WO2023046093A1 - 一种双特异性多肽复合物 - Google Patents

一种双特异性多肽复合物 Download PDF

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WO2023046093A1
WO2023046093A1 PCT/CN2022/120973 CN2022120973W WO2023046093A1 WO 2023046093 A1 WO2023046093 A1 WO 2023046093A1 CN 2022120973 W CN2022120973 W CN 2022120973W WO 2023046093 A1 WO2023046093 A1 WO 2023046093A1
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antigen
antibody
ligand
binding portion
fragment
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French (fr)
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芦迪
霍永庭
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广东菲鹏制药股份有限公司
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    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • 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
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the invention relates to the field of biomedicine, in particular to a bispecific polypeptide complex comprising interleukin 21 and its receptor.
  • Bispecific antibodies are currently the most popular new biomacromolecular drug structure in clinical practice.
  • Bispecific Antibodies refer to antibodies that can simultaneously bind two different antigens or different epitopes of an antigen, and can exert biological functions that cannot be achieved by monoclonal antibodies through a unique mode of action.
  • two natural heavy chains and two natural light chains can randomly generate 10 possible combinations, only one of which is the target bispecific antibody product.
  • the biochemical properties of 10 different double antibody products are similar, and it is extremely difficult to isolate the target double antibody from them, resulting in low yield and low purity of the target double antibody, which increases the cost and affects the curative effect.
  • bispecific antibodies With the advancement of recombinant protein expression and genetic engineering technology, the forms of bispecific antibodies are becoming more and more diverse. Up to now, more than 20 forms of bispecific antibodies have been developed into technical platforms.
  • the core value of the double-antibody technology platform is to solve the problems of heavy chain and heavy chain mismatch, light chain and heavy chain mismatch.
  • the technology platforms for solving heavy chain and heavy chain mismatch mainly include: Knob-into-Holes (KiH), ART-Ig, Strand Exchange Engineered Domain (SEED) technology, XmAb.
  • the technology for solving the mismatch between the heavy chain and the heavy chain is relatively mature, and the technology for solving the mismatch between the light chain and the heavy chain still has room for improvement. In view of this, the present invention is proposed.
  • the present invention aims to solve one of the technical problems in the related art to a certain extent.
  • the inventors proposed a new idea for the development of bispecific antibodies, using the specific affinity of interleukin 21 and its receptor to replace CH1 and CL in the antibody or its functional fragments, thereby avoiding or reducing the heavy chain and light Chain mismatches occur; further, the substitutions may be selected from CH2, CH3 and optionally CH4, simultaneously or independently, thereby promoting the formation of heavy chain heterodimers.
  • the bispecific antibody proposed by the present invention is a multifunctional fusion protein, which can not only exert dual target specificity, but also exert the biological function of interleukin 21 and its receptor transduction. active.
  • the invention provides a bispecific fusion polypeptide comprising a first antigen-binding portion comprising:
  • a first polypeptide comprising from N-terminus to C-terminus the first heavy chain variable domain VH1 of the first antibody operably linked to the first conjugated fragment
  • said first conjugate fragment and said second conjugate fragment are capable of specific binding
  • the first conjugated fragment is a receptor
  • the second conjugated fragment is a ligand
  • the first conjugated fragment is a ligand
  • the second conjugated fragment is a receptor
  • the second antigen binding moiety comprises:
  • a third polypeptide comprising from N-terminus to C-terminus the second heavy chain variable domain VH2 of the second antibody operably linked to a third conjugated fragment
  • a fourth polypeptide comprising from N-terminus to C-terminus the second light chain variable domain VL2 of the second antibody operably linked to the fourth conjugated fragment;
  • the third conjugated fragment and the fourth conjugated fragment can specifically bind; and the third conjugated fragment is a receptor, the fourth conjugated fragment is a ligand, or the the third conjugate fragment is a ligand and the fourth conjugate fragment is a receptor; and
  • the third conjugate fragment and/or the fourth conjugate fragment and the first conjugate fragment and/or the second conjugate fragment are selected from different receptors and ligands.
  • the second antigen binding moiety comprises:
  • a third polypeptide comprising from N-terminus to C-terminus the second heavy chain variable domain VH2 of the second antibody operably linked to the antibody heavy chain constant region CH1, and
  • a fourth polypeptide comprising, from N-terminus to C-terminus, the second light chain variable domain VL2 of the second antibody, which is operably linked to the antibody light chain constant region CL.
  • the receptor only includes an active site that recognizes and binds a ligand, and does not include a functional active site that generates a response.
  • At least one non-natural interchain bond is included between the receptor and the ligand, and the non-natural interchain bond can enhance the specific binding force between the receptor and the ligand; in some embodiments, the non-native interchain bond is formed between a first mutated residue comprised by the receptor and a second mutated residue comprised by the ligand; in some embodiments, at least one non-native interchain bond is formed between the VH1 and VL1 A natural interchain bond formed between a first mutated residue comprised in the first heavy chain variable domain VH1 and a second mutated residue comprised in the first light chain variable domain VL1 ; in some embodiments, at least one of said first and said second mutated residues is a cysteine residue; in some embodiments, said non-natural interchain bond is a disulfide bond.
  • At least one native glycosylation site is absent in said receptor and/or ligand.
  • the receptor and its ligand are IL21/IL21R.
  • the IL21 is selected from any of the sequences shown in SEQ ID NO.44 to SEQ ID NO.53, and the IL21R is selected from any of the sequences shown in SEQ ID NO.54 to SEQ ID NO.60.
  • the IL21 is selected from the sequence shown in SEQ ID NO.51, and the IL21R is selected from the sequence shown in SEQ ID NO.55; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.48 In the sequence shown, the IL21R is selected from the sequence shown in SEQ ID NO.57; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.50, and the IL21R is selected from the sequence shown in SEQ ID NO.58 Sequence; In some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.53, and the IL21R is selected from the sequence shown in SEQ ID NO.59; In some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.
  • the IL21R is selected from the sequence shown in SEQ ID NO.59; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.49, and the IL21R is selected from the sequence shown in SEQ ID NO.58 The sequence shown; In some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.45, and the IL21R is selected from the sequence shown in SEQ ID NO.58; In some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.58; In the sequence shown in ID NO.46, the IL21R is selected from the sequence shown in SEQ ID NO.56; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.47, and the IL21R is selected from the sequence shown in SEQ ID NO.
  • the IL21 is selected from a sequence having at least 80% (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86% %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity, the IL21R Selected from any one of SEQ ID NO.54-60 sequence with at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the bispecific fusion polypeptide comprises an antibody Fc constant region; in some embodiments, the antibody Fc constant region is a heterodimer; in some embodiments, the antibody Fc constant region For association into heterodimers based on KiH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and/or increased flexibility; in some embodiments, the antibody Fc constant region comprises CH2, CH3, and any Selecting CH4, the CH2, CH3 and/or optional CH4 are replaced by the receptor and its ligand.
  • the first antigen-binding moiety binds to different antigens or to different epitopes of the same antigen than the second antigen-binding moiety; in some embodiments, the first antigen-binding moiety targets immune cells, the second antigen-binding moiety targets tumor cells; in some embodiments, both the first antigen-binding moiety and the second antigen-binding moiety target tumor cells; in some embodiments, the second antigen-binding moiety targets tumor cells; Both an antigen binding portion and said second antigen binding portion target immune cells.
  • T cells and tumor antigens can be engaged after binding to the first antigen and the second antigen; NK cells and tumor antigens; in some embodiments, combined with the first antigen and the second antigen can synergistically inhibit signaling pathways; in some embodiments, combined with the first antigen and the second antigen After binding, protein complexes can be formed.
  • the first antigen-binding portion targets human PD-L1
  • the second antigen-binding portion targets human TIGIT
  • the first antigen-binding portion targets human TIGIT
  • the second antigen-binding portion targets Human PD-L1.
  • the invention also relates to an isolated nucleic acid encoding a bispecific fusion polypeptide or a multifunctional fusion polypeptide as described above.
  • the present invention also relates to a vector comprising a nucleic acid as described above.
  • the present invention also relates to host cells containing a nucleic acid as described above or a vector as described above.
  • the present invention also relates to a method for preparing a bispecific fusion polypeptide or a multifunctional fusion polypeptide, comprising:
  • the bispecific fusion polypeptide or multifunctional fusion polypeptide expressed in the host cells is collected.
  • the present invention also relates to a pharmaceutical composition, which comprises the above-mentioned bispecific fusion polypeptide or multifunctional fusion polypeptide, and a pharmaceutically acceptable carrier, excipient, or stabilizer.
  • the present invention also relates to the application of any one of the above-mentioned bispecific fusion polypeptides or multifunctional fusion polypeptides or pharmaceutical compositions in the preparation of medicines for treating diseases.
  • the present invention also relates to a bispecific fusion polypeptide or a multifunctional fusion polypeptide or a pharmaceutical composition as described in any one of the above for use as a medicament, in some embodiments, the medicament is used to treat a disease or condition.
  • the present invention also relates to a method for treating diseases, the method comprising administering to a subject a therapeutically effective amount of the above-mentioned bispecific fusion polypeptide or multifunctional fusion polypeptide or a pharmaceutical composition.
  • Figure 1 shows four classic double antibody platforms: Figure 1A shows the KiH heterodimerization Fc engineering technology; Figure 1B shows the CrossMab bispecific antibody technology; Figure 1C shows Wuhan Youzhiyou YBody double antibody technology (asymmetric scFv double antibody technology) anti); Figure 1D is a symmetric scFv double antibody;
  • Fig. 2 is a novel bispecific antibody FiBody provided by the present invention, in which CH1 and CL of Fab on one side are replaced by ligand receptors with specific affinity;
  • Figure 3 is an exemplary display of the 4 feasible schemes of FiBody:
  • Figure 3-1 is a modified ligand receptor with non-naturally occurring interchain bonds between ligand receptors;
  • Figure 3-2 is CH1, Fab on both sides. CL is replaced by receptors and ligands, and both sides are selected from different ligand receptors;
  • Figure 3-3 shows that CH1 and CL of the Fab on one side of the antibody are replaced by ligand receptors, and the CH3 segment in the Fc dimer is also replaced by ligand receptors.
  • Figure 3-4 shows that CH1 and CL of the Fab on one side of the antibody are replaced by ligand receptors, and CH2 in the Fc dimer is also replaced by ligand receptors; there are many other feasible transformation methods;
  • Figure 4 is an example of when the bispecific antibody of the present invention is used to treat tumors, the targeted binding of the antigen-binding part of the bispecific antibody includes exemplary three types: Figure 4-A The first antigen-binding part target To T cells, the second antigen-binding part targets tumor cells; Figure 4-B The first antigen-binding part and the second antigen-binding part both target tumor cells; Figure 4-C The first antigen-binding part and the second antigen-binding part Both target T cells; Figure 4-D exemplarily reflects that the bispecific antibody of the present invention can optionally be a trifunctional fusion protein, which can not only play different antigen binding, but also activate the ligand receptor pathway and stimulate the biological activity of the ligand receptor ;
  • Figure 5 is a three-dimensional conformation diagram of interleukin and its receptors, which can be divided into four categories: Type A is lifting type, Type B is bow-tie type, Type C is baseball player type, and Type D is pincer type;
  • Figure 6 is an example of four types of interleukins and their receptors in three-dimensional configurations.
  • Class A holds IL2/IL2R, B bow-tie IL22/IL22R, C bow-tie IL18/IL18R, and D clamps IL21/IL21R;
  • Figure 7 is the FCM method in the embodiment of the present invention to detect the binding activity of the double antibody TIGIT end and CHO-Tigit cells (R1116 ⁇ R1117 ⁇ R1119 ⁇ R1121 ⁇ R1123 ⁇ R1124);
  • Figure 8 is the FCM method in the embodiment of the present invention to detect the binding activity of double antibody PDL1 end and CHO-Tigit cells (R1116 ⁇ R1119 ⁇ R1121 ⁇ R1123 ⁇ R1124 ⁇ R0919);
  • Fig. 9 is the detection of binding force (CHO) at both ends of FiBody constructed based on IL21/IL21R in the embodiment of the present invention.
  • Figure 10 is the detection of the binding force at both ends of the FiBody based on IL21/IL21R in the embodiment of the present invention (Jurkat);
  • FIG. 12 Example 9 Disulfide Bond Modified IL21/IL21R ⁇ FiBody (Complex 1-10) Gel Electrophoresis Detection Results;
  • Complex 1 represents R1267
  • Complex 2 represents R1268
  • Complex 3 represents R1269
  • Complex 4 Represents R1270
  • complex 5 represents R1271
  • complex 6 represents R1272
  • complex 7 represents R1273
  • complex 8 represents R1274, complex 9 represents R1275
  • complex 10 represents R1123, the same below;
  • Fig. 15 Example 9 Disulfide bond modified and unmodified IL21/IL21R ⁇ FiBody pairs and the blocking detection results of the binding ability of the target region (@TIGIT).
  • antigen-binding portion or "antigen-binding domain” means the portion of an antigen-binding molecule that confers its binding specificity for an antigenic determinant.
  • the "antigen-binding portion” is a functional fragment of an antibody.
  • amino acid means one of the 20 naturally occurring amino acids encoded by DNA and RNA.
  • wild type or WT means the amino acid sequence or nucleotide sequence found in nature, including allelic variations.
  • a WT protein has an amino acid sequence or nucleotide sequence that has not been intentionally modified.
  • antibody encompasses any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, bispecific (bivalent) antibody or bispecific fusion polypeptide that binds a particular antigen.
  • a natural intact antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region ("HCVR” or VH) and first, second, and third constant regions (CH1, CH2, CH3, respectively), while each light chain consists of a variable region ("LCVR" or VL) and a constant region (CL).
  • HCVR variable region
  • CH1, CH2, CH3, respectively first, second, and third constant regions
  • LCVR variable region
  • Mammalian heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇
  • mammalian light chains can be classified as ⁇ or ⁇ .
  • Antibodies have a "Y" shape, with a backbone consisting of the second (CH2), third (CH3), and optionally fourth (CH4) constant regions of the two heavy chains, which are joined by disulfide bonds .
  • Each arm of the "Y"-shaped structure contains the variable region (VH) and the first constant region (CH1) of one of the heavy chains, which are combined with the variable region (VL) and constant region (CL) of one of the light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDR), (the CDR of the light (L) chain includes LCDR1, LCDR2, LCDR3, and the CDR of the heavy (H) chain includes HCDR1, HCDR2, HCDR3.
  • CDR complementarity determining regions
  • L light
  • H heavy
  • HCDR three CDRs are separated by lateral continuous parts called framework regions (FR), which are more highly conserved than CDRs and form a scaffold to support hypervariable loops.
  • HCVR and LCVR each contain 4 FRs, and CDRs and FR are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant regions of the heavy and light chains are not involved in antigen binding, but have various effector functions.
  • Antibodies can be classified into several classes based on the amino acid sequence of the heavy chain constant region. Antibodies can be divided into five major classes or isoforms according to the presence or absence of alpha, delta, epsilon, gamma, and mu heavy chains: IgA, IgD, IgE, IgG, and IgM, respectively.
  • IgG1 ⁇ 1 heavy chain
  • IgG2 ⁇ 2 heavy chain
  • IgG3 ⁇ 3 heavy chain
  • IgG4 ⁇ 4 heavy chain
  • IgA1 ⁇ 1 heavy chain
  • IgA2 ⁇ 2 heavy chain
  • the hypervariable region typically comprises amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in the variable region of the light chain and 31-35B (HCDR1) in the variable region of the heavy chain , 50-65 (HCDR2) and 95-102 (HCDR3) amino acid residues (Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed.
  • residues that form hypervariable loops such as residue 26 in the light chain variable region -32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region (Chothia and Lesk (1987) J. Mol. Biol. 196:901-917).
  • the antibody is a bispecific antibody (BiAb).
  • BiAb bispecific antibody
  • the term "bispecific” herein refers to two different antigens, or when the two are the same antigen, each of which has binding specificities for different epitopes. The epitopes may be derived from different antigens or from the same antigen.
  • the terms "bispecific fusion polypeptide” and “bispecific antibody” herein refer to all products made with full-length antibodies or fragments with antigen-binding sites.
  • the antibody can be a human antibody, a non-human antibody (such as a mouse-derived antibody), a humanized antibody, or a chimeric antibody (such as a human-mouse chimeric antibody or a chimeric antibody of different subtypes).
  • antibody variants are obtained by conservative modifications or conservative substitutions or substitutions on the antibody sequences provided by the present invention.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p.
  • nucleotide and amino acid sequences indicate the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate insertions or deletions.
  • Fab is a Fab fragment of an immunoglobulin that contains no or a small portion of the residual Fc fragment, eg, a Fab fragment includes the variable domains of the heavy and light chains, and all or part of the first constant domain.
  • Fab hereinafter may also refer to fragments such as F(ab)2.
  • Fc or “Fc region” or “Fc domain” means all or a portion comprising the constant region of an antibody, in some cases excluding the first constant region immunoglobulin domain (e.g. CH1) or a portion thereof, and Polypeptides of all or a portion of the hinge are further excluded in some cases.
  • Fc may refer to the last two constant region immunoglobulin domains of IgA, IgD and IgG (e.g. CH2 and CH3), the last three constant region immunoglobulin domains of IgE and IgM, and optionally these domains All or part of the N-terminus of the flexible hinge.
  • Fc may contain a J chain.
  • the Fc domain comprises the immunoglobulin domains CH2 and CH3 (C ⁇ 2 and C ⁇ 3) and a lower hinge region located between CH1 (C ⁇ 1) and CH2 (C ⁇ 2).
  • the human IgG heavy chain Fc region is generally defined to include residues E216, C226 or A231 at its carboxy-terminus, with numbering according to the EU index as in Kabat.
  • amino acid modifications are made to the Fc region, eg, the Fc is a heterodimer.
  • Modification herein refers to amino acid substitutions, insertions and/or deletions in the polypeptide sequence or changes in the part chemically linked to the protein.
  • Amino acid modification herein refers to amino acid substitutions, insertions and/or deletions in a polypeptide sequence. For clarity, amino acid modifications are always DNA-encoded amino acids, eg, the 20 amino acids with codons in DNA and RNA, unless otherwise indicated.
  • Epitope means herein a determinant of interaction with a particular antigen-binding domain, eg, the variable region of an antibody molecule, called a paratope.
  • An epitope is a grouping of molecules such as amino acids or sugar side chains, and usually has specific structural characteristics as well as specific charge characteristics. A single molecule may have more than one epitope.
  • An epitope may comprise amino acid residues that are directly involved in binding (also referred to as the immunodominant component of the epitope) and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked by specific antigen-binding peptides; In other words, the amino acid residues are within the coverage area of the specific antigen binding peptide.
  • Epitopes can be either conformational or linear. Conformational epitopes result from the spatial juxtaposition of amino acids from different segments of a linear polypeptide chain.
  • a linear epitope is one that arises from contiguous amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes can be distinguished by the loss of binding to the former but not to the latter in the presence of denaturing solvents.
  • An epitope usually includes at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation.
  • Antigen-binding molecules that recognize the same epitope can be validated in a simple immunoassay showing the ability of one antigen-binding molecule to block the binding of another antigen-binding molecule to the target antigen.
  • the present invention includes not only the antigen-binding molecules and antigen-binding domains recited herein, but also antigen-binding molecules and antigen-binding structures that compete for binding with epitopes that bind to the recited antigen-binding molecules or antigen-binding domains. area.
  • binding refers to a certain ligand that is directed and can be competitively blocked by the corresponding substance in vitro or in vivo.
  • Biological binding processes of structural site interactions Such as the binding between antigen and antibody or receptor and ligand.
  • KD dissociation constant
  • Binding properties can be determined by methods well known in the art, such as biolayer interferometry and surface plasmon resonance based methods.
  • One such method entails measuring the rates of association and dissociation of antigen-binding site/antigen or receptor/ligand complexes, where the rates depend on the concentration of the complex partner, the affinity of the interaction, and the equivalent in both directions. Geometric parameters that affect the velocity. Therefore, the association rate (ka) and the dissociation rate (kd) can be determined, and the ratio of kd/ka is equal to the dissociation constant KD ("Nature (Nature)" 361:186-187 (1993) and Davies et al. (1990) Annual Rev Biochem 59:439-473).
  • Immune cell includes cells of the immune system that participate in the defense of the body against infectious disease or foreign substances.
  • Immune cells can include, for example, neutrophils, eosinophils, basophils, lymphocytes, such as B cells and T cells, and monocytes.
  • T cells can include, for example, CD4+, CD8+, T helper cells, cytotoxic T cells, ⁇ T cells, regulatory T cells, suppressor T cells, and natural killer cells.
  • multifunctional fusion polypeptide means a non-naturally occurring binding molecule designed to target two or more antigens.
  • a “multifunctional fusion polypeptide” as described herein is generally a genetically engineered fusion protein designed to bring two different desired biological functions into a single binding molecule.
  • a multifunctional fusion polypeptide can be a multifunctional binding molecule.
  • FiBody refers to a bispecific antibody obtained through recombination by replacing the CL and CH1 on one side of the bispecific antibody by using the specific affinity between the ligand and its receptor, which can avoid the light chain and heavy binding of the bispecific antibody. chain mismatch.
  • the "YBody” technology mentioned in this invention was developed by Wuhan Youzhiyou Company in 2012. This technology is based on the "Knob-into-Holes” technology to form heterodimers. One of them is a normal heavy chain, and the other is an N-terminal link scFv of the Fc functional region, forming an asymmetric bispecific antibody.
  • the words “comprising”, “including” and “containing” will be understood to mean the inclusion of stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements.
  • Consisting of means including and being limited to what the phrase “consisting of” follows. Thus, the phrase “consisting of” indicates that the listed elements are required or required and that no other elements may be present.
  • Consisting essentially of means including any element listed after the phrase, and being limited to other elements that contribute to or do not interfere with the activity or effect of the listed element as specified in the present invention. Thus, the phrase “consisting essentially of” means that the listed elements are required or essential, but that other elements are optional and may be present or absent depending on whether they affect the activity or action of the listed elements. exist.
  • references throughout this disclosure to "one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “another embodiment,” or “further embodiments” ” or a combination thereof means that a particular feature, structure or characteristic described in relation to said embodiment is included in at least one embodiment of the present invention. Therefore, appearances of the foregoing terms in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.
  • the present invention provides a novel bispecific fusion polypeptide, which comprises a ligand (or a fragment thereof) and a receptor (or a fragment thereof), and the ligand (or a fragment thereof) and a receptor (or a fragment thereof) are respectively independently replacing CH1 and CL of Fab on one side of the antibody, specifically, the bispecific fusion polypeptide comprises a first antigen-binding portion, and the first antigen-binding portion comprises: a first polypeptide, the first polypeptide is from N-terminal to C-terminal comprises the first heavy chain variable domain VH1 of the first antibody, which is operably linked to the first conjugated fragment;
  • the first conjugated fragment is a receptor
  • the second conjugated fragment is a ligand
  • the first conjugated fragment is a ligand
  • the second conjugated fragment is a receptor
  • the bispecific fusion polypeptide has: the first polypeptide, which is sequenced from N-terminus to C-terminus: [VH1]-[Linker 1]-[IL21]-[Linker 2]- [CH2]-[CH3], the second polypeptide, from N-terminus to C-terminus: [VL1]-[Linker 3]-[[IL21R];
  • the bispecific fusion The polypeptide has: the first polypeptide, which from the N-terminal to the C-terminal is: [VH1]-[Linker 1]-[IL21R]-[Linker 2]-[CH2]-[CH3], the second polypeptide , and its sequence from N-terminal to C-terminal is: [VL1]-[Linker 3]-[[IL21].
  • the CH2 and CH3 are heavy chain constant region subunits, and the linker 1, linker 2 and linker 3 are linkers connecting polypeptides, which may or may not be the same; in some embodiments,
  • the linker 1, linker 2 and linker 3 are independently selected from (GxS)y linkers, wherein x is selected from an integer of 1-5, and y is selected from an integer of 0-6.
  • the bispecific fusion polypeptide also comprises a second antigen binding portion that is different from the first antigen binding portion.
  • Alternative polypeptide fusions for the second antigen-binding moiety include those selected from:
  • CH1 and CL of the Fab on the other side of the antibody are replaced by another ligand (or its fragment) and its receptor (or its fragment), namely
  • the second antigen binding portion comprises: a third polypeptide comprising from N-terminus to C-terminus the second heavy chain variable domain VH2 of the second antibody operably linked to a third conjugated fragments, and
  • a fourth polypeptide comprising from N-terminus to C-terminus the second light chain variable domain VL2 of the second antibody operably linked to the fourth conjugated fragment;
  • the third conjugated fragment is a receptor and the fourth conjugated fragment is a ligand; or the third conjugated fragment is a ligand and the fourth conjugated fragment is a receptor; and
  • the third conjugate fragment and/or the fourth conjugate fragment and the first conjugate fragment and/or the second conjugate fragment are selected from different receptors and ligands, the the third conjugate fragment and said fourth conjugate fragment are capable of specific binding; or
  • the Fab on the other side of the antibody retains the original CH1 and CL, that is,
  • the second antigen binding portion comprises: a third polypeptide comprising from N-terminus to C-terminus the second heavy chain variable domain VH2 of the second antibody operably linked to the antibody heavy chain chain constant region CH1, and
  • a fourth polypeptide comprising, from N-terminus to C-terminus, the second light chain variable domain VL2 of the second antibody, which is operably linked to the antibody light chain constant region CL.
  • the present invention utilizes the specific binding force unique to the ligand and its receptor itself, and creatively connects it to the antigen-binding region (antibody variable region), and the connection includes connecting to one of the antigen-binding regions, Another antigen-binding region is still connected to CH1 and CL; or both antigen-binding regions are connected to ligand receptors, but the two antigen-binding regions are connected to different kinds of ligand receptors, so as to avoid mismatching of different antigen-binding regions.
  • the bispecific fusion polypeptide provided by the present invention is a multifunctional fusion polypeptide comprising two antibody Fabs, characterized in that CH1 and CL of one side of the Fab are independently controlled by the ligand and its receptor The CH1 and CL of the Fab on the other side are not substituted.
  • the receptor contains not only the active site for recognizing and binding the ligand, but also the functional active site for generating a response; the light part of the first antigen-binding part chain does not mismatch with the heavy chain of the second antigen binding moiety.
  • the CH1 and CL of one side of the Fab are independently replaced by the first ligand and its receptor, and the CH1 and CL of the Fab on the other side are independently replaced by the second ligand and its receptor, so The first ligand and its receptor are different from the second ligand and its receptor.
  • the multifunctional fusion protein can not only exert dual target specificity, but also exert the biological activity of ligand receptor transmission.
  • the ligand and its receptors are IL21 and IL21R, and the multifunctional fusion polypeptide not only has a dual-target targeting effect, but also binds to IL21 and IL21R to activate downstream signaling pathways, exert corresponding biological functions.
  • the bispecific fusion polypeptide has: the first polypeptide, which is sequenced from N-terminus to C-terminus: [VH1]-[Linker 1]-[IL21]-[Linker 2]- [Fc1], the second polypeptide, its sequence from N-terminal to C-terminal is: [VL1]-[Linker 3]-[[IL21R], the third polypeptide, its sequence from N-terminal to C-terminal is: [ VH2]--[Fc2], and the fourth polypeptide, which are: [VL2]-[CL] from N-terminus to C-terminus; in some embodiments, the bispecific fusion polypeptide has: the first polypeptide Peptide, from N-terminus to C-terminus: [VH1]-[Linker 1]-[IL21R]-[Linker 2]-[Fc1], the second polypeptide, from N-terminus to C-terminus: : [VL1]-[Link
  • the Fc1 and Fc2 are two subunits of the heavy chain constant region Fc, which may be the same or different, and preferably the Fc constant region is a heterodimer (heterodimer Fc fusion protein); In some embodiments, the Fc constant regions are associated into heterodimers based on KiH, hydrophobic interactions, electrostatic interactions, hydrophilic interactions, and/or increased flexibility.
  • the linker 1, linker 2 and linker 3 are linkers connecting polypeptides, which may or may not be the same; in some embodiments, the linker 1, linker 2 and linker 3 are independently selected From (GxS)y linker, wherein, x is selected from the integer of 1-5, and y is selected from the integer of 0-6.
  • the VH1 and VL1 cooperate to form an antigen binding site that specifically binds TIGIT
  • the VH2 and VL2 cooperate to form an antigen binding site that specifically binds PD-L1.
  • the VH1 and VL1 cooperate to form an antigen binding site that specifically binds PD-L1
  • the VH2 and VL2 cooperate to form an antigen binding site that specifically binds TIGIT.
  • the antigen-binding portion that binds to TIGIT includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes TIGIT_VH in complex 1 (that is, the first sequence of SEQ ID NO.24 1 to 118 residues) or at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity, light chain variable region including TIGIT_VL in complex 1 (also That is, a polypeptide consisting of the 1st to 107th residues of SEQ ID NO.25) or at least 80% thereof (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%) %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 9
  • the heavy chain variable region of the antigen-binding portion that binds to TIGIT includes HCDR1, HCDR2 and HCDR3 regions, and the HCDR1, HCDR2 and HCDR3 include the 1st to 118th positions of SEQ ID NO.24 respectively HCDR1, HCDR2 and HCDR3 in the variable region consisting of residues, in some embodiments, wherein said light chain variable region comprises LCDR1, LCDR2 and LCDR3 regions, said LCDR1, LCDR2 and LCDR3 respectively comprising SEQ ID NO.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, Either by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.
  • the antigen-binding portion that binds PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the PD-L1 heavy chain variable region in complex 1.
  • the light chain can be
  • the variable region includes the PD-L1 light chain variable region in complex 1 (that is, the polypeptide consisting of the 1st to 108th residues of SEQ ID NO.42) or has at least 80% (such as at least 80%, 81%) , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity
  • the light chain can be
  • the variable region includes the PD-L1 light chain variable region in complex 1 (that is, the polypeptide consisting of the 1st to 108th residues of SEQ ID NO.42) or has at least 80% (such as at least 80%, 81%) , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or
  • the IL21 is selected from any of the sequences shown in SEQ ID NO.44 to SEQ ID NO.53, and the IL21R is selected from any of the sequences shown in SEQ ID NO.54 to SEQ ID NO.60.
  • the IL21 is selected from the sequence shown in SEQ ID NO.51, and the IL21R is selected from the sequence shown in SEQ ID NO.55; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.48
  • the IL21R is selected from the sequence shown in SEQ ID NO.57; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.50, and the IL21R is selected from the sequence shown in SEQ ID NO.58 Sequence;
  • the IL21 is selected from the sequence shown in SEQ ID NO.53, and the IL21R is selected from the sequence shown in SEQ ID NO.59; In some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.59; In some embodiments,
  • the IL21R is selected from the sequence shown in SEQ ID NO.59; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.49, and the IL21R is selected from the sequence shown in SEQ ID NO.58 The sequence shown; In some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.45, and the IL21R is selected from the sequence shown in SEQ ID NO.58; In some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.58; In the sequence shown in ID NO.46, the IL21R is selected from the sequence shown in SEQ ID NO.56; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.47, and the IL21R is selected from the sequence shown in SEQ ID NO.
  • the IL21 is selected from a sequence having at least 80% (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86% %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity, the IL21R Selected from any one of SEQ ID NO.54-60 sequence with at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the bispecific fusion polypeptide is the FiBody molecule shown in Table 3 of the present invention; in some embodiments, the bispecific fusion polypeptide is the complex 1, Complex 2, Complex 3, Complex 4, Complex 5, Complex 6, Complex 7, Complex 8 or Complex 9.
  • Receptor is a substance on the cell membrane or in the cell that can recognize and bind to bioactive molecules, and the bioactive substances that can bind to receptors are collectively referred to as "ligands”.
  • the receptor itself contains at least two active sites: one is the active site that recognizes and binds to the ligand; the other is the functional active site that is responsible for generating a response. Only after that can a response reaction be generated, thereby initiating a series of biochemical reactions, which eventually lead to biological effects in target cells.
  • Receptors are generally glycoproteins, and the binding between wild-type receptors and ligands is not mediated by covalent bonds, but mainly by ionic bonds, hydrogen bonds, van der Waals forces and hydrophobic interactions. When the receptor binds to the ligand, it has the characteristics of saturation, high affinity and specificity.
  • Cooperating receptors and ligands have relatively specific binding affinities, and optionally biological effects.
  • the receptor only contains an active site that recognizes and binds a ligand, and does not contain a functional active site that produces a response (such as the function of activating a biological effect of a downstream signaling pathway).
  • the receptor and/or ligand is a natural ligand structure, and the receptor not only includes an active site that recognizes and binds a ligand, but also includes a functional active site that is responsible for generating a response, and can exert According to the corresponding biological function, the bispecific fusion protein is a multifunctional fusion protein, which not only has bispecificity, but also can exert the function of ligand receptor.
  • the receptor and/or ligand has been modified on the basis of the natural sequence, and the modification includes but is not limited to: truncation, insertion and/or mutation; the purpose of these modifications includes but is not limited to : Increase or reduce the binding force between ligand and receptor; enhance, reduce or eliminate the biological function of ligand receptor; increase, reduce or eliminate the glycosylation site in receptor and or ligand protein; reduce or eliminate Ligand toxicity.
  • the receptor (or fragment thereof) and its corresponding ligand (or fragment thereof) can be bound by covalent binding, non-covalent interaction, or a combination thereof; examples of non-covalent bonds include, but are not limited to, hydrogen bonds , hydrophobic, ionic, and van der Waals bonds.
  • the affinity between the inserted or replaced conjugated fragments when the affinity between the inserted or replaced conjugated fragments is lower than expected (for example, the two variable regions in the antigen-binding moiety cannot be brought together to allow them to specifically recognize the antigen, or Inability to prevent heavy chain mismatches between two or more heavy chain constant regions, or inability to prevent mismatches between antigen-binding moieties to achieve specific VL-VH moiety combinations), can be achieved by targeting the ligand and/or or receptors engineered to increase affinity.
  • At least one non-natural interchain bond is included between the receptor and the ligand, and the non-natural interchain bond can enhance the specific binding force between the receptor and the ligand; in some embodiments, the non-native interchain bond is formed between a first mutated residue of the receptor and a second mutated residue of the ligand; in some embodiments, between the first and the second mutated residues At least one of is a cysteine residue; in some embodiments, the non-natural interchain bond is a disulfide bond.
  • Non-natural interchain linkages refers to interchain linkages not found in wild-type polypeptide polymers.
  • a non-natural interchain bond can be formed between a mutated amino acid residue of one polypeptide and a mutated amino acid residue of another polypeptide.
  • At least one native glycosylation site is absent in said receptor and/or ligand.
  • the receptor and ligand are selected from interleukins and their receptors.
  • the ligand and its receptor are selected from class D interleukins and their receptors, such as IL21/IL21R.
  • Insertion or replacement positions for cooperating receptors (or fragments thereof) and ligands (or fragments thereof) may be located, for example:
  • a receptor or fragment thereof is inserted into or replaces the CL region, and a ligand or fragment thereof is inserted into or replaces the CH1 region;
  • Receptors or fragments thereof are inserted into or replace the CH1 region, and ligands or fragments thereof are inserted into or replace the CL region.
  • the IL21 is selected from any of the sequences shown in SEQ ID NO.44 to SEQ ID NO.53, and the IL21R is selected from any of the sequences shown in SEQ ID NO.54 to SEQ ID NO.60.
  • the IL21 is selected from the sequence shown in SEQ ID NO.51, and the IL21R is selected from the sequence shown in SEQ ID NO.55; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.48
  • the IL21R is selected from the sequence shown in SEQ ID NO.57; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.50, and the IL21R is selected from the sequence shown in SEQ ID NO.58 Sequence;
  • the IL21 is selected from the sequence shown in SEQ ID NO.53, and the IL21R is selected from the sequence shown in SEQ ID NO.59; In some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.59; In some embodiments,
  • the IL21R is selected from the sequence shown in SEQ ID NO.59; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.49, and the IL21R is selected from the sequence shown in SEQ ID NO.58 The sequence shown; In some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.45, and the IL21R is selected from the sequence shown in SEQ ID NO.58; In some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.58; In the sequence shown in ID NO.46, the IL21R is selected from the sequence shown in SEQ ID NO.56; in some embodiments, the IL21 is selected from the sequence shown in SEQ ID NO.47, and the IL21R is selected from the sequence shown in SEQ ID NO.
  • the IL21 is selected from a sequence having at least 80% (for example, at least 80%, 81%, 82%, 83%, 84%, 85%, 86% %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity, the IL21R Selected from any one of SEQ ID NO.54-60 sequence with at least 80% (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity.
  • the bispecific fusion polypeptide provided by the present invention comprises a first antigen-binding portion and a second antigen-binding portion, and has two antigen specificities, the first antigen-binding portion and the second antigen-binding portion are different, and may be the first antigen
  • the binding moiety and the second antigen-binding moiety may bind different antigens, or the first antigen-binding moiety and the second antigen-binding moiety may bind different epitopes of the same antigen.
  • the target of the bispecific fusion protein is a tumor.
  • both the first antigen-binding moiety and the second antigen-binding moiety bind to targets expressed on tumor cells; in some embodiments, the first antigen-binding moiety binds to tumor cells, and the second antigen-binding moiety binds to tumor cells The portion binds to an immune cell; in some embodiments, both the first antigen-binding moiety and the second antigen-binding moiety bind to an immune cell.
  • T cell redirected killing is a desirable mechanism of action in many therapeutic areas.
  • Various bispecific antibody formats are involved in T cell redirection in preclinical and clinical trials (May C et al. (2012) Biochem Pharmacol, 84(9)): 1105-1112, pp.; Frankel SR, and Baeuerle PA, (2013) CURR OPIN Chemical Biology, Vol. 17(3): 385-92, pp.).
  • All T cell retargeting bispecific antibodies or fragments thereof have been engineered to have at least two antigen binding sites, one of which binds to a surface antigen on the target cell and the other binds to a T cell surface antigen .
  • T cell surface antigens the ⁇ subunit of human CD3 derived from the TCR protein complex is most commonly targeted as a target for redirected T cell killing.
  • Tumor-associated antigens that can be targeted include, but are not limited to: ⁇ -fetoprotein (AFP), ⁇ -actinin-4, A3, antigen specific to A33 antibody, ART-4, B7, Ba 733 , BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, carbonic anhydrase IX, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a- e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147,
  • T-cell antigens include, but are not limited to, CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69, and CD90.
  • Immune checkpoints are inhibitory pathways in the immune system that are critical for maintaining self-tolerance and regulating the duration and magnitude of physiological immune responses in peripheral tissues to minimize collateral tissue damage.
  • the targets of the first antigen-binding moiety and the second antigen-binding moiety are both immune checkpoints or their ligands, and the immune checkpoint molecules include but are not limited to: TIGIT, PD-1, TIM- 3.
  • the target to which the first antigen-binding moiety binds is PD-1, and the target to which the second antigen-binding moiety binds is PD-L1; in some embodiments, the target to which the first antigen-binding moiety binds is PD-1, the target of the second antigen-binding part is TIGIT; in some embodiments, the target of the first antigen-binding part is PD-1, and the target of the second antigen-binding part is GTLA4; in some In some embodiments, the target to which the first antigen-binding moiety binds is PD-1, and the target to which the second antigen-binding moiety binds is LAG3; in some embodiments, the target to which the first antigen-binding moiety binds is PD-1, The target that the second antigen-binding portion binds to is TIM-3; in some embodiments, the target that the first antigen-binding portion binds to is PD-1, and the target that
  • the target to which the antigen-binding moiety binds is 4-1-BB; in some embodiments, the target to which the first antigen-binding moiety binds is PD-L1, and the target to which the second antigen-binding moiety binds is 4-1-BB; In some embodiments, the target to which the first antigen-binding moiety binds is PD-L1, and the target to which the second antigen-binding moiety binds is TIGIT.
  • the first antigen binding moiety targets a tumor-associated antigen and the second antigen binding moiety targets an immune checkpoint.
  • the first antigen-binding portion targets HER2, and the second antigen-binding portion targets PD-1; in some embodiments, the first antigen-binding portion targets VEGF, and the second antigen-binding portion targets PD-1 L1; in some embodiments, the first antigen-binding moiety targets Claudin18.2 and the second antigen-binding moiety targets PD-L1; in some embodiments, the first antigen-binding moiety targets HER2 and the second antigen-binding moiety targets Targets CTLA-4; in some embodiments, the first antigen-binding moiety targets CD20 and the second antigen-binding moiety targets CD47; in some embodiments, the first antigen-binding moiety targets HER2 and the second antigen-binding moiety Targets CD47.
  • the first antigen binding moiety and the second antigen binding moiety target tumor heterogeneity simultaneously.
  • exemplary co-targets for tumors include, but are not limited to, HGF and VEGF, IGF-IR and VEGF, Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 and Fc ⁇ RIIIa, CD20 and Fc ⁇ RIIIa, Her2 and Fc ⁇ RIIIa.
  • the bispecific fusion polypeptide of the present invention can bind VEGF and phosphatidylserine; VEGF and ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; ERB3; HER-2 and BSG2; HER-2 and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 and CD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 and PDGFRa and VEGFR2; PDGFRa and PLGF; PDGFRa and
  • exemplary co-targets for autoimmune and inflammatory disorders include, but are not limited to, IL-1 and TNF ⁇ , IL-6 and TNF ⁇ , IL-6 and IL-1, IgE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13, IL-9 and IL-13, CD19 and Fc ⁇ RIIb, and CD79 and Fc ⁇ RIIb.
  • Exemplary targets for the treatment of inflammatory diseases include, but are not limited to: TNF and IL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF and IL-6; TNF and SOST; TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13 and IL23p19; IgE and IL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R and RANKL; IL-17A and IL-1 ⁇ ; IL-1 ⁇ and RANKL;
  • Targets involved in rheumatoid arthritis include, but are not limited to: TNF and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1 ⁇ ; TNF and MIF; TNF and IL-17 ; and TNF and IL-15.
  • Targets for the treatment of systemic lupus erythematosus include but are not limited to: CD20, CD22, CD19, CD28, CD4, CD24, CD37, CD38, CD40, CD69, CD72, CD74, CD79A, CD79B, CD80, CD81, CD83, CD86, IL-4, IL-6, IL10, IL2, IL4, IL11, TNFRSF5, TNFRSF6, TNFRSF8, C5, TNFRSF7, TNFSF5, TNFSF6, TNFSF7, BLR1, HDAC4, HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, IFNB1, AICDA, BLNK, GALNAC4S-6ST, INHA, INHBA, KLF6, DPP4, FCER2,, R2, ILIR2, ITGA2, ITGA3, MS4A1, ST6GALI, CDIC, CHSTIO, HLA-A, HLA-DR
  • MS multiple sclerosis
  • MS including but not limited to: IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200 , IFN ⁇ , GM-CSF, FGF, C5, CD52 and CCR2.
  • Targets for treating sepsis include but not limited to: TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-10, IL-23, FasL, LPS, Toll-like receptor, TLR-4, tissue factor, MIP-2, ADORA2A, IL-1B, CASP1, CASP4, NF ⁇ B1, PROC, TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NF ⁇ B1, PTAFR, TLR2, TLR4, GPR44 , HMOX1, Midkine, IRAK1, NF ⁇ B2, SERPINA1, SERPINE1, and TREM1.
  • antibodies may be prepared against any combination of these antigens; that is, each of these antigens may optionally and independently be included or excluded by the multispecific antibody according to the invention .
  • the first antigen binding portion and the second antigen binding portion target different epitopes of the same antigen.
  • At least one antigen-binding fragment may also include a secretion signal sequence.
  • the secretory signal sequence refers to a sequence that induces the secretion of expressed protein or peptide by linking to the N-terminus of the coding sequence outside the cell membrane or outside the cell, and the signal sequence may be a peptide sequence consisting of about 18-30 amino acids . All proteins that can be transported to the outside of the cell membrane have different signal sequences that are cleaved by signal peptidases on the cell membrane. Typically, for a foreign protein that is not natively expressed by the host cell, a secretion signal sequence that enables secretion of the protein into the periplasm or culture medium, or a modified sequence, may be used.
  • the first antigen binding portion and the second antigen binding portion bind to TIGIT and PD-L1, respectively.
  • the antigen-binding portion that binds to TIGIT includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes TIGIT_VH in complex 1 (that is, the first sequence of SEQ ID NO.24 1 to 118 residues) or at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity, light chain variable region including TIGIT_VL in complex 1 (also That is, a polypeptide consisting of the 1st to 107th residues of SEQ ID NO.25) or at least 80% thereof (such as at least 80%, 81%, 82%, 83%, 84%, 85%, 8
  • the heavy chain variable region of the antigen-binding portion that binds to TIGIT includes HCDR1, HCDR2 and HCDR3 regions, and the HCDR1, HCDR2 and HCDR3 include the 1st to 118th positions of SEQ ID NO.24 respectively HCDR1, HCDR2 and HCDR3 in the variable region consisting of residues, in some embodiments, wherein said light chain variable region comprises LCDR1, LCDR2 and LCDR3 regions, said LCDR1, LCDR2 and LCDR3 respectively comprising SEQ ID NO.
  • the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are defined by the IMGT numbering system, Either by the Kabat numbering system, or by the Chothia numbering system, or by the Contact numbering system, or by the AbM numbering system.
  • the antigen-binding portion that binds PD-L1 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the PD-L1 heavy chain variable region in complex 1.
  • the light chain can be
  • the variable region includes the PD-L1 light chain variable region in complex 1 (that is, the polypeptide consisting of the 1st to 108th residues of SEQ ID NO.42) or has at least 80% (such as at least 80%, 81%) , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity
  • the light chain can be
  • the variable region includes the PD-L1 light chain variable region in complex 1 (that is, the polypeptide consisting of the 1st to 108th residues of SEQ ID NO.42) or has at least 80% (such as at least 80%, 81%) , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or
  • it comprises a heavy chain constant region, Fc, which is a heterodimer (heterodimer Fc fusion protein).
  • the Fc includes but is not limited to the following combinations:
  • the Fc constant region was mutated to avoid heavy chain mismatches.
  • the introduction of mutations into the Fc constant region is based on KiH technology (Knob-into-Holes), that is, amino acid mutations are introduced into a heavy chain of the Fc constant region, and the volume of the introduced amino acids is greater than the volume of the original amino acid residues, A protruding "knob"-like structure (Knob) is formed, and another mutation is introduced in the other chain region of the Fc constant region.
  • the volume of the introduced amino acid is smaller than the volume of the original amino acid residue, forming a depression, similar to the "hole" structure ( Hole), so that the convex heavy chain is more inclined to pair with the concave heavy chain, so as to avoid heavy chain mispairing.
  • KiH technology KiH technology
  • the introduction of mutations in the Fc constant region is based on electrostatic interactions, such as ART-Ig technology, which promotes the pairing of heterologous heavy chains by specifically changing the charge of the Fc constant region domain, which is equivalent to The KiH technology of the electric charge version is described in the patent application WO2006106905, which is incorporated into the present invention in its entirety.
  • the introduction of mutations in the Fc constant region is based on SEED technology.
  • SEED heterodimerization is another design strategy based on spatial mutation, which utilizes the CH3 domain of IgG and IgA (also known as AG SEED). Complementarity of alternate sequences derived from CH3 and GA SEED (CH3). IgG and IgA CH3 derivatives generate complementary sequences, thus excluding the assembly of homodimers lacking complementarity while assembling two complementary heavy chain heterodimers. This technology is described in patent application WO2007110205, which is incorporated in the present invention in its entirety.
  • the introduction of mutations into the Fc constant region is based on the change of the isoelectric point, which facilitates the modification of improving the formation rate of heterodimers and maintaining the stability of the Fc region.
  • This technology is described in WO2014145806, which is incorporated in this patent in its entirety. .
  • the Fc constant regions associate as heterodimers based on hydrophilic interactions or increased flexibility.
  • the Fc constant regions are associated into heterodimers based on any combination of the above techniques, for example, in some embodiments, the Fc constant regions are mutated based on a combination of KIH and electrostatic interactions .
  • the XmAb bispecific platform approach can improve the thermal stability of bispecific antibodies by combining electrostatic interactions, CH3 domain conformation, and hydrogen bonding. Specifically, this strategy swaps the Fc side chain mutations of native IgG1 for S364K and K370S heterodimers to form hydrogen bonds between the two, followed by L368D/K370S substitutions to drive salt bridge interactions to promote heterodimers.
  • all or part of the CH2, CH3 or CH4 region is inserted or replaced with a receptor and its ligand.
  • the region to be inserted or replaced is independently located in the CH2, CH3 or CH4 region, or any position between adjacent regions (such as CH1-CH2 junction, CH2-CH3 junction, CH3- CH4 junction);
  • the two matching suffixes of the replacement region Affinity between combined fragments, K D ⁇ 1 ⁇ 10 -3 (M), such as x ⁇ 10 -4 (M), xx10 -5 (M), xx10 -6 (M), xx 10 -7 (M), x ⁇ 10 -8 (M), x ⁇ 10 -9 (M), x ⁇ 10 -10 (M), x ⁇ 10 -11 (M); the value of x can be selected from 1 ⁇ 9, eg 2, 3, 4, 5, 6, 7, 8.
  • the N-terminal and/or C-terminal of the conjugated fragment is linked to the antigen-binding fragment via a linker peptide.
  • operably linked means that parts (eg, two polypeptides) are linked by a covalent bond, either directly or via one or more linkers (connecting peptides).
  • the number of amino acids of the connecting peptide is 1 to 30; , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; preferably 5-20.
  • the amino acids of the linking peptide are nonsense polypeptides that do not have additional functions (such as protein localization, enzyme cleavage sites, etc.) other than linking.
  • the connecting peptide is a flexible connecting peptide
  • the amino acid sequence of the connecting peptide is selected from one or more of Gly, Ser, Pro, Ala and Glu.
  • the amino acid sequence of the connecting peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, wherein n is selected from 1, 2, 3, 4, 5 or 6.
  • the connecting peptide is usually flexible, which can reduce the steric hindrance between the fusion protein and the target protein, which is more conducive to the correct folding of the protein.
  • the connecting peptide is a rigid linker peptide; ie a relatively inflexible peptide linker.
  • a rigid linker peptide does not require complete lack of flexibility, but is less flexible than a flexible linker peptide such as a glycine-rich peptide linker. Due to their relative lack of flexibility, the rigid linker peptide reduces the movement of two protein domains (in the present case the stabilizer protein and the thermostable reverse transcriptase) linked together by the rigid linker peptide.
  • Linker peptides that provide ordered chains eg, alpha helical structures
  • arginine, leucine, glutamic acid, glutamine, and methionine all show a relatively high propensity for helical linker structures.
  • non-helical linkers containing many proline residues can also exhibit significant rigidity.
  • rigid linking peptides include polylysine and poly-DL-alanine polylysine.
  • rigid linker peptides are described in the linker database described by George et al., Protein Engineering, 15, pp. 871-79 (2003).
  • the rigid linker peptide is also a non-cleavable linker peptide, ie a non-cleavable rigid linker peptide.
  • the invention also relates to an isolated nucleic acid encoding a bispecific fusion polypeptide or a multifunctional fusion protein as described above.
  • isolated nucleic acid refers herein to a polymer of deoxyribonucleic acid or ribonucleic acid in single- or double-stranded form.
  • Isolated nucleic acids include RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and, unless otherwise specified, polypeptides include native polynucleotides, sugar, or base altered analogs.
  • said polynucleotide is a light chain polynucleotide.
  • the isolated nucleic acid includes the nucleotide sequence encoding the amino acid sequence of the protein complex, as well as the nucleotide sequence complementary thereto.
  • the complementary sequence includes a fully complementary sequence and a substantially complementary sequence, which refers to a sequence that can hybridize to a nucleotide sequence encoding an amino acid sequence of a protein complex under stringent conditions known in the art.
  • nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. Such changes include additions, deletions, or non-conservative or conservative substitutions.
  • a polynucleotide encoding an amino acid sequence of a protein complex may be construed to include nucleotide sequences having substantial identity relative to the isolated nucleic acid. The substantial identity is when the nucleotide sequence is aligned with another random sequence in such a way that they correspond maximally, which may show greater than 80% identity when the aligned sequences are analyzed using algorithms common in the art. % homology, greater than 90% homology, or greater than 95% homology.
  • the present invention also relates to a vector comprising a nucleic acid as described above.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses adeno-associated viruses
  • herpesviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • the vector may contain a selection marker (such as a tag that is convenient for enrichment, such as his tag; or a tag that is convenient for detection, such as GFP), and an origin of replication that matches the cell type specified by the cloning vector, and the expression vector then contain regulatory elements such as enhancers, promoters, internal ribosomal entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly U sequence, etc.).
  • the vector can be a cloning vector and an expression vector. When expressing or preparing antibodies or fragments, prokaryotic expression vectors and eukaryotic expression vectors are often involved.
  • Prokaryotic expression vectors are commonly used in PET series and pGEX series, and eukaryotic expression vectors are commonly used in pcDNA3.1, pcDNA3.4, pcDNA4, pEGFP-N1, pEGFP-N1, pSV2, etc.
  • the carrier can be a composition, such as a mixture of various plasmids, and different plasmids carry a part of the antibody or its fragment.
  • the present invention also relates to host cells containing a nucleic acid as described above or a vector as described above.
  • a variety of cultured host cells that can be used include, for example, prokaryotic cells, eukaryotic cells, bacterial cells (such as Escherichia coli or Bacilis stearothermophilus), fungal cells (such as Saccharomyces cerevisiae or Pichia pastoris), insect Cells (such as Lepidoptera cells including Spodoptera cells) or mammalian cells (such as Chinese hamster ovary (CHO) cells, NSO cells, baby hamster kidney (BHK) cells, monkey kidney cells, Hela cells, human Hepatocellular carcinoma cells or 293 cells, etc.).
  • prokaryotic cells such as Escherichia coli or Bacilis stearothermophilus
  • fungal cells such as Saccharomyces cerevisiae or Pichia pastoris
  • insect Cells such as Lepidoptera cells including Spodoptera cells
  • mammalian cells such as Chinese hamster ovary (CHO) cells, NSO cells, baby hamster kidney (B
  • the bispecific fusion polypeptide or multifunctional fusion protein of the present invention can be prepared by any method known in the art.
  • the bispecific fusion polypeptide or multifunctional fusion protein expressed in the host cells is collected.
  • bispecific antibodies Early methods for constructing bispecific antibodies include chemical cross-linking or hybrid hybridoma or tetravalent tumor method (for example, Staerz UD et al., Nature, 314:628-31, 1985; Milstein C et al., Nature, 305:537 -540, 1983; Karpovsky B et al., J. Exp. Med., 160:1686-1701, 1984).
  • the chemical coupling method is to link two different monoclonal antibodies together by chemical coupling to prepare bispecific monoclonal antibodies. For example the chemical combination of two different monoclonal antibodies, or for example the chemical combination of two antibody fragments such as two Fab fragments.
  • the heterozygous-hybridoma method is to produce bispecific monoclonal antibodies by means of cell hybridization or ternary hybridomas. These cell hybridomas or ternary hybridomas are fused by established hybridomas, or established hybridomas and hybridomas from childhood obtained from the fusion of mouse lymphocytes. Although these techniques are used to make BiAbs, various production issues make the use of such complexes difficult, such as generation of mixed populations containing different combinations of antigen-binding sites, difficulties in protein expression, need to purify the BiAb of interest, low yields, production The cost is high.
  • More recent approaches utilize genetically engineered constructs capable of producing a homogeneous product of a single BiAb without extensive purification to remove unwanted by-products.
  • Such constructs include tandem scFv, diabodies, tandem diabodies, dual variable domain antibodies, and heterodimerization using motifs such as Ch1/Ck domains or DNLTM (Chames & Baty, Curr. Opin. Drug. Discov. Devel., 12:276-83, 2009; Chames & Baty, mAbs, 1:539-47).
  • the relevant purification techniques are well known.
  • Antibodies can also be produced using the monolymphocyte antibody approach by cloning and expressing immunoglobulin variable region cDNA produced by a single lymphocyte selected for production of specific antibodies, e.g. by Babcook J et al., Proc. Natl. Acad. Sci. USA. 93:7843-7848, 1996; WO 92/02551; WO 2004/051268 and methods described in WO 2004/106377.
  • Antigenic polypeptides for the production of antibodies can be obtained from genetically engineered hosts comprising expression systems by methods well known in the art. Cells are prepared, or they may be recovered from natural biological sources.
  • nucleic acids encoding one or both polypeptide chains of bispecific antibodies can be introduced into cultured host cells by various known methods (eg, transformation, transfection, electroporation, bombardment with nucleic acid-coated particles, etc.).
  • the nucleic acid encoding the bispecific antibody can be inserted into a vector suitable for expression in the host cell before being introduced into the host cell.
  • vectors will contain sequence elements that enable the expression of the inserted nucleic acid at the RNA and protein levels.
  • the bispecific antibodies of the invention can be used to detect any or all of these antigens by conventional immunological assay methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry (eg in biological samples such as serum or plasma).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • tissue immunohistochemistry eg in biological samples such as serum or plasma.
  • the present invention provides a method for detecting an antigen in a biological sample, the method comprising: contacting the biological sample with the bispecific antibody of the present invention that can specifically recognize the antigen, or part of the antigen of the antibody, and detecting the antigen-binding An antibody (or antibody portion), or a non-binding antibody (or antibody portion), thereby detecting said antigen in said biological sample.
  • the antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody.
  • Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
  • suitable enzymes include, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, acetylcholinesterase;
  • suitable repair group complexes include streptavidin/biotin and Avidin/biotin;
  • suitable fluorescent substances include 7-hydroxycoumarin, fluorescein, fluorescein isothiocyanate, basalin B, dichlorotriazinylamine fluorescein, dan Sulfonyl chloride or phycoerythrin;
  • luminescent substances include 3-aminophthaloyl cyclic hydrazine;
  • suitable radioactive substances include I 125 , I 131 , 35 S or 3 H.
  • the bispecific polypeptide complex of the present invention or the nucleic acid encoding it can be applied to the preparation of pharmaceutical compositions or sterile compositions, for example, combining bispecific fusion polypeptides or multifunctional fusion proteins with pharmaceutically acceptable carriers, excipients agent or stabilizer mixture.
  • a pharmaceutical composition may comprise one or a combination (eg, two or more different) functional fragments of the antibodies of the present invention.
  • a pharmaceutical composition of the invention may comprise a combination of antibodies or antibody fragments (or immunoconjugates) with complementary activities that bind to different epitopes on a target antigen.
  • Formulations of therapeutic and diagnostic agents can be prepared by mixing with pharmaceutically acceptable carriers, excipients or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions or suspensions.
  • the present invention also relates to the application of the above-mentioned bispecific fusion polypeptide or multifunctional fusion protein in the preparation of medicines for treating diseases.
  • the present invention also relates to a bispecific fusion polypeptide or a multifunctional fusion protein as described above for use as a medicine; said medicine is used for the treatment of diseases.
  • the disease may be, for example, cancer, immune disorders, metabolic diseases, and microbial infections.
  • cancer refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body.
  • Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases.
  • the present invention also relates to a method of prophylaxis and/or treatment and administration of a therapeutically effective amount of the pharmaceutical composition to prevent and/or treat diseases as described above.
  • the methods of the invention can be used in human clinical and veterinary applications.
  • the host animal carrying the pathogenic population and being treated with the ligand-immunogen conjugate may be a human, or in the case of veterinary applications, a laboratory animal, an agricultural animal, a domesticated animal or a wild animal.
  • the present invention may be applicable to host animals including, but not limited to: humans; laboratory animals such as rodents (e.g.
  • mice rats, hamsters, etc.
  • rabbits monkeys, chimpanzees
  • domesticated animals such as dogs, cats and rabbits
  • farm animals such as cattle, horses, pigs, sheep, goats
  • captive wild animals such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins and whales.
  • compositions can be injected into entities, including rats, mice, domestic animals, and/or humans, by a variety of routes. All injection methods are contemplated, for example, oral, rectal, intravenous, nasal, abdominal, subcutaneous, or local injections are all possible.
  • the compositions can be injected by other methods known in the art.
  • a “therapeutically effective amount” herein refers to an amount sufficient to treat a disease with a reasonable benefit to loss ratio.
  • a therapeutically effective amount may vary depending on the patient for a variety of reasons, for example, type of disease, severity, onset, age of the entity, body weight, rate of excretion, susceptibility to reactions, health status, and/or complications; And/or drug activity, injection route, injection cycle and injection times, and/or drug combination; it can also be appropriately selected by those of ordinary skill in the art according to the purpose of treatment.
  • the injection amount can be randomly divided into multiples so that the amount is about 0.001-100 mg/kg body weight of an adult.
  • a bispecific fusion polypeptide or multifunctional fusion protein of the invention, or a nucleic acid or polynucleotide encoding an antibody of the invention can also be administered in combination with, for example, standard cancer treatments (eg, surgery, radiation, and chemotherapy).
  • standard cancer treatments eg, surgery, radiation, and chemotherapy
  • antitumor therapy using the compositions of the invention and/or effector cells equipped with these compositions is used in combination with chemotherapy.
  • Non-limiting examples of antibody combination therapy of the invention include surgery, chemotherapy, radiation therapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy, and combinations thereof.
  • Embodiment 1 FiBody design
  • FiBody is a bispecific antibody obtained through recombination by replacing CL and CH1 on one side of the bispecific antibody by using the specific affinity between the ligand and its receptor, which can avoid or reduce the occurrence of light chain and heavy chain of the bispecific antibody. mismatch.
  • Bispecific antibodies were constructed based on the above four types of interleukins and their receptors.
  • the three-dimensional conformation classification is mainly based on the summary analysis of various IL cytokines published on the PDB. Due to the complexity of cytokine structural complexes, some cytokines can be split into multiple structural types, such as IL2, and the complete IL2/IL2R ⁇ /IL2R ⁇ /IL2R ⁇ structural complex is similar to a support (A) structure, but Looking at IL2/IL2R ⁇ alone, it has a clamp-type (D) structure.
  • IL2 as a lifting type (A) structure based on the fact that IL2/IL2R ⁇ alone has a weak affinity, while IL2/IL2R ⁇ has a strong affinity, so IL2R ⁇ is more needed to assist in the formation of the structural complex.
  • Another example is IL21, the complete complex of IL21/IL21R/IL2R ⁇ is a clamp (A) structure, but because IL21/IL21R has a strong affinity, it can be classified as a clamp (D) structure alone.
  • Example 2 construction based on interleukin and its receptor FiBody
  • the VH targeting the first antibody is selected to be connected to the receptor protein through a Linker, and then connected to the Fc of the antibody through a Hinge; the VL targeting the first antibody is connected to the ligand protein through a Linker to reduce or avoid light chains and heavy chains.
  • the chain is mismatched; the other end is the complete Fab structure targeting the second antibody, and the Fc that makes up the first antibody and the Fc that make up the second antibody have conventional KiH modifications to reduce or avoid heavy chain mismatches. or
  • the VH targeting the first antibody is selected to be connected to the ligand protein through a Linker, and then connected to the Fc of the antibody through a Hinge; the VL targeting the second antibody is connected to the receptor protein through a Linker to reduce or avoid light chains and heavy chains.
  • the chain is mismatched; the other end is the complete Fab structure targeting the first antibody, and the Fc of the first antibody and the Fc of the second antibody have a conventional KiH modification to reduce or avoid heavy chain mismatch.
  • the scFv structure double antibody Based on the construction method of the scFv structure double antibody, specifically select the VH of the targeting second antibody (anti-TIGIT) to connect to the VL of the second antibody through a Linker to form a scFv structure, and then connect to the Fc of the antibody through a Hinge;
  • the complete Fab structure of the first antibody (this double antibody platform was developed by Wuhan Youzhiyou and named YBody), the Fc of the first antibody and the Fc of the second antibody have conventional KiH modification to avoid heavy chain mismatch .
  • Table 4 Table 4:
  • the VH of the targeting second antibody is selected to connect to the VL of the second antibody through a Linker to form a scFv structure, and then through the Linker and the complete Fc targeting the first antibody
  • the C-terminal connection form a symmetrical structure.
  • the VH of the targeting second antibody is specifically selected to be connected to the CL domain, and then connected to the Fc of the antibody through Hinge, and the VL of the targeting second antibody (anti-TIGIT) is connected to
  • the CH1 domain forms the light chain; the other end is the complete Fab structure targeting the first antibody, and the Fc that makes up the first antibody and the Fc that make up the second antibody have conventional KiH modifications to avoid heavy chain mismatches.
  • Table 6 Table 6:
  • the construction method of double antibodies based on the scFv structure is to select the VH of the targeting second antibody (for example, anti-TIGIT antibody) to be linked to the VL of the second antibody through a Linker to form a scFv structure, and then through the Linker and the complete Targeting the VH-CH1 of the first antibody (exemplarily selecting an anti-PD-L1 antibody), which is then linked to Fc;
  • the light chain is the VL-CL of the first antibody (exemplarily selecting an anti-PD-L1 antibody), consisting of a symmetrical structure. See Figure 11 for details, and the sequence is shown in Table 7:
  • VH of the targeting second antibody exemplarily select anti-PD-L1 antibody to connect to the receptor protein (IL21R) through Linker, and then connect to the Fc of the antibody through Hinge; target the first antibody (exemplarily select The VL of the anti-TIGIT antibody) is connected to the ligand protein (IL21) through a Linker; the other end is connected to the CL by the VL of the targeting second antibody (anti-PD-L1 antibody is selected as an example), and the targeting structure of the first antibody (Exemplary selection of anti-TIGIT antibody) VH is connected to CH1, and then connected to the Fc of the antibody through Hinge, and the Fc at both ends has a conventional KiH modification.
  • Table 8 For example, Table 8:
  • Embodiment 5 the preparation of FiBody sample
  • the plasmid containing the target gene is formed into a cationic complex with the transfection reagent PEI, it is introduced into the host cell Expi293.
  • the exogenous gene on the plasmid is transcribed and translated in the cell to obtain the target protein.
  • Expi293 was cultured at 37°C, 8% carbon dioxide, and 130rpm, and the cells were counted before transfection.
  • the 2E6 cells were inoculated into a 1L shake flask, and the culture system was about 300ml.
  • the transient cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22 ⁇ m filter membrane.
  • Purification using ProA affinity chromatography The process is as follows, use AKTA york 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatography column, make the target protein adsorb on the chromatography column and Other impurities are separated by breakthrough.
  • Embodiment 6 FiBody physical and chemical detection
  • FiBody class A molecules and class D molecular platforms prepared bispecific antibodies (including each Such engineered and optimized antibodies) have higher expression and/or higher purity.
  • a bispecific antibody with a mispaired form (sample R1124) was also expressed and had an expression level similar to the normal molecule, but with significantly lower purity.
  • the binding activity of the double antibody molecule (TIGIT end) to CHO-TIGIT cells was detected by FCM assay.
  • R1124 is a mismatch test molecule with significantly reduced TIGIT binding activity.
  • the binding activity of the double antibody molecule (PD-L1 end) to CHO-PD-L1 cells was detected by FCM assay.
  • CHO-S-hPDL1 cells (CHO-S recombinant cells expressing human PDL1 on the cell membrane by lentiviral transfection) were labeled with CFSE (BD BioSicences, Cat: 565082), and Jurkat-Tigit cells (via Lentiviral transfection method, recombinant Jurkat cells expressing TIGIT on the cell membrane) or CHO-TIGIT labeled with CellTraceTM Violet (Thermo Fisher, Cat: C34557);
  • the bispecific sample R1123 (0.84%/59.69) can bind CHO-PDL1 (CFSE labeled) and CHO-TIGIT significantly better at the same time [ Figure 9 ] or CHO-PD-L1 (CFSE-labeled) and Jurkat-Tigit (Violet-labeled) [ Figure 10], indicating that the bispecific antibody Fibody of the present invention can exert a very strong Crosslinking effect.
  • the bispecific antibody is subjected to disulfide bond modification.
  • disulfide bond modification of IL21/IL21R ⁇ is shown in Table 10 and Table 11.
  • the IL21 mutation site is numbered according to the natural sequence of human IL21 (sequence SEQ ID NO.44), and the IL21R ⁇ mutation site is numbered according to the natural sequence of human IL21R ⁇ (sequence SEQ ID NO.54), for example
  • the R81C mutation of IL21 refers to the mutation of the 81st position of SEQ ID NO.44 from R to C.
  • Ligand receptor disulfide bond modification Select the VH of the targeting second antibody (for example, anti-TIGIT antibody) to connect to the receptor protein (IL21R ⁇ ) through Linker, and then connect to the Fc of the antibody through Hinge; targeting the second The VL of the antibody is connected to the ligand protein (IL21) through a Linker; the other end is the complete Fab structure targeting the first antibody (anti-PD-L1 antibody selected as an example), the Fc of the first antibody and the second The Fc of the antibody has conventional KiH engineering to avoid heavy chain mismatches.
  • the receptor and ligand proteins are mutated to form an intermolecular disulfide bond to further improve the stability of the molecule.
  • Table 12 the sequence of the constructed FiBody bispecific antibody is shown in Table 12:
  • Polypeptide light chain of PD-L1 Polypeptide light chain of PD-L1:
  • the plasmid containing the target gene is formed into a cationic complex with the transfection reagent PEI, it is introduced into the host cell Expi293.
  • the exogenous gene on the plasmid is transcribed and translated in the cell to obtain the target protein.
  • Expi293 was cultured at 37°C, 8% carbon dioxide, and 130rpm, and the cells were counted before transfection.
  • the 2E6 cells were inoculated into 1L shake flasks, and the culture system was about 300mL.
  • the transient cell expression solution was centrifuged at 9000rpm/20min, the supernatant was collected, and then sterilized and filtered through a 0.22 ⁇ m filter membrane.
  • Purification using ProA affinity chromatography The process is as follows, use AKTA york 150 chromatography equipment, equilibrate the chromatography column (such as MabSelectSuRe LX, GE) with at least 5CV equilibration buffer (10mM PBS), load the sample to the chromatography column, make the target protein adsorb on the chromatography column and Other impurities are separated by breakthrough.
  • the binding activity of the double antibody molecule (TIGIT end) to CHO-TIGIT cells was detected by FCM assay.
  • the binding activity of the double antibody molecule (PD-L1 end) to CHO-PD-L1 cells was detected by FCM assay.
  • the binding activity of the double antibody molecule (TIGIT end) blocking ligand to CHO-TIGIT cells was detected by FCM assay.

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Abstract

涉及生物医药领域，提供一种双特异性融合多肽及其应用，所述双特异性融合多肽包括第一抗原结合部分，所述第一抗原结合部分包含能够特异性结合第一缀合片段和第二缀合物片段，所述第一缀合物与第二缀合片段为配体/受体对。

Description

一种双特异性多肽复合物
本申请要求申请日为2022年1月21日的中国专利申请(申请号：202210072619.8，发明名称：一种双特异性多肽复合物)以及申请日为2021年9月24日的中国专利申请(申请号：202111123525.0，发明名称：一种双特异性多肽复合物)的优先权，这2件中国专利申请的全文通过引用方式整体并入到本申请。
技术领域
本发明涉及生物医药领域，具体而言，涉及一种包含白细胞介素21及其受体的双特异性多肽复合物。
背景技术
双特异性抗体是目前临床上最热门的新型生物大分子药物结构。双特异性抗体(Bispecific Antibodies，BsAb)是指可以同时结合两个不同抗原或一个抗原不同表位的抗体，可通过特有的作用方式，发挥单抗无法实现的生物学功能。
双特异性抗体在组装过程中，两条天然重链和两条天然轻链可随机产生10种可能的组合，其中只有一种是目标双抗产物。10种不同双抗产物的生化特性类似，从中分离出目标双抗的难度极大，导致目标双抗产率低、纯度低，增加成本影响疗效。
随着重组蛋白表达和基因工程技术的进步，双特异性抗体形式越来越多样化，截止目前，已有超过20种的双特异性抗体形式开发成技术平台。
双抗技术平台的核心价值在于解决重链和重链错配、轻链和重链错配的问题，解决重链与重链错配的技术平台主要有：Knob-into-Holes(KiH)、ART-Ig、链交换工程结构域(SEED)技术、XmAb。
解决重链与重链错配的技术已相对成熟，解决轻链与重链错配的技术仍有改进空间，有鉴于此，特提出本发明。
发明内容
本发明旨在一定程度上解决相关技术中的技术问题之一。
发明人提出了一种新的双特异性抗体开发思路，利用白细胞介素21及其受体的特异性亲合力，替换抗体或其功能片段中的CH1和CL，从而避免或减少重链和轻链发生错配；进一步地，所述替换可同时或独立地选自CH2、CH3以及任选地CH4，从而促进重链异二聚体的形成。另一方面，本发明提出的双特异性抗体是一种多功能融合蛋白，所述多功能融合蛋白不仅能发挥双靶点特异性，且能发挥白细胞介素21及其受体传导的生物学活性。
在一个方面，本发明提供一种双特异性融合多肽，其包含第一抗原结合部分，所述第一抗原结合部分包含：
第一多肽，所述第一多肽自N末端至C末端包含第一抗体的第一重链可变结构域VH1，其可操作性地连接至第一缀合片段，和
第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至第二缀合片段，
所述第一缀合片段和所述第二缀合物片段能够特异性结合；
其中，所述第一缀合物片段为受体，所述第二缀合片段为配体；或者所述第一缀合物片段为配体，所述第二缀合片段为受体。
在一些实施方式中，还包括第二抗原结合部分，所述第二抗原结合部分不同于所述第一抗原结合部分；
所述第二抗原结合部分包括：
第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至第三缀合片段，和
第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至第四缀合片段；
其中，所述第三缀合片段和所述第四缀合物片段能够特异性结合；并且所述第三缀合物片段为受体，所述第四缀合片段为配体，或者所述第三缀合物片段为配体，所述第四缀合片段为受体；和
所述第三缀合片段和/或所述第四缀合物片段与所述第一缀合物片段和/或所述第二缀合物片段选自不同的受体和配体。
在一些实施方式中，还包括第二抗原结合部分，所述第二抗原结合部分不同于所述第一抗原结合部分；
所述第二抗原结合部分包括：
第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至抗体重链恒定区CH1，和
第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至抗体轻链恒定区CL。
在一些实施方式中，所述受体仅包含识别并结合配体的活性部位，不包含产生应答反应的功能活性部位。
在一些实施方式中，所述受体和配体之间包含至少一个非天然的链间键，所述非天然链间键能够增强受体和配体间的特异性结合力；在一些实施方式中，所述非天然链间键形成于受体包含的第一突变残基和配体包含的第二突变残基之间；在一些实施方式中，所述VH1和VL1之间包含至少一个非天然的链间键，所述非天然链间键形成于第一重链可变结构域VH1包含的第一突变残基和第一轻链可变结构域VL1包含的第二突变残基之间；在一些实施方式中，所述第一和所述第二突变残基中的至少一个为半胱氨酸残基；在一些实施方式中，所述非天然链间键为二硫键。
在一些实施方式中，其中至少一个天然糖基化位点在所述受体和/或配体中不存在。
在一些实施方式中，所述受体及其配体为IL21/IL21R。在一些实施方式中，所述IL21选自SEQ ID NO.44至SEQ ID NO.53任一所示序列，所述IL21R选自SEQ ID NO.54至SEQ ID NO.60任一所示序列。在一些实施方式中，所述IL21选自SEQ ID NO.51所示序列，所述IL21R选自SEQ ID NO.55所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.48所示序列，所述IL21R选自SEQ ID NO.57所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.50所示序列，所述IL21R选自SEQ ID NO.58所示序列；在一些实施方式中，所述IL21 选自SEQ ID NO.53所示序列，所述IL21R选自SEQ ID NO.59所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.52所示序列，所述IL21R选自SEQ ID NO.59所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.49所示序列，所述IL21R选自SEQ ID NO.58所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.45所示序列，所述IL21R选自SEQ ID NO.58所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.46所示序列，所述IL21R选自SEQ ID NO.56所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.47所示序列，所述IL21R选自SEQ ID NO.60所示序列。在一些实施方式中，所述IL21选自与SEQ ID NO.44-53任一所示序列具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列，所述IL21R选自与SEQ ID NO.54-60任一所示序列具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列。
在一些实施方式中，所述双特异性融合多肽包含抗体Fc恒定区；在一些实施方式中，所述抗体Fc恒定区是异源二聚体；在一些实施方式中，所述抗体Fc恒定区为基于KiH、疏水相互作用、静电相互作用、亲水相互作用和/或增加的柔性而缔合成为异源二聚体；在一些实施方式中，所述抗体Fc恒定区包含CH2、CH3以及任选的CH4，所述CH2、CH3和/或任选的CH4被替换成所述受体及其配体。
在一些实施方式中，所述第一抗原结合部分与所述第二抗原结合部分结合不同的抗原或者结合同一抗原的不同表位；在一些实施方式中，所述第一抗原结合部分靶向免疫细胞，所述第二抗原结合部分靶向肿瘤细胞；在一些实施方式中，所述第一抗原结合部分和所述第二抗原结合部分均靶向肿瘤细胞；在一些实施方式中，所述第一抗原结合部分与所述第二抗原结合部分均靶向免疫细胞。在一些实施方式中，与所述第一抗原与所述第二抗原结合后能够衔接T细胞和肿瘤抗原；在一些实施方式中，与所述第一抗原与所述第二抗原结合后能够衔接NK细胞和肿瘤抗原；在一些实施方式中，与所述第一抗原与所述第二抗原结合后能够协同抑制信号通路；在一些实施方式中，与所述第一抗原与所述第二抗原结合后能够形成蛋白复合物。在一些实施方式中，所述第一抗原结合部分靶向人PD-L1，第二抗原结合部分靶向人TIGIT；或者所述第一抗原结合部分靶向人TIGIT，第二抗原结合部分靶向人PD-L1。
本发明还涉及分离的核酸，其编码如上所述的双特异性融合多肽或多功能融合多肽。
本发明还涉及含有如上所述核酸的载体。
本发明还涉及含有如上所述核酸或者如上所述载体的宿主细胞。
本发明还涉及制备双特异性融合多肽或多功能融合多肽的方法，包括：
用如上所述的载体转化宿主细胞；
培养所转化的宿主细胞；和
收集宿主细胞中表达的双特异性融合多肽或多功能融合多肽。
本发明还涉及药物组合物，其包含如上所述的双特异性融合多肽或多功能融合多肽，和药学上可接受的载体，赋形剂，或稳定剂。
本发明还涉及如上任一所述的双特异性融合多肽或多功能融合多肽或药物组合物在制备 用于治疗疾病的药物中的应用。
本发明还涉及用作药物的如上任一项所述的双特异性融合多肽或多功能融合多肽或药物组合物，在一些实施方式中，所述药物用于治疗疾病或病症。
本发明还涉及一种治疗疾病的方法，所述方法包括向受试者施用治疗有效量的如上所述的双特异性融合多肽或多功能融合多肽或药物组合物。
附图说明
图1为4种经典的双抗平台：图1A为KiH异源二聚Fc改造技术；图1B为CrossMab双特异性抗体技术；图1C为武汉友芝友YBody双抗技术(非对称型scFv双抗)；图1D为对称型scFv双抗；
图2为本发明提供的一种新型双特性抗体FiBody，由具有特异性亲和力的配受体替换一侧Fab的CH1、CL；
图3为示例性的展示FiBody的4中可行方案：图3-1为改造的配受体，配受体之间具有非天然存在的链间键；图3-2为两侧Fab的CH1、CL均被受体、配体取代，两侧选自不同的配受体；图3-3为抗体除一侧Fab的CH1、CL被配受体替换，Fc二聚体中CH3段也被配受体替换；图3-4为抗体除一侧Fab的CH1、CL被配受体替换，Fc二聚体中CH2也被配受体替换；其他的可行性改造方式还有很多；
图4为示例性的当本发明的双特异性抗体用于治疗肿瘤时，双特异性抗体的抗原结合部分的靶向结合包括示例性的3种类型：图4-A第一抗原结合部分靶向T细胞，第二抗原结合部分靶向肿瘤细胞；图4-B第一抗原结合部分和第二抗原结合部分均靶向肿瘤细胞；图4-C第一抗原结合部分与第二抗原结合部分均靶向T细胞；图4-D示例性的体现本发明双特性抗体可选的为三功能融合蛋白，除发挥不同的抗原结合，还能激活配受体通路，激发配受体生物学活性；
图5为白细胞介素及其受体的立体构像图，可以分为四类：A类为托举型，B类为蝴蝶结型，C类为棒球手型，D类为钳型；
图6为四类立体构像的白介素及其受体的举例，A类托举型为IL2/IL2R，B类蝴蝶结型为IL22/IL22R,C类蝴蝶结型为IL18/IL18R,D类钳型为IL21/IL21R；
图7为本发明实施例中FCM法检测双抗TIGIT端与CHO-Tigit细胞结合活性(R1116\R1117\R1119\R1121\R1123\R1124)；
图8为本发明实施例中FCM法检测双抗PDL1端与CHO-Tigit细胞结合活性(R1116\R1119\R1121\R1123\R1124\R0919)；
图9为本发明实施例中基于IL21/IL21R构建的FiBody两端结合力检测(CHO)；
图10为本发明实施例中基于IL21/IL21R构建的FiBody两端结合力检测(Jurkat)；
图11实施例8中R1155、R1160结构示意图；
图12实施例9二硫键改造IL21/IL21RαFiBody(复合物1～10)凝胶电泳检测结果；附图中，复合物1表示R1267，复合物2表示R1268，复合物3表示R1269，复合物4表示R1270，复合物5表示R1271，复合物6表示R1272，复合物7表示R1273，复合物8表示R1274，复合物9表示R1275，复合物10表示R1123，下同；
图13实施例9二硫键改造与未改造IL21/IL21RαFiBody对靶向区结合力(@TIGIT)的检测结果；
图14实施例9二硫键改造与未改造IL21/IL21RαFiBody对靶向区结合力(@PD-L1)的检测结果；
图15实施例9二硫键改造与未改造IL21/IL21RαFiBody对与靶向区结合力(@TIGIT)的阻断检测结果。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。在本发明中引用的所有文献，包括公开出版物、专利和专利申请，都通过引用的方式全文并入本文。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
术语解释：
术语“抗原结合部分”或“抗原结合结构域”意指对抗原决定簇赋予其结合特异性的抗原结合分子的部分。在一些实施方案中，所述“抗原结合部分”为抗体功能片段。
术语“氨基酸”意指由DNA和RNA编码的20个天然存在的氨基酸之一。
术语“野生型或WT”意指在自然界发现的氨基酸序列或核苷酸序列，包含等位变异。WT蛋白质具有未经过有意修饰的氨基酸序列或核苷酸序列。
术语“抗体”涵盖任意可结合某特定抗原的免疫球蛋白、单克隆抗体、多克隆抗体、多特异性抗体、双特异性(双价)抗体或双特异性融合多肽。一个天然的完整抗体包含两条重链和两条轻链。每条重链由一个可变区(“HCVR”或VH)以及第一、第二和第三恒定区(分别为CH1、CH2、CH3)组成，而每条轻链由一个可变区(“LCVR”或VL)以及一个恒定区(CL)组成。哺乳动物的重链可分为α、δ、ε、γ和μ，哺乳动物的轻链可分为λ或κ。
抗体呈“Y”型，主干由两条重链的第二(CH2)、第三(CH3)以及任选地第四(CH4)恒定区组成，两条重链恒定区其通过二硫键结合。“Y”型结构的每条臂包含其中一条重链的可变区(VH)和第一恒定区(CH1)，其与一条轻链的可变区(VL)和恒定区(CL)结合。轻链和重链的可变区负责抗原的结合。每条链的可变区均含有三个高变区，称互补决定区(CDR)，(轻(L)链的CDR包含LCDR1、LCDR2、LCDR3，重(H)链的CDR包含HCDR1、HCDR2、HCDR3。其中，三个CDR由被称为框架区(FR)的侧面连续部分间隔开，框架区比CDR更加高度保守并形成一个支架支撑超变环。HCVR和LCVR各包含4个FR，并且CDR和FR自氨基端至羧基端依以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
重链和轻链的恒定区不参与抗原结合，但具有多种效应功能。抗体依据重链恒定区的氨基酸序列可以分成几类。根据是否含有α、δ、ε、γ和μ重链，抗体可分别分为五个主要的分 类或异形体：IgA、IgD、IgE、IgG和IgM。几个主要的抗体分类还可分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)等。
高变区通常包含来自轻链可变区中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)以及重链可变区中的31-35B(HCDR1)、50-65(HCDR2)和95-102(HCDR3)的氨基酸残基(Kabat等人，《免疫学相关蛋白质的序列(SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST)》，第5版.马里兰州贝塞斯达美国国家卫生研究院公共卫生服务部(Public Health Service，National Institutes of Health，Bethesda，Md.)(1991))，或那些形成高变环的残基，例如轻链可变区中的残基26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)以及重链可变区中的26-32(HCDR1)、53-55(HCDR2)和96-101(HCDR3)(Chothia和Lesk(1987)《分子生物学杂志(J.Mol.Biol.)》196：901-917)。
在一些实施方式中，所述抗体为双特异抗体(BiAb)。术语“双特异性”在本文中是指两种不同抗原，或当这两者是相同抗原时，它们每一个都具有针对不同表位的结合特异性。所述表位可以源自不同抗原或相同抗原。术语“双特异性融合多肽”和“双特异性抗体”在本文中是指所有制得的具有全长抗体或带抗原结合位点的片段的产物。所述抗体可以是人抗体，非人抗体(如小鼠来源抗体)，人源化抗体，或嵌合抗体(如人-小鼠嵌合抗体或不同亚型抗体的嵌合)。在一些情况下，抗体的变体是在本发明所提供的抗体序列上发生保守修饰或保守置换或取代所得到的。“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。所属领域技术人员将能够使用熟知的技术确定如本文所阐明的抗原结合分子的合适变体。对于核苷酸和氨基酸序列，术语“同一性”表明当具有适当的插入或缺失的情况下最佳比对和比较时两个核酸或两个氨基酸序列之间的同一性程度。
术语“Fab”为免疫球蛋白中不含或含一小部分残余Fc片段的Fab片段，例如，Fab片断包括重链和轻链的可变区、以及所有或部分的第一恒定域。出于简单，后文中的的术语“Fab”也可指诸如F(ab)2这样的片段。
术语“Fc”或“Fc区”或“Fc结构域”意指包含抗体的恒定区，在一些情况下排除第一恒定区免疫球蛋白结构域(例如CH1)或其一部分的全部或一部分，并且在一些情况下进一步排除铰链的全部或一部分的多肽。因此，Fc可指IgA、IgD和IgG的最后两个恒定区免疫球蛋白结构域(例如CH2和CH3)，IgE和IgM的最后三个恒定区免疫球蛋白结构域，以及任选地这些结构域的柔性铰链N端的全部或一部分。对于IgA和IgM，Fc可以包含J链。对于IgG来说，Fc结构域包含免疫球蛋白结构域CH2和CH3(Cγ2和Cγ3)以及位于CH1(Cγ1)与CH2(Cγ2)之间的较低铰链区。尽管Fc区的边界可以变化，但是人类IgG重链Fc区通常被定义为包括其羧基端的残基E216、C226或A231，其中编号根据如Kabat中的EU索引。在一些实施方案中，如下文更全面地描述，对Fc区进行氨基酸修饰，例如所述Fc为异源二聚体。
本文的“修饰”是指多肽序列中的氨基酸取代、插入和/或缺失或与蛋白质化学连接的部分 的改变。本文的“氨基酸修饰”是指多肽序列中的氨基酸取代、插入和/或缺失。为清楚起见，除非另外指出，否则氨基酸修饰始终是由DNA编码的氨基酸，例如在DNA和RNA中具有密码子的20个氨基酸。
“表位”在本文中意指与特定抗原结合结构域，例如抗体分子的可变区(称为互补位)相互作用的决定子。表位是例如氨基酸或糖侧链的分子的分组，并且通常具有特定的结构特征以及特定的电荷特征。单个分子可具有超过一个表位。表位可以包含直接参与结合的氨基酸残基(也称为表位的免疫显性组分)和不直接参与结合的其它氨基酸残基，例如被特异性抗原结合肽有效阻断的氨基酸残基；换句话说，氨基酸残基在特异性抗原结合肽的覆盖面积内。表位可以是构形的也可以是线性的。构形表位是由来自线性多肽链的不同区段的氨基酸空间并置而产生。线性表位是由多肽链中的相邻氨基酸残基产生的表位。构形和非构形表位的区别可以在于在变性溶剂存在下，与前者而非后者的结合丧失。表位通常包括独特空间构象中的至少3个，并且更通常至少5个或8-10个氨基酸。识别相同表位的抗原结合分子可以在简单的免疫分析中验证，显示一种抗原结合分子阻断另一种抗原结合分子与靶抗原结合的能力。如下所概述，本发明不仅包括本文中所列举的抗原结合分子和抗原结合结构域，还包括与所列举的抗原结合分子或抗原结合结构域结合的表位竞争结合的抗原结合分子和抗原结合结构域。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指有指向的、能被相应物质竞争阻断的某种配基在体外或体内与特异结构位点相互作用的生物结合过程。如抗原和抗体或受体和配体之间的结合。
特异性结合的强度或亲和力可以根据相互作用的解离常数(KD)表示，其中较小的KD表示较大的亲和力，较大的KD表示较低的亲和力。例如KD为至少约10 -4M、至少约10 -5M、至少约10 -6M、至少约10 -7M、至少约10 -8M、至少约10 -9M、替代地至少约10 -10M、至少约10 -11M、至少约10 -12M、或更大的抗原结合力来展现。结合特性可以通过所属领域众所周知的方法，例如生物层干涉测量法和基于表面等离振子共振的方法来确定。一种这样的方法需要测量抗原结合位点/抗原或受体/配体复合物缔合和解离的速率，其中速率取决于复合物搭配物的浓度、相互作用的亲和力以及在两个方向上同等地影响速率的几何参数。因此，可以确定缔合速率(ka)和解离速率(kd)，并且kd/ka的比例等于解离常数KD(《自然(Nature)》361：186-187(1993)和Davies等人(1990)《生物化学年鉴(Annual RevBiochem)》59：439-473)。
术语“免疫细胞”包括参与保护机体抵抗传染性疾病或外来物质的免疫系统的细胞。免疫细胞可以包括例如嗜中性粒细胞，嗜酸性粒细胞，嗜碱性粒细胞，淋巴细胞，如B细胞和T细胞，和单核细胞。T细胞可以包括例如，CD4+、CD8+、T辅助细胞、细胞毒性T细胞、γδT细胞、调节性T细胞、抑制性T细胞和天然杀伤细胞。
术语“多功能融合多肽”意指设计来靶向两个或更多个抗原的非天然存在的结合分子。本文所述的“多功能融合多肽”通常是遗传工程化的融合蛋白，其经设计以将两个不同的所需的生物学功能带入单个结合分子。例如，多功能融合多肽可以是多功能结合分子。
术语“FiBody”,是利用配体及其受体之间特异性亲和力，取代双特异性抗体一侧的CL和CH1，重组获得的双特异性抗体，其能够避免双特异性抗体轻链与重链发生错配。
本发明中提到的“YBody”技术由武汉友芝友公司于2012年开发，该技术是在“Knob-into- Holes”技术的基础上，形成异源二聚体。其中一条为正常重链，另外一条为Fc功能区的N端链接scFv，形成了不对称的双特异性抗体。
术语“约”或“大约”是指与参照定量、水平、值、数量、频率、百分比、维度、大小、量、重量或长度相差30、25、20、25、10、9、8、7、6、5、4、3、2或1％的定量、水平、值、数量、频率、百分比、维度、大小、量、重量或长度。在特定实施方式中，当术语“约”或“大约”位于数值之前时，表示所述值加上或减去15％、10％、5％或1％的范围。
除非上下文另有规定，词语“包含”、“包括”和“含有”将被理解为表示包括所述的步骤或要素或一组步骤或要素，但不排除任何其他步骤或要素或一组步骤或要素。“由……组成”所表示的是包括并且限于短语“由……组成”所接的内容。因此，短语“由……组成”表示所列出的要素是需要的或必需的，并且没有其他要素可存在。“基本由……组成”所表示的是包括列于此短语之后的任意要素，并且限于有助于或不妨碍所列的要素的如在本发明中详述的活性或作用的其他要素。因此，短语“基本由……组成”表示所列出的要素是需要的或必需的，但其他要素是可选地并可取决于其是否影响所列出的要素的活性或作用而存在或不存在。
在本发明全文中提及的“一个实施方式”、“实施方式”、“特定实施方式”、“相关实施方式”、“某种实施方式”、“另外的实施方式”或“进一步的实施方式”或其组合表示所描述的与所述实施方式相关的特定特征、结构或特性包含于本发明的至少一个实施方式中。因此，在本说明书全文各处出现前述用语未必都指同一实施方式。此外，所述特定特征、结构或特性可在一个或多个实施方式中以任意适宜方式组合。
术语“任选地”仅用于描述目的，而不能理解为指示或暗示相对重要性。由此，限定有“任选地”的特征可以明示或者隐含地包括或不包括该特征。
在说明书和权利要求中的术语“第一”、“第二”用于区分相似元素，而不一定用于描述顺序或时间次序。应当理解，如此使用的术语在合适环境下是可互换的，并且本文描述的本发明的实施方案能够以与本文描述或举例说明不同的其他顺序操作。
双特异性融合多肽
本发明提供了新的双特异性融合多肽，其包含配体(或其片段)及其受体(或其片段)，所述配体(或其片段)及其受体(或其片段)分别独立地替换抗体一侧Fab的CH1和CL，具体地，所述双特异性融合多肽包含第一抗原结合部分，所述第一抗原结合部分包含：第一多肽，所述第一多肽自N末端至C末端包含第一抗体的第一重链可变结构域VH1，其可操作性地连接至第一缀合片段；
第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至第二缀合片段，
其中，所述第一缀合物片段为受体，所述第二缀合片段为配体；或者所述第一缀合物片段为配体，所述第二缀合片段为受体。
在一些实施方式中，所述双特异性融合多肽具有：第一多肽，其从N端到C端依次为：[VH1]-[连接子1]-[IL21]-[连接子2]-[CH2]-[CH3],第二多肽，其从N端到C端依次为：[VL1]-[连接子3]-[[IL21R]；在一些实施方式中，所述双特异性融合多肽具有：第一多肽，其从N端到C端依次为：[VH1]-[连接子1]-[IL21R]-[连接子2]-[CH2]-[CH3],第二多肽，其从N端到C 端依次为：[VL1]-[连接子3]-[[IL21]。其中，所述CH2和CH3为重链恒定区亚基，所述连接子1、连接子2和连接子3为连接多肽的连接子，其可以相同，也可以不相同；在一些实施方案中，所述连接子1、连接子2和连接子3独立选自(GxS)y连接子，其中，x选自1-5的整数，y选自0-6的整数。
所述双特异性融合多肽还包含第二抗原结合部分，所述第二抗原结合部分与第一抗原结合部分不同。对于第二抗原结合部分可选的多肽融合方式包括选自：
1.抗体另一侧Fab的CH1和CL被另一种配体(或其片段)及其受体(或其片段)替换，即
所述第二抗原结合部分包括：第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至第三缀合片段，和
第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至第四缀合片段；
所述第三缀合物片段为受体，所述第四缀合片段为配体；或者所述第三缀合物片段为配体，所述第四缀合片段为受体；和
所述第三缀合片段和/或所述第四缀合物片段与所述第一缀合物片段和/或所述第二缀合物片段选自不同的受体和配体，所述第三缀合片段和所述第四缀合物片段能够特异性结合；或者
2.抗体另一侧Fab保留原来的CH1和CL，即，
所述第二抗原结合部分包括：第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至抗体重链恒定区CH1，和
第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至抗体轻链恒定区CL。
本发明利用配体及其受体本身特有的特异性结合力，将其创造性地与抗原结合区(抗体可变区)可操作性地连接，所述连接包括与其中之一抗原结合区连接，另一抗原结合区仍与CH1和CL连接；或者两种抗原结合区都与配受体连接，只是两种抗原结合区连接不同种类地配受体，从而避免不同抗原结合区发生错配。
在一些实施方式中，本发明提供的双特异性融合多肽是一种多功能融合多肽，其包含2种抗体Fab，其特征在于，其中一侧Fab的CH1和CL独立地被配体及其受体所取代，另一侧Fab的CH1和CL未被取代，所述受体既包含识别并结合配体的活性部位，也包含产生应答反应的功能活性部位；所述第一抗原结合部分的轻链不会与所述第二抗原结合部分的重链错配。在一些实施方式中，其中一侧Fab的CH1和CL独立地被第一配体及其受体所取代，另一侧Fab的CH1和CL独立地被第二配体及其受体取代，所述第一配体及其受体与所述第二配体及其受体不同。
在一些实施方式中，所述多功能融合蛋白不仅能发挥双靶点特异性，且能发挥配受体传导的生物学活性。例如，在某个特定的实施方式中，所述配体及其受体为IL21和IL21R，所述多功能融合多肽除具有双靶点靶向作用外，IL21和IL21R结合，激活下游信号通路，发挥相应生物学功能。
在一些实施方式中，所述双特异性融合多肽具有：第一多肽，其从N端到C端依次为：[VH1]-[连接子1]-[IL21]-[连接子2]-[Fc1],第二多肽，其从N端到C端依次为：[VL1]-[连接子3]-[[IL21R],第三多肽，其从N端到C端依次为：[VH2]--[Fc2],和第四多肽，其从N端到C端依次为：[VL2]-[CL]；在一些实施方式中，所述双特异性融合多肽具有：第一多肽，其从N端到C端依次为：[VH1]-[连接子1]-[IL21R]-[连接子2]-[Fc1],第二多肽，其从N端到C端依次为：[VL1]-[连接子3]-[[IL21],第三多肽，其从N端到C端依次为：[VH2]--[Fc2],和第四多肽，其从N端到C端依次为：[VL2]-[CL]。其中，所述Fc1和Fc2为重链恒定区Fc的2个亚基，可以相同，也可以不相同，优选的所述Fc恒定区是异源二聚体(异二聚体Fc融合蛋白)；在一些实施方案中，所述Fc恒定区为基于KiH、疏水相互作用、静电相互作用、亲水相互作用和/或增加的柔性而缔合成为异源二聚体。所述连接子1、连接子2和连接子3为连接多肽的连接子，其可以相同，也可以不相同；在一些实施方案中，所述连接子1、连接子2和连接子3独立选自(GxS)y连接子，其中，x选自1-5的整数，y选自0-6的整数。
在一些实施方式中，所VH1和VL1配合形成特异性结合TIGIT的抗原结合位点，所VH2和VL2配合形成特异性结合PD-L1的抗原结合位点。在一些实施方式中，所VH1和VL1配合形成特异性结合PD-L1的抗原结合位点，所VH2和VL2配合形成特异性结合TIGIT的抗原结合位点。在一些实施方式中，所述结合TIGIT的抗原结合部分包括重链可变区和轻链可变区，其中重链可变区包括复合物1中的TIGIT_VH(也即SEQ ID NO.24的第1至第118位残基组成的多肽)或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列，轻链可变区包括复合物1中的TIGIT_VL(也即SEQ ID NO.25的第1至第107位残基组成的多肽)或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列。在一些实施方式中，其中所述结合TIGIT的抗原结合部分的重链可变区包括HCDR1、HCDR2和HCDR3区，所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.24的第1至第118位残基组成的可变区中的HCDR1、HCDR2和HCDR3，在一些实施方式中,其中所述轻链可变区包括LCDR1、LCDR2和LCDR3区，所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.25的第1至第107位残基组成的轻链可变区中的LCDR1、LCDR2和LCDR3；在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。在一些实施方式中，所述结合PD-L1的抗原结合部分包括重链可变区和轻链可变区，其中所述重链可变区包括复合物1中的PD-L1重链可变区(也即SEQ ID NO.43的第1至第119位残基组成的多肽)或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列，轻链可变区包括复合物1中PD-L1轻链可变区(也即SEQ ID NO.42的第1至第108位残基组成的多肽)或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列；在一些实施方式中，所述 结合PD-L1的抗原结合部分的重链可变区包括HCDR1、HCDR2和HCDR3区，所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.43中的HCDR1、HCDR2和HCDR3，在一些实施方式中,其中所述轻链可变区包括LCDR1、LCDR2和LCDR3区，所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.42中的LCDR1、LCDR2和LCDR3；在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。
在一些实施方式中，所述IL21选自SEQ ID NO.44至SEQ ID NO.53任一所示序列，所述IL21R选自SEQ ID NO.54至SEQ ID NO.60任一所示序列。在一些实施方式中，所述IL21选自SEQ ID NO.51所示序列，所述IL21R选自SEQ ID NO.55所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.48所示序列，所述IL21R选自SEQ ID NO.57所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.50所示序列，所述IL21R选自SEQ ID NO.58所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.53所示序列，所述IL21R选自SEQ ID NO.59所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.52所示序列，所述IL21R选自SEQ ID NO.59所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.49所示序列，所述IL21R选自SEQ ID NO.58所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.45所示序列，所述IL21R选自SEQ ID NO.58所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.46所示序列，所述IL21R选自SEQ ID NO.56所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.47所示序列，所述IL21R选自SEQ ID NO.60所示序列。在一些实施方式中，所述IL21选自与SEQ ID NO.44-53任一所示序列具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列，所述IL21R选自与SEQ ID NO.54-60任一所示序列具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列。
在一些实施方式中，所述双特异性融合多肽为本发明表3所示的FiBody分子；在一些实施方式中，所述双特异性融合多肽为本发明实施例9所述的复合物1、复合物2、复合物3、复合物4、复合物5、复合物6、复合物7、复合物8或复合物9。
ⅰ.配体和受体
“受体(receptor)”是细胞膜上或细胞内能识别生物活性分子并与之结合的物质，能与受体结合的生物活性物质统称为“配体(ligand)”。
根据受体在细胞中的位置，将其分为细胞表面受体和细胞内受体两大类。受体本身至少含有两个活性部位：一个是识别并结合配体的活性部位；另一个是负责产生应答反应的功能活性部位，这一部位只有在与配体结合形成二元复合物并变构后才能产生应答反应，由此启动一系列的生化反应，最终导致靶细胞产生生物效应。
受体一般为糖蛋白，野生型受体与配体之间的结合不通过共价键介导，主要靠离子键、氢键、范德华力和疏水作用而相互结合。受体在与配体结合时，具有饱和性、高亲和性、专一性等特性。
互相配合的受体和配体具有相对特异结合的亲和力，以及任选的生物学效应。在一些实 施方式中，所述受体仅包含识别并结合配体的活性部位，不包含产生应答反应的功能活性部位(例如激活下游信号通路的生物学效应的功能)。在一些实施方式中，所述受体和/或配体为天然的受配体结构，所述受体既包含识别结合配体的活性部位，又包含负责产生应答反应的功能活性部位，能够发挥相应的生物学功能，所述双特异性融合蛋白是一种多功能融合蛋白，不仅具有双特异性，而且能发挥配受体功能。
在一些实施方式中，所述受体和/或配体在天然序列的基础上做了修饰，所述修饰包括但不限于:截短、插入和/或突变；这些修饰的目的包括但不限于：增加或降低配体和受体的结合力；增强、降低或消除配体受体的生物学功能；增加、减少或消除受体和或配体蛋白中的糖基化位点；降低或消除受配体毒性。
受体(或其片段)和其相应的配体(或其片段)的结合方式可以是共价结合、非共价相互作用或其组合；非共价键的例子包括，但不限于，氢键、疏水键、离子键、和范德华键。在一些实施方式中，当被插入或替换的缀合片段之间的亲和力低于预期时(例如不能拉近抗原结合部分中的两个可变区以使其获得特异性识别抗原的功能，或者不能防止2个或多个重链恒定区之间的重链错配，或者不能防止抗原结合部分之间错配以实现特定VL-VH部分的组合)，可以通过对抗体所述配体和/或受体进行改造以增加亲和力。在一些实施方式中，所述受体和配体之间包含至少一个非天然的链间键，所述非天然链间键能够增强受体和配体间的特异性结合力；在一些实施方式中，所述非天然链间键形成于受体的第一突变残基和配体的第二突变残基之间；在一些实施方式中，所述第一和所述第二突变残基中的至少一个为半胱氨酸残基；在一些实施方式中，所述非天然链间键为二硫键。
“非天然的链间键”是指在野生型多肽聚合物中未发现的链间键。例如，非天然链间键可以在一条多肽的突变的氨基酸残基和另一条多肽的突变氨基酸残基之间形成。
在一些实施方式中，其中至少一个天然糖基化位点在所述受体和/或配体中不存在。
在一些实施方式中，所述受体和配体选自白细胞介素及其受体。
发明人对大量的白细胞介素及其受体进行了立体构像研究，发现大量的白细胞介素或IFN类分子立体构像可以分为4类：A类-托举型、B类-蝴蝶结型、C-棒球手型、D类-钳型。
在一些实施例中，所述配体及其受体选自D类白细胞介素及其受体，例如IL21/IL21R。
互相配合的受体(或其片段)以及配体(或其片段)的插入或替换位置可以位于，例如：
受体或其片段插入或替换CL区，配体或其片段插入或替换CH1区；或
受体或其片段插入或替换CH1区，配体或其片段插入或替换CL区。
在一些实施方式中，所述IL21选自SEQ ID NO.44至SEQ ID NO.53任一所示序列，所述IL21R选自SEQ ID NO.54至SEQ ID NO.60任一所示序列。在一些实施方式中，所述IL21选自SEQ ID NO.51所示序列，所述IL21R选自SEQ ID NO.55所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.48所示序列，所述IL21R选自SEQ ID NO.57所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.50所示序列，所述IL21R选自SEQ ID NO.58所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.53所示序列，所述IL21R选自SEQ ID NO.59所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.52所示序列，所述IL21R选自SEQ ID NO.59所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.49所示 序列，所述IL21R选自SEQ ID NO.58所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.45所示序列，所述IL21R选自SEQ ID NO.58所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.46所示序列，所述IL21R选自SEQ ID NO.56所示序列；在一些实施方式中，所述IL21选自SEQ ID NO.47所示序列，所述IL21R选自SEQ ID NO.60所示序列。在一些实施方式中，所述IL21选自与SEQ ID NO.44-53任一所示序列具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列，所述IL21R选自与SEQ ID NO.54-60任一所示序列具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列。
ⅱ.抗原结合部分
本发明提供的双特异性融合多肽，包含第一抗原结合部分和第二抗原结合部分，具有两种抗原特异性，第一抗原结合部分与第二抗原结合部分是不同的，可以是第一抗原结合部分与第二抗原结合部分结合不同的抗原，也可以是第一抗原结合部分与第二抗原结合部分结合相同抗原的不同表位。
在一些实施方式中，所述双特异性融合蛋白针对的靶标是肿瘤。在一些实施方式中，第一抗原结合部分与第二抗原结合部分结合的靶点都在肿瘤细胞表达；在一些实施方式中，第一抗原结合部分结合的靶点在肿瘤细胞，第二抗原结合部分结合的靶点在免疫细胞；在一些实施方式中，第一抗原结合部分与第二抗原结合部分结合的靶点都在免疫细胞。
T细胞重定向杀伤是许多治疗领域中理想的作用机制。在临床前和临床试验中，各种双特异性抗体形式参与T细胞重定向(May C等人(2012)Biochem Pharmacol，84(9))：1105年至1112年，第；弗兰克尔SR，和Baeuerle PA，(2013)CURR OPIN化学生物学，第17卷(3)：385-92，页)。所有T细胞重新靶向的双特异性抗体或其片段已被工程化以具有至少两个抗原结合位点，其中一个位点与靶细胞上的表面抗原结合另一个位点与T细胞表面抗原结合。在T细胞表面抗原中，源自TCR蛋白质复合物的人CD3的ε亚基最常被靶向作为重定向T细胞杀伤的靶标。
可被靶向的肿瘤相关联抗原包括但不限于：α-胎蛋白(AFP)、α-辅肌动蛋白-4、A3、对A33抗体有特异性的抗原、ART-4、B7、Ba 733、BAGE、BrE3-抗原、CA125、CAMEL、CAP-1、碳酸酐酶IX、CASP-8/m、CCCL19、CCCL21、CD1、CD1a、CD2、CD3、CD4、CD5、CD8、CD11A、CD14、CD15、CD16、CD18、CD19、CD20、CD21、CD22、CD23、CD25、CD29、CD30、CD32b、CD33、CD37、CD38、CD40、CD40L、CD44、CD45、CD46、CD52、CD54、CD55、CD59、CD64、CD66a-e、CD67、CD70、CD70L、CD74、CD79a、CD80、CD83、CD95、CD126、CD132、CD133、CD138、CD147、CD154、CDC27、CDK-4/m、CDKN2A、CTLA-4、CXCR4、CXCR7、CXCL12、HIF-1α、结肠特异性抗原p(CSAp)、CEA(CEACAM5)、CEACAM6、c-Met、DAM、EGFR、EGFRvIII、EGP-1(TROP-2)、EGP-2、ELF2-M、Ep-CAM、成纤维细胞生长因子(FGF)、Flt-1、Flt-3、叶酸盐受体、G250抗原、Claudin18.2、GAGE、gp100、GRO-β、HLA-DR、HM1.24、人绒毛膜BCMA促性腺激素(HCG)和其亚基、HER2/neu、HMGB-1、缺氧诱导因子(HIF-1)、HSP70-2M、HST-2、Ia、IGF-1R、 IFN-γ、IFN-α、IFN-β、IFN-λ、IL-4R、IL-6R、IL-13R、IL-15R、IL-17R、IL-18R、IL-2、IL-6、IL-8、IL-12、IL-15、IL-17、IL-18、IL-23、IL-25、胰岛素样生长因子-1(IGF-1)、KC4-抗原、KS-1-抗原、KS1-4、Le-Y、LDR/FUT、巨噬细胞迁移抑制因子(MIF)、MAGE、MAGE-3、MART-1、MART-2、NY-ESO-1、TRAG-3、mCRP、MCP-1、MIP-1A、MIP-1B、MIF、MUC1、MUC2、MUC3、MUC4、MUC5ac、MUC13、MUC16、MUM-1/2、MUM-3、NCA66、NCA95、NCA90、PAM4抗原、胰腺癌粘蛋白、PD-1受体、胎盘生长因子、p53、PLAGL2、前列腺酸性磷酸酶、PSA、PRAME、PSMA、PlGF、ILGF、ILGF-1R、IL-6、IL-25、RS5、RANTES、T101、SAGE、S100、存活素、存活素-2B、TAC、TAG-72、腱生蛋白、TRAIL受体、TNF-α、Tn抗原、Thomson-Friedenreich抗原、肿瘤坏死抗原、VEGFR、ED-B纤连蛋白、WT-1、17-1A-抗原、补体因子C3、C3a、C3b、C5a、C5、血管生成标记物、bcl-2、bcl-6、Kras、致癌基因标记物以及致癌基因产物(参见，例如Sensi等人，Clin Cancer Res2006，12：5023-32；Parmiani等人，JImmunol2007，178：1975-79；Novellino等人Cancer Immunol Immunother2005，54：187-207)。
虽然对于效应T细胞具有特异性的抗体或其它结合分子优选地结合至CD3抗原，但是在效应T细胞上表达的其它抗原是已知的并且可由T-细胞重定向复合物靶向。示例性T-细胞抗原包括但不限于，CD2、CD3、CD4、CD5、CD6、CD8、CD25、CD28、CD30、CD40、CD40L、CD44、CD45、CD69和CD90。
免疫检查点是免疫系统中的抑制途径，其对维持自身耐受性和调节外周组织中生理性免疫应答的持续时间和幅度以使附带组织损伤最小化至关重要。在一些实施方式中，第一抗原结合部分与第二抗原结合部分结合的靶点均为免疫检查点或其配体，所述免疫检查点分子包括但不限于：TIGIT、PD-1、TIM-3、LAG3、GTLA4、BTLA、BTN1A1、VISTA、LAIR、CD96、PVRIG、LILRA3、LILRA4、LILRB1、LILRB2、LILRB3、LLRB4、NKG-2A、CD47、CD200R1、CD300、Dectin-1、ICOS、NKp30、CD28、CD28H、CRTAM、DNAM-1、4-1-BB、BAFF、CD27、CD30、CD40、DR3、GITR、HVEM、LIGHT、OX40、TACI、2B4、CD2、CD48、CD229、SLAM、SLAMF5、GRAAC、TIM1、TIM4、CD7、DPPIV。
在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为PD-L1；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为TIGIT；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为GTLA4；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为LAG3；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为TIM-3；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为CD47；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为GTLA4；在一些实施方式中，第一抗原结合部分结合的靶点为PD-1，第二抗原结合部分结合的靶点为4-1-BB；在一些实施方式中，第一抗原结合部分结合的靶点为PD-L1，第二抗原结合部分结合的靶点为4-1-BB；在一些实施方式中，第一抗原合部分结合的靶点为PD-L1，第二抗原结合部分结合的靶点为TIGIT。
在一些实施方式中，第一抗原结合部分靶向肿瘤相关抗原，第二抗原结合部分靶向免疫检查点。在一些实施方式中，第一抗原结合部分靶向HER2,第二抗原结合部分靶向PD-1；在一些实施方式中，第一抗原结合部分靶向VEGF,第二抗原结合部分靶向PD-L1；在一些实施方式中，第一抗原结合部分靶向Claudin18.2,第二抗原结合部分靶向PD-L1；在一些实施方式中，第一抗原结合部分靶向HER2,第二抗原结合部分靶向CTLA-4；在一些实施方式中，第一抗原结合部分靶向CD20,第二抗原结合部分靶向CD47；在一些实施方式中，第一抗原结合部分靶向HER2,第二抗原结合部分靶向CD47。
在一些实施方式中，第一抗原结合部分和第二抗原结合部分同时靶向肿瘤异质性。用于肿瘤的示例性共同靶标包括但不限于HGF和VEGF,IGF-1R和VEGF,Her2和VEGF,CD19和CD3,CD20和CD3,Her2和CD3,CD19和FcγRIIIa,CD20和FcγRIIIa,Her2和FcγRIIIa。本发明的双特异性融合多肽能够结合VEGF和磷脂酰丝氨酸；VEGF和ErbB3；VEGF和PLGF；VEGF和ROBO4；VEGF和BSG2；VEGF和CDCP1；VEGF和ANPEP；VEGF和c-MET；HER-2和ERB3；HER-2和BSG2；HER-2和CDCP1；HER-2和ANPEP；EGFR和CD64；EGFR和BSG2；EGFR和CDCP1；EGFR和ANPEP；IGF1R和PDGFR；IGF1R和VEGF；IGF1R和CD20；CD20和CD74；CD20和CD30；CD20和DR4；CD20和VEGFR2；CD20和CD52；CD20和CD4；HGF和c-MET；HGF和NRP1；HGF和磷脂酰丝氨酸；ErbB3和IGF1R；ErbB3和IGF1,2；c-Met和Her-2；c-Met和NRP1；c-Met和IGF1R；IGF1,2和PDGFR；IGF1,2和CD20；IGF1,2和IGF1R；IGF2和EGFR；IGF2和HER2；IGF2和CD20；IGF2和VEGF；IGF2和IGF1R；IGF1和IGF2；PDGFRa和VEGFR2；PDGFRa和PLGF；PDGFRa和VEGF；PDGFRa和c-Met；PDGFRa和EGFR；PDGFRb和VEGFR2；PDGFRb和c-Met；PDGFRb和EGFR；RON和c-Met；RON和MTSP1；RON和MSP；RON和CDCP1；VGFR1和PLGF；VGFR1和RON；VGFR1和EGFR；VEGFR2和PLGF；VEGFR2和NRP1；VEGFR2和RON；VEGFR2和DLL4；VEGFR2和EGFR；VEGFR2和ROBO4；VEGFR2和CD55；LPA和S1P；EPHB2和RON；CTLA4和VEGF；CD3和EPCAM；CD40和IL6；CD40和IGF；CD40和CD56；CD40和CD70；CD40和VEGFR1；CD40和DR5；CD40和DR4；CD40和APRIL；CD40和BCMA；CD40和RANKL；CD28和MAPG；CD80和CD40；CD80和CD30；CD80和CD33；CD80和CD74；CD80和CD2；CD80和CD3；CD80和CD19；CD80和CD4；CD80和CD52；CD80和VEGF；CD80和DR5；CD80和VEGFR2；CD22和CD20；CD22和CD80；CD22和CD40；CD22和CD23；CD22和CD33；CD22和CD74；CD22和CD19；CD22和DR5；CD22和DR4；CD22和VEGF；CD22和CD52；CD30和CD20；CD30和CD22；CD30和CD23；CD30和CD40；CD30和VEGF；CD30和CD74；CD30和CD19；CD30和DR5；CD30和DR4；CD30和VEGFR2；CD30和CD52；CD30和CD4；CD138和RANKL；CD33和FTL3；CD33和VEGF；CD33和VEGFR2；CD33和CD44；CD33和DR4；CD33和DR5；DR4和CD137；DR4和IGF1,2；DR4和IGF1R；DR4和DR5；DR5和CD40；DR5和CD137；DR5和CD20；DR5和EGFR；DR5和IGF1,2；DR5和IGFR,DR5和HER-2,以及EGFR和DLL4。其他靶标组合包括EGF/erb-2/erb-3家族的一个或多个成员。
此外，用于自身免疫病症和炎性病症的示例性共同靶标包括但不限于IL-1和TNFα,IL-6 和TNFα,IL-6和IL-1,IgE和IL-13,IL-1和IL-13,IL-4和IL-13,IL-5和IL-13,IL-9和IL-13,CD19和FcγRIIb,以及CD79和FcγRIIb。
用于治疗炎性疾病的示例性靶点包括但不限于：TNF和IL-17A；TNF和RANKL；TNF和VEGF；TNF和SOST；TNF和DKK；TNF和αVβ3；TNF和NGF；TNF和IL-23p19；TNF和IL-6；TNF和SOST；TNF和IL-6R；TNF和CD-20；IgE和IL-13；IL-13和IL23p19；IgE和IL-4；IgE和IL-9；IgE和IL-9；IgE和IL-13；IL-13和IL-9；IL-13和IL-4；IL-13和IL-9；IL-13和IL-9；IL-13和IL-4；IL-13和IL-23p19；IL-13和IL-9；IL-6R和VEGF；IL-6R和IL-17A；IL-6R和RANKL；IL-17A和IL-1β；IL-1β和RANKL；IL-1β和VEGF；RANKL和CD-20；IL-1α和IL-1β；IL-1α和IL-1β。
参与类风湿性关节炎(RA)的靶点包括但不限于：TNF和IL-18；TNF和IL-12；TNF和IL-23；TNF和IL-1β；TNF和MIF；TNF和IL-17；和TNF和IL-15。
治疗系统性红斑狼疮(SLE)的靶点包括但不限于：CD20,CD22,CD19,CD28,CD4,CD24,CD37,CD38,CD40,CD69,CD72,CD74,CD79A,CD79B,CD80,CD81,CD83,CD86,IL-4,IL-6,IL10,IL2,IL4,IL11,TNFRSF5,TNFRSF6,TNFRSF8,C5,TNFRSF7,TNFSF5,TNFSF6,TNFSF7,BLR1,HDAC4,HDAC5,HDAC7A,HDAC9,ICOSL,IGBP1,MS4A1,RGSI,SLA2,IFNB1,AICDA,BLNK,GALNAC4S-6ST,INHA,INHBA,KLF6,DPP4,FCER2,,R2,ILIR2,ITGA2,ITGA3,MS4A1,ST6GALI,CDIC,CHSTIO,HLA-A,HLA-DRA,NT5E,CTLA4,B7.1,B7.2,BlyS,BAFF,IFN-α和TNF-α。
用于治疗多发性硬化症(MS)的靶点，包括但不限于：IL-12,TWEAK,IL-23,CXCL13,CD40,CD40L,IL-18,VEGF,VLA-4,TNF,CD45RB,CD200,IFNγ,GM-CSF,FGF,C5,CD52和CCR2。
用于治疗脓毒症的靶点包括但不限于：TNF,IL-1,MIF,IL-6,IL-8,IL-18,IL-12,IL-10,IL-23,FasL,LPS,Toll-样受体,TLR-4,组织因子，MIP-2,ADORA2A,IL-1B,CASP1,CASP4,NFκB1,PROC,TNFRSFIA,CSF3,CCR3,ILIRN,MIF,NFκB1,PTAFR,TLR2,TLR4,GPR44,HMOX1,中期因子,IRAK1,NFκB2,SERPINA1,SERPINE1,和TREM1。
为了形成本发明的双特异性融合蛋白，可以制备针对这些抗原的任意组合的抗体；即，这些抗原中的每一个可以任选地和独立地被根据本发明的多特异性抗体包括或不包括。
在一些实施方式中，第一抗原结合部分和第二抗原结合部分靶向同一抗原的不同表位。
在一些实施方式中，至少一个抗原结合片段还可以包括分泌信号序列。
分泌信号序列是指，通过连接至编码序列位于细胞膜外侧或细胞外侧的N端而诱导所表达的蛋白或肽的分泌的序列，所述信号序列可以是由约18-30个氨基酸组成的肽序列。所有能转运到细胞膜外侧的蛋白有不同的信号序列，所述信号序列被细胞膜上的信号肽酶切割。通常，对于并非宿主细胞天然表达的外来蛋白而言，可以采用能将该蛋白分泌到细胞周质或培养基中的分泌信号序列，或采用修饰的序列。
在一些实施方式中，所第一抗原结合部分和第二抗原结合部分分别与TIGIT和PD-L1结合。在一些实施方式中，所述结合TIGIT的抗原结合部分包括重链可变区和轻链可变区，其中重链可变区包括复合物1中的TIGIT_VH(也即SEQ ID NO.24的第1至第118位残基组成 的多肽)或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列，轻链可变区包括复合物1中的TIGIT_VL(也即SEQ ID NO.25的第1至第107位残基组成的多肽)或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列。在一些实施方式中，其中所述结合TIGIT的抗原结合部分的重链可变区包括HCDR1、HCDR2和HCDR3区，所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.24的第1至第118位残基组成的可变区中的HCDR1、HCDR2和HCDR3，在一些实施方式中,其中所述轻链可变区包括LCDR1、LCDR2和LCDR3区，所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.25的第1至第107位残基组成的轻链可变区中的LCDR1、LCDR2和LCDR3；在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。在一些实施方式中，所述结合PD-L1的抗原结合部分包括重链可变区和轻链可变区，其中所述重链可变区包括复合物1中的PD-L1重链可变区(也即SEQ ID NO.43的第1至第119位残基组成的多肽)或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列，轻链可变区包括复合物1中PD-L1轻链可变区(也即SEQ ID NO.42的第1至第108位残基组成的多肽)或与其具有至少80％(例如至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性的序列；在一些实施方式中，所述结合PD-L1的抗原结合部分的重链可变区包括HCDR1、HCDR2和HCDR3区，所述HCDR1、HCDR2和HCDR3分别包括SEQ ID NO.43中的HCDR1、HCDR2和HCDR3，在一些实施方式中,其中所述轻链可变区包括LCDR1、LCDR2和LCDR3区，所述LCDR1、LCDR2和LCDR3分别包括SEQ ID NO.42中的LCDR1、LCDR2和LCDR3；在一些实施方案中，所述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3由IMGT编号系统定义，或由Kabat编号系统定义，或由Chothia编号系统定义，或由Contact编号系统定义，或由AbM编号系统定义。
ⅲ.异二聚体Fc融合蛋白
在一些实施方式中，其包含重链恒定区Fc，所述Fc恒定区是异源二聚体(异二聚体Fc融合蛋白)。
所述Fc包括但不限于如下组合：
CH2；
CH2+CH3；或
CH2+CH3+CH4；
所述Fc恒定区引入突变以避免重链错配。
在一些实施方式中，所述Fc恒定区引入突变为基于KiH技术(Knob-into-Holes),即在其中Fc恒定区一条重链中引入氨基酸突变，引入的氨基酸体积大于最初氨基酸残基体积，形成一个突起的类似“杵”的型结构(Knob)，在Fc恒定区另一条链区引入另一突变，引入的氨基酸 体积小于最初氨基酸残基体积，形成一个凹陷，类似“臼”的结构(Hole)，从而凸型重链更倾向和凹型重链配对，从而避免重链发生错配。该技术记载于专利申请WO1996027011中，该专利全文引入本发明。
在一些实施方式中，所述Fc恒定区引入突变为基于静电相互作用，例如ART-lg技术，该技术通过特异性的改变Fc恒定区结构域的电荷，促进异源重链的配对，相当于电荷版的KiH技术，该技术记载于专利申请WO2006106905中，该专利全文引入本发明。
在一些实施方式中，所述Fc恒定区引入突变为基于SEED技术，SEED异二聚化是另一种基于空间突变的设计策略，该策略利用了从IgG和IgA CH3域(也称为AG SEED CH3和GA SEED CH3)衍生的交替序列的互补性。IgG和IgA CH3衍生物产生互补序列，因此在组装两个互补的重链异源二聚体的同时，排除了缺乏互补性的同源二聚体的组装。该技术记载于专利申请WO2007110205中，该专利全文引入本发明。
在一些实施方式中，所述Fc恒定区引入突变为基于等电点改变，便于提高异源二聚体形成率以及保持Fc区域稳定性的改造，该技术记载于WO2014145806，该专利全文引入本专利。
在一些实施方式中，所述Fc恒定区基于亲水相互作用或增加的柔性而缔合成为异源二聚体。
在一些实施方式中，所述Fc恒定区基于以上技术的任意组合缔合成为异源二聚体，例如，在一些实施方式中，所述Fc恒定区基于KIH和静电相互作用的组合进行了突变。例如，XmAb双特异性平台方法可以通过结合静电相互作用，CH3域构象和氢键提高双特异性抗体的热稳定性。具体的，该策略将天然IgG1的Fc侧链突变交换为S364K和K370S异二聚体，以在两者之间形成氢键，然后进行L368D/K370S取代驱动盐桥相互作用以促进异二聚体的形成，专利申请WO2014145907，该专利全文引入本专利。
在一些实施方式中，所述CH2、CH3或CH4区域的全长或部分被插入或替换成受体及其配体。
在一些实施方式中，被插入或替换的区域独立地位于CH2、CH3或CH4区，或任意像个相邻的区之间的位置(如CH1-CH2交界处、CH2-CH3交界处、CH3-CH4交界处)；
在一些实施方式中，当上述任意两个恒定区(例如CL-CH1、CH2-CH2、CH3-CH3或CH4-CH4区之任一项)被插入或替换时，替换区域两个互相配合的缀合片段之间的亲和力，K D<1×10 -3(M)，例如如x×10 -4(M)、x×10 -5(M)、x×10 -6(M)、x×10 -7(M)、x×10 -8(M)、x×10 -9(M)、x×10 -10(M)、x×10 -11(M)；x的值可选自1～9，例如2、3、4、5、6、7、8。
在一些实施方式中，所述缀合片段的N端和/或C端通过连接肽与所述抗原结合片段连接。
术语“可操作连接”是指部件(例如两条多肽)直接地或经由一个或多个连接子(连接肽)通过共价键连接。
在一些实施方式中，所述连接肽的氨基酸数目为1～30个；可以是1，2，3，4，5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个；优选5～20个。
在一些实施方式中，所述连接肽的氨基酸是不具有除连接以外的额外功能(例如蛋白定位、酶切位点等)的无意义多肽。
在一些实施方式中，所述连接肽为柔性连接肽；
在一些实施方式中，所述连接肽的氨基酸序列选自Gly、Ser、Pro、Ala以及Glu中的一种或多种。
在一些实施方式中，所述连接肽的氨基酸序列选自(GGGGS)n、(GGGS)n、(GGS)n、(GS)n或(G)n，其中n选自1，2，3，4，5或6。
连接肽通常是柔性的，可以减少融合蛋白与目的蛋白之间的空间位阻，从而更有利于蛋白正确折叠。
在另外的实施方案中，连接肽是刚性接头肽；即相对非柔性肽接头。刚性连接肽不要求完全缺乏柔性，而是比柔性接头肽如富甘氨酸肽接头柔性少。由于其相对缺乏柔性，刚性连接肽降低通过刚性连接肽连接在一起的两个蛋白结构域(在当前情况下是稳定剂蛋白和热稳定逆转录酶)的运动。提供有序链(例如α螺旋结构)的连接肽可提供刚性接头肽。例如，精氨酸、亮氨酸、谷氨酸、谷氨酰胺和甲硫氨酸都显示出相对高的螺旋接头结构倾向。然而，包含许多脯氨酸残基的非螺旋接头也可表现显著的刚性。刚性连接肽的实例包括聚赖氨酸和聚-DL-丙氨酸聚赖氨酸。刚性肽接头的进一步描述由Wriggers等，Biopolymers，80，第736-46页(2005)提供。此外，刚性接头肽在由George等，Protein Engineering，15，第871-79页(2003)描述的接头数据库中描述。优选地，刚性连接肽也是非可切割接头肽，即非可切割刚性连接肽。
分离的核酸
本发明还涉及分离的核酸，其编码如上所述的双特异性融合多肽或多功能融合蛋白。
术语“分离的核酸”在本文中是指以单链或双链形式存在的脱氧核糖核酸或核糖核酸聚合物。所述分离的核酸包括RNA基因组序列，DNA(gDNA和cDNA)或从DNA转录的RNA序列，而且，除非特别指明，所述多肽还包括天然多核苷酸、糖、或碱基改变的类似物。根据本发明一个方面，所述多核苷酸是轻链多核苷酸。
所述分离的核酸包括编码蛋白复合物氨基酸序列的核苷酸序列，也包括与其互补的核苷酸序列。所述互补序列包括完全互补的序列和基本上互补的序列，这是指能在本领域已知的严谨条件下与编码蛋白复合物氨基酸序列的核苷酸序列杂交的序列。
而且，编码蛋白复合物氨基酸序列的核苷酸序列可以被改变或突变。所述改变包括添加、缺失、或非保守取代或保守取代。编码蛋白复合物氨基酸序列的多核苷酸可以被解释为，包括相对于该分离的核酸有实质性同一性的核苷酸序列。所述实质性同一性将该核苷酸序列与另外的随机序列以使得它们最大对应的方式进行比对，当用本领域常见的算法分析所比对的序列时，所述序列可显示大于80％的同源性，大于90％的同源性，或大于95％的同源性。
载体
本发明还涉及含有如上所述核酸的载体。
术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转 染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。所述载体可以包含选择标记(例如便于富集的标签，例如his tag；或便于被检测的标签，例如GFP)，以及与所述克隆载体所指定的细胞类型相匹配的复制起点，而表达载体则包含对于影响指定靶细胞中的表达必要的调节元件例如增强子、启动子、内部核糖体进入位点(IRES)和其他表达控制元件(例如转录终止信号，或者多腺苷酸化信号和多聚U序列等)。所述载体可以是克隆载体与表达载体。在表达或是制备抗体或片段时，常涉及原核表达载体和真核表达载体，原核表达载体常用PET系列、pGEX系列，真核表达载体常用pcDNA3.1、pcDNA3.4、pcDNA4、pEGFP-N1、pEGFP-N1、pSV2等。
在本发明中，载体可以为组合物，例如为多种质粒的混合物，不同质粒负载抗体或其片段的一部分。
宿主细胞
本发明还涉及含有如上所述核酸或者如上所述载体的宿主细胞。
可以使用的多种培养的宿主细胞包括，例如，原核细胞、真核细胞、细菌细胞(如大肠杆菌或嗜热脂肪芽胞杆菌(Bacilisstearothermophilus))、真菌细胞(如酿酒酵母或毕赤酵母)、昆虫细胞(如包括草地夜蛾细胞在内的鳞翅目昆虫细胞)或哺乳动物细胞(如中国仓鼠卵巢(CHO)细胞、NS0细胞、小仓鼠肾(BHK)细胞、猴肾细胞、Hela细胞、人肝细胞癌细胞或293细胞等等)。
制备双特异性融合多肽或多功能融合蛋白的方法
可采用本领域任何已知的方法制备本发明的双特异性融合多肽或多功能融合蛋白。
例如：用如上所述的载体转化宿主细胞；
培养所转化的宿主细胞；和
收集宿主细胞中表达的双特异性融合多肽或多功能融合蛋白。
特别的，可采用如下方法。
早期构建双特异性抗体的方法有化学交联法或杂合杂交瘤或四价体瘤法(例如，Staerz UD等,Nature,314:628-31,1985；Milstein C等,Nature,305:537-540,1983；Karpovsky B等,J.Exp.Med.,160:1686-1701,1984)。化学偶联法是将2个不同的单克隆抗体用化学偶联的方式连接在一起，制备出双特异性单克隆抗体。例如两种不同单克隆抗体的化学结合，或例如两个抗体片段如两个Fab片段的化学结合。杂合—杂交瘤法是通过细胞杂交法或者三元杂交瘤的方式产生双特异性单克隆抗体，这些细胞杂交瘤或者三元杂交瘤是通过建成的杂交瘤融合，或者建立的杂交瘤和从小鼠得到的淋巴细胞融合而得到的。虽然这些技术用于制造BiAb，但各种产生问题使得此类复合物难以使用，诸如产生含有抗原结合位点的不同组合的混合群体、蛋白质表现方面的困难、需要纯化目标BiAb、低产率、生产费用高等。
最近的方法利用经过基因工程改造的构建体，其能够产生单一BiAb的均质产物而无需 彻底纯化以去除不需要的副产物。此类构建体包括串联scFv、二抗体、串联二抗体、双可变结构域抗体和使用诸如Ch1/Ck结构域或DNLTM的基元的异源二聚(Chames&Baty,Curr.Opin.Drug.Discov.Devel.,12:276-83,2009；Chames&Baty,mAbs,1:539-47)。相关纯化技术是公知的。
还可以使用单淋巴细胞抗体方法通过克隆和表达由选择用于产生特异性抗体的单个淋巴细胞产生的免疫球蛋白可变区cDNA来产生抗体，例如由Babcook J等人,Proc.Natl.Acad.Sci.USA.93:7843-7848,1996；WO 92/02551；WO 2004/051268和WO2004/106377所述的方法。
用于产生例如用于免疫宿主或用于淘选诸如用于噬菌体展示(或酵母细胞或细菌细胞表面表达)的抗体的抗原多肽可以通过本领域熟知的方法从包含表达系统的遗传工程改造的宿主细胞制备，或者它们可以是从天然生物来源回收。例如，可将编码双特异性抗体的一条或两条多肽链的核酸通过多种已知的方法(如转化、转染、电穿孔、用核酸包被的微粒轰击等)引入培养的宿主细胞。在一些实施方案中，编码双特异性抗体的核酸在被引入宿主细胞前可先插入至适于在宿主细胞中表达的载体中。典型的所述载体可包含使插入的核酸能够在RNA和蛋白质水平上表达的序列元件。
本发明的双特异性抗体，或其部分可通过常规的免疫学分析方法，例如酶联免疫吸附试验(ELISA)，放射免疫分析(RIA)或组织免疫组织化学用于检测任一或所有这些抗原(例如在生物样品，如血清或血浆中)。本发明提供检测生物样品中的抗原的方法，该方法包括：使所述生物样品与本发明的可特异识别所述抗原的双特异性抗体，或抗体部分抗原相接触，并检测与抗原结合的抗体(或抗体部分)，或非结合抗体(或抗体部分)，由此检测所述生物样品中的所述抗原。所述抗体用可检测的物质进行直接或间接的标记，以便于检测结合或非结合抗体。合适的可检测物质包括多种酶，修复基团，荧光物质，发光物质和放射性物质。合适的酶的例子包括，辣根过氧化物酶，碱性磷酸酶，β-半乳糖苷酶，乙酰胆碱酯酶；合适的修复基团复合物的例子包括链霉抗生物素蛋白/生物素和抗生物素蛋白/生物素；合适的荧光物质的例子包括7-羟基香豆素，荧光素，荧光素异硫氰酸盐，硷性蕊香红B，二氯三嗪基胺荧光素，丹磺酰氯或藻红蛋白；发光物质的例子包括3-氨基邻苯二甲酰环肼；合适的放射性物质的例子包括I 125、I 131、 35S或 3H。
药物组合物
本发明的双特异性多肽复合物或编码其的核酸可以应用于制备药物组合物或无菌组合物，例如，将双特异性融合多肽或多功能融合蛋白与药学上可接受的载体、赋形剂或稳定剂混合。药物组合物可包括一种或组合的(如两种或更多不同的)本发明的抗体其功能片段。例如，本发明的药物组合物可包含与靶抗原上的不同表位结合的具有互补活性的抗体或抗体片段(或免疫缀合物)的组合。治疗和诊断剂的制剂可通过以例如冻干粉末、浆液、水性溶液或悬浮液的形式与药学可接受的载体、赋形剂或稳定剂混合来制备。
医药用途与治疗方法
本发明还涉及如上所述的双特异性融合多肽或多功能融合蛋白在制备用于治疗疾病的药物中的应用。
本发明还涉及用作药物的如上所述的双特异性融合多肽或多功能融合蛋白；所述药物用于治疗疾病。
根据本发明一个方面，所述疾病可以是例如，癌症、免疫性病症、代谢性疾病以及微生物感染。
术语“癌症”是指以体内异常细胞的不受控生长为特征的一大类疾病。“癌症”包括良性和恶性癌症以及休眠肿瘤或微转移。
本发明还涉及一种预防和/或治疗和施用治疗有效量的药物组合物以预防和/或治疗如上所述疾病的方法。
本发明的方法可以用于人类临床医学和兽医学应用。因此，带有致病生物群体并且用配体-免疫原缀合物治疗的宿主动物可以是人类，或者在兽医学应用的情况下，可以是实验室动物、农用动物、驯养动物或野生动物。本发明可以适用于包括但不限于以下的宿主动物：人类；实验室动物，诸如啮齿动物(例如小鼠、大鼠、仓鼠等)、兔、猴、黑猩猩；驯养动物，例如狗、猫和兔；农用动物，例如牛、马、猪、绵羊、山羊；和关养的野生动物，例如熊、熊猫、狮、虎、豹、大象、斑马、长颈鹿、大猩猩、海豚和鲸。
药物组合物可通过多种途径注射至实体中，所述实体包括大鼠、小鼠、家养动物、和/或人类。所有注射方法都可以预期，例如，口服，直肠，静脉，鼻，腹部，皮下，或局部注射都是有可能的。组合物可以用本领域已知的其它方法来注射。
“治疗有效量”在本文中是指，根据合理的益损比来看，能治疗疾病的足够量。治疗有效量可以因患者引起的多种原因而有不同，所述原因例如，疾病类型、严重程度、发作、实体的年龄、体重、排泄速度、反应易感性、健康状态、和/或并发症；和/或药物活性、注射途径、注射周期和注射次数、和/或药物组合；也可以由本领域普通技术人员根据治疗目的进行适当选择。例如，注射量可以随机分为多次，使得该量为约0.001-100mg/kg成人体重。
本发明的双特异性融合多肽或多功能融合蛋白或编码本发明抗体的核酸或多核苷酸还可与例如标准癌症治疗(例如，手术、放射和化学疗法)组合施用。例如，使用本发明的组合物和/或装备了这些组合物的效应细胞的抗肿瘤疗法与化学疗法联合使用。本发明抗体组合治疗的非限制性实例包括手术、化疗、放疗、免疫疗法、基因疗法、DNA疗法、RNA疗法、纳米疗法、病毒疗法、辅助疗法及其组合。
下面将结合实施例对本发明的实施方案进行详细描述。
实施例1、FiBody设计
FiBody是利用配体及其受体之间特异性亲和力，取代双特异性抗体一侧的CL和CH1，重组获得的双特异性抗体，其能够避免或减少双特异性抗体轻链与重链发生错配。
本实施例以白细胞介素及其受体为例构建FiBody，根据白细胞介素及其受体的立体构像将其分为四类，见图5及表1：
表1.FiBody分类
Figure PCTCN2022120973-appb-000001
Figure PCTCN2022120973-appb-000002
基于以上4类白细胞介素及其受体分别构建双特异性抗体。
备注：关于立体构象分类，主要是基于对PDB上已公布的各种IL细胞因子进行归纳总结分析。由于细胞因子结构复合物的复杂性，有些细胞因子可以拆分为多种结构类型，比如IL2，完整的IL2/IL2Rα/IL2Rβ/IL2Rγ结构复合物是类似于一个托举(A)的结构，但是单独看IL2/IL2Rβ，则是钳型(D)的结构。我们将IL2划归为托举型(A)结构是基于单独的IL2/IL2Rβ亲和力较弱，而IL2/IL2Rα亲和力较强，所以更多需要IL2Rα来辅助形成结构复合体。再比如IL21，IL21/IL21R/IL2Rγ的完整复合物是托举(A)的结构，但是由于IL21/IL21R具有较强的亲和力，所以可以单独划归为钳型(D)的结构。
实施例2、基于白细胞介素及其受体FiBody的构建
选取靶向第一抗体的VH通过Linker连接在受体蛋白上，再通过Hinge与抗体的Fc连接；靶向第一抗体的VL通过Linker连接在配体蛋白上，以降低或避免轻链与重链发生错配；另一端为靶向第二抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以降低或避免重链发生错配。或
选取靶向第一抗体的VH通过Linker连接在配体蛋白上，再通过Hinge与抗体的Fc连接；靶向第二抗体的VL通过Linker连接在受体蛋白上，以降低或避免轻链与重链发生错配；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以降低或避免重链错配。构建的一些示例性FiBody分子结构及序列表见表2及表3
表2.FiBody分子结构
Figure PCTCN2022120973-appb-000003
表3.示例性FiBody分子序列表
Figure PCTCN2022120973-appb-000004
Figure PCTCN2022120973-appb-000005
Figure PCTCN2022120973-appb-000006
Figure PCTCN2022120973-appb-000007
实施例3、构建基于scFv、CrossMab结构的双特异性抗体作为实验对照
正如前文所描述，scFv和CrossMab都是常用的双特异性抗体构建技术手段，在这里作为设计对照，跟我们的分子进行对比：
基于scFv结构双抗的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH通过Linker连接至第二抗体的VL上形成scFv结构，再通过Hinge与抗体的Fc连接；另一端为靶向第一抗体的完整Fab结构，(此双抗平台为武汉友芝友开发，命名为YBody),组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。具体例如表4：
表4.Y-Body分子序列
Figure PCTCN2022120973-appb-000008
Figure PCTCN2022120973-appb-000009
描述基于scFv结构双抗的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH通过Linker连接至第二抗体的VL上形成scFv结构，再通过Linker与完整的靶向第一抗体的Fc的C端连接；组成一个对称的结构。具体例如表5：
表5.scFv结构双抗分子序列
Figure PCTCN2022120973-appb-000010
基于CrossMab结构双抗的构建方法，具体选取靶向第二抗体(抗TIGIT)的VH连接至CL结构域，再通过Hinge与抗体的Fc连接，靶向第二抗体(抗TIGIT)的VL连接至CH1结构域，形成轻链；另一端为靶向第一抗体的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。具体例如表6：
表6.CrossMab结构双抗分子序列
Figure PCTCN2022120973-appb-000011
Figure PCTCN2022120973-appb-000012
基于scFv结构双抗的构建方法，具体重链为选取靶向第二抗体(示例性地选取抗TIGIT抗体)的VH通过Linker连接至第二抗体的VL上形成scFv结构，再通过Linker与完整的靶向第一抗体(示例性地选取抗PD-L1抗体)的VH-CH1，其再与Fc相连；轻链为第一抗体(示例性地选取抗PD-L1抗体)的VL-CL,组成一个对称的结构。具体见图11，序列例如表7：
表7.scFv结构双抗分子序列
Figure PCTCN2022120973-appb-000013
Figure PCTCN2022120973-appb-000014
实施例4、抗体重-轻链错配测试
轻链错配是双抗平台面临的一个难点问题。为了验证本平台防错配性能，我们专门设计了受体、配体分布在抗体两边的Fab，故意设计错配的重、轻链结构，并进行表达验证。
描述Fab-IL21/IL21R_Fc错配的构建方法：
选取靶向第二抗体(示例性地选取抗PD-L1抗体)的VH通过Linker连接在受体蛋白(IL21R)上，再通过Hinge与抗体的Fc连接；靶向第一抗体(示例性地选取抗TIGIT抗体)的VL通过Linker连接在配体蛋白(IL21)上；另一端为靶向第二抗体(示例性地选取抗PD-L1抗体)的VL连接在CL上，靶向结构第一抗体(示例性地选取抗TIGIT抗体)的VH连接在CH1上，再通过Hinge与抗体的Fc连接，两端的Fc具有常规的KiH改造。具体例如表8：
表8.错配的FiBody抗体序列
Figure PCTCN2022120973-appb-000015
Figure PCTCN2022120973-appb-000016
实施例5、FiBody样品的制备
蛋白瞬转表达：
将含有目的基因的质粒通过与转染试剂PEI形成阳离子复合物后，导入到宿主细胞Expi293，质粒在细胞内期间，质粒上的外源基因在细胞内发生转录翻译，从而得到目的蛋白。
Expi293在37℃、8％二氧化碳、130rpm条件培养，并在转染前通过细胞计数，将2E6的细胞接种至1L摇瓶中，培养体系约为300ml。配制转染复合物准备转染：首先将750μg目标质粒加入到含有15mlOpti-MEM试剂的50ml离心管中，轻轻混匀，标记为A管；将1.5mg转染试剂PEI加入到含有15mlOpti-MEM试剂的50ml离心管中，轻轻混匀后，室温孵育5min，标记为B管；将B管PEI稀释液逐滴加入到A管DNA稀释液中，轻轻混匀后，室温孵育15min，孵育结束后，将PEI-目标质粒复合物加入到Expi293细胞，置于37℃摇床中继续培养。直到D7-D10后收样。
蛋白纯化：
瞬转细胞表达液经过9000rpm/20min离心，收集上清，再经过0.22μm滤膜除菌过滤。纯化采用ProA亲和层析。过程如下，使用AKTA avant 150层析设备，用至少5CV平衡缓冲液(10mM PBS)平衡层析柱(如MabSelectSuRe LX，GE)，加载样品至层析柱，使目标蛋白吸附在层析柱上而其他杂质穿透分离。完成上样后使用至少5CV平衡缓冲液(10mM PBS)再次冲洗层析柱，随后使用洗脱缓冲液(20mM NaAc，pH＝3.4)洗脱目标蛋白，收集管中预先加入中和缓冲液(1M Tris，pH8.0)，中和缓冲液的加入体积根据洗脱样品的预估含量而定，一般加入10％洗脱体积量。
实施例6、FiBody理化检测
样品经过一步纯化后通过HPLC-SEC进行检测(分析柱TOSOH，TSKgel G2000)纯度，各个样品的表达量及纯度结果见表9。
表9.双特异性抗体理化检测结果
Figure PCTCN2022120973-appb-000017
结果显示,相比于非对称scFv(Y-Body，R0809)、对称ScFv(R0810)、CrossMab(R0959)结构的双特异性抗体，FiBodyA类分子和D类分子平台制备双特异性抗体(包括各种改造优化抗体)具有更高的表达量和/或更高的纯度。
意外的是具有错误配对形式的双特异性抗体(样品R1124)也能表达并具有类似正常分 子的表达量，但是具有明显低的纯度。
实施例7、FiBody抗原亲和力检测
TIGIT端结合活性分析
通过FCM实验方法检测双抗分子(TIGIT端)与CHO-TIGIT细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL,3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL,2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h,再加入50μL配体，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1:500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测。
结果如图7所示：白细胞介素21及其受体替换CH1和CL效果最好，靶向区结合力并未受到影响，与阳性对照(R0774,重链可变区和轻链可变区与R1116的TIGIT_VH/TIGIT_VL相同的抗Tigit的hIgG1单克隆抗体)表现出了相当的亲和力；C类分子R1119的替换CH1和CL后靶向区受到严重影响。
R1124为错配测试分子，其TIGIT结合活性显著降低。
PD-L1端结合活性分析
通过FCM实验方法检测双抗分子(PD-L1端)与CHO-PD-L1细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL,3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL,2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h,再加入50μL配体，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1:500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测。
结果如图8所示，各类FiBody均显示了与阳性抗体(R0919，重链可变区和轻链可变区与R1116的PD-L1_VH/PD-L1_VL相同的抗PD-L1的hIgG1单克隆抗体)相当的亲和力，错配分子R1124与PDL1端的靶向亲和力明显降低。
实施例8、基于IL21/IL21R构建的FiBody两端结合力检测
Crosslinking of PDL1+cells and Tigit+cells(PDL1/Tigit与细胞交联)：
1.先将CHO-S-hPDL1细胞(通过慢病毒转染方式，在细胞膜上重组表达human PDL1的CHO-S重组细胞)用CFSE(BD BioSicences,Cat:565082)标记，Jurkat-Tigit细胞(通过慢病毒转染方式，在细胞膜上重组表达TIGIT的Jurkat重组细胞)或者CHO-TIGIT用CellTraceTM  Violet(Thermo Fisher,Cat:C34557)标记；
2.将标记好的细胞按照1:1的比例(分别1E5)混合并加入抗体(20nM)在96孔U型板一起孵育，室温孵育1h；
3.孵育好的细胞轻柔混匀后流式上机检测，FITC+(CFSE)/PB450+(Violet)双阳性细胞即为交联细胞。
相比较于scFv双抗(R1155-3.12％、R1160-3.72％)，双特异性样品R1123(30.84％/59.69)可以显著更好的同时结合CHO-PDL1(CFSE标记)与CHO-TIGIT【图9】或者CHO-PD-L1(CFSE标记)与Jurkat-Tigit(Violet标记)[图10],说明本发明双特异性抗体Fibody能发挥非常强的Crosslinking效应。
实施例9具有二硫键改造的的双特异性抗体
为了进一步加强双特异性抗体稳定性并且延长双特异性抗体的半衰期，对双特异性抗体进行二硫键改造，示例性地，IL21/IL21Rα的二硫键改造见表10和表11。
表10.IL21/IL21Rα二硫键改造
组合 IL21 IL21Rα
1 R81C E38C
2 Q17C A71C
3 R14C D72C
4 S5C S92C
5 S4C S92C
6 R10C D72C
7 I13C D72C
8 I71C A127C
9 K82C Y36C
备注：表中，所述IL21突变的位点依据人IL21(序列SEQ ID NO.44)的自然顺序编号，IL21Rα突变的位点依据人IL21Rα(序列SEQ ID NO.54)的自然顺序编号，例如IL21的R81C突变是指SEQ ID NO.44的第81位由R突变为C。
表11.IL21/IL21Rα氨基酸序列
Figure PCTCN2022120973-appb-000018
Figure PCTCN2022120973-appb-000019
Figure PCTCN2022120973-appb-000020
配受体二硫键改造：选取靶向第二抗体(示例性地选取抗TIGIT抗体)的VH通过Linker连接在受体蛋白(IL21Rα)上，再通过Hinge与抗体的Fc连接；靶向第二抗体的VL通过Linker连接在配体蛋白(IL21)上；另一端为靶向第一抗体(示例性地选取的抗PD-L1抗体)的完整Fab结构，组成第一抗体的Fc与组成第二抗体的Fc具有常规的KiH改造，以避免重链错配。同时，对受体、配体蛋白进行突变，目的形成分子间二硫键，进一步提高分子的稳定性，示例性地，构建的FiBody双特异性抗体的序列见表12：
表12.FiBody双特异性抗体中IL21/IL21Rα一侧序列
Figure PCTCN2022120973-appb-000021
Figure PCTCN2022120973-appb-000022
Figure PCTCN2022120973-appb-000023
Figure PCTCN2022120973-appb-000024
复合物1～9另一侧为靶向PD-L1的多肽，其序列如下:
PD-L1的多肽轻链：
Figure PCTCN2022120973-appb-000025
重链：
Figure PCTCN2022120973-appb-000026
(1)蛋白瞬转表达：
将含有目的基因的质粒通过与转染试剂PEI形成阳离子复合物后，导入到宿主细胞Expi293，质粒在细胞内期间，质粒上的外源基因在细胞内发生转录翻译，从而得到目的蛋白。
Expi293在37℃、8％二氧化碳、130rpm条件培养，并在转染前通过细胞计数，将2E6的细胞接种至1L摇瓶中，培养体系约为300mL。配制转染复合物准备转染：首先将750μg目标质粒加入到含有15mlOpti-MEM试剂的50mL离心管中，轻轻混匀，标记为A管；将1.5mg转染试剂PEI加入到含有15mlOpti-MEM试剂的50ml离心管中，轻轻混匀后，室温孵育5min，标记为B管；将B管PEI稀释液逐滴加入到A管DNA稀释液中，轻轻混匀后，室温孵育15min，孵育结束后，将PEI-目标质粒复合物加入到Expi293细胞，置于37℃摇床中继续培养。直到D7-D10后收样。
(2)二硫键改造样品的纯化：
瞬转细胞表达液经过9000rpm/20min离心，收集上清，再经过0.22μm滤膜除菌过滤。纯化采用ProA亲和层析。过程如下，使用AKTA avant 150层析设备，用至少5CV平衡缓冲液(10mM PBS)平衡层析柱(如MabSelectSuRe LX，GE)，加载样品至层析柱，使目标蛋白吸附在层析柱上而其他杂质穿透分离。完成上样后使用至少5CV平衡缓冲液(10mM PBS)再次冲洗层析柱，随后使用洗脱缓冲液(20mM NaAc，pH＝3.4)洗脱目标蛋白，收集管中预先加入中和缓冲液(1M Tris，pH8.0)，中和缓冲液的加入体积根据洗脱样品的预估含量而定，一般加入10％洗脱体积量。
(3)二硫键改造IL21/IL21RαFiBody凝胶电泳检测:
对二硫键改造IL21/IL21Rα复合物进行SDS-PAGE电泳检测，检测结果如图12所示，未进行二硫键改造复合物(复合物10,同R1123)在分子量25KD～35KD之间有条带，说明存在游离轻链；复合物1～复合物9在分子量25KD～35KD之间无条带，说明二硫键改造成功。
(4)靶向部分亲和力检测:
TIGIT端结合活性分析
通过FCM实验方法检测双抗分子(TIGIT端)与CHO-TIGIT细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL,3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL,2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1:500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测，检测结果见图13与未进行二硫键改造的分子相比亲和力相当，说明二硫键改造未减弱了靶向区的亲和力。
PD-L1端结合活性分析
通过FCM实验方法检测双抗分子(PD-L1端)与CHO-PD-L1细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL,3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL,2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1:500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测。检测结果见图14与未进行二硫键改造的分子相比亲和力相当，说明二硫键改造不会影响靶向区的亲和力。
(5)TIGIT端阻断活性分析
通过FCM实验方法检测双抗分子(TIGIT端)阻断配体与CHO-TIGIT细胞结合活性。配置3％BSA缓冲液：称取4.5gBSA到150mL 1XPBS中，混匀后放置冰上备用；抗体稀释：将受试抗体、阳性对照用3％BSA稀释成初始浓度为800nM，亚型对照稀释成初始浓度为20μg/mL，体积300μL,3倍梯度稀释(100+200)共10个点；结合活性检测：细胞计数并铺板：将R0254-3细胞计数后，按100μL,2E+05/孔分到96孔V型板中；先将不同浓度抗体50μL加入到细胞中，2-8度孵育0.5h,再加入50μL配体，2-8度孵育0.5h；350xg离心5min后，去掉上清，按200μL/孔3％BSA；350xg离心5min后，去掉上清，3％BSA配制荧光抗体PE Goat anti-human IgG Fc和PE Goat anti-mouse IgG Fc(1:500x稀释)，按100μL/孔加入对应的96孔板中，2-8度孵育30min；350g离心5min，去上清，3％BSA洗一遍细胞；350xg离心5min后，去掉上清，按100μL/孔加入1XPBS重悬细胞；按照CytoFLEX流式细胞仪标准操作规程上机检测，检测结果见图15与未进行二硫键改造的分子相比亲和力相当，说明二硫键改造未减弱靶向区的亲和力。以上所述实施例的各技术特征可以进行任意的组合，为使描述 简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (15)

  1. 一种双特异性融合多肽，其包含第一抗原结合部分，所述第一抗原结合部分包含：
    第一多肽，所述第一多肽自N末端至C末端包含第一抗体的第一重链可变结构域VH1，其可操作性地连接至第一缀合片段，和
    第二多肽，所述第二多肽自N末端至C末端包含第一抗体的第一轻链可变结构域VL1，其可操作地连接至第二缀合片段，
    所述第一缀合片段和所述第二缀合物片段能够特异性结合；
    其中，所述第一缀合物片段为受体，所述第二缀合片段为配体；或者所述第一缀合物片段为配体，所述第二缀合片段为受体；
    可选地，所述配体和受体为IL21/IL21R；可选的，所述受体和配体之间包含至少一个非天然的链间键。
  2. 根据权利要求1所述的双特异性融合多肽，还包括第二抗原结合部分，所述第二抗原结合部分不同于所述第一抗原结合部分，所述第二抗原结合部分包括：
    第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至第三缀合片段，和
    第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至第四缀合片段；
    其中，
    a)所述第三缀合片段和所述第四缀合物片段能够特异性结合；
    b)所述第三缀合物片段为受体，所述第四缀合片段为配体；或者所述第三缀合物片段为配体，所述第四缀合片段为受体；和
    c)所述第三缀合片段和/或所述第四缀合物片段与所述第一缀合物片段和/或所述第二缀合物片段选自不同的受体和配体。
  3. 根据权利要求1所述的双特异性融合多肽，还包括第二抗原结合部分，所述第二抗原结合部分不同于所述第一抗原结合部分，所述第二抗原结合部分包括：
    第三多肽，所述第三多肽自N末端至C末端包含第二抗体的第二重链可变结构域VH2，其可操作性地连接至抗体重链恒定区CH1，和
    第四多肽，所述第四多肽自N末端至C末端包含第二抗体的第二轻链可变结构域VL2，其可操作地连接至抗体轻链恒定区CL。
  4. 根据权利要求1至3任一项所述的双特异性融合多肽，所述受体和配体之间包含至少一个非天然的链间键，所述非天然链间键能够增强受体和配体间的特异性结合力；可选地，所述非天然链间键形成于受体包含的第一突变残基和配体包含的第二突变残基之间；可选地，所述第一突变残基和所述第二突变残基中的至少一个为半胱氨酸残基；可选地，所述非天然链间键为二硫键。
  5. 根据权利要求1至4任一项所述的双特异性融合多肽，所述第一重链可变结构域VH1和第一轻链可变结构域VL1之间包含至少一个非天然的链间键；所述非天然链间键形成于第一重链可变结构域VH1包含的第一突变残基和第一轻链可变结构域VL1包含的第二突变残基之间；可选地，所述第一突变残基和所述第二突变残基中的至少一个为半胱氨酸残基；可 选地，所述非天然链间键为二硫键。
  6. 根据权利要求1至5任一项所述的双特异性融合多肽，其中至少一个天然糖基化位点在所述受体和/或配体中不存在。
  7. 根据权利要求1至6任一项所述的双特异性融合多肽，所述IL21选自SEQ ID NO.44至SEQ ID NO.53任一所示序列，所述IL21R选自SEQ ID NO.54至SEQ ID NO.60任一所示序列。
  8. 根据权利要求1至7任一项所述的双特异性融合多肽，其包含抗体Fc恒定区；可选地，所述抗体Fc恒定区是异源二聚体；可选地，所述抗体Fc恒定区为基于KiH、疏水相互作用、静电相互作用、亲水相互作用和/或增加的柔性而缔合成为异源二聚体。
  9. 根据权利要求8所述的双特异性融合多肽，所述抗体Fc恒定区包含CH2和CH3或所述抗体Fc恒定区包含CH2、CH3和CH4，且所述Fc恒定区上的两个CH2、两个CH3和/或两个CH4被所述受体及其配体替换。
  10. 根据权利要求1至9任一项所述的双特异性融合多肽，所述第一抗原结合部分与所述第二抗原结合部分结合不同的抗原或者结合同一抗原的不同表位；
    可选地，所述第一抗原结合部分靶向免疫细胞，所述第二抗原结合部分靶向肿瘤细胞；
    可选地，所述第一抗原结合部分和所述第二抗原结合部分均靶向肿瘤细胞；
    可选地，所述第一抗原结合部分与所述第二抗原结合部分均靶向免疫细胞；
    可选地，所述第一抗原结合部分靶向人PD-L1，第二抗原结合部分靶向人TIGIT；或者所述第一抗原结合部分靶向人TIGIT，第二抗原结合部分靶向人PD-L1。
  11. 分离的核酸，其编码权利要求1至10任一项所述的双特异性融合多肽。
  12. 含有权利要求11所述核酸的载体。
  13. 含有权利要求11所述核酸或者权利要求12所述载体的宿主细胞。
  14. 药物组合物，其包含权利要求1至10任一项所述的双特异性融合多肽，和药学上可接受的载体，赋形剂，或稳定剂。
  15. 根据权利要求1至10任一项所述的双特异性融合多肽在制备用于治疗疾病的药物中的应用。
PCT/CN2022/120973 2021-09-24 2022-09-23 一种双特异性多肽复合物 WO2023046093A1 (zh)

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