragment does not contain a functional group that can affect the coupling between the drug conjugate and the connecting peptide at Cys1.
  • the first antigen-binding fragment and/or the second antigen-binding fragment do not contain functional groups that can affect the coupling between the drug conjugate and the connecting peptide at Cys1.
  • the first antigen-binding fragment and/or the second antigen-binding fragment does not contain a covalent bond such as a disulfide bond capable of forming a covalent bond with the side chain sulfhydryl group of Cys1 of the first connecting peptide and/or the second connecting peptide The functional group.
  • the first antigen-binding fragment and/or the second antigen-binding fragment does not include a light chain or light chain fragment containing the constant region Cys-214 (Eu numbering).
  • the antigen-binding protein may further include a third antigen-binding fragment and/or a fourth antigen-binding fragment.
  • the third antigen-binding fragment and/or the fourth antigen-binding fragment further included are further connected to the N-terminus or C-terminus of the antigen-binding protein of (a) or (b) or (c) or (d) above:
  • the C-terminus of the third antigen-binding fragment may be directly or indirectly connected to the N-terminus of the first antigen-binding fragment, and the C-terminus of the first antigen-binding fragment may be connected to the N-terminus of the first connecting peptide.
  • the C-terminus of the fourth antigen-binding fragment may be directly or indirectly connected to the N-terminus of the second antigen-binding fragment, and the C-terminus of the second antigen-binding fragment may be connected to the first
  • the N-terminal of the two-linker peptide is connected; it has a structure as shown in Figure 42(B).
  • the third antigen-binding fragment and/or the fourth antigen-binding fragment may include any protein functional region that can specifically bind to an antigen, which can be either "Fab", It can also be "VH” or single-chain antibody scFv; it can also be other antigen binding forms (such as the soluble extracellular domain of receptor proteins, ligand proteins, lipocalins, neural cell adhesion molecules ( NCAM), fibronectin (fibronectin), ankyrin repeat fragment protein (DARPins) and other derivative protein structures.
  • the antigen binding protein may further include a pairing unit capable of pairing the first connecting peptide and the second connecting peptide
  • the pairing unit may include a first pairing subunit and a second pairing subunit
  • the The first pairing subunit can interact with the second pairing subunit through a covalent bond or a non-covalent bond.
  • the "pairing unit” may be a dimeric form of an antibody Fc fragment or a variant thereof;
  • the "pairing unit” may be in the form of an asymmetric dimer, for example, the first pairing subunit is an Fc fragment monomer with a "knob” mutation (T366W), and the second pairing subunit has a "knob” mutation (T366W). Fc fragment monomer of hole” mutation (T366S/L368A/Y407V);
  • the "pairing unit” may be in the form of an asymmetric dimer, for example, the first pairing subunit is the ⁇ chain of CD8, and the second pairing subunit is the ⁇ chain of CD8;
  • the "pairing unit” may be in the form of an asymmetric dimer, for example, the first pairing subunit is the ⁇ chain of CD79, and the second pairing subunit is the ⁇ chain of CD79;
  • the "pairing unit” may be in the form of an asymmetric dimer, for example, the first pairing subunit is the ⁇ chain of the T cell receptor, and the second pairing subunit is the ⁇ chain of the T cell receptor.
  • the N-terminus of the first pairing subunit may be connected to the C-terminus of the first connecting peptide, and the N-terminus of the second pairing subunit may be connected to the C-terminus of the second connecting peptide.
  • the N-terminus of the first pairing subunit may be connected to the C-terminus of the second connecting peptide, and the N-terminus of the second pairing subunit may be connected to the C-terminus of the first connecting peptide .
  • the present application provides an antigen binding protein with the structure shown in Figure 42(A), which has the following structural features:
  • the antigen-binding protein may include a first connecting peptide and a second connecting peptide, a first antigen-binding fragment and a second antigen-binding fragment, and a pairing unit;
  • the amino acid sequence of the first connecting peptide and the second connecting peptide may be the same or different;
  • the targets of the first antigen-binding fragment and the second antigen-binding fragment may be the same or different; the amino acid sequences of the first antigen-binding fragment and the second antigen-binding fragment may be the same or different;
  • the pairing unit may be an Fc fragment homologous or heterodimer
  • the C-terminus of the first antigen-binding fragment may be directly or indirectly connected to the N-terminus of the first connecting peptide, and the C-terminus of the second antigen-binding fragment may be directly or indirectly connected to the N-terminus of the second connecting peptide. Indirect connection
  • the C-terminus of the first connecting peptide and the second connecting peptide can be respectively connected to the N-terminus of the Fc fragment;
  • the first connecting peptide may include at least two cysteines Cys1 and Cys2, and the Cys1 and Cys2 are separated by at least 3 arbitrary amino acids that are not Cys;
  • the second connecting peptide may include at least two cysteines Cys1 and Cys2, and the Cys1 and Cys2 are separated by at least 3 arbitrary amino acids that are not Cys;
  • a disulfide bond may be formed between Cys1 of the first connecting peptide and Cys1 of the second connecting peptide;
  • a disulfide bond may be formed between Cys1 of the first connecting peptide and Cys2 of the second connecting peptide;
  • the first connecting peptide and/or the second connecting peptide may be derived from a human IgG1 hinge region sequence (SEQ ID NO: 73) or derived sequences (SEQ ID NOs: 74-105, 145-146) or derived from Hinge region sequence of mouse IgG2c (SEQ ID NO: 147);
  • the first antigen-binding fragment and/or the second antigen-binding fragment can refer to any protein functional region that can specifically bind to an antigen, and can be a VHH domain derived from a camel or an HCAb transgenic mouse, or a single chain Antibody scFv; or other antigen-binding forms (such as receptor protein soluble extracellular domain, ligand protein, lipocalin lipocalins, neural cell adhesion molecule NCAM, fibronectin fibronectin, ankyrin repeat fragment protein DARPins and other derivative proteins structure);
  • the first antigen-binding fragment and the second antigen-binding fragment do not contain functional groups that can affect the coupling between the drug conjugate and the connecting peptide at Cys1; for example, they do not include functional groups that can interact with the first connecting peptide and the connecting peptide.
  • the side chain sulfhydryl group of Cys1 in the second connecting peptide forms a covalent bond functional group; for example, it does not include a functional group capable of forming a disulfide bond with the side chain sulfhydryl group of Cys1 in the first connecting peptide and the second connecting peptide; for example, , Does not contain the light chain or light chain fragments containing the constant region Cys-214 (Eu numbering);
  • the one or more antigen-binding fragments do not include a functional group that can affect the coupling of the drug conjugate with the first connecting peptide and/or the second connecting peptide at Cys1, or
  • the effect is manifested in significantly reducing the coupling probability of the drug conjugate at the Cys1 site, and the significant reduction may include one or more of the following:
  • the probability of coupling the Cys1 of the connecting peptide with the drug conjugate may be reduced to 50%, 40%, 30%, 20%, 10% Or lower,
  • the purity of the prepared protein-drug conjugate is less than 50%, for example, less than 40%, 30%, 20%, 10% Or lower,
  • the one or more antigen-binding fragments may not include a functional group capable of forming a disulfide bond with the Cys1 of the first connecting peptide and/or the Cys1 of the second connecting peptide Or a conjugate formed by the aforementioned functional group and other drug conjugates;
  • the one or more antigen-binding fragments may not include a half capable of forming a disulfide bond with the Cys1 of the first connecting peptide and/or the Cys1 of the second connecting peptide. Cystine or a conjugate formed by the aforementioned cysteine and other drug conjugates.
  • the present application also provides an antigen binding protein having a structure as shown in Figure 42(B), which has the following structural characteristics:
  • the antigen-binding protein On the basis of the antigen-binding protein with the structure shown in Figure 42(A), it further comprises a third antigen-binding fragment and a fourth antigen-binding fragment;
  • the C-terminus of the third antigen-binding fragment may be directly or indirectly connected to the N-terminus of the first antigen-binding fragment, and the C-terminus of the first antigen-binding fragment may be connected to the N-terminus of the first connecting peptide ;
  • the C-terminus of the fourth antigen-binding fragment may be directly or indirectly connected to the N-terminus of the second antigen-binding fragment, and the C-terminus of the second antigen-binding fragment may be connected to the N-terminus of the second connecting peptide ;
  • the first antigen-binding fragment, the second antigen-binding fragment, the third antigen-binding fragment and/or the fourth antigen-binding fragment may be partly or completely the same or different; for example, the first and second antigen binding The fragments are the same, the third and fourth binding fragments are the same, the first and third antigen binding fragments are different, and the second and fourth antigen binding fragments are different;
  • the third antigen-binding fragment and/or the fourth antigen-binding fragment may include any protein functional region that can specifically bind to an antigen, which can be either "Fab", "VH", or single-chain antibody scFv; can also be other antigen binding forms (such as the soluble extracellular domain of receptor proteins, ligand proteins, lipocalins, neural cell adhesion molecules (NCAM), fibronectin, fibronectin, ankyrin repeats Derivative protein structures such as fragment proteins (DARPins);
  • the third antigen-binding fragment and/or the fourth antigen-binding fragment also do not include a functional group that can affect the coupling between the drug conjugate and the connecting peptide at Cys1 or the conjugate formed by the aforementioned functional group and other drug conjugates.
  • it does not include a functional group capable of forming a disulfide bond with the side chain sulfhydryl group of Cys1 in the first connecting peptide and the second connecting peptide;
  • the third antigen-binding fragment and/or the fourth antigen-binding fragment does not include a functional group capable of forming a disulfide bond with the Cys1 of the first connecting peptide and/or the Cys1 of the second connecting peptide Or a conjugate formed by the aforementioned functional group and other drug conjugates;
  • the third antigen-binding fragment and/or the fourth antigen-binding fragment does not include a half capable of forming a disulfide bond with the Cys1 of the first connecting peptide and/or the Cys1 of the second connecting peptide Cystine or a conjugate formed by the aforementioned cysteine and other drug conjugates.
  • the antigen binding protein of the present application can target a specific antigen.
  • the antigen-binding protein may comprise one or more antigen-binding fragments, which can bind to a specific antigen on the target cell.
  • the specific antigen on the target cell may be a tumor antigen.
  • the tumor antigen may be CD19, BCMA, TSHR, CD171, CS-1, CLL-1, GD3, Tn Ag, FLT3, CD38, CD123, CD44v6, B7H3, B7H4, KIT, IL-13Ra2, IL -11Ra, PSCA, PSMA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, MUC1, EGFR, NCAM, CAIX, LMP2, EphA2, Fucosyl GM1 (fucosyl GM1), sLe, GM3, TGS5 , HMWMAA, GD2, FOLR1, FOLR2, TEM1/CD248, TEM7R, CLDN6, CLDN18.2, GPRC5D, CXORF61, CD
  • the tumor antigen may be MSLN; for example, the tumor antigen may be BCMA; for example, the tumor antigen may be 5T4; for example, the tumor antigen may be ROR1.
  • the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof.
  • the antigen-binding fragment may include the variable region of an antibody heavy chain VH.
  • the antigen-binding fragment may include a single-chain antibody variable region scFv.
  • the antigen-binding fragment may include a receptor protein soluble cell. Outer region fusion protein.
  • the antigen-binding protein may comprise an antibody or antigen-binding fragment thereof that targets CTLA4, an antibody or antigen-binding fragment thereof that targets MSLN, an antibody or antigen-binding fragment thereof that targets BCMA, an antibody or antigen-binding fragment thereof that targets 5T4. Binding fragments, ROR1-targeting antibodies or antigen-binding fragments thereof, and/or soluble fusion proteins derived from the extracellular region of SIRP ⁇ targeting CD47.
  • the CTLA4 antigen-binding fragment may include a heavy chain variable region; its heavy chain variable region VH includes HCDR1, HCDR2, and HCDR3, which are the amino acid sequences shown in SEQ ID NO: 7, 22, and 38, respectively.
  • the amino acid sequences of the listed CDRs are shown in accordance with Chothia's definition rules.
  • the VH of the heavy chain variable region may include the amino acid sequence shown in SEQ ID NO:55.
  • the CTLA4 antigen binding protein may comprise a polypeptide chain, such as the amino acid sequence shown in SEQ ID NO:64.
  • the CTLA4 antigen-binding fragment may include a heavy chain variable region; its heavy chain variable region VH may include HCDR1, HCDR2, and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 8, 23, and 39, respectively .
  • the amino acid sequences of the listed CDRs are shown in accordance with Chothia's definition rules.
  • the VH of the heavy chain variable region may include the amino acid sequence shown in SEQ ID NO:56.
  • the CTLA4 antigen binding protein may comprise a polypeptide chain, such as the amino acid sequence shown in SEQ ID NO:65.
  • the MSLN antigen-binding fragment may include a heavy chain variable region; its heavy chain variable region VH may include HCDR1, HCDR2, and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 10, 25, and 41, respectively .
  • the amino acid sequences of the listed CDRs are shown in accordance with Chothia's definition rules.
  • the VH of the heavy chain variable region may include the amino acid sequence shown in SEQ ID NO:58.
  • the MSLN antigen binding protein may include a polypeptide chain, such as the amino acid sequence shown in SEQ ID NO: 67.
  • the 5T4 antigen-binding fragment may include a heavy chain variable region; its heavy chain variable region VH may include HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 13, 28 and 44, respectively .
  • the amino acid sequences of the listed CDRs are shown in accordance with Chothia's definition rules.
  • the VH of the heavy chain variable region may include the amino acid sequence shown in SEQ ID NO: 61.
  • the 5T4 antigen binding protein may include a polypeptide chain, such as the amino acid sequence shown in SEQ ID NO: 69.
  • the BCMA antigen-binding fragment may include a heavy chain variable region; its heavy chain variable region VH may include HCDR1, HCDR2, and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 9, 24, and 40, respectively .
  • the amino acid sequences of the listed CDRs are shown in accordance with Chothia's definition rules.
  • the VH of the heavy chain variable region may include the amino acid sequence shown in SEQ ID NO: 57.
  • the BCMA antigen binding protein may include a polypeptide chain, such as the amino acid sequence shown in SEQ ID NO: 66.
  • the BCMA antigen-binding fragment may include a heavy chain variable region; its heavy chain variable region VH may include HCDR1, HCDR2, and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 11, 26 and 42, respectively .
  • the amino acid sequences of the listed CDRs are shown in accordance with Chothia's definition rules.
  • the VH of the heavy chain variable region may include the amino acid sequence shown in SEQ ID NO:59.
  • the BCMA antigen binding protein may comprise a polypeptide chain, such as the amino acid sequence shown in SEQ ID NO: 68.
  • the BCMA antigen-binding fragment may include a heavy chain variable region; its heavy chain variable region VH may include HCDR1, HCDR2, and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 14, 29 and 42, respectively .
  • the amino acid sequences of the listed CDRs are shown in accordance with Chothia's definition rules.
  • the VH of the heavy chain variable region may include the amino acid sequence shown in SEQ ID NO: 62.
  • the BCMA antigen binding protein may include a polypeptide chain, such as the amino acid sequence shown in SEQ ID NO: 70.
  • the BCMA antigen binding protein may comprise a polypeptide chain, such as the amino acid sequence shown in any one of SEQ ID NOs: 106-129, 148.
  • the ROR1 antigen-binding fragment may include a light chain variable region and a heavy chain variable region; its light chain variable region VL may include LCDR1, LCDR2, and LCDR3, which are SEQ ID NOs: 49, 51, and The amino acid sequence shown in 53; its heavy chain variable region VH may include HCDR1, HCDR2 and HCDR3, which are the amino acid sequences shown in SEQ ID NOs: 12, 27 and 43, respectively.
  • the amino acid sequences of the listed CDRs are shown in accordance with Chothia's definition rules.
  • the light chain variable region VL may include the amino acid sequence shown in SEQ ID NO: 63
  • the heavy chain variable region VH may include the amino acid sequence shown in SEQ ID NO: 60.
  • the ROR1 antigen binding protein may include a polypeptide chain, such as the amino acid sequence shown in SEQ ID NO: 71.
  • the CD47 antigen binding protein may comprise a polypeptide chain, such as the amino acid sequence shown in SEQ ID NO:149.
  • the antigen binding protein may also include heavy chain constant regions CH2 and CH3 and a hinge region.
  • the sequence of the heavy chain constant region includes the amino acid sequence shown in SEQ ID NO: 72,
  • the hinge region sequence is preferably a human IgG1 antibody heavy chain hinge region sequence (SEQ ID NO: 73).
  • the antigen-binding protein may also have a symmetrical structure as shown in Figure 59, containing four antigen-binding fragments and four polypeptide chains; the antigen-binding protein includes antigen-binding fragment A and antigen.
  • Binding fragment B The antigen-binding fragment A and the antigen-binding fragment B target different antigens or antigen epitopes; the antigen-binding fragment A is a Fab structure, and the antigen-binding fragment B is a VH structure; In the antigen-binding protein, the number of the antigen-binding fragment A is two, and the number of the antigen-binding fragment B is two; the antigen-binding protein consists of antigen-binding fragment A and antigen-binding fragment in sequence from the N-terminus to the C-terminus B and Fc, wherein the antigen-binding fragment A and the antigen-binding fragment B are directly connected, and the antigen-binding fragment B and the Fc are connected through a connecting peptide (or hinge region).
  • the antigen binding protein has four polypeptide chains, and the four polypeptide chains include a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain; the first polypeptide chain and the The second polypeptide chain has the same amino acid sequence and is called a "long chain”; the third polypeptide chain and the fourth polypeptide chain have the same amino acid sequence and are called a "short chain”; the short chain From the N-terminus to the C-terminus, it includes VH_A-CH1, and the long chain from the N-terminus to the C-terminus includes VL_A-CL-VH_B-L-CH2-CH3 in turn; wherein, VH_A and VL_A are respectively The heavy chain variable region and the light chain variable region, VH_B is the heavy chain variable region of the antigen-binding fragment B, and the L is the connecting peptide.
  • the connecting peptide has an amino acid sequence as shown in SEQ ID NO:73.
  • the antigen-binding fragment A includes a light chain variable region and a heavy chain variable region, and the light chain variable region VL includes the amino acid sequence shown in SEQ ID NO: 131, and the heavy chain can
  • the variable region VH includes the amino acid sequence shown in SEQ ID NO: 130;
  • the antigen-binding fragment B includes the heavy chain variable region, and the heavy chain variable region VH includes the amino acid sequence shown in SEQ ID NO: 62.
  • the antigen binding protein includes two identical “short chains” and two identical “long chains” ( Figure 59), and the “short chains” include those shown in SEQ ID NO: 134
  • the "long chain” includes the amino acid sequence shown in SEQ ID NO: 135.
  • all the CDRs mentioned can include the case of mutation based on the restricted sequence.
  • the mutation is an insertion, deletion or substitution of 3, 2 or 1 amino acid based on the amino acid sequence of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, VL CDR3, respectively.
  • amino acid mutations in similar "insertions, deletions or substitutions with 3, 2 or 1 amino acids” refer to the amino acid mutations in the variant sequence compared to the original amino acid sequence, including those in the original amino acid sequence. Amino acid insertions, deletions or substitutions occur on the basis of the sequence.
  • the CDR mutations can include 3, 2 or 1 amino acid mutations, and the same or different numbers of amino acid residues can optionally be selected for mutations between these CDRs.
  • CDR1 can be mutated.
  • the VH, VL, or the polypeptide chain may all include the situation where mutations are made on the basis of the defined sequence.
  • the mutation refers to the deletion, substitution or addition of one or more amino acid residues in the defined amino acid sequence, and the mutated amino acid sequence has at least 85% sequence homology with the defined amino acid sequence and maintains Or improve the binding activity of the antibody or its antigen-binding fragment or binding protein; the at least 85% sequence homology is preferably at least 90% sequence homology; more preferably at least 95% sequence homology; most preferably It is at least 99% sequence homology.
  • the homology generally refers to the similarity, similarity or association between two or more sequences.
  • the "percentage of sequence homology" can be calculated in the following way: the two sequences to be aligned are compared in the comparison window to determine the presence of the same nucleic acid base (for example, A, T, C, G) or Positions of the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) To get the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to generate the sequence homology percentage.
  • the same nucleic acid base for example, A, T, C, G
  • Positions of the same amino acid residues e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu,
  • the alignment to determine the percent sequence homology can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithms required to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.
  • the homology can also be determined by the following methods: FASTA and BLAST.
  • FASTA for a description of the FASTA algorithm, please refer to WRPearson and DJ Lipman's "Improved Tools for Biological Sequence Comparison", Proc. Natl. Acad.
  • the protein-drug conjugate described in the present application may include at least one drug conjugate; the drug conjugate may be covalently attached to the protein portion of the protein-drug conjugate.
  • the drug conjugate may include at least one loaded drug.
  • the loaded drug may include active drug ingredients and/or labeling molecules.
  • the loaded drug is a type of small molecule compound or toxin or other drug molecule form with pharmacological activity, which can be but not limited to small molecule compound, toxin molecule, antibiotic, oligonucleotide, protein degradation targeting agent.
  • Protac PROTAC
  • affinity ligand for example, fluorescent group, nuclide group, polypeptide, immunomodulatory molecule (for example, Toll-like receptor agonist molecule, STING agonist molecule), etc.
  • Various drug loads are known in the art.
  • small molecule compounds usually refer to a class of substances with strong cytotoxicity.
  • the small molecule compound can exert such cytotoxic and cytostatic effects through mechanisms including but not limited to tubulin binding, DNA binding, RNA polymerase inhibition, protein synthesis, or topoisomerase inhibition.
  • the small molecule compound may be a tubulin inhibitor; for example, the tubulin inhibitor may be maytansine (for example, DM1 or DM4), auristatin (for example, MMAE or MMAF), and the like.
  • the small molecule compound may be a DNA damaging agent; for example, the DNA damaging agent may be calicheamicins, pyrrolobenzodiazepines (PBD, pyrrolobenzodiazepines), and the like.
  • protein degradation targeting chimera is a class of compounds capable of inducing the polyubiquitination of the target protein to cause the degradation of the target protein; for example, the PROTAC may be a BET protein degrading agent.
  • the drug conjugate may also include at least one linker.
  • the linker may include a cleavable linker or a non-cleavable linker.
  • the linker is used to connect one or more loaded drugs to the antigen binding protein.
  • the chain-scissible linker may be a "cleavable" linker that facilitates drug release.
  • cleavable linkers may include, but are not limited to, acid-sensitive linkers, protease-sensitive linkers, light-sensitive linkers, or disulfide-containing linkers.
  • the linker may have a functional group capable of reacting with free cysteine present on the antigen binding protein to form a covalent bond.
  • reactive functional groups may include, but are not limited to, maleimide, haloacetamide, ⁇ -haloacetyl, activated esters such as succinimide ester, 4-nitrophenyl ester, five Fluorophenyl esters, tetrafluorophenyl esters, acid anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.
  • the linker may have a functional group capable of reacting with an electrophilic group present on the antigen binding protein.
  • reactive functional groups may include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate ), and arylhydrazide.
  • the linker may include one or more linker members, and a variety of linker members are known in the art, for example, maleimidocaproyl ("MC”, maleimidocaproyl), maleimide Propionyl ("MP”), valine-citrulline ("val-cit” or “vc”, valine-citrulline), p-aminobenzyloxycarbonyl (“PAB”, p-aminobenzyloxycarbonyl).
  • MC maleimidocaproyl
  • MP maleimide Propionyl
  • val-cit or "vc", valine-citrulline
  • PAB p-aminobenzyloxycarbonyl
  • the drug conjugate is mc-vc-PAB-MMAE (also known as VcMMAE, the structure is shown in Figure 6, CAS No.: 646502-53-6), which contains the linker mc-vc -PAB and load drug MMAE.
  • the drug conjugate can also be a BRD4 protein degrading agent (the structure is shown in Figure 64), which includes PROTAC GNE-987 targeting BET and a disulfide-containing linker.
  • the present application provides a protein-drug conjugate.
  • the protein-drug conjugate includes at least one antigen binding protein portion and at least one drug conjugate portion.
  • the antigen-binding protein portion is the antigen-binding protein that targets a specific antigen and contains one or more antigen-binding fragments and at least two connecting peptides as described in the "antigen-binding protein" above.
  • the drug conjugate contains at least one loaded drug.
  • the loaded drug may include, but is not limited to, small molecule compounds, toxin molecules, antibiotics, oligonucleotides, protein degradation targeted chimeras (PROTAC), affinity ligands, fluorescent labeling groups, nuclide labeling groups, Polypeptides, immunomodulatory molecules (for example, Toll-like receptor agonists), etc.
  • the drug conjugate may also include at least one linker.
  • the drug-loaded drug can be coupled to the antigen binding protein by covalent bonding through the linker; in this application, the drug-loaded drug can be covalently bonded via the linker It is coupled to the connecting peptide in the antigen binding protein.
  • the drug conjugate may be linked to the antigen binding protein through the Cys1 of the first connecting peptide and/or the Cys1 of the second connecting peptide, thereby producing the protein -Drug conjugates.
  • a reactive sulfhydryl functional group is formed by breaking the disulfide bond between the first connecting peptide and the second connecting peptide of the antigen-binding protein to form a reactive sulfhydryl functional group to realize the covalent binding of the drug conjugate at Cys1.
  • the one or more antigen-binding fragments do not include a functional group that can affect the coupling of the drug conjugate and the connecting peptide at Cys1; for example, it does not include a side chain sulfhydryl group capable of interacting with Cys1 in the connecting peptide.
  • a functional group that forms a covalent bond for example, it does not include a functional group capable of forming a disulfide bond with the side chain sulfhydryl group of Cys1 in the connecting peptide.
  • connection between the drug conjugate and the antigen binding protein mainly occurs at the Cys1 site of the first connecting peptide and/or the second connecting peptide, and hardly occurs at other cysteine residues.
  • Acid site e.g., Cys2 site.
  • the probability of Cys1 of the connecting peptide being bound by the drug conjugate is at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%.
  • % Can also be at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99%.
  • the probability of Cys2 of the connecting peptide being bound by the drug conjugate is at most 50%, or at most 45%, or at most 40%, or at most 35%, or at most 30.
  • % Can also be up to 25%, up to 20%, up to 15%, up to 10%, or up to 5%.
  • Cys1 in the first connecting peptide and/or the second connecting peptide is used as the coupling site to connect the drug conjugate described in this application to produce a protein-drug conjugate with a uniform DAR value .
  • the content of the component also used as D2) is at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75% It can also be at least 80%, or at least 85%, or at least 90%, or at least 95%.
  • the Cys1; without further purification steps, a protein-drug conjugate with a uniform DAR value can be obtained.
  • the cysteine Cys-220 (Eu number) in the natural sequence (SEQ ID NO: 73) of the human IgG1 antibody heavy chain hinge region is used as the coupling site to connect the drug coupling described in this application It can produce protein-drug conjugates with uniform DAR value.
  • the content of the component also used as D2) is at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75% It can also be at least 80%, or at least 85%, or at least 90%, or at least 95%.
  • the protein-drug conjugate may have a structure as shown in Figure 41 or Figure 43.
  • the protein-drug conjugate may be an antibody-conjugated drug (ADC).
  • ADC antibody-conjugated drug
  • This application also provides a protein-drug conjugate having the structure shown in Figure 43(A) obtained by using the antigen-binding protein with the structure shown in Figure 42(A) for drug coupling, which has the following Structure:
  • the protein-drug conjugate comprises a first connecting peptide and the second connecting peptide, a first antigen-binding fragment and a second antigen-binding fragment;
  • the amino acid sequence of the first connecting peptide and the second connecting peptide may be the same or different;
  • the targets of the first antigen-binding fragment and the second antigen-binding fragment may be the same or different; the amino acid sequences of the first antigen-binding fragment and the second antigen-binding fragment may be the same or different;
  • the protein-drug conjugate also includes a pairing unit, such as an Fc fragment;
  • the first connecting peptide and the second connecting peptide comprise at least two cysteines, which are named Cys1 and Cys2 from N-terminus to C-terminus, respectively;
  • the sequence of the first connecting peptide and/or the second connecting peptide may be a human IgG1 hinge region sequence (SEQ ID NO: 73) or its derivative sequence (SEQ ID NOs: 74-105, 145-146).
  • the Cys1 of the first connecting peptide and/or the second connecting peptide may be Cys-220 (Eu numbering) of the hinge region sequence of human IgG1 or Cys-220 (Eu numbering) of the derived sequence corresponding to the hinge region sequence of human IgG1 ;
  • the Cys2 may be Cys-226 (Eu numbering) of the hinge region sequence of human IgG1 or Cys of the derived sequence corresponding to Cys-226 (Eu numbering) of the hinge region sequence of human IgG1;
  • the sequence of the first connecting peptide and/or the second connecting peptide may also be the hinge region sequence of mouse IgG2c (SEQ ID NO: 147) or a derivative sequence thereof, the first connecting peptide and/or the second connecting peptide
  • the Cys1 may be the first cysteine of the hinge region sequence of mouse IgG2c or its derivative sequence
  • the Cys2 may be the second cysteine of the hinge region sequence of mouse IgG2c or its derivative sequence acid
  • the first antigen-binding fragment and/or the second antigen-binding fragment do not affect the coupling of the drug conjugate at the Cys1 site of the first connecting peptide and/or the second connecting peptide;
  • the first antigen-binding fragment and/or the second antigen-binding fragment does not contain the drug conjugate that can affect the coupling of the drug conjugate on the Cys1 of the first connecting peptide and/or the second connecting peptide
  • the functional group
  • the first antigen-binding fragment and/or the second antigen-binding fragment does not include a functional group capable of forming a covalent bond with the side chain sulfhydryl group of Cys1;
  • the first antigen-binding fragment and/or the second antigen-binding fragment does not include a functional group capable of forming a disulfide bond with Cys1.
  • This application also provides a protein-drug conjugate having the structure shown in Figure 43(B) obtained by using the antigen-binding protein with the structure shown in Figure 42(B) for drug coupling, which has the following Structure:
  • the protein-drug conjugate includes a first connecting peptide and the second connecting peptide, and includes a first antigen-binding fragment, the second antigen-binding fragment, the third antigen-binding fragment and the fourth antigen-binding Fragment
  • the amino acid sequence of the first connecting peptide and the second connecting peptide may be the same or different;
  • the first antigen-binding fragment, the second antigen-binding fragment, the third antigen-binding fragment and/or the fourth antigen-binding fragment may be partly or completely the same or different; for example, the first and second antigen binding The fragments are the same, the third and fourth binding fragments are the same, the first and third antigen-binding fragments are different, and the second and fourth antigen-binding fragments are different; the same or different parts may be the target and/or Amino acid sequence.
  • the protein-drug conjugate also includes a pairing unit, such as an Fc fragment;
  • the first connecting peptide and the second connecting peptide comprise at least two cysteines, which are named Cys1 and Cys2 from N-terminus to C-terminus, respectively;
  • the sequence of the first connecting peptide and/or the second connecting peptide may be a human IgG1 hinge region sequence (SEQ ID NO: 73) or its derivative sequence (SEQ ID NOs: 74-105, 145-146).
  • the Cys1 of the first connecting peptide and/or the second connecting peptide may be Cys-220 (Eu numbering) of the hinge region sequence of human IgG1 or Cys-220 (Eu numbering) of the derived sequence corresponding to the hinge region sequence of human IgG1 ;
  • the Cys2 may be Cys-226 (Eu numbering) of the hinge region sequence of human IgG1 or Cys of the derived sequence corresponding to Cys-226 (Eu numbering) of the hinge region sequence of human IgG1;
  • the sequence of the first connecting peptide and/or the second connecting peptide may also be the hinge region sequence of mouse IgG2c (SEQ ID NO: 147) or a derivative sequence thereof, the first connecting peptide and/or the second connecting peptide
  • the Cys1 may be the first cysteine of the hinge region sequence of mouse IgG2c or its derivative sequence
  • the Cys2 may be the second cysteine of the hinge region sequence of mouse IgG2c or its derivative sequence acid
  • the first antigen-binding fragment and/or the second antigen-binding fragment and/or the third antigen-binding fragment and/or the fourth antigen-binding fragment do not contain the ability to affect the drug conjugate and the connecting peptide in Cys1 Functional groups for site-specific coupling;
  • the first antigen-binding fragment and/or the second antigen-binding fragment and/or the third antigen-binding fragment and/or the fourth antigen-binding fragment do not contain covalently capable of forming a side chain sulfhydryl group of Cys1 The functional group of the bond;
  • the first antigen-binding fragment and/or the second antigen-binding fragment and/or the third antigen-binding fragment and/or the fourth antigen-binding fragment do not contain a disulfide capable of forming a disulfide with the side chain sulfhydryl group of Cys1 The functional group of the bond.
  • the application also provides a protein-drug conjugate.
  • the drug conjugate may include a first polypeptide chain and a second polypeptide chain.
  • the first polypeptide chain from N-terminus to C-terminus may sequentially include a first antigen-binding fragment, a first connecting peptide, and a first pairing subunit.
  • the second polypeptide chain is from N-terminus to C-terminus, and may include a second antigen-binding fragment, a second connecting peptide and a second pairing subunit in sequence. Wherein, the first paired subunit and the second paired subunit can interact to form a dimer.
  • the first polypeptide chain from the N-terminus to the C-terminus may sequentially include the VH of the first antigen-binding fragment, the VL of the first antigen-binding fragment, and the first connecting peptide (for example, a human IgG1 hinge region or a mouse IgG2c Hinge region or derived sequence), CH 2 and CH 3 , and the drug conjugate is connected to the first polypeptide chain through Cys1 of the first connecting peptide (for example, Cys220 (EU code) of the IgG hinge region).
  • the first connecting peptide for example, a human IgG1 hinge region or a mouse IgG2c Hinge region or derived sequence
  • the second polypeptide chain from the N-terminus to the C-terminus may sequentially include the VH of the second antigen-binding fragment, the VL of the second antigen-binding fragment, and the second connecting peptide (for example, a human IgG1 hinge region or a mouse IgG2c The hinge region or derived sequence), CH 2 and CH 3 , and the drug conjugate is connected to the second polypeptide chain through Cys1 of the second connecting peptide (for example, Cys220 (EU code) of the IgG hinge region).
  • the second connecting peptide for example, a human IgG1 hinge region or a mouse IgG2c The hinge region or derived sequence
  • CH 2 and CH 3 The hinge region or derived sequence
  • the drug conjugate is connected to the second polypeptide chain through Cys1 of the second connecting peptide (for example, Cys220 (EU code) of the IgG hinge region).
  • the first polypeptide chain from the N-terminus to the C-terminus may sequentially comprise the VH of the first antigen-binding fragment, the first connecting peptide (for example, the hinge region of human IgG1 or the hinge region of mouse IgG2c or a derivative sequence), CH 2 and CH 3 , and the drug conjugate is connected to the first polypeptide chain through Cys1 of the first connecting peptide (for example, Cys220 (EU code) of the IgG hinge region).
  • the first connecting peptide for example, the hinge region of human IgG1 or the hinge region of mouse IgG2c or a derivative sequence
  • CH 2 and CH 3 a derivative sequence
  • the drug conjugate is connected to the first polypeptide chain through Cys1 of the first connecting peptide (for example, Cys220 (EU code) of the IgG hinge region).
  • the second polypeptide chain from the N-terminus to the C-terminus may sequentially comprise the VH of the second antigen-binding fragment, the second connecting peptide (for example, the hinge region of human IgG1 or the hinge region of mouse IgG2c or a derivative sequence), CH 2 and CH 3 , and the drug conjugate is connected to the second polypeptide chain through Cys1 of the second connecting peptide (for example, Cys220 (EU code) of the IgG hinge region).
  • the second connecting peptide for example, the hinge region of human IgG1 or the hinge region of mouse IgG2c or a derivative sequence
  • CH 2 and CH 3 a derivative sequence
  • the drug conjugate is connected to the second polypeptide chain through Cys1 of the second connecting peptide (for example, Cys220 (EU code) of the IgG hinge region).
  • the first polypeptide chain from the N-terminus to the C-terminus may sequentially include the soluble extracellular region of the receptor protein of the first antigen-binding fragment, and the first connecting peptide (for example, the hinge region of human IgG1 or the hinge region of mouse IgG2c). Or derived sequence), CH 2 and CH 3 , and the drug conjugate is connected to the first polypeptide chain through Cys1 of the first connecting peptide (for example, Cys220 (EU code) of the IgG hinge region).
  • the first connecting peptide for example, the hinge region of human IgG1 or the hinge region of mouse IgG2c.
  • CH 2 and CH 3 the drug conjugate is connected to the first polypeptide chain through Cys1 of the first connecting peptide (for example, Cys220 (EU code) of the IgG hinge region).
  • the second polypeptide chain from the N-terminus to the C-terminus may sequentially include the soluble extracellular region of the receptor protein of the second antigen-binding fragment, and the second connecting peptide (for example, the hinge region of human IgG1 or the hinge region of mouse IgG2c). Or derived sequence), CH 2 and CH 3 , and the drug conjugate is connected to the second polypeptide chain through Cys1 of the second connecting peptide (for example, Cys220 (EU encoding) of the IgG hinge region).
  • the second connecting peptide for example, the hinge region of human IgG1 or the hinge region of mouse IgG2c.
  • CH 2 and CH 3 the drug conjugate is connected to the second polypeptide chain through Cys1 of the second connecting peptide (for example, Cys220 (EU encoding) of the IgG hinge region).
  • the protein-drug conjugate may include a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain.
  • the first polypeptide chain from the N-terminus to the C-terminus may sequentially include a third antigen-binding fragment, a first antigen-binding fragment, a first connecting peptide, and a first pairing subunit.
  • the second polypeptide chain from N-terminus to C-terminus may sequentially include a fourth antigen-binding fragment, a second antigen-binding fragment, a second connecting peptide, and a second pairing subunit. Wherein, the first paired subunit and the second paired subunit can interact to form a dimer.
  • the third polypeptide chain may include a third antigen-binding fragment.
  • the fourth polypeptide chain may include a fourth antigen-binding fragment.
  • the first polypeptide chain from the N-terminus to the C-terminus may sequentially include the VL of the third antigen-binding fragment, the CL of the third antigen-binding fragment, the VH of the first antigen-binding fragment, and the first connecting peptide (for example, Human IgG1 hinge region or mouse IgG2c hinge region or derived sequence), CH 2 and CH 3 , and the drug conjugate passes through the Cys1 of the first connecting peptide (for example, Cys220 (EU code) of the IgG hinge region) and the The first polypeptide chain is connected.
  • the first connecting peptide for example, Human IgG1 hinge region or mouse IgG2c hinge region or derived sequence
  • the second polypeptide chain from the N-terminus to the C-terminus may sequentially include the VL of the fourth antigen-binding fragment, the CL of the fourth antigen-binding fragment, the VH of the second antigen-binding fragment, and the second connecting peptide (for example, Human IgG1 hinge region or mouse IgG2c hinge region or derived sequence), CH 2 and CH 3 , and the drug conjugate passes through the Cys1 of the second connecting peptide (for example, Cys220 (EU code) of the IgG hinge region) and the The second polypeptide chain is connected.
  • the second connecting peptide for example, Human IgG1 hinge region or mouse IgG2c hinge region or derived sequence
  • a third polypeptide chain from the N-terminus to C-terminus may in turn comprise a third antigen-binding fragment of the VH and CH third antigen binding fragment 1.
  • the fourth polypeptide chain from the N-terminus to C-terminus may in turn comprise a fourth antigen binding fragment CH 1 and VH fourth antigen-binding fragments.
  • the third antigen-binding fragment and/or the fourth antigen-binding fragment may be Fab.
  • the present application provides one or more nucleic acid molecules, which can encode the protein-drug conjugate as described in the present application.
  • each nucleic acid molecule in the one or more nucleic acid molecules may encode the complete protein-drug conjugate, or may encode a part of it (for example, HCDR1-3, LCDR1-3, VL, VH , One or more of polypeptide chain or heavy chain).
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, for example, can be amplified by polymerase chain reaction (PCR), (ii) can be produced by clonal recombination, (iii) ) Purified, for example, fractionation by enzyme digestion and gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.
  • PCR polymerase chain reaction
  • the isolated nucleic acid may be a nucleic acid molecule prepared by recombinant DNA technology.
  • this application provides one or more vectors, which comprise one or more nucleic acid molecules described in this application.
  • Each vector may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcriptional expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • the expression control sequence may also include an enhancer sequence or an upstream activator sequence.
  • suitable promoters can include, for example, promoters for SP6, T3 and T7 polymerases, human U6 RNA promoters, CMV promoters and artificial hybrid promoters (such as CMV), wherein A certain part can be fused with a certain part of a gene promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase), which may or may not contain additional introns.
  • One or more nucleic acid molecules described in this application can be operably linked to the expression control element.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering.
  • the vector is an expression vector.
  • the expression vector may include a eukaryotic cell expression vector and/or a prokaryotic cell expression vector.
  • the application provides a host cell, which may contain one or more nucleic acid molecules described in this application and/or one or more vectors described in this application.
  • each or each host cell may contain one or one of the nucleic acid molecules or vectors described in this application.
  • each or each host cell may contain multiple (e.g., two or more) or multiple (e.g., two or more) nucleic acid molecules or vectors described in the present application.
  • the vector described in this application can be introduced into the host cell, such as prokaryotic cells (for example, bacterial cells), mammalian eukaryotic cells or other eukaryotic cells, such as cells from plants, fungi or yeast cells.
  • the vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, and the like.
  • the host cell can be COS, CHO, NSO, sf9, sf21, DH5a, BL21(DE3) or TG1.
  • the prokaryotic cells can be E. coli cells such as TG1 and BL21.
  • the eukaryotic cells can be selected from CHO cells, CHO-K1 cells, CHOZN cells, CHO-S cells, ExpiCHO-S cells, NS/0 cells, HEK293 cells, HEK293-T cells, HEK293-F cells, or HEK293 cells. -6E cells.
  • the present application also provides a method for preparing the protein-drug conjugate, which comprises making the drug conjugate and the specific cysteine of the connecting peptide (for example, the first Cys1 of a connecting peptide and/or Cys1 of the second connecting peptide are covalently bonded to obtain the protein-drug conjugate described in the present application.
  • the preparation method can be a process of drug coupling based on the reactive sulfhydryl functional group of free cysteine at a specific site, and the preparation method can produce a coupling site specificity and homogeneity.
  • the preparation method may include one or a combination of multiple steps including “reduction”, “reoxidation”, “coupling” and “purification”.
  • the preparation method may include steps such as “reduction”, “coupling” and “purification”; for another example, the preparation method may include steps such as “reduction” and “coupling”.
  • the implementation step may include one or more reaction processes. During the reaction process, reaction conditions such as reaction temperature, reaction time, reaction buffer type and pH, reducing agent type and dosage, conjugate molecule type and dosage, etc., may all have a significant impact on the result of the preparation method. , For example, have a major impact on the efficiency of drug coupling and the quality of the final product.
  • the reaction process may also include a combination of different reaction conditions and parameters; for example, the reaction process for reducing the disulfide bond may be combined with different reducing agent types, different reducing agent dosages, different reduction reaction temperatures, different reaction buffers, Multiple parameter conditions such as different pH and different reaction time.
  • the reducing agent DTT dithiothreitol
  • TCEP Tris(2-carboxyethyl)phosphine
  • the amount of reducing agent used in the "reduction" step of the preparation method is about 1 to 50 mole multiples, for example, about 1 to 30 mole multiples, about 1 to 20 mole multiples, and about 1 to 10 mole multiples.
  • the mole ratio is about 1 to 7 mole ratio, about 1 to 6 mole ratio, about 1 to 5 mole ratio, about 1.5 to 6 mole ratio, or about 1.5 to 5 mole ratio.
  • reaction time of the "reduction" step of the preparation method is about 1 to 17 hours, for example, about 1 to 10 hours, about 1 to 7 hours, about 1 to 5 hours, and about 1 hour. ⁇ 4 hours, about 1 to 3 hours or about 1.5 to 2 hours.
  • the reaction temperature in the "reduction" step of the preparation method is about 0-40°C, for example, 0-37°C, about 0-30°C, about 20-37°C, about 20-37°C. 30°C, or room temperature (about 25-30°C).
  • the reaction temperature is 0°C, room temperature (25-30°C), or 37°C.
  • the pH of the reaction buffer in the "reduction" step of the preparation method is about 4.0 to 9.0, about 5.0 to 8.0, or about 5.0 to 7.0.
  • the reducing agent TCEP can be used in the "reduction" step of the preparation method, the amount of the reducing agent is 1 to 7 mole multiples, the reaction time is 1 to 3 hours, and the reaction temperature is 23 to 30°C.
  • the pH of the reaction buffer is 5.0 to 7.0.
  • the reducing agent DTT dithiothreitol
  • TCEP Tris(2-carboxyethyl)phosphine
  • the amount of the reducing agent is 1-50 mole multiples, and the reaction time is long. It is 1-17 hours, the reaction temperature is 0-40°C, and the pH of the reaction buffer is 5.0-8.0.
  • the reducing agent is TCEP
  • the amount of TCEP can be 1 to 7 mole multiples
  • the reaction time can be 1 to 3 hours
  • the pH of the reaction buffer is 5.0 to 7.0
  • the reaction temperature is 0°C or room temperature (25-30°C) Or 37°C.
  • the amount of the reducing agent TCEP can be 1.5-6 mole multiples, the reaction time can be 1.5-2 hours, the pH of the reaction buffer is 5.0-6.0, and the reaction temperature is 0°C or room temperature (25-30°C) or 37°C .
  • the "coupling" step of the preparation method may use mc-vc-PAB-MMAE ( Figure 6) as a drug conjugate.
  • the "coupling" step of the preparation method may use a BRD4 protein degrading agent ( Figure 64) as a drug conjugate.
  • the amount of the drug conjugate used in the "coupling" step of the preparation method is about 1-50 mole multiples, for example, about 1-30 mole multiples, about 1-20 mole multiples, about 10-15 molar multiples, about 1-10 molar multiples, about 1-7 molar multiples, or about 3-7 molar multiples.
  • the reaction time of the "coupling" step of the preparation method is about 0.5 to 10 hours, for example, about 0.5 to 10 hours, about 0.5 to 3 hours, about 0.5 to 2 hours, about 1 to 2 hours or about 0.5 to 1.
  • the reaction temperature in the "coupling" step of the preparation method is about 0-40°C, for example, 0-37°C, about 0-30°C, about 20-37°C, about 20°C. ⁇ 30°C, or room temperature (about 25-30°C).
  • the reaction temperature is room temperature (25-30°C).
  • the pH of the reaction buffer in the "coupling" step of the preparation method is about 4.0 to 9.0, about 5.0 to 8.0, or about 5.0 to 7.0.
  • the "coupling" step of the preparation method can use mc-vc-PAB-MMAE ( Figure 6) as the drug conjugate, and the amount of the drug conjugate is 3-7 molar multiples.
  • the reaction time is 0.5-2 hours, the reaction temperature is 23-30°C, and the pH of the reaction buffer is 5.0-7.0.
  • the "coupling" step of the preparation method can use mc-vc-PAB-MMAE ( Figure 6) as the drug conjugate, and the amount of the drug conjugate is 1-50 molar multiples.
  • the reaction time is 0.5-10 hours, the reaction temperature is 0-40°C, and the pH of the reaction buffer is 5.0-8.0.
  • the amount of mc-vc-PAB-MMAE can be 3-18 mole multiples, the reaction time can be 0.5-3 hours, the pH of the reaction buffer is 5.0-7.0, and the reaction temperature is room temperature (25-30°C). More preferably, the amount of mc-vc-PAB-MMAE can be 3-7 molar multiples, the reaction time can be 0.5 hours, the pH of the reaction buffer is 5.0-6.0, and the reaction temperature is room temperature (25-30°C).
  • the "coupling" step of the preparation method can use the BRD4 protein degrading agent (Figure 64) as the drug conjugate, the amount of the drug conjugate is 10-15 mole multiples, and the reaction time is For 1 to 3 hours, the reaction temperature is 23-30°C, and the pH of the reaction buffer is 7.0-9.0.
  • the preparation method may also provide a combination of steps including "reduction” and "coupling” and a combination of reaction condition parameters.
  • a preferred combination of the reaction conditions and parameters can be: the reaction buffer is a pH 6.0 buffer containing 20 mM His-HCl, the amount of the reducing agent TCEP is 1.5 to 6.0 molar multiples, and the reduction reaction time is 1.5 to 2.0 hours
  • the reduction reaction temperature is room temperature (25-30°C)
  • the conjugate is mc-vc-PAB-MMAE, the amount of conjugate is 3.0-7.0 mole multiples, the coupling reaction time is 0.5 hours, and the coupling reaction temperature is room temperature (25-30°C).
  • the preparation method may also provide a "purification” step.
  • HIC hydrophobic interaction chromatography
  • the preparation method may also provide a combination of steps including "reduction” and “coupling” and a combination of reaction condition parameters; under the combination of the reaction steps and reaction condition parameters, The disulfide bond of the first cysteine (Cys1) of the connecting peptide can be effectively opened while the other disulfide bonds remain intact, making Cys1 almost the only free cysteine residue, which then serves as a specificity The coupling site.
  • the present application provides a pharmaceutical composition, which may include the protein-drug conjugate and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may also include other antigen binding proteins or therapeutic agents.
  • the present application provides an application of the protein-drug conjugate and/or the pharmaceutical composition in the preparation of drugs for diagnosis, prevention and/or treatment of diseases.
  • the drugs can be used to treat tumors or other diseases.
  • this application provides a use of the protein-drug conjugate in combination with other therapies or drugs in the preparation of medicines, which can be used to treat tumors or other diseases.
  • the other therapies or drugs can be selected from the group consisting of chemotherapy, radiotherapy, miRNA and oligonucleotides.
  • the present application provides a method for detecting specific antigens in vitro or in vivo, which may include the use of the protein-drug conjugate and/or the pharmaceutical composition for detection. In some applicable situations, the method may be for non-diagnostic purposes.
  • the present application provides a drug delivery device for the administration of the protein-drug conjugate or pharmaceutical composition.
  • the present invention will be further described by way of examples below, but the present invention is not limited to the scope of the described examples.
  • the examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are relevant to the present Those of ordinary skill in the field are well known and described in many publications.
  • the experimental methods without specific conditions are selected according to conventional methods and conditions, or according to the product specification.
  • the Harbour HCAb mouse (Harbour Antibodies BV, WO2010/109165A2) is a transgenic mouse carrying a human immunoglobulin immune library, capable of producing antibodies with only heavy chains, which are only half the size of traditional IgG antibodies.
  • the antibody produced by the mouse has a human antibody heavy chain variable domain and a mouse constant domain.
  • After obtaining the VH sequence of the heavy chain antibody against a specific target use conventional molecular biology methods to compare the VH sequence with the human IgG antibody heavy chain Fc sequence (preferably those containing the hinge region of human IgG1 and CH2 and CH3 regions).
  • the sequence such as the sequence shown in SEQ ID NO: 72) is fused and expressed to obtain a fully human recombinant HCAb antibody molecule.
  • the Harbour H2L2 mouse (Harbour Antibodies BV) is a transgenic mouse carrying a human immunoglobulin immune library.
  • the antibodies produced by it have complete human antibody variable domains and rat constant domains.
  • the VL and VH sequences of the antibody are fused and expressed with the corresponding human kappa light chain constant region and IgG1 heavy chain constant region sequences to obtain recombinant fully human antibody molecules.
  • the soluble recombinant human CTLA4 protein (ACRO Biosystems, #CT4-H5229) was used to immunize Harbour HCAb mice for multiple rounds.
  • CTLA4 specific antibody titer in the serum of the detected mouse reaches a certain level, the spleen cells of the mouse are removed to separate the B cells, and the mouse plasma cell sorting kit (Miltenyi, #130-092-530) is used for sorting CD138-positive plasma cells.
  • the human VH gene was amplified from plasma cells by conventional molecular biology methods, and the amplified human VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the sequence of the Fc region of the human IgG1 antibody heavy chain.
  • the plasmid is transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression, and a fully human HCAb antibody supernatant is obtained.
  • ELISA was used to test the binding of the HCAb antibody supernatant to the recombinant human CTLA4 protein, and the positive HCAb antibody was identified.
  • These HCAb antibodies were further identified, and several candidate HCAb antibody molecules were selected based on their binding ability to human CTLA4, the binding ability of cynomolgus CTLA4, and the ability to inhibit the binding of CTLA4 to B7-1. Then the candidate HCAb antibody molecule is sequenced and optimized, and several variant sequences are obtained.
  • the VH sequence of the HCAb antibody is fused with the human IgG1 heavy chain hinge region and Fc sequence to obtain a fully human recombinant HCAb antibody molecule.
  • the recombinant fully human HCAb antibodies against CTLA4 are listed in Table 1-1.
  • the soluble recombinant human BCMA-ECD-Fc fusion protein (ACRO Biosystems, #BC7-H82F0) was used to immunize Harbour HCAb mice for multiple rounds.
  • the antigen protein is mixed with an immune adjuvant to form an immunogenic reagent, and then injected subcutaneously through the inguinal or through the abdominal cavity.
  • the total injection dose received by each mouse is 100 microliters.
  • each mouse was immunized with an immunogen reagent prepared by mixing 50 micrograms of antigen protein and complete Freund's adjuvant (Sigma, #F5881) at a volume ratio of 1:1.
  • each mouse was immunized with immunogenic reagents mixed with 25 micrograms of antigen protein and Sigma Adjuvant System adjuvant (Sigma, #S6322).
  • the interval between each round of booster immunization is at least two weeks, usually no more than five rounds of booster immunization.
  • the immunization time was 0, 14, 28, 42, 56, and 70 days; and on the 49th and 77th days, the mouse serum antibody titers were detected.
  • Five days before the isolation of Harbour HCAb mouse spleen B cells the final booster immunization was performed at a dose of 25 micrograms of antigen protein per mouse.
  • the spleen cells of the mouse are taken out to separate B cells, and the CD138-positive plasma cells and the BCMA antigen-positive cells are sorted using the BD FACSAria TM III cell sorter.
  • B cell population RNA was extracted, cDNA was reverse transcribed, and human VH gene was amplified by PCR. The amplified VH gene fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the human IgG1 antibody heavy chain Fc domain sequence.
  • the plasmid was transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression, and the expressed HCAb
  • mammalian host cells such as human embryonic kidney cells HEK293
  • the antibody supernatant was combined with recombinant human BCMA-Fc, Avitag recombinant protein (ACRO Biosystems, #BC7-H82F0) for Mirrorball (SPT Labtech, Fluorescence cytometer) screening, the obtained positive monoclonal antibody supernatant was further identified by flow cytometry FACS.
  • FACS was used to test the antibody supernatant with the HEK293T cell line HEK293T/hBCMA (Beijing Kangyuan Bochuang, KC-0233) with high expression of human BCMA, and the HEK293T cell line HEK293T/cynoBCMA (Beijing Kangyuan Bochuang, with high expression of cynomolgus monkey BCMA).
  • KC-0979 and high-expressing human BCMA cell line NCI-H929 (ATCC, CRL-9068) and other cells.
  • the nucleotide sequence encoding the variable domain of the antibody molecule is obtained by conventional sequencing methods.
  • the VH sequence of the HCAb antibody is fused with the human IgG1 heavy chain hinge region and Fc sequence to obtain a fully human recombinant HCAb antibody molecule.
  • the soluble recombinant human MSLN protein (ACRO Biosystems, #MSN-H5223) was used to immunize Harbour HCAb mice for multiple rounds. The method similar to that described in Example 1.2 was used to screen and obtain fully human HCAb antibodies against MSLN. The recombinant fully human HCAb antibodies against MSLN are listed in Table 1-1.
  • HCAb mice were immunized with soluble recombinant human 5T4 protein (NovoProtein, #C678) for multiple rounds. The method similar to that described in Example 1.2 was used to screen and obtain a fully human HCAb antibody against 5T4. The recombinant fully human HCAb antibodies against 5T4 are listed in Table 1-1.
  • mice were immunized for multiple rounds with soluble recombinant human ROR1 protein (AcroBiosystems, #RO1-H5250).
  • the anti-ROR1 antibody clone 1015M2-H4 was obtained by a method similar to that described in Example 1.2 and combined with phage display screening.
  • the 1015M2-H4 VH and VL were prepared by overlapping PCR to obtain a single-chain variable region fragment scFv; then the scFv and a sequence containing the restriction endonuclease BamHI restriction site (the corresponding amino acid sequence is GGGAS) and human
  • the IgG heavy chain Fc (including the hinge region of human IgG1 and the CH2 and CH3 regions, as shown in SEQ ID NO: 72) was fused and expressed to obtain an anti-ROR1 antigen binding protein molecule PR002129 with a scFv-Fc dimer structure.
  • Anti-ROR1 antigen binding proteins are listed in Table 1-1.
  • This example introduces a general method for preparing antibodies using mammalian host cells (e.g., human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and its derived cells), transient transfection expression and affinity capture and isolation techniques.
  • mammalian host cells e.g., human embryonic kidney cells HEK293 or Chinese hamster ovary cells CHO and its derived cells
  • This method is suitable for target antibodies containing Fc region; the target antibody can be composed of one or more protein polypeptide chains; it can be derived from one or more expression plasmids.
  • the amino acid sequence of the antibody polypeptide chain is converted into a nucleotide sequence through a codon optimization method; the encoded nucleotide sequence is synthesized and cloned into an expression vector compatible with the host cell.
  • Plasmids encoding antibody polypeptide chains are simultaneously transfected into mammalian host cells according to a specific ratio, and conventional recombinant protein expression and purification techniques can be used to obtain recombinant proteins with correct folding and polypeptide chain assembly. Specifically, FreeStyle TM 293-F cells (Thermo, #R79007) were expanded in FreeStyle TM F17 Expression Medium (Thermo, #A1383504).
  • the cell concentration is 1.2 ⁇ 10 6 cells/ml.
  • the plasmid encoding the antibody polypeptide chain was mixed according to a certain ratio, a total of 30 ⁇ g plasmid (the ratio of plasmid to cell is 1 ⁇ g:1ml) was dissolved in 1.5ml Opti-MEM reduced serum medium (Thermo, #31985088), and filtered with a 0.22 ⁇ m filter. bacteria.
  • the antigen binding proteins used in this application are summarized in Table 1-1, and the sequence numbers of the amino acid sequences of the corresponding polypeptide chains and CDR regions SEQ ID NOs are listed in Table 1-2, Table 1-3, Table 1-4 and Table 1-5.
  • Table 1-1 Some exemplary antigen binding proteins used in this application.
  • Table 1-3 The sequence numbering list of the antigen binding protein of the scFv-Fc structure in this application.
  • This example uses analytical size exclusion chromatography (SEC) to analyze the purity and aggregate form of the protein sample.
  • SEC analytical size exclusion chromatography
  • Example 2.2 Using HIC-HPLC (Hydrophobic Interaction-High Performance Liquid Chromatography) to analyze protein purity, hydrophobicity and coupling product components
  • This example uses analytical hydrophobic interaction chromatography (HIC) to analyze the purity and hydrophobicity of protein samples. Connect the analytical column TSKge1 Buty1-NPR (Tosoh Bioscience, #14947, 4.6mm ⁇ 3.5cm, 2.5 ⁇ m) to a high performance liquid chromatograph (HPLC) (model: Agilent Technologies, Agilent 1260 Infinity II), buffered with PBS Equilibrate the solution for at least 1 hour at room temperature.
  • HPLC high performance liquid chromatograph
  • the setting method ranges from 100% buffer A (25mM phosphate buffer, 1.5M ammonium sulfate ((NH4)2SO4), pH 7.0 ⁇ 7.2) to 100% buffer B (20mM phosphate buffer, 20% isopropanol (IPA), pH 7.0 ⁇ 7.2) linear gradient, flow rate set to 0.7ml/min, protein sample concentration 1mg/ml, injection volume 20 ⁇ l, detection wavelength 280nm, running at room temperature.
  • IPA isopropanol
  • This example uses analytical reversed-phase high performance liquid chromatography (RP) to analyze the purity and hydrophobicity of protein samples.
  • RP reversed-phase high performance liquid chromatography
  • the analytical chromatographic column Zorbax RRHD 300-Diphenyl (Agilent Technologies, #858750-944, 2.1 ⁇ 100mm, 1.8 ⁇ m) is connected to the high performance liquid chromatograph (HPLC) (Model: Agilent Technologies, Agilent 1260 Infinity II), balance the system according to the instructions.
  • HPLC high performance liquid chromatograph
  • the setting method is a linear gradient from 100% buffer A (1% trifluoroacetic acid TFA dissolved in water) to 100% buffer B (1% trifluoroacetic acid TFA dissolved in acetonitrile) within 60 minutes, and the flow rate is set to 0.35 ml/min, operating temperature is 50°C, detection wavelength is 280nm.
  • ChemStation software uses the ChemStation software to integrate the chromatogram and calculate the relevant data, generate an analysis report, and report the residence time of the components of different molecular sizes in the sample.
  • the analytical chromatographic column PLRP-S 1000A (Agilent Technologies, #PL1912-1502, 2.1 ⁇ 50mm, 5.0 ⁇ m) is connected to the high performance liquid chromatograph (HPLC) (Model: Agilent Technologies, Agilent 1260 Infinity II), balance the system according to the instructions.
  • HPLC high performance liquid chromatograph
  • the setting method is a linear gradient from 100% buffer A (0.05% trifluoroacetic acid TFA dissolved in water) to 100% buffer B (0.05% trifluoroacetic acid TFA dissolved in acetonitrile) within 40 minutes, and the flow rate is set to 0.25 ml/min, operating temperature is 60°C, detection wavelength is 280nm.
  • ChemStation software to integrate the chromatogram and calculate the relevant data, generate an analysis report, and report the residence time of the components of different molecular sizes in the sample.
  • the number of coupled molecules carried by the different components of the coupling product can be estimated through different residence times.
  • liquid chromatography-mass spectrometry was used to analyze the molecular weight of protein samples.
  • LC-MS liquid chromatography-mass spectrometry
  • MS mass spectrometer
  • the determination of the molecular weight of a protein sample requires a sugar-removing treatment to remove sugar chains and other modifications.
  • the sample is treated with the glycosidase PNGase F (NEB, #P0705L) at 37°C for at least 4 hours.
  • the protein samples to be tested are all treated with glycosidase PNGase F for sugar removal.
  • determining the reduced molecular weight of a protein sample requires a reduction treatment of the sample, for example, treating the sample with 10 nM DTT (dithiothreitol) at 37° C. for 10 minutes.
  • a reduction treatment of the sample for example, treating the sample with 10 nM DTT (dithiothreitol) at 37° C. for 10 minutes.
  • a sample that has undergone reduction treatment is referred to as a reduced sample, and a sample that has not been subjected to reduction treatment is referred to as a non-reduced sample.
  • the chromatographic column bioZen 3.6 ⁇ m Intact C4 (Phenomenex, #00F-4767-AN, 2.1 ⁇ 150mm, 3.6 ⁇ m) is connected to an ultra-high performance liquid chromatograph (UPLC) (model: Agilent Technologies, Agilent 1290 UPLC), and balance the system according to the instructions.
  • Buffer A is 0.1% formic acid FA dissolved in water
  • buffer B is 0.1% formic acid FA dissolved in acetonitrile.
  • the injection is about 1 ⁇ g, the flow rate is set to 0.3ml/min, and the operating temperature is 80°C.
  • the chromatographic column ACQUITY UPLC Protein BEH C4 (Waters, #186004495, 2.1 ⁇ 50mm, 1.7 ⁇ m, ) Connect to an ultra high performance liquid chromatograph (UPLC) (model: Waters Acquity UPLC), and balance the system according to the instructions.
  • Buffer A is 0.1% formic acid FA dissolved in water
  • buffer B is 0.1% formic acid FA dissolved in acetonitrile.
  • the injection is about 10 ⁇ L
  • the flow rate is set to 0.2 ⁇ 0.5ml/min
  • the operating temperature is 80°C
  • the detection wavelength is 214nM.
  • mix buffers A and B into a linear gradient within 20 minutes use 25-90% B for 14 minutes, and use 25% B for the next 6 minutes.
  • mix buffers A and B into a linear gradient within 8 minutes use 5-95% B for the first 4 minutes, and use 95-5% B for the last 4 minutes.
  • the mass spectrometer model used is AB Sceix X500B, and the operating mode is TOF-MS; when testing reduced samples, the scanning range is 600-4000m/z; when testing non-reduced samples, the scanning range is 900 -6000m/z.
  • the model of the mass spectrometer used is Waters Xevo G2-XS Q-TOF, and the molecular weight scanning range is 500-4000 Da.
  • LC conditions and MS conditions are used to realize the combination of LC-MS to determine the molecular weight of protein samples.
  • the combination of conditions used is (chromatographic column: ACQUITY UPLC Protein BEH C4; LC instrument: Waters Acquity UPLC; MS instrument: Waters Xevo G2-XS Q-TOF).
  • Example 3.2 Example 8.2, Example 9.2, Example 10.1, Example 11.2, Example 12.1, Example 13.2, Example 14.2, and Example 16.1
  • the combination of conditions used is (chromatographic column: bioZen 3.6 ⁇ m Intact C4; LC instrument: Agilent 1290 UPLC; MS instrument: AB Sceix X500B).
  • Example 6.2 and Example 7.2 the combination of conditions used is (chromatographic column: Agilent PLRP-S 1000A; LC instrument: Agilent 1290 UPLC; MS instrument: Waters Xevo G2-XS Q-TOF).
  • Uncle Unchained Labs is a multifunctional one-stop protein stability analysis platform. It uses full fluorescence, static light scattering (SLS) and dynamic light scattering (DLS) detection methods to characterize protein stability. The same set of samples can obtain melting temperature (Tm), aggregation temperature (Tagg) and particle size (diameter) at the same time.
  • SLS static light scattering
  • DLS dynamic light scattering
  • Tm melting temperature
  • Tagg aggregation temperature
  • particle size particle size at the same time.
  • select Uncle's "Tm&Tagg with optional DLS” application program for operation take 9 ⁇ L of sample into the Uni tube, and set a gradient of 0.3°C/min to gradually increase the temperature from 25°C to 95°C. Perform the initial and final DLS measurements four acquisitions, each acquisition for 5 seconds.
  • the Uncle analysis software uses the barycentric mean (BCM) formula to calculate the Tm value of each sample; calculates the Tagg value through the SLS fluorescence intensity curve (aggregation curve) at the wavelength of 266nm or 473nm; the particle size of the sample
  • BCM barycentric mean
  • a Malvern VP-capillary DSC instrument was used to analyze the melting temperature (Tm) of the protein sample by differential scanning calorimetry (DSC). Dilute the sample with buffer to the detection concentration (0.5 mg/mL) and place it in the DSC sample cell, and place the buffer in the reference cell. The DSC sample cell and the reference cell are subjected to exactly the same isothermal heating control.
  • the scanning temperature range is 25-100°C
  • the scanning speed is 1°C/min
  • the equilibration time before scanning is 3 minutes, and each sample to be tested is analyzed once.
  • the peak diagram composed of the heat that compensates for the temperature difference of the sample cell is the thermodynamic curve diagram of the sample, and the peak top temperature of the curve is the Tm value of the sample.
  • the curve of protein samples containing multiple domains may show multiple peaks due to the different stability of different domains.
  • DSF Differential Scanning Fluorimetry
  • the human IgG1 antibody is a tetrameric structure containing two identical heavy chains and two identical light chains. Two interchain disulfide bonds are formed between the two heavy chains, one heavy chain and the corresponding light chain. An interchain disulfide bond is formed between the chains; several sulfhydryl groups formed after reduction of the natural interchain disulfide bonds of these antibodies become available cysteine coupling sites.
  • Figure 44(B) shows the hinge region sequence of human IgG1 (SEQ ID NO: 73), which contains three cysteines, and the corresponding positions are Cys-220, Cys-226 and Cys-229 according to Eu numbering.
  • Cys-226 and Cys-229 of the two heavy chain hinge regions each form an inter-heavy chain disulfide bond, while Cys-220 forms a heavy chain and a light chain with Cys-214 near the C'end of the light chain The disulfide bond between.
  • Figure 2 shows the structure of a heavy chain antibody HCAb with the sequence of the hinge region of a human IgG1 antibody, and the difference between the disulfide bond structure of the hinge region between human IgG1 and HCAb. Since there is no light chain, Cys-220 located in the hinge region obviously can no longer form a disulfide bond with the light chain Cys-214.
  • the state of Cys-220 has two possibilities as shown in Figure 3: (1) Cys-220 does not form a disulfide bond between heavy chains, and its side chain sulfhydryl group (-SH) will interact with the sulfhydryl group in the environment (such as cytoplasm). Molecules (such as glutathione) form disulfide bonds; (2) Like Cys-226 or Cys-229, Cys-220 forms interchain disulfide bonds between two heavy chains, so the hinge region will form three Disulfide bond.
  • -SH side chain sulfhydryl group
  • This example uses different experimental methods to identify the status of Cys-220 in HCAb PR000020 (see Table 1-1 for molecular information).
  • Cys-220 does not form disulfide bonds between heavy chains (Assumption 1), but its side chain sulfhydryl groups form disulfide bonds with other sulfhydryl-containing molecules in the environment, then the non-reduced molecular weight (ie, complete molecular weight) of the HCAb will be greater than The theoretical value of molecular weight when disulfide bonds between heavy chains are formed (Assumption 2).
  • the theoretical non-reduced molecular weight of PR000020 is 77,640 Da; according to Hypothesis 1, its non-reduced molecular weight may be 77,878 Da (with an increase of 2 cysteines (+119Da ⁇ 2)) or 78,250 Da (with an increase of 2 Glutathione (+305Da ⁇ 2)).
  • the purified HCAb PR000020 protein was treated with glycosidase PNGase F for sugar removal, and its non-reduced molecular weight was determined by the method described in Example 2.4 (LC instrument: Waters Acquity UPLC; MS instrument: Waters Xevo G2-XS Q-TOF), and the result was It is 77,643 Da (see Figure 4 for the deconvoluted molecular weight map), which is almost consistent with the predicted molecular weight according to Hypothesis 2. From this, it can be inferred that Cys-220 forms an inter-heavy chain disulfide bond.
  • Papain can specifically hydrolyze the peptide bond between His-224 and Thr-225 in the hinge region (see Endo, S., & Arata, Y. (1985).).
  • the purified HCAb PR000020 protein was digested with Papain.
  • Figure 5(A) if Cys-220 does not form an inter-heavy chain disulfide bond (hypothesis 1), then Papain hydrolyzing HCAb will produce about 13KDa fragments (single VH domain) and about 50KDa Fc fragments; if Cys-220 forms an inter-heavy chain disulfide bond (hypothesis 2), then Papain hydrolysis of HCAb will produce a fragment of about 27KDa (two VH domains connected by the disulfide bond of Cys-220) and an Fc fragment of about 50KDa .
  • Figure 5(B) shows the result of non-reducing SDS-PAGE of the digested product; the digested product of HCAb (corresponding to lanes 3 and 4) has a clear band near 28KDa, but no band near 14KDa, so The establishment of hypothesis 2 is confirmed, that is, Cys-220 forms a disulfide bond between heavy chains.
  • PR002129 is a dimer structure containing heavy chain Fc, and each polypeptide chain (heavy chain) contains a single-chain variable region fragment scFv, a connecting peptide sequence and human IgG1 heavy chain Fc sequence; among them, scFv is derived from anti- The VH and VL sequences of ROR1 antibody 1015M2-H4, the connecting peptide sequence contains the hinge region sequence of human IgG1 and a GGGAS sequence (in order to introduce suitable restriction endonuclease sites).
  • Cys-220 In PR002129 and other similar scFv-Fc structures, the state of Cys-220 also has two possibilities as shown in Figure 37(A): formation or non-formation of interchain disulfide bonds. If Cys-220 does not form an inter-heavy chain disulfide bond, its side chain sulfhydryl group (-SH) will form a disulfide bond with sulfhydryl-containing molecules in the environment (such as cysteine or glutathione), making it practical The molecular weight is significantly greater than the theoretical molecular weight.
  • -SH side chain sulfhydryl group
  • the purified PR002129 protein was treated with glycosidase PNGase F for sugar removal, and its non-reduced molecular weight was determined by the method described in Example 2.4 (LC instrument: Agilent 1290 UPLC; MS instrument: AB Sceix X500B) (shown in Figure 37(B)) The deconvoluted molecular weight map), the result is 104,510Da. Compared with the predicted molecular weight of different hypotheses (Table 3-2), it is inferred that Cys-220 is not connected to other sulfhydryl-containing molecules, so Cys-220 forms an inter-heavy chain two Sulfur bond.
  • human IgG1 passes through the last Cys of the light chain (ie, Cys-214 encoded by Eu). It forms an interchain disulfide bond with the fifth Cys of the heavy chain (Cys-220 encoded by Eu), while IgG2, IgG3, and IgG4 form an interchain disulfide bond through the last Cys of the light chain and the third Cys of the heavy chain. Disulfide bonds (see, Liu, H. et al. mAbs (2012), 4(1), 17-23).
  • the inventor of the present application further analyzed the crystal structure of IgG1 (PDB accession number 1HZH). Cys-220 and the light chain Cys-214 formed a stable disulfide bond, and the C ⁇ atoms of Cys-220 in the two heavy chain hinge regions were one of the The distance between the two Cys-220s is relatively far, and it is inferred from the structure of the IgG1 that it is difficult to form a disulfide bond between the two Cys-220. On the other hand, the aforementioned studies have verified the fact that Cys-220 forms an inter-heavy chain disulfide bond in a structure lacking the light chain.
  • Cys-220 can form inter-heavy chain disulfide bonds like Cys-226 or Cys-229, but its disulfide bond stability may be weaker than Cys-226 or Cys-229. Therefore, through optimization, it can specifically open the disulfide bond of Cys-220 while keeping other disulfide bonds unchanged; in this way, Cys-220 may provide an anchor point for cysteine coupling for site-directed coupling.
  • the inventor of the present application further studied the hinge region sequence and disulfide bond structure of IgG antibodies of different species ( Figure 44) and found that the hinge region sequence of human and mouse and alpaca are very different, the length of the hinge region sequence and the Cys contained in it are very different. The numbers are not the same ( Figure 44(A)). The inventors used the known IgG full-length antibody crystal structure to analyze the disulfide bond structure of the hinge region.
  • Figure 44(B) shows the human IgG1 hinge region (PDB accession number 1HZH); Figure 44(C) shows the human IgG4 hinge Region (PDB accession number 5DK3); Figure 44(D) shows the mouse IgG1 hinge region (PDB accession number 1IGY); Figure 44(E) shows the mouse IgG2a hinge region (PDB accession number 1IGT).
  • a hinge region of human IgG1 and mouse IgG1 contribute to the disulfide bond between the heavy and light chains, and human IgG1 uses Cys-220 (Eu numbering) as the first Cys of the hinge region. It forms a disulfide bond with the light chain.
  • Mouse IgG1 uses Cys-235 (residue numbering) as the first Cys of the hinge region to form a disulfide bond with the light chain; however, the difference is that in the hinge region of human IgG1, the first Cys forms a disulfide bond with the light chain.
  • One Cys (Cys-220, Eu numbering) and the second Cys (Cys-226, Eu numbering) are separated by 5 other types of amino acid residues.
  • the first Cys ( Cys-235, residue numbering) and the second Cys (Cys-237, residue numbering) are separated by 1 other kind of amino acid residue.
  • Cys-220 of the human IgG1 heavy chain can form weaker inter-heavy chain disulfide bonds when the light chain is deleted, and this feature may be related to the distance between Cys-220 and Cys-226 ( That is, the distance between the first Cys and the second Cys) is related; the first Cys and the second Cys may need to be separated by a certain number of other types of amino acid residues in order for the two Cys to form different disulfide bonds and disulfide There is a difference in the stability (or bond energy) of the bond.
  • This example is to study the influence of the disulfide bond of Cys-220 on the stability of the molecular structure.
  • several HCAb molecules were selected to prepare their C220S mutant derivatives (Cys-220 in the hinge region was mutated to serine Ser to disrupt the formation of disulfide bonds), or the drug conjugate was prepared by the method described in Example 4 Derivatives (ADC); then use the Uncle method described in Example 2.5 or the DSC method described in Example 2.6 to determine the molecular melting temperature (Tm) to characterize its thermal stability.
  • ADC Derivatives
  • Tm molecular melting temperature
  • FIG 38 shows the DSC thermodynamic analysis curve of HCAb PR000184 and its derivatives
  • Figure 39 shows the DSC thermodynamic analysis curve of HCAb PR000453 and its derivatives
  • Figure 40 shows the Uncle thermodynamic analysis curve of HCAb PR004432 and its derivatives ;
  • Tm1 minimum melting temperature
  • Tm 1 Melting temperature of HCAb and derivative molecules
  • the drug coupling process based on cysteine residues includes a combination of one or more steps consisting of different reaction processes such as “reduction”, “reoxidation” and “coupling”.
  • reaction conditions such as reaction temperature, reaction time, reaction buffer type and pH, reducing agent type and dosage, conjugate molecule type and dosage, etc.
  • reaction conditions include combinations of different parameters, and this example only lists representative methods; those skilled in the art can also use different parameter conditions according to the method taught in this example and experiment to establish a similar coupling reaction process. .
  • This example tried to use the strong reducing agent DTT (dithiothreitol) or TCEP (Tris(2-carboxyethyl)phosphine) with moderate reducing ability.
  • This embodiment may also try to use the oxidant DHAA (dehydroascorbic acid) to process the sample.
  • DHAA dehydroascorbic acid
  • the drug conjugate that this example also tried to use was mc-vc-PAB-MMAE (also known as VcMMAE, the molecular structure is shown in Figure 6, the molecular weight is 1315.78Da; manufacturer: Lianning Biopharmaceutical, product number: SET0201), the The compound contains a maleimide active reactive group, which can undergo addition reaction with sulfhydryl to form a stable thioether bond.
  • hydrophobic interaction is used to separate the components in the coupling product.
  • the sample obtained by coupling is purified by the AKTA pure chromatography system (Cytiva Life Sciences, AKTA pure) using a ToyoScreen Phenyl-600M hydrophobic interaction chromatography column (Tosoh Bioscience, #21892). Within 30 column volumes, the hydrophobic phase was linearly increased from 0% to 100%, during which each group of peaks was collected separately. After the collected components are detected by HIC-HPLC (the method described in Example 2.2), they are combined, concentrated, and exchanged to obtain the coupling product.
  • This example studied the anti-CTLA4 HCAb antibody PR000020 (SEQ ID NO: 64) and mc-vc-PAB-MMAE using cysteine to couple to the Cys-220 site to prepare antibody-conjugated drugs with uniform DAR value .
  • Example 1.6 the method described in Example 1.6 was used to produce and purify the HCAb PR000020 recombinant protein; then, the method described in Example 4.1 was used to use a number of different combinations of reduction, reoxidation and coupling conditions.
  • HCAb PR000020 conducts coupling reaction (Table 5-1), trying to obtain better reaction conditions.
  • the reducing agent used is TCEP (Tris(2-carboxyethyl)phosphine) or DTT (dithiothreitol); the oxidizing agent is DHAA (dehydroascorbic acid); the drug conjugate is mc-vc-PAB-MMAE (manufacturer: Lianning Biopharmaceutical , Item No.: SET0201).
  • HCAb PR000020 was coupled in parallel with 6 sets of conditions, and trastuzumab (the 7th experiment) was used as an IgG1 control.
  • the pH of the reduction or coupling reaction is 6.5-7.0, and the temperature is room temperature (25-30°C).
  • the coupled products of each group of experiments were analyzed by the HIC-HPLC analysis method described in Example 2.2 for the components of the coupled products, which are listed in Table 5-2.
  • the purified recombinant HCAb PR000020 and its coupled product PR000020-ADC were quantitatively analyzed using high performance liquid chromatography and mass spectrometry techniques to determine their coupling Composition of product components.
  • Table 5-3 shows that the purity of the sample before coupling of HCAb PR000020 is as high as 95%;
  • Table 5-5 shows that 99% of the product obtained after the coupling product is purified by one-step HIC (Example 4.2) is the product of coupling 2 compounds.
  • LC-MS was used to analyze ADC coupling sites.
  • the purified samples of the uncoupled HCAb PR000020 and the coupled product PR000020-ADC are reduced with DTT (dithiothreitol); then, the active sulfhydryl group of cysteine is processed with iodoacetamide (IAM). Block; then, digest the sample with trypsin; analyze the peptide fragment of the digested product with LC-MS.
  • IAM iodoacetamide
  • Block digest the sample with trypsin; analyze the peptide fragment of the digested product with LC-MS.
  • use the software Peaks Studio Bioinformatics Solutions Inc.
  • Each heavy chain of HCAb PR000020 has 9 Cys (numbered according to the sequence position of the amino acid sequence, respectively: C23, C96, C124, C130, C133, C165, C225, C271, C329), where C124 corresponds to Eu numbering 220. After analysis, in the unconjugated PR000020 sample, in all 9 Cys, except for C225, which was not detected due to the denser pancreatin digestion site distribution before and after, the Cys at the remaining 8 sites were all detected.
  • Figure 11(C) lists the peptides containing IAM modified Cys (Cys-IAM) identified in uncoupled samples.
  • C124 ie Cys-220 with Eu number
  • Figure 11(B) A list of peptides containing IAM-modified Cys (Cys-IAM) identified in the coupled sample is listed. These peptides are marked with a gray background on the amino acid sequence of PR000020-ADC in Figure 11(A). It can be seen that in the PR000020-ADC sample, C124 (i.e., Cys-220) did not undergo IAM modification.
  • This example is to study the activity of the HCAb antibody PR000020 and its conjugate product PR000020-ADC in binding to cells that highly express CTLA4.
  • Flow cytometry FACS was used to test the binding ability of antibody molecules with HEK293T cell line HEK293/hCTLA4 (constructed with platinum medicine) that highly express human CTLA4.
  • HEK293/hCTLA4 cells were digested and resuspended in DMEM medium; the cell density was adjusted to 1 ⁇ 10 6 cells/mL.
  • HEK293 cells that did not express CTLA4 served as a negative control. Then, the cells were seeded in a 96-well V bottom plate (Corning, #3894) at 100 ⁇ L/well, centrifuged at 4° C. for 5 minutes, and the supernatant was discarded.
  • the antibody molecules diluted in gradients were added to a 96-well plate and mixed uniformly.
  • the antibody molecules can be diluted to a total of 12 concentrations from the highest final concentration of 500 nM in a 3-fold concentration gradient. Place the cells at 4°C and incubate for 1 hour in the dark. Then, 100 ⁇ L/well of pre-cooled FACS buffer (PBS buffer containing 0.5% BSA) was added to rinse the cells twice, centrifuged at 500 g at 4° C. for 5 minutes, and the supernatant was discarded.
  • pre-cooled FACS buffer PBS buffer containing 0.5% BSA
  • the software GraphPad Prism 8 is used for data processing and mapping analysis, and through four-parameter nonlinear fitting, parameters such as the binding curve of the antibody to the target cell and EC50 value are obtained.
  • the antibody PR000020 and its purified conjugate product PR000020-ADC have strong specific binding activity with HEK293/hCTLA4 cells.
  • This example studied the anti-MSLN HCAb antibody PR000759 (SEQ ID NO: 67) and mc-vc-PAB-MMAE using cysteine to couple to the Cys-220 site to prepare an antibody-conjugated drug with a uniform DAR value .
  • Example 5.1 Based on the preliminarily determined optimal coupling reaction conditions obtained in Example 5.1, further optimization is made for the antibody molecule.
  • the purified HCAb PR000759 recombinant protein was conjugated in parallel using 5 sets of conditions; the reducing agent was TCEP, and the drug conjugate selected was mc-vc-PAB-MMAE, and the drug pair used for coupling The amount of the conjugate is 5-18 mole multiples, and the reaction time is 1-2 hours.
  • the pH of the reduction or coupling reaction is 6.5-7.0, and the temperature is room temperature (25-30°C).
  • the coupled products of each group of experiments were analyzed by the HIC-HPLC analysis method described in Example 2.2 for the components of the coupled products, which are listed in Table 6-1.
  • the purified recombinant HCAb PR000759 and its coupled product PR000759-ADC were quantitatively analyzed using high performance liquid chromatography and mass spectrometry techniques to determine their coupling Composition of product components.
  • the heavy chain (+1D) coupled with one compound is the main component, and the average DAR value of the PR000759-ADC product is calculated to be 1.98.
  • LC-MS was used to analyze ADC coupling sites.
  • the first step is to preprocess the sample PR000759-ADC to be tested.
  • the second step is to analyze the sample to be tested.
  • the sample was digested with trypsin and then identified by LC-MS/MS, and the software Peaks Studio (Bioinformatics Solutions Inc.) was used to search the database.
  • the parameter setting was: trypsin digestion; fragment ion mass tolerance: 0.05Da; precursor ion Mass tolerance: 10ppm; Maximum missing cuts: 2; Variable modifications: Carbamidomethylation 57.02, Oxidation (M) 15.99, Demand (NQ) 0.98, Pyroglutamate formation (N-term E)-18.01, ADC drug 1315.78.
  • the search results are filtered by strict card values to obtain credible peptides (-10lgP ⁇ 20), with a sequence coverage of nearly 100%. From the peptide list, three peptides with mc-vc-PAB-MMAE coupling sites were screened out ( Figure 14), and the results showed that PR000759-ADC was at position 127 (Eu numbering 220) and position 136 ( The coupling modification was identified on the cysteine at position 168 (Eu number 229) and position 168 (Eu number 261) (shown in bold C in Figure 14).
  • This example studied the anti-BCMA HCAb antibody PR001046 (SEQ ID NO: 68) and mc-vc-PAB-MMAE using cysteine to couple to the Cys-220 site to prepare an antibody-conjugated drug with a uniform DAR value .
  • Example 5.1 Based on the preliminarily determined optimal coupling reaction conditions obtained in Example 5.1, further optimization is made for the antibody molecule.
  • Table 7-1 the purified HCAb PR001046 recombinant protein was conjugated with 6 sets of conditions in parallel; TCEP was selected as the reducing agent, and the drug conjugate selected was mc-vc-PAB-MMAE. The amount of conjugate is 5-8 mole multiples, and the reaction time is 1-3 hours.
  • the pH of the reduction or coupling reaction is 6.5-7.0, and the temperature is room temperature (25-30°C).
  • the coupled products of each set of experiments were analyzed by the HIC-HPLC analysis method described in Example 2.2 for the components of the coupled products, which are listed in Table 7-1.
  • the purified recombinant HCAb PR001046 and its coupled product PR001046-ADC were quantitatively analyzed using high performance liquid chromatography and mass spectrometry techniques to determine their coupling Composition of product components.
  • the heavy chain (+1D) coupled with 1 compound is the main component, and the average DAR value of the PR001046-ADC product is calculated to be 2.20.
  • Example 6.3 the method described in Example 6.3 was used to analyze the coupling site of PR001046-ADC.
  • Figure 18 lists three peptides with mc-vc-PAB-MMAE coupling sites.
  • PR001046-ADC is at position 127 (Eu numbering 220), position 136 (Eu numbering 229), and A coupling modification was identified on the cysteine at position 168 (Eu number 261) (shown in bold C in Figure 18).
  • This example is to study the activity of HCAb antibody PR001046 and its conjugate product PR001046-ADC in binding to cells with high BCMA expression.
  • Flow cytometry FACS was used to test the binding ability of antibody molecules with HEK293T cell line HEK293T-hBCMA (Beijing Kangyuan Bochuang, KC-0233) which highly expresses human BCMA.
  • the cells were digested and resuspended in DMEM complete medium, and the cell density was adjusted to 1 ⁇ 10 6 cells/mL.
  • 100 ⁇ L cells/well were seeded on 96-well V bottom plate (Corning, #3894), and then 100 ⁇ L/well, 3 times concentration gradient dilution of 2 times the final concentration of the test antibody was added. Place the cells at 4°C and incubate for 1 hour in the dark.
  • the software GraphPad Prism 8 is used for data processing and mapping analysis, and through four-parameter nonlinear fitting, parameters such as the binding curve of the antibody to the target cell and EC50 value are obtained.
  • the antibody PR001046 and its purified coupling product PR001046-ADC have strong specific binding activity with HEK293T-hBCMA cells.
  • This example is to study the specific cell killing activity of the anti-BCMA antibody conjugate product PR001046-ADC on the cell line NCI-H929 (ATCC, CRL-9068) that highly expresses human BCMA, and to SNU-16 that does not express human BCMA. (ATCC, CRL-5974) cells have no killing activity.
  • NCI-H929 cells and SNU-16 cells were seeded in 96-well plates (Perkin Elmer, #6005225), the number of seeded cells were: 10000 cells/50 ⁇ L/well (NCI-H929), 5000 cells/50 ⁇ L/well (SNU -16), 10000 cells NCI-H929 and 5000 cells SNU-16/100 ⁇ L/well mix (NCI-H929+SNU-16).
  • the concentration of PR001046 and PR001046-ADC are 1 ⁇ g/ml, 0.1 ⁇ g/ml and 0.01 ⁇ g/ml, and the concentration of mc-vc-PAB-MMAE is 10nM, 1nM And 0.1nM.
  • incubate at 37°C and 5% CO2 for 72 hours add CellTiter-Glo cell viability detection reagent (Promega, #G7573), incubate at room temperature for at least 10 minutes to obtain a stable luminescence signal, and detect the luminescence value with a microplate reader.
  • PR001046-ADC at a concentration of 0.1 ⁇ g/ml and above can specifically kill BCMA-positive cells NCI-H929 and mixed cells of NCI-H929 and SNU-16, but cannot kill BCMA-negative cells SNU-16.
  • unconjugated PR001046 or mc-vc-PAB-MMAE compounds cannot effectively kill cells.
  • This example studied the anti-5T4 HCAb antibody PR004432 (SEQ ID NO: 69) and mc-vc-PAB-MMAE using cysteine to couple to the Cys-220 site to prepare antibody-conjugated drugs with uniform DAR value .
  • Example 5.1 Based on the preliminarily determined optimal coupling reaction conditions obtained in Example 5.1, further optimization is made for the antibody molecule. As shown in Table 8-1, two sets of conditions were used in parallel to couple the purified HCAb PR004432 recombinant protein; TCEP was selected as the reducing agent, the amount of TCEP was 4 molar multiples, and the drug conjugate selected was mc-vc-PAB-MMAE The amount of drug conjugate used for coupling is 12 mole multiples, and the reaction time is 1-2 hours. In this experiment, the pH of the reduction or coupling reaction is 6.5-7.0, and the temperature is room temperature (25-30°C).
  • the fractional yield ('D2%') is about 60%.
  • the purified recombinant HCAb PR004432 and its coupled product PR004432-ADC were quantitatively analyzed using high performance liquid chromatography and mass spectrometry techniques to determine their coupling Composition of product components.
  • the heavy chain (+1D) coupled with 1 compound is the main component (accounting for 96.5%), and the average product of PR004432-ADC is calculated The DAR value is 1.93.
  • Example 6.3 the method described in Example 6.3 was used to analyze the coupling site of PR004432-ADC.
  • the PR004432-ADC sample was digested with trypsin and identified by LC-MS/MS, and the data obtained was searched with the software BioPharmView (SCIEX).
  • the search parameters were set to: trypsin digestion; fragment ion mass tolerance: 0.05Da ; Parent ion mass tolerance: 10ppm; maximum missed cut number: 1; variable modification: Carbamidomethylation 57.02, Oxidation (M) 15.99, Deamidation (NQ) 0.98, mc-vc-PAB-MMAE 1315.78.
  • Find the relevant target peptide with compound coupling modification molecular weight difference 1315.78
  • the occupancy rate of the coupling site is calculated using the following formula:
  • Figure 24 shows the analysis of the coupling sites of PR004432-ADC using LC-MS peptide spectra, including the 3 selected peptides with coupling sites and the corresponding coupling site coverage. Analyze all peptides that contain Cys sites and undergo coupling modifications, and the secondary spectra corresponding to each peptide have also been checked one by one to confirm that the coupling modifications are highly reliable. For example, peptides containing SC(+1315.78)DK accounted for 97.12%, which means that 97.12% of Cys-220 sites are coupled with compounds. Similarly, only 1.5% of Cys-226 or Cys-229 sites are coupled.
  • This example is to study the activity of HCAb antibody PR004432 and its conjugate product PR004432-ADC in binding to cells that express BCMA.
  • Flow cytometry FACS was used to test the binding ability of the antibody molecule to the human breast cancer cell line HCC1954 (ATCC, CRL-2338) that highly expresses human 5T4. Specifically, adjust the cell density to 1x10 6 cells/mL, inoculate 100 ⁇ L cells/well on 96-well V bottom plate (Corning, #3894), and then add 100 ⁇ L/well, 3 times the final concentration of the final concentration of the dilution Measure antibodies. Place the cells at 4°C and incubate for 1 hour in the dark. After that, 100 ⁇ L/well of pre-cooled PBS was added to rinse the cells twice, centrifuged at 500 g for 5 minutes at 4° C., and the supernatant was discarded.
  • the software GraphPad Prism 8 is used for data processing and mapping analysis, and through four-parameter nonlinear fitting, parameters such as the binding curve of the antibody to the target cell and EC50 value are obtained.
  • the antibody PR004432 and its purified coupling product PR004432-ADC both have strong specific binding activity with HCC1954 cells.
  • This example is to study the cell killing activity of the anti-5T4 antibody conjugate product PR004432-ADC on the human breast cancer cell line HCC1954 (ATCC, CRL-2338) that highly expresses human 5T4.
  • HCC1954 cells were seeded in a 96-well plate (Perkin Elmer, #6005225) at 4000 cells/50 ⁇ L/well, and incubated overnight at 37°C and 5% CO2. Then, add 50 ⁇ L/well of the pre-diluted sample to be tested.
  • PR004432, PR004432-ADC and PR004433-ADC are diluted from the highest final concentration of 30nM to a 3-fold concentration gradient, and mc-vc-PAB-MMAE is from the highest final concentration to 60nM Dilute with a 3-fold concentration gradient; another well without the sample to be tested is set as a control well.
  • the software GraphPad Prism 8 is used for data processing and mapping analysis, and through four-parameter nonlinear fitting, parameters such as the binding curve of the antibody to the target cell and EC50 value are obtained.
  • Figure 27 shows the anti-5T4 HCAb PR004432 and its coupling product PR004432-ADC, the anti-BCMA HCAb PR004433 coupling product PR004433-ADC, and the uncoupled cytotoxic compound mc-vc-PAB-MMAE waiting for the test sample Cytotoxic killing of HCC1954 cells expressing human 5T4.
  • PR004432-ADC Only PR004432-ADC can produce effective and specific cytotoxic killing of HCC1954, and it is antibody concentration-dependent; mc-vc-PAB-MMAE only has the killing effect at the highest concentration (60nM), and other molecules cannot kill cells. Produce effective killing. This result confirms the high specificity and high potency of PR004432-ADC's target dependence.
  • This example studied the anti-BCMA HCAb antibody PR004433 (SEQ ID NO: 70) and mc-vc-PAB-MMAE conjugated to Cys-220 site using cysteine to prepare antibody-conjugated drugs with uniform DAR value .
  • Example 5.1 Based on the preliminarily determined optimal coupling reaction conditions obtained in Example 5.1, further optimization is made for the antibody molecule.
  • Table 9-1 the purified HCAb PR004433 recombinant protein was conjugated with 6 sets of conditions in parallel; TCEP was selected as the reducing agent, and the amount of TCEP was 1.7-2.1 molar multiples, and the drug conjugate selected was mc-vc-PAB -MMAE, the amount of drug conjugate used for coupling is 7 mole multiples, and the reaction time is 2 hours.
  • the pH of the reduction or coupling reaction is 6.5-7.0, and the temperature is room temperature (25-30°C).
  • the purified recombinant HCAb PR004433 and its coupled product PR004433-ADC were quantitatively analyzed using high performance liquid chromatography and mass spectrometry techniques to determine their coupling Composition of product components.
  • Figure 30 and the table below respectively show the HIC-HPLC analysis results of HCAb PR004433 and its coupling product PR004433-ADC: (A) PR004433 before coupling (Table 9-4); (B) After coupling, before purification (Table 9-5); (C) After coupling and one-step HIC purification (Table 9-6).
  • Example 2.4 The method described in Example 2.4 (LC instrument: Agilent 1290 UPLC; MS instrument: AB Sceix X500B) was used to analyze the molecular weight of the purified sample of PR004433-ADC. Before the LC-MS analysis, the sample to be tested is subjected to sugar removal treatment.
  • Figure 31 shows the mass spectrometry deconvolution processing profile of molecular weight analysis: (A) non-reduced sample of PR004433-ADC; (B) reduced sample of PR004433-ADC.
  • the molecular weight analysis method described in Example 5.2 the molecular weight of the components coupled with different numbers of compounds and their content in the sample were calculated (the following table).
  • the heavy chain (+1D) coupled with 1 compound is the main component (accounting for 90.5%), and the average product of PR004433-ADC is calculated The DAR value is 2.02.
  • Example 8.3 the method described in Example 8.3 was used to analyze the coupling site of PR004433-ADC.
  • Figure 34 shows the analysis of the coupling sites of PR004433-ADC using LC-MS peptide spectra, including the 3 peptides with coupling sites selected and the corresponding coupling site coverage. Analyze all peptides that contain Cys sites and undergo coupling modifications, and the secondary spectra corresponding to each peptide have also been checked one by one to confirm that the coupling modifications are highly reliable. For example, peptides containing SC(+1315.78)DK accounted for 98%, which means that 98% of Cys-220 sites are coupled with compounds. Similarly, only 4% of Cys-226 or Cys-229 sites are coupled.
  • Example 7.4 the method described in Example 7.4 was used to study the HCAb antibody PR004433 or its conjugate product PR004433-ADC and the HEK293T cell line HEK293T-hBCMA (Beijing Kangyuan Bochuang, KC-0233), high-expressing human BCMA.
  • BCMA cell line NCI-H929 ATCC, CRL-9068
  • cell line that does not express human BCMA SNU-16 ATCC, CRL-5974
  • both PR004433 and PR004433-ADC can specifically bind to HEK293T-hBCMA cells (Figure 32(A)) and NCI-H929 cells ( Figure 32(B)); moreover, PR004433 cannot bind BCMA negative cells SNU-16 ( Figure 32(C)).
  • This example is to study the effect of the anti-BCMA antibody conjugate product PR004433-ADC on the HEK293T cell line HEK293T-hBCMA (Beijing Kangyuan Bochuang, KC-0233) with high expression of human BCMA and the tumor cell line NCI-H929 with high expression of human BCMA (ATCC, CRL-9068) specific cell killing activity, and no killing activity on HEK293T (ATCC, CRL-11268) and SNU-16 (ATCC, CRL-5974) cells that do not express human BCMA.
  • HEK293T-hBCMA, NCI-H929, HEK293T and SNU-16 cells were seeded in 96-well plates (Perkin Elmer, #6005225), the number of seeded cells were: 2500 cells/50 ⁇ L/well (HEK293T-hBCMA), 5000 cells/well 50 ⁇ L/well (NCI-H929), 2500 cells/50 ⁇ L/well (HEK293T), 5000 cells/50 ⁇ L/well (SNU-16). Then incubate overnight at 37°C and 5% CO2. Then, add 50 ⁇ L/well of the pre-diluted sample to be tested.
  • PR004433, PR004433-ADC and PR004432-ADC are diluted from the highest final concentration of 10nM to 3 times the concentration, and mc-vc-PAB-MMAE is from the highest final concentration to 20nM Dilute with a 3-fold concentration gradient; another well without the sample to be tested is set as a control well. Then, incubate at 37°C and 5% CO2 for 72 hours, add CellTiter-Glo cell viability detection reagent (Promega, #G7573), incubate at room temperature for at least 10 minutes to obtain a stable luminescence signal, and detect the luminescence value with a microplate reader. Calculate the cell viability according to the following formula:
  • the software GraphPad Prism 8 is used for data processing and mapping analysis, and through four-parameter nonlinear fitting, parameters such as the binding curve of the antibody to the target cell and EC50 value are obtained.
  • Figure 33 shows the anti-BCMA HCAb PR004433 and its coupling product PR004433-ADC, the anti-5T4 HCAb PR004432 coupling product PR004432-ADC, and the uncoupled cytotoxic compound mc-vc-PAB-MMAE waiting for the test sample to BCMA Cytotoxic killing of positive cells (HEK293T-hBCMA and NCI-H929) and BCMA negative cells (HEK293T and SNU-16).
  • PR004433-ADC Only PR004433-ADC can produce effective and specific cytotoxicity to BCMA positive cells HEK293T-hBCMA ( Figure 33(A)) and NCI-H929 ( Figure 33(C)), which is antibody concentration-dependent, and is negative for BCMA Cells are not cytotoxic; other molecules cannot effectively kill cells. This result confirms the target-dependent high specificity and high efficiency of PR004433-ADC.
  • the anti-BCMA HCAb antibody PR004433 (SEQ ID NO: 70) and mc-vc-PAB-MMAE have been studied in Example 9 by coupling cysteine to the Cys-220 site to prepare an antibody with a uniform DAR value.
  • this example designed a series of experiments, fixing some of the experimental condition parameters, and changing other experimental condition parameters to try to obtain better reaction conditions. Specifically, this example uses 17 sets of different combinations of reduction and coupling conditions to carry out the coupling reaction of HCAb PR004433 and analyze the coupling products. Compared with Example 9, this example further examines the reaction buffer. The influence of parameters such as pH and reduction reaction temperature on the coupling.
  • the concentration of PR004433 is 5mg/ml; the reaction buffer is 20mM His-HCl, the pH of which can be adjusted; the reducing agent is TCEP, whose molar equivalent is variable; the reduction reaction temperature and reaction time are variable; coupled drugs
  • the molecule is mc-vc-PAB-MMAE (manufacturer: Lianning Biopharmaceutical, product number: SET0201), and its molar equivalent is variable; the coupling reaction temperature is room temperature, and the reaction time is 30 minutes.
  • Table 10-1 lists the 17 groups of experiments and their reaction conditions and parameters.
  • the coupling product produced in experiment #12 was further analyzed by the method described in Example 9.3 to analyze the coupling site of the antibody coupled to the drug molecule. The result showed that 96.22% of the Cys-220 site was coupled; similarly, only 6% of Cys-226 or Cys-229 sites are coupled. Therefore, the coupling product produced by Experiment #12 is a highly homogenous antibody-conjugated drug molecule conjugated to Cys-220.
  • This example studied the gap between the first Cys (Cys1) and the second Cys (Cys2) in the connecting peptide variant sequence generated based on the natural sequence (SEQ ID NO: 73) of the human IgG1 antibody heavy chain hinge region.
  • randomized primers of different lengths were designed for the hinge region to produce a series of antigen binding proteins with different hinge region connecting peptides through protein engineering.
  • 3 4, 5, 6, 7, 8, 9 or 10 arbitrary amino acids can be inserted between Cys1 and Cys2 to generate SEQ ID NOs: 74- 81 any of the connecting peptide sequences shown.
  • each length of connecting peptide randomly selected three different mutation sequences to further study the different amino acid composition between Cys1 and Cys2, and the amino acid between Cys1 and Cys2 could not be cysteine.
  • the antigen binding protein (PR004433 variant sequence) with linker peptides of different length and amino acid composition was expressed and purified using the aforementioned method, and coupled with the drug conjugate mc-vc-PAB-MMAE, Analyze the components of the coupling product and the coupling site.
  • Table 11-2 lists a series of connecting peptide sequences of different lengths generated by randomization. Replace the hinge region sequence of HCAb PR004433 with the connecting peptide sequence in Table 11-2 to generate a variety of PR004433-derived antigen binding protein variant sequences (Table 11-3), the variable regions and Fc constant regions of these antigen binding proteins
  • the connecting peptides have different lengths and amino acid compositions.
  • the PR004433-derived antigen binding protein in Table 11-3 was expressed and purified according to the method described in Example 1.6, and the purity was analyzed by the SEC-HPLC method described in Example 2.1 and the DSF method described in Example 2.7 was used to determine its purity. The lowest melting temperature (Tm1).
  • Tm1 The lowest melting temperature
  • Table 11-4 The results of antigen-binding protein expression and purification and thermal stability analysis are listed in Table 11-4; it can be seen that when the hinge region sequence of HCAb PR004433 is replaced with the variant sequence of various connecting peptides, and when the interval between Cys1 and Cys2 is With at least 3 other amino acids, PR004433-derived variant sequences can be effectively expressed, and the expression yield and thermal stability Tm1 of most variant molecules are not affected.
  • This example further uses the LC-MS molecular weight analysis method described in Example 3.2 to determine the status of Cys1 in different connecting peptide variant sequences.
  • the state of Cys1 (or Cys-220) has two possibilities as shown in Figure 3: formation of interchain disulfide bonds or not. If Cys1 does not form an inter-heavy chain disulfide bond, its side chain sulfhydryl group (-SH) will form a disulfide bond with sulfhydryl-containing molecules in the environment (such as cysteine or glutathione), making its actual molecular weight obvious Greater than theoretical molecular weight.
  • -SH side chain sulfhydryl group
  • the purified antigen-binding protein was treated with glycosidase PNGase F for sugar removal, and the method described in Example 2.4 (LC instrument: Agilent 1290 UPLC; MS instrument: AB Sceix X500B) was used to determine its complete (non-reduced) molecular weight. The theoretical molecular weights are compared; Table 11-5 shows that the theoretical and measured molecular weights are very consistent. Therefore, the Cys1 of the antigen-binding protein is not connected to other sulfhydryl-containing molecules.
  • Cys1 can form inter-heavy chain disulfide bonds (that is, disulfide bonds are formed between Cys1 of the first connecting peptide and Cys1 of the second connecting peptide. key).
  • This example further uses the method described in Example 9.4 to study the binding ability of the HCAb antibody PR004433 and its variant molecules with NCI-H929 (ATCC, CRL-9068) cells that highly express human BCMA.
  • NCI-H929 ATCC, CRL-9068
  • the ability of the PR004433 variant molecule to bind to NCI-H929 is almost the same as that of the parent molecule PR004433, indicating that changes in the hinge region (connecting peptide) sequence of PR004433 will not affect its antigen-binding fragment and target. The combination.
  • This example further uses the coupling method described in Example 10 to perform coupling reactions on the PR004433-derived variant molecules listed in Table 11-4, based on the optimal reaction conditions and parameters obtained in Example 10.1 Above, by changing the amount of reducing agent TCEP, to produce different coupling reaction products.
  • the concentration of PR004433-derived variant molecules (PR006031, PR006034, PR006037, PR006040, PR006312, PR006314, PR006317, and PR006320) with different connecting peptides is 5 mg/ml
  • the reaction buffer contains 20 mM His-HCl pH 6.0 buffer
  • the amount of reducing agent TCEP varies from 1.5 to 6.0 molar equivalents
  • the reduction reaction is at room temperature for 2 hours
  • the amount of the conjugate mc-vc-PAB-MMAE is 7.0 mole multiples
  • the coupling reaction is at room temperature for 0.5 hours ; Its coupling product does not go through the HIC purification step.
  • the coupled product also uses the molecular weight analysis method used in Example 10.1 to calculate the molecular weight of the components coupled with different numbers of compounds and their content in the sample.
  • the analysis results are listed in Table 11-6; for example, The Cys1 and Cys2 of the connecting peptide in PR006031 are separated by 3 amino acids.
  • the average DAR value of the coupling product is 2.12; for another example, the Cys1 and Cys2 of the connecting peptide in PR006320 are separated by 10 amino acids.
  • 3.0 equivalents of TCEP is used as a reducing agent and the above reaction parameters are used for coupling, the coupling product
  • the average DAR value of the coupling product is 1.99.
  • Cys1 and Cys2 of the connecting peptide are separated by at least 3 other amino acids
  • Cys1 can form an inter-heavy chain disulfide bond (that is, between Cys1 of the first connecting peptide and Cys1 of the second connecting peptide) Disulfide bond, this disulfide bond is called “Cys1-Cys1”
  • Cys2 can also form an inter-heavy chain disulfide bond (that is, a disulfide bond is formed between Cys2 of the first connecting peptide and Cys2 of the second connecting peptide, This disulfide bond is called "Cys2-Cys2").
  • the stability of the disulfide bonds Cys1-Cys1 may be weaker than other disulfide bonds.
  • the disulfide bond Cys1-Cys1 can be effectively opened while other disulfide bonds (for example, Cys2-Cys2) remain intact, making Cys1 almost the only free cysteine residue, which then serves as a specific Sexual coupling site.
  • the protein-drug conjugate produced by using the antigen binding protein containing the linking peptide has a coupling site at Cys1 and a CAR (or DAR) about 2.
  • mouse IgG2c antibody heavy chain hinge region SEQ ID NO: 147) and mc-vc-PAB-MMAE through cysteine coupling to prepare antibody conjugate drugs.
  • the hinge region of mouse IgG2c has three cysteines, and there are 5 other amino acid residues between the first cysteine (Cys1) and the second cysteine (Cys2).
  • HCAb PR004433 On the basis of HCAb PR004433 (SEQ ID NO: 70), part of its human IgG1 hinge region (SEQ ID NO: 73) is replaced with mouse IgG2c antibody hinge region sequence (SEQ ID NO: 147); that is, to replace PR004433's hinge region (SEQ ID NO: 73).
  • VH SEQ ID NO: 62
  • mouse IgG2c antibody hinge region SEQ ID NO: 147)
  • human IgG1 heavy chain constant region CH2 and CH3 sequences were fused and expressed to obtain a new HCAb molecule PR006468 (SEQ ID NO: 148) .
  • PR006468 was expressed and purified according to the method described in Example 1.6, and the purity was analyzed by the SEC-HPLC method described in Example 2.1. Then, the purified PR006468 recombinant protein was further subjected to a coupling reaction using the coupling method described in Example 10.
  • the concentration of PR006468 is 1.75mg/ml
  • the reaction buffer is 20mM His-HCl pH 6.0 buffer
  • the amount of reducing agent TCEP is 7.0 times the molar equivalent
  • the reduction reaction is at 37°C for 2 hours
  • the conjugate The dosage of mc-vc-PAB-MMAE is 7.0 molar equivalents
  • the coupling reaction is carried out at room temperature for 0.5 hours.
  • the product was passed through a desalting column to remove excess drug conjugates, and the coupling product PR006468-ADC was purified using the method described in Example 4.2.
  • Example 2.4 The method described in Example 2.4 (LC instrument: Agilent 1290 UPLC; MS instrument: AB Sceix X500B) was used to analyze the molecular weight of the purified sample of PR006468-ADC. Before the LC-MS analysis, the sample to be tested is subjected to sugar removal treatment.
  • Figure 47 shows the mass spectrometry deconvolution processing map of the non-reduced molecular weight analysis of PR006468-ADC. According to the molecular weight analysis method described in Example 5.2, the molecular weight of the components coupled with different numbers of compounds and their content in the sample were calculated.
  • Example 8.3 the method described in Example 8.3 was used to analyze the coupling site of PR006468-ADC.
  • Figure 48 shows the use of LC-MS peptide spectrogram to analyze the coupling sites of PR006468-ADC, including the 3 peptides with coupling sites selected and the corresponding coupling site coverage. Analyze all peptides that contain Cys sites and undergo coupling modifications, and the secondary spectra corresponding to each peptide have also been checked one by one to confirm that the coupling modifications are highly reliable. For example, peptides containing C(+1315.78)PPLK accounted for 90.8% ( Figure 71 shows the secondary spectrum of the peptide), which shows that 90.8% of the mouse hinge region Cys1 site is coupled with the compound. Similarly, only 5.8% of Cys2 sites are coupled.
  • mouse IgG2c hinge region sequence can also be used for the site-specific cysteine coupling described in the present invention.
  • This example uses the method described in Example 7.4 to study the antigen-binding proteins PR006468 (including mouse IgG2c hinge region), PR004433 (including human IgG1 hinge region) and their coupling products PR006468-ADC and PR004433-ADC and highly expressed human BCMA The binding ability of the tumor cell NCI-H929 (ATCC, CRL-9068).
  • PR006468 and PR004433 have similar binding capabilities.
  • PR006468-ADC and PR004433-ADC also have similar binding capabilities. This shows that replacing the human IgG1 hinge region sequence in the antigen binding protein with the mouse IgG2c hinge region sequence will not affect the target binding ability of the antigen binding protein and its coupling products.
  • This example uses the method described in Example 9.5 to study the effects of antigen binding proteins PR006468 (including mouse IgG2c hinge region), PR004433 (including human IgG1 hinge region) and their coupling products PR006468-ADC and PR004433-ADC on high expression of human BCMA Specific cell killing activity of the tumor cell NCI-H929 (ATCC, CRL-9068).
  • both PR006468-ADC and PR004433-ADC have effective and specific cytotoxic killing to NCI-H929 and are antibody concentration-dependent, while other molecules cannot effectively kill cells; and PR006468-ADC
  • the target cell killing effect of PR004433-ADC is almost the same. This also shows that the coupling product with mouse IgG2c hinge region sequence can also retain the original function.
  • This example studied the anti-ROR1 antigen binding protein PR002129 (SEQ ID NO: 71) and mc-vc-PAB-MMAE using cysteine to couple to the Cys-220 site to prepare an antibody coupling with a uniform DAR value drug.
  • the sequence of PR002129 is derived from Example 1.5, which contains the sequence of the hinge region of human IgG1.
  • Example 5.1 Based on the preliminarily determined coupling reaction conditions obtained in Example 5.1, combined with the better reaction conditions parameters obtained in Example 10.1, the antigen binding protein was coupled. As shown in Table 13-1, the purified scFv-Fc PR002129 recombinant protein was coupled in parallel with multiple sets of conditions.
  • the reaction conditions used in experiment #20210202-2 are: the reaction buffer contains 20mM His-HCl pH 6.0 buffer, the amount of reducing agent TCEP is 2.3 times the molar equivalent, the reduction reaction is at room temperature for 2 hours, and the conjugate is mc- The amount of vc-PAB-MMAE is 7.0 molar equivalents, and the coupling reaction is carried out at room temperature for 0.5 hours.
  • the purified recombinant PR002129 and its coupled product PR002129-ADC were quantitatively analyzed using high performance liquid chromatography and mass spectrometry techniques to determine the coupled product The composition of the components.
  • Example 2.4 The method described in Example 2.4 (LC instrument: Agilent 1290 UPLC; MS instrument: AB Sceix X500B) was used to analyze the molecular weight of the samples before and after purification of PR002129-ADC. Before the LC-MS analysis, the sample to be tested is subjected to sugar removal treatment.
  • Figure 52 shows the mass spectrometry deconvolution processing pattern of molecular weight analysis: (A) the non-reduced molecular weight of the sample before HIC purification; (B) the reduced molecular weight of the sample after HIC purification; (C) the non-reduced molecular weight of the sample after HIC purification.
  • the molecular weight of the components coupled with different numbers of compounds and their content in the sample were calculated (the following table).
  • the heavy chain (+1D) of each compound is the main component (88%), and the average DAR value of the PR002129-ADC product is calculated to be 1.9.
  • Example 8.3 the method described in Example 8.3 was used to analyze the coupling site of PR002129-ADC.
  • Figure 53 shows the analysis of PR002129-ADC compound coupling sites using LC-MS peptide spectroscopy, including the selected 3 peptides with coupling sites and the corresponding coupling site coverage. Analyze all peptides that contain Cys sites and undergo coupling modifications, and the secondary spectra corresponding to each peptide have also been checked one by one to confirm that the coupling modifications are highly reliable. For example, peptides containing SC(+1315.78)DK accounted for 88%, which means that 88% of Cys-220 sites are coupled with compounds.
  • the purpose of this example is to study the activity of the antigen binding protein PR002129 and its coupling product PR002129-ADC in binding to cells that highly express ROR1.
  • Flow cytometry FACS was used to test the binding ability of the antigen-binding protein to the tumor cell PanC-1 (ATCC, CRL-1469) that highly expresses human ROR1. Specifically, the cells were digested and resuspended in DMEM complete medium to adjust the cell density to 1 ⁇ 10 6 cells/mL. 100 ⁇ L cells/well were seeded on 96-well V bottom plate (Corning, #3894), and then 100 ⁇ L/well, 2 times the final concentration of 3 times the concentration gradient dilution of the test binding protein was added. Place the cells at 4°C and incubate for 1 hour in the dark.
  • the software GraphPad Prism 8 is used for data processing and mapping analysis, and through four-parameter nonlinear fitting, parameters such as the binding curve of the antibody to the target cell and EC50 value are obtained.
  • PR002129 and its coupling product PR002129-ADC both have strong specific binding activity with PanC-1 cells, and their binding ability is comparable.
  • the drug coupling method applied by the present invention is applied to non-antibody fusion proteins, and the first Cys of the connecting peptide (for example, Cys-220 in the hinge region of human IgG1) is used to position the fusion protein.
  • the first Cys of the connecting peptide for example, Cys-220 in the hinge region of human IgG1
  • Specific coupling This is a brand new attempt.
  • the drug coupling is not limited to antibodies, but is extended to any fusion protein, only the structure containing the connecting peptide is required; this shows that the drug coupling method of the present invention is versatile .
  • ALX148 is a high-affinity variant fusion protein of human SIRPa (Kauder SE et al., PLoS ONE (2016) 13(8): e0201832), and its sequence is disclosed in patent US10829771B2. Compared with the human SIRPa wild-type sequence, the affinity of ALX148 with CD47 is increased by about 3000 times.
  • the partial sequence of the SIRPa variant in ALX148 was fused and expressed with the sequence containing the hinge region of human IgG1 and the Fc (SEQ ID NO: 72) to obtain the SIRPa-Fc fusion protein PR006345 (SEQ ID NO: 149). Then PR006345 was coupled and analyzed.
  • the coupling method described in Example 10 was used to perform a coupling reaction on the SIRPa-Fc fusion protein PR006345. Based on the optimal reaction conditions and parameters obtained in Example 10.1, by changing the dosage of the reducing agent TCEP, To produce different coupling reaction products.
  • the concentration of PR006345 is 4.1mg/ml
  • the reaction buffer is 20mM His-HCl pH 6.0
  • the amount of reducing agent TCEP varies from 1.5 to 3.0 times the molar equivalent
  • the reduction reaction is at room temperature for 2 hours.
  • the amount of the conjugate mc-vc-PAB-MMAE is 7.0 mole multiples, and the coupling reaction is at room temperature for 0.5 hours; the coupling product does not undergo HIC purification steps. Furthermore, the coupled product also uses the molecular weight analysis method used in Example 10.1 to calculate the molecular weight of the components coupled with different numbers of compounds and their content in the sample. The analysis results are listed in Table 14-1.
  • Example 8.3 the method described in Example 8.3 was used to analyze the coupling site of PR006345-ADC.
  • Figure 57 shows the use of LC-MS peptide spectrogram to analyze the coupling sites of PR006345-ADC compounds, including the selected 3 peptides with coupling sites and the corresponding coupling site coverage. Analyze all peptides that contain Cys sites and undergo coupling modifications, and the secondary spectra corresponding to each peptide have also been checked one by one to confirm that the coupling modifications are highly reliable. For example, peptides containing SC(+1315.78)DK accounted for 92.45%, which means that 92.45% of Cys-220 sites are coupled with compounds.
  • This example is to study the activity of SIRPa-Fc fusion protein PR006345 and its coupling product PR006345-ADC in binding to cells that express CD47.
  • Flow cytometry FACS was used to test the binding ability of the antigen binding protein to CHOK1-hCD47 cells (GenScript, M00581) that highly express human CD47. Specifically, the cells were digested and resuspended in DMEM complete medium to adjust the cell density to 1 ⁇ 10 6 cells/mL. 100 ⁇ L cells/well were seeded on 96-well V bottom plate (Corning, #3894), and then 100 ⁇ L/well, 2 times the final concentration of 3 times the concentration gradient dilution of the test binding protein was added. Place the cells at 4°C and incubate for 1 hour in the dark.
  • the software GraphPad Prism 8 is used for data processing and mapping analysis, and through four-parameter nonlinear fitting, parameters such as the binding curve of the antibody to the target cell and EC50 value are obtained.
  • PR006345 and its coupling product PR006345-ADC have strong specific binding activity with CD47, and the binding ability is equivalent.
  • the antigen-binding fragment VH and the antigen-binding fragment Fab are used to construct a tetravalent bispecific Fab-HCAb structure as shown in FIG. 59, and the drug coupling method of the present application is applied to the structure, using the The first Cys of the connecting peptide (for example, Cys-220 in the hinge region of human IgG1) is site-specifically coupled to it.
  • the first Cys of the connecting peptide for example, Cys-220 in the hinge region of human IgG1
  • This structure belongs to the bispecific symmetric structure shown in Figure 42(B), in which one antigen-binding fragment is Fab and the other antigen-binding fragment is VH.
  • this structure has two different polypeptide chains (called short chain and long chain)
  • the short chain does not form a disulfide bond with the first Cys (Cys-220) on the connecting peptide of the long chain; therefore, in some Under the combination of reaction conditions, the disulfide bond of the Cys can be effectively opened while the other disulfide bonds remain intact, so that the Cys can be used as a site-directed coupling site.
  • This example uses the VH sequence (SEQ ID NO: 62) of the anti-BCMA HCAb PR004433 and the VH (SEQ ID NO: 130) and VL sequences (SEQ ID NO: 131) of the anti-BCMA IgG antibody PR000892 as shown in Figure 59.
  • the bispecific (dual binding epitope) antigen binding protein PR005744 with a tetravalent Fab-HCAb structure is shown.
  • the sequence of PR000892 is disclosed in the invention patent CN111234020B.
  • the serial numbers of PR004433, PR000892 and PR005744 are listed in Table 15-1 and Table 15-2.
  • the antigen binding protein PR005744 was prepared using the method described in Example 1.6. Subsequently, it was tested for its ability to bind BCMA and its ability to internalize on the BCMA highly expressing cell NCI-H929 (ATCC, CRL-9068).
  • NCI-H929 (ATCC, CRL-9068) cells were seeded into a 96-well plate (Beyotime, #FT018) at 2 ⁇ 10 5 cells/well; then 200 nM test antigen binding protein diluted with FACS buffer was added ; Then incubate at 4°C for 1 hour; then, take samples and incubate at 37°C for different times (such as 30 minutes, 1 hour, 2 hours, and 4 hours); then, centrifuge and resuspend the cells, and add fluorescent secondary antibody (Jackson ImmunoResearch Inc., #109-545-098) and then incubated at 4°C for 30 minutes.
  • fluorescent secondary antibody Jackson ImmunoResearch Inc., #109-545-098
  • the bispecific antigen binding protein PR005744 showed a strong internalization effect, which can internalize more than 60% of BCMA within 30 minutes.
  • Example 9.1 or Example 10.1 was used to couple PR005744.
  • the reaction buffer is 20mM His-HCl pH 6.0; the reducing agent is TCEP, and its molar equivalent is variable; the reduction reaction is at room temperature for 2 hours, and the amount of the conjugate mc-vc-PAB-MMAE is 7.0 molar multiples , The coupling reaction was at room temperature for 0.5 hours.
  • the coupling products were analyzed using the method described in Example 2.4 (LC instrument: Agilent 1290 UPLC; MS instrument: AB Scix X500B) for the complete molecular weight analysis of the non-reducing sample de-sugar treatment, and using the The total ion current diagram or deconvolution diagram in the mass spectrum is used to calculate the molecular weight of the components coupled with different numbers of compounds and their content in the sample; the analysis results are listed in Table 15-3.
  • the reaction buffer is a pH 6.0 buffer containing 20 mM His-HCl
  • the amount of the reducing agent TCEP is 3.5 molar multiples
  • the reduction reaction is at room temperature for 2 hours
  • Example 8.3 the method described in Example 8.3 was used to analyze the coupling site of PR005744-ADC.
  • Figure 61 shows the analysis of the coupling sites of PR005744-ADC using LC-MS peptide spectroscopy, including the 3 selected peptides with coupling sites and the corresponding coupling site coverage. Analyze all peptides that contain Cys sites and undergo coupling modifications, and the secondary spectra corresponding to each peptide have also been checked one by one to confirm that the coupling modifications are highly reliable. For example, peptides containing SC(+1315.78)DK accounted for 99.07%, which shows that almost all Cys-220 sites are coupled with compounds. This confirmed that drug conjugation occurred specifically in the first Cys (ie Cys-220) of the connecting peptide in PR005744.
  • Example 7.4 the method described in Example 7.4 was used to study the binding ability of PR005744 and its coupling product PR005744-ADC with the tumor cell NCI-H929 (ATCC, CRL-9068) that highly expresses human BCMA.
  • both PR005744 and its conjugate product PR005744-ADC can specifically bind to NCI-H929 cells, and the binding ability is equivalent.
  • Example 9.5 the method described in Example 9.5 was used to study the specific cell killing activity of PR005744 and its coupling product PR005744-ADC on tumor cells NCI-H929 (ATCC, CRL-9068) that highly express human BCMA.
  • PR005744-ADC produced effective and specific cytotoxic killing of NCI-H929, and showed a concentration-dependent, while PR005744 could not produce effective killing of cells.
  • This example studied the anti-BCMA HCAb antibody PR004433 (SEQ ID NO: 70) and the protein degradation targeting chimera (PROTAC) using cysteine to couple to the Cys-220 site to prepare antibody-conjugated drugs.
  • PROTAC is a class of compounds that can induce the target protein to degrade by inducing its polyubiquitination.
  • the mechanism of action of PROTAC is very different from traditional cytotoxic small molecule compounds such as MMAE, and the molecular structure is also more complex.
  • MMAE cytotoxic small molecule compounds
  • the tissue selectivity is relatively limited, and it is difficult to distinguish different cell types. It has been reported in the literature that coupling PROTAC to an antibody that binds to HER2 can enhance the selectivity of PROTAC so that it can only degrade the target in HER2-positive tumor cells (Maneiro MA et al., ACS Chem Biol. 2020; 15( 6):1306-1312.).
  • the PROTAC used in this example is a BRD4 protein degrading agent (the structure is shown in Figure 64), which includes PROTAC GNE-987 targeting BET and a disulfide-containing linker.
  • the PROTAC is used as a drug conjugate, and the HCAb antibody PR004433 is specifically coupled to the HCAb antibody PR004433 by using the drug coupling method of the present invention. This is a brand-new attempt.
  • the drug conjugate is not limited to traditional cytotoxic compounds such as MMAE, but is extended to other types of loaded drugs; this shows that the drug coupling method of the present invention is versatile.
  • This example uses the coupling method described in Example 10 and further changed conditions to perform coupling reaction on HCAb PR004433.
  • the drug conjugate is a BRD4 protein degrading agent compound (see Figure 64 for the structure, with a molecular weight of 1306.58 Da; manufacturer) : MedChemExpress, article number: HY-129938), the compound is referred to as PROTAC in this example.
  • the concentration of PR004433 is 5.0mg/ml
  • the reaction buffer is 20mM His-HCl pH 6.0 buffer
  • the amount of reducing agent TCEP is 3.0 molar equivalents
  • the reduction reaction is at room temperature for 2 hours
  • use Replace the product with a 30kd ultrafiltration tube into a pH 8.0 buffer containing 50mM Tris-HCl
  • the amount of conjugate PROTAC is 10.0 to 15.0 molar equivalents
  • the coupling reaction is at room temperature for 2 hours
  • the methylsulfone (Methylsulfonyl, CH3SO2) group of the PROTAC molecule is broken, exposing the reactive sulfhydryl group, and then forming a disulfide bond with the reactive sulfhydryl group on the antibody. Therefore, coupling of each PROTAC to the antibody increased the total molecular weight by 1226 Da.
  • Example 2.4 LC instrument: Agilent 1290 UPLC; MS instrument: AB Sceix X500B was used to analyze the molecular weight of the coupling product PR004433-PROTAC.
  • Figure 65 shows the mass spectrometry deconvolution processing map of PR004433-PROTAC for non-reducing molecular weight analysis, and based on this, the molecular weight of the components coupled with different numbers of PROTAC and their content in the sample are calculated.
  • Example 7.4 the method described in Example 7.4 was used to study the binding ability of HCAb PR004433 and its coupling product PR004433-PROTAC with the tumor cell NCI-H929 (ATCC, CRL-9068) that highly expresses human BCMA.
  • both PR004433 and its coupling product PR004433-PROTAC can specifically bind to NCI-H929 cells.
  • This example uses the VH sequence of the anti-BCMA HCAb PR004433 (SEQ ID NO: 62) and the scFv sequence of the anti-ROR1 antibody PR002129 (with the VH shown in SEQ ID NO: 60 and the VL shown in SEQ ID NO: 63 ) Construct a bivalent bispecific antigen binding protein 2129/4433 with an asymmetric structure as shown in Figure 67.
  • the drug coupling method of the present invention is applied to the structure, and the first Cys of the connecting peptide (for example, Cys-220 in the hinge region of human IgG1) is used for site-specific coupling to it.
  • the first antigen-binding fragment and the second antigen-binding fragment have different structures and amino acid sequences.
  • the first antigen-binding fragment is a VH structure
  • the second antigen-binding fragment is a VH structure.
  • the antigen-binding fragment is a scFv structure, so it is an asymmetric structure containing two different polypeptide chains.
  • the first polypeptide chain contains a first connecting peptide (comprising a human IgG1 hinge region sequence)
  • the second polypeptide chain contains a second connecting peptide (comprising a human IgG1 hinge region sequence).
  • the first Cys (Cys-220) on the first connecting peptide and the first Cys (Cys-220) on the second connecting peptide form a disulfide bond; therefore, under some combination of reaction conditions, the disulfide of Cys-220 The bond can be effectively opened while the other disulfide bonds remain intact, allowing the Cys to serve as a site-directed coupling site.
  • the plasmid encoding PR004433 polypeptide chain (SEQ ID NO: 70) and the plasmid encoding PR002129 polypeptide chain (SEQ ID NO: 71) were co-transfected and expressed and one-step affinity purification was performed according to the method described in Example 1.6.
  • the heterodimeric protein 2129/4433 is the target protein of this experiment.
  • the purified heterodimeric protein 2129/4433 was coupled using the method described in Example 10.1.
  • the concentration of 2129/4433 was 3.2 mg/ml
  • the reaction buffer was a buffer containing 20 mM His-HCl pH 6.0
  • the amount of the reducing agent TCEP was 2.1 to 2.5 molar equivalents.
  • the reduction reaction was carried out at room temperature for 2 hours.
  • the amount of the conjugate mc-vc-PAB-MMAE is 7.0 molar equivalents
  • the coupling reaction is at room temperature for 0.5 hours.
  • the product is passed through a desalting column to remove excess drug conjugates to obtain the coupled product 2129/4433-ADC.
  • the coupling product is further purified by the HIC method described in Example 4.2.
  • Example 8.3 the method described in Example 8.3 was used to analyze the coupling site of 2129/4433-ADC.
  • TLR9 Toll-like receptor 9
  • CpG oligonucleotides are natural agonistic ligands of TLR9, and TLR9 can activate immune cells after binding to CpG.
  • the CpG oligonucleotide is coupled to the antigen binding protein in the foregoing embodiment through a maleimide group or sulfhydryl-containing linker using cysteine to obtain a coupling product.
  • the anti-5T4 HCAb PR004432 and the CpG oligonucleotide CpG-1826 are coupled to the Cys1 position of the connecting peptide using the method of the present invention Point (for example, Cys-220) to prepare the conjugated drug PR004432-CpG; the anti-ROR1 binding protein PR002129 and CpG are coupled to the Cys-220 site to prepare the conjugated drug PR002129-CpG; SIRPa-Fc fusion protein PR006345 and CpG Coupling to Cys-220 site to prepare conjugated drug PR006345-CpG.
  • Point for example, Cys-220
  • the anti-ROR1 binding protein PR002129 and CpG are coupled to the Cys-220 site to prepare the conjugated drug PR002129-CpG
  • SIRPa-Fc fusion protein PR006345 and CpG Coupling to Cys-220 site to prepare conjugated drug PR006345-CpG.
  • CpG-conjugated drugs specifically recognize antigen targets on tumors, use Fc receptors on immune cells in the tumor microenvironment to mediate internalization and activate TLR9 through CpG to further activate immune cells, and use multiple mechanisms to improve anti-tumor effects .

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Abstract

本申请涉及一种蛋白-药物偶联物，其包含抗原结合蛋白部分和药物偶联物部分；所述抗原结合蛋白部分包括一个或多个抗原结合片段，以及与所述抗原结合片段直接或间接连接的至少两个连接肽；所述两个连接肽可各自独立地含有第一半胱氨酸Cys1和第二半胱氨酸Cys2；所述药物偶联物部分共价结合于所述Cys1上。本申请还涉及一种蛋白-药物偶联物的制备方法，以及所述蛋白-药物偶联物在预防或治疗肿瘤或其他疾病中的用途。

Description

蛋白-药物偶联物和定点偶联方法
本申请要求申请日为2020年12月08日的中国专利申请202011423832.6的优先权。
技术领域
本发明一般涉及生物医药领域，具体的涉及一种蛋白-药物偶联物以及一种制备所述蛋白-药物偶联物的方法。
背景技术
抗体偶联药物(Antibody-Drug Conjugate，ADC)是由靶向肿瘤抗原的抗体通过连接子与高效细胞毒性的小分子化学药物偶联产生的，利用抗体与靶抗原特异性结合的特点，将小分子药物靶向递送至肿瘤细胞进而发挥杀伤肿瘤的作用。近年来，随着新的连接子、小分子毒素、抗体以及靶点发现技术的不断发展，ADC早期的问题不断被克服，陆续有ADC药物的上市，在血液瘤、实体瘤展现良好的临床效果。另一方面，近年来，ADC技术也被应用于非肿瘤领域；例如，AbbVie公司将类固醇偶联于抗TNFα的抗体上用于治疗多种TNFα介导的自身免疫性疾病。
偶联方法和连接子结构对于ADC药物的稳定性至关重要。半胱氨酸偶联技术是早期的化学偶联方法的代表；其利用IgG1抗体天然的4个链间二硫键作为偶联用的活性巯基，相较于赖氨酸酰胺偶联技术，该技术具有可控的DAR值(药物/抗体比)，但是仍产生DAR值为0到8的分布的混合物。由于此类技术产生的偶联产物成分不均一，其稳定性和药学性能有较大不足。近年来，随着细胞毒性药物与定点偶联方法的不断发展，第三代ADC技术通过药物与抗体经特定位点偶联获得稳定的DAR值，提高了抗体偶联药物的稳定性和药代动力学性能。第三代ADC代表的偶联技术主要包括ThioMab、ThioBridge、引入非天然氨基酸、转肽反应、N-糖链偶联等。ThioMab技术最早由Genentech公司开发，在半胱氨酸偶联技术基础上，其在抗体中的除天然二硫键以外的特定位点引入两个或多个半胱氨酸作为偶联位点，用以产生位点专一的DAR为2的高均质性的抗体偶联药物；但是由于其引入的突变不是天然存在的氨基酸序列，因而面临潜在的分子稳定性和免疫原性的风险。ThioBridge技术则是将单抗本身的二硫键还原，利用二溴马来酰 亚胺或二溴吡啶二苯醚等试剂与还原的链间半胱氨酸反应，使其重新桥接，得到主要组分是DAR为4的产物，但是该技术仍有发生二硫键错配的风险。非天然氨基酸偶联技术使用一种特殊的编码非天然氨基酸的tRNA合成酶，使得细胞可以合成带有对乙酰苯丙氨酸(p-acetylphenylalanine)残基的各种抗体，利用对乙酰苯丙氨酸残基的羰基与带有烷氧基胺基团的连接子发生偶联反应，得到主要组分是DAR为2的产物；由于该技术需要使用经过改造的宿主细胞来产生非天然氨基酸，因而其明显的不足是抗体生产的产率较低而成本较高。利用转肽反应的偶联技术是在抗体的重链或轻链的C’末端引入一段序列LPETG，转肽酶Sortase A特异识别该序列并水解苏氨酸和甘氨酸间的肽键，然后再把含有甘氨酸的偶联物连接到苏氨酸上；但是该技术产生的偶联产物中引入了额外的肽段序列，因而也面临潜在的免疫原性的风险。N-糖链偶联技术如GlycoConnect技术使用内切糖苷酶Endo S2修剪位于抗体CH2区的Asn297(Eu编号)的糖链，然后使用突变的半乳糖转移酶GalT(Y289L)和N-叠氮基乙酰半乳糖胺(GalNAz)引入叠氮基，利用点击化学(Click Chemistry)将该叠氮基与小分子药物偶联，产生稳定且均匀的DAR为2的抗体偶联药物；由于该技术需要使用特殊的突变的酶，其工艺开发成本较高。
目前，尽管已经开发了多种可以定点偶联产生具有可控的DAR值的偶联技术，但是这些技术或是引入氨基酸突变、或是引入额外的氨基酸序列、或是改变Fc上的糖链结构、或是引入非天然氨基酸，其工艺开发过程繁杂且成本较高。而且现有技术中没有通用的针对重链抗体、单链抗体或者双特异性抗体或者融合蛋白来制备蛋白-药物偶联物的偶联技术。因而仍亟需开发新的无需复杂的制备工艺的定点偶联技术，以提高偶联产物的稳定性，减少免疫原性。
发明内容
本申请提供了蛋白-药物偶联物的定点偶联方法及由该方法制备所得的蛋白药物偶联物。具有下列性质中的一种或多种：1)可以定点偶联产生均一DAR值(药物/抗体比)或均一CAR值(偶联物/抗体比)的偶联产物；2)相较于其他定点偶联技术如THIOMAB技术等，可利用天然铰链区氨基酸序列，不引入氨基酸突变或额外肽段序列，也不改变Fc上的糖链结构；3)可对天然铰链区的氨基酸序列进行长度或特定位点氨基酸种类的优化，提高产物的表达量和均一度；4)无需特别复杂的偶联技术或工艺；5)该定点偶联方法的副产物少，可开发性强；6)抗原结合片段可扩展到多种形式，全人VH结构域、骆驼科VHH结构域、单链抗体scFv、可溶性受体融合蛋白等；7)抗原结合片段可扩展成 多价多特异性形式；8)有良好的稳定性和半衰期；9)有更好的靶细胞内化作用；10)有更好的组织穿透性。
一方面，本申请提供了一种蛋白-药物偶联物，其包含抗原结合蛋白部分和药物偶联物部分；所述抗原结合蛋白部分包括一个或多个抗原结合片段，以及与所述抗原结合片段直接或间接连接的至少两个连接肽；所述至少两个连接肽包括第一连接肽和第二连接肽；其中，所述第一连接肽包含第一半胱氨酸Cys1和第二半胱氨酸Cys2，所述药物偶联物部分通过所述Cys1偶联至所述第一连接肽；且所述第二连接肽包含第一半胱氨酸Cys1和第二半胱氨酸Cys2，所述药物偶联物部分通过所述Cys1偶联至所述第二连接肽。
在某些实施方式中，所述第一连接肽的所述Cys2与所述第二连接肽的的所述Cys2之间通过二硫键连接。
在某些实施方式中，所述一个或多个抗原结合片段中不包含能够影响药物偶联物与所述第一连接肽和/或所述第二连接肽在Cys1处进行偶联的官能团或者前述官能团与其他药物偶联物形成的缀合物，所述影响表现为显著降低药物偶联物在Cys1位点的偶联几率。
在某些实施方式中，所述一个或多个抗原结合片段中不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的官能团或者前述官能团与其他药物偶联物形成的缀合物。
在某些实施方式中，所述一个或多个抗原结合片段中不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的半胱氨酸或者前述半胱氨酸与其他药物偶联物形成的缀合物。
在某些实施方式中，所述第一连接肽和所述第二连接肽的氨基酸序列可以相同或不同。
在某些实施方式中，所述第一连接肽和/或所述第二连接肽自N端至C端包含Cys1-(X)n-Cys2所示的氨基酸序列；其中，n为大于或等于3的整数，X是不为Cys的任意氨基酸。
在某些实施方式中，所述第一连接肽和/或所述第二连接肽源自抗体铰链区序列或其衍生序列。其中，所述衍生序列为通过对抗体铰链区序列进行改造获得，所述改造不涉及抗体铰链区中Cys1和Cys2的改变。例如，所述改造为调整抗体铰链区序列中Cys1和Cys2之间的氨基酸种类、数目和/或在Cys1前进行柔性连接氨基酸的添加。
在某些实施方式中，所述连接肽源自人IgG铰链区，例如，所述抗体铰链区为人IgG1 铰链区。
在某些实施方式中，所述连接肽为人IgG1的铰链区，所述第一连接肽和所述第二连接肽的序列各自独立地如SEQ ID NO:73所示，连接肽中所述Cys1为人IgG1铰链区根据EU编码的Cys-220，所述Cys2为人IgG1铰链区根据EU编码的Cys-226。
在某些实施方式中，所述第一连接肽和所述第二连接肽可以各自独立地源自人IgG1的铰链区的衍生序列，其氨基酸序列如SEQ ID NOs:74-105、145-146中任一项所示；所述第一连接肽中所述Cys1为对应于人IgG1铰链区Cys-220的Cys，所述第一连接肽中所述Cys2为对应于人IgG1铰链区Cys-226的Cys；所述第二连接肽中所述Cys1为对应于人IgG1铰链区Cys-220的Cys，所述第二连接肽中所述Cys2为对应于人IgG1铰链区Cys-226的Cys。
在某些实施方式中，所述连接肽为小鼠IgG2c的铰链区，所述第一连接肽和所述第二连接肽的序列各自独立地如SEQ ID NO:147所示，所述连接肽中的所述Cys1为小鼠IgG2c的铰链区序列的第一个半胱氨酸，所述Cys2为其序列的第二个半胱氨酸。
在某些实施方式中，蛋白-药物偶联物的抗原结合蛋白部分包括的多个抗原结合片段连接于同一所述连接肽或分别连接于不同的所述连接肽。
在某些实施方式中，所述多个抗原结合片段可以部分或全部相同或彼此均不同。
在某些实施方式中，所述多个抗原结合片段包括第一抗原结合片段和第二抗原结合片段。第一抗原结合片段和第二抗原结合片段可以相同或不同。例如，所述第一抗原结合片段和所述第二抗原结合片段结合相同的靶点且氨基酸序列相同；例如，所述第一抗原结合片段和所述第二抗原结合片段结合相同的靶点但氨基酸序列不相同；例如，所述第一抗原结合片段和所述第二抗原结合片段结合不同的靶点。
在某些实施方式中，所述多个抗原结合片段包含第一抗原结合片段、第二抗原结合片段、第三抗原结合片段和第四抗原结合片段。第一抗原结合片段、第二抗原结合片段、第三抗原结合片段和第四抗原结合片段可以部分或全部相同或彼此均不同。例如，所述第一抗原结合片段和所述第二抗原结合片段结合相同的靶点且氨基酸序列相同；例如，所述第一抗原结合片段和所述第二抗原结合片段结合相同的靶点但氨基酸序列不相同；例如，所述第一抗原结合片段和所述第二抗原结合片段结合不同的靶点。例如，所述第三抗原结合片段和所述第四抗原结合片段结合相同的靶点且氨基酸序列相同；例如，所述第三抗原结合片段和所述第四抗原结合片段结合相同的靶点但氨基酸序列不相同；例如，所述第三抗原结合片段和所述第四抗原结合片段结合不同的靶点。例如，所述第一 抗原结合片段和所述第二抗原结合片段和所述第三抗原结合片段和所述第四抗原结合片段结合不同的靶点。
在某些实施方式中，所述一个或多个抗原结合片段可以各自独立地为VHH结构域、VH、VL、Fab、ScFv、受体蛋白可溶性胞外区、配体蛋白、脂质运载蛋白lipocalins、神经细胞粘附分子NCAM、纤维结合蛋白fibronectin和/或锚蛋白重复片段蛋白DARPins等衍生蛋白结构。
在某些实施方式中，所述一个或多个抗原结合片段能够特异性结合肿瘤抗原或非肿瘤抗原。
在某些实施方式中，其中所述肿瘤抗原包括CD19、BCMA、TSHR、CD171、CS-1、CLL-1、GD3、Tn Ag、FLT3、CD38、CD123、CD44v6、B7H3、B7H4、KIT、IL-13Ra2、IL-11Ra、PSCA、PSMA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-beta、SSEA-4、MUC1、EGFR、NCAM、CAIX、LMP2、EphA2、岩藻糖基GM1(fucosyl GM1)、sLe、GM3、TGS5、HMWMAA、GD2、FOLR1、FOLR2、TEM1/CD248、TEM7R、CLDN6、CLDN18.2、GPRC5D、CXORF61、CD97、CD179a、ALK、多聚唾液酸(polysialic acid)、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TAARP、WT1、ETV6-AML、SPA17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、FOSL1、hTERT、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、AR(雄激素受体)、细胞周期蛋白B1(Cyclin B1)、MYCN、RhoC、CYP1B1、BORIS、SART3、PAX5、OY-TES1、LCK、AKAP-4、SSX2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、CD20、CD30、HER2、ROR1、FLT3、TAAG72、CD22、CD33、GD2、gp100Tn、FAP、酪氨酸酶(tyrosinase)、EPCAM、CEA、IGF-1R、EphB2、间皮素(mesothelin)、钙黏蛋白17(Cadherin17)、CD32b、EGFRvIII、GPNMB、GPR64、HER3、LRP6、LYPD8、NKG2D、SLC34A2、SLC39A6、SLITRK6、GUCY2C、5T4和/或TACSTD2等。
在某些实施方式中，所述第一抗原结合片段和/或所述第二抗原结合片段各自独立地抗原结合片段包含HCDR1、HCDR2和HCDR3，且所述抗原结合片段包含选自下述的任意一组氨基酸序列：(1)HCDR1：SEQ ID NO:7，HCDR2：SEQ ID NO:22，HCDR3：SEQ ID NO:38；(2)HCDR1：SEQ ID NO:10，HCDR2：SEQ ID NO:25，HCDR3：SEQ ID NO:41；(3)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:26，HCDR3：SEQ ID NO:42；(4)HCDR1：SEQ ID NO:13，HCDR2：SEQ ID NO:28，HCDR3：SEQ ID NO: 44；(5)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:29，HCDR3：SEQ ID NO:42；(6)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:24，HCDR3：SEQ ID NO:40；和(7)HCDR1：SEQ ID NO:8，HCDR2：SEQ ID NO:23，HCDR3：SEQ ID NO:39。
在某些实施方式中，所述抗原结合片段包含VH，且所述VH包括SEQ ID NOs:55-59、61和62中任一项所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合片段和/或所述第二抗原结合片段各自独立地抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别依次包含SEQ ID NO:12，SEQ ID NO:27，SEQ ID NO:43，SEQ ID NO:49，SEQ ID NO:51和SEQ ID NO:53所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合片段和/或所述第二抗原结合片段各自独立地包含VL和VH，且所述VL包括SEQ ID NO:63所示的氨基酸序列，所述VH包括SEQ ID NO:60所示的氨基酸序列。
在某些实施方式中，所述第一抗原结合片段和/或所述第二抗原结合片段各自独立地包含一对VH和VL和/或另一种VH，且所述一对VH和VL分别包括SEQ ID NO:60和SEQ ID NO:63所示的氨基酸序列，和所述另一种VH包括SEQ ID NO:62所示的氨基酸序列。例如，所述一对VH和VL可以形成scFv。
在某些实施方式中，所述第一抗原结合片段和/或所述第二抗原结合片段各自独立地包含VH，且所述VH包括SEQ ID NO:62所示的氨基酸序列；和，所述第三抗原结合片段和/或所述第四抗原结合片段各自独立地包含一对VH和VL，且所述一对VH和VL分别包括SEQ ID NO:130和SEQ ID NO:131所示的氨基酸序列。例如，所述第三抗原结合片段和/或所述第四抗原结合片段可以包含Fab。
在某些实施方式中，所述蛋白-药物偶联物中的抗原结合蛋白部分还可以包含能够使得第一连接肽和第二连接肽配对的配对单元部分，所述配对单元可以包含至少第一配对亚单元和第二配对亚单元，且所述第一配对亚单元可以通过共价键或者非共价键与所述第二配对亚单元相互作用。
在某些实施方式中，所述配对单元为Fc片段或突变的Fc片段。
在某些实施方式中，所述第一配对亚单元的N端与所述第一连接肽的C端连接，且所述第二配对亚单元的N端与所述第二连接肽的C端连接，或者，所述第一配对亚单元的N端与所述第二连接肽的C端连接，且所述第二配对亚单元的N端与所述第一连接肽 的C端连接。
在某些实施方式中，所述的蛋白-药物偶联物包含SEQ ID NOs:64-71、106-129、148-149中任一项所示的氨基酸序列。
在某些实施方式中，所述蛋白-药物偶联物的所述药物偶联物部分包括分别与第一连接肽和第二连接肽连接的两个药物偶联物，所述药物偶联物包含载荷药物以及任选地连接子。
在某些实施方式中，所述的蛋白-药物偶联物中所述药物偶联物包含至少一个载荷药物。所述载荷药物可以包含但不局限于小分子化合物、毒素分子、抗生素、寡核苷酸、蛋白降解靶向嵌合体(PROTAC)、亲和配体、荧光或核素标记基团、多肽、免疫调节剂等。
在某些实施方式中，所述连接子包含可断裂的连接子或非可断裂的连接子；其中，所述可断裂的连接子包含蛋白酶可切割的连接子。
在某些实施方式中，所述药物偶联物包括mc-vc-PAB-MMAE(如图6所示)。
在某些实施方式中，所述药物偶联物包括PROTAC，例如一种BRD4的蛋白降解剂(如图64所示)。
在某些实施例中，所述蛋白-药物偶联物为抗体-药物偶联药物(ADC)。
另一方面，本申请提供了一种制备本申请所述的蛋白-药物偶联物的方法，其包括使所述药物偶联物与所述第一连接肽和/或所述第二连接肽上的所述Cys1的反应性巯基进行共价结合。
另一方面，本申请提供了一种制备蛋白-药物偶联物的方法。所述蛋白-药物偶联物包含抗原结合蛋白部分和药物偶联物部分；所述抗原结合蛋白部分包括一个或多个抗原结合片段，以及与所述抗原结合片段直接或间接连接的至少两个连接肽；所述两个连接肽包含第一连接肽和第二连接肽，其中所述第一连接肽包含第一半胱氨酸Cys1和第二半胱氨酸Cys2，且所述第二连接肽包含第一半胱氨酸Cys1和第二半胱氨酸Cys2；所述方法包括使所述药物偶联物部分通过所述第一连接肽的所述Cys1与所述第一连接肽联结，和/或使所述药物偶联物部分通过所述第二连接肽的所述Cys1与所述第二连接肽联结。
在某些实施方式中，所述一个或多个抗原结合片段中不包含能够影响药物偶联物与所述第一连接肽和/或所述第二连接肽在Cys1处进行偶联的官能团或者前述官能团与其他药物偶联物形成的缀合物，所述影响表现为显著降低药物偶联物在Cys1位点的偶联几率。
在某些实施方式中，所述方法中的所述一个或多个抗原结合片段不包含能够与所述 第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的官能团或者前述官能团与其他药物偶联物形成的缀合物。
在某些实施方式中，所述一个或多个抗原结合片段不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的半胱氨酸或者前述半胱氨酸与其他药物偶联物形成的缀合物。
在某些实施方式中，所述方法中的所述第一连接肽和所述第二连接肽的氨基酸序列可以相同或不同。
在某些实施方式中，所述方法中的所述第一连接肽和/或所述第二连接肽自N端至C端包含Cys1-(X)n-Cys2所示的氨基酸序列；其中，n为大于或等于3的整数，X是指不为Cys的任意氨基酸。
在某些实施方式中，所述第一连接肽和/或所述第二连接肽源自抗体铰链区序列或其衍生序列。
在某些实施方式中，所述方法中的所述衍生序列为通过对抗体铰链区序列进行改造获得，所述改造不涉及抗体铰链区中Cys1和Cys2的改变。在某些实施方式中，所述改造为调整抗体铰链区序列中Cys1和Cys2之间的氨基酸种类、数目和/或在Cys1前进行柔性连接氨基酸的添加。
在某些实施方式中，所述方法中的所述连接肽为人IgG铰链区，优选人IgG1铰链区。
在某些实施方式中，所述方法中的所述第一连接肽为人IgG1的铰链区，所述第一连接肽序列如SEQ ID NO:73所示，所述第一连接肽中所述Cys1为人IgG1铰链区根据EU编码的Cys-220，所述Cys2为人IgG1铰链区根据EU编码的Cys-226。
在某些实施方式中，所述方法中的所述第二连接肽为人IgG1的铰链区，所述第二连接肽序列如SEQ ID NO:73所示，所述第二连接肽中所述Cys1为人IgG1铰链区根据EU编码的Cys-220，所述Cys2为人IgG1铰链区根据EU编码的Cys-226。
在某些实施方式中，所述方法中的所述第一连接肽可以源自人IgG1的铰链区的衍生序列，其氨基酸序列如SEQ ID NOs:74-105、145-146中任一项所示的序列，所述第一连接肽中所述Cys1为对应于人IgG1铰链区Cys-220的Cys，连接肽中所述Cys2为对应于人IgG1铰链区Cys-226的Cys。
在某些实施方式中，所述方法中的所述第二连接肽可以源自人IgG1的铰链区的衍生序列，其氨基酸序列如SEQ ID NOs:74-105、145-146中任一项所示的序列，所述第二连 接肽中所述Cys1为对应于人IgG1铰链区Cys-220的Cys，连接肽中所述Cys2为对应于人IgG1铰链区Cys-226的Cys。
在某些实施方式中，所述方法中的所述第一连接肽为小鼠IgG2c的铰链区，其氨基酸序列如SEQ ID NO:147所示，所述第一连接肽中所述Cys1为其序列的第一个半胱氨酸，所述Cys2为其序列的第二个半胱氨酸。
在某些实施方式中，所述方法中的所述第二连接肽为小鼠IgG2c的铰链区，其氨基酸序列如SEQ ID NO:147所示，所述第二连接肽中所述Cys1为其序列的第一个半胱氨酸，所述Cys2为其序列的第二个半胱氨酸。
在某些实施方式中，所述方法包括还原、偶联以及任选地纯化步骤。
在某些实施方式中，所述还原包括在还原剂的条件下使所述第一连接肽的所述Cys1和所述第二连接肽的所述Cys1之间的二硫键还原。
在某些实施方式中，所述还原包括使用还原剂DTT和/或TCEP；在某些实施方式中，所述还原剂为TCEP。
在某些实施方式中，所述还原剂的用量为1至50摩尔倍数；在某些实施方式中，所述还原剂的用量为1至6摩尔倍数；在某些实施方式中，还原剂用量为1.5至3摩尔倍数。
在某些实施方式中，所述还原的反应时长为1至17小时；在某些实施方式中，所述还原反应时长为1至3小时；在某些实施方式中，还原反应时长为1.5至2小时。
在某些实施方式中，所述还原的反应温度为0至40℃；在某些实施方式中，所述还原的反应温度为0℃或室温(25-30℃)或37℃。
在某些实施方式中，所述还原的反应缓冲液pH为4.0至9.0；在某些实施方式中，所述还原反应缓冲液pH为5.0至8.0；在某些实施方式中，所述pH为5.0至7.0；在某些实施方式中，所述pH为5.0至6.0。
在某些实施方式中，所述偶联包括使所述药物偶联物与所述第一连接肽和/或所述第二连接肽上的所述Cys1的反应性巯基进行共价结合。
在某些实施方式中，所述药物偶联物包括mc-vc-PAB-MMAE或PROTAC或CpG寡核苷酸。
在某些实施方式中，所述药物偶联物的用量为1至50摩尔倍数；在某些实施方式中，所述药物偶联物的用量为1至10摩尔倍数；在某些实施方式中，药物偶联物用量为3至7摩尔倍数。
在某些实施方式中，所述药物偶联物是mc-vc-PAB-MMAE，mc-vc-PAB-MMAE的用量为1至50摩尔倍数；在某些实施方式中，mc-vc-PAB-MMAE的用量为3至18摩尔倍数；在某些实施方式中，mc-vc-PAB-MMAE的用量为3至7摩尔倍数。
在某些实施方式中，所述药物偶联物PROTAC的用量为10至15摩尔倍数。
在某些实施方式中，所述偶联的反应时长为0.5至10小时；在某些实施方式中，所述偶联反应时长为0.5至3小时；在某些实施方式中，偶联反应时长为0.5至1小时。
在某些实施方式中，所述偶联的反应温度为0至40℃；在某些实施方式中，所述偶联的反应温度为室温(25-30℃)。
在某些实施方式中，所述偶联的反应缓冲液pH为4.0至9.0；在某些实施方式中，所述偶联的反应缓冲液pH为5.0至8.0；在某些实施方式中，所述pH为5.0至7.0；在某些实施方式中，所述pH为7.0至8.0；在某些实施方式中，所述pH为5.0至6.0。
在某些实施方式中，所述蛋白-药物偶联物的纯度大于80％。例如，所述蛋白-药物偶联物的纯度大于82％、大于85％、大于88％或大于90％。
在某些实施方式中，所述制备方法可以提供一种包括“还原”和“偶联”的反应步骤和反应条件参数的组合；所述的反应步骤和反应条件参数的组合可以进一步提高偶联产物中单一组分的含量，减少额外的“纯化”步骤的需求；在某些实施方式中，经过偶联步骤后无需额外的纯化步骤即可以得到纯度大于90％的高均质性的偶联产物；进一步在某些实施方式中，所述高均质性的偶联产物是位点特异性地偶联于Cys1的CAR＝2的蛋白-药物偶联物。
另一方面，本申请提供了包含所述的蛋白-药物偶联物和药学上可接受的载体的药物组合物。
另一方面，本申请提供了预防和/或治疗疾病的方法，其包括施用所述的蛋白-药物偶联物和/或所述的药物组合物，任选地，所述蛋白-药物偶联物和/或所述药物组合物与其他疗法或药物联用。
另一方面，本申请提供了所述的蛋白-药物偶联物和/或所述的药物组合物在制备药物中的用途，所述药物用于治疗肿瘤或其他疾病。
另一方面，本申请提供了所述的蛋白-药物偶联物与其他疗法或药物联用在制备药物中的用途，所述药物用于治疗肿瘤或其他疾病。
在某些实施方式中，所述其他疗法或药物选自下组：化疗、放疗、miRNA和寡核苷酸。
在某些实施方式中，所述肿瘤选自下组中的任意一种或多种：淋巴瘤、多发性骨髓瘤、乳腺癌、卵巢癌、肾癌、子宫内膜癌、黑色素瘤、胰腺癌、肺癌、胃癌、肝癌、间皮瘤、食管癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、胸腺癌和结直肠癌。
另一方面，本申请提供了诊断特定疾病的方法，其包括使用所述的蛋白-药物偶联物和/或所述的药物组合物。
另一方面，本申请提供了检测特定靶标的方法，其包括使用所述的蛋白-药物偶联物和/或所述的药物组合物，优选地，所述检测为非疾病诊断目的的。
另一方面，本申请提供了用于检测的试剂盒，其包括所述的蛋白-药物偶联物和/或所述的药物组合物，和任选地使用说明。
另一方面，本申请提供了给药装置，其包括所述的蛋白-药物偶联物和/或所述的药物组合物，以及施用所述蛋白-药物偶联物和/或所述药物组合物的装置。
本申请的发明人意外的发现：在没有轻链的情况下，人IgG1重链铰链区的Cys-220(Eu编码)有机会形成重链间的二硫键，但是这个二硫键没有其他的二硫键稳定，只需要用较弱的还原剂就可以把Cys-220的二硫键和其他二硫键区分开，使Cys-220的二硫键可以被有效地打开而其他二硫键保持完整，进而使得Cys-220成为几乎唯一的游离半胱氨酸残基作为定点偶联位点与药物偶联物进行共价结合。通过对人IgG1天然铰链区的Cys-220和Cys-226之间的氨基酸种类和数量进行调整或在Cys-220前添加柔性连接肽形成的铰链区衍生序列同样能够作为连接肽以实现在对应于天然铰链区Cys-220位点的定点偶联。
在符合本领域常识的基础上，上述各条件，可任意组合，即得本申请各较佳实例。
附图说明
图1显示的是人IgG1抗体的二硫键结构。
图2显示了具有人IgG1抗体铰链区序列的重链抗体HCAb的结构，以及铰链区二硫键结构在人IgG1和HCAb之间的差异。
图3显示了在HCAb结构中，位于铰链区的220位置(Eu编号)的半胱氨酸Cys-220的两种可能状态：Cys-220形成或者未形成重链之间的二硫键。
图4显示了HCAb PR000020的非还原样品分子量分析的质谱去卷积处理图谱。
图5显示了HCAb PR000020的木瓜蛋白酶Papain酶切分析结果：(A)Cys-220是否形成重链之间的二硫键的假设以及相应的酶切产物组分推测；(B)酶切产物的非还原SDS-PAGE的结果。
图6显示了(A)含有药物偶联物mc-vc-PAB-MMAE的ADC示意图和mc-vc-PAB-MMAE(也称为VcMMAE)的结构，mc-vc-PAB-MMAE由连接子mc-vc-PAB和载荷药物MMAE组成，(图中“抗体”和“ADCs”仅作为一般性的示意，不指代本发明的“抗原结合蛋白”或“蛋白-药物偶联物”的特定结构)；和(B)质谱分析中mc-vc-PAB-MMAE的特征碎片结构。
图7显示了HCAb PR000020及其偶联产物PR000020-ADC结合高表达CTLA4的细胞的结合活性。
图8显示了HCAb PR000020及其偶联产物PR000020-ADC的HIC-HPLC分析结果：(A)HCAb偶联前；(B)偶联后，纯化前；(C)偶联及一步HIC纯化后。
图9显示了HCAb PR000020及其偶联产物PR000020-ADC的RP-HPLC分析结果：(A)HCAb偶联前；(B)偶联后，纯化前；(C)偶联及一步HIC纯化后。
图10显示了HCAb PR000020的偶联产物PR000020-ADC的分子量分析的质谱去卷积处理图谱：(A)非还原样品；(B)还原样品。
图11显示了利用LC-MS肽谱图分析PR000020-ADC的化合物偶联位点：(A)PR000020-ADC的氨基酸序列，含有Cys-IAM的多肽片段用灰色背景标识；(B)PR000020-ADC样品中含有Cys-IAM的多肽片段；(C)PR000020样品中含有Cys-IAM的多肽片段；(B)和(C)中表的各列名释义见(C)下方。
图12显示了HCAb PR000759蛋白表达纯化后的样品的SEC-HPLC分析结果。
图13显示了HCAb PR000759的偶联产物PR000759-ADC经一步HIC纯化后的样品的HIC-HPLC分析结果。
图14显示了利用LC-MS肽谱图分析PR000759-ADC的化合物偶联位点，和筛选出的带有偶联位点的肽段；其中表的各列名释义同图11(C)。
图15显示了HCAb PR000759及其偶联产物PR000759-ADC的分子量分析的质谱去卷积处理图谱：(A)PR000759的非还原样品；(B)PR000759-ADC的非还原样品；(C)PR000759-ADC的还原样品。
图16显示了HCAb PR001046蛋白表达纯化后的样品的SEC-HPLC分析结果。
图17显示了HCAb PR001046的偶联产物PR001046-ADC经一步HIC纯化后的样品的HIC-HPLC分析结果。
图18显示了利用LC-MS肽谱图分析PR001046-ADC的化合物偶联位点，和筛选出的带有偶联位点的肽段；其中表的各列名释义同图11(C)。
图19显示了HCAb PR001046及其偶联产物PR001046-ADC的分子量分析的质谱去卷积处理图谱：(A)PR001046的非还原样品；(B)PR001046-ADC的非还原样品；(C)PR001046-ADC的还原样品。
图20显示了HCAb PR001046及其偶联产物PR001046-ADC结合高表达BCMA的细胞的结合活性。
图21显示了PR001046-ADC的特异性的细胞毒性：(A)特异性地杀伤BCMA +细胞NCI-H929以及混合细胞；(B)不同纯度的ADC(D2组分)对于细胞毒性的影响。
图22显示了HCAb PR004432蛋白表达纯化后的样品的SEC-HPLC分析结果。
图23显示了HCAb PR004432的偶联产物PR004432-ADC经一步HIC纯化后的样品的RP-HPLC分析结果。
图24显示了利用LC-MS肽谱图分析PR004432-ADC的化合物偶联位点，包括筛选出的带有偶联位点的肽段，以及对应的偶联位点覆盖率。
图25显示了HCAb PR004432的偶联产物PR004432-ADC的分子量分析的质谱去卷积处理图谱：(A)非还原样品；(B)还原样品。
图26显示了HCAb PR004432及其偶联产物PR004432-ADC结合高表达5T4的细胞的结合活性。
图27显示了PR004432-ADC对高表达5T4的细胞的特异性的细胞毒性。
图28显示了HCAb PR004433蛋白表达纯化后的样品的SEC-HPLC分析结果。
图29显示了HCAb PR004433的偶联产物PR004433-ADC经一步HIC纯化后的样品的HIC-HPLC分析结果。
图30显示了HCAb PR004433及其偶联产物PR004433-ADC的HIC-HPLC分析结果：(A)HCAb偶联前；(B)偶联后，纯化前；(C)偶联及一步HIC纯化后。
图31显示了HCAb PR004433的偶联产物PR004433-ADC的分子量分析的质谱去卷积处理图谱：(A)非还原样品；(B)还原样品。
图32显示了HCAb PR004433及其偶联产物PR004433-ADC结合表达BCMA的细胞的结合活性：(A)结合高表达人BCMA的细胞HEK293T-hBCMA；(B)结合高表达人BCMA的细胞NCI-H929；(C)不结合BCMA阴性细胞SNU-16。
图33显示了PR004433-ADC的特异性的细胞毒性：(A)对HEK293T-hBCMA有特异性的细胞毒性；(B)对HEK293T没有细胞毒性；(C)对NCI-H929有特异性的细胞毒性；(D)对SNU-16没有细胞毒性。
图34显示了利用LC-MS肽谱图分析PR004433-ADC的化合物偶联位点，包括筛选出的带有偶联位点的肽段，以及对应的偶联位点覆盖率。
图35显示了利用LC-MS肽谱图分析PR000020-ADC的化合物偶联位点时，筛选出的带有偶联位点的肽段所对应的二级谱图。
图36显示了利用LC-MS肽谱图分析PR000020-ADC的化合物偶联位点时，酶解肽段PHGSDIWGQGTMVTVSSEPKSC#DK(#表示偶联位点)在ADC样品(上)和单抗样品(下)的XIC图。
图37显示了利用LC-MS分析PR002129中Cys-220的二硫键：(A)显示了在PR002129的结构中，Cys-220的两种可能状态；(B)PR002129的非还原样品分子量分析的质谱去卷积处理图谱。
图38显示了HCAb PR000184及其C220S衍生变体的DSC热力学分析曲线图：(A)PR000184；(B)PR000184(C220S)。
图39显示了HCAb PR000453及其C220S衍生变体的DSC热力学分析曲线图：(A)PR000453；(B)PR000453(C220S)。
图40显示了HCAb PR004432、其C220S衍生变体和其偶联产物利用Uncle分析平台测定熔解温度Tm：(A)PR004432；(B)PR004432(C220S)；(C)PR004432-ADC。
图41举例说明了本发明具体实施方式中所述“蛋白-药物偶联物”的组成部分以及相关术语。
图42显示了示例性的抗原结合蛋白：(A)具有两个抗原结合片段和两个连接肽的抗原结合蛋白；(B)具有四个抗原结合片段和两个连接肽的抗原结合蛋白。
图43显示了利用图42所示的抗原结合蛋白进行偶联产生的示例性的蛋白-药物偶联物：(A)具有两个抗原结合片段和两个连接肽的蛋白-药物偶联物；(B)具有四个抗原结合片段和两个连接肽的蛋白-药物偶联物。
图44显示了不同物种的铰链区序列和二硫键结构：(A)人、小鼠和羊驼的不同IgG同型的铰链区序列，半胱氨酸Cys被标识出；(B)人IgG1铰链区序列和二硫键结构；(C)人IgG4铰链区序列和二硫键结构；(D)小鼠IgG1铰链区序列和二硫键结构；(E)小鼠IgG2a铰链区序列和二硫键结构。
图45显示了HCAb PR004433在不同的还原和偶联反应条件下制备的偶联产物利用LC-MS分析非还原样品的完整分子量所得到的质谱图中的总离子流图(TIC)，分别对应于实施例10.1的表10-1和表10-2中的实验编号：(A)实验#8；(B)实验#9；(C)实验#11；(D)实验#12。
图46显示了HCAb PR004433及其变体分子结合NCI-H929细胞的结合活性。
图47显示了HCAb PR006468的偶联产物PR006468-ADC的非还原样品的完整分子量分析的质谱去卷积处理图谱。
图48显示了利用LC-MS肽谱图分析PR006468-ADC的化合物偶联位点，包括筛选出的带有偶联位点的肽段，以及对应的偶联位点覆盖率。
图49显示了BCMA结合蛋白(HCAb PR004433，HCAb PR006468)及其偶联产物(PR004433-ADC，PR006468-ADC)结合NCI-H929细胞的结合活性。
图50显示了PR006468-ADC和PR004433-ADC对NCI-H929细胞的细胞毒性。
图51显示了PR002129的偶联产物PR002129-ADC的HIC-HPLC分析结果：(A)偶联后，纯化前；(B)偶联及一步HIC纯化后。
图52显示了PR002129的偶联产物PR002129-ADC的分子量分析的质谱去卷积处理图谱：(A)HIC纯化前样品的非还原分子量；(B)HIC纯化后样品的还原分子量；(C)HIC纯化后样品的非还原分子量。
图53显示了利用LC-MS肽谱图分析PR002129-ADC的化合物偶联位点，包括筛选出的带有偶联位点的肽段，以及对应的偶联位点覆盖率。
图54显示了PR002129及其偶联产物PR002129-ADC结合高表达ROR1的细胞的结合活性。
图55显示了SIRPa-Fc融合蛋白PR006345的偶联产物PR006345-ADC的纯化前样品的HIC-HPLC分析结果。
图56显示了SIRPa-Fc融合蛋白PR006345的偶联产物PR006345-ADC的纯化前样品的非还原分子量分析的质谱去卷积处理图谱。
图57显示了利用LC-MS肽谱图分析PR006345-ADC的化合物偶联位点，包括筛选出的带有偶联位点的肽段，以及对应的偶联位点覆盖率。
图58显示了PR006345及其偶联产物PR006345-ADC结合高表达CD47的细胞的结合活性。
图59显示了四价双特异性抗原结合蛋白PR005744的结构。
图60显示了PR005744在NCI-H929细胞上的内化。
图61显示了利用LC-MS肽谱图分析PR005744-ADC的化合物偶联位点，包括筛选出的带有偶联位点的肽段，以及对应的偶联位点覆盖率。
图62显示了PR005744及其偶联产物PR005744-ADC结合NCI-H929细胞的结合活性。
图63显示了PR005744-ADC对NCI-H929细胞的细胞毒性。
图64显示了BRD4蛋白降解剂(PROTAC)的分子结构。
图65显示了HCAb PR004433与PROTAC进行偶联的产物PR004433-PROTAC的纯化前样品的非还原分子量分析的质谱去卷积处理图谱。
图66显示了HCAb PR004433及其偶联产物PR004433-PROTAC结合NCI-H929细胞的结合活性。
图67显示了二价双特异性抗原结合蛋白2129/4433的结构。
图68显示了2129/4433的偶联产物2129/4433-ADC的纯化前样品的HIC-HPLC分析结果。
图69显示了利用LC-MS肽谱图分析2129/4433-ADC的化合物偶联位点，包括筛选出的带有偶联位点的肽段，以及对应的偶联位点覆盖率。
图70显示了利用LC-MS肽谱图分析PR001046-ADC的化合物偶联位点时，筛选出的带有偶联位点的肽段所对应的二级谱图。
图71显示了利用LC-MS肽谱图分析PR006468-ADC的化合物偶联位点时，筛选出的带有偶联位点的肽段所对应的二级谱图。
图72显示了利用LC-MS肽谱图分析2129/4433-ADC的化合物偶联位点时，筛选出的带有偶联位点的肽段所对应的二级谱图。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说 明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“免疫球蛋白”(Ig)与“抗体”在本文中可互换使用。基本的四链抗体单元是异四聚体蛋白，其由两条相同的轻链(L)和两条相同的重链(H)构成。在IgG的情况中，每个L链通过一个共价二硫键连接到H链，而两条H链通过一个或多个二硫键彼此相连，二硫键的数目取决于H链的同种型。每个H和L链还具有规律间隔的链内二硫键。每条H链在氨基端具有可变结构域(VH)，接着是三个(对于每条α和γ链)或四个(对于μ和ε同种型)恒定结构域(CH)。每条L链在氨基端具有可变结构域(VL)，接着是一个恒定结构域(CL)。人IgG有四个亚型：IgG1、IgG2、IgG3和IgG4。人或鼠类的抗体L链还进一步分为κ链和λ链两种，相应地，其可变结构域分为Vκ和Vλ，其恒定结构域分为Cκ和Cλ。
在本申请中，术语“结合蛋白”或者“抗原结合蛋白”通常是指包含结合抗原的部分的蛋白质，以及任选地允许结合抗原的部分采用促进抗原结合蛋白与抗原结合的构象的支架或骨架部分。典型地，所述“结合蛋白”或者“抗原结合蛋白”可以包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区和相对保守的框架区(FR)。每个VH和VL可由三个CDR和四个FR区构成。VH和VL含有与抗原相互作用的结合位点。VH的三个CDR分别表示为HCDR1、HCDR2和HCDR3，也可表示为VH CDR1、VH CDR2和VH CDR3；VL的三个CDR分别表示为LCDR1、LCDR2和LCDR3，也可表示为VL CDR1、VL CDR2和VL CDR3。抗原结合蛋白的实例可以包括但不限于抗体、抗原结合片段(Fab，Fab’，F(ab) 2，Fv片段，F(ab’) 2，scFv，di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物、受体蛋白可溶性胞外区、配体蛋白或其他形式的融合蛋白等，只要它们显示出所需的抗原结合活性即可。
在本申请中，所述CDR的氨基酸序列均是按照Chothia定义规则所示出的。但是，本领域人员公知，在本领域中可以通过多种方法来定义抗体的CDR，例如基于序列可变性的Kabat定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见JMol Biol 273:927-48,1997)。在本发明的技术方案中，还可以使用包含了Kabat定义和Chothia定义的Combined定义规则来确定可变结构域序列中的氨基酸残基。所述Combined定义规则即是将Kabat定义和Chothia定义的范围相结合，基于此取了一个更大的范围，详见下表。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体 或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖如通过本发明描述的上述已知方案中的任何一种界定的互补决定区。虽然本发明中请求保护的范围是基于Chothia定义规则所示出的序列，但是根据其他CDR的定义规则所对应的氨基酸序列也应当在本发明的保护范围中。
  Kabat Chothia Combined
LCDR1 L24--L34 L24--L34 L24-L34
LCDR2 L50--L56 L50--L56 L50-L56
LCDR3 L89--L97 L89--L97 L89-L97
HCDR1 H31--H35 H26--H32 H26-H35
HCDR2 H50--H65 H52--H56 H50-H65
HCDR3 H95--H102 H95--H102 H95-H102
表-I 本申请抗体CDR定义方法
其中，Laa-Lbb可以指从抗体轻链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列；Haa-Hbb可以指从抗体重链的N端开始，第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如，L24-L34可以指从抗体轻链N端开始，按照Chothia编码规则的从第24位至第34位的氨基酸序列；H26-H32可以指从抗体重链N端开始，按照Chothia编码规则的从第26位至第32位的氨基酸序列。本领域技术人员应当知晓的是，在用Chothia编码CDR时，有些位置会有插入位点的情况(可参见http://bioinf.org.uk/abs/)。
在本申请中，术语“单克隆抗体”通常是指从一群基本上同质的细胞中获得的抗体，即集群中的抗体是相同的，除了可能存在的少量的自然突变。单克隆抗体通常针对单个抗原位点具有高度特异性。而且，与常规多克隆抗体制剂(通常具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性之外，单克隆抗体的优点在于它们可以通过杂交瘤培养合成，不受其他免疫球蛋白污染。修饰语“单克隆”表示从基本上同质的抗体群体获得的抗体的特征，但是不被解释为需要通过任何特定方法产生抗体。例如，根据本发明使用的单克隆抗体可以在杂交瘤细胞中制备，或者可以通过重组DNA方法制备。
在本申请中，术语“全人源抗体”通常是指将人类编码抗体的基因全部转移至基因工程改造的抗体基因缺失动物中，使动物表达的抗体。抗体所有部分(包括抗体的可变 区和恒定区)均由人类来源的氨基酸序列所组成。全人源抗体可以大大减少异源抗体对人体造成的免疫副反应。本领域获得全人源抗体的方法可以有噬菌体展示技术、转基因小鼠技术等。
在本申请中，术语“特异性结合”通常是指抗原结合蛋白与抗原表位结合，并且该结合需要抗原结合蛋白和表位之间的一些互补性。根据该定义，当抗体相比于其将结合随机的，不相关的表位而言更容易通过其抗原结合蛋白与表位结合时，抗体被称为“特异性结合”该抗原。“表位”是指抗原上与抗原结合蛋白(如抗体)结合的特定的原子基团(例如，糖侧链、磷酰基、磺酰基)或氨基酸。
在本申请中，术语“Fab”通常指常规抗体(例如IgG)中与抗原结合的部分，包括抗体的重链可变区VH、轻链可变区VL和重链恒定区结构域CH1以及轻链恒定区CL。在常规抗体中，VH的C端与CH1的N端联结形成重链Fd片段，VL的C端与CL的N端联结形成轻链，CH1的C端又进一步与重链的铰链区和其他恒定区结构域联结形成重链。在某些实施方式中，“Fab”也指Fab的变体结构。例如，在某些实施方式中，VH的C端与CL的N端联结形成一个多肽链，VL的C端与CH1的N端联结形成另一个多肽链，形成Fab(cross VH/VL)的结构；在某些实施方式中，Fab的CH1不与铰链区联结，而是CL的C端与重链的铰链区联结，形成Fab(cross Fd/LC)的结构。
在本申请中，术语“VH”通常指抗体的重链可变区VH结构域。在某些实施方式中，“VH”可以是人或者其他动物的常规抗体(H2L2结构)的重链可变区VH。在某些实施方式中，“VH”也可以是骆驼科等动物的重链抗体(HCAb结构)的重链可变区VHH。在某些实施方式中，“VH”还可以是利用Harbour HCAb转基因小鼠产生的全人源重链抗体(HCAb结构)的重链可变区VH。
在本申请中，术语“抗原结合片段”通常指任何可以与抗原特异结合的蛋白功能区域。在某些实施方式中，“抗原结合片段”可以是“Fab”。在某些实施方式中，“抗原结合片段”也可以是“VH”。在某些实施方式中，“抗原结合片段”也可以是单链抗体scFv。在某些实施方式中，“抗原结合片段”还可以是其他抗原结合形式(例如受体蛋白可溶性胞外区、配体蛋白、脂质运载蛋白(lipocalins)、神经细胞粘附分子(NCAM)、纤维结合蛋白(fibronectin)、锚蛋白重复片段蛋白(DARPins)等衍生蛋白结构)。
在本申请中，术语“配对单元”通常是指一组含有至少两个可以互相配对的亚单元的结构。所述互相配对的亚单元之间可以互相结合，该结合需要亚单元之间的互补性；所述互相配对的亚单元之间可以发生共价键或者非共价键相互作用；所述非共价键相互 作用可以包括范德华力、氢键、疏水作用、静电作用、偶极作用等。“配对单元”可以是天然存在的或者经过修饰的具有同源或者异源多聚体形式的多肽链的集合；例如，“配对单元”可以是抗体Fc片段或其变体的二聚体形式；“配对单元”可以是CD79的α链和β链；“配对单元”可以是CD8的α链和β链；“配对单元”可以是T细胞受体(TCR)的α链和β链。
在本申请中，术语“Fc片段”通常是指抗体的可结晶片段(fragment crystallizable,Fc)，即包含由重链恒定结构域CH2和CH3组成的多肽链。在某些实施方式中，Fc片段可以是天然的二聚体形式；在另一些实施方式中，Fc片段可以是修饰的单体形式。
在本申请中，术语“CTLA4”通常是指细胞毒性T淋巴细胞相关蛋白-4(也称为CD152)、其功能变体和/或其功能片段。CTLA4序列是本领域已知的。例如，示例性的全长的人CTLA4序列可以在Uniprot登录号P16410中找到；示例性的全长的食蟹猴CTLA4序列可以在Uniprot登录号G7PL88中找到。针对CTLA4的药物可能应用于治疗黑色素瘤、肺癌、结肠癌、肝癌、头颈癌、肾癌、乳腺癌、胰腺癌、膀胱癌等肿瘤。
在本申请中，术语“BCMA”通常是指肿瘤坏死因子受体超家族成员17(也称为CD269，B细胞成熟蛋白)、其功能变体和/或其功能片段。BCMA序列是本领域已知的。例如，示例性的全长的人BCMA序列可以在Uniprot登录号Q02223中找到；示例性的全长的食蟹猴BCMA序列可以在NCBI登录号XP_005591343中找到。针对BCMA的药物可能应用于治疗多发性骨髓瘤和其他血液系统恶性肿瘤。
在本申请中，术语“MSLN”通常是指间皮素分子(也称为MPF)、其功能变体和/或其功能片段。MSLN序列是本领域已知的。例如，示例性的全长的人MSLN序列可以在Uniprot登录号Q13421中找到；示例性的全长的食蟹猴MSLN序列可以在NCBI登录号XP_005590873中找到。针对MSLN的药物可能应用于治疗间皮瘤、肺癌、胰腺癌、乳腺癌、卵巢癌、子宫内膜癌等肿瘤。
在本申请中，术语“5T4”通常是指滋养细胞糖蛋白(也称为TPBG)、其功能变体和/或其功能片段。5T4序列是本领域已知的。例如，示例性的全长的人5T4序列可以在Uniprot登录号Q13641中找到；示例性的全长的食蟹猴5T4序列可以在Uniprot登录号Q4R8Y9中找到。针对5T4的药物可能应用于治疗胸腺癌、肺癌、食管癌、胃癌、小肠癌、胰腺癌、肝癌、胆囊癌、肾癌、膀胱癌、卵巢癌、子宫内膜癌、宫颈癌等肿瘤。
在本申请中，术语“ROR1”通常是指失活酪氨酸蛋白激酶跨膜受体ROR1(也称为NTRKR1)、其功能变体和/或其功能片段。ROR1序列是本领域已知的。例如，示例性的 全长的人ROR1序列可以在Uniprot登录号Q01973中找到；示例性的全长的食蟹猴ROR1序列可以在NCBI登录号XP_015290264中找到。针对ROR1的药物可能应用于治疗白血病、非霍奇金淋巴瘤、套细胞淋巴瘤、乳腺癌、肺癌、卵巢癌等肿瘤。
在本申请中，术语“CD47”通常是指白细胞表面抗原CD47、其功能变体和/或其功能片段。CD47序列是本领域已知的。例如，示例性的全长的人CD47序列可以在Uniprot登录号Q08722中找到。针对CD47的药物可能应用于治疗白血病、非霍奇金淋巴瘤、多发性骨髓瘤、黑色素瘤、头颈癌等肿瘤。
在本申请中，术语“SIRPα”通常是指信号调节蛋白α(也称为BIT、MFR、MYD1、PTPNS1、SHPS1或SIRP)、其功能变体和/或其功能片段。SIRPα是CD47的受体。SIRPα序列是本领域已知的。例如，示例性的全长的人SIRPα序列可以在Uniprot登录号P78324中找到。
在本申请中，术语“蛋白-药物偶联物”通常是指一类将药物偶联物通过共价键结合到抗原结合蛋白而形成的药物分子。所述药物偶联物包含至少一个载荷药物。本领域已知多种蛋白-药物偶联物及其制备方法。在某些实施例中，所述蛋白-药物偶联物可以是抗体-药物偶联药物(ADC)。例如，示例性的蛋白-药物偶联物可包括但不限于曲妥珠单抗(trastuzumab)，奥瑞珠单抗(ocrelizumab)，帕妥珠单抗(pertuzumab)，利妥昔单抗(rituximab)等抗体可以经由连接子单元将载荷药物模块共价附着至抗体以形成抗体-药物偶联物，以实现靶向治疗效应。在某些实施例中，“蛋白-药物偶联物”可以包含抗原结合片段、连接肽和药物偶联物。在某些实施例中，“蛋白-药物偶联物”的所述药物偶联物可以是mc-vc-PAB-MMAE(也称为VcMMAE)。
在本申请中，术语“药物偶联物”通常是指一类可以通过特定官能团以共价键结合到其他分子的、具有特定结构的原子和基团的集合体；其包含至少一个载荷药物，和任选地包含连接子。本领域已知多种载荷药物、多种连接子或连接子构件。在某些实施例中，所述药物偶联物可以是mc-vc-PAB-MMAE(也称为VcMMAE)。
在本申请中，术语“载荷药物”通常是指一类具有药学活性的小分子化合物或者毒素或其他药物分子形式，可以是但不局限于小分子化合物、毒素分子、抗生素、寡核苷酸、蛋白降解靶向嵌合体(PROTAC)、亲和配体、荧光基团、核素基团、多肽、免疫调节分子等。本领域已知多种载荷药物，例如单甲基奥瑞司他汀E(“MMAE”，monomethyl auristatin E)，美登素(“DM1”，Mertansine)，Toll样受体激动剂分子。
在本申请中，术语“连接肽”或“肽段”通常是指本申请中所述“蛋白-药物偶联物” 中的多肽链的一部分，所述连接肽含有至少一个半胱氨酸以作为与药物偶联物共价结合的位点。在本申请中，所述连接肽可以包括第一连接肽和/或第二连接肽，所述第一连接肽与所述第二连接肽的氨基酸序列可以相同或不同。
在本申请中，术语“连接子”、“连接子单元”、“接头”或“接头单元”通常是指为可用于连接一个或多个载荷药物与抗原结合蛋白以形成“蛋白-药物偶联物”的化学官能模块。连接子可包含一种或多种连接子构件，本领域已知多种连接子构件，例如，马来酰亚胺基己酰基(“MC”，maleimidocaproyl)，缬氨酸-瓜氨酸(“val-cit”或“vc”，valine-citrulline)，对氨基苄氧羰基(“PAB”，p-aminobenzyloxycarbonyl)。在本申请中，所述“连接子”可以包括由氨基酸组成的多肽连接子，但是所述“连接子”与前述“连接肽”不同，前述“连接肽”是抗原结合蛋白的一部分。
在本申请中，术语“缀合物”通常是指本申请中所述“药物偶联物”与特定官能团发生共价结合形成的原子和基团的集合体。在某些实施例中，所述缀合物可以是药物偶联物与巯基发生反应形成的。例如，在某些实施例中，所述缀合物可以是mc-vc-PAB-MMAE的马来酰亚胺活性反应基团与巯基发生加成反应形成的稳定的原子/基团的集合体。
在本申请中，术语“DAR”(Drug-Antibody Ratio)通常是指载荷药物-抗体比例，即抗体所连接载荷药物数量的平均值。目前的蛋白-药物偶联物所带的载药量通常为0～8个载荷药物分子(D0～D8)/抗体。通常，也用“D0”、“D2”、“D4”、“D6”和“D8”分别表示偶联产物中的没有偶联载荷药物分子(DAR＝0)、偶联了2个载荷药物分子(DAR＝2)、偶联了4个载荷药物分子(DAR＝4)、偶联了6个载荷药物分子(DAR＝6)和偶联了8个载荷药物分子(DAR＝8)的蛋白-药物偶联物组分。在某些实施例中，“D0”也称为“裸抗”。HIC-HPLC分析方法是测定蛋白-药物偶联物的DAR和载药量分布的常用分析方法。
在本申请中，术语“CAR”(Conjugate/Antigen binding protein Ratio)来表示药物偶联物-抗原结合蛋白比例，即抗原结合蛋白所连接药物偶联物数量的平均值。通常一个药物偶联物包含至少一个载荷药物；在一些实施方式中，现有技术可以使一个药物偶联物部分只含有一个载荷药物，即此情形中，“CAR”和“DAR”的值可以等同；在另一些实施方式中，现有技术可以使一个药物偶联物部分通过连接子连接多个载荷药物(参见，Kumara等人，Bioorganic&Medicinal Chemistry Letters(2018),28,3617-3621)，即在此情形中，“DAR”的值可以数倍于“CAR”的值；例如，当一个药物偶联物含有两个载荷药物时，DAR值可以为CAR值的2倍。
在本申请的具体实施例中，通过利用人IgG1抗体重链铰链区的天然序列(SEQ ID  NO:73)中的半胱氨酸Cys-220(Eu编号)作为偶联位点，联结本申请所述药物偶联物，可以产生均一CAR值的蛋白-药物偶联物，特别地，所述方法可以产生均一的CAR＝2的蛋白-药物偶联物。在本申请的一些具体实施例中，当使用mc-vc-PAB-MMAE作为药物偶联物进行偶联时，由于一个mc-vc-PAB-MMAE只含有一个载荷药物MMAE，因此CAR值和DAR值可以等同，也可以使用DAR值来指代一个抗原结合蛋白所连接的药物偶联物的数量；特别地，所述方法可以产生均一DAR值的蛋白-药物偶联物，例如，所述蛋白-药物偶联物的DAR＝2。
在本申请中，术语“源自”通常是指从亲本氨基酸或核苷酸序列得到的衍生氨基酸或核苷酸序列，它通常指亲本序列和衍生序列的结构相似性，不暗含或包含对源自亲本序列的衍生序列的过程或来源限制，因此使用“源自”讨论蛋白或多核苷酸时，不管其物理起源。在某些情形中，“源自”可以是指衍生序列是未经修饰的、亲本序列的一部分。例如，在本申请的具体实施例中，Cys1、Cys2可以是未经修饰的天然的抗体铰链区序列的一部分。
一方面，本申请提供了以下实施方案：
1.蛋白-药物偶联物，其基本上由第一多肽链和第二多肽链组成，所述第一多肽链包含第一半胱氨酸(Cys1)和第二半胱氨酸(Cys2)，所述第二多肽链包含第一半胱氨酸(Cys1)第二半胱氨酸(Cys2)，且所述第一多肽链的Cys1处偶联有结构-L1-P1，所述第二多肽链的Cys1处偶联有结构-L2-P2。
2.根据实施方案1所述的蛋白-药物偶联物，其中所述结构-L1-P1可以没有L1。
3.根据实施方案1-2中任一项所述的蛋白-药物偶联物，其中所述结构-L2-P2可以没有L2。
4.根据实施方案1-3中任一项所述的蛋白-药物偶联物，其中所述L1和/或所述L2包含连接子。
5.根据实施方案4所述的蛋白-药物偶联物，其中所述连接子包括可断裂连接子。
6.根据实施方案4-5中任一项所述的蛋白-药物偶联物，其中所述连接子包括蛋白酶敏感型连接子。
7.根据实施方案4-6中任一项所述的蛋白-药物偶联物，其中所述连接子包括mc-vc-PAB。
8.根据实施方案1-7中任一项所述的蛋白-药物偶联物，其中所述P1和/或所述P2包含载荷药物。
9.根据实施方案8所述的蛋白-药物偶联物，其中所述载荷药物包含药物活性成分和标记分子。
10.根据实施方案8-9中任一项所述的蛋白-药物偶联物，其中所述载荷药物包括小分子化合物、毒素分子、寡核苷酸、蛋白降解靶向嵌合体(PROTAC)、亲和配体、标记基团、抗生素、多肽和/或免疫调节分子。
11.根据实施方案1-10中任一项所述的蛋白-药物偶联物，其中所述P1和P2可以相同或不同。
12.根据实施方案1-11中任一项所述的蛋白-药物偶联物，其中所述L1和L2可以相同或不同。
13.根据实施方案1-12中任一项所述的蛋白-药物偶联物，其中所述L1和P1以共价结合方式连接。
14.根据实施方案1-13中任一项所述的蛋白-药物偶联物，其中所述L2和P2以共价结合方式连接。
15.根据实施方案1-14中任一项所述的蛋白-药物偶联物，其中所述第一多肽链包含第一抗原结合片段。
16.根据实施方案1-15中任一项所述的蛋白-药物偶联物，其中所述第二多肽链包含第二抗原结合片段。
17.根据实施方案15或16所述的蛋白-药物偶联物，其中所述第一抗原结合片段和/或第二抗原结合片段靶向肿瘤抗原或非肿瘤抗原。
18.根据实施方案15-17中任一项所述的蛋白-药物偶联物，其中所述抗原结合片段包含VH、VL、scFv、Fab、dAb、受体蛋白可溶性胞外区和/或衍生蛋白结构等。
19.根据实施方案1-18中任一项所述的蛋白-药物偶联物，其还包含配对单元。
20.根据实施方案19所述的蛋白-药物偶联物，其中所述配对单元为Fc结构域。
21.根据实施方案1-20中任一项所述的蛋白-药物偶联物，其中所述第一多肽链包含 第一连接肽，所述第二多肽链包含第二连接肽。
22.根据实施方案15-21中任一项所述的蛋白-药物偶联物，其中所述抗原结合片段通过所述连接肽与所述配对单元连接。
23.根据实施方案15-22中任一项所述的蛋白-药物偶联物，其中所述抗原结合片段的C端与所述第一连接肽和/或第二连接肽的N端连接。
24.根据实施方案15-23中任一项所述的蛋白-药物偶联物，其中所述第一连接肽和/或第二连接肽的C端与所述配对单元的N端连接。
25.根据实施方案1-24中任一项所述的蛋白-药物偶联物，其中所述第一多肽链或第二多肽链各自独立地包含SEQ ID NO:64-77、106-129、148-149中任一项所示的氨基酸序列。
26.根据实施方案1-25中任一项所述的蛋白-药物偶联物，其中所述第一多肽链自N端至C端依次包含所述第一抗原结合片段、所述第一连接肽(例如，IgG的铰链区)和所述配对单元。
27.根据实施方案1-26中任一项所述的蛋白-药物偶联物，其中所述第二多肽链自N端至C端依次包含所述第二抗原结合片段、所述第二连接肽(例如，IgG的铰链区)和所述配对单元。
28.根据实施方案1-27中任一项所述的蛋白-药物偶联物，其中所述第一多肽链自N端至C端依次包含所述第一抗原结合片段的VH、所述第一抗原结合片段的VL、所述第一连接肽(例如，IgG的铰链区)、CH 2和CH 3。
29.根据实施方案28所述的蛋白-药物偶联物，其中所述第二多肽链自N端至C端依次包含所述第二抗原结合片段的VH、所述第二抗原结合片段的VL、所述第二连接肽(例如，IgG的铰链区)、CH 2和CH 3。
30.根据实施方案1-27中任一项所述的蛋白-药物偶联物，其中所述第一多肽链自N端至C端依次包含所述第一抗原结合片段的VL、所述第一抗原结合片段的VH、所述第一连接肽(例如，IgG的铰链区)、CH 2和CH 3。
31.根据实施方案30所述的蛋白-药物偶联物，其中所述第二多肽链自N端至C端 依次包含所述第二抗原结合片段的VL、所述第二抗原结合片段的VH、所述第二连接肽(例如，IgG的铰链区)、CH 2和CH 3。
32.根据实施方案1-27中任一项所述的蛋白-药物偶联物，其中所述第一多肽链自N端至C端依次包含所述第一抗原结合片段的VH、所述第一连接肽(例如，IgG的铰链区)、CH 2和CH 3。
33.根据实施方案32所述的蛋白-药物偶联物，其中所述第二多肽链自N端至C端依次包含所述第二抗原结合片段的VH、所述第二连接肽(例如，IgG的铰链区)、CH 2和CH 3。
34.蛋白-药物偶联物，根据实施方案1-33中任一项所述的蛋白-药物偶联物，以及第三多肽链和/或第四多肽链。
35.根据实施方案1-34中任一项所述的蛋白-药物偶联物，其还包含第三抗原结合片段和/或第四抗原结合片段。
36.根据实施方案34或35所述的蛋白-药物偶联物，其中所述第三抗原结合片段和/或第四抗原结合片段包括抗体或其抗原结合片段。
37.根据实施方案35-36中任一项所述的蛋白-药物偶联物，其中所述第一多肽链自N端至C端依次包含所述第三抗原结合片段、所述第一抗原结合片段、所述第一连接肽(例如，IgG的铰链区)和所述配对单元。
38.根据实施方案35-37中任一项所述的蛋白-药物偶联物，其中所述第二多肽链自N端至C端依次包含所述第四抗原结合片段、所述第二抗原结合片段、所述第二连接肽(例如，IgG的铰链区)和所述配对单元。
39.根据实施方案35-38中任一项所述的蛋白-药物偶联物，其中所述第一多肽链自N端至C端依次包含所述第三抗原结合片段的VL、所述第三抗原结合片段的CL、所述第一抗原结合片段的VH、所述第一连接肽(例如，IgG的铰链区)、CH 2和CH 3。
40.根据实施方案35-39中任一项所述的蛋白-药物偶联物，其中所述第二多肽链自N端至C端依次包含所述第四抗原结合片段的VL、所述第四抗原结合片段的CL、所述第二抗原结合片段的VH、所述第二连接肽(例如，IgG的铰链区)、CH 2和CH 3。
41.根据实施方案35-40中任一项所述的蛋白-药物偶联物，其中所述第三多肽链自N端至C端，依次包含所述第三抗原结合片段的VH和所述第三抗原结合片段的CH 1。
42.根据实施方案35-41中任一项所述的蛋白-药物偶联物，其中所述第四多肽链自N端至C端，依次包含所述第四抗原结合片段的VH和所述第四抗原结合片段的CH 1。
43.根据实施方案35-42中任一项所述的蛋白-药物偶联物，其中所述第三抗原结合片段和/或第四抗原结合片段为Fab。
44.根据实施方案35-43中任一项所述的蛋白-药物偶联物，其中所述第一抗原结合片段和所述第二抗原结合片段的氨基酸序列相同，和/或，所述第三抗原结合片段和所述第四抗原结合片段的氨基酸序列相同。
45.根据实施方案35-44中任一项所述的蛋白-药物偶联物，其中所述第一多肽链和所述第二多肽链的氨基酸序列相同，和/或，所述第三多肽链和所述第四多肽链的氨基酸序列相同。
46.根据实施方案1-27中任一项所述的蛋白-药物偶联物，其中所述第一多肽链自N端至C端依次包含所述第一抗原结合片段的受体蛋白可溶性胞外区、所述第一连接肽(例如，IgG的铰链区)、CH 2和CH 3。
47.根据实施方案46所述的蛋白-药物偶联物，其中所述第二多肽链自N端至C端依次包含所述第二抗原结合片段的受体蛋白可溶性胞外区、所述第二连接肽(例如，IgG的铰链区)、CH 2和CH 3。
抗原结合蛋白
一方面，本申请提供了一种抗原结合蛋白。
在本申请中，所述抗原结合蛋白包含至少两个连接肽、一个或者多个抗原结合片段。
在本申请中，两个连接肽分别为第一连接肽和第二连接肽；第一连接肽和第二连接肽的氨基酸序列可以相同或不同。
在本申请的所述抗原结合蛋白中，所述第一连接肽可以至少包含第一半胱氨酸Cys1和第二半胱氨酸Cys2，且所述第二连接肽可以至少包含第一半胱氨酸Cys1和第二半胱氨酸Cys2。
在本申请的所述抗原结合蛋白中，所述第一连接肽的Cys1和所述第二连接肽的Cys1之间可以形成二硫键，称为“二硫键Cys1-Cys1”。
在本申请的所述抗原结合蛋白中，所述第一连接肽的Cys2和所述第二连接肽的Cys2之间可以形成二硫键，称为“二硫键Cys2-Cys2”。
在本申请的所述抗原结合蛋白中，所述“二硫键Cys1-Cys1”弱于所述“二硫键Cys2-Cys2”。特别地，在具体的实施方式中，二硫键Cys1-Cys1相比二硫键Cys2-Cys2，更容易被还原剂所打开。
在本申请中，所述抗原结合蛋白中的所述第一连接肽的所述Cys1和所述Cys2之间可以相隔至少3个(例如，4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个或更多)不为半胱氨酸的氨基酸。
在本申请中，所述抗原结合蛋白中的所述第二连接肽的所述Cys1和所述Cys2之间可以相隔至少3个(例如，4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个或更多)不为半胱氨酸的氨基酸。
例如，所述第一连接肽自N端至C端可以包含Cys1-(X)n-Cys2所示的氨基酸序列，其中，n可以为大于或等于3的整数(例如n可为3，n可为4，n可为5，n可为6，n可为7，n可为8，n可为9，n可为10，n可为11，n可为12，n可为13，n可为14，n可为15，或n可为大于15的整数)；所述X指的是不为Cys的任意氨基酸。
例如，所述第二连接肽自N端至C端可以包含Cys1-(X)n-Cys2所示的氨基酸序列，其中，n可以为大于或等于3的整数(例如n可为3，n可为4，n可为5，n可为6，n可为7，n可为8，n可为9，n可为10，n可为11，n可为12，n可为13，n可为14，n可为15，或n可为大于15的整数)；所述X指的是不为Cys的任意氨基酸。
在本申请中，所述抗原结合蛋白中的所述连接肽可以是含有至少两个半胱氨酸(Cys1和Cys2)的天然存在的氨基酸序列，也可以是对“含有至少两个半胱氨酸的天然存在的氨基酸序列”进行改造获得的衍生序列。所述“改造”不涉及天然存在的氨基酸序列中Cys1和Cys2的改变；例如，所述改造为调整天然存在的氨基酸序列中Cys1和Cys2之间的氨基酸种类、数目和/或在Cys1前进行柔性连接肽(如GSGS、GGGGS)的添加。
例如，所述第一连接肽或第二连接肽可以为人IgG的铰链区(SEQ ID NO:73)，所述Cys1为人IgG铰链区根据EU编码的Cys-220。
例如，所述第一连接肽可以为人IgG1的铰链区，连接肽序列如SEQ ID NO:73所示，连接肽中所述Cys1为天然IgG1铰链区根据EU编码的Cys-220，所述Cys2为天然IgG1 铰链区根据EU编码的Cys-226。又例如，所述第一连接肽可以为源自人IgG1的铰链区的衍生序列，其氨基酸序列如SEQ ID NOs:74-105、145-146中任一项所示的序列，连接肽中所述Cys1为对应于人IgG1铰链区Cys-220的Cys，连接肽中所述Cys2为对应于人IgG1铰链区Cys-226的Cys。
例如，所述第二连接肽可以为人IgG1的铰链区，连接肽序列如SEQ ID NO:73所示，连接肽中所述Cys1为天然IgG1铰链区根据EU编码的Cys-220，所述Cys2为天然IgG1铰链区根据EU编码的Cys-226。又例如，所述第二连接肽可以为源自人IgG1的铰链区的衍生序列，其氨基酸序列如SEQ ID NOs:74-105、145-146中任一项所示的序列，连接肽中所述Cys1为对应于人IgG1铰链区Cys-220的Cys，连接肽中所述Cys2为对应于人IgG1铰链区Cys-226的Cys。
例如，所述第一连接肽或第二连接肽可以为小鼠IgG2c的铰链区(SEQ ID NO:147)，所述Cys1为其序列的第一个半胱氨酸，所述Cys2为其序列的第二个半胱氨酸。
在本申请中，所述“一个或多个抗原结合片段”中的“多个抗原结合片段”可以部分或全部相同或都不同；所述“相同”是指两种或以上的抗原结合片段的氨基酸序列相同，所述“不同”是指两种或以上抗原结合片段的氨基酸序列不同，两种或以上的抗原结合片段可以识别不同的抗原或表位，也可以是，两种或以上的抗原结合蛋白识别相同的抗原或相同表位，但序列和结构不同。
在本申请中，所述抗原结合蛋白可以包含至少两个所述抗原结合片段。在本申请中，所述抗原结合蛋白可以包含第一抗原结合片段和第二抗原结合片段(如图42(A)所示结构)，第一抗原结合片段和第二抗原结合片段的氨基酸序列可以相同或不同，第一抗原结合片段和第二抗原结合片段的靶点可以相同或不同。
在本申请中，所述抗原结合蛋白还可以进一步包含第三抗原结合片段和/或第四抗原结合片段(如图42(B)所示结构)；第一、二、三、四抗原结合片段可以部分或全部相同或不同。
在本申请中，一个或多个抗原结合片段可以连接于同一连接肽，也可以连接于不同连接肽。所述抗原结合片段可以位于所述连接肽的N端或者C端。
在本申请中，所述抗原结合蛋白包含第一连接肽和所述第二连接肽，包含第一抗原结合片段和第二抗原结合片段，所述抗原结合蛋白可以包括以下结构：
(a)所述第一抗原结合片段的C端可以与所述第一连接肽的N端直接或间接连接，且所述第二抗原结合片段的C端可以与所述第二连接肽的N端直接或间接连接，具有如图42(A)所示结构；
(b)所述第一抗原结合片段的N端可以与所述第一连接肽的C端直接或间接连接，且所述第二抗原结合片段的C端可以与所述第二连接肽的N端直接或间接连接；
(c)所述第一抗原结合片段的N端可以与所述第一连接肽的C端直接或间接连接，且所述第二抗原结合片段的N端可以与所述第二连接肽的C端直接或间接连接；
(d)所述第一抗原结合片段的C端可以与所述第一连接肽的N端直接或间接连接，且所述第二抗原结合片段的N端可以与所述第二连接肽的C端直接或间接连接。
在本申请中，所述第一抗原结合片段和/或所述第二抗原结合片段可以指任何可以与抗原特异结合的蛋白功能区域，既可以是来源于骆驼或者HCAb转基因鼠的VHH结构域，也可以是单链抗体scFv；或者是其他抗原结合形式(如受体蛋白可溶性胞外区、配体蛋白、脂质运载蛋白lipocalins、神经细胞粘附分子NCAM、纤维结合蛋白fibronectin、锚蛋白重复片段蛋白DARPins等衍生蛋白结构)。但是，所述第一抗原结合片段和/或所述第二抗原结合片段中不包含能够影响药物偶联物与连接肽在Cys1处进行偶联的官能团。
在本申请中，所述第一抗原结合片段和/或第二抗原结合片段中不包含能够影响药物偶联物与连接肽在Cys1处进行偶联的官能团。例如，所述第一抗原结合片段和/或所述第二抗原结合片段不包含能够与所述第一连接肽和/或第二连接肽的Cys1的侧链巯基形成共价键例如二硫键的官能团。例如，在某些具体实施例中，所述第一抗原结合片段和/或第二抗原结合片段不包含含有恒定区Cys-214(Eu编号)的轻链或轻链片段。
在本申请中，所述抗原结合蛋白还可以进一步包含第三抗原结片段和/或第四抗原结合片段。进一步包含的第三抗原结合片段和/或第四抗原结合片段进一步连接在上述(a)或(b)或(c)或(d)的所述抗原结合蛋白的N端或者C端：
例如，所述第三抗原结合片段的C端可以与所述第一抗原结合片段的N端直接或者间接相连，且所述第一抗原结合片段的C端可以与所述第一连接肽的N端连接；和/或，所述第四抗原结合片段的C端可以与所述第二抗原结合片段的N端直接或者间接相连，且所述第二抗原结合片段的C端可以与所述第二连接肽的N端连接；具有如图42(B)所示结构。
在本申请的所述抗原结合蛋白的结构中，所述第三抗原结合片段和/或所述第四抗原 结合片段可以包含任何可以与抗原特异结合的蛋白功能区域，既可以是“Fab”，也可以是“VH”，也可以是单链抗体scFv；还可以是其他抗原结合形式(例如受体蛋白可溶性胞外区、配体蛋白、脂质运载蛋白(lipocalins)、神经细胞粘附分子(NCAM)、纤维结合蛋白(fibronectin)、锚蛋白重复片段蛋白(DARPins)等衍生蛋白结构。
在本申请中，所述抗原结合蛋白还可以包含能够使得第一连接肽和第二连接肽配对的配对单元，所述配对单元可以包含第一配对亚单元和第二配对亚单元，且所述第一配对亚单元可以通过共价键或者非共价键与所述第二配对亚单元相互作用。
在某些实施方式中，“配对单元”可以是抗体Fc片段或其变体的二聚体形式；
在某些实施方式中，“配对单元”可以是非对称的二聚体形式，例如第一配对亚单元是具有“knob”突变(T366W)的Fc片段单体，而第二配对亚单元是具有“hole”突变(T366S/L368A/Y407V)的Fc片段单体；
在某些实施方式中，“配对单元”可以是非对称的二聚体形式，例如第一配对亚单元是CD8的α链，而第二配对亚单元是CD8的β链；
在某些实施方式中，“配对单元”可以是非对称的二聚体形式，例如第一配对亚单元是CD79的α链，而第二配对亚单元是CD79的β链；
在某些实施方式中，“配对单元”可以是非对称的二聚体形式，例如第一配对亚单元是T细胞受体的α链，而第二配对亚单元是T细胞受体的β链。
在某些实施方式中，所述第一配对亚单元的N端可以与所述第一连接肽的C端连接，且所述第二配对亚单元的N端与所述第二连接肽的C端连接；或者，所述第一配对亚单元的N端可以与所述第二连接肽的C端连接，且所述第二配对亚单元的N端与所述第一连接肽的C端连接。
在某些具体实施方式中，本申请提供了一种具有如图42(A)所示结构的抗原结合蛋白，其具有如下结构特征：
所述抗原结合蛋白可以包含第一连接肽和第二连接肽，第一抗原结合片段和第二抗原结合片段，和配对单元；
所述第一连接肽和第二连接肽的氨基酸序列可以相同或不同；
所述第一抗原结合片段与所述第二抗原结合片段的靶点可以相同或不同；所述第一抗原结合片段与所述第二抗原结合片段的氨基酸序列可以相同或不同；
所述配对单元可以是Fc片段同源或异源二聚体；
所述第一抗原结合片段的C端与可以所述第一连接肽的N端直接或间接连接，且所述第二抗原结合片段的C端可以与所述第二连接肽的N端直接或间接连接；
所述第一连接肽和第二连接肽的C端分可以别与所述Fc片段的N端连接；
所述第一连接肽可以包含至少两个半胱氨酸Cys1和Cys2，所述的Cys1和Cys2之间相隔至少3个不为Cys的任意氨基酸；
所述第二连接肽可以包含至少两个半胱氨酸Cys1和Cys2，所述的Cys1和Cys2之间相隔至少3个不为Cys的任意氨基酸；
所述第一连接肽的Cys1和所述第二连接肽的Cys1之间可以形成二硫键；
所述第一连接肽的Cys1和所述第二连接肽的Cys2之间可以形成二硫键；
所述第一连接肽和/或所述第二连接肽可以源自人IgG1铰链区序列(SEQ ID NO:73)或其衍生序列(SEQ ID NOs:74-105、145-146)或者源自小鼠IgG2c的铰链区序列(SEQ ID NO:147)；
所述第一抗原结合片段和/或所述第二抗原结合片段可以指任何可以与抗原特异结合的蛋白功能区域，既可以是来源于骆驼或者HCAb转基因鼠的VHH结构域，也可以是单链抗体scFv；或者是其他抗原结合形式(如受体蛋白可溶性胞外区、配体蛋白、脂质运载蛋白lipocalins、神经细胞粘附分子NCAM、纤维结合蛋白fibronectin、锚蛋白重复片段蛋白DARPins等衍生蛋白结构)；
所述第一抗原结合片段和所述第二抗原结合片段中不包含能够影响药物偶联物与连接肽在Cys1处进行偶联的官能团；例如，不包含能够与所述第一连接肽和所述第二连接肽中Cys1的侧链巯基形成共价键官能团；例如，不包含能够与所述第一连接肽和所述第二连接肽中Cys1的侧链巯基形成二硫键的官能团；例如，不包含含有恒定区Cys-214(Eu编号)的轻链或轻链片段；
在某些实施方式中，所述一个或多个抗原结合片段中不包含能够影响药物偶联物与所述第一连接肽和/或所述第二连接肽在Cys1处进行偶联的官能团或者前述官能团与其他药物偶联物形成的缀合物，所述影响表现为显著降低所述药物偶联物在Cys1位点的偶联几率，所述显著降低可以包括下述一项或多项：
1)显著降低所述Cys1与所述药物偶联物进行偶联的几率，
2)当存在所述官能团或所述缀合物时，使得所述连接肽的Cys1与所述药物偶联物偶联的几率可能降低至50％、40％、30％、20％、10％或更低，
3)当存在所述官能团或所述缀合物时，使得制备得到的所述蛋白-药物偶联物的纯度为低于50％，例如，低于40％、30％、20％、10％或更低，
4)当存在所述官能团或所述缀合物时，所述官能团或所述缀合物与所述Cys1之间发生相互作用，使得所述Cys1不能与所述药物偶联物进行偶联，或者，使得所述Cys1不能在本申请所述的方法中与所述药物偶联物进行偶联；
在某些实施方式中，所述一个或多个抗原结合片段可以不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的官能团或者前述官能团与其他药物偶联物形成的缀合物；
在某些实施方式中，所述一个或多个抗原结合片段可以不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的半胱氨酸或者前述半胱氨酸与其他药物偶联物形成的缀合物。
在某些具体实施方式中，本申请还提供了一种具有如图42(B)所示结构的抗原结合蛋白，其具有如下结构特征：
具有如图42(A)所示结构的抗原结合蛋白的结构特征；
在具有如图42(A)所示结构的抗原结合蛋白的基础上进一步包含了第三抗原结合片段和第四抗原结合片段；
所述第三抗原结合片段的C端可以与所述第一抗原结合片段的N端直接或者间接相连，且所述第一抗原结合片段的C端可以与所述第一连接肽的N端连接；
所述第四抗原结合片段的C端可以与所述第二抗原结合片段的N端直接或者间接相连，且所述第二抗原结合片段的C端可以与所述第二连接肽的N端连接；
所述第一抗原结合片段，所述第二抗原结合片段，所述第三抗原结合片段和/或所述第四抗原结合片段可以部分或完全相同或都不同；例如，第一、二抗原结合片段相同，第三、四结合片段相同，第一、三抗原结合片段不同，第二、四抗原结合片段不同；
所述第三抗原结合片段和/或所述第四抗原结合片段可以包含任何可以与抗原特异结合的蛋白功能区域，既可以是“Fab”，也可以是“VH”，也可以是单链抗体scFv；还可以是其他抗原结合形式(例如受体蛋白可溶性胞外区、配体蛋白、脂质运载蛋白(lipocalins)、神经细胞粘附分子(NCAM)、纤维结合蛋白(fibronectin)、锚蛋白重复片段蛋白(DARPins)等衍生蛋白结构；
所述第三抗原结合片段和/或所述第四抗原结合片段中同样不包含能够影响药物偶联物与连接肽在Cys1处进行偶联的官能团或者前述官能团与其他药物偶联物形成的缀 合物；例如不包含能够与所述第一连接肽和所述第二连接肽中Cys1的侧链巯基形成二硫键的官能团；
所述第三抗原结合片段和/或所述第四抗原结合片段不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的官能团或者前述官能团与其他药物偶联物形成的缀合物；
所述第三抗原结合片段和/或所述第四抗原结合片段不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的半胱氨酸或者前述半胱氨酸与其他药物偶联物形成的缀合物。
本申请的抗原结合蛋白可以靶向特定抗原。
所述抗原结合蛋白可包含一个或多个抗原结合片段，可以结合靶细胞上的特定抗原。例如，所述靶细胞上的特定抗原可以为肿瘤抗原。又例如，所述的肿瘤抗原可以是CD19、BCMA、TSHR、CD171、CS-1、CLL-1、GD3、Tn Ag、FLT3、CD38、CD123、CD44v6、B7H3、B7H4、KIT、IL-13Ra2、IL-11Ra、PSCA、PSMA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-beta、SSEA-4、MUC1、EGFR、NCAM、CAIX、LMP2、EphA2、岩藻糖基GM1(fucosyl GM1)、sLe、GM3、TGS5、HMWMAA、GD2、FOLR1、FOLR2、TEM1/CD248、TEM7R、CLDN6、CLDN18.2、GPRC5D、CXORF61、CD97、CD179a、ALK、多聚唾液酸(polysialic acid)、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TAARP、WT1、ETV6-AML、SPA17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、FOSL1、hTERT、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、AR(雄激素受体)、细胞周期蛋白B1(Cyclin B1)、MYCN、RhoC、CYP1B1、BORIS、SART3、PAX5、OY-TES1、LCK、AKAP-4、SSX2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、CD20、CD30、HER2、ROR1、FLT3、TAAG72、CD22、CD33、GD2、gp100Tn、FAP、酪氨酸酶(tyrosinase)、EPCAM、CEA、IGF-1R、EphB2、间皮素(mesothelin)、钙黏蛋白17(Cadherin17)、CD32b、EGFRvIII、GPNMB、GPR64、HER3、LRP6、LYPD8、NKG2D、SLC34A2、SLC39A6、SLITRK6、GUCY2C、5T4和/或TACSTD2等。
例如，所述的肿瘤抗原可以是MSLN；例如，所述的肿瘤抗原可以是BCMA；例如，所述的肿瘤抗原可以是5T4；例如，所述的肿瘤抗原可以是ROR1。
在某些实施方式中，所述的分离的抗原结合蛋白可以包括抗体或其抗原结合片段。 例如，所述抗原结合片段可以包括抗体重链可变区VH，又例如，所述抗原结合片段可以包括单链抗体可变区scFv，又例如，所述抗原结合片段可以包括受体蛋白可溶性胞外区融合蛋白。
例如，所述抗原结合蛋白可以包含靶向CTLA4的抗体或其抗原结合片段、靶向MSLN的抗体或其抗原结合片段、靶向BCMA的抗体或其抗原结合片段、靶向5T4的抗体或其抗原结合片段、靶向ROR1的抗体或其抗原结合片段和/或靶向CD47的SIRPα胞外区衍生的可溶性融合蛋白。
在本申请中，所述CTLA4抗原结合片段可包含重链可变区；其重链可变区VH包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:7、22和38所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。例如，其重链可变区VH可包括如SEQ ID NO:55所示的氨基酸序列。又例如，所述CTLA4抗原结合蛋白可包含一个多肽链，如SEQ ID NO:64所示的氨基酸序列。
在本申请中，所述CTLA4抗原结合片段可包含重链可变区；其重链可变区VH可包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:8、23和39所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。例如，其重链可变区VH可包括如SEQ ID NO:56所示的氨基酸序列。又例如，所述CTLA4抗原结合蛋白可包含一个多肽链，如SEQ ID NO:65所示的氨基酸序列。
在本申请中，所述MSLN抗原结合片段可包含重链可变区；其重链可变区VH可包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:10、25和41所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。例如，其重链可变区VH可包括如SEQ ID NO:58所示的氨基酸序列。又例如，所述MSLN抗原结合蛋白可包含一个多肽链，如SEQ ID NO:67所示的氨基酸序列。
在本申请中，所述5T4抗原结合片段可包含重链可变区；其重链可变区VH可包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:13、28和44所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。例如，其重链可变区VH可包括如SEQ ID NO:61所示的氨基酸序列。又例如，所述5T4抗原结合蛋白可包含一个多肽链，如SEQ ID NO:69所示的氨基酸序列。
在本申请中，所述BCMA抗原结合片段可包含重链可变区；其重链可变区VH可包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:9、24和40所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。例如，其重链可变区VH可包括如 SEQ ID NO:57所示的氨基酸序列。又例如，所述BCMA抗原结合蛋白可包含一个多肽链，如SEQ ID NO:66所示的氨基酸序列。
在本申请中，所述BCMA抗原结合片段可包含重链可变区；其重链可变区VH可包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:11、26和42所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。例如，其重链可变区VH可包括如SEQ ID NO:59所示的氨基酸序列。又例如，所述BCMA抗原结合蛋白可包含一个多肽链，如SEQ ID NO:68所示的氨基酸序列。
在本申请中，所述BCMA抗原结合片段可包含重链可变区；其重链可变区VH可包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:14、29和42所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。例如，其重链可变区VH可包括如SEQ ID NO:62所示的氨基酸序列。又例如，所述BCMA抗原结合蛋白可包含一个多肽链，如SEQ ID NO:70所示的氨基酸序列。又例如，所述BCMA抗原结合蛋白可包含一个多肽链，如SEQ ID NOs:106-129、148中任一项所示的氨基酸序列。
在本申请中，所述ROR1抗原结合片段可包含轻链可变区和重链可变区；其轻链可变区VL可包含LCDR1、LCDR2和LCDR3，分别为SEQ ID NO:49、51和53所示的氨基酸序列；其重链可变区VH可包含HCDR1、HCDR2和HCDR3，分别为SEQ ID NO:12、27和43所示的氨基酸序列。所列CDR的氨基酸序列按照Chothia定义规则所示。例如，其轻链可变区VL可包括如SEQ ID NO:63所示的氨基酸序列，其重链可变区VH可包括如SEQ ID NO:60所示的氨基酸序列。又例如，所述ROR1抗原结合蛋白可包含一个多肽链，如SEQ ID NO:71所示的氨基酸序列。
在本申请中，所述CD47抗原结合蛋白可包含一个多肽链，如SEQ ID NO:149所示的氨基酸序列。
在本申请中，所述抗原结合蛋白还可以包括重链恒定区CH2和CH3以及铰链区，所述重链恒定区(含铰链区)的序列包括如SEQ ID NO:72所示的氨基酸序列，所述铰链区序列优选人IgG1抗体重链铰链区序列(SEQ ID NO:73)。
在本申请中，所述抗原结合蛋白还可以是具有如图59所示的对称的、含有四个抗原结合片段的且含有四条多肽链的结构；所述抗原结合蛋白包括抗原结合片段A和抗原结合片段B；所述抗原结合片段A和所述抗原结合片段B靶向不同的抗原或抗原表位；所述抗原结合片段A为Fab结构，所述抗原结合片段B为VH结构；在所述抗原结合蛋白中，所述抗原结合片段A的数量为二个，所述抗原结合片段B的数量为二个；所述抗原 结合蛋白从N端至C端依次为抗原结合片段A、抗原结合片段B和Fc，其中所述抗原结合片段A与所述抗原结合片段B之间直接相连，所述抗原结合片段B与所述Fc通过连接肽(或铰链区)连接。所述抗原结合蛋白具有四条多肽链，所述四条多肽链包含第一多肽链、第二多肽链、第三多肽链和第四多肽链；所述第一多肽链和所述第二多肽链具有相同的氨基酸序列，称为“长链”；所述第三多肽链和所述第四多肽链具有相同的氨基酸序列，称为“短链”；所述短链从N端至C端依次包括VH_A-CH1，所述长链从N端至C端依次包括VL_A-CL-VH_B-L-CH2-CH3；其中，VH_A和VL_A分别为所述抗原结合片段A的重链可变区和轻链可变区，VH_B为所述抗原结合片段B的重链可变区，所述L为连接肽。在某些实施方式中，所述连接肽具有如SEQ ID NO:73所示的氨基酸序列。在某些实施方式中，所述抗原结合片段A包含轻链可变区和重链可变区，其轻链可变区VL包括如SEQ ID NO:131所示的氨基酸序列，其重链可变区VH包括如SEQ ID NO:130所示的氨基酸序列；所述抗原结合片段B包含重链可变区，其重链可变区VH包括如SEQ ID NO:62所示的氨基酸序列。在某些实施方式中，所述抗原结合蛋白包含两条相同的“短链”和两条相同的“长链”(图59)，所述“短链”包括如SEQ ID NO:134所示的氨基酸序列，所述“长链”包括如SEQ ID NO:135所示的氨基酸序列。
本申请中，所述的CDR均可包含在所限序列的基础上进行突变的情形。所述突变为在所述的VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2、VL CDR3的氨基酸序列的基础上分别具有3、2或1个氨基酸的插入、缺失或替换。本申请中，在类似“具有3、2或1个氨基酸的插入、缺失或替换”中“氨基酸突变”是指相较于原氨基酸序列而言，变体的序列存在氨基酸的突变，包括在原氨基酸序列的基础上发生氨基酸的插入、缺失或替换。示例性的解释是对CDR的突变可以包含3个、2个或1个氨基酸的突变，这些CDR之间可以任选地选择相同或不同数目的氨基酸残基进行突变，例如可以是对CDR1进行1个氨基酸的突变，对CDR2和CDR3不进行氨基酸突变。
本申请中，所述的VH、VL、或所述的多肽链均可包含在所限定的序列的基础上进行突变的情形。所述突变为所限定的氨基酸序列上发生了一个或多个氨基酸残基的缺失、取代或添加，且所述突变的氨基酸序列与所限定的氨基酸序列具有至少85％序列同源性，并保持或改善了所述抗体或其抗原结合片段、结合蛋白的结合活性；所述至少85％序列同源性优选为至少90％序列同源性；更优选为至少95％序列同源性；最优选为至少99％序列同源性。
在本申请中，所述同源性通常是指两个或多个序列之间的相似性、类似或关联。可 以通过以下方式计算“序列同源性百分比”：将两条待比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同源性百分比。为了确定序列同源性百分数而进行的比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。所述同源性也可以通过以下的方法测定：FASTA和BLAST。对FASTA算法的描述可以参见W.R.Pearson和D.J.Lipman的“用于生物学序列比较的改进的工具”，美国国家科学院院刊(Proc.Natl.Acad.Sci.)，85：2444-2448，1988；和D.J.Lipman和W.R.Pearson的“快速灵敏的蛋白质相似性搜索”，Science，227：1435-1441，1989。对BLAST算法的描述可参见S.Altschul、W.Gish、W.Miller、E.W.Myers和D.Lipman的“一种基本的局部对比(alignment)搜索工具”，分子生物学杂志，215：403-410，1990。
药物偶联物
本申请所述蛋白-药物偶联物可以包含至少一个药物偶联物；所述药物偶联物可以以共价结合形式联结在所述蛋白-药物偶联物中的蛋白部分上。
所述药物偶联物可以包含至少一个载荷药物。所述载荷药物可以包含药物活性物成分和/或标记分子。
在本申请中，载荷药物是一类具有药学活性的小分子化合物或者毒素或其他药物分子形式，可以是但不局限于小分子化合物、毒素分子、抗生素、寡核苷酸、蛋白降解靶向嵌合体(PROTAC)、亲和配体、荧光基团、核素基团、多肽、免疫调节分子(例如，Toll样受体激动剂分子，STING激动剂分子)等。本领域已知多种载荷药物。例如，小分子化合物通常是指具有较强细胞毒性的一类物质。示例性的小分子化合物可通过包括但不限于微管蛋白结合、DNA结合、抑制RNA聚合酶、蛋白质合成或抑制拓扑异构酶等机制来发挥此类细胞毒性和细胞抑制性效应。例如，小分子化合物可以是微管蛋白抑制剂； 例如，所述微管蛋白抑制剂可以是美登素(例如，DM1或DM4)和奥瑞司他汀(例如，MMAE或MMAF)等等。例如，所述小分子化合物可以是DNA损伤剂；例如，所述DNA损伤剂可以是卡奇霉素(Calicheamicins)、吡咯并苯并二氮杂卓(PBD，pyrrolobenzodiazepines)等等。例如，蛋白降解靶向嵌合体(PROTAC)是一类能够通过诱导靶蛋白的多聚泛素化而导致靶蛋白降解的化合物；例如，所述PROTAC可以是BET蛋白降解剂。
在本申请中，所述药物偶联物还可以包含至少一个连接子。例如，所述连接子可包含可断裂连接子或非可断裂连接子。所述连接子用于将一个或多个载荷药物连接到抗原结合蛋白。在本申请中，可断链的连接子可以是便于释放药物的“可切割”连接子。例如，可切割连接子可以包括但不局限于酸敏感连接子，蛋白酶敏感连接子，光敏感连接子，或含二硫化物连接子。
在本申请中，所述连接子可具有能够与抗原结合蛋白上存在的游离半胱氨酸反应以形成共价键的官能团。例如，此类反应性官能团可以包括但不局限于马来酰亚胺，卤代乙酰胺，α-卤代乙酰基，活化的酯诸如琥珀酰亚胺酯，4-硝基苯基酯，五氟苯基酯，四氟苯基酯，酸酐，酰氯，磺酰氯，异氰酸酯，和异硫氰酸酯。例如，所述连接子可具有能够与抗原结合蛋白上存在的亲电子基团反应的官能团。例如，此类反应性官能团可以包括但不局限于酰肼(hydrazide)，肟(oxime)，氨基(amino)，肼(hydrazine)，硫代半卡巴腙(thiosemicarbazone)，肼羧酸酯(hydrazine carboxylate)，和芳酰肼(arylhydrazide)。
在本申请中，连接子可包含一种或多种连接子构件，本领域已知多种连接子构件，例如，马来酰亚胺基己酰基(“MC”，maleimidocaproyl)，马来酰亚胺基丙酰基(“MP”)，缬氨酸-瓜氨酸(“val-cit”或“vc”，valine-citrulline)，对氨基苄氧羰基(“PAB”，p-aminobenzyloxycarbonyl)。
在本申请中，所述药物偶联物是mc-vc-PAB-MMAE(也称为VcMMAE，结构如图6所示，CAS No.:646502-53-6)，其包含连接子mc-vc-PAB和载荷药物MMAE。
在本申请中，所述药物偶联物还可以是BRD4蛋白降解剂(结构如图64所示)，其包含靶向BET的PROTAC GNE-987和含二硫化物的连接子。
蛋白-药物偶联物
另一方面，本申请提供了一种蛋白-药物偶联物。
在本申请中，所述蛋白-药物偶联物包含至少一个抗原结合蛋白部分和至少一个药物 偶联物部分。
在本申请中，所述抗原结合蛋白部分是前面“抗原结合蛋白”描述的靶向特定抗原的、包含一个或多个抗原结合片段的和至少两个连接肽的抗原结合蛋白。
在本申请中，所述药物偶联物包含至少一个载荷药物。所述载荷药物可以包含但不局限于小分子化合物、毒素分子、抗生素、寡核苷酸、蛋白降解靶向嵌合体(PROTAC)、亲和配体、荧光标记基团、核素标记基团、多肽、免疫调节分子(例如，Toll样受体激动剂)等。
在本申请中，所述药物偶联物还可以包含至少一个连接子。
在本申请中，所述载荷药物可以通过所述连接子以共价键结合方式偶联到所述抗原结合蛋白；在本申请中，所述载荷药物可以通过所述连接子以共价键结合方式偶联到所述抗原结合蛋白中的所述连接肽上。
在本申请中，所述药物偶联物可以通过所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1与所述抗原结合蛋白联结，进而产生所述的蛋白-药物偶联物。
在本申请中，通过断裂抗原结合蛋白第一连接肽和第二连接肽的Cys1间的二硫键形成反应性巯基官能团实现与药物偶联物在Cys1处的共价结合。
在本申请中，所述一个或多个抗原结合片段不包含能够影响药物偶联物与连接肽在Cys1处进行偶联的官能团；例如，不包含能够与所述连接肽中Cys1的侧链巯基形成共价键官能团；例如，不包含能够与所述连接肽中Cys1的侧链巯基形成二硫键的官能团。
在本申请中，所述药物偶联物与所述抗原结合蛋白的联结主要发生在所述第一连接肽和/或所述第二连接肽的Cys1位点，几乎不发生在其他半胱氨酸位点(例如，Cys2位点)。例如，所述连接肽的Cys1被所述药物偶联物所结合的几率至少为50％，也可以至少为55％，也可以至少为60％，也可以至少为65％，也可以至少为70％，也可以至少为75％，也可以至少为80％，也可以至少为85％，也可以至少为90％，也可以至少为95％，也可以至少为99％。例如，所述连接肽的Cys2被所述药物偶联物所结合的几率至多为50％，也可以至多为45％，也可以至多为40％，也可以至多为35％，也可以至多为30％，也可以至多为25％，也可以至多为20％，也可以至多为15％，也可以至多为10％，也可以至多为5％。
在本申请中，以所述第一连接肽和/或第二连接肽中的Cys1作为偶联位点，联结本申请所述药物偶联物，可以产生均一DAR值的蛋白-药物偶联物。在一些具体的实施例中，产生含有两个药物偶联物(即CAR＝2；当所述药物偶联物的一个单元仅含有一个载 荷药物时，也是DAR＝2，也作D2)的均一偶联产物。在一些具体的实施例中，偶联产物中的具有CAR＝2(或DAR＝2，也作D2)的蛋白-药物偶联物分子为主要组分，该所述CAR＝2(或DAR＝2，也作D2)的组分的含量至少为50％，也可以至少为55％，也可以至少为60％，也可以至少为65％，也可以至少为70％，也可以至少为75％，也可以至少为80％，也可以至少为85％，也可以至少为90％，也可以至少为95％。在一些具体的实施例中，偶联产物中含有超过80％的CAR＝2(或DAR＝2，也作D2)的组分，且偶联主要发生于第一连接肽和第二连接肽中的所述Cys1；无需经过进一步纯化步骤，即可得到均一DAR值的蛋白-药物偶联物。
在本申请中，利用人IgG1抗体重链铰链区的天然序列(SEQ ID NO:73)中的半胱氨酸Cys-220(Eu编号)作为偶联位点，联结本申请所述药物偶联物，可以产生均一DAR值的蛋白-药物偶联物。在一些具体的实施例中，产生含有两个药物偶联物(即CAR＝2；当所述药物偶联物的一个单元仅含有一个载荷药物时，也是DAR＝2，也作D2)的均一偶联产物。在一些具体的实施例中，偶联产物中的具有CAR＝2(或DAR＝2，也作D2)的蛋白-药物偶联物分子为主要组分，该所述CAR＝2(或DAR＝2，也作D2)的组分的含量至少为50％，也可以至少为55％，也可以至少为60％，也可以至少为65％，也可以至少为70％，也可以至少为75％，也可以至少为80％，也可以至少为85％，也可以至少为90％，也可以至少为95％。在一些具体的实施例中，偶联产物中含有超过80％的CAR＝2(或DAR＝2，也作D2)的组分，且偶联主要发生于人IgG1铰链区中的Cys-220(Eu编号)；无需经过进一步纯化步骤，即可得到均一DAR值的蛋白-药物偶联物。
在本申请中，所述蛋白-药物偶联物可以具有如图41或图43所示结构。
在本申请中，所述蛋白-药物偶联物可以是抗体偶联药物(ADC)。
本申请还提供了一种利用具有如图42(A)所示结构的抗原结合蛋白进行药物偶联，得到的具有如图43(A)所示结构的蛋白-药物偶联物，其具有如下结构特征：
具有如图42(A)所示结构的抗原结合蛋白的结构特征；
所述蛋白-药物偶联物包含第一连接肽和所述第二连接肽，第一抗原结合片段和二抗原结合片段；
所述第一连接肽和所述第二连接肽的氨基酸序列可以相同或不同；
所述第一抗原结合片段与所述第二抗原结合片段的靶点可以相同或不同；所述第一抗原结合片段和所述第二抗原结合片段的氨基酸序列可以相同或不同；
所述蛋白-药物偶联物还包含配对单元，例如Fc片段；
所述第一连接肽和所述第二连接肽包含至少两个半胱氨酸，从N端到C端分别命名为Cys1和Cys2；
所述第一连接肽和/或第二连接肽的序列可以是人IgG1铰链区序列(SEQ ID NO:73)或其衍生序列(SEQ ID NOs:74-105、145-146)，所述第一连接肽和/或第二连接肽的所述Cys1可以是人IgG1铰链区序列的Cys-220(Eu编号)或衍生序列中对应于人IgG1铰链区序列的Cys-220(Eu编号)的Cys；且所述Cys2可以是人IgG1铰链区序列的Cys-226(Eu编号)或衍生序列中对应于人IgG1铰链区序列的Cys-226(Eu编号)的Cys；
所述第一连接肽和/或第二连接肽的序列还可以是小鼠IgG2c的铰链区序列(SEQ ID NO:147)或其衍生序列，所述第一连接肽和/或第二连接肽的所述Cys1可以是小鼠IgG2c的铰链区序列或其衍生序列的第一个半胱氨酸，且所述Cys2可以是小鼠IgG2c的铰链区序列或其衍生序列的第二个半胱氨酸；
所述蛋白-药物偶联物在制备过程中，所述第一连接肽和所述第二连接肽的Cys2间的二硫键(即图42(A)所示结构的抗原结合蛋白的“二硫键Cys2-Cys2”)被保留；
所述蛋白-药物偶联物在制备过程中，所述第一连接肽和所述第二连接肽的Cys1间的二硫键(即图42(A)所示结构的抗原结合蛋白的“二硫键Cys1-Cys1”)被断裂用于药物偶联物的偶联；
所述第一抗原结合片段和/或所述第二抗原结合片段不影响所述药物偶联物在所述第一连接肽和/或第二连接肽的所述Cys1位点的偶联；
所述第一抗原结合片段和/或所述第二抗原结合片段中不包含能够影响所述药物偶联物在所述第一连接肽和/或所述第二连接肽的Cys1上发生偶联的官能团；
所述第一抗原结合片段和/或所述第二抗原结合片段中不包含能够与Cys1的侧链巯基形成共价键的官能团；
所述第一抗原结合片段和/或所述第二抗原结合片段中不包含能够与Cys1形成二硫键的官能团。
本申请还提供了一种利用具有如图42(B)所示结构的抗原结合蛋白进行药物偶联，得到的具有如图43(B)所示结构的蛋白-药物偶联物，其具有如下结构特征：
具有如图42(B)所示结构的抗原结合蛋白的结构特征；
所述蛋白-药物偶联物包含第一连接肽和所述第二连接肽，包含第一抗原结合片段，所述第二抗原结合片段，所述第三抗原结合片段和所述第四抗原结合片段；
所述第一连接肽和所述第二连接肽的氨基酸序列可以相同或不同；
所述第一抗原结合片段，所述第二抗原结合片段，所述第三抗原结合片段和/或所述第四抗原结合片段可以部分或完全相同或都不同；例如，第一、二抗原结合片段相同，第三、四结合片段相同，第一、三抗原结合片段不同，第二、四抗原结合片段不同；所述相同或不同的部分可以是所述抗原结合片段结合的靶点和/或氨基酸序列。
所述蛋白-药物偶联物还包含配对单元，例如Fc片段；
所述第一连接肽和所述第二连接肽包含至少两个半胱氨酸，从N端到C端分别命名为Cys1和Cys2；
所述第一连接肽和/或第二连接肽的序列可以是人IgG1铰链区序列(SEQ ID NO:73)或其衍生序列(SEQ ID NOs:74-105、145-146)，所述第一连接肽和/或第二连接肽的所述Cys1可以是人IgG1铰链区序列的Cys-220(Eu编号)或衍生序列中对应于人IgG1铰链区序列的Cys-220(Eu编号)的Cys；且所述Cys2可以是人IgG1铰链区序列的Cys-226(Eu编号)或衍生序列中对应于人IgG1铰链区序列的Cys-226(Eu编号)的Cys；
所述第一连接肽和/或第二连接肽的序列还可以是小鼠IgG2c的铰链区序列(SEQ ID NO:147)或其衍生序列，所述第一连接肽和/或第二连接肽的所述Cys1可以是小鼠IgG2c的铰链区序列或其衍生序列的第一个半胱氨酸，且所述Cys2可以是小鼠IgG2c的铰链区序列或其衍生序列的第二个半胱氨酸；
所述蛋白-药物偶联物在制备过程中，所述第一连接肽和所述第二连接肽的Cys2间的二硫键(即图42(B)所示结构的抗原结合蛋白的“二硫键Cys2-Cys2”)被保留；
所述蛋白-药物偶联物在制备过程中，所述第一连接肽和所述第二连接肽的Cys1间的二硫键(即图42(B)所示结构的抗原结合蛋白的“二硫键Cys1-Cys1”)被断裂用于药物偶联物的偶联；
所述第一抗原结合片段和/或所述第二抗原结合片段和/或所述第三抗原结合片段和/或所述第四抗原结合片段不包含能够影响药物偶联物与连接肽在Cys1处进行定点偶联的官能团；
所述第一抗原结合片段和/或所述第二抗原结合片段和/或所述第三抗原结合片段和/或所述第四抗原结合片段中不包含能够与Cys1的侧链巯基形成共价键的官能团；
所述第一抗原结合片段和/或所述第二抗原结合片段和/或所述第三抗原结合片段和/或所述第四抗原结合片段中不包含能够与Cys1的侧链巯基形成二硫键的官能团。
本申请还提供了一种蛋白-药物偶联物。
在某些情形中，所述药物偶联物可以包括第一多肽链和第二多肽链。所述第一多肽链自N端至C端，可依次包含第一抗原结合片段、第一连接肽和第一配对亚单元。所述第二多肽链自N端至C端，可依次包含第二抗原结合片段、第二连接肽和第二配对亚单元。其中，所述第一配对亚单元和所述第二配对亚单元可以相互作用以形成二聚体。
例如，所述第一多肽链自N端至C端，可依次包含第一抗原结合片段的VH、第一抗原结合片段的VL、第一连接肽(例如，人IgG1铰链区或小鼠IgG2c铰链区或衍生序列)、CH 2和CH 3，且药物偶联物通过所述第一连接肽的Cys1(例如，IgG铰链区的Cys220(EU编码))与所述第一多肽链连接。例如，所述第二多肽链自N端至C端，可依次包含第二抗原结合片段的VH、第二抗原结合片段的VL、第二连接肽(例如，人IgG1铰链区或小鼠IgG2c铰链区或衍生序列)、CH 2和CH 3，且药物偶联物通过所述第二连接肽的Cys1(例如，IgG铰链区的Cys220(EU编码))与所述第二多肽链连接。
例如，所述第一多肽链自N端至C端，可依次包含第一抗原结合片段的VH、第一连接肽(例如，人IgG1铰链区或小鼠IgG2c铰链区或衍生序列)、CH 2和CH 3，且药物偶联物通过所述第一连接肽的Cys1(例如，IgG铰链区的Cys220(EU编码))与所述第一多肽链连接。例如，所述第二多肽链自N端至C端，可依次包含第二抗原结合片段的VH、第二连接肽(例如，人IgG1铰链区或小鼠IgG2c铰链区或衍生序列)、CH 2和CH 3，且药物偶联物通过所述第二连接肽的Cys1(例如，IgG铰链区的Cys220(EU编码))与所述第二多肽链连接。
例如，所述第一多肽链自N端至C端，可依次包含第一抗原结合片段的受体蛋白可溶性胞外区、第一连接肽(例如，人IgG1铰链区或小鼠IgG2c铰链区或衍生序列)、CH 2和CH 3，且药物偶联物通过所述第一连接肽的Cys1(例如，IgG铰链区的Cys220(EU编码))与所述第一多肽链连接。例如，所述第二多肽链自N端至C端，可依次包含第二抗原结合片段的受体蛋白可溶性胞外区、第二连接肽(例如，人IgG1铰链区或小鼠IgG2c铰链区或衍生序列)、CH 2和CH 3，且药物偶联物通过所述第二连接肽的Cys1(例如，IgG铰链区的Cys220(EU编码))与所述第二多肽链连接。
在某些情形中，所述蛋白-药物偶联物可包含第一多肽链、第二多肽链、第三多肽链和第四多肽链。所述第一多肽链自N端至C端，可依次包含第三抗原结合片段、第一抗原结合片段、第一连接肽和第一配对亚单元。所述第二多肽链自N端至C端，可依次包含第四抗原结合片段、第二抗原结合片段、第二连接肽和第二配对亚单元。其中，所述第 一配对亚单元和所述第二配对亚单元可以相互作用以形成二聚体。所述第三多肽链可包含第三抗原结合片段。所述第四多肽链可包含第四抗原结合片段。
例如，所述第一多肽链自N端至C端，可依次包含第三抗原结合片段的VL、第三抗原结合片段的CL、第一抗原结合片段的VH、第一连接肽(例如，人IgG1铰链区或小鼠IgG2c铰链区或衍生序列)、CH 2和CH 3，且药物偶联物通过所述第一连接肽的Cys1(例如，IgG铰链区的Cys220(EU编码))与所述第一多肽链连接。例如，所述第二多肽链自N端至C端，可依次包含第四抗原结合片段的VL、第四抗原结合片段的CL、第二抗原结合片段的VH、第二连接肽(例如，人IgG1铰链区或小鼠IgG2c铰链区或衍生序列)、CH 2和CH 3，且药物偶联物通过所述第二连接肽的Cys1(例如，IgG铰链区的Cys220(EU编码))与所述第二多肽链连接。例如，所述第三多肽链自N端至C端，可依次包含第三抗原结合片段的VH和第三抗原结合片段的CH 1。例如，所述第四多肽链自N端至C端，可依次包含第四抗原结合片段的VH和第四抗原结合片段的CH 1。例如，所述第三抗原结合片段和/或第四抗原结合片段可以为Fab。
核酸、载体、宿主细胞
另一方面，本申请提供了一种或多种核酸分子，其可以编码如本申请所述的蛋白-药物偶联物。例如，所述一种或多种核酸分子中的每一个核酸分子可以编码完整的所述蛋白-药物偶联物，也可以编码其中的一部分(例如，HCDR1-3、LCDR1-3、VL、VH、多肽链或重链中的一种或多种)。
本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如可以通过聚合酶链式反应(PCR)扩增产生的，(ii)可以通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如可以通过化学合成。
例如，所述分离的核酸可以是通过重组DNA技术制备的核酸分子。
另一个方面，本申请提供了一种或多种载体，其包含本申请所述的一种或多种核酸分子。每种载体中可包含一种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转 录或mRNA翻译的其他控制元件等。例如，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。所述表达控制序列还可包括增强子序列或上游活化子序列。在本申请中，适当的启动子可包括，例如用于SP6、T3和T7聚合酶的启动子、人U6RNA启动子、CMV启动子及其人工杂合启动子(如CMV)，其中启动子的某部分可与其他细胞蛋白(如人GAPDH，甘油醛-3-磷酸脱氢酶)基因启动子的某部分融合，其可包含或不包含另外的内含子。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。
所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体为表达载体。例如，所述表达载体可以包含真核细胞表达载体和/或原核细胞表达载体。
另一方面，本申请提供了宿主细胞，所述宿主细胞可包含本申请所述的一种或多种核酸分子和/或本申请所述的一种或多种载体。例如，每种或每个宿主细胞可包含一个或一种本申请所述的核酸分子或载体。在某些实施方式中，每种或每个宿主细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的核酸分子或载体。例如，可将本申请所述的载体引入所述宿主细胞中，例如原核细胞(例如，细菌细胞)，哺乳动物真核细胞或者其他真核细胞，如来自植物的细胞、真菌或酵母细胞等。可通过本领域已知的方法将本申请所述的载体引入所述宿主细胞中，例如电穿孔、lipofectine转染、lipofectamin转染等。例如，所述宿主细胞可以为COS、CHO、NSO、sf9、sf21、DH5a、BL21(DE3)或TG1。例如，所述原核细胞可以选择E.coli细胞如TG1、BL21。例如，所述真核细胞可以选择如CHO细胞、CHO-K1细胞、CHOZN细胞、CHO-S细胞、ExpiCHO-S细胞、NS/0细胞、HEK293细胞、HEK293-T细胞、HEK293-F细胞或HEK293-6E细胞。
方法、药物组合物和用途
另一方面，本申请还提供了一种制备所述蛋白-药物偶联物的方法，其包括使所述药物偶联物与所述连接肽的特定的半胱氨酸(例如，所述第一连接肽的Cys1和/或所述第二连接肽的Cys1)以共价键方式结合，以得到本申请所述的蛋白-药物偶联物。例如，所 述制备方法可以是一种基于特定位点的游离半胱氨酸的反应性巯基官能团进行药物偶联的过程，所述制备方法可以产生出具有偶联位点特异性和均质性的蛋白-药物偶联物。又例如，在某些具体实施方式中，所述制备方法可以产生出高均质性的CAR＝2(一个抗原结合蛋白携带两个偶联物)的蛋白-药物偶联物。
所述制备方法可以包括由“还原”、“再氧化”、“偶联”和“纯化”等多种实施步骤组成的一个或多个步骤的组合。例如，所述制备方法可以包括“还原”、“偶联”和“纯化”等步骤；又例如，所述制备方法可以包括“还原”和“偶联”等步骤。所述实施步骤可以包含一个或多个反应过程。在所述反应过程中，反应条件例如反应温度、反应时间、反应缓冲液类型及pH、还原剂类型及用量、偶联物分子类型及用量等，都可能对所述制备方法的结果产生重大影响，例如对药物偶联的效率和终产物的质量产生重大影响。所述反应过程还可以包含不同反应条件参数的组合；例如，使二硫键还原的反应过程可以结合不同的还原剂类型、不同的还原剂用量、不同的还原反应温度、不同的反应缓冲液、不同的pH和不同的反应时长等多个参数条件。
在一些具体实施方式中，所述制备方法的所述“还原”步骤可以使用还原剂DTT(dithiothreitol)或TCEP(Tris(2-carboxyethyl)phosphine)。
在一些具体实施方式中，所述制备方法的所述“还原”步骤的还原剂用量为约1～50摩尔倍数，例如，约1～30摩尔倍数，约1～20摩尔倍数，约1～10摩尔倍数，约1～7摩尔倍数，约1～6摩尔倍数，约1～5摩尔倍数，约1.5～6摩尔倍数或约1.5～5摩尔倍数。
在一些具体实施方式中，所述制备方法的所述“还原”步骤的反应时长为约1～17小时，例如，约1～10小时，约1～7小时，约1～5小时，约1～4小时，约1～3小时或约1.5～2小时。
在一些具体实施方式中，所述制备方法的所述“还原”步骤的反应温度为约0～40℃，例如，0～37℃，约0～30℃，约20～37℃，约20～30℃，或室温(约25-30℃)。例如，反应温度为0℃、室温(25-30℃)或37℃。
在一些具体实施方式中，所述制备方法的所述“还原”步骤的反应缓冲液pH为约4.0～9.0、约5.0～8.0或约5.0～7.0。
在一些具体实施方式中，所述制备方法的所述“还原”步骤可以使用还原剂TCEP，还原剂用量为1～7摩尔倍数，反应时长为1～3小时，反应温度为23～30℃，反应缓冲液pH为5.0～7.0。
在一些具体实施方式中，所述制备方法的所述“还原”步骤可以使用还原剂DTT(dithiothreitol)或TCEP(Tris(2-carboxyethyl)phosphine)，还原剂用量为1～50摩尔倍数，反应时长为1～17小时，反应温度为0～40℃，反应缓冲液pH为5.0～8.0。较佳地，还原剂为TCEP，TCEP用量可以为1～7摩尔倍数，反应时长可以为1～3小时，反应缓冲液pH为5.0～7.0，反应温度为0℃或室温(25-30℃)或37℃。更佳地，还原剂TCEP用量可以为1.5～6摩尔倍数，反应时长可以为1.5～2小时，反应缓冲液pH为5.0～6.0，反应温度为0℃或室温(25-30℃)或37℃。
在一些具体实施方式中，所述制备方法的所述“偶联”步骤可以使用mc-vc-PAB-MMAE(图6)作为药物偶联物。
在一些具体实施方式中，所述制备方法的所述“偶联”步骤可以使用BRD4蛋白降解剂(图64)作为药物偶联物。
在一些具体实施方式中，所述制备方法的所述“偶联”步骤的药物偶联物用量为约1～50摩尔倍数，例如，约1～30摩尔倍数，约1～20摩尔倍数，约10～15摩尔倍数，约1～10摩尔倍数，约1～7摩尔倍数或约3～7摩尔倍数。
在一些具体实施方式中，所述制备方法的所述“偶联”步骤的反应时长为约0.5～10小时，例如，约0.5～10小时，约0.5～3小时，约0.5～2小时，约1～2小时或约0.5～1。
在一些具体实施方式中，所述制备方法的所述“偶联”步骤的反应温度为约0～40℃，例如，0～37℃，约0～30℃，约20～37℃，约20～30℃，或室温(约25-30℃)。例如，反应温度为室温(25-30℃)。
在一些具体实施方式中，所述制备方法的所述“偶联”步骤的反应缓冲液pH为约4.0～9.0、约5.0～8.0或约5.0～7.0。
在一些具体实施方式中，所述制备方法的所述“偶联”步骤可以使用mc-vc-PAB-MMAE(图6)作为药物偶联物，药物偶联物用量为3～7摩尔倍数，反应时长为0.5～2小时，反应温度为23～30℃，反应缓冲液pH为5.0～7.0。
在一些具体实施方式中，所述制备方法的所述“偶联”步骤可以使用mc-vc-PAB-MMAE(图6)作为药物偶联物，药物偶联物用量为1～50摩尔倍数，反应时长为0.5～10小时，反应温度为0～40℃，反应缓冲液pH为5.0～8.0。较佳地，mc-vc-PAB-MMAE用量可以为3～18摩尔倍数，反应时长可以为0.5～3小时，反应缓冲液pH为5.0～7.0，反应温度为室温(25-30℃)。更佳地，mc-vc-PAB-MMAE用量可以为3～7摩尔倍 数，反应时长可以为0.5小时，反应缓冲液pH为5.0～6.0，反应温度为室温(25-30℃)。
在一些具体实施方式中，所述制备方法的所述“偶联”步骤可以使用BRD4蛋白降解剂(图64)作为药物偶联物，药物偶联物用量为10～15摩尔倍数，反应时长为1～3小时，反应温度为23～30℃，反应缓冲液pH为7.0～9.0。
在一些具体实施方式中，所述制备方法还可以提供一种包括“还原”和“偶联”等步骤的组合和反应条件参数的组合。一种较佳的所述反应条件参数的组合可以是：反应缓冲液为含20mM His-HCl的pH 6.0的缓冲液，还原剂TCEP用量为1.5～6.0摩尔倍数，还原反应时长为1.5～2.0小时，还原反应温度为室温(25-30℃)，偶联物为mc-vc-PAB-MMAE，偶联物用量为3.0～7.0摩尔倍数，偶联反应时长为0.5小时，偶联反应温度为室温(25-30℃)。
在一些具体实施方式中，所述制备方法还可以提供一种“纯化”步骤。所述“纯化”步骤利用疏水相互作用色谱层析方法(HIC)将偶联反应产物中各组分进行分离，以获取某一特定组分，例如，利用HIC纯化分离偶联产物得到CAR＝2的蛋白-药物偶联物。
在一些具体实施方式中，所述制备方法还可以进一步提高偶联产物中单一组分的含量，减少进一步的纯化的需求；更佳地，经过偶联步骤后无需额外纯化步骤即可以得到纯度大于90％的CAR＝2高均质性的偶联产物。
在一些具体实施方式中，所述制备方法还可以提供一种包括“还原”和“偶联”等步骤的组合和反应条件参数的组合；在所述的反应步骤和反应条件参数的组合下，所述的连接肽的第一半胱氨酸(Cys1)的二硫键可以被有效地打开而其他二硫键保持完整，使得Cys1成为几乎唯一的游离半胱氨酸残基，进而作为特异性的偶联位点。
另一方面，本申请提供了一种药物组合物，所述药物组合物可以包含所述蛋白-药物偶联物，以及药学上可接受的载体。例如，所述药物组合物还可以包括其他抗原结合蛋白或治疗剂。
另一方面，本申请提供了一种所述的蛋白-药物偶联物和/或所述的药物组合物在制备诊断、预防和/或治疗疾病的药物中的应用。例如，所述的药物可以用于治疗肿瘤或其他疾病。
另一方面，本申请提供了一种所述蛋白-药物偶联物与其他疗法或药物联用在制备药物中的用途，所述药物可以用于治疗肿瘤或其他疾病。例如，所述其他疗法或药物可以选自下组：化疗、放疗、miRNA和寡核苷酸。
另一方面，本申请提供了一种体外或体内检测特异性抗原的方法，其可以包括使用 如所述的蛋白-药物偶联物和/或所述的药物组合物进行检测。在某些适用情形中，所述方法可以为非诊断目的的。
另一方面，本申请提供了用于所述蛋白-药物偶联物或药物组合物施用的给药装置。
实施例
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法.这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1 抗体的制备
Harbour HCAb小鼠(Harbour Antibodies BV，WO2010/109165A2)是一种携带人免疫球蛋白免疫库的转基因小鼠，能够产生仅有重链的抗体，该抗体的大小只有传统IgG抗体的一半。该小鼠产生的抗体具有人的抗体重链可变结构域和小鼠恒定结构域。在获得针对某一特定靶点的重链抗体的VH序列以后，利用常规的分子生物学手段将VH序列和人的IgG抗体重链Fc序列(优选为含有人IgG1铰链区和CH2以及CH3区的序列，如SEQ ID NO:72所示序列)进行融合表达，得到全人源重组HCAb抗体分子。
Harbour H2L2小鼠(Harbour Antibodies BV)是一种携带人免疫球蛋白免疫库的转基因小鼠，其产生的抗体具有完整的人的抗体可变结构域和大鼠恒定结构域。利用杂交瘤技术或者单B细胞分选技术或者其他技术筛选出针对某一特定靶点的抗体。将抗体的VL和VH序列与相应的人的κ轻链恒定区和IgG1重链恒定区序列进行融合表达，得到重组全人源抗体分子。
实施例1.1 获得抗CTLA4的全人源HCAb抗体
用可溶的重组人CTLA4蛋白(ACRO Biosystems,#CT4-H5229)对Harbour HCAb小鼠进行多轮免疫。当检测小鼠血清中CTLA4特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出分离B细胞，用小鼠浆细胞分选试剂盒(Miltenyi,#130-092-530)分选 CD138阳性的浆细胞。用常规的分子生物学手段从浆细胞中扩增人VH基因，并将扩增的人VH基因片段构建到编码人IgG1抗体重链Fc区域序列的哺乳动物细胞表达质粒pCAG载体中。质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达，得到全人源HCAb抗体上清。用ELISA测试HCAb抗体上清与重组人CTLA4蛋白的结合，鉴定出阳性HCAb抗体。对这些HCAb抗体进行进一步的鉴定，根据其对人CTLA4的结合能力、食蟹猴CTLA4的结合能力、抑制CTLA4与B7-1结合能力等参数，优选出数个候选HCAb抗体分子。然后对候选HCAb抗体分子进行序列分析和优化，得到数个变体序列。将HCAb抗体的VH序列和人的IgG1重链铰链区及Fc序列进行融合表达，得到全人源重组HCAb抗体分子。抗CTLA4的重组全人源HCAb抗体列于表1-1。
实施例1.2 获得抗BCMA的全人源HCAb抗体
用可溶的重组人BCMA-ECD-Fc融合蛋白(ACRO Biosystems,#BC7-H82F0)对Harbour HCAb小鼠进行多轮免疫。抗原蛋白与免疫佐剂混合成免疫原试剂，然后通过皮下经腹股沟注射或通过腹腔注射。在每一轮免疫中，每只小鼠接受的总注射剂量是100微升。在首轮免疫中，每只小鼠接受用50微克抗原蛋白与完全弗氏佐剂(Sigma,#F5881)以体积比1:1混合配制的免疫原试剂的免疫。在随后的每轮增强免疫中，每只小鼠接受用25微克抗原蛋白与Sigma Adjuvant System佐剂(Sigma,#S6322)混合配制的免疫原试剂的免疫。每轮增强免疫的间隔时间至少为两周，通常不超过五轮增强免疫。免疫时间为第0、14、28、42、56、70天；并且在第49、77天，检测小鼠血清抗体滴度。在进行Harbour HCAb小鼠脾B细胞分离前5天，以每只小鼠25微克抗原蛋白的剂量进行最后一次增强免疫。
当检测小鼠血清中BCMA特异的抗体滴度达到一定的水平后，将小鼠的脾细胞取出分离B细胞，用BD FACSAria TM III细胞分选仪分选CD138阳性的浆细胞和BCMA抗原阳性的B细胞群。提取RNA，反转录cDNA后PCR扩增人VH基因。扩增的VH基因片段构建到编码人IgG1抗体重链Fc结构域序列的哺乳动物细胞表达质粒pCAG载体中，质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达，表达的HCAb的抗体上清与重组人BCMA-Fc,Avitag重组蛋白(ACRO Biosystems,#BC7-H82F0)进行Mirrorball(SPT Labtech,
Figure PCTCN2021083024-appb-000001
fluorescence cytometer)筛选，获得的阳性单克隆抗体上清用流式细胞术FACS进一步的鉴定。用FACS测试抗体上清与高表达人BCMA的HEK293T细胞株HEK293T/hBCMA(北京康源博创,KC-0233)、高表达食蟹猴BCMA的 HEK293T细胞株HEK293T/cynoBCMA(北京康源博创,KC-0979)和高表达人BCMA的细胞系NCI-H929(ATCC,CRL-9068)等细胞的结合能力。通过多轮筛选，获得了数个阳性候选HCAb抗体分子。利用常规的测序手段获得编码抗体分子可变结构域的核苷酸序列。将HCAb抗体的VH序列和人的IgG1重链铰链区及Fc序列进行融合表达，得到全人源重组HCAb抗体分子。
针对抗BCMA的HCAb抗体PR001046的可变区VH的CDR区进行两轮定点突变，以获得结合BCMA的亲和力提高的突变体，如PR001046_R2_4G10(即PR004433)。抗BCMA的重组全人源HCAb抗体列于表1-1。
实施例1.3 获得抗MSLN的全人源HCAb抗体
用可溶的重组人MSLN蛋白(ACRO Biosystems,#MSN-H5223)对Harbour HCAb小鼠进行多轮免疫。用类似实施例1.2所述方法筛选并获得抗MSLN的全人源HCAb抗体。抗MSLN的重组全人源HCAb抗体列于表1-1。
实施例1.4 获得抗5T4的全人源HCAb抗体
用可溶的重组人5T4蛋白(NovoProtein,#C678)对Harbour HCAb小鼠进行多轮免疫。用类似实施例1.2所述方法筛选并获得抗5T4的全人源HCAb抗体。抗5T4的重组全人源HCAb抗体列于表1-1。
实施例1.5 获得抗ROR1的抗原结合蛋白
用可溶的重组人ROR1蛋白(AcroBiosystems,#RO1-H5250)对Harbour H2L2小鼠进行多轮免疫。用类似实施例1.2所述方法并结合噬菌体展示筛选获得抗ROR1的抗体克隆1015M2-H4。将1015M2-H4的VH和VL利用重叠PCR制备得到单链可变区片段scFv；然后把scFv和一段含有核酸限制性内切酶BamHI的酶切位点的序列(对应氨基酸序列为GGGAS)以及人的IgG重链Fc(包含人IgG1铰链区和CH2以及CH3区，如SEQ ID NO:72所示序列)进行融合表达，得到具有scFv-Fc二聚体结构的抗ROR1的抗原结合蛋白分子PR002129。抗ROR1的抗原结合蛋白列于表1-1。
实施例1.6 抗体的表达和纯化
本实施例介绍了利用哺乳动物宿主细胞(例如，人胚肾细胞HEK293或中国仓鼠卵巢细胞CHO及其衍生细胞)、瞬时转染表达和亲和捕获分离等技术来制备抗体的一般方 法。本方法适用于含有Fc区的目标抗体；目标抗体可以由一条或多条蛋白质多肽链组成；可以来源于一个或多个表达质粒。
将抗体多肽链的氨基酸序列通过密码子优化方法转换成核苷酸序列；合成编码的核苷酸序列并克隆到与宿主细胞兼容的表达载体上。将编码抗体多肽链的质粒按照特定比例同时转染哺乳动物宿主细胞，利用常规的重组蛋白表达和纯化技术，可以得到具有正确折叠和多肽链组装的重组蛋白。具体地，将FreeStyle TM 293-F细胞(Thermo,#R79007)在FreeStyle TM F17 Expression Medium培养基(Thermo,#A1383504)中扩培。瞬时转染开始之前，调节细胞浓度至6-8x10 5细胞/ml，于37℃8％CO 2摇床中培养24小时，细胞浓度在1.2x10 6细胞/ml。准备30ml培养的细胞。将编码抗体多肽链的质粒按照一定比例混合共计30μg质粒(质粒与细胞的比例为1μg:1ml)溶解于1.5ml Opti-MEM减血清培养基(Thermo,#31985088)，并用0.22μm滤膜过滤除菌。再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,#23966-2)120μl，静置5分钟。把PEI缓慢加入质粒中，室温孵育10分钟，边摇晃培养瓶边缓慢滴入质粒PEI混合溶液，于37℃8％CO 2摇床中培养5天。5天后观测细胞活率。收集培养物，以3300g转速离心10分钟后取上清；然后将上清高速离心去除杂质。用PBS pH7.4缓冲液平衡含有MabSelect TM(GE Healthcare,#71-5020-91)的重力柱(Bio-Rad,#7311550)，2-5倍柱体积冲洗。将上清样品过柱；用5-10倍柱体积的PBS缓冲液冲洗柱子，再用pH3.5的0.1M甘氨酸洗脱目的蛋白，随后用pH 8.0的Tris-HCl调节至中性，最后用超滤管(Millipore,#UFC901024)浓缩换液至PBS缓冲液或者含有其他成分的缓冲液，得到纯化的重组蛋白溶液。最后用NanoDrop(Thermo,NanoDrop TM One)测定浓度，分装、存储备用。
本申请所使用的抗原结合蛋白总结于表1-1，其对应的多肽链和CDR区的氨基酸序列的序列编号SEQ ID NOs列于表1-2、表1-3、表1-4和表1-5。
靶点 克隆号 蛋白编号 序列来源
CTLA4 CL5v3 PR000184 实施例1.1
CTLA4 CL24 PR000020 实施例1.1
MSLN 1018P004B9 PR000759 实施例1.3
BCMA 1005P63B7 PR001046 实施例1.2
5T4 1041P014H2 PR004432 实施例1.4
BCMA PR001046_R2_4G10 PR004433 实施例1.2
ROR1 1015M2-H4 PR002129 实施例1.5
BCMA 269A37917 PR000453 专利WO2017025038A1
CD47 ALX148 PR006345 专利US10829771B2
表1-1 本申请所使用的部分示例性的抗原结合蛋白。
蛋白编号 重链 VH HCDR1 HCDR2 HCDR3
PR000020 64 55 7 22 38
PR000184 65 56 8 23 39
PR000759 67 58 10 25 41
PR001046 68 59 11 26 42
PR004432 69 61 13 28 44
PR004433 70 62 14 29 42
PR000453 66 57 9 24 40
表1-2 本申请中HCAb抗体的序列编号表。
蛋白编号 多肽链 VL VH LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3
PR002129 71 63 60 49 51 53 12 27 43
表1-3 本申请中scFv-Fc结构的抗原结合蛋白的序列编号表。
蛋白编号 多肽链
PR006345 149
表1-4 本申请中可溶性受体融合蛋白的序列编号表。
蛋白编号 多肽链1 多肽链2
PR005744 134 135
2129/4433 70 71
表1-5 本申请中双特异性抗原结合蛋白的序列编号表。
实施例2 分析方法
实施例2.1 利用SEC-HPLC(分子尺寸排阻-高效液相色谱)分析蛋白纯度和聚体
本实施例使用分析型分子尺寸排阻层析色谱法(SEC)来分析蛋白样品的纯度和聚体 形式。将分析型色谱柱TSKgel G3000SWxl(Tosoh Bioscience,#08541,5μm,7.8mm×30cm)连接到高效液相色谱仪HPLC(Agilent Technologies,Agilent 1260 Infinity II)，用PBS缓冲液室温下平衡至少1小时。适量蛋白样品(至少10μg)用0.22μm滤膜过滤后注射入系统，并设定HPLC程序：用PBS缓冲液将样品以1.0ml/min的流速流过色谱柱，最长时间为25分钟，检测波长280nm，室温运行。采集后用ChemStation软件对色谱图进行积分并计算相关数据，生成分析报告，报告出样品内不同分子尺寸组分的滞留时间。
实施例2.2 利用HIC-HPLC(疏水相互作用-高效液相色谱)分析蛋白纯度、疏水性和偶联产物组分
本实施例使用分析型疏水相互作用层析色谱法(HIC)来分析蛋白样品的纯度和疏水性。将分析型色谱柱TSKge1 Buty1-NPR(Tosoh Bioscience,#14947,4.6mm×3.5cm,2.5μm)连接到高效液相色谱仪(HPLC)(型号：Agilent Technologies,Agilent 1260 Infinity II)，用PBS缓冲液室温下平衡至少1小时。设定方法由约22分钟内从100％缓冲液A(25mM磷酸盐缓冲液，1.5M硫酸铵((NH4)2SO4)，pH 7.0～7.2)至100％缓冲液B(20mM磷酸盐缓冲液，20％异丙醇(IPA)，pH 7.0～7.2)的线性梯度，流速设定为0.7ml/min,蛋白样品浓度1mg/ml,进样体积20μl,检测波长280nm，室温运行。采集后用ChemStation软件对色谱图进行积分并计算相关数据，生成分析报告，报告出样品内不同分子尺寸组分的滞留时间；可以通过不同的滞留时间来推测偶联产物的不同组分所携带的偶联分子数目。
实施例2.3 利用RP-HPLC(反相-高效液相色谱)分析蛋白纯度、疏水性和偶联产物组分
本实施例使用分析型反相高效液相色谱法(RP)来分析蛋白样品的纯度和疏水性。本领域技术人员可以使用不同型号的高效液相色谱仪和分析型色谱柱按照技术说明书并经过实验摸索以建立相似的分析手段，本实施例仅列举其中代表性方法。
在一些具体实施方式中，例如在本发明的实施例5.2中，将分析型色谱柱Zorbax RRHD 300-Diphenyl(Agilent Technologies,#858750-944,2.1×100mm,1.8μm)连接到高效液相色谱仪(HPLC)(型号：Agilent Technologies,Agilent 1260 Infinity II)，按照说明书对系统进行平衡。设定方法由60分钟内从100％缓冲液A(1％三氟乙酸TFA溶于水)至100％缓冲液B(1％三氟乙酸TFA溶于乙腈)的线性梯度，流速设定为0.35ml/min,运行温度为50℃，检测波长280nm。采集后用ChemStation软件对色谱图进行 积分并计算相关数据，生成分析报告，报告出样品内不同分子尺寸组分的滞留时间。
在另一些具体实施方式中，例如在本发明的实施例8.2中，将分析型色谱柱PLRP-S 1000A(Agilent Technologies,#PL1912-1502,2.1×50mm,5.0μm)连接到高效液相色谱仪(HPLC)(型号：Agilent Technologies,Agilent 1260 Infinity II)，按照说明书对系统进行平衡。设定方法由40分钟内从100％缓冲液A(0.05％三氟乙酸TFA溶于水)至100％缓冲液B(0.05％三氟乙酸TFA溶于乙腈)的线性梯度，流速设定为0.25ml/min,运行温度为60℃，检测波长280nm。采集后用ChemStation软件对色谱图进行积分并计算相关数据，生成分析报告，报告出样品内不同分子尺寸组分的滞留时间。
随后，可以通过不同的滞留时间来推测偶联产物的不同组分所携带的偶联分子数目。
实施例2.4 利用LC-MS(液相色谱质谱联用)测定分子量
本实施例使用液相色谱质谱联用方法(LC-MS)来分析蛋白样品的分子量。本领域技术人员可以使用不同型号的液相色谱仪(LC)和质谱仪(MS)按照技术说明书并经过实验摸索以建立相似的分析手段，本实施例仅列举其中代表性方法。
样本预处理
在一些具体实施方式中，测定蛋白样品的分子量需要对样品进行去糖处理以去除糖链等修饰，例如用糖苷酶PNGase F(NEB,#P0705L)在37℃处理样品至少4小时。例如，在本发明的所有实施例中测定蛋白样品分子量的时候，如无特殊强调，待测蛋白样品都利用糖苷酶PNGase F进行去糖处理。
在一些具体实施方式中，测定蛋白样品的还原分子量需要对样品进行还原处理，例如用10nM DTT(dithiothreitol)在37℃处理样品10分钟。例如，在本发明的所有实施例中，经过还原处理的样品称为还原样品，未经还原处理的样品称为非还原样品。
LC条件设定
在一些具体实施方式中，将色谱柱bioZen 3.6μm Intact C4(Phenomenex,#00F-4767-AN,2.1×150mm,3.6μm)连接到超高效液相色谱仪(UPLC)(型号：Agilent Technologies,Agilent 1290 UPLC)，按照说明书对系统进行平衡。缓冲液A为0.1％甲酸FA溶于水，缓冲液B为0.1％甲酸FA溶于乙腈。进样约1μg，流速设定为0.3ml/min,运行温度为80℃。当测试还原样品时，在12分钟内用缓冲液A和B混合成线性梯度:前8分钟使用10-60％B,后4分钟使用80％B。当测试非还原样品时，在9分钟内用缓冲液A和B混合成线性梯度:前6分钟使用15-60％B,后3分钟使用80％B。
在另一些具体实施方式中，将色谱柱ACQUITY UPLC Protein BEH C4(Waters,#186004495,2.1×50mm,1.7μm,
Figure PCTCN2021083024-appb-000002
)连接到超高效液相色谱仪(UPLC)(型号：Waters Acquity UPLC)，按照说明书对系统进行平衡。缓冲液A为0.1％甲酸FA溶于水，缓冲液B为0.1％甲酸FA溶于乙腈。进样约10μL，流速设定为0.2～0.5ml/min,运行温度为80℃，检测波长214nM。当测试还原样品时，在20分钟内用缓冲液A和B混合成线性梯度:14分钟使用25-90％B,后6分钟使用25％B。当测试非还原样品时，在8分钟内用缓冲液A和B混合成线性梯度:前4分钟使用5-95％B,后4分钟使用95-5％B。
MS条件设定
在一些具体实施方式中，使用的质谱仪型号为AB Sceix X500B，运行模式为TOF-MS；当测试还原样品时，扫描范围是600-4000m/z；当测试非还原样品时，扫描范围是900-6000m/z。使用Sceix OS软件对原始质谱图进行去卷积处理得到分子量图。
在另一些具体实施方式中，使用的质谱仪型号为Waters Xevo G2-XS Q-TOF，分子量扫描范围是500～4000Da。使用Water MaxEnt或者Agilent MassHunter BioConfirm软件对原始质谱图进行去卷积处理。
LC-MS联用
本发明申请的多个具体实施方式中使用了不同的LC条件和MS条件的组合设定，以实现LC-MS联用来测定蛋白样品的分子量。例如，在实施例3.1和实施例5.2中，使用的条件组合为(色谱柱：ACQUITY UPLC Protein BEH C4；LC仪器：Waters Acquity UPLC；MS仪器：Waters Xevo G2-XS Q-TOF)。又例如，在实施例3.2、实施例8.2、实施例9.2、实施例10.1、实施例11.2、实施例12.1、实施例13.2、实施例14.2和实施例16.1中，使用的条件组合为(色谱柱：bioZen 3.6μm Intact C4；LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix X500B)。又例如，在实施例6.2、实施例7.2中，使用的条件组合为(色谱柱：Agilent PLRP-S 1000A；LC仪器：Agilent 1290 UPLC；MS仪器：Waters Xevo G2-XS Q-TOF)。
实施例2.5 利用Uncle测定蛋白的分子稳定性和分子聚集性
Uncle(Unchained Labs)是一个多功能一站式的蛋白稳定性分析平台，它通过全荧光，静态光散射(SLS)和动态光散(DLS)检测方法来表征蛋白质的稳定性。同一组样品 可同时得到熔解温度(Tm)，聚集温度(Tagg)和粒径(diameter)等参数。在本实施例中，选择Uncle的“Tm&Tagg with optional DLS”应用程序进行操作，取9μL样品加入Uni管中，设置以0.3℃/分钟的梯度逐渐从25℃升温至95℃。进行初始和最终DLS测量四次采集，每次采集5秒。实验运行结束后，Uncle分析软件采用重心均值(BCM)公式来计算每个样品的Tm值；通过SLS在266nm或473nm波长下的荧光强度的曲线(聚集曲线)来计算Tagg值；样品的粒径和分散度则通过DLS相关的函数来计算。
实施例2.6 利用DSC(差示扫描量热法)测定蛋白的熔解温度
本实施例使用Malvern VP-capillary DSC仪器通过差示扫描量热法(DSC)分析蛋白样品的熔融温度(Tm)。将样品用缓冲液稀释至检测浓度(0.5mg/mL)后置于DSC样品池，将缓冲液置于参比池。对DSC样品池和参比池进行完全一样的等温升温控制，其中扫描温度区间为25–100℃，扫描速度为1℃/分钟,扫描前平衡时间为3分钟，每个待测样品分析一次。由补偿样品池温差的热量组成的峰图即为样品的热力学曲线图，曲线峰顶温度即为样品的Tm值。含有多个结构域的蛋白样品由于不同结构域的稳定性不同，其曲线可能呈现多个峰。
实施例2.7 利用DSF(差示扫描荧光法)测定蛋白的熔解温度
差示扫描荧光法(Differential Scanning Fluorimetry,DSF)是一种常用的高通量的测定蛋白质热稳定性的方法。它使用实时荧光定量PCR仪器通过监测与去折叠的蛋白分子结合的染料的荧光强度的变化，来反映蛋白质的变性的过程，从而反映出蛋白分子的热稳定性。本实施例利用DSF方法来测定蛋白分子熔融温度(Tm)。将10μg蛋白加入96-孔PCR板(Thermo,#AB-0700/W)，接着加入2μl 100×稀释的染料SYPROTM(Invitrogen,#2008138)，然后加入缓冲液使得终体积为40μl每孔。将PCR板密封，放置于实时荧光定量PCR仪器(Bio-Rad CFX96 PCR System)，先于25℃孵育5分钟，然后以0.2℃/0.2分钟的梯度逐渐从25℃升温至95℃，在测试结束时将温度降至25℃。使用FRET扫描模式并使用Bio-Rad CFX Maestro软件进行数据分析并计算出样品的Tm。
实施例3 鉴定Cys-220位点二硫键的形成
如图1所示，人IgG1抗体是含有两条相同重链和两条相同轻链的四聚体结构，两 个重链之间形成了两个链间二硫键，一个重链和对应轻链之间形成了一个链间二硫键；这些抗体的天然链间二硫键经还原后形成的数个巯基成为了可用的半胱氨酸偶联位点。图44(B)显示了人IgG1的铰链区序列(SEQ ID NO:73)，其含有三个半胱氨酸，其对应位点按照Eu编号分别为Cys-220、Cys-226和Cys-229；其中，两个重链铰链区的Cys-226和Cys-229分别各自形成一个重链间二硫键，而Cys-220则与靠近轻链C’末端的Cys-214形成重链和轻链之间的二硫键。
图2显示了具有人IgG1抗体铰链区序列的重链抗体HCAb的结构，以及铰链区二硫键结构在人IgG1和HCAb之间的差异。由于没有轻链的存在，位于铰链区的Cys-220显然无法再和轻链Cys-214形成二硫键。Cys-220的状态有如图3所示的两种可能性：(一)Cys-220没有形成重链间二硫键，其侧链巯基(-SH)会与环境中(如细胞质)的含巯基分子(如谷胱甘肽)形成二硫键；(二)如同Cys-226或Cys-229那样，Cys-220形成两条重链之间的链间二硫键，因此铰链区会形成三个二硫键。
本实施例研究了数个含有Cys-220的抗原结合蛋白(包括重链抗体HCAb和单链抗体scFv-Fc等结构)分子中Cys-220位点二硫键的形成。
实施例3.1 研究HCAb PR000020中Cys-220
本实施例采用不同的实验方法来鉴定在HCAb PR000020(分子信息见表1-1)中Cys-220的状态。
利用LC-MS分子量分析来鉴定Cys-220
如果Cys-220没有形成重链间二硫键(假设1)，而是其侧链巯基与环境中的其他含巯基分子形成二硫键，那么该HCAb的非还原分子量(即完整分子量)会大于形成重链间二硫键时的分子量理论值(假设2)。按照假设2，PR000020的非还原分子量理论值为77,640Da；按照假设1，其非还原分子量可能为77,878Da(增加了2个半胱氨酸(+119Da×2))或78,250Da(增加了2个谷胱甘肽(+305Da×2))。
将纯化的HCAb PR000020蛋白用糖苷酶PNGase F作去糖处理，并用实施例2.4所述方法(LC仪器：Waters Acquity UPLC；MS仪器：Waters Xevo G2-XS Q-TOF)测定其非还原分子量，结果为77,643Da(去卷积分子量图谱见图4)，与按照假设2的预测分子量几乎一致。由此，可以推断Cys-220形成了重链间二硫键。
Figure PCTCN2021083024-appb-000003
Figure PCTCN2021083024-appb-000004
表3-1 HCAb PR000020不同的假设的分子量
利用木瓜蛋白酶Papain酶切分析来鉴定Cys-220
木瓜蛋白酶Papain可以较特异地水解位于铰链区的His-224和Thr-225之间的肽键(参见Endo,S.,&Arata,Y.(1985).)。将纯化的HCAb PR000020蛋白用Papain进行酶切。由图5(A)所示，如果Cys-220没有形成重链间二硫键(假设1)，那么Papain水解HCAb将会产生约13KDa的片段(单个VH结构域)和约50KDa的Fc片段；如果Cys-220形成重链间二硫键(假设2)，那么Papain水解HCAb将会产生约27KDa的片段(由Cys-220的二硫键连接在一起的两个VH结构域)和约50KDa的Fc片段。图5(B)显示了酶切产物的非还原SDS-PAGE的结果；HCAb的酶切产物(对应第3和第4泳道)在28KDa附近有明显条带，而在14KDa附近没有条带，因此证实了假设2的成立，即Cys-220形成了重链间二硫键。
综上所述，利用LC-MS分子量分析方法和Papain酶切分析方法都证明了Cys-220形成了重链间二硫键。
实施例3.2 研究scFv-Fc PR002129中Cys-220
本实施例研究在scFv-Fc PR002129(表1-1)中Cys-220的状态。PR002129的氨基酸序列如SEQ ID NO:71所示，其含有人IgG1铰链区序列(SEQ ID NO:73)。PR002129是含有重链Fc的二聚体结构，其每一条多肽链(重链)包含一个单链可变区片段scFv，一个连接肽序列和人的IgG1重链Fc序列；其中，scFv来源于抗ROR1的抗体1015M2-H4的VH和VL序列，连接肽序列包含人IgG1铰链区序列和一段GGGAS序列(为了引入合适的限制性内切酶位点)。
在PR002129及其他类似的scFv-Fc结构中，Cys-220的状态也有如图37(A)中所示的两种可能性：形成或者不形成链间二硫键。如果Cys-220没有形成重链间二硫键，其侧链巯基(-SH)会与环境中的含巯基分子(如，半胱氨酸或者谷胱甘肽)形成二硫键， 使其实际分子量明显大于理论分子量。
利用LC-MS分子量分析来鉴定Cys-220
将纯化的PR002129蛋白用糖苷酶PNGase F作去糖处理，并用实施例2.4所述方法(LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix X500B)测定其非还原分子量(图37(B)所示其去卷积分子量图谱)，结果为104,510Da，与不同假设的预测分子量进行比较(表3-2)，推断出Cys-220没有联结其他含巯基分子，因此Cys-220形成了重链间二硫键。
组分 理论值(Da) 实验值(Da) 偏差(Da)
PR002129 104,508 104,510 +2
PR002129联结2个半胱氨酸 104,746    
PR002129联结2个谷胱甘肽 105,118    
表3-2 PR002129非还原分子量LC-MS分析
实施例3.3 铰链区序列分析和Cys-220的二硫键
人的不同IgG同种型的铰链区序列是已知的，铰链区结构和二硫键也做了较充分的研究，例如人IgG1通过轻链的最后一个Cys(即Eu编码的Cys-214)和重链的第五个Cys(即Eu编码的Cys-220)形成链间二硫键，而IgG2、IgG3和IgG4则是通过轻链的最后一个Cys和重链的第三个Cys形成链间二硫键(参见，Liu,H.等人mAbs(2012),4(1),17–23)。
本申请发明人进一步对IgG1的晶体结构(PDB登录号1HZH)进行分析，Cys-220与轻链Cys-214形成稳定的二硫键，而且两个重链铰链区的Cys-220的Cβ原子之间位置相对较远，从该IgG1结构上推测这两个Cys-220之间难以形成二硫键。但是另一方面，前述研究已经验证了在缺失轻链的结构中Cys-220形成重链间二硫键的事实。因此，推测为：Cys-220可以像Cys-226或Cys-229那样形成重链间二硫键，但是其二硫键的稳定性可能比Cys-226的或Cys-229的要弱。因此通过优化可以使其特定地打开Cys-220的二硫键而保持其他二硫键不变；这样，Cys-220有可能为半胱氨酸偶联提供了定点偶联用的锚点。
本申请发明人再进一步研究不同物种的IgG抗体铰链区序列和二硫键结构(图44)发现，人和小鼠以及羊驼的铰链区序列差异很大，其铰链区序列长度和所含Cys的数量 都不相同(图44(A))。发明人利用已知的IgG全长抗体晶体结构分析铰链区的二硫键结构，如图44(B)显示了人IgG1铰链区(PDB登录号1HZH)；图44(C)显示了人IgG4铰链区(PDB登录号5DK3)；图44(D)显示了小鼠IgG1铰链区(PDB登录号1IGY)；图44(E)显示了小鼠IgG2a铰链区(PDB登录号1IGT)。在已知的晶体结构中，只有人IgG1和小鼠IgG1的铰链区贡献了重链和轻链之间的二硫键，人IgG1通过Cys-220(Eu编号)作为铰链区的第一个Cys与轻链形成二硫键，小鼠IgG1通过Cys-235(残基编号)作为铰链区的第一个Cys与轻链形成二硫键；但是，不同的是，在人IgG1铰链区中，第一个Cys(Cys-220，Eu编号)和第二个Cys(Cys-226，Eu编号)之间相隔了5个其他种类氨基酸残基，而在小鼠IgG1铰链区中，第一个Cys(Cys-235，残基编号)和第二个Cys(Cys-237，残基编号)之间相隔了1个其他种类氨基酸残基。
本申请发明人进一步推测人IgG1重链的Cys-220在缺失轻链时可以形成较弱的重链间二硫键，而这一特性可能与Cys-220相对于Cys-226之间的距离(即第一Cys和第二Cys之间的距离)相关；第一Cys和第二Cys之间可能需要间隔一定数目的其他种类氨基酸残基才能使两个Cys分别形成不同的二硫键且二硫键的稳定性(或键能)有差异。在后续的实施例中，进一步研究了两个Cys之间的间隔和由铰链区序列衍生的变体连接肽序列，以及利用第一个Cys进行药物偶联的应用。在图44(A)所示铰链区序列中，只有小鼠IgG2c的铰链区中Cys的数目和排列与人IgG1的相似，可以推测小鼠IgG2c的铰链区序列中第一个Cys可能和人IgG1铰链区的Cys-220有相似的能力。
实施例3.4 Cys-220的二硫键缺失并不影响分子稳定性
本实施例为了研究Cys-220的二硫键对于分子结构稳定性的影响。本实施例选取了数个HCAb分子，制备其C220S突变衍生分子(将铰链区Cys-220突变成丝氨酸Ser以破坏其二硫键的形成)，或者以实施例4所述方法制备药物偶联衍生物(ADC)；然后利用实施例2.5所述Uncle方法或实施例2.6所述DSC方法测定分子熔解温度(Tm)，以表征其热稳定性。所述HCAb分子来自表1-1。
图38显示了HCAb PR000184及其衍生分子的DSC热力学分析曲线图；图39显示了HCAb PR000453及其衍生分子的DSC热力学分析曲线图；图40显示了HCAb PR004432及其衍生分子的Uncle热力学分析曲线图；结果总结于表3-3，其列出了每个样品的最低熔解温度(Tm1)。由此可见，C220S突变并没有对分子结构的热稳定性带来明显的影响；而且，利用Cys-220的偶联也没有对热稳定性造成显著的改变。
Figure PCTCN2021083024-appb-000005
表3-3 HCAb及衍生分子的熔解温度，Tm 1为最低熔解温度。
由此可见，Cys-220的二硫键缺失并不影响分子稳定性。
实施例4 偶联反应、纯化
实施例4.1 偶联反应
基于半胱氨酸残基的药物偶联过程包括由“还原”、“再氧化”和“偶联”等不同反应过程组成的一个或多个步骤的组合。在这些反应过程中，反应条件如反应温度、反应时间、反应缓冲液类型及pH、还原剂类型及用量、偶联物分子类型及用量等都对整个偶联反应的效率和终产物的质量产生重大影响。这些反应条件包括不同参数的组合，本实施例仅列举其中代表性方法；本领域技术人员也可以根据本实施例所教导方法结合采用不同的参数条件并经过实验摸索以建立相似的偶联反应过程。
还原
本实施例尝试使用还原剂为强还原剂DTT(dithiothreitol)或还原能力适中的TCEP (Tris(2-carboxyethyl)phosphine)。还原剂的用量为x摩尔倍数(x＝1～50)；还原反应时长为y小时(y＝1～17)；反应的温度为0～40℃，反应的缓冲液pH为4.0～9.0。
再氧化
本实施例还可能尝试使用氧化剂DHAA(dehydroascorbic acid)对样品进行处理。
偶联
本实施例还尝试使用的药物偶联物为mc-vc-PAB-MMAE(也称为VcMMAE，分子结构见图6，分子量为1315.78Da；生产商：联宁生物制药，货号：SET0201)，该化合物含有一个马来酰亚胺活性反应基团，可以与巯基发生加成反应，形成稳定的硫醚键。本实施例尝试的药物偶联物用量为z摩尔倍数(z＝1～20)；偶联反应时长为h小时(h＝0.5～3)；反应温度为25～30℃，pH为4.0～9.0。偶联结束之后，通过脱盐柱将产物去除多余药物偶联物，同时置换到合适的缓冲液中保存。
本申请的多个具体实施方式可能使用了多种偶联反应条件和步骤，包括但不限于本实施例所述方法中的一个或者多个步骤的组合。
实施例4.2 利用HIC(疏水相互作用)纯化偶联产物
本实施例使用疏水相互作用将偶联产物中各组分进行分离。将偶联所得样品，通过AKTA pure层析系统(Cytiva Life Sciences,AKTA pure)，利用ToyoScreen Phenyl-600M疏水作用色谱层析柱(Tosoh Bioscience,#21892)进行纯化。在30个柱体积内，将疏水相从0％线性提升到100％，期间对各组峰分别收集。所收集组分，经过HIC-HPLC检测(实施例2.2所述方法)后，进行合并、浓缩、换液得到偶联产物。
实施例5 HCAb PR000020的偶联和分析
本实施例研究了抗CTLA4的HCAb抗体PR000020(SEQ ID NO:64)与mc-vc-PAB-MMAE利用半胱氨酸偶联到Cys-220位点来制备具有均一DAR值的抗体偶联药物。
实施例5.1 偶联反应条件优化
本实施例先用实施例1.6中所述方法生产和纯化HCAb PR000020重组蛋白；然后利用实施例4.1中所述方法，使用多个不同的还原、再氧化和偶联条件的组合条件，对纯化的HCAb PR000020进行偶联反应(表5-1)，试图得到较优的反应条件。所使用的 还原剂为TCEP(Tris(2-carboxyethyl)phosphine)或DTT(dithiothreitol)；氧化剂为DHAA(dehydroascorbic acid)；药物偶联物为mc-vc-PAB-MMAE(生产商：联宁生物制药，货号：SET0201)。平行用6组条件对HCAb PR000020进行偶联，以及使用trastuzumab(第7组实验)作为IgG1对照。在本实验中，还原或偶联反应的pH为6.5-7.0，温度为室温(25-30℃)。每组实验的偶联的产物用实施例2.2所述HIC-HPLC分析方法分析其偶联产物的组分，列于表5-2。
Figure PCTCN2021083024-appb-000006
表5-1 尝试的不同的偶联反应条件
Figure PCTCN2021083024-appb-000007
表5-2 不同的偶联反应条件的产物的HIC-HPLC分析结果
结合表5-1和表5-2，实验条件5和6产生的主要产物是偶联了2个化合物分子的抗体偶联药物(DAR＝2)。因此，初步确定的优化偶联反应条件为：还原剂选择还原能力适中的TCEP，还原剂的用量在1.5-2.5摩尔倍数之间，还原反应的温度为25-30℃，反应 pH为6.5-7.0，反应时长为3小时；偶联所用的药物偶联物mc-vc-PAB-MMAE的用量为7-10摩尔倍数之间，温度为25-30℃，pH为6.5-7.0，反应时长为1-2小时。
偶联结束之后，通过脱盐柱将产物去除多余药物偶联物，并用实施例4.2所述HIC方法纯化偶联产物，得到DAR＝2的主要产物。
实施例5.2 偶联产物分析
本实施例利用高效液相色谱和质谱等技术对纯化的重组HCAb PR000020和其偶联产物PR000020-ADC(包括偶联后产物和经过一步HIC纯化后的产物)进行定量分析，以确定其偶联产物组分的构成。
利用实施例2.2中所述HIC-HPLC方法对上述样品进行分析。图8和下表分别显示了HCAb PR000020及其偶联产物PR000020-ADC的HIC-HPLC分析结果：(A)HCAb偶联前；(B)偶联后，纯化前；(C)偶联及一步HIC纯化后。表5-3显示HCAb PR000020偶联前的样品纯度高达95％；表5-4显示HCAb PR000020经实施例5.1偶联细胞毒化合物后的产物中，75％的组分是偶联了2个化合物的产物(DAR＝2)；表5-5显示了该偶联产物经过一步HIC纯化(实施例4.2)后得到的产物中，99％的组分是偶联了2个化合物的产物。
滞留时间(分钟) 峰面积％ 峰说明
7.22 95.76 HCAb主峰
8.26 4.00 抗体杂质
9.56 0.24 抗体杂质
表5-3 HCAb PR000020(lot.#HSP302)的HIC-HPLC分析结果
滞留时间(分钟) 峰面积％ 峰说明
7.53 6.87 HCAb裸抗峰
8.53 1.61 DAR＝1组分峰
9.54 75.15 DAR＝2组分峰
11.25 3.29 聚体杂质或DAR>2组分峰
12.14 13.07 未反应的细胞毒化合物峰
表5-4 PR000020-ADC纯化前样品(lot.#HSP302-170814)的HIC-HPLC分析结果
滞留时间(分钟) 峰面积％ 峰说明
7.73 0.31 HCAb裸抗峰
9.44 99.69 DAR＝2组分峰
表5-5 PR000020-ADC纯化后样品(lot.#HSP302-170814PA0815)的HIC-HPLC分析结果
利用实施例2.3中所述RP-HPLC方法(使用分析型色谱柱Zorbax RRHD 300-Diphenyl)对上述样品进行分析。图9和下表分别显示了HCAb PR000020及其偶联产物PR000020-ADC的RP-HPLC分析结果：(A)HCAb偶联前；(B)偶联后，纯化前；(C)偶联及一步HIC纯化后。表5-6显示HCAb PR000020偶联前的样品纯度高达99％；表5-7显示HCAb PR000020经实施例5.1偶联细胞毒化合物后的产物中，75％的组分是偶联了2个化合物的产物(DAR＝2)；表5-8显示了该偶联产物经过一步HIC纯化(实施例4.2)后得到的产物中，96％的组分是偶联了2个化合物的产物。
滞留时间(分钟) 峰面积％ 峰说明
35.46 99.06 HCAb主峰
36.99 0.94 抗体杂质
表5-6 HCAb PR000020(lot.#HSP302)的RP-HPLC分析结果
滞留时间(分钟) 峰面积％ 峰说明
35.48 8.71 HCAb裸抗峰
36.22 2.89 DAR＝1组分峰
37.10 75.88 DAR＝2组分峰
38.34 1.12 偶联产物杂质峰
39.39 7.86 未反应细胞毒化合物峰
41.96 3.54 未知
表5-7 PR000020-ADC纯化前样品(lot.#HSP302-170814)的RP-HPLC分析结果
滞留时间(分钟) 峰面积％ 峰说明
35.68 0.73 HCAb裸抗峰
36.54 3.09 DAR＝1组分峰
37.22 96.18 DAR＝2组分峰
表5-8 PR000020-ADC纯化后样品(lot.#HSP302-170814PA0815)的RP-HPLC分析结果
利用实施例2.4中所述方法(LC仪器：Waters Acquity UPLC；MS仪器：Waters Xevo  G2-XS Q-TOF)对偶联产物PR000020-ADC的纯化后样品进行分子量分析。在进行LC-MS分析之前，待测样品经过去糖处理。图10分别显示了偶联产物PR000020-ADC的分子量分析的质谱去卷积处理图谱：(A)非还原样品；(B)还原样品。用PR000020的氨基酸序列计算其完整分子量(非还原分子量)和重链分子量(还原分子量)的理论值；偶联物mc-vc-PAB-MMAE的分子量为1315.78Da；推算出偶联了不同数目的化合物的组分的分子量理论值。对图谱中的峰和对应分子量进行分析，并通过积分计算峰面积以推测相应组分的含量(下表)。表5-9显示了DAR＝2的组分是主要成分；相应地，如表5-10所示，在还原样品中，近90％的重链偶联有1个化合物(+1D)。
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR000020裸抗 77,640      
PR000020 DAR＝1 78,956      
PR000020 DAR＝2 80,272 80,281 +9 主要成分
PR000020 DAR＝3 81,588      
表5-9 PR000020-ADC纯化后样品非还原分子量LC-MS分析
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR000020重链 38,823 38,827 +4 9.5％
PR000020重链+1D 40,139 40,145 +6 87.6％
PR000020重链+2D 41,455 41,460 +5 2.9％
表5-10 PR000020-ADC纯化后样品还原分子量LC-MS分析
综合以上多种分析手段的结果，可以推论出：利用半胱氨酸偶联，偶联产物中主要成分是DAR＝2的组分，经过进一步HIC纯化，可以得到纯度>90％的DAR＝2高均质性的产物。
实施例5.3 用LC-MS肽图分析化合物偶联位点
本实施例利用LC-MS来分析ADC偶联位点。简言之，将未偶联的HCAb PR000020和偶联产物PR000020-ADC的纯化后样品，用DTT(dithiothreitol)还原；然后，半胱氨酸的活性巯基用碘代乙酰胺(iodoacetamide,IAM)进行封闭；接着，用胰蛋白酶trypsin消化酶切样品；将酶切产物肽段用LC-MS分析。最后，将所得到的数据采用软件Peaks Studio(Bioinformatics Solutions Inc.)进行搜库，通过将偶联药物可能发生于半胱氨酸Cys 上设置为可变修饰，结合原始的二级质谱图信息，并通过提取离子流色谱图(XIC)进行比对，最终确定药物偶联位点。
如果Cys与mc-vc-PAB-MMAE发生反应形成稳定的硫醚键，那么该Cys位点就不能通过前述的还原烷基化处理方法形成IAM修饰。HCAb PR000020的每一条重链上有9个Cys(按照氨基酸序列顺序位置编号，分别为：C23,C96,C124,C130,C133,C165,C225,C271,C329)，其中C124对应Eu编号是220。经分析，在未偶联的PR000020样品中，所有9个Cys中，除C225由于前后较为密集的胰酶酶切位点分布导致未被检出，其余8个位点上的Cys均被检测到由于还原烷基化处理带来较高程度的IAM修饰，图11(C)列出了未偶联样品中所鉴定到的含有IAM修饰的Cys(Cys-IAM)的肽段列表。在偶联产物PR000020-ADC样品中，总共检测到1个药物偶联位点，为C124(即Eu编号的Cys-220)，而其它8个Cys均被检测到IAM修饰，图11(B)列出了偶联样品中所鉴定到的含有IAM修饰的Cys(Cys-IAM)的肽段列表，这些肽段在图11(A)的PR000020-ADC的氨基酸序列上用灰色背景标识。由此可见，在PR000020-ADC样品中，C124(即Cys-220)没有发生IAM修饰。
采用Peaks Studio进行搜库时，将偶联药物(mc-vc-PAB-MMAE，质量差异为1315.78Da)可能发生于Cys上的情况设置为可变修饰。搜库结果显示仅在偶联样品中鉴定到含C124(即Cys-220)的肽段
Figure PCTCN2021083024-appb-000008
(#表示修饰位点)发生了修饰(即药物偶联)。该肽段的二级质谱图如图35所示(m/z＝966.2409,z＝4,RT＝83.77min)。由于在实验过程中mc-vc-PAB-MMAE会发生碎裂(如图6(B)所示特征碎片结构)，导致了y离子的缺失，因此可以在该肽段二级谱图的低质量端观察到其特征碎片离子的存在，也由此进一步证实了C124(即Cys-220)的偶联修饰。通过比对PR000020-ADC样品与PR000020单抗样品中该肽段的提取离子流色谱图(XIC图，图36)发现，发生药物偶联的肽段(RT＝83.77min)的信号仅在ADC样品中检出，而作为对照的单抗样品中没有检测到相应的信号，这就进一步证明了该半胱氨酸残基为药物偶联位点。
综上所述，利用LC-MS肽图分析PR000020-ADC偶联产物证实了药物偶联发生在位点Cys-220。
实施例5.4 结合CTLA4高表达细胞
本实施例是为了研究HCAb抗体PR000020和其偶联产物PR000020-ADC结合高表达CTLA4的细胞的活性。
利用流式细胞术FACS测试抗体分子与高表达人CTLA4的HEK293T细胞株HEK293/hCTLA4(和铂医药构建)等细胞的结合能力。具体地，消化HEK293/hCTLA4细胞并用DMEM培养基重悬；将细胞密度调整为1x10 6细胞/mL。未表达CTLA4的HEK293细胞作为阴性对照。接着将细胞以100μL/孔接种于96孔V底板(Corning,#3894)，4℃下离心5分钟，弃上清。随后将梯度稀释的抗体分子以100μL/孔加入96孔板并混合均匀，抗体分子可以从最高终浓度为500nM按照3倍浓度梯度稀释的共12个浓度。将细胞放置于4℃，避光孵育1小时。然后，加入100μL/孔预冷的FACS缓冲液(含有0.5％BSA的PBS缓冲液)漂洗细胞两次，4℃下500g离心5分钟，弃上清。接着，再加入100μL/孔荧光二抗(Alexa Fluor 488anti-human IgG Fc,Biolegend,#409322,1:1000稀释)，放置于4℃，避光孵育1小时。随后以200μL/孔加入预冷的FACS缓冲液漂洗细胞两次，然后于4℃下500g离心5分钟，弃上清。最后，以200μL/孔加入预冷的FACS缓冲液重悬细胞。使用BD FACS CANTOII流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
图7中所示，抗体PR000020及其纯化后的偶联产物PR000020-ADC都与HEK293/hCTLA4细胞有较强的特异性结合活性。
实施例6 HCAb PR000759的偶联和分析
本实施例研究了抗MSLN的HCAb抗体PR000759(SEQ ID NO:67)与mc-vc-PAB-MMAE利用半胱氨酸偶联到Cys-220位点来制备具有均一DAR值的抗体偶联药物。
实施例6.1 偶联反应条件优化
以实施例5.1所得到的初步确定的优化偶联反应条件为基础，针对该抗体分子做进一步优化。如表6-1所示，平行用5组条件对纯化的HCAb PR000759重组蛋白进行偶联；还原剂选择TCEP，选择的药物偶联物为mc-vc-PAB-MMAE，偶联所用的药物偶联物用量为5-18摩尔倍数，反应时长为1-2小时。在本实验中，还原或偶联反应的pH为6.5-7.0，温度为室温(25-30℃)。每组实验的偶联的产物用实施例2.2所述HIC-HPLC分析方法分析其偶联产物的组分，列于表6-1。在本实施例中，实验组#2的偶联反应条件 可以实现DAR＝2组分(表6-1中的’D2％’)占比最高。偶联结束之后，通过脱盐柱将产物去除多余药物偶联物，并用实施例4.2所述HIC方法纯化偶联产物，得到DAR＝2的主要产物。
Figure PCTCN2021083024-appb-000009
表6-1 HCAb PR000759的偶联反应条件
实施例6.2 偶联产物分析
本实施例利用高效液相色谱和质谱等技术对纯化的重组HCAb PR000759和其偶联产物PR000759-ADC(包括偶联后产物和经过一步HIC纯化后的产物)进行定量分析，以确定其偶联产物组分的构成。
利用实施例2.1中所述SEC-HPLC方法和实施例2.2中所述HIC-HPLC方法对上述样品进行分析。图12和表6-2显示了HCAb PR000759重组蛋白偶联前的样品的SEC-HPLC分析结果，显示其纯度高达95％。图13和表6-3显示了偶联产物PR000759-ADC经过一步HIC纯化(实施例4.2)后得到的产物的HIC-HPLC分析结果，显示其94％的组分是偶联了2个化合物的产物(DAR＝2)。
滞留时间(分钟) 峰面积％ 峰说明
5.68 3.14  
7.22 1.43  
8.38 95.43 HCAb主峰
表6-2 HCAb PR000759(lot.#HSP307A PA1222F3-4)的SEC-HPLC分析结果
Figure PCTCN2021083024-appb-000010
Figure PCTCN2021083024-appb-000011
表6-3 PR000759-ADC纯化后样品(lot.#HSP307A-181229MIX)的HIC-HPLC分析结果
利用实施例2.4中所述方法对HCAb PR000759和PR000759-ADC的纯化后样品进行分子量分析。在进行LC-MS分析之前，待测样品经过去糖处理。图15显示了分子量分析的质谱去卷积处理图谱：(A)PR000759的非还原样品；(B)PR000759-ADC的非还原样品；(C)PR000759-ADC的还原样品。按照实施例5.2中所述分子量分析方法过程推算出偶联了不同数目的化合物的组分的分子量及其在样品中的含量(下表)。表6-4显示了HCAb PR000759的实际测定分子量。表6-5显示了在PR000759-ADC的非还原样品中，DAR＝2的组分是主要成分(占91％)，推算出PR000759-ADC产物的平均DAR值为2.10。相应地，如表6-6所示，在PR000759-ADC的还原样品中，偶联有1个化合物的重链(+1D)是主要成分，推算出PR000759-ADC产物的平均DAR值为1.98。
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR000759单抗 78,645 78,648～78,776 +3～+131* 主要成分
表6-4 HCAb PR000759(lot.#HSP307A PA1222F3-4)非还原分子量LC-MS分析
(*可能含有1个C’末端Lys未剪切(+128Da))
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR000759裸抗 78,645 78,650 +5 0.8％
PR000759DAR＝1 79,961 79,971 +10 2.4％
PR000759DAR＝2 81,277 81,284 +7 90.6％
PR000759DAR＝3 82,593 82,607 +14 1.8％
PR000759DAR＝4 83,909 83,922 +13 2.3％
PR000759DAR＝6 86,541 86,554 +13 2.0％
表6-5 PR000759-ADC纯化后样品(lot.#HSP307A-181229MIX)非还原分子量LC-MS分析
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR000759重链 39,324 39,330 +6 10.2％
PR000759重链+1D 40,639 40,646 +7 83.0％
PR000759重链+2D 41,955 41,964 +9 4.5％
PR000759重链+3D 43,271 43,280 +9 2.3％
表6-6 PR000759-ADC纯化后样品(lot.#HSP307A-181229MIX)还原分子量LC-MS分析
综合以上多种分析手段的结果，可以推论出：利用半胱氨酸偶联，偶联产物中主要成分是DAR＝2的组分，经过进一步HIC纯化，可以得到纯度>90％的DAR＝2高均质性的产物。
实施例6.3 用LC-MS肽图分析化合物偶联位点
本实施例利用LC-MS来分析ADC偶联位点。第一步，预处理待测样本PR000759-ADC。将样品转移至10KD超滤管中，经三次超滤后将样品置换到含8M尿素的缓冲液；再加入终浓度为20mM的DTT(dithiothreitol)，于37℃反应1小时；随后加入终浓度为50mM的吲哚乙酸(indole-3-acetic acid,IAA)，于室温下避光反应30分钟；再次超滤后置换到含50mM碳酸氢铵的缓冲液，并以样品:酶＝50:1的比例加入胰蛋白酶trypsin，于37℃过夜酶解。第二步，分析待测样品。样品经胰酶酶切后经由LC-MS/MS进行鉴定，采用软件Peaks Studio(Bioinformatics Solutions Inc.)进行搜库，其参数设置为：trypsin酶解；碎片离子质量容许误差：0.05Da；母离子质量容许误差：10ppm；最大漏切数：2；可变修饰为：Carbamidomethylation 57.02，Oxidation(M)15.99，Deamidation(NQ)0.98，Pyroglutamate formation(N-term E)-18.01，ADC drug 1315.78。
搜库结果经严格卡值过滤后得到可信肽段(-10lgP≥20)，其序列覆盖率近100％。从肽段列表中，筛选出带有mc-vc-PAB-MMAE偶联位点的三条肽段(图14)，结果显示PR000759-ADC在第127位(Eu编号为220)、第136位(Eu编号为229)和第168位(Eu编号为261)的半胱氨酸上鉴定到带有偶联修饰(如图14中粗体的C所示)。进一步对这三条肽段的对应的二级谱图进行了逐一核对，证实在其发生Δmass＝1315.78Da的修饰均为高度可信的；另外，将二级谱图的低质量端进行放大，可以清楚的看到mc-vc-PAB-MMAE发生碎裂后产生的特征碎片离子的存在，也进一步证实了偶联修饰位点。
在该肽图分析中，无法精确定量每一种偶联位点在产物中所占比例；但是结合之前的HIC-HPLC和LC-MS分子量分析，证明绝大多数偶联发生在PR000759的第127位的半胱氨酸(即Cys-220)以形成DAR＝2的偶联产物。
实施例7 HCAb PR001046的偶联和分析
本实施例研究了抗BCMA的HCAb抗体PR001046(SEQ ID NO:68)与mc-vc-PAB-MMAE利用半胱氨酸偶联到Cys-220位点来制备具有均一DAR值的抗体偶联药物。
实施例7.1 偶联反应条件优化
以实施例5.1所得到的初步确定的优化偶联反应条件为基础，针对该抗体分子做进一步优化。如表7-1所示，平行用6组条件对纯化的HCAb PR001046重组蛋白进行偶联；还原剂选择TCEP，选择的药物偶联物为mc-vc-PAB-MMAE，偶联所用的药物偶联物用量为5-8摩尔倍数，反应时长为1-3小时。在本实验中，还原或偶联反应的pH为6.5-7.0，温度为室温(25-30℃)。每组实验的偶联的产物用实施例2.2所述HIC-HPLC分析方法分析其偶联产物的组分，列于表7-1。当TCEP用量在1.3-1.5摩尔倍数时，DAR＝2组分得率(表7-1中的’D2％’)最高。偶联结束之后，通过脱盐柱将产物去除多余药物偶联物，并用实施例4.2所述HIC方法纯化偶联产物，得到DAR＝2的主要产物。
Figure PCTCN2021083024-appb-000012
表7-1 HCAb PR001046的偶联反应条件
实施例7.2 偶联产物分析
本实施例利用高效液相色谱和质谱等技术对纯化的重组HCAb PR001046和其偶联产物PR001046-ADC(包括偶联后产物和经过一步HIC纯化后的产物)进行定量分析，以确定其偶联产物组分的构成。
利用实施例2.1中所述SEC-HPLC方法和实施例2.2中所述HIC-HPLC方法对上述样品进行分析。图16和表7-2显示了HCAb PR001046重组蛋白偶联前的样品的SEC- HPLC分析结果，显示其纯度高达98％。图17和表7-3显示了偶联产物PR001046-ADC经过一步HIC纯化(实施例4.2)后得到的产物的HIC-HPLC分析结果，显示其88％的组分是偶联了2个化合物的产物(DAR＝2)。
滞留时间(分钟) 峰面积％ 峰说明
<7 0.28  
7.31 1.59  
8.30 98.13 HCAb主峰
表7-2 HCAb PR001046(lot.#HSP307B PA1222F11-12)的SEC-HPLC分析结果
Figure PCTCN2021083024-appb-000013
表7-3 PR001046-ADC纯化后样品(lot.#HSP307B-181229MIX)的HIC-HPLC分析结果
利用实施例2.4中所述方法对HCAb PR001046和PR001046-ADC的纯化后样品进行分子量分析。在进行LC-MS分析之前，待测样品经过去糖处理。图19显示了分子量分析的质谱去卷积处理图谱：(A)PR001046的非还原样品；(B)PR001046-ADC的非还原样品；(C)PR001046-ADC的还原样品。按照实施例5.2中所述分子量分析方法过程推算出偶联了不同数目的化合物的组分的分子量及其在样品中的含量(下表)。表7-4显示了HCAb PR001046的实际测定分子量。表7-5显示了在PR001046-ADC的非还原样品中，DAR＝2的组分是主要成分(占81％)，推算出PR001046-ADC产物的平均DAR值为2.39。相应地，如表7-6所示，在PR001046-ADC的还原样品中，偶联有1个化合物的重链(+1D)是主要成分，推算出PR001046-ADC产物的平均DAR值为2.20。
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR001046单抗 78,735 78,738～78,866 +3～+131* 主要成分
表7-4 HCAb PR001046(lot.#HSP307B PA1222F11-12)非还原分子量LC-MS分析
(*可能含有1个C’末端Lys未剪切(+128Da))
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR001046裸抗 78,735 78,739 +4 1.3％
PR001046DAR＝1 80,051 80,057 +6 0.5％
PR001046DAR＝2 81,367 81,373 +6 81.2％
PR001046DAR＝3 82,683 82,691 +8 3.9％
PR001046DAR＝4 83,999 84,009 +10 6.7％
PR001046DAR＝5 85,315 85,331 +16 0.4％
PR001046DAR＝6 86,631 86,643 +12 6.0％
表7-5 PR001046-ADC纯化后样品(lot.#HSP307B-181229MIX)非还原分子量LC-MS分析
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR001046重链 39,369 39,375 +6 7.8％
PR001046重链+1D 40,685 40,692 +7 79.3％
PR001046重链+2D 42,001 42,009 +8 8.3％
PR001046重链+3D 43,317 43,325 +8 4.7％
表7-6 PR001046-ADC纯化后样品(lot.#HSP307B-181229MIX)还原分子量LC-MS分析
综合以上多种分析手段的结果，可以推论出：利用半胱氨酸偶联，偶联产物中主要成分是DAR＝2的组分，经过进一步HIC纯化，可以得到纯度>80％的DAR＝2高均质性的产物。
实施例7.3 用LC-MS肽图分析化合物偶联位点
本实施例利用实施例6.3中所述方法来分析PR001046-ADC的偶联位点。
图18列出了带有mc-vc-PAB-MMAE偶联位点的三条肽段，结果显示PR001046-ADC在第127位(Eu编号为220)、第136位(Eu编号为229)和第168位(Eu编号为261)的半胱氨酸上鉴定到带有偶联修饰(如图18中粗体的C所示)。进一步对这三条肽段的对应的二级谱图进行了逐一核对，证实在其发生Δmass＝1315.78Da的修饰均为高度可信的；例如，图70显示了含有C220偶联位点的肽段的二级谱图。
在该肽图分析中，无法精确定量每一种偶联位点在产物中所占比例；但是结合之前的HIC-HPLC和LC-MS分子量分析，证明绝大多数偶联发生在PR001046的第127位的半胱氨酸(即Cys-220)以形成DAR＝2的偶联产物。
实施例7.4 结合BCMA高表达细胞
本实施例是为了研究HCAb抗体PR001046和其偶联产物PR001046-ADC结合高表达BCMA的细胞的活性。
利用流式细胞术FACS测试抗体分子与高表达人BCMA的HEK293T细胞株HEK293T-hBCMA(北京康源博创,KC-0233)的结合能力。具体地，消化细胞并用DMEM完全培养基重悬，调整细胞密度为1x10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson ImmunoResearch Inc.,#109-545-098,1:500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
图20中所示，抗体PR001046及其纯化后的偶联产物PR001046-ADC都与HEK293T-hBCMA细胞有较强的特异性结合活性。
实施例7.5 细胞毒性杀伤实验
本实施例为了研究抗BCMA的抗体偶联产物PR001046-ADC对高表达人BCMA的细胞系NCI-H929(ATCC,CRL-9068)的特异性细胞杀伤活性，并且对不表达人BCMA的SNU-16(ATCC,CRL-5974)细胞没有杀伤活性。
将NCI-H929细胞和SNU-16细胞分别接种于96孔板(Perkin Elmer,#6005225)，接种细胞数量分别为：10000细胞/50μL/孔(NCI-H929)、5000细胞/50μL/孔(SNU-16)、10000细胞NCI-H929和5000细胞SNU-16/100μL/孔混合(NCI-H929+SNU-16)。然后，以50μL/孔加入预先梯度稀释的待测样品，PR001046和PR001046-ADC的浓度为1μg/ml、0.1μg/ml和0.01μg/ml，mc-vc-PAB-MMAE的浓度为10nM、1nM和0.1nM。接着，在37℃和5％CO2环境下孵育72小时，加入CellTiter-Glo细胞活力检测试剂(Promega,#G7573)，室温下孵育至少10分钟得到稳定发光信号，酶标仪检测发光值。应用软件 GraphPad Prism 8进行数据处理和作图分析。本实施例中，使用了多组不同的PR001046-ADC样品，包括纯化前或纯化后的含有不同DAR＝2(D2)组分百分比的样品。
图21中所示，0.1μg/ml及以上浓度的PR001046-ADC可以特异性地杀伤BCMA阳性细胞NCI-H929以及NCI-H929与SNU-16的混合细胞，但是不能杀伤BCMA阴性细胞SNU-16。如图21(A)所示，未偶联的PR001046或mc-vc-PAB-MMAE化合物不能对细胞产生有效的杀伤。另一方面，如图21(B)所示，偶联产物主要成分(DAR＝2)的纯度对于杀伤能力没有显著影响。
实施例8 HCAb PR004432的偶联和分析
本实施例研究了抗5T4的HCAb抗体PR004432(SEQ ID NO:69)与mc-vc-PAB-MMAE利用半胱氨酸偶联到Cys-220位点来制备具有均一DAR值的抗体偶联药物。
实施例8.1 偶联反应条件优化
以实施例5.1所得到的初步确定的优化偶联反应条件为基础，针对该抗体分子做进一步优化。如表8-1所示，平行用2组条件对纯化的HCAb PR004432重组蛋白进行偶联；还原剂选择TCEP，TCEP用量为4摩尔倍数，选择的药物偶联物为mc-vc-PAB-MMAE，偶联所用的药物偶联物用量为12摩尔倍数，反应时长为1-2小时。在本实验中，还原或偶联反应的pH为6.5-7.0，温度为室温(25-30℃)。每组实验的偶联的产物用实施例2.3所述RP-HPLC分析方法(使用分析型色谱柱PLRP-S 1000A)分析其偶联产物的组分，列于表8-1；DAR＝2组分得率(’D2％’)约60％。偶联结束之后，通过脱盐柱将产物去除多余药物偶联物，并用实施例4.2所述HIC方法纯化偶联产物，得到DAR＝2的主要产物。
Figure PCTCN2021083024-appb-000014
表8-1 HCAb PR004432的偶联反应条件
实施例8.2 偶联产物分析
本实施例利用高效液相色谱和质谱等技术对纯化的重组HCAb PR004432和其偶联产物PR004432-ADC(包括偶联后产物和经过一步HIC纯化后的产物)进行定量分析，以确定其偶联产物组分的构成。
利用实施例2.1中所述SEC-HPLC方法和实施例2.3中所述RP-HPLC方法(使用分析型色谱柱PLRP-S 1000A)对上述样品进行分析。图22和表8-2显示了HCAb PR004432重组蛋白偶联前的样品的SEC-HPLC分析结果，显示其纯度高达98％。图23和表8-3显示了偶联产物PR004432-ADC经过一步HIC纯化(实施例4.2)后得到的产物的RP-HPLC分析结果，显示其90.6％的组分是偶联了2个化合物的产物(DAR＝2)。
滞留时间(分钟) 峰面积％ 峰说明
6.90 0.59  
7.32 0.76  
7.70 98.64 HCAb主峰
表8-2 HCAb PR004432(lot.#HSP314-01K20200427-0507PB0507)的SEC-HPLC分析结果
Figure PCTCN2021083024-appb-000015
表8-3 PR004432-ADC纯化后样品(lot.#HSP31401-ADC-20200609)的RP-HPLC分析结果
利用实施例2.4中所述方法(LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix X500B)对PR004432-ADC的纯化后样品进行分子量分析。在进行LC-MS分析之前，待测样品经过去糖处理。图25显示了分子量分析的质谱去卷积处理图谱：(A)PR004432-ADC的非还原样品；(B)PR004432-ADC的还原样品。按照实施例5.2中所述分子量分析方法过程推算出偶联了不同数目的化合物的组分的分子量及其在样品中的含量(下表)。表8-4显示了在PR004432-ADC的非还原样品中，DAR＝2的组分是主要成分(占98.3％)，推算出PR004432-ADC产物的平均DAR值为1.97。相应地，如表8-5所示，在PR004432- ADC的还原样品中，偶联有1个化合物的重链(+1D)是主要成分(占96.5％)，推算出PR004432-ADC产物的平均DAR值为1.93。
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR004432裸抗 80,531 80,534～80,549 +3～+18 1.7％
PR004432DAR＝1 81,847      
PR004432DAR＝2 83,163 83,138～83,397 -25～+234* 98.3％
PR004432DAR＝3 84,479      
表8-4 PR004432-ADC纯化后样品(lot.#HSP31401-ADC-20200609)非还原分子量LC-MS分析
(*可能包含C’端未剪切Lys(+128Da)或其他修饰带来的分子量偏差)
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR004432重链 40,266 40,199～40,399 -67～+133 3.5％
PR004432重链+1D 41,582 41,448～41,714 -134～+132* 96.5％
PR004432重链+2D 42,898      
表8-5 PR004432-ADC纯化后样品(lot.#HSP31401-ADC-20200609)还原分子量LC-MS分析
(*可能包含C’端未剪切Lys(+128Da)或其他修饰带来的分子量偏差)
综合以上多种分析手段的结果，可以推论出：利用半胱氨酸偶联，偶联产物中主要成分是DAR＝2的组分，经过进一步HIC纯化，可以得到纯度>90％的DAR＝2高均质性的产物。
实施例8.3 用LC-MS肽图分析化合物偶联位点
本实施例利用实施例6.3中所述方法来分析PR004432-ADC的偶联位点。简言之，PR004432-ADC样品经trypsin酶解后用LC-MS/MS鉴定，所得数据用软件BioPharmView(SCIEX)搜库，搜库参数设置为：trypsin酶解；碎片离子质量容许误差：0.05Da；母离子质量容许误差：10ppm；最大漏切数：1；可变修饰：Carbamidomethylation 57.02，Oxidation(M)15.99，Deamidation(NQ)0.98，mc-vc-PAB-MMAE 1315.78。找到带有化合物偶联修饰(分子量差异1315.78)的相关目标肽段，查看相应二级谱图进一步确认。偶联位点占有率(该位点联结有化合物的比例)用下面公式计算：
Figure PCTCN2021083024-appb-000016
图24显示了利用LC-MS肽谱图分析PR004432-ADC的化合物偶联位点，包括筛 选出的带有偶联位点的3条肽段，以及相应的偶联位点覆盖率。分析所有含有Cys位点并发生偶联修饰的肽段，每一条肽段对应的二级谱图也经过逐一核对，以证实其发生的偶联修饰均为高度可信的。例如，含有SC(+1315.78)DK的肽段占比97.12％，这说明，97.12％的Cys-220位点被偶联有化合物。类似地，只有1.5％的Cys-226或Cys-229位点发生偶联。
实施例8.4 结合5T4高表达细胞
本实施例是为了研究HCAb抗体PR004432和其偶联产物PR004432-ADC结合高表达BCMA的细胞的活性。
利用流式细胞术FACS测试抗体分子与高表达人5T4的人乳腺癌细胞株HCC1954(ATCC,CRL-2338)的结合能力。具体地，调整细胞密度为1x10 6细胞/mL，以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson ImmunoResearch Inc.,#109-545-098,1:500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
图26中所示，抗体PR004432及其纯化后的偶联产物PR004432-ADC都与HCC1954细胞有较强的特异性结合活性。
实施例8.5 细胞毒性杀伤实验
本实施例为了研究抗5T4的抗体偶联产物PR004432-ADC对高表达人5T4的人乳腺癌细胞株HCC1954(ATCC,CRL-2338)的细胞杀伤活性。
将HCC1954细胞以4000细胞/50μL/孔接种于96孔板(Perkin Elmer,#6005225)，于37℃和5％CO2下孵育过夜。然后，以50μL/孔加入预先梯度稀释的待测样品，PR004432、PR004432-ADC和PR004433-ADC从最高终浓度为30nM以3倍浓度梯度稀释，mc-vc-PAB-MMAE从最高终浓度为60nM以3倍浓度梯度稀释；另设置不加待测样 品的孔为对照孔。接着，在37℃和5％CO2环境下孵育72小时，加入CellTiter-Glo细胞活力检测试剂(Promega,#G7573)，室温下孵育至少10分钟得到稳定发光信号，酶标仪检测发光值。将发光值按如下公式计算细胞活率：
细胞活率(％)＝(A/B)×100％
其中：A＝实验孔(靶细胞+待测样品+培养基)；B＝对照孔(靶细胞+培养基)
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
图27显示了抗5T4的HCAb PR004432及其偶联产物PR004432-ADC、抗BCMA的HCAb PR004433的偶联产物PR004433-ADC、未偶联的细胞毒化合物mc-vc-PAB-MMAE等待测样品对高表达人5T4的细胞HCC1954的细胞毒性杀伤。仅PR004432-ADC能够对HCC1954产生有效的特异性的细胞毒性杀伤，且呈现抗体浓度依赖性；mc-vc-PAB-MMAE仅在最高浓度(60nM)时有杀伤效果，而其他分子都不能对细胞产生有效的杀伤。此结果证实了PR004432-ADC的靶点依赖的高特异性和高效力。
实施例9 HCAb PR004433的偶联和分析
本实施例研究了抗BCMA的HCAb抗体PR004433(SEQ ID NO:70)与mc-vc-PAB-MMAE利用半胱氨酸偶联到Cys-220位点来制备具有均一DAR值的抗体偶联药物。
实施例9.1 偶联反应条件优化
以实施例5.1所得到的初步确定的优化偶联反应条件为基础，针对该抗体分子做进一步优化。如表9-1所示，平行用6组条件对纯化的HCAb PR004433重组蛋白进行偶联；还原剂选择TCEP，TCEP用量在1.7–2.1摩尔倍数，选择的药物偶联物为mc-vc-PAB-MMAE，偶联所用的药物偶联物用量为7摩尔倍数，反应时长为2小时。在本实验中，还原或偶联反应的pH为6.5-7.0，温度为室温(25-30℃)。每组实验的偶联的产物用实施例2.2所述HIC-HPLC分析方法分析其偶联产物的组分，尤其是DAR＝2的组分含量(’D2％’)，列于表9-1。偶联结束之后，通过脱盐柱将产物去除多余药物偶联物，并用实施例4.2所述HIC方法纯化偶联产物，得到DAR＝2的主要产物。
Figure PCTCN2021083024-appb-000017
Figure PCTCN2021083024-appb-000018
表9-1 HCAb PR004433的偶联反应条件
实施例9.2 偶联产物分析
本实施例利用高效液相色谱和质谱等技术对纯化的重组HCAb PR004433和其偶联产物PR004433-ADC(包括偶联后产物和经过一步HIC纯化后的产物)进行定量分析，以确定其偶联产物组分的构成。
利用实施例2.1中所述SEC-HPLC方法和实施例2.2中所述HIC-HPLC方法对上述样品进行分析。图28和表9-2显示了HCAb PR004433重组蛋白偶联前的样品的SEC-HPLC分析结果，显示其纯度高达98％。图29和表9-3显示了偶联产物PR004433-ADC经过一步HIC纯化(实施例4.2)后得到的产物的HIC-HPLC分析结果，显示其90.8％的组分是偶联了2个化合物的产物(DAR＝2)。图30和下表分别显示了HCAb PR004433及其偶联产物PR004433-ADC的HIC-HPLC分析结果：(A)PR004433偶联前(表9-4)；(B)偶联后，纯化前(表9-5)；(C)偶联及一步HIC纯化后(表9-6)。
滞留时间(分钟) 峰面积％ 峰说明
<6.5 0.86  
6.86 1.12  
7.73 98.02 HCAb主峰
表9-2 HCAb PR004433(lot.#HSP314-02K20200330-0409PA0409)的SEC-HPLC分析结果
Figure PCTCN2021083024-appb-000019
Figure PCTCN2021083024-appb-000020
表9-3 PR004433-ADC纯化后样品(lot.#HSP31402-ADC-20200521)的HIC-HPLC分析结果
滞留时间(分钟) 峰面积％ 峰说明
7.07 0.11  
7.77 99.89 HCAb主峰
表9-4 HCAb PR004433(lot.#HSP314-02K20200330-0409PA0409)的HIC-HPLC分析结果
Figure PCTCN2021083024-appb-000021
表9-5 PR004433-ADC纯化前样品(lot.#HSP31402-20041601)的HIC-HPLC分析结果
滞留时间(分钟) 峰面积％ 峰说明
8.45 1.77 DAR＝1组分峰
9.37 93.85 DAR＝2组分峰
11.02 4.38 杂质或DAR>2组分峰
表9-6 PR004433-ADC纯化后样品(lot.#HSP31402-20041601PA0416)的HIC-HPLC分析结果
利用实施例2.4中所述方法(LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix X500B)对PR004433-ADC的纯化后样品进行分子量分析。在进行LC-MS分析之前，待测样品经过去糖处理。图31显示了分子量分析的质谱去卷积处理图谱：(A)PR004433-ADC的非还原样品；(B)PR004433-ADC的还原样品。按照实施例5.2中所述分子量分析方法过程推算出偶联了不同数目的化合物的组分的分子量及其在样品中的含量(下表)。表9-7显示了在PR004433-ADC的非还原样品中，DAR＝2的组分是主要成分(占98％)，推算出PR004433-ADC产物的平均DAR值为2.02。相应地，如表9-8所示，在PR004433-ADC的还原样品中，偶联有1个化合物的重链(+1D)是主要成分(占90.5％)，推算出PR004433-ADC产物的平均DAR值为2.02。
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR004433裸抗 78,797     0％
PR004433DAR＝1 80,113 80,119 +6 0.8％
PR004433DAR＝2 81,429 81,405～81,693 -24～+264* 98.1％
PR004433DAR＝4 84,065 84,070 +5 0.5％
PR004433DAR＝6 86,701 86,704 +3 0.5％
表9-7 PR004433-ADC纯化后样品(lot.#HSP31402-ADC-20200521)非还原分子量LC-MS分析
(*可能包含C’端未剪切Lys(+128Da)或其他修饰带来的分子量偏差)
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR004433重链 39,400 39,403～39,503 +3～+103 4.7％
PR004433重链+1D 40,716 40,585～40,847 -131～+131* 90.5％
PR004433重链+2D 42,032 42,037～42,164 +5～+127 4.1％
PR004433重链+3D 43,348 43,353 +5 0.7％
表9-8 PR004433-ADC纯化后样品(lot.#HSP31402-ADC-20200521)还原分子量LC-MS分析
(*可能包含C’端未剪切Lys(+128Da)或其他修饰带来的分子量偏差)
综合以上多种分析手段的结果，可以推论出：利用半胱氨酸偶联，偶联产物中主要成分是DAR＝2的组分，经过进一步HIC纯化，可以得到纯度>90％的DAR＝2高均质性的产物。
实施例9.3 用LC-MS肽图分析化合物偶联位点
本实施例利用实施例8.3中所述方法来分析PR004433-ADC的偶联位点。
图34显示了利用LC-MS肽谱图分析PR004433-ADC的化合物偶联位点，包括筛选出的带有偶联位点的3条肽段，以及相应的偶联位点覆盖率。分析所有含有Cys位点并发生偶联修饰的肽段，每一条肽段对应的二级谱图也经过逐一核对，以证实其发生的偶联修饰均为高度可信的。例如，含有SC(+1315.78)DK的肽段占比98％，这说明，98％的Cys-220位点被偶联有化合物。类似地，只有4％的Cys-226或Cys-229位点发生偶联。
实施例9.4 结合BCMA高表达细胞
本实施例利用实施例7.4中所述方法研究HCAb抗体PR004433或其偶联产物PR004433-ADC与高表达人BCMA的HEK293T细胞株HEK293T-hBCMA(北京康源博创,KC-0233)、高表达人BCMA的细胞系NCI-H929(ATCC,CRL-9068)或不表达人 BCMA的细胞系SNU-16(ATCC,CRL-5974)等细胞的结合能力。
图32中所示，PR004433和PR004433-ADC都可以与HEK293T-hBCMA细胞(图32(A))和NCI-H929细胞(图32(B))有较强的特异性结合；而且，PR004433不能结合BCMA阴性细胞SNU-16(图32(C))。
实施例9.5 细胞毒性杀伤实验
本实施例为了研究抗BCMA的抗体偶联产物PR004433-ADC对高表达人BCMA的HEK293T细胞株HEK293T-hBCMA(北京康源博创,KC-0233)和高表达人BCMA的肿瘤细胞系NCI-H929(ATCC,CRL-9068)的特异性细胞杀伤活性，并且对不表达人BCMA的细胞HEK293T(ATCC,CRL-11268)和SNU-16(ATCC,CRL-5974)没有杀伤活性。
将HEK293T-hBCMA、NCI-H929、HEK293T和SNU-16细胞分别接种于96孔板(Perkin Elmer,#6005225)，接种细胞数量分别为：2500细胞/50μL/孔(HEK293T-hBCMA)，5000细胞/50μL/孔(NCI-H929)，2500细胞/50μL/孔(HEK293T)，5000细胞/50μL/孔(SNU-16)。随后于37℃和5％CO2下孵育过夜。然后，以50μL/孔加入预先梯度稀释的待测样品，PR004433、PR004433-ADC和PR004432-ADC从最高终浓度为10nM以3倍浓度梯度稀释，mc-vc-PAB-MMAE从最高终浓度为20nM以3倍浓度梯度稀释；另设置不加待测样品的孔为对照孔。接着，在37℃和5％CO2环境下孵育72小时，加入CellTiter-Glo细胞活力检测试剂(Promega,#G7573)，室温下孵育至少10分钟得到稳定发光信号，酶标仪检测发光值。将发光值按如下公式计算细胞活率：
细胞活率(％)＝(A/B)×100％
其中：A＝实验孔(靶细胞+待测样品+培养基)；B＝对照孔(靶细胞+培养基)
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
图33显示了抗BCMA的HCAb PR004433及其偶联产物PR004433-ADC、抗5T4的HCAb PR004432的偶联产物PR004432-ADC、未偶联的细胞毒化合物mc-vc-PAB-MMAE等待测样品对BCMA阳性细胞(HEK293T-hBCMA和NCI-H929)和BCMA阴性细胞(HEK293T和SNU-16)的细胞毒性杀伤。仅PR004433-ADC能够对BCMA阳性细胞HEK293T-hBCMA(图33(A))和NCI-H929(图33(C))产生有效的特异性的细胞毒性杀伤，且呈现抗体浓度依赖性，对BCMA阴性细胞没有细胞毒性；而其他分子都不能对细胞产生有效的杀伤。此结果证实了PR004433-ADC的靶点依赖的高特异性和高效力。
实施例10 HCAb PR004433的偶联反应条件优化
在实施例9中已经研究了抗BCMA的HCAb抗体PR004433(SEQ ID NO:70)与mc-vc-PAB-MMAE利用半胱氨酸偶联到Cys-220位点来制备具有均一DAR值的抗体偶联药物；但是如实施例9.1所述实验过程和表9-1所列出的反应条件和结果所示，其经过一步偶联后的产物中，DAR＝2的组分含量(’D2％’)约为50-60％，需要经过一步HIC纯化(实施例4.2所述方法)来去除非主要组分得到高纯度的DAR＝2组分。
本实施例进一步优化偶联反应条件，使得经过一步偶联后的产物中，DAR＝2的组分含量提高，减少了进一步纯化的需求；更佳地，经过偶联步骤后无需额外纯化步骤即可以得到纯度大于90％的DAR＝2高均质性的产物。
实施例10.1 偶联反应条件优化
由于在还原和偶联等反应过程中，多种反应条件如反应温度、反应时间、反应缓冲液类型及pH、还原剂类型及用量、偶联物分子类型及用量等都可能带来很大影响，因此在实施例4.1中所述方法的基础上，本实施例设计了一系列实验，固定其中一些实验条件参数，而对另一些实验条件参数进行改变，尝试得到更优的反应条件。具体地，本实施例使用了17组不同的还原和偶联条件的组合对HCAb PR004433进行偶联反应并对偶联产物进行分析，相较于实施例9，本实施例还进一步考察了反应缓冲液pH和还原反应温度等参数对偶联的影响。在本实验中，PR004433的浓度为5mg/ml；反应缓冲液为20mM His-HCl，其pH可调节；还原剂为TCEP，其摩尔当量可变；还原反应温度和反应时长为变量；偶联药物分子为mc-vc-PAB-MMAE(生产商：联宁生物制药，货号：SET0201)，其摩尔当量可变；偶联反应温度为室温，反应时长为30分钟。表10-1列出了所述17组实验及其各反应条件参数。对于每组实验，其偶联产物利用实施例2.4中所述方法(LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix X500B；样品未经去糖预处理)进行非还原样品的完整分子量分析，并利用每个样品的质谱图中的总离子流图(TIC)并结合实施例5.2使用的分子量分析过程来推算出偶联了不同数目的化合物的组分的分子量及其在 样品中的含量；分析结果列于表10-2。在表10-2中，“D0％”、“D2％”、“D4％”和“D6％”分别表示没有偶联化合物(DAR＝0)、偶联了2个化合物(DAR＝2)、偶联了4个化合物(DAR＝4)和偶联了6个化合物(DAR＝6)的组分的含量，并据此计算出平均DAR值。其中，实验#8、#9、#11和#12产生的偶联产物中DAR＝2组分含量超过了85％，其平均DAR值也非常接近2.0；这四组实验的偶联产物样品的质谱图的TIC图分别对应于图45的(A)-(D)。
Figure PCTCN2021083024-appb-000022
表10-1 PR004433的偶联反应条件
Figure PCTCN2021083024-appb-000023
Figure PCTCN2021083024-appb-000024
表10-2 PR004433偶联后产物(不经过HIC纯化)利用LC-MS分析其各组分的结果
特别地，实验#12产生的偶联产物中DAR＝2组分含量超过了90％，其他组分含量极低，说明HCAb PR004433利用实验#12的反应条件可以产生高纯度的偶联产物，无需经过额外纯化步骤，即可得到DAR值确定的高均一性的产物。这相较于实施例9中的偶联方法是显著的进步。
对实验#12产生的偶联产物进一步利用实施例9.3中所述方法来分析抗体偶联药物分子的偶联位点，结果表明，96.22％的Cys-220位点被偶联；类似地，只有6％的Cys-226或Cys-229位点发生偶联。因此，实验#12产生的偶联产物中是高均一性的定点偶联于Cys-220的抗体偶联药物分子。
本实施例充分地说明了，本申请的发明人对整个偶联过程的各种反应条件进行了多次的尝试和优化，找到了更优化的反应条件组合，在一些反应条件组合下，Cys-220的二 硫键可以被有效地打开而其他二硫键保持完整，使得Cys-220成为几乎唯一的游离半胱氨酸残基，进而作为特异性的偶联位点。
实施例11 人IgG1铰链区序列的变化
本实施例研究了基于人IgG1抗体重链铰链区的天然序列(SEQ ID NO:73)产生的连接肽变体序列中第一个Cys(Cys1)和第二个Cys(Cys2)之间所间隔的氨基酸的数目和种类，和对第一个Cys作为特异性定点偶联位点产生均一CAR值(偶联物/抗原结合蛋白比)的偶联产物的影响。
实施例11.1 改变HCAb PR004433的铰链区连接肽以产生变体序列
本实施例在HCAb PR004433的基础上，针对铰链区设计了不同长度的随机化引物通过蛋白工程改造产生一系列具有不同铰链区连接肽的抗原结合蛋白。例如，利用表11-1中所述引物，可以在Cys1和Cys2之间插入分别为3、4、5、6、7、8、9或10个任意氨基酸，以产生如SEQ ID NOs:74-81任一所示连接肽序列。然后，每一种长度的连接肽随机挑选3种不同突变序列进一步研究Cys1和Cys2之间的不同氨基酸组成，且Cys1和Cys2之间的氨基酸不能是半胱氨酸。随后，在后续实施例中，具有不同长度和氨基酸组成的连接肽的抗原结合蛋白(PR004433变体序列)利用前述方法进行表达纯化，并与药物偶联物mc-vc-PAB-MMAE偶联，分析偶联产物的组分和偶联位点。表11-2列出了一系列由随机化产生的不同长度的连接肽序列。将HCAb PR004433的铰链区序列替换成表11-2中的连接肽序列以产生多种PR004433衍生的抗原结合蛋白变体序列(表11-3)，这些抗原结合蛋白的可变区和Fc恒定区之间的连接肽具有不同的长度和氨基酸组成。
Figure PCTCN2021083024-appb-000025
Figure PCTCN2021083024-appb-000026
表11-1 随机化引物列表(正向引物序列中N表示A/T/G/C中任一种碱基，K表示G/T中任一种碱基)
Figure PCTCN2021083024-appb-000027
Figure PCTCN2021083024-appb-000028
表11-2 不同长度的连接肽
抗原结合蛋白 连接肽标识 连接肽SEQ ID NO 多肽链SEQ ID NO
PR004433 hIgG1_hinge 73 70
PR006031 Hinge3_5 82 106
PR006032 Hinge3_6 83 107
PR006033 Hinge3_18 84 108
PR006034 Hinge4_1 85 109
PR006035 Hinge4_6 86 110
PR006036 Hinge4_15 87 111
PR006037 Hinge5_2 88 112
PR006038 Hinge5_12 89 113
PR006039 Hinge5_13 90 114
PR006040 Hinge6_4 91 115
PR006041 Hinge6_15 92 116
PR006042 Hinge6_18 93 117
PR006310 Hinge7_2 94 118
PR006311 Hinge7_3 95 119
PR006312 Hinge7_19 96 120
PR006313 Hinge8_8 97 121
PR006314 Hinge8_18 98 122
PR006315 Hinge8_20 99 123
PR006316 Hinge9_5 100 124
PR006317 Hinge9_7 101 125
PR006318 Hinge9_14 102 126
PR006319 Hinge10_3 103 127
PR006320 Hinge10_5 104 128
PR006321 Hinge10_7 105 129
表11-3 HCAb PR004433的铰链区序列替换成多种连接肽序列以产生不同衍生变体序列
实施例11.2 HCAb PR004433衍生的变体分子的表达和鉴定
表11-3中的PR004433衍生的抗原结合蛋白按照实施例1.6中所述方法进行表达纯化，并利用实施例2.1中所述SEC-HPLC方法分析纯度和利用实施例2.7中所述DSF方法确定其最低熔解温度(Tm1)。抗原结合蛋白的表达纯化和热稳定性分析结果列于表11-4；由此可见，当HCAb PR004433的铰链区序列替换成各种连接肽的变体序列后，且当Cys1和Cys2之间间隔至少3个其他氨基酸时，PR004433衍生的变体序列都能够有效的表达，且绝大多数变体分子的表达产量和热稳定性Tm1没有受到影响。
Figure PCTCN2021083024-appb-000029
Figure PCTCN2021083024-appb-000030
表11-4 PR004433及其变体分子的表达纯化和热稳定性分析
本实施例进一步利用实施例3.2中所述LC-MS分子量分析方法来确定不同的连接肽变体序列中Cys1的状态。在PR004433衍生的变体序列中，Cys1(或Cys-220)的状态有如图3所示的两种可能性：形成或者不形成链间二硫键。如果Cys1没有形成重链间二硫键，其侧链巯基(-SH)会与环境中的含巯基分子(如，半胱氨酸或者谷胱甘肽)形成二硫键，使其实际分子量明显大于理论分子量。将纯化的抗原结合蛋白用糖苷酶PNGase F作去糖处理，并用实施例2.4所述方法(LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix X500B)测定其完整(非还原)分子量，与相应的理论分子量进行比较；表11-5显示了分子量的理论值和测定值是非常一致的，因此，所述抗原结合蛋白的Cys1没有联结其他含巯基分子。由此推断出，当Cys1和Cys2之间间隔3个至10个其他氨基酸时，Cys1能够形成重链间二硫键(即第一连接肽的Cys1和第二连接肽的Cys1之间形成二硫键)。
抗原结合蛋白 连接肽标识 理论值(Da) 测定值(Da) 偏差(Da)
PR006031 Hinge3_5 78,395 78,395.6 0.6
PR006032 Hinge3_6 78,261 78,261.4 0.4
PR006033 Hinge3_18 78,201 78,201.5 0.5
PR006034 Hinge4_1 78,467 78,467.8 0.8
PR006035 Hinge4_6 78,629 78,629.9 0.9
PR006036 Hinge4_15 78,487 78,487.9 0.9
PR006037 Hinge5_2 78,775 78,776.0 1
PR006038 Hinge5_12 78,871 78,872.2 1.2
PR006039 Hinge5_13 78,769 78,770.2 1.2
PR006040 Hinge6_4 79,046 79,046.7 0.7
PR006041 Hinge6_15 78,717 78,718.2 1.2
PR006042 Hinge6_18 78,966 78,966.9 0.9
PR006310 Hinge7_2 78,992 78,991.6 -0.4
PR006312 Hinge7_19 79,278 79,278.0 0
PR006313 Hinge8_8 79,596 79,596.3 0.3
PR006314 Hinge8_18 79,148 79,147.7 -0.3
PR006316 Hinge9_5 79,364 79,364.4 0.4
PR006317 Hinge9_7 79,208 79,207.8 -0.2
PR006318 Hinge9_14 79,486 79,486.5 0.5
PR006319 Hinge10_3 80,035 80,035.0 0
PR006320 Hinge10_5 79,760 79,760.7 0.7
PR006321 Hinge10_7 80,089 80,088.9 -0.1
表11-5 PR004433变体分子的纯化后样品的完整分子量LC-MS测定结果
本实施例还进一步利用实施例9.4中所述方法研究HCAb抗体PR004433及其变体分子与高表达人BCMA的NCI-H929(ATCC,CRL-9068)细胞的结合能力。如图46中所示，PR004433变体分子结合NCI-H929的能力与亲本分子PR004433的结合能力几乎相同，说明对PR004433的铰链区(连接肽)序列的改变不会影响其抗原结合片段与靶点的结合。
实施例11.3 HCAb PR004433衍生的变体分子的偶联产物分析
本实施例还进一步利用实施例10中所述偶联方法对表11-4中所列的PR004433衍 生的变体分子进行偶联反应，在实施例10.1所得到的较优的反应条件参数的基础上，通过改变还原剂TCEP的用量，来产生不同的偶联反应产物。在本实验中，具有不同连接肽的PR004433衍生的变体分子(PR006031、PR006034、PR006037、PR006040、PR006312、PR006314、PR006317和PR006320)的浓度为5mg/ml，反应缓冲液为含20mM His-HCl的pH 6.0缓冲液，还原剂TCEP的用量从1.5到6.0倍摩尔当量变化，还原反应于室温2小时，偶联物mc-vc-PAB-MMAE的用量为7.0摩尔倍数，偶联反应于室温0.5小时；其偶联产物不经过HIC纯化步骤。进一步地，偶联产物也利用实施例10.1所使用的分子量分析方法来推算出偶联了不同数目的化合物的组分的分子量及其在样品中的含量，分析结果列于表11-6；例如，PR006031中的连接肽的Cys1和Cys2间隔了3个氨基酸，当使用2.0当量的TCEP作为还原剂并利用上述反应参数进行偶联，其偶联产物中的DAR＝2组分含量为84.3％，偶联产物的平均DAR值为2.12；又例如，PR006320中的连接肽的Cys1和Cys2间隔了10个氨基酸，当使用3.0当量的TCEP作为还原剂并利用上述反应参数进行偶联，其偶联产物中的DAR＝2组分含量为94.8％，偶联产物的平均DAR值为1.99。
Figure PCTCN2021083024-appb-000031
Figure PCTCN2021083024-appb-000032
表11-6 PR004433变体分子的偶联产物(不经过HIC纯化)利用LC-MS分析其各组分的结果
本实施例说明了，当连接肽的Cys1和Cys2之间相隔至少3个其他氨基酸时，Cys1能够形成重链间二硫键(即第一连接肽的Cys1和第二连接肽的Cys1之间形成二硫键，此二硫键称为“Cys1-Cys1”)，而且Cys2也能够形成重链间二硫键(即第一连接肽的Cys2和第二连接肽的Cys2之间形成二硫键，此二硫键称为“Cys2-Cys2”)。但是，二硫键Cys1-Cys1的稳定性可能弱于其他二硫键。在一些反应条件组合下，二硫键Cys1-Cys1可以被有效地打开而其他二硫键(例如，Cys2-Cys2)保持完整，使得Cys1成为几乎唯一的游离半胱氨酸残基，进而作为特异性的偶联位点。使用含该连接肽的抗原结合蛋白产生的蛋白-药物偶联物，其偶联位点在Cys1且CAR(或DAR)约为2。
实施例12 位于小鼠IgG2c铰链区的偶联和分析
本实施例研究了利用小鼠IgG2c抗体重链铰链区(SEQ ID NO:147)与mc-vc-PAB-MMAE通过半胱氨酸偶联来制备抗体偶联药物。小鼠IgG2c的铰链区具有三个半胱氨酸，第一半胱氨酸(Cys1)和第二半胱氨酸(Cys2)之间间隔了5个其他氨基酸残基。
在HCAb PR004433(SEQ ID NO:70)的基础上，将其人IgG1铰链区(SEQ ID NO:73)部分替换成小鼠IgG2c抗体铰链区序列(SEQ ID NO:147)；即，将PR004433的VH(SEQ ID NO:62)与小鼠IgG2c抗体铰链区(SEQ ID NO:147)以及人IgG1重链恒定区CH2和CH3序列进行融合表达，得到新的HCAb分子PR006468(SEQ ID NO:148)。
实施例12.1 PR006468的偶联和分析
将PR006468按照实施例1.6中所述方法进行表达纯化，并利用实施例2.1中所述SEC-HPLC方法分析纯度。然后，进一步利用实施例10中所述偶联方法对纯化的PR006468重组蛋白进行偶联反应。
在本实验中，PR006468的浓度为1.75mg/ml，反应缓冲液为含20mM His-HCl pH 6.0缓冲液，还原剂TCEP的用量为7.0倍摩尔当量，还原反应于37℃ 2小时，偶联物mc-vc-PAB-MMAE的用量为7.0摩尔当量，偶联反应于室温0.5小时。偶联结束之后，通过脱盐柱将产物去除多余药物偶联物，并用实施例4.2所述方法纯化偶联产物PR006468-ADC。
利用实施例2.4中所述方法(LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix  X500B)对PR006468-ADC的纯化后样品进行分子量分析。在进行LC-MS分析之前，待测样品经过去糖处理。图47显示了PR006468-ADC的非还原分子量分析的质谱去卷积处理图谱。按照实施例5.2中所述分子量分析方法过程推算出偶联了不同数目的化合物的组分的分子量及其在样品中的含量。表12-1显示了在PR006468-ADC的非还原样品中，DAR＝2的组分是主要成分(占95.5％)，并推算出PR006468-ADC产物的平均DAR值为2.09。
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR006468裸抗 80,091     0％
PR006468DAR＝2 82,723 81,965～83,675 -758～+952[*1] 95.5％
PR006468DAR＝4 85,354 84,624 -730[*2] 4.5％
表12-1 PR006468-ADC纯化后样品(lot.#CY20210203)非还原分子量LC-MS分析
[*1]可能存在O糖基化(+947Da)；[*2]mc-vc-PAB-MMAE可能发生部分断裂(-762Da)。
实施例12.2 用LC-MS肽图分析化合物偶联位点
本实施例利用实施例8.3中所述方法来分析PR006468-ADC的偶联位点。
图48显示了利用LC-MS肽谱图分析PR006468-ADC的化合物偶联位点，包括筛选出的带有偶联位点的3条肽段，以及相应的偶联位点覆盖率。分析所有含有Cys位点并发生偶联修饰的肽段，每一条肽段对应的二级谱图也经过逐一核对，以证实其发生的偶联修饰均为高度可信的。例如，含有C(+1315.78)PPLK的肽段占比90.8％(图71显示了该肽段的二级谱图)，这说明，90.8％的小鼠铰链区Cys1位点被偶联有化合物。类似地，仅有5.8％的Cys2位点发生偶联。
因而，小鼠IgG2c铰链区序列也可以用于本发明所述的位点特异的半胱氨酸偶联。
实施例12.3 结合BCMA高表达细胞
本实施例利用实施例7.4中所述方法研究抗原结合蛋白PR006468(包含小鼠IgG2c铰链区)、PR004433(包含人IgG1铰链区)及其偶联产物PR006468-ADC和PR004433-ADC与高表达人BCMA的肿瘤细胞NCI-H929(ATCC,CRL-9068)的结合能力。
图49中所示，PR006468和PR004433有相似的结合能力，相应地，PR006468-ADC和PR004433-ADC也有相似的结合能力。这说明了，将抗原结合蛋白中的人IgG1铰链区序列替换成小鼠IgG2c铰链区序列，不会影响抗原结合蛋白及其偶联产物的靶点结合能 力。
实施例12.4 细胞毒性杀伤实验
本实施例利用实施例9.5中所述方法研究抗原结合蛋白PR006468(包含小鼠IgG2c铰链区)、PR004433(包含人IgG1铰链区)及其偶联产物PR006468-ADC和PR004433-ADC对高表达人BCMA的肿瘤细胞NCI-H929(ATCC,CRL-9068)的特异性细胞杀伤活性。
图50中所示，PR006468-ADC和PR004433-ADC都对NCI-H929产生有效的特异性的细胞毒性杀伤，且呈现抗体浓度依赖性，而其他分子不能对细胞产生有效的杀伤；且PR006468-ADC和PR004433-ADC的靶细胞杀伤效力几乎一致。这也说明了，具有小鼠IgG2c铰链区序列的偶联产物也能保留原有的功能。
实施例13 scFv-Fc PR002129的偶联和分析
本实施例研究了抗ROR1的抗原结合蛋白PR002129(SEQ ID NO:71)与mc-vc-PAB-MMAE利用半胱氨酸偶联到Cys-220位点来制备具有均一DAR值的抗体偶联药物。PR002129的序列来源于实施例1.5，其含有人IgG1铰链区序列。
实施例13.1 偶联反应条件优化
以实施例5.1所得到的初步确定的偶联反应条件为基础，并结合实施例10.1所得到的较优的反应条件参数，对该抗原结合蛋白进行偶联。如表13-1所示，平行多组条件对纯化的scFv-Fc PR002129重组蛋白进行偶联。每组实验的偶联的产物用实施例2.2所述HIC-HPLC分析方法分析其偶联产物的组分，尤其是DAR＝2的组分含量(’D2％’)。偶联结束之后，通过脱盐柱将产物去除多余药物偶联物；并可选地，使用实施例4.2所述方法纯化偶联产物，得到DAR＝2的主要产物。其中，实验#20210202-2使用的反应条件为：反应缓冲液为含20mM His-HCl pH 6.0缓冲液，还原剂TCEP的用量为2.3倍摩尔当量，还原反应于室温2小时，偶联物mc-vc-PAB-MMAE的用量为7.0摩尔当量，偶联反应于室温0.5小时。
Figure PCTCN2021083024-appb-000033
Figure PCTCN2021083024-appb-000034
表13-1 PR002129的偶联反应条件
实施例13.2 偶联产物分析
本实施例利用高效液相色谱和质谱等技术对纯化的重组PR002129和其偶联产物PR002129-ADC(包括偶联后产物和经过一步HIC纯化后的产物)进行定量分析，以确定其偶联产物组分的构成。
利用实施例2.2中所述HIC-HPLC方法对上述样品进行分析。图51和下表分别显示了PR002129的偶联产物PR002129-ADC的HIC-HPLC分析结果：(A)偶联后，纯化前(表13-2)；(B)偶联及一步HIC纯化后(表13-3)。偶联产物PR002129-ADC在HIC纯化前，其DAR＝2的组分占比73.2％，推算出的平均DAR值为2.07；经过一步HIC纯化后，其97.9％的组分是DAR＝2。
滞留时间(分钟) 峰面积％ 峰说明
11.86 10.28 DAR＝0组分峰
13.34 1.36 DAR＝1组分峰
15.54 73.2 DAR＝2组分峰
19.99 15.17 DAR＝4组分峰
表13-2 PR002129-ADC纯化前样品(lot.#CY20210202)的HIC-HPLC分析结果
滞留时间(分钟) 峰面积％ 峰说明
8.30 97.9 DAR＝2组分峰
表13-3 PR002129-ADC纯化后样品(lot.#HSP31405-20200820)的HIC-HPLC分析结果
利用实施例2.4中所述方法(LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix X500B)对PR002129-ADC的纯化前和纯化后样品进行分子量分析。在进行LC-MS分析之前，待测样品经过去糖处理。图52显示了分子量分析的质谱去卷积处理图谱：(A)HIC纯化前样品的非还原分子量；(B)HIC纯化后样品的还原分子量；(C)HIC纯化后样品的 非还原分子量。按照实施例5.2中所述分子量分析方法过程推算出偶联了不同数目的化合物的组分的分子量及其在样品中的含量(下表)。表13-4显示了在HIC纯化前的PR002129-ADC的非还原样品中，DAR＝2的组分占比74.1％，推算出的平均DAR值为2.03。表13-5显示了在纯化后的PR002129-ADC的非还原样品中，DAR＝2的组分是主要成分；相应地，如表13-6所示，在其还原样品中，偶联有1个化合物的重链(+1D)是主要成分(占88％)，推算出PR002129-ADC产物的平均DAR值为1.9。
Figure PCTCN2021083024-appb-000035
表13-4 PR002129-ADC纯化前样品(lot.#CY20210202)非还原分子量LC-MS分析
[*1]可能有部分糖化(+162Da)修饰存在；[*2]由于链间的三个二硫键全部打开，在变性条件下以单条重链形式存在，因此重链+3D即对应为DAR＝6的组分。
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR002129裸抗 104,508      
PR002129DAR＝1 105,824 105,848 +24*  
PR002129DAR＝2 107,140 107,163 +23* 主要
表13-5 PR002129-ADC纯化后样品(lot.#HSP31405-20200820)非还原分子量LC-MS分析
(*可能包含某些基团的氧化修饰带来的分子量偏差)
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR002129重链 52,255 52,258 +3 10％
PR002129重链+1D 53,571 53,575 +4 88％
PR002129重链+2D 54,887 54,892 +5 2％
表13-6 PR002129-ADC纯化后样品(lot.#HSP31405-20200820)还原分子量LC-MS分析
综合以上多种分析手段的结果，可以推论出：利用半胱氨酸偶联，偶联产物中主要 成分是DAR＝2的组分，经过进一步HIC纯化，可以得到纯度约为90％的DAR＝2高均质性的产物。
实施例13.3 用LC-MS肽图分析化合物偶联位点
本实施例利用实施例8.3中所述方法来分析PR002129-ADC的偶联位点。
图53显示了利用LC-MS肽谱图分析PR002129-ADC的化合物偶联位点，包括筛选出的带有偶联位点的3条肽段，以及相应的偶联位点覆盖率。分析所有含有Cys位点并发生偶联修饰的肽段，每一条肽段对应的二级谱图也经过逐一核对，以证实其发生的偶联修饰均为高度可信的。例如，含有SC(+1315.78)DK的肽段占比88％，这说明，88％的Cys-220位点被偶联有化合物。
实施例13.4 结合ROR1高表达细胞
本实施例是为了研究抗原结合蛋白PR002129及其偶联产物PR002129-ADC结合高表达ROR1的细胞的活性。
利用流式细胞术FACS测试抗原结合蛋白与高表达人ROR1的肿瘤细胞PanC-1(ATCC,CRL-1469)的结合能力。具体地，消化细胞并用DMEM完全培养基重悬，调整细胞密度为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测结合蛋白。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson ImmunoResearch Inc.,#109-545-098,1:500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
图54中所示，PR002129及其偶联产物PR002129-ADC都与PanC-1细胞有很强的特异性结合活性，且结合能力相当。
实施例14 SIRPa-Fc融合蛋白的偶联和分析
本实施例将本发明申请的药物偶联方法应用到非抗体类的融合蛋白上，利用所述连接肽的第一个Cys(例如，人IgG1铰链区的Cys-220)对融合蛋白进行位点特异性的偶联。这是一种全新的尝试，将药物偶联不局限于抗体，而是扩展到任意融合蛋白，只需要其含有所述连接肽的结构；这说明了本发明申请的药物偶联方法具有通用性。
ALX148是人SIRPa的高亲和力变体融合蛋白(Kauder SE等人，PLoS ONE(2018)13(8):e0201832)，其序列公开于专利US10829771B2。与人SIRPa野生型序列相比，ALX148与CD47亲和力提高了约3000倍。
本实施例将ALX148中SIRPa变体部分序列与含有人IgG1铰链区和Fc的序列(SEQ ID NO:72)进行融合表达，得到SIRPa-Fc融合蛋白PR006345(SEQ ID NO:149)。随后对PR006345进行偶联和分析。
实施例14.1 偶联反应条件优化
本实施例利用实施例10中所述偶联方法对SIRPa-Fc融合蛋白PR006345进行偶联反应，在实施例10.1所得到的较优的反应条件参数的基础上，通过改变还原剂TCEP的用量，来产生不同的偶联反应产物。在本实验中，PR006345的浓度为4.1mg/ml，反应缓冲液为含20mM His-HCl pH 6.0缓冲液，还原剂TCEP的用量从1.5到3.0倍摩尔当量变化，还原反应于室温2小时，偶联物mc-vc-PAB-MMAE的用量为7.0摩尔倍数，偶联反应于室温0.5小时；其偶联产物不经过HIC纯化步骤。进一步地，偶联产物也利用实施例10.1所使用的分子量分析方法来推算出偶联了不同数目的化合物的组分的分子量及其在样品中的含量，分析结果列于表14-1。
实验# TCEP当量 D2％ 平均DAR
1 1.5 88％ 1.89
2 2.0 89％ 2.23
3 3.0 74％ 2.91
表14-1融合蛋白PR006345在不同反应条件的偶联产物(不经过HIC纯化)利用LC-MS分析其各组分的结果
实验#2产生的偶联产物利用LC-MS分子量分析方法测定出DAR＝2组分含量为89％， 偶联产物的平均DAR值为2.23。
实施例14.2 偶联产物分析
本实施例利用高效液相色谱和质谱等技术对SIRPa-Fc融合蛋白PR006345和实验#2产生的偶联产物PR006345-ADC(不经过HIC纯化)进行分析，以确定其偶联产物组分的构成。
利用实施例2.2中所述HIC-HPLC方法对上述样品进行分析。图54和表14-2显示了未纯化的偶联产物PR006345-ADC的HIC-HPLC分析结果；其DAR＝2的组分占比75.4％，推算出的平均DAR值为1.83。
利用实施例2.4中所述方法(LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix X500B)对未纯化的偶联产物PR006345-ADC进行分子量分析。在进行LC-MS分析之前，待测样品经过去糖处理。图56显示了分子量分析的质谱去卷积处理图谱。按照实施例5.2中所述分子量分析方法过程推算出偶联了不同数目的化合物的组分的分子量及其在样品中的含量(表14-3)；其DAR＝2的组分占比89％，推算出的平均DAR值为2.23。
滞留时间(分钟) 峰面积％ 峰说明
10.36 14.66 DAR＝0组分峰
12.27 2.67 DAR＝1组分峰
14.82 75.37 DAR＝2组分峰
19.68 7.30 DAR＝4组分峰
表14-2 PR006345-ADC纯化前样品(lot.#CY20210202)的HIC-HPLC分析结果
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR006345DAR＝0 78,640 78,640 0 3％
PR006345DAR＝2 81,272 81,152～81,439 -120～+167[*1] 89％
PR006345DAR＝4 83,904 83,910 +6 2％
PR006345重链+3D[*2] 43,268 43,272 +4 7％
表14-3 PR006345-ADC纯化前样品(lot.#CY20210202)非还原分子量LC-MS分析
[*1]可能有部分糖化(+162Da)修饰存在；[*2]由于链间的三个二硫键全部打开，在变性条件下以单条重链形式存在，因此重链+3D即对应为DAR＝6的组分。
综合以上多种分析手段的结果，可以推论出：利用半胱氨酸偶联，SIRPa-Fc融合蛋 白的偶联产物中主要成分是DAR＝2的组分。
实施例14.3 用LC-MS肽图分析化合物偶联位点
本实施例利用实施例8.3中所述方法来分析PR006345-ADC的偶联位点。
图57显示了利用LC-MS肽谱图分析PR006345-ADC的化合物偶联位点，包括筛选出的带有偶联位点的3条肽段，以及相应的偶联位点覆盖率。分析所有含有Cys位点并发生偶联修饰的肽段，每一条肽段对应的二级谱图也经过逐一核对，以证实其发生的偶联修饰均为高度可信的。例如，含有SC(+1315.78)DK的肽段占比92.45％，这说明，92.45％的Cys-220位点被偶联有化合物。
实施例14.4 结合CD47高表达细胞
本实施例是为了研究SIRPa-Fc融合蛋白PR006345及其偶联产物PR006345-ADC结合高表达CD47的细胞的活性。
利用流式细胞术FACS测试抗原结合蛋白与高表达人CD47的CHOK1-hCD47细胞(GenScript,M00581)的结合能力。具体地，消化细胞并用DMEM完全培养基重悬，调整细胞密度为1×10 6细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,#3894)，随后加入100μL/孔，2倍于终浓度的3倍浓度梯度稀释的待测结合蛋白。将细胞放置于4℃，避光孵育1小时。之后，加入100μL/孔预冷PBS漂洗细胞两次，于500g、4℃下离心5分钟，弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson ImmunoResearch Inc.,#109-545-098,1:500稀释)，4℃，避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次，于500g、4℃下离心5分钟，弃上清。最后，200μL/孔预冷PBS重悬细胞，使用BD FACS CANTOII流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。
应用软件GraphPad Prism 8进行数据处理和作图分析，通过四参数非线性拟合，得到抗体对靶细胞的结合曲线及EC50值等参数。
图58中所示，PR006345及其偶联产物PR006345-ADC都与CD47有很强的特异性结合活性，且结合能力相当。
实施例15 双特异性抗原结合蛋白PR005744的偶联和分析
本实施例利用抗原结合片段VH和抗原结合片段Fab构建了具有如图59所示的四价双特异性Fab-HCAb结构，并将本申请的药物偶联方法应用到该结构上，利用所述连接肽的第一个Cys(例如，人IgG1铰链区的Cys-220)对其进行位点特异性的偶联。
该结构属于如图42(B)所示的双特异性对称结构，其中一种抗原结合片段是Fab，而另一种抗原结合片段是VH。该结构虽然有两条不同的多肽链(称为短链和长链)，但是短链并不与长链的连接肽上的第一个Cys(Cys-220)形成二硫键；因而在一些反应条件组合下，该Cys的二硫键可以被有效地打开而其他二硫键保持完整，使得该Cys可以作为定点偶联位点。
实施例15.1 PR005744的构建和制备
本实施例利用抗BCMA的HCAb PR004433的VH序列(SEQ ID NO:62)和抗BCMA的IgG抗体PR000892的VH(SEQ ID NO:130)和VL序列(SEQ ID NO:131)构建如图59所示的具有四价Fab-HCAb结构的双特异性(双结合表位)的抗原结合蛋白PR005744。其中，PR000892的序列公开于发明专利CN111234020B。PR004433、PR000892和PR005744的序列编号列于表15-1和表15-2。
抗原结合蛋白 类型 VH VL 重链 轻链
PR004433 HCAb 62   70  
PR000892 IgG1 130 131 132 133
表15-1 本实施例所使用的单抗的序列编号表。
抗原结合蛋白 结构 短链 长链
PR005744 Fab-HCAb 134 135
表15-2 本实施例所构建的双特异性抗体的序列编号表。
抗原结合蛋白PR005744利用实施例1.6中所述方法进行制备。随后，测试其结合BCMA的能力和其在BCMA高表达细胞NCI-H929(ATCC,CRL-9068)上内化的能力。
实施例15.2 PR005744的内化作用
本实施例是为了研究靶向BCMA的抗原结合蛋白内化介导对表达人BCMA的细胞的杀伤。具体地，将NCI-H929(ATCC,CRL-9068)细胞以2×10 5个/孔接种至96-孔板 (Beyotime,#FT018)；随后加入用FACS缓冲液稀释的200nM待测抗原结合蛋白；然后置于4℃孵育1小时；接着，取样品于37℃分别孵育不同时间(如30分钟、1小时、2小时和4小时)；然后，离心并重悬细胞，加入荧光二抗(Jackson ImmunoResearch Inc.,#109-545-098)后再于4℃孵育30分钟。最后，使用流式细胞仪读取荧光发光信号值，并用软件FlowJo v10(FlowJo,LLC)处理和分析数据。以37℃孵育0分钟(T0)时的荧光信号MFI为基线，不同孵育时间的样品的MFI与T0的基线进行扣减并计算相对减少量，以此反映抗原结合蛋白的内化作用的效率。应用软件GraphPad Prism 8进行数据处理和作图分析。
图60中所示，双特异性抗原结合蛋白PR005744显示很强的内化作用，其在30分钟内可以使超过60％的BCMA被内化。
实施例15.3 PR005744的偶联产物分析
进一步地，利用实施例9.1或实施例10.1中所述方法对PR005744进行偶联。在本实验中，反应缓冲液为20mM His-HCl pH 6.0；还原剂为TCEP，其摩尔当量可变；还原反应于室温2小时，偶联物mc-vc-PAB-MMAE的用量为7.0摩尔倍数，偶联反应于室温0.5小时。对于每组实验，其偶联产物利用实施例2.4中所述方法(LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix X500B)进行非还原样品去糖处理的完整分子量分析，并利用每个样品的质谱图中的总离子流图或去卷积图来推算出偶联了不同数目的化合物的组分的分子量及其在样品中的含量；分析结果列于表15-3。例如，当使用所述反应条件参数的组合(反应缓冲液为含20mM His-HCl的pH 6.0缓冲液，还原剂TCEP用量为3.5摩尔倍数，还原反应于室温2小时，偶联物mc-vc-PAB-MMAE的用量为7.0摩尔倍数，偶联反应于室温0.5小时)时，其偶联产物中约84％的组分是偶联有2个药物偶联物的(即DAR＝2，D2％)。
TCEP当量 D0％ D2％ D4％ D6％ 平均DAR
2.0 23.1％ 71.8％ 4.0％ 1.1％ 1.66
2.5 21.2％ 72.6％ 5.1％ 1.1％ 1.72
3.5 6.4％ 84.1％ 7.1％ 2.4％ 2.11
表15-3 PR005744偶联后产物(不经过HIC纯化)利用LC-MS分析其各组分的结果
实施例15.4 用LC-MS肽图分析化合物偶联位点
本实施例利用实施例8.3中所述方法来分析PR005744-ADC的偶联位点。
图61显示了利用LC-MS肽谱图分析PR005744-ADC的化合物偶联位点，包括筛选出的带有偶联位点的3条肽段，以及相应的偶联位点覆盖率。分析所有含有Cys位点并发生偶联修饰的肽段，每一条肽段对应的二级谱图也经过逐一核对，以证实其发生的偶联修饰均为高度可信的。例如，含有SC(+1315.78)DK的肽段占比99.07％，这说明，几乎所有的Cys-220位点被偶联有化合物。这证实了药物偶联特异地发生于PR005744中连接肽的第一个Cys(即Cys-220)。
实施例15.5 结合BCMA高表达细胞
本实施例利用实施例7.4中所述方法研究PR005744及其偶联产物PR005744-ADC与高表达人BCMA的肿瘤细胞NCI-H929(ATCC,CRL-9068)的结合能力。
图62中所示，PR005744及其偶联产物PR005744-ADC都能够与NCI-H929细胞特异性地结合，且结合能力相当。
实施例15.6 细胞毒性杀伤实验
本实施例利用实施例9.5中所述方法研究PR005744及其偶联产物PR005744-ADC对高表达人BCMA的肿瘤细胞NCI-H929(ATCC,CRL-9068)的特异性细胞杀伤活性。
图63中所示，PR005744-ADC对NCI-H929产生有效的特异性的细胞毒性杀伤，且呈现浓度依赖性，而PR005744不能对细胞产生有效的杀伤。
实施例16 HCAb PR004433-PROTAC的偶联和分析
本实施例研究了抗BCMA的HCAb抗体PR004433(SEQ ID NO:70)与蛋白降解靶向嵌合体(PROTAC)利用半胱氨酸偶联到Cys-220位点来制备抗体偶联药物。
PROTAC是一类能够通过诱导靶蛋白的多聚泛素化而导致靶蛋白降解的化合物。PROTAC的作用机理与传统的细胞毒性小分子化合物如MMAE有很大的不同，分子结构也更为复杂。目前，PROTAC虽然对不少靶点有较好的药效，但是其组织选择性却比较有限，而且难以区分不同的细胞类型。有文献报道，将PROTAC偶联到结合HER2的抗体上，可以增强PROTAC的选择性，使其只能降解HER2阳性的肿瘤细胞中的靶点 (Maneiro MA等人，ACS Chem Biol.2020；15(6):1306-1312.)。
本实施例使用的PROTAC是一种BRD4蛋白降解剂(结构如图64所示)，其包含靶向BET的PROTAC GNE-987和含二硫化物的连接子。将该PROTAC作为药物偶联物，利用本发明申请的药物偶联方法对HCAb抗体PR004433进行位点特异性的偶联。这是一种全新的尝试，将药物偶联物不局限于传统的细胞毒性化合物如MMAE，而是扩展到其他载荷药物类型；这说明了本发明申请的药物偶联方法具有通用性。
实施例16.1 PROTAC的偶联反应和分析
本实施例利用实施例10中所述偶联方法及进一步改变的条件对HCAb PR004433进行偶联反应，药物偶联物为一种BRD4蛋白降解剂化合物(结构见图64，分子量1306.58Da；生产商：MedChemExpress，货号：HY-129938)，该化合物在本实施例中简称为PROTAC。在偶联实验中，PR004433的浓度为5.0mg/ml，反应缓冲液为含20mM His-HCl pH 6.0缓冲液，还原剂TCEP的用量为3.0摩尔当量，还原反应于室温2小时；还原反应后用30kd超滤管将产物置换到含50mM Tris-HCl的pH 8.0缓冲液中；偶联物PROTAC的用量为10.0到15.0摩尔当量，偶联反应于室温2小时；其偶联产物(PR004433-PROTAC)不经过HIC纯化步骤。
在偶联反应时，该PROTAC分子的甲砜基(Methylsulfonyl，CH3SO2)断裂，暴露出反应性巯基，进而与抗体上的反应性巯基形成二硫键。因此，每一个PROTAC偶联到抗体上使得总分子量增加1226Da。
进一步地，利用实施例2.4中所述方法(LC仪器：Agilent 1290 UPLC；MS仪器：AB Sceix X500B)对偶联产物PR004433-PROTAC进行分子量分析。在进行LC-MS分析之前，待测样品经过去糖处理。图65显示了PR004433-PROTAC的非还原分子量分析的质谱去卷积处理图谱，并据此推算出偶联了不同数目的PROTAC的组分的分子量及其在样品中的含量。表16-1显示了在PR004433-PROTAC的非还原样品中，DAR＝2的组分是主要成分(占66.4％)，并推算出偶联产物的平均DAR值为1.6。
组分 理论值(Da) 实验值(Da) 偏差(Da) 含量
PR004433裸抗 78,797 78,798 +1 28.4％
PR004433DAR＝2 81,249 81,225～81,414 -24～+165[*] 66.4％
PR004433DAR＝4 83,701 83,708 +7 3.5％
表16-1 PR004433-PROTAC纯化前样品(lot.#CY20210203)非还原分子量LC-MS分析
[*]可能有部分糖化(+162Da)修饰存在。
实施例16.2 结合BCMA高表达细胞
本实施例利用实施例7.4中所述方法研究HCAb PR004433及其偶联产物PR004433-PROTAC与高表达人BCMA的肿瘤细胞NCI-H929(ATCC,CRL-9068)的结合能力。
图66中所示，PR004433及其偶联产物PR004433-PROTAC都能够与NCI-H929细胞特异性地结合。
实施例17 双特异性抗原结合蛋白2129/4433的偶联和分析
本实施例利用抗BCMA的HCAb PR004433的VH序列(SEQ ID NO:62)和抗ROR1的抗体PR002129的scFv序列(具有如SEQ ID NO:60所示的VH和SEQ ID NO:63所示的VL)构建如图67所示的具有非对称结构的二价双特异性的抗原结合蛋白2129/4433。并将本发明申请的药物偶联方法应用到该结构上，利用所述连接肽的第一个Cys(例如，人IgG1铰链区的Cys-220)对其进行位点特异性的偶联。
该结构属于如图42(A)所示的结构，但是其第一抗原结合片段和第二抗原结合片段具有不同的结构和氨基酸序列，所述第一抗原结合片段是VH结构，所述第二抗原结合片段是scFv结构，因而它是含有两条不同多肽链的非对称结构。第一多肽链含有第一连接肽(包含人IgG1铰链区序列)，第二多肽链含有第二连接肽(包含人IgG1铰链区序列)。第一连接肽上的第一个Cys(Cys-220)和第二连接肽上的第一个Cys(Cys-220)形成二硫键；因而在一些反应条件组合下，Cys-220的二硫键可以被有效地打开而其他二硫键保持完整，使得该Cys可以作为定点偶联位点。
实施例17.1 2129/4433的构建和制备
将编码PR004433多肽链(SEQ ID NO:70)的质粒和编码PR002129多肽链(SEQ ID NO:71)的质粒共转染并按照实施例1.6中所述方法进行表达和一步亲和纯化，其产物中有PR004433的同源二聚体(4433/4433)和PR002129的同源二聚体(2129/2129)以及PR004433和PR002129的各一条链形成的异源二聚体(2129/4433，图67)。其中，异源二聚体蛋白2129/4433是本实验的目的蛋白。由于这三个组分(4433/4433，2129/2129，2129/4433)有不同的分子量和不同的等电点(表17-1)，因而可以利用离子交换层析或 者分子尺寸排阻层析等方法对产物做进一步的分离，以得到纯化的异源二聚体目的蛋白2129/4433。
进一步地，利用实施例3.2中所述LC-MS分子量分析方法确认了异源二聚体蛋白2129/4433中2129-链和4433-链上的Cys-220之间形成了重链间二硫键。
组分 分子量理论值(Da) 等电点预测值
2129/2129 104,508 9.40
4433/4433 78,797 7.14
2129/4433 91,652 8.92
表17-1 计算不同二聚体组分的分子量和等电点
实施例17.2 2129/4433的偶联产物分析
利用实施例10.1中所述方法对纯化的异源二聚体蛋白2129/4433进行偶联。在本实验中，2129/4433的浓度为3.2mg/ml，反应缓冲液为含20mM His-HCl pH 6.0缓冲液，还原剂TCEP的用量为2.1～2.5摩尔当量，还原反应于室温2小时，偶联物mc-vc-PAB-MMAE的用量为7.0摩尔当量，偶联反应于室温0.5小时。偶联结束之后，通过脱盐柱将产物去除多余药物偶联物，得到偶联产物2129/4433-ADC。可选地，进一步用实施例4.2所述HIC方法纯化偶联产物。
然后，利用实施例2.2中所述HIC-HPLC方法对未经HIC纯化的2129/4433-ADC样品进行分析。图68和表17-2显示了2129/4433-ADC纯化前样品的HIC-HPLC分析结果：在HIC纯化前，其DAR＝2的组分占比68.7％，推算出的平均DAR值为2.26。
滞留时间(分钟) 峰面积％ 峰说明
12.88 7.05 DAR＝0组分峰
15.65 3.05 DAR＝1组分峰
17.12 68.72 DAR＝2组分峰
20.81 18.43 DAR＝4组分峰
表17-2 偶联产物2129/4433-ADC纯化前样品(lot.#CY20210202)的HIC-HPLC分析结果
实施例17.3 用LC-MS肽图分析化合物偶联位点
本实施例利用实施例8.3中所述方法来分析2129/4433-ADC的偶联位点。
图69显示了利用LC-MS肽谱图分析2129/4433-ADC经HIC纯化后样品(DAR＝2的组分占比100％)的化合物偶联位点，包括筛选出的带有偶联位点的3条肽段，以及相应的偶联位点覆盖率。分析所有含有Cys位点并发生偶联修饰的肽段，每一条肽段对应的二级谱图也经过逐一核对，以证实其发生的偶联修饰均为高度可信的。例如，含有SC(+1315.78)DK的肽段占比99.6％(图72显示了该肽段的二级谱图)；这说明，几乎所有的Cys-220位点被偶联有化合物。
实施例18 偶联免疫调节激动剂分子
Toll样受体9(TLR9)是广泛表达于树突状细胞和巨噬细胞等免疫细胞中的重要受体分子，在人体先天免疫中发挥重要作用。CpG寡核苷酸是TLR9的天然的激动型配体，TLR9结合CpG后可以激活免疫细胞。本实施例将CpG寡核苷酸通过含马来酰亚胺基团或巯基的连接子利用半胱氨酸偶联到前述实施例中的抗原结合蛋白上得到偶联产物。例如，抗5T4的HCAb PR004432与含3’-端巯基修饰的CpG寡核苷酸CpG-1826(序列5'-tccatgacgttcctgacgtt-3')利用本发明所述方法偶联到所述连接肽的Cys1位点(例如，Cys-220)来制备偶联药物PR004432-CpG；抗ROR1的结合蛋白PR002129与CpG偶联到Cys-220位点来制备偶联药物PR002129-CpG；SIRPa-Fc融合蛋白PR006345与CpG偶联到Cys-220位点来制备偶联药物PR006345-CpG。这些CpG偶联药物特异地识别肿瘤上的抗原靶点，利用肿瘤微环境内的免疫细胞上Fc受体介导内化并通过CpG激活TLR9，进一步激活免疫细胞，利用多种机制提高抗肿瘤效果。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (73)

  1. 蛋白-药物偶联物，其包含抗原结合蛋白部分和药物偶联物部分；所述抗原结合蛋白部分包括一个或多个抗原结合片段，以及与所述抗原结合片段直接或间接连接的至少两个连接肽；所述至少两个连接肽包括第一连接肽和第二连接肽；其中所述第一连接肽包含第一半胱氨酸Cys1和第二半胱氨酸Cys2，所述药物偶联物部分通过所述Cys1偶联至所述第一连接肽；且所述第二连接肽包含第一半胱氨酸Cys1和第二半胱氨酸Cys2，所述药物偶联物部分通过所述Cys1偶联至所述第二连接肽。
  2. 根据权利要求1所述的蛋白-药物偶联物，其中所述第一连接肽的所述Cys2与所述第二连接肽的所述Cys2之间通过二硫键连接。
  3. 根据权利要求1或2所述的蛋白-药物偶联物，其中所述一个或多个抗原结合片段中不包含能够影响药物偶联物与所述第一连接肽和/或所述第二连接肽在Cys1处进行偶联的官能团或者前述官能团与其他药物偶联物形成的缀合物，所述影响表现为显著降低药物偶联物在Cys1位点的偶联几率。
  4. 根据权利要求1-3中任一项所述的蛋白-药物偶联物，其中所述一个或多个抗原结合片段不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的官能团或者前述官能团与其他药物偶联物形成的缀合物。
  5. 根据权利要求1-4中任一项所述的蛋白-药物偶联物，其中所述一个或多个抗原结合片段不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的半胱氨酸或者前述半胱氨酸与其他药物偶联物形成的缀合物。
  6. 根据权利要求1-5中任一项所述的蛋白-药物偶联物，其中所述第一连接肽与所述第二连接肽相同或不同。
  7. 根据权利要求1-6中任一项所述的蛋白-药物偶联物，其中所述第一连接肽的所述Cys1和所述Cys2之间相隔至少3个除半胱氨酸以外的任意氨基酸，和/或所述第二连接肽的所述Cys1和所述Cys2之间相隔至少3个除半胱氨酸以外的任意氨基酸。
  8. 根据权利要求1-7中任一项所述的蛋白-药物偶联物，其中所述第一连接肽和/或所述第二连接肽自N端至C端包含Cys1-(X)n-Cys2所示的氨基酸序列；其中，n为大于或等于3的整数，X是除半胱氨酸以外的任意氨基酸。
  9. 根据权利要求1-8中任一项所述的蛋白-药物偶联物，其中所述第一连接肽和/或所述第二连接肽源自抗体铰链区序列或其衍生序列。
  10. 根据权利要求9所述的蛋白-药物偶联物，其中所述抗体铰链区为IgG铰链区。
  11. 根据权利要求9或10所述的蛋白-药物偶联物，其中所述抗体铰链区为人IgG1铰链区(SEQ ID NO:73)或者小鼠IgG2c铰链区(SEQ ID NO:147)。
  12. 根据权利要求1-11中任一项所述的蛋白-药物偶联物，其中所述Cys1为人IgG1铰链区根据EU编码的Cys-220，或与之对应的半胱氨酸，或者所述Cys1为小鼠IgG2c铰链区的第一个半胱氨酸。
  13. 根据权利要求1-12中任一项所述的蛋白-药物偶联物，其中所述第一连接肽和所述第二连接肽的序列各自独立地如SEQ ID NOs:73-105、145-147中任一项所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的蛋白-药物偶联物，其中所述多个抗原结合片段连接于同一所述连接肽或分别连接于不同的所述连接肽。
  15. 根据权利要求1-14中任一项所述的蛋白-药物偶联物，其中所述多个抗原结合片段可以部分或全部相同或彼此均不同。
  16. 根据权利要求1-15中任一项所述的蛋白-药物偶联物，其中所述多个抗原结合片段包括第一抗原结合片段和第二抗原结合片段。
  17. 根据权利要求1-16中任一项所述的蛋白-药物偶联物，其中所述多个抗原结合片段进一步包含第三抗原结合片段和/或第四抗原结合片段。
  18. 根据权利要求1-17中任一项所述的蛋白-药物偶联物，其中所述一个或多个抗原结合片段可以各自独立地为VHH结构域、VH、VL、Fab、ScFv、受体蛋白可溶性胞外区、配体蛋白、脂质运载蛋白lipocalins、神经细胞粘附分子NCAM、纤维结合蛋白fibronectin和/或锚蛋白重复片段蛋白DARPins。
  19. 根据权利要求1-18中任一项所述的蛋白-药物偶联物，其中所述一个或多个抗原结合片段能够特异性结合肿瘤抗原或非肿瘤抗原。
  20. 根据权利要求19所述的蛋白-药物偶联物，其中所述肿瘤抗原包括CD19、BCMA、TSHR、CD171、CS-1、CLL-1、GD3、Tn Ag、FLT3、CD38、CD123、CD44v6、B7H3、B7H4、KIT、IL-13Ra2、IL-11Ra、PSCA、PSMA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-beta、SSEA-4、MUC1、EGFR、NCAM、CAIX、LMP2、EphA2、岩藻糖基GM1(fucosyl GM1)、sLe、GM3、TGS5、HMWMAA、GD2、FOLR1、FOLR2、TEM1/CD248、TEM7R、CLDN6、CLDN18.2、GPRC5D、CXORF61、CD97、CD179a、ALK、多聚唾液酸(polysialic acid)、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TAARP、WT1、ETV6-AML、SPA17、XAGE1、Tie 2、MAD-CT-1、MAD-CT-2、FOSL1、 hTERT、ML-IAP、ERG(TMPRSS2 ETS融合基因)、NA17、PAX3、AR(雄激素受体)、细胞周期蛋白B1(Cyclin B1)、MYCN、RhoC、CYP1B1、BORIS、SART3、PAX5、OY-TES1、LCK、AKAP-4、SSX2、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5、IGLL1、CD20、CD30、HER2、ROR1、FLT3、TAAG72、CD22、CD33、GD2、gp100Tn、FAP、酪氨酸酶(tyrosinase)、EPCAM、CEA、IGF-1R、EphB2、间皮素(mesothelin)、钙黏蛋白17(Cadherin17)、CD32b、EGFRvIII、GPNMB、GPR64、HER3、LRP6、LYPD8、NKG2D、SLC34A2、SLC39A6、SLITRK6、GUCY2C、5T4和/或TACSTD2。
  21. 根据权利要求1-20中任一项所述的蛋白-药物偶联物，其中所述一个或多个抗原结合片段各自独立地包含HCDR1、HCDR2和HCDR3，且所述抗原结合片段包含选自下述的任意一组氨基酸序列：(1)HCDR1：SEQ ID NO:7，HCDR2：SEQ ID NO:22，HCDR3：SEQ ID NO:38；(2)HCDR1：SEQ ID NO:10，HCDR2：SEQ ID NO:25，HCDR3：SEQ ID NO:41；(3)HCDR1：SEQ ID NO:11，HCDR2：SEQ ID NO:26，HCDR3：SEQ ID NO:42；(4)HCDR1：SEQ ID NO:13，HCDR2：SEQ ID NO:28，HCDR3：SEQ ID NO:44；(5)HCDR1：SEQ ID NO:14，HCDR2：SEQ ID NO:29，HCDR3：SEQ ID NO:42；(6)HCDR1：SEQ ID NO:9，HCDR2：SEQ ID NO:24，HCDR3：SEQ ID NO:40；和(7)HCDR1：SEQ ID NO:8，HCDR2：SEQ ID NO:23，HCDR3：SEQ ID NO:39。
  22. 根据权利要求1-21中任一项所述的蛋白-药物偶联物，其中所述一个或多个抗原结合片段各自独立地包含VH，且所述VH包括SEQ ID NOs:55-59、61和62中任一项所示的氨基酸序列。
  23. 根据权利要求1-22中任一项所述的蛋白-药物偶联物，其中所述一个或多个抗原结合片段各自独立地包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，且所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3分别依次包含SEQ ID NO:12，SEQ ID NO:27，SEQ ID NO:43，SEQ ID NO:49，SEQ ID NO:51和SEQ ID NO:53所示的氨基酸序列。
  24. 根据权利要求1-23中任一项所述的蛋白-药物偶联物，其中所述一个或多个抗原结合片段包含至少一对VH和VL，且所述一对VH和VL分别包括SEQ ID NO:60和SEQ ID NO:63所示的氨基酸序列；或者所述一个或多个抗原结合片段包含至少一对VH和VL和/或另一种VH，且所述一对VH和VL分别包括SEQ ID NO:130和SEQ ID  NO:131所示的氨基酸序列，和所述另一种VH包括SEQ ID NO:62所示的氨基酸序列。
  25. 根据权利要求1-24中任一项所述的蛋白-药物偶联物，其还包含与所述连接肽直接或间接连接的配对单元部分，所述配对单元包含至少第一配对亚单元和第二配对亚单元，且所述第一配对亚单元可以通过共价键或者非共价键与所述第二配对亚单元相互作用。
  26. 根据权利要求25所述的蛋白-药物偶联物，其中所述配对单元为抗体Fc片段或突变的Fc片段。
  27. 根据权利要求25或26所述的蛋白-药物偶联物，所述第一配对亚单元的N端与所述第一连接肽的C端连接，且所述第二配对亚单元的N端与所述第二连接肽的C端连接；或者，所述第一配对亚单元的N端与所述第二连接肽的C端连接，且所述第二配对亚单元的N端与所述第一连接肽的C端连接。
  28. 根据权利要求1-27中任一项所述的蛋白-药物偶联物，其包含SEQ ID NOs:64-71、106-129、148和149中任一项所示的氨基酸序列，或SEQ ID NOs:134和135所示的氨基酸序列的组合。
  29. 根据权利要求1-28中任一项所述的蛋白-药物偶联物，所述药物偶联物部分包括分别与第一连接肽和第二连接肽连接的两个药物偶联物，所述药物偶联物包含载荷药物以及任选地连接子。
  30. 根据权利要求29所述的蛋白-药物偶联物，其中所述载荷药物包含小分子化合物、寡核苷酸、蛋白降解靶向嵌合体(PROTAC)、亲和配体、荧光标记基团、核素标记基团、毒素分子、抗生素分子、免疫调节剂和/或多肽等。
  31. 根据权利要求29或30所述的蛋白-药物偶联物，其中所述连接子包含可断裂的连接子或非可断裂的连接子。
  32. 根据权利要求29-31中任一项所述的蛋白-药物偶联物，其中所述可断裂的连接子包含蛋白酶可切割的连接子。
  33. 根据权利要求1-32中任一项所述的蛋白-药物偶联物，其中所述药物偶联物包括mc-vc-PAB-MMAE和/或PROTAC。
  34. 根据权利要求1-33中任一项所述的蛋白-药物偶联物，其为抗体-药物偶联物。
  35. 制备权利要求1-34中任一项所述的蛋白-药物偶联物的方法，其包括使所述药物偶联物与所述第一连接肽和/或所述第二连接肽上的所述Cys1的反应性巯基进行共价结合。
  36. 一种制备蛋白-药物偶联物的方法，所述蛋白-药物偶联物包含抗原结合蛋白部分和药物偶联物部分，所述抗原结合蛋白部分包括一个或多个抗原结合片段，以及与所述抗原结合片段直接或间接连接的至少两个连接肽；所述两个连接肽包含第一连接肽和第二连接肽，其中所述第一连接肽包含第一半胱氨酸Cys1和第二半胱氨酸Cys2，所述第二连接肽包含第一半胱氨酸Cys1和第二半胱氨酸Cys2；所述方法包括使所述药物偶联物部分通过所述第一连接肽的所述Cys1与所述第一连接肽联结，和/或使所述药物偶联物部分通过所述第二连接肽的所述Cys1与所述第二连接肽联结。
  37. 根据权利要求36所述的方法，其中所述一个或多个抗原结合片段中不包含能够影响药物偶联物与所述第一连接肽和/或所述第二连接肽在Cys1处进行偶联的官能团或者前述官能团与其他药物偶联物形成的缀合物，所述影响表现为显著降低药物偶联物在Cys1位点的偶联几率。
  38. 根据权利要求36-37中任一项所述的方法，其中所述一个或多个抗原结合片段不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的官能团或者前述官能团与其他药物偶联物形成的缀合物。
  39. 根据权利要求36-38中任一项所述的方法，其中所述一个或多个抗原结合片段不包含能够与所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1形成二硫键的半胱氨酸或者前述半胱氨酸与其他药物偶联物形成的缀合物。
  40. 根据权利要求36-39中任一项所述的方法，所述第一连接肽和所述第二连接肽相同或不同。
  41. 根据权利要求36-40中任一项所述的方法，其中所述第一连接肽和/或所述第二连接肽自N端至C端包含Cys1-(X)n-Cys2所示的氨基酸序列；其中，n为大于或等于3的整数，X是指除半胱氨酸以外的任意氨基酸。
  42. 根据权利要求36-41中任一项所述的方法，其中所述第一连接肽和/或所述第二连接肽源自抗体铰链区序列或其衍生序列。
  43. 根据权利要求42所述的方法，其中所述衍生序列为通过对抗体铰链区序列进行改造获得，所述改造不涉及抗体铰链区中Cys1和Cys2的改变。
  44. 根据权利要求43所述的方法，其中所述改造为调整抗体铰链区序列中Cys1和Cys2之间的氨基酸种类、数目和/或在Cys1前进行柔性连接氨基酸的添加。
  45. 根据权利要求42-44中任一项所述的方法，其中所述抗体铰链区为IgG铰链区。
  46. 根据权利要求42-45中任一项所述的方法，其中所述抗体铰链区为人IgG1铰链区(SEQ ID NO:73)或者小鼠IgG2c铰链区(SEQ ID NO:147)。
  47. 根据权利要求36-46中任一项所述的方法，其中所述Cys1为人IgG1铰链区根据EU编码的Cys-220，或与之对应的半胱氨酸，或者所述Cys1为小鼠IgG2c铰链区的第一个半胱氨酸。
  48. 根据权利要求36-47中任一项所述的方法，其中所述第一连接肽和所述第二连接肽的序列各自独立地如SEQ ID NOs:73-105、145-147中任一项所示的氨基酸序列。
  49. 根据权利要求36-48中任一项所述的方法，其包括还原、偶联以及任选地纯化步骤。
  50. 根据权利要求49所述的方法，其中所述还原包括在还原剂的条件下使所述抗原结合蛋白部分中所述第一连接肽的所述Cys1和/或所述第二连接肽的所述Cys1之间形成的二硫键被断裂形成反应性巯基，而所述第一连接肽的Cys2和所述第二连接肽的Cys2之间形成的二硫键被保留。
  51. 根据权利要求49或50中所述的方法，其中所述还原包括使用还原剂DTT和/或TCEP；优选地，所述还原剂为TCEP。
  52. 根据权利要求50或51中所述的方法，其中所述还原剂的用量为1至50摩尔倍数；优先地，所述还原剂的用量为1至6摩尔倍数。
  53. 根据权利要求49-52中任一项所述的方法，其中所述还原的反应时长为1至17小时；优选地，所述还原的反应时长为1.5至3小时；进一步优选地，所述还原的反应时长为2小时。
  54. 根据权利要求49-53中任一项所述的方法，其中所述还原的反应温度为0至40℃；优选地，所述还原的反应温度为0℃或室温(25-30℃)或37℃；进一步优选地，所述还原的反应温度为室温(25-30℃)。
  55. 根据权利要求49-54中任一项所述的方法，其中所述还原的反应缓冲液pH为5.0至8.0；优选地，所述还原的反应缓冲液pH为5.0至7.0；进一步优选地，所述还原的反应缓冲液pH为5.0至6.0。
  56. 根据权利要求49-55中任一项所述的方法，其中所述偶联包括使所述药物偶联物与所述第一连接肽的所述Cys1和/或所述第二连接肽上的所述Cys1的反应性巯基进行共价结合。
  57. 根据权利要求36-56中任一项所述的方法，其中所述药物偶联物包括mc-vc-PAB-MMAE或PROTAC。
  58. 根据权利要求36-57中任一项所述的方法，其中所述药物偶联物mc-vc-PAB-MMAE的用量为1至50摩尔倍数；优选地，所述药物偶联物的用量为3至18摩尔倍数；进一步优先地，所述药物偶联物的用量为3至7摩尔倍数。
  59. 根据权利要求36-58中任一项所述的方法，其中所述药物偶联物PROTAC的用量为10至15摩尔倍数。
  60. 根据权利要求49-49中任一项所述的方法，其中所述偶联的反应时长为0.5至10小时；优选地，所述偶联的反应时长为0.5至3小时；进一步优选地，所述偶联的反应时长为0.5至2小时。
  61. 根据权利要求49-60中任一项所述的方法，其中所述偶联的反应温度为0至40℃；优先地，所述偶联的反应温度为室温(25-30℃)。
  62. 根据权利要求49-61中任一项所述的方法，其中所述偶联的反应缓冲液pH为5.0至8.0；优选地，所述偶联的反应缓冲液pH为5.0至7.0；进一步优选地，所述偶联的反应缓冲液pH为5.0至6.0。
  63. 根据权利要求36-62中任一项所述的方法，其中制备所得的蛋白-药物偶联物的纯度大于80％。
  64. 药物组合物，其包含权利要求1-34中任一项所述的蛋白-药物偶联物，以及药学上可接受的载体。
  65. 预防和/或治疗疾病的方法，其包括施用权利要求1-34中任一项所述的蛋白-药物偶联物和/或权利要求64中所述的药物组合物，任选地，所述蛋白-药物偶联物和/或所述药物组合物与其他疗法或药物联用；所述疾病为肿瘤或其他疾病。
  66. 权利要求1-34中任一项所述的蛋白-药物偶联物和/或权利要求64中所述的药物组合物在制备药物中的用途，所述药物用于治疗肿瘤或其他疾病。
  67. 权利要求1-34中任一项所述的蛋白-药物偶联物和/或权利要求64中所述的药物组合物与其他疗法或药物联用在制备药物中的用途，所述药物用于治疗肿瘤或其他疾病。
  68. 根据权利要求78或80所述的用途，其中所述其他疗法或药物选自下组：化疗、放疗、miRNA和寡核苷酸。
  69. 根据权利要求65-64中任一项所述的方法或用途，其中所述肿瘤选自下组中的任意一种或多种：淋巴瘤、多发性骨髓瘤、乳腺癌、卵巢癌、肾癌、子宫内膜癌、黑色素瘤、胰腺癌、肺癌、胃癌、肝癌、间皮瘤、食管癌、头颈部肿瘤、胆管癌、胆囊癌、膀胱癌、胸腺癌和结直肠癌。
  70. 诊断特定疾病的方法，其包括使用权利要求1-34中任一项所述的蛋白-药物偶联物和/或权利要求64中所述的药物组合物。
  71. 检测特定靶标的方法，其包括使用权利要求1-34中任一项所述的蛋白-药物偶联物和/或权利要求64中所述的药物组合物，优选地，所述检测为非疾病诊断目的的。
  72. 检测试剂盒，其包括权利要求1-34中任一项所述的蛋白-药物偶联物和/或权利要求64中所述的药物组合物，和任选地使用说明。
  73. 给药装置，其包括要求1-34中任一项所述的蛋白-药物偶联物和/或权利要求64中所述的药物组合物，以及施用所述蛋白-药物偶联物和/或所述药物组合物的装置。
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