WO2022016972A1 - 融合蛋白及其用途 - Google Patents

融合蛋白及其用途 Download PDF

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WO2022016972A1
WO2022016972A1 PCT/CN2021/094655 CN2021094655W WO2022016972A1 WO 2022016972 A1 WO2022016972 A1 WO 2022016972A1 CN 2021094655 W CN2021094655 W CN 2021094655W WO 2022016972 A1 WO2022016972 A1 WO 2022016972A1
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cells
sequence
fusion protein
segment
lpetgg
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刘玉
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斯普迈(北京)生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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]
    • C07K14/55—IL-2
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4633—Antibodies or T cell engagers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4635—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to the field of proteins, in particular to fusion proteins suitable for linking the Fc region of an antibody to a cell.
  • Cell therapy is a new disease treatment technology that has emerged in recent years. It refers to the use of the characteristics of certain cells with specific functions (such as stem cells and immune cells), and the cells with enhanced functions produced after specific treatment, and then enter the body to achieve the treatment of diseases. the goal of. With the continuous development of basic theories, technical means and clinical medical exploration research such as stem cell therapy, immune cell therapy and gene editing, cell therapy products have provided new treatment ideas and methods for some serious and refractory diseases, showing that more and more higher application value.
  • Targeted therapy is a drug therapy that treats diseases by interfering with specific molecules.
  • Targeted therapy is a combination of targeted therapy and cell therapy.
  • Targeted cell therapy such as CAR-T cells (chimeric antigen receptor T cells), TCR-T cells (T cell receptor T cells), CAR-NK cells (chimeric antigen receptor NK cells), cells are targeted by expressing the antibody scFv segment on the cell surface, and shown in the treatment of malignant diseases such as tumors The excellent clinical effect has been proven to be a very promising disease treatment strategy.
  • CAR-T cells chimeric antigen receptor T cells
  • TCR-T cells T cell receptor T cells
  • CAR-NK cells chimeric antigen receptor NK cells
  • Antibodies also known as immunoglobulins (Ig for short), can be divided into five categories: IgG, IgM, IgA, IgE, and IgD according to their physicochemical properties and biological functions.
  • the Fab segment of an antibody fragment of antigen binding, Fab
  • the Fc segment is a crystallizable segment (fragment crystallizable, Fc), according to Depending on the type of antibody, this region consists of 2 or 3 constant domains of the heavy chain, eg, the Fc domain of IgG contains the heavy chain CH2 and CH3 domains.
  • the Fc segment can bind to various Fc receptors (Fc receptors, FcRs) and other immune molecules, and this process can trigger different target cell killing effects, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) [Woof J, Burton D. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat Rev Immunol. 2004, 4(2): 89–99. Heyman B.Complement and Fc-receptors in regulation of the antibody response.Immunol Lett.1996,54(2–3):195–199.], is a key effector segment for therapeutic antibodies to function in vivo [Seidel UJ, Schlegel P, Lang P.
  • Fc fusion protein refers to the use of genetic engineering technology to fuse the Fc segment of an antibody with a protein molecule with a specific biological function, which not only has the original activity of the functional protein, but also has certain characteristics of the antibody, such as ADCC, CDC and ADCP functions.
  • a protein containing an Fc segment (such as an antibody or an Fc fusion protein with an antigen-binding region) is directly linked to the surface of the effector cell, it can not only make the cell have the targeting ability to recognize a specific antigen, but also solve the problem of the preparation of existing therapeutic cells. It is a complex problem, and after the protein is combined with the corresponding antigen, its Fc segment can further effectively stimulate the killing activity of effector cells.
  • transpeptidase A performs a ligation reaction with a substrate containing an oligomeric Gly sequence at the N-terminus by recognizing and cleaving the peptide bond between T/G in the LPXTG substrate sequence [Kruger RG, Otvos B, Frankel BA, et al .Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA. Biochemistry, 2004, 43(6): 1541–1551; Suree N, Liew CK, Villareal VA, et al.
  • the purpose of the present invention is to address the problems and deficiencies in the prior art, to provide a protein molecule containing an Fc segment and a method for directly linking the protein molecule containing an Fc segment to the cell surface.
  • the effector cells obtained by this method can bind to the corresponding soluble antigen and cell surface antigen through the protein containing the Fc segment attached to its surface, and then the Fc segment of the protein can bind to the Fc receptor on the surface of the effector cell and trigger the effector cell.
  • Activation of related signaling pathways ultimately achieves specific antigen clearance and target cell killing, which can be used to prevent and treat diseases caused by cell proliferation and/or dysfunction in the body, such as tumors, autoimmune diseases, and infectious diseases.
  • this paper first provides fusion proteins containing Fc segments (such as antibodies and Fc fusion proteins), which can be directly linked to the cell surface mediated by transpeptidase A. Also provided herein are methods of directly linking Fc segment-containing fusion proteins (eg, antibodies and Fc fusion proteins) to the cell surface via the transpeptidase A protein.
  • Fc segment-containing fusion proteins eg, antibodies and Fc fusion proteins
  • the invention provides an Fc segment-containing fusion protein comprising a first portion, an Fc segment, a linker portion, and a substrate portion of transpeptidase A from N to C-terminus.
  • the moieties may be directly linked or linked through one or more amino acid residues.
  • the linking moiety comprises a moiety or region selected from a linker or a protein or polypeptide.
  • the linking moiety is a linker alone, a protein or polypeptide alone, or a portion comprising a protein or polypeptide and a linker from the N-terminus to the C-terminus.
  • the protein or polypeptide is selected from scFvs.
  • the linker comprises a sequence selected from the group consisting of:
  • the substrate moiety comprises the sequence shown as LPXTG.
  • the first moiety may be selected from the group consisting of F(ab') 2 , F(ab'), Fab, Fv, scFv, receptors and ligands.
  • the first moiety can also be any other binding partner of the relevant pathogen.
  • the Fc segment is a wild-type Fc segment or a variant Fc segment.
  • the Fc segment is selected from the Fc segments of IgG, IgM, IgA, IgD and IgE.
  • the Fc segment is selected from the Fc segments of IgGl, IgG2, IgG3 and IgG4.
  • the fusion protein comprises the structure of any of the following:
  • the present invention provides a nucleic acid encoding a fusion protein according to the present invention.
  • the nucleic acid comprises SEQ ID No. 4 and 12, SEQ ID No. 4 and 14, SEQ ID No. 4 and 16, SEQ ID No. 4 and 18, SEQ ID No. 4 and 20, SEQ ID No. 4 and 18 The coding sequence of No. 28, SEQ ID No. 30, or SEQ ID No. 32.
  • the present invention provides a vector comprising a nucleic acid according to the present invention.
  • the present invention provides a host cell comprising a vector according to the present invention.
  • the present invention provides a method for generating a fusion protein according to the present invention, comprising:
  • the invention provides a method for attaching an Fc segment-containing fusion protein according to the invention to a cell surface, comprising contacting the cell with an Fc segment-containing fusion protein and transpeptidase A A step of.
  • the cells are effector cells.
  • the cells are NK cells or T cells.
  • the cells are peripheral blood NK cells, peripheral blood T cells, and cord blood NK cells.
  • the cells are NK92-FcyRIII cells.
  • the present invention provides cells or progeny thereof prepared according to the methods of the present invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a cell according to the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides the use of a cell according to the present invention or a pharmaceutical composition according to the present invention in the manufacture of a medicament for a disease.
  • the disease is a disease caused by abnormal cell proliferation and/or function.
  • the disease is tumor, autoimmune disease and/or infectious disease.
  • the connecting portion comprising scFv or IL2, IL2 wherein the amino acid sequence may be GGGGSGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTRT (sequence 33); the amino acid sequence of scFv may DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSA SFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHW
  • the present invention successfully linked the fusion protein containing the Fc segment directly to the cell surface.
  • the resulting effector cells are precisely targeted.
  • an effector cell expresses an Fc receptor
  • the effector cell to which the Fc segment-containing protein is linked can be activated by the corresponding target cell.
  • NK cells NK92-FcyRIII
  • Fc ⁇ RIII CD16A
  • the precise targeting effector cells prepared by directly linking the Fc segment-containing protein molecule to the effector cell surface can be simpler than the existing method of preparing specific targeting cells by cell transfection, At the same time, it can also reduce the possible risk of genome manipulation of effector cells; on the other hand, after binding to the antigen, the Fc segment of the fusion protein can activate the killing function of effector cells by interacting with Fc receptors. Therefore, the effector cells prepared based on the present invention have the advantages of simple preparation method, good safety, enhanced activity, etc., and can be used to prevent and/or treat diseases caused by abnormal cell proliferation and/or function in the body, such as tumors, autoimmunity, etc. It has important application value for diseases and infectious diseases.
  • Figure 1 Schematic representation of the structure of fusion proteins containing Fc segments (including recombinant antibodies and Fc fusion proteins, ID: RP1-RP14).
  • Figure 2 Polyacrylamide gel electrophoresis of recombinant antibody and Fc fusion protein solutions.
  • Figure 3 Graph of assay of recombinant antibody and Fc fusion protein binding to antigen activity.
  • Figure 4 Flow cytometry plot showing the use of transpeptidase A to link recombinant antibody or Fc fusion protein to NK92-FcyRIII cells.
  • Figure 5 Flow cytometry chart showing the specific binding of cells with recombinant antibodies and Fc fusion proteins to the corresponding target antigens.
  • Figure 6 Flow cytometry chart showing that NK92-Fc ⁇ RIII cells successfully linked with recombinant antibody and Fc fusion protein can be further activated by target cells expressing specific antigens.
  • Figure 7 Graph of cell killing effect showing that NK92-FcyRIII cells to which recombinant antibodies and Fc fusion proteins have been linked can kill target cells expressing specific antigens.
  • Figure 8 Flow cytometry plot showing the use of transpeptidase A to attach recombinant protein or Fc fusion protein to the surface of peripheral blood NK cells, peripheral blood T cells and cord blood NK cells.
  • the present invention is based on the following discovery of the inventor: adopt the protein modification method (US20160122707A1; Jeong HJ, et al., Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling.
  • PLoS introduced in existing patents and documents One.2017Dec 4;12(12):e0189068;Chen I,et al.,A general strategy for the evolution of bond-forming enzymes using yeast display.Proc Natl Acad Sci U S A.2011 Jul 12;108(28) :11399-404) Directly fuse the LPXTG segment specifically recognized by transpeptidase A at the C-terminus of the complete antibody or Fc fusion protein, that is, without adding any linking sequence between the complete antibody or Fc fusion protein and the LPXTG sequence, which cannot be achieved. Intact antibodies or Fc fusion proteins are directly attached to the cell surface by transpeptidase A. In contrast, the use of the specifically constructed fusion proteins of the present invention allows the direct attachment of intact antibodies or Fc fusion proteins to the cell surface via transpeptidase A.
  • transpeptidase A is a membrane-bound enzyme that covalently attaches a protein containing an enzyme substrate recognition sequence to the bacterial cell membrane.
  • the specific substrate recognition motif of transpeptidase A is LPXTG, the enzyme cleaves between residues threonine (T) and glycine (G) of this substrate sequence, and further cleaves with the N-terminal containing oligoglycine sequence.
  • the substrate undergoes a ligation reaction.
  • the kind and source of the transpeptidase A may not be limited as long as the transpeptidase A retains its above-mentioned functional properties.
  • transpeptidase A may be native transpeptidase A, or may be a variant of transpeptidase A (see, eg, CN201610726374.0).
  • first part refers to the binding region in the fusion protein.
  • the binding region may be an antigen binding region of an antibody, a receptor for a ligand, or a ligand for a receptor, so long as it is capable of binding a target, eg, a target on a target cell.
  • the first part and the Fc region may be homologous or heterologous.
  • the first portion and the Fc region are naturally occurring antibodies.
  • the first portion can be an antibody fragment.
  • the first part can bind cancer antigens, infectious disease antigens, autoimmune disease antigens.
  • the first moiety can bind the HER2 protein.
  • Antibody which encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.
  • Antibodies also known as immunoglobulins (abbreviated as Ig), can be divided into five categories according to their physicochemical properties and biological functions: IgG, IgM, IgA, IgE, and IgD.
  • the Fab segment of an antibody is an antigen-binding fragment, consisting of a complete light chain and heavy chain VH and CH1 domains; the Fc segment is a crystallizable segment (fragment crystallizable, Fc), according to Depending on the type of antibody, this region consists of 2 or 3 constant domains of the heavy chain, eg, the Fc domain of IgG contains the heavy chain CH2 and CH3 domains.
  • Fc segment is used to define the C-terminal region of an immunoglobulin heavy chain, which contains at least part of the constant region.
  • the term includes native sequence Fc segments and variant Fc segments.
  • a "variant Fc segment” comprises an amino acid sequence that differs from that of a "native” or “wild-type” sequence Fc segment due to at least one "amino acid modification".
  • the variant Fc region may have at least one amino acid substitution, eg, from about one to about ten substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide, compared to the native sequence Fc region or to the Fc region of the parent polypeptide Amino acid substitutions, and preferably from about one to about five amino acid substitutions.
  • the variant Fc segments herein will preferably have at least about 80% homology, and most preferably at least about 90% homology, with native sequence Fc regions and/or with the Fc segment of the parent polypeptide, and more preferably with have at least about 95% homology.
  • the Fc region can bind various Fc receptors (Fc receptors, FcR) and other immune molecules, and this process can trigger different target cell killing effects, including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent Cell phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) are key effector segments for therapeutic antibodies to function in vivo.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent Cell phagocytosis
  • CDC complement-dependent cytotoxicity
  • the effector function of an antibody is the function contributed by the Fc effector domain of an IgG (eg, the Fc region of an immunoglobulin). This function can be accomplished, for example, by binding of Fc effector domains to Fc receptors on immune cells with phagocytic or lytic activity, or by binding of Fc effector domains to components of the complement system.
  • Typical effector functions are ADCC, ADCP and CDC.
  • Effective functions refer to those biological activities attributable to the Fc region of an antibody, which vary by antibody isotype.
  • antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis (ADCP); cell surface receptors such as Downregulation of B cell receptors); and B cell activation.
  • linking moiety refers to the moiety that links between the Fc region and the transpeptidase A substrate LPXTG. This part may contain only linkers. In this case, the connecting portion may be the joint itself. Alternatively, the linking moiety may comprise only proteins or polypeptides, such as IL2 or scFv. The linking moiety may also comprise both proteins or polypeptides and linkers. For example, the linking moiety comprises an IL2 or scFv and a linker from the N-terminus to the C-terminus. The type of linker is not particularly limited as long as the Fc-containing fusion protein can be linked to the cell surface.
  • the linker can be a flexible linking sequence, such as G(n)S(n), where n ⁇ 1, preferably, the linking sequence is GGGGSGGGGSGGGGS.
  • the linker can also be a rigid linking sequence, such as (EAAAK)n, n ⁇ 1, preferably, the linking sequence is EAAAK.
  • the linking moiety comprises a protein or polypeptide and a linker
  • the linker is (GGGGS)n, n ⁇ 1
  • the linker may also be a rigid linker sequence (EAAAK)n, n ⁇ 1.
  • the linker is (GGGGS)n, where n ⁇ 3.
  • the linker is (EAAAK) n , with n ⁇ 1.
  • n ⁇ 3 means, for example, that n is an integer of 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • n ⁇ 1 means, for example, n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • portion is equivalent to "region" in the context used in the first moiety, the linker moiety and the substrate moiety of transpeptidase A.
  • the first portion, the linker portion and the substrate portion may be referred to as the first region, the linker region and the substrate region.
  • Fc receptor or "FcR” is used to describe a receptor that binds to the Fc region of an antibody.
  • Preferred FcRs are native sequence human FcRs.
  • an FcR can be an FcR (gamma receptor) that binds an IgG antibody, and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcyRII receptors include FcyRIIA (activating receptor) and FcyRIIB ("inhibiting receptor”), which have similar amino acid sequences, differing primarily in their cytoplasmic domains.
  • the activating receptor FcyRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain.
  • the inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine inhibitory motif (ITIM) in its cytoplasmic domain.
  • FcR encompasses other FcRs, including those to be identified in the future.
  • the term also includes the neonatal receptor FcRn responsible for the transfer of maternal IgG to the fetus.
  • the effector cells may express FcyRIII.
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to cell-mediated responses in which FcR-expressing non-specific cytotoxic cells (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize The antibody bound to the target cell subsequently causes lysis of the target cell.
  • cytotoxic cells eg, natural killer (NK) cells, neutrophils, and macrophages
  • NK cells The primary cells used to mediate ADCC (NK cells) express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9 (1991) 457-492.
  • antibody-dependent phagocytosis and “ADCP” refer to the process in which antibody-coated cells are destroyed by phagocytic immune cells (eg, macrophages, neutrophils, and dendrites) that bind to the Fc region of an immunoglobulin. cells) are internalized in whole or in part.
  • phagocytic immune cells eg, macrophages, neutrophils, and dendrites
  • Fc fusion protein or “fusion protein” refers to the use of genetic engineering technology to fuse the Fc region of an antibody with a protein molecule with a specific biological function, which not only has the original activity of the functional protein, but also It has certain properties of antibodies, such as functions such as ADCC, CDC and ADCP. It can be seen from this that if an antigen-binding molecule with an Fc segment (such as an antibody and an antigen-binding region-Fc fusion protein) is directly linked to the surface of the effector cell, the cell can not only have the targeting ability to recognize a specific antigen, but also the Fc region The triggered physiological effects can further enhance the killing activity of effector cells.
  • the Fc fusion protein can have the following structure from the N-terminus to the C-terminus:
  • the full length antibodies in these Fc fusion proteins can be IgGl, IgG2, IgG3 or IgG4 antibodies.
  • antibody fragment refers to a molecule that is not an intact antibody, comprising a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (eg, scFv); and formed from antibody fragments of multispecific antibodies.
  • antibody fragments that bind to a specific antigen may be used as the first part of the invention.
  • sequence of the transpeptidase A gene is shown in sequence 1, and primer F was designed and synthesized according to sequence 1: 5'-CGGCAGC CATATG GCTAAACCTCAAATTCCGA-3' (underlined is the restriction endonuclease NdeI restriction enzyme recognition sequence, sequence 35) and primer R: 5'-GTGGTG CTCGAG TTATTTGACTTCTGTAGCTAC-3' (underlined the restriction endonuclease XhoI recognition sequence, sequence 36).
  • step 3 Take the PCR amplification product recovered in step 2, digest with restriction endonuclease NdeI and restriction endonuclease XhoI, and recover the digested DNA fragment.
  • the vector pET-28(a) was digested with restriction endonuclease NdeI and restriction endonuclease XhoI, and the digested vector backbone was recovered.
  • the structure of the recombinant expression plasmid pET- Srt A is described as follows: The small fragment between the restriction endonucleases NdeI and XhoI recognition sequences of the vector pET-28(a) is replaced with the DNA molecule shown in SEQ ID NO: 1 in the sequence listing. According to the sequencing results, the sequence of SEQ ID No. 1 of transpeptidase A was correctly inserted into the vector pET-28(a).
  • the recombinant expression plasmid pET- Srt A expresses the protein shown in sequence 2 in the sequence listing (hereinafter referred to as transpeptidase A protein).
  • the recombinant expression plasmid pET- Srt A was introduced into Escherichia coli BL21(DE3) to obtain a recombinant strain named BL21(DE3)-pET- Srt A.
  • a single colony of BL21(DE3)-pET- Srt A was inoculated into LB medium containing 100 mg/mL kanamycin, and cultured with shaking at 37° C. and 200 rpm to obtain a culture liquid 1 with an OD600 of 0.6.
  • IPTG was added to the culture solution 1 to obtain a culture solution 2 (in the culture solution 2, the IPTG concentration was 0.1 mM); then, the culture solution 3 was obtained by shaking culture at 37° C. and 200 rpm for 6 hours.
  • step 4 Take the bacteria collected in step 3 and resuspend with 10 mL of pH7.4, TBS buffer (10 mmol/L, Tris, 0.9% NaCl) to obtain a bacterial suspension; then ultrasonically treat the bacterial suspension.
  • Ultrasound parameters ultrasound frequency 30%; ultrasound for 10s, stop for 5s, and total ultrasound time for 30min.
  • step 5 Take the system completed in step 4, centrifuge at 12000rpm for 10min, and collect the supernatant.
  • step 6 Mix the supernatant collected in step 5 with Ni-NTA Resin and incubate for 10 min, then discard the supernatant and wash 3 times with pH 7.4 and TBS buffer containing 20 mM imidazole.
  • step 6 After completing step 6, elute with pH 7.4 and TBS buffer containing 500 mM imidazole, and collect the post-column solution, which is the transpeptidase A protein.
  • fusion protein containing Fc segment (including recombinant antibody and Fc fusion protein, code: RP1-RP14) is shown in Figure 1.
  • the construction of the expression plasmid of the fusion protein is as described in Example 1. That is, as in the cloning technique described in Example 1, the synthesized nucleic acid was cloned with primers containing HindIII and XhoI restriction sites, and the synthesized sequence was ligated into the vector pCDNA3.1(+) digested with the corresponding enzymes.
  • the light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 3.
  • nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-LPETGG (sequence 4).
  • sequence 5 The DNA molecule shown in sequence 5 was used to replace the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) to obtain a heavy chain expression vector.
  • nucleotides 61-1428 encode the full-length heavy chain of the recombinant antibody Ab-CH-LPETGG (sequence 6).
  • the light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 3.
  • nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-GGGGS-LPETGG (sequence 4).
  • the heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 7.
  • nucleotides 61-1443 encode the full-length heavy chain of the recombinant antibody Ab-CH-GGGGS-LPETGG (sequence 8).
  • the light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 3.
  • nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-GGGGS(x2)-LPETGG (sequence 4).
  • nucleotides 61-1473 encode the full-length heavy chain of the recombinant antibody Ab-CH-GGGGS(x2)-LPETGG (sequence 10).
  • the light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 3.
  • nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-GGGGS(x3)-LPETGG (sequence 4).
  • the heavy chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 11.
  • nucleotides 61-1458 encode the full-length heavy chain of the recombinant antibody Ab-CH-GGGGS(x3)-LPETGG (sequence 12).
  • the light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 3.
  • nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-EAAAK-LPETGG (sequence 4).
  • nucleotides 61-1443 encode the full-length heavy chain of the recombinant antibody Ab-CH-EAAAK-LPETGG (sequence 14).
  • the light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 3.
  • nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab-CH-IL2-LPETGG (sequence 4).
  • nucleotides 61-1854 encode the full-length heavy chain of the recombinant antibody Ab-CH-IL2-LPETGG (sequence 16).
  • the light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 3.
  • nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab1-CH-scFv.Ab2-GGGGS-LPETGG (sequence 4).
  • nucleotides 61-2178 encode the full-length heavy chain of the recombinant antibody Abl-CH-scFv.Ab2-GGGGS-LPETGG (sequence 18).
  • the light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 3.
  • nucleotides 61-702 encode the full-length light chain of the recombinant antibody Abl-CH-scFv.Ab2-EAAAK-LPETGG (sequence 4).
  • nucleotides 61-2178 encode the full-length heavy chain of the recombinant antibody Abl-CH-scFv.Ab2-EAAAK-LPETGG (sequence 20).
  • the Fc fusion protein expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 21.
  • nucleotides 61-1506 encode the scFv-Fc-LPETGG full-length protein (sequence 22).
  • nucleotides 61-1521 encode the full-length protein of scFv-Fc-GGGGS-LPETGG (sequence 24).
  • nucleotides 61-1536 encode the full-length protein of scFv-Fc-(GGGGS)x2-LPETGG (sequence 26).
  • nucleotides 61-1550 encode the full-length protein of scFv-Fc-(GGGGS)x3-LPETGG (sequence 28).
  • nucleotides 61-1521 encode the full-length protein of scFv-Fc-EAAAK-LPETGG (sequence 30).
  • the light chain expression vector was obtained by replacing the fragment between the HindIII and XhoI restriction sites in the vector pCDNA3.1(+) with the DNA molecule shown in sequence 3.
  • nucleotides 61-702 encode the full-length light chain of the recombinant antibody Ab(IgG4)-EAAAK-LPETGG (sequence 4).
  • nucleotides 61-1800 encode the full-length heavy chain of the recombinant antibody Ab(IgG4)-EAAAK-LPETGG (sequence 32).
  • step 2.2 Take the supernatant obtained in step 2.1, carry out protein A purification, and collect the purified product.
  • step 2.3 Take the purified product obtained in step 2.2, carry out ultrafiltration, concentrate, and change the solution, and replace the system with PBS buffer to obtain recombinant antibody and Fc fusion protein solution.
  • the protein concentration of the solution was detected by A280nm ultraviolet light absorption method.
  • Figure 2 shows the polyacrylamide gel electrophoresis pattern of the fusion protein (including recombinant antibody and Fc fusion protein) solution containing the Fc segment.
  • non-reducing electrophoresis means that no reducing agent 2-mercaptoethanol is added
  • reducing electrophoresis means adding 1% reducing agent 2-mercaptoethanol.
  • the apparent molecular weights of the recombinant antibodies and the Fc fusion protein matched their predicted molecular weights, and the fusion protein containing the Fc segment was correctly expressed.
  • the coating solution is composed of a coating source and a coating buffer, and the concentration of the coating source in the coating solution is 1 ⁇ g/mL.
  • the coating was originally HER2 protein (purchased from Yiqiao Shenzhou Company, item number: 10004-H08H4).
  • Coating buffer pH 9.6: Na 2 CO 3 1.59 g, NaHCO 3 2.94 g, and the balance is water.
  • step 2 After completing step 1, take the ELISA plate and wash it three times with PBST buffer.
  • step 3 After completing step 2, take the ELISA plate, add PBST buffer containing 5 g/100 mL of skim milk powder, and block at 37° C. for 1 h.
  • fusion protein solution containing the Fc segment prepared above, prepare a stock solution with an antibody concentration of 10 ⁇ g/mL in PBST buffer containing 5g/100mL nonfat dry milk, and then use PBST buffer containing 5g/100mL nonfat dry milk to carry out Three-fold serial dilutions were obtained to obtain fusion protein solutions containing the Fc segment at different concentrations.
  • step 5 Take the ELISA plate that has been completed in step 3, add the fusion protein solutions (100 ⁇ L per well) with different concentrations of the Fc segment obtained in step 4, and incubate at 37° C. for 1 h. Three replicate wells were set up for each concentration.
  • step 5 After completing step 5, take the ELISA plate and wash it three times with PBST buffer (250 ⁇ L per well each time).
  • step 6 take the ELISA plate, add HRP-labeled goat anti-human IgG secondary antibody dilution (dilute the HRP-labeled goat anti-human IgG secondary antibody at 1:40000 in 5g/100mL nonfat milk powder) PBST buffer) and incubated at 37°C for 30 min.
  • step 7 take the ELISA plate, add TMB chromogenic reagent (100 ⁇ L per well), and develop color at room temperature for 10 min.
  • step 8 After completing step 8, take the ELISA plate, add 2N H 2 SO 4 solution to stop color development (50 ⁇ L per well), and then detect the OD value at a wavelength of 450 nm.
  • Example 3 Using transpeptidase A to link the fusion protein containing the Fc segment to NK92-Fc ⁇ RIII cells and detection of cell activity
  • transpeptidase A to connect the fusion protein containing the Fc segment to NK92-Fc ⁇ RIII cells (purchased Bought from ATCC, Item No.: pta-8837)
  • NK92-Fc ⁇ RIII cell suspension with a concentration of 1.0x10 6 /mL, add transpeptidase A to a final concentration of 20ug/ml, and add fusion protein containing Fc segment to a final concentration of 10ug/ml to obtain incubation system.
  • the incubation system was incubated at 25°C for 90min.
  • the cells were collected by centrifugation, and washed thoroughly with pH7.4, 0.01 mol/L PBS buffer.
  • the cells obtained above were named: recombinant protein-SrtA-NK.
  • NK92-Fc ⁇ RIII cell suspension Take 100 ⁇ L of NK92-Fc ⁇ RIII cell suspension with a concentration of 1.0 ⁇ 10 6 /mL, and add only the fusion protein containing the Fc segment to a final concentration of 10ug/ml to obtain an incubation system.
  • the incubation system was incubated at 25°C for 90min.
  • the cells were collected by centrifugation, and washed thoroughly with pH7.4, 0.01 mol/L PBS buffer.
  • the cells obtained above were named: recombinant protein-NK.
  • step 3 centrifuge, collect cells, and wash thoroughly with pH7.4, 0.01mol/L PBS buffer.
  • step 4 the untreated cells were used as blank cells, and flow cytometry was used to detect the level of cell-labeled fusion protein containing Fc segment with or without transpeptidase A.
  • transpeptidase A could not link Fc segment-containing fusion proteins (such as recombinant antibody RP1 or Fc fusion protein RP9) that only added LPXTG sequence at the C-terminus of the Fc segment to the cell surface;
  • Recombinant antibodies RP4, RP5, RP6, RP7, RP8, RP14
  • Fc fusion proteins RP12, RP13
  • the cells linked with the fusion protein containing the Fc segment can specifically bind to the corresponding target antigen
  • NK92-Fc ⁇ RIII cell suspension Take 200 ⁇ L of NK92-Fc ⁇ RIII cell suspension with a concentration of 1.0x10 6 /mL, add transpeptidase A to a final concentration of 20ug/ml, and add fusion protein containing Fc segment to a final concentration of 10ug/ml to obtain incubation system.
  • the incubation system was incubated at 25°C for 90min.
  • the cells were collected by centrifugation, and washed thoroughly with pH7.4, 0.01 mol/L PBS buffer.
  • the cells obtained above were named: recombinant protein-SrtA-NK.
  • NK92-Fc ⁇ RIII cell suspension Take 200 ⁇ L of NK92-Fc ⁇ RIII cell suspension with a concentration of 1.0 ⁇ 10 6 /mL, and add only the fusion protein containing the Fc segment to a final concentration of 10ug/ml to obtain an incubation system.
  • the incubation system was incubated at 25°C for 90min.
  • the cells were collected by centrifugation, and washed thoroughly with pH7.4, 0.01 mol/L PBS buffer.
  • the cells obtained above were named: recombinant protein-NK.
  • step 3 centrifuge, collect cells, and wash thoroughly with pH7.4, 0.01mol/L PBS buffer.
  • step 4 After completing step 4, add FITC-labeled streptavidin (FITC Streptavidin, purchased from Biolegend, Item No.: 405201) according to the instructions to obtain an incubation system. The incubation system was incubated at 25°C for 30 min.
  • FITC Streptavidin purchased from Biolegend, Item No.: 405201
  • step 6 centrifuge to collect cells and wash thoroughly with pH7.4, 0.01mol/L PBS buffer.
  • step 6 the untreated cells are used as blank cells, and the level of cell-binding target antigen HER2 is detected by flow cytometry.
  • the NK92-Fc ⁇ RIII cells successfully connected to the fusion protein containing the Fc segment can be further expressed Target Cell Activation of Alloantigens
  • NK92-Fc ⁇ RIII cell suspension with a concentration of 1.0x10 6 /mL, add transpeptidase A to a final concentration of 20ug/ml, and add fusion protein containing Fc segment to a final concentration of 10ug/ml to obtain incubation system.
  • the incubation system was incubated at 25°C for 90min.
  • the cells were collected by centrifugation, and washed thoroughly with pH7.4, 0.01 mol/L PBS buffer.
  • the cells obtained above were named: recombinant protein-SrtA-NK.
  • NK92-Fc ⁇ RIII cell suspension Take 200 ⁇ L of NK92-Fc ⁇ RIII cell suspension with a concentration of 1.0 ⁇ 10 6 /mL, and add only the fusion protein containing the Fc segment to a final concentration of 10ug/ml to obtain an incubation system.
  • the incubation system was incubated at 25°C for 90min.
  • the cells were collected by centrifugation, and washed thoroughly with pH7.4, 0.01 mol/L PBS buffer.
  • the cells obtained above were named: recombinant protein-NK.
  • SKOV3 cell suspension (the cell is a cell line with high antigen HER2 protein expression) at a concentration of 4.0 ⁇ 10 5 /mL, and then add 200 ⁇ L of step 1 or 2 at a concentration of 2 ⁇ 10 6 /mL After the cell suspension is completed, an incubation system is obtained. The incubation system was incubated at 37°C for 60 min.
  • step 4 Take the system completed in step 3, centrifuge at 4°C, collect cells, and wash thoroughly with ice-bath pH7.4, 0.01mol/L PBS buffer.
  • step 4 follow the instructions to join Alexa 488-labeled anti-human CD107a antibody (Alexa 488 anti-human CD107a, purchased from Biolegend Company, Item No.: 328610) and APC-labeled anti-human CD56 antibody (APC anti-human CD56(NCAM), purchased from Biolegend Company, Item No.: 318310), to obtain an incubation system.
  • the incubation system was incubated at 4°C for 30 min.
  • step 5 centrifuge at 4°C to collect the cells and wash thoroughly with ice-bath pH7.4, 0.01mol/L PBS buffer.
  • step 6 use the untreated cells as blank cells, and use flow cytometry to detect the level of CD107a in cells.
  • the NK92-Fc ⁇ RIII cells that have been linked to the fusion protein containing the Fc segment can kill the specific antigen epitope target cells
  • NK92-Fc ⁇ RIII cell suspension with a concentration of 1.0x10 6 /mL, add transpeptidase A to a final concentration of 20ug/ml, and add fusion protein containing Fc segment to a final concentration of 10ug/ml to obtain incubation system.
  • the incubation system was incubated at 25°C for 90min.
  • the cells were collected by centrifugation, and washed thoroughly with pH7.4, 0.01 mol/L PBS buffer.
  • the cells obtained above were named: recombinant protein-SrtA-NK.
  • NK92-Fc ⁇ RIII cell suspension Take 200 ⁇ L of NK92-Fc ⁇ RIII cell suspension with a concentration of 1.0 ⁇ 10 6 /mL, and add only the fusion protein containing the Fc segment to a final concentration of 10ug/ml to obtain an incubation system.
  • the incubation system was incubated at 25°C for 90min.
  • the cells were collected by centrifugation, and washed thoroughly with pH7.4, 0.01 mol/L PBS buffer.
  • the cells obtained above were named: recombinant protein-NK.
  • NK92-Fc ⁇ RIII cells (RP5-SrtA-NK, RP7-SrtA-NK, RP8-SrtA-NK, RP13-SrtA-NK) that had been linked with fusion proteins containing Fc segment could kill SKOV3 cells expressing specific antigens. However, there is no killing effect on MCF-7 cells that do not express the antigen.
  • the control group NK92-Fc ⁇ RIII cells (RP5-NK, RP7-NK, RP8-NK, RP13-NK,) not linked to the fusion protein containing the Fc segment had no killing effect on the target cells expressing specific antigens.
  • transpeptidase A to link the fusion protein containing the Fc segment to peripheral blood NK cells/peripheral blood T cells/cord blood NK cells
  • Peripheral blood NK cells were purchased from Junda Kean Technology Co., Ltd.
  • T cell negative selection kit purchased from Stemcell, Item No.: 710410 (operate according to the instructions) to enrich the T cells.
  • the cord blood NK cells were donated by Shandong Qilu Stem Cell Engineering Co., Ltd.
  • peripheral blood NK cells or peripheral blood T cells or umbilical cord blood NK cell suspension with a concentration of 1.0 ⁇ 10 6 /mL, and add only the fusion protein containing the Fc segment to a final concentration of 10ug/ml to obtain an incubation system.
  • the incubation system was incubated at 25°C for 90min.
  • the cells were collected by centrifugation, and washed thoroughly with pH7.4, 0.01 mol/L PBS buffer.
  • the cells obtained above were named: recombinant protein-cells.
  • step 6 Take the cells collected in step 6 and add APC-labeled donkey anti-human IgG antibody (APC AffiniPure F(ab') 2 Fragment Donkey Anti-Human IgG, Fc ⁇ fragment specific, purchased from Jackson ImmunoResearch Company, Item No.: 709- 136-098) to obtain an incubation system. The incubation system was incubated at 25°C for 30 min.
  • APC-labeled donkey anti-human IgG antibody APC AffiniPure F(ab') 2 Fragment Donkey Anti-Human IgG, Fc ⁇ fragment specific, purchased from Jackson ImmunoResearch Company, Item No.: 709- 136-098
  • step 8 use untreated cells as blank cells, and use flow cytometry to detect the level of cell markers.
  • the experimental results are shown in Figure 8.
  • the results show that the fusion protein containing the Fc segment modified by adding a linker sequence between the Fc segment and the LPXTG segment can be successfully directly linked to peripheral blood NK cells and peripheral blood T cells under the mediation of transpeptidase A. and the surface of umbilical cord blood NK cells.
  • constructs obtained by ligating the C-terminus of an Fc segment in an intact antibody or an Fc fusion protein with a LPXTG segment recognized by transpeptidase A through a specific linker sequence are capable of direct ligation by transpeptidase A to the cell surface.
  • the constructs were RP4, RP5, RP6, RP7, RP8, RP12, RP13 and RP14 in the above examples.

Abstract

本发明涉及融合蛋白及其用途。融合蛋白从N至C端包含第一部分、Fc区段、连接部分和转肽酶A的底物部分，所述连接部分包含选自接头和蛋白质或多肽的部分，该蛋白质或多肽选自IL2或scFv；所述接头包含选自以下的序列：(1)(GGGGS)n，其中当所述连接部分包含所述蛋白质或多肽和接头时，n≥1；当所述连接部分仅包含接头时，n≥3；和(2)(EAAAK)n，n≥1；所述底物部分包含以LPXTG所示的序列。使用本发明的融合蛋白可以直接连接至细胞使细胞具有靶向性，比现有的通过细胞转染制备靶向性细胞的方法更为简单，同时也能够降低对效应细胞基因组操作可能产生的风险。

Description

融合蛋白及其用途
本申请要求申请日为2020年7月24日的发明名称为“融合蛋白及其用途”的中国专利申请的优先权，该申请的内容通过引用完整并入本文。
技术领域
本发明涉及蛋白质领域，具体地涉及适合于将抗体Fc区连接到细胞的融合蛋白。
背景技术
细胞治疗是近几年兴起的疾病治疗新技术，是指利用某些具有特定功能的细胞的特性(如干细胞和免疫细胞)，经过特定处理后产生的功能增强的细胞，继而输入体内达到治疗疾病的目的。随着干细胞治疗、免疫细胞治疗和基因编辑等基础理论、技术手段和临床医疗探索研究的不断发展，细胞治疗产品为一些严重及难治性疾病提供了新的治疗思路与方法，显示出越来越高的应用价值。
靶向治疗是通过干扰特定分子来治疗疾病的药物疗法，靶向治疗与细胞治疗结合所产生的靶向性细胞治疗，如CAR-T细胞(嵌合抗原受体T细胞)、TCR-T细胞(T细胞受体T细胞)、CAR-NK细胞(嵌合抗原受体NK细胞)，通过将抗体scFv区段表达在细胞表面使细胞具有靶向性，在肿瘤等恶性疾病的治疗中显示出卓越的临床效果，已被证实是非常有前景的疾病治疗策略。2018年，两款CAR-T药物已获美国FDA批准上市。但是，对于现有的细胞疗法来说，细胞制备周期和成本问题直接导致细胞治疗时效性和广泛性受到极大限制[Cornetta K.,Pollok K.E.,Miller A.D.Transduction of Primary Hematopoietic Cells by Retroviral Vectors.Cold Spring Harbor Protocols 2008,2008,4884.10.1101；Shearer R.F.,Saunders D.N.Experimental Design for Stable Genetic Manipulation in Mammalian Cell Lines:Lentivirus and Alternatives.Genes Cells 2015,20,1–10.10.1111]。
抗体(antibody)，又称免疫球蛋白(immunoglobulin，简称Ig)，按理化性质和生物学功能，可将其分为IgG、IgM、IgA、IgE、IgD五类。抗体的Fab区段(fragment of antigen binding，Fab)为抗原结合片段，由一个完整的 轻链和重链的VH和CH1结构域组成；Fc区段为可结晶段(fragment crystallizable，Fc)，根据抗体类型不同，该区域由重链的2个或者3个恒定结构域组成，如IgG的Fc结构域包含重链CH2和CH3结构域。Fc区段可结合各种Fc受体(Fc receptors,FcRs)和其它免疫分子，这一过程可引发不同的靶细胞杀伤效果，包括抗体依赖的细胞介导的细胞毒性作用(ADCC)、抗体依赖的细胞吞噬作用(ADCP)和补体依赖的细胞毒性作用(CDC)[Woof J,Burton D.Human antibody-Fc receptor interactions illuminated by crystal structures.Nat Rev Immunol.2004,4(2):89–99.Heyman B.Complement and Fc-receptors in regulation of the antibody response.Immunol Lett.1996,54(2–3):195–199.]，是治疗性抗体在体内发挥效用的关键效应区段[Seidel UJ,Schlegel P,Lang P.Natural killer cell mediated antibody-dependent cellular cytotoxicity in tumor immunotherapy with therapeutic antibodies；Hatjiharissi,E,Xu,L,Santos,D et al.Increased natural killer cell expression of CD16,augmented binding and ADCC activity to rituximab among individuals expressing the Fc RIIIa-158 V/V and V/F polymorphism.Blood 2007；110:2561–4；Musolino,A,Naldi,N,Bortesi,B et al.Immunoglobulin G fragment C receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with HER-2/neu–positive metastatic breast cancer.J Clin Oncol 2008；26:1789–96；Lo Nigro C,Macagno M,Sangiolo D,Bertolaccini L,Aglietta M,Merlano MC.NK-mediated antibody-dependent cell-mediated cytotoxicity in solid tumors:biological evidence and clinical perspectives.Ann Transl Med.2019 Mar；7(5):105]。Fc融合蛋白是指利用基因工程技术将抗体的Fc区段与具有特定生物学功能的蛋白分子融合，其不仅具有该功能蛋白的原有活性，而且还具有抗体的某些特性，如ADCC、CDC和ADCP等功能。由此可知，如果将含Fc区段的蛋白(如抗体或具有抗原结合区域的Fc融合蛋白)直接连接至效应细胞表面，不仅可以使细胞具有识别特定抗原的靶向性，解决现存治疗细胞制备复杂的问题，而且该蛋白与相应抗原结合后，其Fc区段可以进一步有效激发效应细胞的杀伤活性。
目前有多种方法将各种分子(如核酸、蛋白)连接至细胞表面，如利用细胞膜表面氨基、巯基或硫醇基团将含有相容基团的分子与细胞相连[Lee DY,Park SJ,Nam JH,Byun Y.Tissue Engineering.2006；12:615–623；Hsiao SC, Shum BJ,Onoe H,Douglas ES,Gartner ZJ,Mathies RA,et al.Langmuir.2009；25:6985–6991；Krishnamachari Y,Pearce ME,Salem AK.Advanced Materials.2008；20:989–993；Murciano JC,Medinilla S,Eslin D,Atochina E,Cines DB,Muzykantov VR.Nature Biotechnology.2003；21:891–896；Stephan MT,Moon JJ,Um SH,Bershteyn A,Irvine DJ.Nature Medicine.2010；16:1035–1041]，将含疏水性糖磷脂酰肌醇(GPI)的目的蛋白锚定至细胞膜[Notohamiprodjo M,Djafarzadeh R,Mojaat A,von Luttichau I,Grone HJ,Nelson PJ.Protein Eng Des Sel.2006；19:27–35；Ko IK,Kean TJ,Dennis JE.Biomaterials.2009；30:3702–3710；Kim SA,Peacock JS.Journal of Immunological Methods.1993；158:57–65]，利用带阳离子的PEG或纳米材料将目标分子吸附至细胞[Wilson JT,Krishnamurthy VR,Cui WX,Qu Z,Chaikof EL.Journal of the American Chemical Society.2009；131:18228–18229；Stephan MT,Moon JJ,Um SH,Bershteyn A,Irvine DJ.Nature Medicine.2010；16:1035–1041；Gao H,Shi W,Freund LB.Proc Natl Acad Sci U S A.2005；102:9469–9474]，利用细胞表面受体-配体连接细胞-目标分子[Swiston AJ,Cheng C,Um SH,Irvine DJ,Cohen RE,Rubner MF.Nano Letters.2008；8:4446–4453]，另外还有点击化学方法[Swee LK,Lourido S,Bell GW,Ingram JR,Ploegh HL.One-step enzymatic modification of the cell surface redirects cellular cytotoxicity and parasite tropism.ACS Chem Biol.2015 Feb 20；10(2):460-5；
Figure PCTCN2021094655-appb-000001
I,Kang J,Girona GE,Aramburu IV,Lemke EA.Labeling proteins on live mammalian cells using click chemistry.Nat Protoc.2015 May；10(5):780-91；Horisawa K.Front Physiol.Specific and quantitative labeling of biomolecules using click chemistry.2014 Nov 24；5:457；Uttamapinant C,Sanchez MI,Liu DS,Yao JZ,Ting AY.Site-Specific Labeling of Proteins in Live Mammalian Cells using Click Chemistry.Nat Protoc.2013 Aug；8(8):1620-34；Li J,Chen M,Liu Z,Zhang L,Felding BH,Moremen KW,Lauvau G,Abadier M,Ley K,Wu P.A Single-Step Chemoenzymatic Reaction for the Construction of Antibody-Cell Conjugates.ACS Cent Sci.2018 Dec 26；4(12):1633-1641]，转肽酶A(Sortase A，简称SrtA)催化方法[Pishesha N,et al.,Engineered Erythrocytes Covalently Linked to Antigenic Peptides Can Protect Against Autoimmune Disease.Proc.Natl.Acad.Sci.U.S.A.2017,114,3157– 3162；Chen I,Dorr BM,Liu DR.A general strategy for the evolution of bond-forming enzymes using yeast display.Proc Natl Acad Sci U S A.2011 Jul 12；108(28):11399-404；Tanaka T,Yamamoto T,Tsukiji S,Nagamune T.Site-specific protein modification on living cells catalyzed by Sortase.Chembiochem.2008 Mar 25；9(5):802-7；US 2016.0122707A1]等等。
其中转肽酶A通过识别并切割LPXTG底物序列中的T/G之间的肽键，与N端含有寡聚Gly序列的底物进行连接反应[Kruger RG,Otvos B,Frankel BA,et al.Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA.Biochemistry,2004,43(6):1541–1551；Suree N,Liew CK,Villareal VA,et al.The structure of the Staphylococcus aureus sortase-substrate complex reveals how the universally conserved LPXTG sorting signal is recognized.J Biol Chem,2009,284(36):24465–24477]。尽管已发现转肽酶A可以将多种分子(如生物素、多肽、抗体重链可变区VHH片段)直接连接至细胞表面(US20160122707A1；Jeong HJ,et al.,Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling.PLoS One.2017 Dec 4；12(12):e0189068；Chen I,et al.,A general strategy for the evolution of bond-forming enzymes using yeast display.Proc Natl Acad Sci U S A.2011 Jul 12；108(28):11399-404.),但是，由于Fc区段的分子量较大且结构复杂，目前尚未有研究报道能成功通过转肽酶A将含Fc区段(fragment crystallizable region)的完整抗体或Fc融合蛋白直接连接至细胞表面。
鉴于将含Fc区段的蛋白连接至细胞不仅可以使细胞具有靶向性，而且Fc区段可以进一步增强效应细胞的细胞毒活性，本领域需要将含Fc区段的蛋白直接连接至细胞表面的方法。
发明内容
本发明的目的是针对现有技术中存在的问题和不足，提供一种含Fc区段的蛋白分子和将含Fc区段的蛋白分子直接连接至细胞表面的方法。该方法获得的效应细胞可以通过其表面连接的含Fc区段的蛋白与相应的可溶性抗原以及细胞表面抗原结合，继而该蛋白的Fc区段能够通过与效应细胞表面Fc受体结合并触发效应细胞活化相关信号通路，最终实现特异性的抗原清除和靶 细胞杀伤，可以用于预防和治疗机体因细胞增殖和/或功能异常导致的疾病，如肿瘤、自身免疫病和感染性疾病等。
为解决上述技术问题，本文首先提供了含Fc区段的融合蛋白(如抗体和Fc融合蛋白)，该融合蛋白可以由转肽酶A介导直接连接到细胞表面。本文还提供了将含Fc区段的融合蛋白(如抗体和Fc融合蛋白)通过转肽酶A蛋白直接连接至细胞表面的方法。
在一方面，本发明提供了含Fc区段的融合蛋白，该融合蛋白从N至C端包含第一部分、Fc区段、连接部分和转肽酶A的底物部分。各部分之间可以是直接连接的或者通过一个或多个氨基酸残基连接。
在一个实施方案中，连接部分包含选自接头或蛋白质或多肽的部分或区域。例如，连接部分是单独的接头、单独的蛋白质或多肽、或者从N端至C端包含蛋白质或多肽和接头的部分。优选地，蛋白质或多肽选自scFv。
在一个实施方案中，接头包含选自以下的序列：
(1)(GGGGS)n，其中当所述连接部分包含所述蛋白质或多肽和接头时，n≥1；当所述连接部分仅包含接头时，n≥3的整数；和
(2)(EAAAK) n，n≥1的整数。
在一个实施方案中，底物部分包含以LPXTG所示的序列。
在一个实施方案中，第一部分可以选自F(ab′) 2、F(ab′)、Fab、Fv、scFv、受体和配体。第一部分还可以是相关致病原的任何其它结合配偶体。
在一个实施方案中，Fc区段是野生型Fc区段或变体Fc区段。
在一个实施方案中，Fc区段选自IgG、IgM、IgA、IgD和IgE的Fc区段。
在一个实施方案中，Fc区段选自IgG1、IgG2、IgG3和IgG4的Fc区段。
在一个实施方案中，融合蛋白包含以下任一项的结构：
全长抗体-GGGGS-GGGGS-GGGGS-LPETGG；
全长抗体-EAAAK-LPETGG；
全长抗体-IL2-LPETGG；
全长抗体-scFv-GGGGS-LPETGG；
全长抗体-scFv-EAAAK-LPETGG；
scFv-Fc区段-GGGGS-GGGGS-GGGGS-LPETGG；或
scFv-Fc区段-EAAAK-LPETGG。
在另一方面，本发明提供了核酸，其编码根据本发明所述的融合蛋白。
在一个实施方案中，核酸包含SEQ ID No.4和12、SEQ ID No.4和14、SEQ ID No.4和16、SEQ ID No.4和18、SEQ ID No.4和20、SEQ ID No.28、SEQ ID No.30、或SEQ ID No.32的编码序列。
在另一方面，本发明提供了载体，其包含根据本发明所述的核酸。
在另一方面，本发明提供了宿主细胞，其包含根据本发明所述的载体。
在另一方面，本发明提供了生成根据本发明所述的融合蛋白的方法，其包括：
(1)将表达重链的载体和在有轻链的情况下表达轻链的载体等摩尔混合；
(2)将载体混合物导入宿主细胞并且在适合于融合蛋白表达的条件下表达合适的时间；和
(3)回收培养基上清液，并且纯化融合蛋白。
在另一方面，本发明提供了用于将根据本发明所述的含Fc区段的融合蛋白连接到细胞表面的方法，其包括使细胞与含Fc区段的融合蛋白和转肽酶A接触的步骤。
在一个实施方案中，细胞是效应细胞。在一个实施方案中，细胞是NK细胞或T细胞。例如，细胞是外周血NK细胞、外周血T细胞和脐带血NK细胞。例如，细胞是NK92-FcγRIII细胞。
在另一方面，本发明提供了根据本发明的方法制备的细胞或其后代。
在另一方面，本发明提供了药物组合物，其包含根据本发明所述的细胞和药学可接受的载体。
在另一方面，本发明提供了根据本发明所述的细胞或根据本发明所述的药物组合物在制备用于疾病的药物中的用途。
在一个实施方案中，疾病是由细胞增殖和/或功能异常引起的疾病。例如，疾病是肿瘤、自身免疫病和/或传染病。
在本文中，连接部分包含IL2或scFv，其中IL2的氨基酸序列可以为GGGGSGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTRT(序列33)；scFv的氨基酸序列可以为DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSA SFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS(序列34)。
本发明成功将含Fc区段的融合蛋白直接连接至细胞表面。所获得的效应细胞具有精准靶向性。当效应细胞表达Fc受体时，连接了含Fc区段的蛋白的效应细胞能够被相应靶细胞激活。实验证明，含Fc区段的蛋白分子修饰的表达FcγRIII(CD16A)的NK细胞(NK92-FcγRIII)能够特异性杀伤靶细胞。因此，通过将含Fc区段的蛋白分子直接连接至效应细胞表面制备的精准靶向性效应细胞，一方面可以比现有的通过细胞转染制备特异性靶向性细胞的方法更为简单，同时也能够降低对效应细胞基因组操作可能产生的风险；另一方面，与抗原结合后，该融合蛋白Fc区段可以通过与Fc受体相互作用，激活效应细胞的杀伤功能。因此，基于本发明制备的效应细胞具有制备方法简单、安全性好、活性增强等优点，可以用于预防和/或治疗机体因细胞增殖和(或)功能异常导致的疾病，如肿瘤、自身免疫病和感染性疾病等，具有重要的应用价值。
附图说明
图1：含Fc区段的融合蛋白(包括重组抗体和Fc融合蛋白，编号:RP1-RP14)结构的示意图。
图2：重组抗体和Fc融合蛋白溶液的聚丙烯酰胺凝胶电泳图。
图3：重组抗体和Fc融合蛋白结合抗原活性检测的曲线图。
图4：流式细胞仪检测图，显示了使用转肽酶A将重组抗体或Fc融合蛋白连接至NK92-FcγRIII细胞的情况。
图5：流式细胞仪检测图，显示了连接了重组抗体和Fc融合蛋白的细胞能与相应靶抗原特异性结合的情况。
图6：流式细胞仪检测图，显示了成功连接重组抗体和Fc融合蛋白的NK92-FcγRIII细胞能进一步被表达特异性抗原的靶细胞激活。
图7：细胞杀伤效果的曲线图，显示了已连接重组抗体和Fc融合蛋白的NK92-FcγRIII细胞可以杀伤特异性抗原表达的靶细胞。
图8：流式细胞仪检测图，显示了使用转肽酶A将重组蛋白或Fc融合蛋白连接至外周血NK细胞、外周血T细胞和脐带血NK细胞表面。
具体实施方式
本发明基于发明人的以下发现：采用现有专利和文献中介绍的蛋白改造方法(US20160122707A1；Jeong HJ,et al.,Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling.PLoS One.2017Dec 4；12(12):e0189068；Chen I,et al.,A general strategy for the evolution of bond-forming enzymes using yeast display.Proc Natl Acad Sci U S A.2011 Jul 12；108(28):11399-404)在完整抗体或Fc融合蛋白的C端直接融合表达转肽酶A特异识别的LPXTG区段，即完整抗体或Fc融合蛋白与LPXTG序列之间不添加任何连接序列，并不能实现通过转肽酶A将完整抗体或Fc融合蛋白直接连接至细胞表面。相反，采用本发明的具备特定构造的融合蛋白可以通过转肽酶A将完整抗体或Fc融合蛋白直接连接至细胞表面。
如本文所用，转肽酶A是使含有酶底物识别序列的蛋白质与细菌细胞膜共价连接的膜结合酶。转肽酶A特异性底物识别基序是LPXTG，该酶在此底物序列的残基苏氨酸(T)和甘氨酸(G)之间进行切割，进一步与N端含有寡聚甘氨酸序列的底物进行连接反应。在本文中，转肽酶A的种类和来源可以不受限制，只要转肽酶A保留其上述功能特性。例如，转肽酶A可以是天然的转肽酶A，或者可以是转肽酶A的变体(例如参见CN201610726374.0)。
如本文所用，“第一部分”是指融合蛋白中的结合区。该结合区可以是抗体的抗原结合区、针对配体的受体、或针对受体的配体，只要其能够结合靶物，例如靶细胞上的靶物。在本文中，第一部分和Fc区可以是同源的或异源的。例如，第一部分和Fc区为天然存在的抗体。第一部分可以是抗体片段。第一部分可以结合癌症抗原、传染病抗原、自身免疫性疾病抗原。例如第一部分可以结合HER2蛋白。
“抗体”，其涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)和抗体片段，只要它们显示希望的抗原结合活性。抗体，又称免疫球蛋白(简称Ig)，按理化性质和生物学功能，可将其分为IgG、IgM、IgA、IgE、IgD五类。抗体的Fab区段(fragment of antigen  binding，Fab)为抗原结合片段，由一个完整的轻链和重链的VH和CH1结构域组成；Fc区段为可结晶段(fragment crystallizable，Fc)，根据抗体类型不同，该区域由重链的2个或者3个恒定结构域组成，如IgG的Fc结构域包含重链CH2和CH3结构域。
术语“Fc区段”用来定义免疫球蛋白重链的C端区域，其含有至少部分恒定区。该术语包括天然序列Fc区段和变体Fc区段。“变体Fc区段”包含由于至少一个“氨基酸修饰”而与“天然”或“野生型”序列Fc区段的氨基酸序列不同的氨基酸序列。与天然序列Fc区或与亲本多肽的Fc区段相比，变体Fc区可以具有至少一个氨基酸取代，例如在天然序列Fc区段中或在亲本多肽的Fc区中从约一个至约十个氨基酸取代，且优选从约一个至约五个氨基酸取代。本文的变体Fc区段将优选与天然序列Fc区和/或与亲本多肽的Fc区段具有至少约80％同源性，且最优选与之具有至少约90％同源性，更优选与之具有至少约95％同源性。Fc区可结合各种Fc受体(Fc receptors，FcR)和其它免疫分子，这一过程可引发不同的靶细胞杀伤效果，包括抗体依赖的细胞介导的细胞毒性作用(ADCC)、抗体依赖的细胞吞噬作用(ADCP)和补体依赖的细胞毒性作用(CDC)，是治疗性抗体在体内发挥效用的关键效应区段。抗体的效应子功能是由IgG的Fc效应子结构域(例如免疫球蛋白的Fc区)贡献的功能。这种功能可以例如通过Fc效应子结构域与具有吞噬或溶胞活性的免疫细胞上的Fc受体的结合、或通过Fc效应子结构域与补体***成分的结合来实现。典型的效应子功能是ADCC、ADCP和CDC。“效应子功能”指可归因于抗体的Fc区的那些生物学活性，其随抗体同种型而不同。抗体效应子功能的实例包括：C1q结合和依赖补体的细胞毒性(CDC)；Fc受体结合；依赖抗体的细胞介导的细胞毒性(ADCC)；吞噬作用(ADCP)；细胞表面受体(例如B细胞受体)的下调；及B细胞活化。
如本文所用，“连接部分”是指连接Fc区与转肽酶A底物LPXTG之间的部分。该部分可以仅包含接头。在此种情况中，连接部分可以是接头本身。或者，连接部分可以仅包含蛋白质或多肽，例如IL2或scFv。连接部分还可以包含蛋白质或多肽和接头两者。例如，连接部分从N端至C端包含IL2或scFv和接头。接头的种类没有特别限制，只要能够将含有Fc的融合蛋白连接到细胞表面上。例如，接头可以是柔性连接序列，如G(n)S(n)，n≥1，优选的是，该连接序列为GGGGSGGGGSGGGGS。接头也可以是刚性连接序列，如 (EAAAK)n，n≥1，优选的是，该连接序列为EAAAK。在连接部分包含蛋白质或多肽和接头的实施方案中，接头是(GGGGS)n，n≥1，接头也可以是刚性连接序列(EAAAK)n，n≥1。在连接部分仅包含接头的实施方案中，接头是(GGGGS)n，n≥3。在一个实施方案中，接头是(EAAAK) n，n≥1。在本文中，n≥3指例如n为3、4、5、6、7、8、9、10、或11的整数。n≥1指例如n为1、2、3、4、5、6、7、8、9、10、或11的整数。
术语“部分”在第一部分、连接部分和转肽酶A的底物部分中使用的上下文中等同于“区域”。第一部分、连接部分和底物部分可以称为第一区域、连接区域和底物区域。
术语“Fc受体”或“FcR”用来描述与抗体的Fc区结合的受体。优选的FcR是天然序列人FcR。此外，FcR可以是结合IgG抗体的FcR(γ受体)，且包括FcγRI、FcγRII和FcγRIII亚类的受体，包括这些受体的等位基因变体和可变剪接形式。FcγRII受体包括FcγRIIA(活化性受体)和FcγRIIB(“抑制性受体”)，其具有相似的氨基酸序列，区别主要在于其胞质结构域。活化性受体FcγRIIA在其胞质结构域中包含免疫受体酪氨酸活化基序(ITAM)。抑制性受体FcγRIIB在其胞质结构域中包含免疫受体酪氨酸抑制基序(ITIM)。“FcR”涵盖其他FcR，包括有待将来鉴定的那些。该术语还包括负责将母体IgG转移至胎儿的新生儿受体FcRn。在本文中，效应细胞可以表达FcγRIII。
“依赖抗体的细胞介导的细胞毒性”和“ADCC”指细胞介导的反应，其中表达FcR的非特异性细胞毒性细胞(例如天然杀伤(NK)细胞、中性粒细胞和巨噬细胞)识别靶细胞上结合的抗体，随后引起靶细胞的裂解。用于介导ADCC的主要细胞(NK细胞)仅表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。造血细胞上的FcR表达总结在Ravetch和Kinet,Annu.Rev.Immunol9(1991)457-492的464页上表3中。
术语“依赖抗体的细胞吞噬作用”和“ADCP”指这样的过程，其中抗体包被的细胞被与免疫球蛋白Fc区结合的吞噬性免疫细胞(例如巨噬细胞、中性粒细胞和树突细胞)全部或部分地内化。
如本文中使用，“Fc融合蛋白”或“融合蛋白”是指利用基因工程技术将抗体的Fc区与具有特定生物学功能的蛋白分子融合，其不仅具有该功能蛋白的原有活性，而且还具有抗体的某些特性，如ADCC、CDC和ADCP等功能。由此可知，如果将具有Fc区段的抗原结合分子(如抗体和抗原结合区-Fc 融合蛋白)直接连接至效应细胞表面，不仅可以使细胞具有识别特定抗原的靶向性，而且，Fc区所触发的生理学效应可以进一步增强效应细胞的杀伤活性。在本文中，Fc融合蛋白从N端至C端可以具备以下结构：
全长抗体-GGGGS-GGGGS-GGGGS-LPETGG；
全长抗体-EAAAK-LPETGG；
全长抗体-IL2-LPETGG；
全长抗体-scFv-GGGGS-LPETGG；
全长抗体-scFv-EAAAK-LPETGG；
scFv-Fc区段-GGGGS-GGGGS-GGGGS-LPETGG；
scFv-Fc区段-EAAAK-LPETGG。
这些Fc融合蛋白中的全长抗体可以是IgG1、IgG2、IgG3或IgG4抗体。
“抗体片段”指非完整抗体的分子，其包含完整抗体的部分，该部分结合完整抗体所结合的抗原。抗体片段的实例包括但不限于Fv、Fab、Fab'、Fab’-SH、F(ab')2；双抗体(diabody)；线性抗体；单链抗体分子(例如scFv)；及从抗体片段形成的多特异性抗体。在本文中，可以使用结合特定抗原的抗体片段作为本发明的第一部分。
实施例
下面结合具体实施例对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1：转肽酶A蛋白的表达和纯化
一、重组表达质粒pET-SrtA的构建
1、转肽酶A基因序列如序列1所示，根据序列1设计并合成引物F：5’-CGGCAGC CATATGGCTAAACCTCAAATTCCGA-3’(下划线为限制性内切酶NdeI的酶切识别序列，序列35)和引物R：5’-GTGGTG CTCGAGTTATTTGACTTCTGTAGCTAC-3’(下划线为限制性内切酶XhoI的酶切识别序列，序列36)。
序列1:
Figure PCTCN2021094655-appb-000002
2、合成上述序列1所示DNA片段并以其为模板(参考文献:Chen I,et al.,A general strategy for the evolution of bond-forming enzymes using yeast display.Proc Natl Acad Sci U S A.2011 Jul 12；108(28):11399-404.)，采用步骤1合成的引物F和引物R进行PCR扩增(反应程序：95℃5min；95℃30s，55℃30s，72℃2min，30个循环)，然后利用PCR产物回收试剂盒回收约457bp的PCR扩增产物。
3、取步骤2回收的PCR扩增产物，用限制性内切酶NdeI和限制性内切酶XhoI酶切，回收酶切后的DNA片段。
4、用限制性内切酶NdeI和限制性内切酶XhoI酶切载体pET-28(a)，回收酶切后的载体骨架。
5、将DNA片段和载体骨架连接，得到重组表达质粒pET -SrtA。
对重组表达质粒pET- SrtA进行结构描述如下：将载体pET-28(a)的限制性内切酶NdeI和XhoI识别序列间的小片段替换为序列表中序列1所示的DNA分子。根据测序结果，转肽酶A的序列SEQ ID No.1正确***载体pET-28(a)中。重组表达质粒pET- SrtA表达序列表中序列2所示的蛋白质(以下简称为转肽酶A蛋白)。
序列2:
Figure PCTCN2021094655-appb-000003
二、转肽酶A蛋白的表达和纯化
1、将重组表达质粒pET- SrtA导入大肠杆菌BL21(DE3)中，得到重组菌，命名为BL21(DE3)-pET- SrtA。
2、将BL21(DE3)-pET- SrtA单菌落接种于含100mg/mL卡那霉素的LB培养基，37℃、200rpm振荡培养，得到OD600为0.6的培养菌液1。向培养菌液1中加入IPTG，得到培养菌液2(培养菌液2中，IPTG的浓度为0.1mM)；然后 37℃、200rpm振荡培养6h，得到培养菌液3。
3、取培养菌液3，5000rpm离心10min，收集菌体。
4、取步骤3收集的菌体，用10mL pH7.4、TBS缓冲液(10mmol/L，Tris,0.9％NaCl)重悬，得到菌悬液；然后对该菌悬液进行超声处理。超声参数：超声频率30％；超声10s，停5s，超声总时间为30min。
5、取完成步骤4的体系，12000rpm离心10min，收集上清液。
6、将步骤5收集的上清液和Ni-NTA Resin混合并孵育10min，然后弃上清，用含20mM咪唑的pH7.4、TBS缓冲液洗涤3次。
7、完成步骤6后，用含500mM咪唑的pH7.4、TBS缓冲液进行洗脱，收集过柱后溶液，即为转肽酶A蛋白。
实施例2：含Fc区段的融合蛋白的制备和结合活性鉴定
一、含Fc区段的融合蛋白的制备
1、含Fc区段的融合蛋白质粒的构建
含Fc区段的融合蛋白(包括重组抗体和Fc融合蛋白，编号:RP1-RP14)结构见图1。融合蛋白的表达质粒的构建如实施例1所述。即，如实施例1所述的克隆技术，用含有HindIII和XhoI酶切位点的引物克隆合成的核酸，并且将合成的序列连接到用相应酶消化的载体pCDNA3.1(+)上。
1.1.重组抗体Ab-CH-LPETGG(编号:RP1)的表达质粒的构建:
用序列3所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到轻链表达载体。序列3中，第61-702位核苷酸编码重组抗体Ab-CH-LPETGG的全长轻链(序列4)。
序列3：
ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGGTCGAC CGGTGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAACACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGACCTTTGGCCAGGGCACCAAACTCGAGATCAAACGTACGGTGGCGGCGCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGTACCGCTAGCGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TGA(第1-60位核苷酸编码 信号肽，最后三个核苷酸TGA为终止密码子)
序列4:
Figure PCTCN2021094655-appb-000004
用序列5所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到重链表达载体。序列5中，第61-1428位核苷酸编码重组抗体Ab-CH-LPETGG的全长重链(序列6)。
序列5:
ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGGTCGAC CGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAACTGCCCGAGACCGGCGGC TGA(第1-60位核苷酸编码信号肽，最后三个核苷酸TGA为终止密码子)
序列6:
Figure PCTCN2021094655-appb-000005
1.2.重组抗体Ab-CH-GGGGS-LPETGG(编号:RP2)的表达质粒的构建:
用序列3所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到轻链表达载体。序列3中，第61-702位核苷酸编码重组抗体Ab-CH-GGGGS-LPETGG的全长轻链(序列4)。
用序列7所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到重链表达载体。序列7中，第61-1443位核苷酸编码重组抗体Ab-CH-GGGGS-LPETGG的全长重链(序列8)。
序列7:
ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGGTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAGGCGGCGGCGGCAGCCTGCCCGAGACCGGCGGCTGA(第1-60位核苷酸编码信号肽，最后三个核苷酸TGA为终止密码子)
序列8:
Figure PCTCN2021094655-appb-000006
1.3.重组抗体Ab-CH-GGGGS(x2)-LPETGG(编号:RP3)的表达质粒的构 建:
用序列3所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到轻链表达载体。序列3中，第61-702位核苷酸编码重组抗体Ab-CH-GGGGS(x2)-LPETGG的全长轻链(序列4)。
用序列9所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到重链表达载体。序列9中，第61-1473位核苷酸编码重组抗体Ab-CH-GGGGS(x2)-LPETGG的全长重链(序列10)。
序列9:
ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGGTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCTGCCCGAGACCGGCGGCTGA(第1-60位核苷酸编码信号肽，最后三个核苷酸TGA为终止密码子)
序列10:
Figure PCTCN2021094655-appb-000007
1.4.重组抗体Ab-CH-GGGGS(x3)-LPETGG(编号:RP4)的表达质粒的构 建:
用序列3所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到轻链表达载体。序列3中，第61-702位核苷酸编码重组抗体Ab-CH-GGGGS(x3)-LPETGG的全长轻链(序列4)。
用序列11所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到重链表达载体。序列11中，第61-1458位核苷酸编码重组抗体Ab-CH-GGGGS(x3)-LPETGG的全长重链(序列12)。
序列11:
ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGGTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC CTGCCCGAGACCGGCGGCTGA(第1-60位核苷酸编码信号肽，最后三个核苷酸TGA为终止密码子)
序列12:
Figure PCTCN2021094655-appb-000008
Figure PCTCN2021094655-appb-000009
1.5.重组抗体Ab-CH-EAAAK-LPETGG(编号:RP5)的表达质粒的构建:
用序列3所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到轻链表达载体。序列3中，第61-702位核苷酸编码重组抗体Ab-CH-EAAAK-LPETGG的全长轻链(序列4)。
用序列13所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到重链表达载体。序列13中，第61-1443位核苷酸编码重组抗体Ab-CH-EAAAK-LPETGG的全长重链(序列14)。
序列13:
Figure PCTCN2021094655-appb-000010
序列14:
Figure PCTCN2021094655-appb-000011
Figure PCTCN2021094655-appb-000012
1.6.重组抗体Ab-CH-IL2-LPETGG(编号:RP6)的表达质粒的构建:
用序列3所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到轻链表达载体。序列3中，第61-702位核苷酸编码重组抗体Ab-CH-IL2-LPETGG的全长轻链(序列4)。
用序列15所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到重组重链表达载体。序列15中，第61-1854位核苷酸编码重组抗体Ab-CH-IL2-LPETGG的全长重链(序列16)。
序列15:
Figure PCTCN2021094655-appb-000013
序列16:
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGS AP TSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHL QCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETA TIVEFLNRWITFCQSIISTLTRTLPETGG(加下划线的部分为IL2的氨基酸序列)
1.7.重组抗体Ab1-CH-scFv.Ab2-GGGGS-LPETGG(编号:RP7)的表达质 粒的构建:
用序列3所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到轻链表达载体。序列3中，第61-702位核苷酸编码重组抗体Ab1-CH-scFv.Ab2-GGGGS-LPETGG的全长轻链(序列4)。
用序列17所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到重组重链表达载体。序列17中，第61-2178位核苷酸编码重组抗体Ab1-CH-scFv.Ab2-GGGGS-LPETGG的全长重链(序列18)。
序列17:
ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGGTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAGGCGGCGGCGGCAGC GACATCCAGATGA CCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTG CAGGGCCAGCCAGGACGTGAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGC AAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCA GCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCT GCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACCTGTACCACCCCGCCA CCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCC TGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTT CAGCGACAGCTGGATCCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGG GTGGCCTGGATCAGCCCCTACGGCGGCAGCACCTACTACGCCGACAGCGTGAAGG GCAGGTTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAA CAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCACTGGCCC GGCGGCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGCAGCCTGCCCGAGACCGGCGGCTGA(scFv.Ab2序列的编码序列加下划线)
序列18：
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS DIQM TQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGG GSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEW VAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHW PGGFDYWGQGTLVTVSSGGGGS LPETGG(scFv.Ab2序列为粗体加下划线)
1.8.重组抗体Ab1-CH-scFv.Ab2-EAAAK-LPETGG(编号:RP8)的表达质 粒的构建:
用序列3所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到轻链表达载体。序列3中，第61-702位核苷酸编码重组抗体Ab1-CH-scFv.Ab2-EAAAK-LPETGG的全长轻链(序列4)。
用序列19所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到重组重链表达载体。序列19中，第61-2178位核苷酸编码重组抗体Ab1-CH-scFv.Ab2-EAAAK-LPETGG的全长重链(序列20)。
序列19:
ATGGAGACCGACACCCTGCTGCTCTGGGTGCTGCTGCTCTGGGTGCCCGGGTCGACCGGTGAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCG CCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAAGGCGGCGGCGGCAGC GACATCCAGATGA CCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTG CAGGGCCAGCCAGGACGTGAGCACCGCCGTGGCCTGGTACCAGCAGAAGCCCGGC AAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCA GCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCT GCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGTACCTGTACCACCCCGCCA CCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCG GCGGCAGCGGCGGCGGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCC TGGTGCAGCCCGGCGGCAGCCTGAGGCTGAGCTGCGCCGCCAGCGGCTTCACCTT CAGCGACAGCTGGATCCACTGGGTGAGGCAGGCCCCCGGCAAGGGCCTGGAGTGG GTGGCCTGGATCAGCCCCTACGGCGGCAGCACCTACTACGCCGACAGCGTGAAGG GCAGGTTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAA CAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGAGGCACTGGCCC GGCGGCTTCGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGAGGCCGCCGCCAAGCTGCCCGAGACCGGCGGCTGA(scFv.Ab2序列加下划线，接头序列为GAGGCCGCCGCCAAG)
序列20:
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGS DIQM TQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKGGGGSGGG GSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEW VAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHW PGGFDYWGQGTLVTVSS
Figure PCTCN2021094655-appb-000014
LPETGG(scFv.Ab2序列为粗体加下划线，接头序列为EAAAK)
1.9.Fc融合蛋白scFv-Fc-LPETGG(编号:RP9)的表达质粒的构建:
用序列21所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到Fc融合蛋白表达载体。序列21中，第61-1506位核苷 酸编码scFv-Fc-LPETGG全长蛋白(序列22)。
序列21:
Figure PCTCN2021094655-appb-000015
序列22:
Figure PCTCN2021094655-appb-000016
1.10.Fc融合蛋白scFv-Fc-GGGGS-LPETGG(编号:RP10)的表达质粒的 构建:
用序列23所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到Fc融合蛋白表达载体。序列23中，第61-1521位核苷酸编码scFv-Fc-GGGGS-LPETGG的全长蛋白(序列24)。
序列23:
Figure PCTCN2021094655-appb-000017
序列24:
Figure PCTCN2021094655-appb-000018
1.11.Fc融合蛋白scFv-Fc-(GGGGS)x2-LPETGG(编号:RP11)的表达质粒 的构建:
用序列25所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到Fc融合蛋白表达载体。序列25中，第61-1536位核苷酸编码scFv-Fc-(GGGGS)x2-LPETGG的全长蛋白(序列26)。
序列25:
Figure PCTCN2021094655-appb-000019
Figure PCTCN2021094655-appb-000020
序列26:
Figure PCTCN2021094655-appb-000021
1.12.Fc融合蛋白scFv-Fc-(GGGGS)x3-LPETGG(编号:RP12)的表达质粒 的构建:
用序列27所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到Fc融合蛋白表达载体。序列27中，第61-1550位核苷酸编码scFv-Fc-(GGGGS)x3-LPETGG的全长蛋白(序列28)。
序列27:
Figure PCTCN2021094655-appb-000022
Figure PCTCN2021094655-appb-000023
序列28:
Figure PCTCN2021094655-appb-000024
1.13.Fc融合蛋白scFv-Fc-EAAAK-LPETGG(编号:RP13)的表达质粒的 构建:
用序列29所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到Fc融合蛋白表达载体。序列29中，第61-1521位核苷酸编码scFv-Fc-EAAAK-LPETGG的全长蛋白(序列30)。
序列29:
Figure PCTCN2021094655-appb-000025
Figure PCTCN2021094655-appb-000026
序列30:
Figure PCTCN2021094655-appb-000027
1.14.重组抗体Ab(IgG4)-EAAAK-LPETGG(编号:RP14)的表达质粒的构 建:
用序列3所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到轻链表达载体。序列3中，第61-702位核苷酸编码重组抗体Ab(IgG4)-EAAAK-LPETGG的全长轻链(序列4)。
用序列31所示的DNA分子取代载体pCDNA3.1(+)中HindIII和XhoI酶切位点之间的片段，得到重链表达载体。序列31中，第61-1800位核苷酸编码重组抗体Ab(IgG4)-EAAAK-LPETGG的全长重链(序列32)。
序列31:
Figure PCTCN2021094655-appb-000028
Figure PCTCN2021094655-appb-000029
序列32:
Figure PCTCN2021094655-appb-000030
2、含Fc区段的融合蛋白的制备
2.1使用上述重链表达载体和轻链表达载体(若有轻链的话)等摩尔混合后，采用ExpiFectamine TM CHO Transfection Kit(购买自Therno Fisher公司，货号:A29129)(按说明书操作)共转染CHO细胞表达重组抗体；使用上述Fc融合蛋白表达质粒，采用ExpiFectamine TM CHO Transfection Kit(货号:A29129)(按说明书操作)转染CHO细胞表达Fc融合蛋白；然后采用ExpiCHO TM Expression Medium培养基在37℃、5％CO 2条件下培养8天，收集上清液。
2.2取步骤2.1得到的上清液，进行蛋白A纯化，收集纯化产物。
2.3取步骤2.2得到的纯化产物，进行超滤浓缩换液，将体系更换为PBS缓冲液，得到重组抗体和Fc融合蛋白溶液。
采用A280nm紫外光吸收法检测溶液的蛋白浓度。
含Fc区段的融合蛋白(包括重组抗体和Fc融合蛋白)溶液的聚丙烯酰氨凝胶电泳图见图2。图2中，非还原电泳为未加入还原剂2-巯基乙醇，还原电泳 为加入1％还原剂2-巯基乙醇。如图2中所述，重组抗体和Fc融合蛋白的表观分子量与它们的预测分子量相符，含Fc区段的融合蛋白得到正确表达。
二、ELISA检测含Fc区段的融合蛋白的结合活性
1、取酶标板，加入包被液(100μl/孔)，4℃过夜。
包被液有包被原和包被缓冲液组成，包被原在包被液中的浓度为1μg/mL。包被原为HER2蛋白(购买自义翘神州公司，货号:10004-H08H4)。包被缓冲液(pH9.6)：Na 2CO 3 1.59g、NaHCO 3 2.94g，余量为水。
2、完成步骤1后，取所述酶标板，用PBST缓冲液洗涤三次。
3、完成步骤2后，取所述酶标板，加入含5g/100mL脱脂奶粉的PBST缓冲液，37℃封闭1h。
4、取上文制备的含Fc区段的融合蛋白溶液，采用含5g/100mL脱脂奶粉的PBST缓冲液制备抗体浓度为10μg/mL的母液，然后采用含5g/100mL脱脂奶粉的PBST缓冲液进行三倍梯度稀释，得到不同浓度的含Fc区段的融合蛋白溶液。
5、取完成步骤3的酶标板，加入步骤4得到的不同浓度的含Fc区段的融合蛋白溶液(每孔100μL)，37℃孵育1h。每种浓度设置3个复孔。
6、完成步骤5后，取所述酶标板，用PBST缓冲液洗涤三次(每次每孔250μL)。
7、完成步骤6后，取所述酶标板，加入HRP标记的山羊抗人IgG二抗稀释液(将HRP标记的山羊抗人IgG二抗按1:40000稀释于含5g/100mL脱脂奶粉的PBST缓冲液)，37℃孵育30min。
8、完成步骤7后，取所述酶标板，加入TMB显色试剂(每孔100μL)，室温显色10min。
9、完成步骤8后，取所述酶标板，加入2N H 2SO 4溶液终止显色(每孔50μL)，然后检测在450nm波长下OD值。
结果见图3。结果显示，上文所制备的含Fc区段的融合蛋白可以与相应抗原结合，且结合活性较高。表明上述对于含Fc区段的融合蛋白的改造没有改变其抗原结合活性。
实施例3 使用转肽酶A将含Fc区段的融合蛋白连接至NK92-FcγRIII细胞及细胞活性检测
一、使用转肽酶A将含Fc区段的融合蛋白连接至NK92-FcγRIII细胞(购 买自ATCC，货号:pta-8837)
1、取100μL浓度为1.0x10 6/mL的NK92-FcγRIII细胞悬浮液，加入转肽酶A至终浓度为20ug/ml，加入含Fc区段的融合蛋白至终浓度为10ug/ml，获得孵育体系。将该孵育体系在25℃的条件下孵育90min。离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。将上述获得的细胞命名为：重组蛋白-SrtA-NK。
2、取100μL浓度为1.0x10 6/mL的NK92-FcγRIII细胞悬浮液，只加入含Fc区段的融合蛋白至终浓度为10ug/ml，获得孵育体系。将该孵育体系在25℃的条件下孵育90min。离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。将上述获得的细胞命名为：重组蛋白-NK。
3、取步骤1和2收集的细胞，按照说明书加入APC标记的驴抗人IgG抗体(APC AffiniPure F(ab') 2Fragment Donkey Anti-Human IgG,Fcγ fragment specific，购买自Jackson ImmunoResearch公司，货号:709-136-098)，获得孵育体系。将该孵育体系在25℃的条件下孵育30min。
4、完成步骤3后，离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。
5、完成步骤4后，以未进行处理的细胞做为空白组细胞，使用流式细胞仪检测加或不加转肽酶A条件下细胞标记含Fc区段的融合蛋白的水平。
实验结果见图4。结果表明，转肽酶A不能将仅在Fc区段C末端加入LPXTG序列的含Fc区段的融合蛋白(如重组抗体RP1或Fc融合蛋白RP9)连接至细胞表面；经过进一步在Fc区段和LPXTG区段之间添加连接序列改造后的重组抗体(RP4,RP5,RP6,RP7,RP8，RP14)和Fc融合蛋白(RP12,RP13)，在转肽酶A的介导下能够成功连接至细胞表面。
二、连接了含Fc区段的融合蛋白的细胞能与相应靶抗原特异结合
1、取200μL浓度为1.0x10 6/mL的NK92-FcγRIII细胞悬浮液，加入转肽酶A至终浓度为20ug/ml，加入含Fc区段的融合蛋白至终浓度为10ug/ml，获得孵育体系。将该孵育体系在25℃的条件下孵育90min。离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。将上述获得的细胞命名为：重组蛋白-SrtA-NK。
2、取200μL浓度为1.0x10 6/mL的NK92-FcγRIII细胞悬浮液，只加入含Fc区段的融合蛋白至终浓度为10ug/ml，获得孵育体系。将该孵育体系在25℃的条件下孵育90min。离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。将上述获得的细胞命名为：重组蛋白-NK。
3、取100μL浓度为1.0x10 6/mL的步骤1和2完成后的细胞悬浮液，加入生物素标记的HER2蛋白(购买自ACRObiosysterms，货号:H822R)，获得孵育体系。将该孵育体系在25℃的条件下孵育60min。
4、完成步骤3后，离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。
5、完成步骤4后，按照说明书加入FITC标记的链霉亲合素(FITC Streptavidin，购买自Biolegend公司，货号:405201)，获得孵育体系。将该孵育体系在25℃的条件下孵育30min。
6、完成步骤5后，离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。
7、完成步骤6后，以未进行处理的细胞做为空白组细胞，使用流式细胞仪检测细胞结合靶抗原HER2的水平。
实验结果见图5。结果表明，成功连接了含Fc区段融合蛋白的细胞能与靶抗原特异结合。
三、成功连接含Fc区段融合蛋白的NK92-FcγRIII细胞能进一步被表达特 异抗原的靶细胞激活
1、取400μL浓度为1.0x10 6/mL的NK92-FcγRIII细胞悬浮液，加入转肽酶A至终浓度为20ug/ml，加入含Fc区段的融合蛋白至终浓度为10ug/ml，获得孵育体系。将该孵育体系在25℃的条件下孵育90min。离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。将上述获得的细胞命名为：重组蛋白-SrtA-NK。
2、取200μL浓度为1.0x10 6/mL的NK92-FcγRIII细胞悬浮液，只加入含Fc区段的融合蛋白至终浓度为10ug/ml，获得孵育体系。将该孵育体系在25℃的条件下孵育90min。离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。将上述获得的细胞命名为：重组蛋白-NK。
3、取24孔板，先加入200μL浓度为4.0×10 5/mL的SKOV3细胞(该细胞为 抗原HER2蛋白高表达细胞系)悬浮液，再加入200μL浓度为2x10 6/mL的步骤1或2完成后的细胞悬浮液，获得孵育体系。将该孵育体系在37℃的条件下培养60min。
4、取完成步骤3的体系，4℃，离心，收集细胞，用冰浴的pH7.4、0.01mol/L PBS缓冲液充分洗涤。
5、完成步骤4后，按照说明书加入Alexa
Figure PCTCN2021094655-appb-000031
488标记的抗人CD107a抗体(Alexa
Figure PCTCN2021094655-appb-000032
488 anti-human CD107a，购买自Biolegend公司，货号:328610)和APC标记的抗人CD56抗体(APC anti-human CD56(NCAM)，购买自Biolegend公司，货号:318310)，获得孵育体系。将该孵育体系在4℃的条件下孵育30min。
6、完成步骤5后，4℃，离心，收集细胞，用冰浴的pH7.4、0.01mol/L PBS缓冲液充分洗。
7、完成步骤6后，以未进行处理的细胞做为空白组细胞，使用流式细胞仪检测细胞CD107a的水平。
实验结果见图6。结果表明，成功连接含Fc区段融合蛋白的NK92-FcγRIII细胞能进一步被表达特异抗原的靶细胞激活。
四、已连接含Fc区段融合蛋白的NK92-FcγRIII细胞可以杀伤特异抗原表 达的靶细胞
1、取400μL浓度为1.0x10 6/mL的NK92-FcγRIII细胞悬浮液，加入转肽酶A至终浓度为20ug/ml，加入含Fc区段的融合蛋白至终浓度为10ug/ml，获得孵育体系。将该孵育体系在25℃的条件下孵育90min。离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。将上述获得的细胞命名为：重组蛋白-SrtA-NK。
2、取200μL浓度为1.0x10 6/mL的NK92-FcγRIII细胞悬浮液，只加入含Fc区段的融合蛋白至终浓度为10ug/ml，获得孵育体系。将该孵育体系在25℃的条件下孵育90min。离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。将上述获得的细胞命名为：重组蛋白-NK。
3、取96孔板，先加入50μL浓度为4.0x10 5/mL的SKOV3细胞(该细胞为抗原HER2蛋白高表达细胞系)悬浮液，再加入50μL浓度为2x10 6/mL的步骤1或2完成后的细胞悬浮液，获得孵育体系。将该孵育体系在37℃的条件下培 养4hr。
4、取96孔板，先加入50μL浓度为4.0x10 5/mL的MCF-7细胞(该细胞为抗原HER2蛋白阴性细胞系)悬浮液，再加入50μL浓度为2x10 6/mL的步骤1或2完成后的细胞悬浮液，获得孵育体系。将该孵育体系在37℃的条件下培养4hr。
5、取完成步骤3和4的体系，加入检测乳酸脱氢酶(LDH)活性的工作液100ul(购自东仁化学公司，货号:CK12)，避光显色15min，然后加入50ul终止液并测定0D490nm吸光值。
实验结果见图7。结果表明，已连接含Fc区段融合蛋白的NK92-FcγRIII细胞(RP5-SrtA-NK,RP7-SrtA-NK,RP8-SrtA-NK,RP13-SrtA-NK)可以杀伤特异抗原表达的SKOV3细胞，而对不表达该抗原的MCF-7细胞则没有杀伤作用。未连接含Fc区段融合蛋白的对照组NK92-FcγRIII细胞(RP5-NK,RP7-NK,RP8-NK,RP13-NK,)对特异抗原表达的靶细胞没有杀伤作用。
五、使用转肽酶A将含Fc区段的融合蛋白连接至外周血NK细胞/外周血 T细胞/脐带血NK细胞
1、外周血NK细胞购买自君达科安科技有限公司。
2、外周血T细胞制备:首先从人外周血通过密度梯度离心分离获得单个核细胞，然后使用T细胞阴选试剂盒(购自Stemcell公司，货号:710410)(按说明书操作)富集其中的T细胞。
3、脐带血NK细胞由山东省齐鲁干细胞工程有限公司赠送。
4、取100μL浓度为1.0x10 6/mL的外周血NK细胞或外周血T细胞或脐带血NK细胞悬浮液，加入转肽酶A至终浓度为20ug/ml，加入含Fc区段的融合 蛋白至终浓度为10ug/ml，获得孵育体系。将该孵育体系在25℃的条件下孵育90min。离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。将上述获得的细胞命名为：重组蛋白-SrtA-细胞。
5、取100μL浓度为1.0x10 6/mL的外周血NK细胞或外周血T细胞或脐带血NK细胞悬浮液，仅加入含Fc区段的融合蛋白至终浓度为10ug/ml，获得孵育体系。将该孵育体系在25℃的条件下孵育90min。离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。将上述获得的细胞命名为：重组蛋白-细胞。
6、完成步骤4和5后，离心收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。
7、取步骤6收集的细胞，按照说明书加入APC标记的驴抗人IgG抗体(APC AffiniPure F(ab') 2Fragment Donkey Anti-Human IgG,Fcγ fragment specific，购买自Jackson ImmunoResearch公司，货号:709-136-098)，获得孵育体系。将该孵育体系在25℃的条件下孵育30min。
8、完成步骤7后，离心，收集细胞，用pH7.4、0.01mol/L PBS缓冲液充分洗涤。
9、完成步骤8后，以未进行处理的细胞做为空白组细胞，使用流式细胞仪检测细胞标记水平。
实验结果见图8。结果表明，在Fc区段和LPXTG区段之间添加连接序列改造后的含Fc区段的融合蛋白，在转肽酶A的介导下能够成功直接连接至外周血NK细胞、外周血T细胞和脐带血NK细胞表面。
结论
我们的实验结果表明，采用现有专利和文献中介绍的蛋白改造方法(US20160122707A1；Jeong HJ,et al.,Generation of Ca2+-independent sortase A mutants with enhanced activity for protein and cell surface labeling.PLoS One.2017 Dec 4；12(12):e0189068；Chen I,et al.,A general strategy for the evolution of bond-forming enzymes using yeast display.Proc Natl Acad Sci U S A.2011 Jul 12；108(28):11399-404)仅在含Fc区段的融合蛋白(如完整抗体或Fc融合蛋白)的C端直接融合表达转肽酶A特异识别的LPXTG区段(即完整抗体或Fc融合蛋白中的Fc区段与LPXTG序列之间不添加任何连接序列)所获得的融合蛋白并不能由转肽酶A介导直接连接至细胞表面。我们证实，将完整抗体或Fc融合蛋白中Fc区段的C端通过特定连接序列与转肽酶A识别的LPXTG区段连接后的构建体能够由转肽酶A介导直接连接至细胞表面。构建体为上述实施例中的RP4、RP5、RP6、RP7、RP8、RP12、RP13和RP14。

Claims (15)

  1. 融合蛋白，该融合蛋白从N至C端包含第一部分、Fc区段、连接部分和转肽酶A的底物部分，其中：
    所述连接部分包含选自接头和蛋白质或多肽的区域，该蛋白质或多肽选自IL2或scFv，优选SEQ ID No.33或34；
    所述接头包含选自以下的序列：
    (1)(GGGGS)n，其中当所述连接部分包含所述蛋白质或多肽和接头时，n≥1；当所述连接部分仅包含接头时，n≥3；
    (2)(EAAAK) n，n≥1；
    所述底物部分包含以LPXTG所示的序列。
  2. 根据权利要求1所述的融合蛋白，其中所述第一部分选自F(ab′)、F(ab′) 2、Fab、Fv、scFv、受体和配体。
  3. 根据权利要求1或2所述的融合蛋白，其中所述Fc区段是野生型Fc区段或变体Fc区段，
    优选地，所述Fc区段选自IgG、IgM、IgA、IgD和IgE的Fc区段，优选选自IgG1、IgG2、IgG3和IgG4的Fc区段。
  4. 根据权利要求1-3中任一项所述的融合蛋白，其包含以下任一项的结构：
    全长抗体-GGGGS-GGGGS-GGGGS-LPETGG；
    全长抗体-EAAAK-LPETGG；
    全长抗体-IL2-LPETGG；
    全长抗体-scFv-GGGGS-LPETGG；
    全长抗体-scFv-EAAAK-LPETGG；
    scFv-Fc区段-GGGGS-GGGGS-GGGGS-LPETGG；或
    scFv-Fc区段-EAAAK-LPETGG。
  5. 核酸，其编码根据权利要求1-4中任一项所述的融合蛋白。
  6. 根据权利要求5所述的核酸，其包含SEQ ID No.4和12、SEQ ID No.4和14、SEQ ID No.4和16、SEQ ID No.4和18、SEQ ID No.4和20、SEQ ID No.28、SEQ ID No.30、或SEQ ID No.32的编码序列。
  7. 载体，其包含根据权利要求5或6所述的核酸。
  8. 宿主细胞，其包含根据权利要求7所述的载体。
  9. 生成根据权利要求1-4中任一项所述的融合蛋白的方法，其包括：
    (1)将权利要求7中的表达重链的载体和在有轻链的情况下表达轻链的载体等摩尔混合；
    (2)将载体混合物导入宿主细胞并且在适合于融合蛋白表达的条件下表达合适的时间；和
    (3)回收培养基上清液，并且纯化融合蛋白。
  10. 用于将根据权利要求1-4中任一项所述的融合蛋白连接到细胞表面的方法，其包括使细胞与融合蛋白和转肽酶A接触的步骤。
  11. 根据权利要求10所述的方法，其中所述细胞是效应细胞，优选NK细胞或T细胞，优选外周血NK细胞、外周血T细胞和脐带血NK细胞，优选NK92-FcγRIII细胞。
  12. 根据权利要求10或11的方法制备的细胞。
  13. 药物组合物，其包含根据权利要求12所述的细胞和药学可接受的载体。
  14. 根据权利要求12所述的细胞或根据权利要求13所述的药物组合物在制备用于疾病的药物中的用途。
  15. 根据权利要求14所述的用途，其中所述疾病是由细胞增殖和/或功能异常引起的疾病，优选肿瘤、自身免疫病和/或传染病。
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CN111848816B (zh) * 2020-07-24 2021-06-08 斯普迈(北京)生物科技有限公司 融合蛋白及其用途
CN114163536B (zh) * 2021-12-08 2023-07-04 宁波大学 一种基于乳酸菌LPxTG基序重组蛋白的构建及应用
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