WO2020186974A1 - 一种双特异性抗体及其制备方法与应用 - Google Patents

一种双特异性抗体及其制备方法与应用 Download PDF

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WO2020186974A1
WO2020186974A1 PCT/CN2020/076516 CN2020076516W WO2020186974A1 WO 2020186974 A1 WO2020186974 A1 WO 2020186974A1 CN 2020076516 W CN2020076516 W CN 2020076516W WO 2020186974 A1 WO2020186974 A1 WO 2020186974A1
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antibody
chain
seq
monoclonal antibody
bispecific
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PCT/CN2020/076516
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English (en)
French (fr)
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袁清安
孟庆武
白丽莉
赵立坤
李延虎
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益科思特(北京)医药科技发展有限公司
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Priority to EP20773644.8A priority Critical patent/EP3882276A4/en
Priority to US17/290,957 priority patent/US20220002408A1/en
Priority to JP2021547639A priority patent/JP7262597B2/ja
Publication of WO2020186974A1 publication Critical patent/WO2020186974A1/zh

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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/7051—T-cell receptor (TcR)-CD3 complex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present invention relates to the technical fields of biotechnology and immunology, in particular to a bispecific antibody that binds CD19 and CD3, and a preparation method and application thereof.
  • Antibody drugs are biomacromolecule drugs prepared by antibody engineering technology with cell engineering technology and genetic engineering technology as the main body. They have the advantages of high specificity, uniform properties, and targeted preparation for specific targets. Monoclonal antibodies are mainly used clinically in the following three aspects: tumor treatment, immune disease treatment and anti-infection treatment. Among them, tumor therapy is currently the most widely used field of monoclonal antibodies. Currently, monoclonal antibody products that have entered clinical trials and are on the market, and the number of products used for tumor therapy accounts for about 50%. Monoclonal antibody therapy for tumors is an immunotherapy aimed at specific targets of diseased cells to stimulate the immune system to kill target cells.
  • bispecific Sex antibodies are one of the development directions to improve the therapeutic effect of antibodies, and have become a hot spot in the field of antibody engineering research.
  • Bispecific antibodies are two kinds of artificial antibodies that can specifically recognize and bind to different antigens or different antigenic sites. If the two antigens are located on different cell surfaces, the bispecific antibody can bridge between the two antigen molecules, thereby forming cross-links between cells and mediating the cells to produce targeted effector functions. BsAb has broad application prospects in biomedicine, especially in tumor immunotherapy.
  • Bispecific antibodies (immune double antibodies) used for immunotherapy are artificial antibodies containing two specific cell receptor antigen binding sites, which can bridge diseased cells (target cells) and functional cells (immune cells) , To stimulate a directed immune response.
  • BsAb-mediated immune cells such as T cells, NK cells, etc.
  • the mechanism of action is that BsAb can simultaneously bind to tumor-related antigens and target molecules on immune effector cells. While immunizing cells, it directly leads to the specific killing of tumor cells by immune effector cells.
  • Bispecific antibodies can be obtained through a variety of ways, and their preparation methods mainly include: chemical coupling method, hybrid-hybridoma method and genetic engineering antibody preparation method.
  • the chemical coupling method is a bispecific monoclonal antibody prepared by chemically coupling two different monoclonal antibodies together, which is the earliest bispecific monoclonal antibody.
  • the hybrid-hybridoma method is to produce bispecific monoclonal antibodies by cell hybridization or ternary hybridomas. These cell hybridomas or ternary hybridomas are fused through the established hybridomas, or the established hybridomas and the mice The fusion of lymphocytes can only be used to produce bispecific antibodies of murine origin. Therefore, its application is greatly restricted.
  • bispecific microbodies diabodies
  • single-chain diabodies single-chain diabodies.
  • multivalent bispecific antibodies multivalent bispecific antibodies.
  • genetically engineered bispecific antibody drugs have entered clinical trials in the world, and they have shown good application prospects.
  • the CD3 molecule on the surface of T cells is composed of 4 subunits: ⁇ , ⁇ , ⁇ , and ⁇ . Its molecular masses are 18.9kDa, 23.1kDa, 20.5kDa and 18.7kDa, and their lengths are 171, 207, 182, and 164, respectively. Amino acid residues. Together, they form 6 peptide chains, which are often tightly combined with T cell receptors (TCR) to form a TCR-CD3 complex containing 8 peptide chains.
  • TCR T cell receptors
  • the schematic diagram of the structure is shown in Figure 1. The complex has the functions of T cell activation, signal transduction and stabilization of the TCR structure.
  • the cytoplasmic segment of CD3 contains immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • TCR recognizes and binds to antigen peptides presented by MHC (major histo-compatibility complex) molecules, resulting in the ITAM of CD3.
  • Tyrosine residues in the conserved sequence are phosphorylated by the tyrosine protein kinase p56lck in T cells, and then other tyrosine protein kinases (such as ZAP-70) containing the SH2 (Scrhomology 2) domain are recruited.
  • ZAP-70 tyrosine protein kinases
  • the phosphorylation of ITAM and its binding to ZAP-70 are one of the important biochemical reactions in the early stages of the signal transduction process of T cell activation. Therefore, the function of the CD3 molecule is to transduce the activation signal generated by the TCR to recognize the antigen.
  • CD19 also known as B4 or Leu-12, belongs to the immunoglobulin (Ig) superfamily. Its molecular weight is 95kDa. It is located on the short arm of chromosome 16. It contains 15 exons and encodes a type I transmembrane with 556 amino acids. When glycoprotein and immunoglobulin genes are recombined, they are first expressed in late progenitor B cells and early pre-B cells. CD19 is highly expressed throughout the development and maturation of B cells, until the B cells differentiate into plasma cells, the expression is down-regulated, and its expression in mature B cells is three times that of immature cells.
  • Ig immunoglobulin
  • CD19 establishes the B cell signal threshold by simultaneously regulating the B cell receptor (B cell receptor, BCR) dependent and independent signals, and plays an important regulatory role in the development, proliferation and differentiation of B cells.
  • B cell receptor B cell receptor
  • CD19 forms a complex with receptors CD21 (CD2), CD81 (TAPA-1) and CD225, and reduces trigger B by regulating endogenous and receptor-induced signals.
  • CD21 CD2
  • CD81 TAPA-1
  • CD225 The threshold of antigen concentration required for cell division and differentiation.
  • CD81 provides a molecular docking site for signaling pathways and regulates the expression of CD19.
  • CD19 activates protein tyrosine kinase (PTK) by recruiting and amplifying the activation of Src family protein tyrosine kinases and activates BCR signaling. At the same time, when BCR signal is activated, CD19 can also activate PI3K and downstream Akt kinase to enhance BCR signal and promote B cell proliferation.
  • PTK protein tyrosine kinase
  • CD19 is expressed in both normal and malignant B lymphocytes and is regarded as one of the most reliable surface markers covering a long period of time during the development of B cells.
  • CD19 is expressed in pre-B cells, B cells and follicular dendritic cells, mantle cells, and dendritic cells in the inter-follicular T cell area.
  • CD19 can be detected in plasma cells isolated from human tissues by flow cytometry.
  • B lymphocyte tumors including B lymphocyte lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, follicular lymphoma, Burkitt lymphoma, and marginal zone lymphoma.
  • CD19 has become a specific molecular target for the treatment of B cell malignant tumors.
  • immunotherapy strategies targeting CD19 have been extensively developed in preclinical and clinical studies, including monoclonal antibodies, bispecific antibodies, and chimeric antigen receptor modified T cells (CAR-T), and have achieved significant advantages
  • CAR-T chimeric antigen receptor modified T cells
  • Adoptive immunotherapy for tumors is the introduction of autologous or foreign immunocompetent cells into the patient's body after in vitro expansion, directly killing tumor cells, regulating and enhancing the body’s immune function, mainly including LAK cells, TIL cells, activated T lymphocytes and CIK cell immunotherapy.
  • immunotherapy can only remove a small number of scattered tumor cells, and has limited efficacy for advanced solid tumors. Therefore, it is often used as an adjuvant therapy in combination with conventional methods such as surgery, chemotherapy, and radiotherapy: first use conventional methods to clean up a large number of tumor cells, and then use immunotherapy to remove the remaining tumor cells, which can improve the effect of tumor comprehensive treatment.
  • the ideal adoptive immunotherapy for tumors should be: one end of the bispecific antibody binds to the surface antigen of immune cells (such as CD3) and is then imported into the body together, while the other end of the bispecific antibody is It can bind well to the surface antigens of tumor cells; in this way, the bispecific antibody can build a bridge between tumor cells and immune cells in the body, so that the immune cells are concentrated around the tumor cells, thereby killing the tumor cells.
  • immune cells such as CD3
  • This method can effectively solve the metastasis and spread of tumor cells, and overcome the disadvantages of "incompleteness, easy metastasis, and large side effects" after the three traditional treatment methods of surgery and radiotherapy. Therefore, it is of great significance to develop highly efficient bispecific antibodies that bind tumor cells and immune cells.
  • the purpose of the present invention is to provide a bispecific antibody that binds CD19 and CD3, has a specific targeting effect, and can efficiently stimulate a targeted immune response, and a preparation method and application thereof.
  • Bispecific antibodies include (a) a monoclonal antibody unit consisting of two complete light chain-heavy chain pairs, and (b) two identical single chains containing a heavy chain variable region and a light chain variable region
  • the single-chain antibody unit of an antibody wherein the single-chain antibody unit has a specific binding capacity to the surface antigen CD3 of immune cells, and the monoclonal antibody unit has a specific binding capacity to the tumor cell surface antigen CD19; the single-chain antibody unit is connected by a connecting peptide At the N-terminal or C-terminal of
  • the present invention provides a bispecific antibody, the bispecific antibody comprising (a) a monoclonal antibody unit and (b) a single chain antibody unit; the monoclonal antibody unit is 2 complete light
  • the chain-heavy chain pair can specifically bind to CD19;
  • the single-chain antibody unit includes two single-chain antibodies (ScFv), and the single-chain antibody includes a heavy chain variable region and a light chain variable region, which can specifically bind CD3;
  • the bispecific antibody is a symmetric structure connected by any of the following methods:
  • N-terminals of the two single-chain antibodies are respectively connected to the C-terminals of the two heavy chains of the monoclonal antibody through a connecting peptide;
  • the amino acid sequence of the connecting peptide is (GGGGX)n, where X is Gly or Ser, and n is a natural number from 1 to 4 (that is, 1, 2, 3, or 4).
  • X is Gly or Ser
  • n is a natural number from 1 to 4 (that is, 1, 2, 3, or 4).
  • amino acid sequence of the connecting peptide is shown in SEQ ID NO.13.
  • the light chain sequence of the single-chain antibody is shown in SEQ ID NO. 5 or shown in SEQ ID NO. 9.
  • the heavy chain sequence of the single-chain antibody is shown in SEQ ID NO. 6 or shown in SEQ ID NO. 10.
  • Both the light chain and the heavy chain of the single-chain antibody can specifically bind to the immune cell surface antigen CD3.
  • the present invention expresses single-chain antibodies in the form of fusion peptides.
  • specific antibody structure and sequence design it is found that when single-chain antibodies and monoclonal antibodies are connected in different ways, specific single-chain antibody fusion peptide sequences can be better Improve the stability of the antibody structure and the binding of two antigens.
  • the light chain and the heavy chain of the single-chain antibody constitute a fusion peptide
  • the sequence of the fusion peptide is any one of the following:
  • the sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 18 or the amino acid sequence shown in SEQ ID No. 18
  • the amino acid sequence of a polypeptide with the same function obtained by the substitution, deletion or insertion of one or more amino acids.
  • the sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 19 or the amino acid sequence shown in SEQ ID NO. 19 is obtained by replacing, deleting or inserting one or more amino acids.
  • the amino acid sequence of the functional polypeptide is shown in SEQ ID NO. 19 or the amino acid sequence shown in SEQ ID NO. 19 is obtained by replacing, deleting or inserting one or more amino acids.
  • the bispecific antibody may be a murine antibody, a humanized antibody, a chimeric antibody or a recombinant antibody.
  • the light chain and heavy chain of the monoclonal antibody are connected by disulfide bonds.
  • the Fc fragment of the monoclonal antibody is the Fc fragment of a human or humanized antibody.
  • the human or humanized antibody includes one of IgG1 antibody, IgG2 antibody, IgG3 antibody, and IgG4 antibody.
  • the Fc fragment of the monoclonal antibody is the Fc fragment of a human or humanized IgG4 antibody.
  • the full-length sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO. 3; the full-length sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO. 1 or SEQ ID Shown in NO.20.
  • amino acid sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more amino acids means that one or more amino acid residues are different from the sequence shown but retained.
  • sequence of the biological activity of the molecule which can be a "conservatively modified variant” or modified by “conservative amino acid substitution", “conservatively modified variant” or “conservative amino acid substitution” refers to the art Amino acid substitutions known to the skilled person, making such substitutions generally does not change the biological activity of the resulting molecule. Generally speaking, those skilled in the art recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially change the biological activity. Exemplary substitutions are preferably made in accordance with the substitutions shown below:
  • the present invention provides a bispecific antibody against human CD3 and CD19, which has the heavy chain variable region, light chain variable region of the single antibody, and the heavy chain and light chain of the monoclonal antibody.
  • the present invention screened two bispecific antibodies that can retain the biological function of the corresponding monoclonal antibody to the greatest extent, and have obvious advantages in terms of production technology and medicinal properties, which bind CD3 and CD19. Its structure and sequence are as follows:
  • the light chain and heavy chain fusion peptide sequence of the single-chain antibody is shown in SEQ ID NO.16, the light chain sequence of the monoclonal antibody is shown in SEQ ID NO.3, and the heavy chain sequence of the monoclonal antibody is shown in SEQ ID NO.1;
  • the antibody structure is a symmetric structure where the C-terminals of two single-chain antibody fusion peptides are respectively connected to the N-terminals of the two heavy chains of monoclonal antibodies through the connecting peptides shown in SEQ ID NO.13 (such as Figure 2 A);
  • the present invention also provides a gene encoding the bispecific antibody.
  • codon coding rules and the degeneracy and preference of the codons those skilled in the art can design the coding gene according to the amino acid sequence of the above-mentioned bispecific antibody.
  • the full-length coding gene sequence of the light chain of the monoclonal antibody is shown in SEQ ID NO.4.
  • the full-length coding gene sequence of the heavy chain of the monoclonal antibody is shown in SEQ ID NO. 2 or shown in SEQ ID NO. 21.
  • the coding gene of the single-chain antibody when the C-terminals of the two single-chain antibodies are respectively connected to the N-terminals of the two heavy chains of the monoclonal antibody through a connecting peptide, the coding gene of the single-chain antibody The sequence is shown in SEQ ID NO.14; when the N-terminals of the two single-chain antibodies are connected to the C-terminals of the two heavy chains of the monoclonal antibody through a connecting peptide, the coding gene of the single-chain antibody The sequence is shown in SEQ ID NO.15.
  • the above-mentioned gene sequences can be used to express the bispecific antibody or be combined with other coding gene sequences of the remaining units of the bispecific antibody to express the bispecific antibody.
  • the present invention also provides biological materials containing the gene.
  • the biological material includes recombinant DNA, expression cassettes, vectors, host cells, engineered bacteria or cell lines.
  • the present invention also provides a method for preparing the bispecific antibody, which includes: constructing an expression vector containing the single-chain antibody and the encoding gene of the monoclonal antibody; introducing the expression vector into a host cell to obtain stable expression of the Host cells for bispecific antibodies; host cells are cultured and separated and purified to obtain the bispecific antibodies.
  • bispecific antibody When preparing the bispecific antibody, those skilled in the art can select host cells, expression vectors, methods for introducing expression vectors into host cells, and methods for separation and purification of antibodies as required in the art.
  • the host cell is a CHO-K1 cell.
  • the expression vector is pG4HK.
  • the construction of the expression vector can adopt conventional methods in the art.
  • the construction method of the expression vector includes: using the light chain encoding gene of the anti-CD19 monoclonal antibody through SalI and BsiWI Double enzyme digestion is connected to the expression vector pG4HK to obtain an anti-CD19 light chain expression vector, the plasmid is named pG4HK19VL; the anti-CD3 single-chain antibody coding gene and the anti-CD19 monoclonal antibody heavy chain gene are digested by Hind III and BstEII The fusion fragment was connected to the vector pG4HK19VL to obtain a bispecific antibody expression vector.
  • the separation and purification can adopt antibody separation and purification methods commonly used in the art.
  • the separation and purification includes the following steps:
  • the present invention also provides a pharmaceutical composition comprising the bispecific antibody of the present invention.
  • the pharmaceutical composition also includes other active ingredients or excipients permitted in the field of pharmacy.
  • the present invention provides any of the following applications of the bispecific antibody or the coding gene of the bispecific antibody or the biological material containing the coding gene:
  • the B cell related diseases expressed by CD19 include but are not limited to B cell related tumors and autoimmune diseases caused by B cells.
  • the B-cell related tumors are not limited to B-lymphocyte tumors and B-cell leukemia.
  • the beneficial effects of the present invention are as follows:
  • the present invention uses genetic engineering and antibody engineering methods to construct a bispecific antibody that binds to CD19 and CD3, which contains a single chain antibody and a complete monoclonal antibody structure, and the bispecific antibody fusion protein retains a complete single chain antibody.
  • the cloned antibody structure and has a highly stable symmetrical structure, better retains the biological functions of anti-CD3 single-chain antibodies and anti-CD19 monoclonal antibodies, realizing a bispecific antibody molecule with excellent anti-CD19 and anti-CD3
  • the biological functions of the two monoclonal antibodies can build a bridge between tumor cells and immune effector cells, effectively activate immune effector cells and directed immune responses, significantly enhance the effectiveness of immune cells to kill tumor cells, and minimize With the ADCC effect, it has high safety.
  • the bispecific antibody provided by the present invention has a completely symmetrical structure, it will not produce protein isomers with other structures during host expression, thereby greatly reducing the difficulty of extraction and purification processes, and has simple preparation, The advantage of high yield has broad application prospects in tumor immunotherapy.
  • Figure 1 is a schematic diagram of the structure of the cell surface antigen CD3 molecule in the background of the present invention.
  • FIG. 2 is a schematic diagram of the molecular structure of two bispecific antibodies YK001 and YK002 obtained through screening in Example 1 of the present invention, wherein A is the bispecific antibody YK001; B is the bispecific antibody YK002.
  • Figure 3 is an SDS-PAGE electrophoresis diagram of the bispecific antibodies YK001 and YK002 in Example 2 of the present invention, where A and C are reduced SDS-PAGE electrophoresis detection; B and D are non-reduced SDS-PAGE electrophoresis detection; A and B SDS-PAGE electrophoresis results of YK001 bispecific antibody; C and D are SDS-PAGE electrophoresis results of YK002 bispecific antibody; M represents protein molecular weight marker, and lane 1 is the target protein.
  • Figure 4 is a HPLC-SEC purity peak graph of bispecific antibodies YK001 and YK002 in Example 2 of the present invention, wherein A is bispecific antibody YK001; B is bispecific antibody YK002.
  • Figure 5 shows the binding efficiency of bispecific antibodies YK001 and YK002 with Raji cells determined based on flow cytometry in Example 3 of the present invention, where A is the negative control NC; B is the YK001 bispecific antibody; C is the positive control antibody (PC) Anti-CD19; D is the negative control NC; E is the YK002 bispecific antibody; F is the positive control antibody (PC) Anti-CD19.
  • Figure 6 shows the binding efficiency of bispecific antibodies YK001 and YK002 to T cells determined based on flow cytometry in Example 3 of the present invention, wherein A is the negative control NC; B is the YK001 bispecific antibody; C is the YK002 bispecific antibody Antibody, D is the positive control (PC) Anti-CD3).
  • Figure 7 is a diagram showing the results of the bispecific antibodies YK001 and YK002 effectively mediating PBMC cells to kill Raji tumor cells in Example 4 of the present invention, wherein ( ⁇ ) represents the YK001 bispecific antibody, It represents YK002 bispecific antibody, ( ⁇ ) represents Anti-CD19 monoclonal antibody ( ⁇ ) represents irrelevant control 0527 ⁇ CD3 bispecific antibody (Her2 ⁇ CD3 bispecific antibody), ( ⁇ ) represents Anti-CD3 monoclonal antibody.
  • Example 1 The structure and sequence design of CD19 ⁇ CD3 bispecific antibody
  • tumor cell surface antigen CD19 and the immune cell surface antigen CD3 were used as targets to design bispecific antibodies.
  • the present invention screens a variety of CD19 and CD3 binding bispecific antibody structures to determine bispecific antibodies with symmetrical structures including single-chain antibody units and monoclonal antibody units. structure.
  • the anti-CD19 monoclonal antibody unit is an IgG antibody, including two complete light chain-heavy chain pairs (that is, containing complete Fab and Fc domains, and the heavy chain and light chain are connected by disulfide bonds).
  • -CD3 single-chain antibody unit includes 2 single-chain antibodies (ScFv), each single-chain antibody contains a heavy chain variable region and a light chain variable region domain, which divides the heavy chain variable region and the light chain variable region through
  • the connecting peptide is constructed as a fusion peptide.
  • Single-chain antibodies and monoclonal antibodies are connected by connecting peptides. For the connection between single-chain antibodies and monoclonal antibodies, two different connection methods are designed to obtain two bispecific antibodies with different symmetric structures:
  • amino acid sequence of each domain of the bispecific antibody is as follows:
  • amino acid sequence of the heavy chain variable region of the anti-CD19 monoclonal antibody of YK001 is shown in SEQ ID NO.19, and the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO.1;
  • amino acid sequence of the heavy chain variable region of the anti-CD19 monoclonal antibody of YK002 is shown in SEQ ID NO.19, and the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO.20;
  • amino acid sequence of the light chain variable region of the anti-CD19 monoclonal antibody is shown in SEQ ID NO. 18, and the full-length amino acid sequence of the light chain is shown in SEQ ID NO. 3 (YK001 and YK002 are the same);
  • amino acid sequence of the anti-CD3 single chain antibody in YK001 is shown in SEQ ID NO.16;
  • amino acid sequence of the anti-CD3 single chain antibody in YK002 is shown in SEQ ID NO.17.
  • the coding genes of the bispecific antibodies were designed.
  • the specific sequences are as follows:
  • nucleotide sequence encoding the heavy chain of the anti-CD19 monoclonal antibody of YK001 is shown in SEQ ID NO.2.
  • nucleotide sequence encoding the heavy chain of the anti-CD19 monoclonal antibody of YK002 is shown in SEQ ID NO. 21;
  • the nucleotide sequence encoding the light chain of the anti-CD19 monoclonal antibody is shown in SEQ ID NO. 4 (YK001 and YK002 are the same);
  • nucleotide sequence encoding the anti-CD3 single-chain antibody in YK001 is shown in SEQ ID NO.14;
  • the nucleotide sequence encoding the anti-CD3 single-chain antibody in YK002 is shown in SEQ ID NO.15.
  • synthetic anti-CD19 monoclonal antibody light chain coding gene fragments (YK001 and YK002 are the same) and anti-CD3 single chain antibody coding gene-anti-CD19 monoclonal antibody heavy chain coding gene fusion fragments (YK001, That is, the C-terminus of the anti-CD3 single-chain antibody is connected to the N-terminus of the heavy chain of the anti-CD19 monoclonal antibody) and the fusion fragment of the heavy chain coding gene of the anti-CD19 monoclonal antibody-anti-CD3 single chain antibody coding gene (YK002, The N-terminus of the anti-CD3 single-chain antibody is connected to the C-terminus of the heavy chain of the anti-CD19 monoclonal antibody).
  • CHO-K1 cells Resuscitate CHO-K1 cells, inoculate 6 ⁇ 10 6 cells into 12ml CD-CHO medium (containing 6mM GlutaMAX) at a density of 0.5 ⁇ 10 6 /ml, culture in 5% CO 2 , 37°C, 135rpm shaker.
  • the cell count is used to determine the cell concentration to ensure cell viability above 95%.
  • the cells were quickly transferred to CD-CHO medium (without GlutaMAX) after the electric shock, spread in a 96-well plate, and cultured in 5% CO 2 at 37°C.
  • each well was supplemented with MSX to a final concentration of 50 ⁇ M, 5% CO 2 , and cultured at 37°C.
  • the supernatant of the fermentation culture was centrifuged at 2000 rpm for 10 min, and then filtered with a 0.22 uM filter membrane.
  • SuRe affinity chromatography column purchased from GE company, article number 18-5438-02 to capture the antibody in the pretreated fermentation broth, and fully equilibrate it with equilibration buffer (10mM PB, 0.1M NaCl, pH7.0) After the chromatography column, pass through the affinity chromatography column and eluted with the elution buffer (0.1M citric acid, pH 3.0).
  • equilibration buffer 10mM PB, 0.1M NaCl, pH7.0
  • the sample prepared by affinity chromatography was further purified by SP cation exchange chromatography.
  • the cation exchange column was purchased from GE Company (17-1014-01, 17-1014-03), and equilibrated buffer (50mM PBS, pH 5.5) After equilibrating the chromatography column, after the SP column is bound, the elution buffer (50mM PBS, 1.0M NaCl, pH 5.5) is linearly eluted with 20 column volumes.
  • the purified bispecific antibodies YK001 and YK002 were tested by SDS-PAGE and HPLC-SEC.
  • the results of SDS-PAGE are shown in Figure 3, the results of YK001 reduction SDS-PAGE electrophoresis detection are shown in Figure 3 A, and the non-reducing SDS-PAGE electrophoresis detection results are shown in Figure 3 B; YK002 reduction SDS-PAGE
  • the result of electrophoresis detection is shown in Figure 3 C, and the result of non-reducing SDS-PAGE electrophoresis detection is shown in Figure 3 D.
  • the HPLC-SEC detection result is shown in Figure 4, where the SEC detection result of YK001 is shown in Figure 4A, and the SEC detection result of YK002 is shown in Figure 4B.
  • the test results showed that the bispecific antibodies YK001 and YK002 were successfully prepared after expression and purification, and the purity of the purified bispecific antibodies was above 95%.
  • Collect Raji cells Collect 1 ⁇ 10 6 cells/tube.
  • Bs-antibody binding add bispecific antibodies YK001 and YK002 to 5 ⁇ g/ml respectively, and incubate on ice for 45min.
  • Rinse cells Add 1ml staining buffer to the cell suspension, mix well, centrifuge at 350 ⁇ g, 4°C for 5 min, remove the supernatant, and rinse again. After centrifugation, resuspend the cells with 100 ⁇ l staining buffer.
  • Rinse cells add 1ml staining buffer to the cell suspension, mix well, centrifuge at 350 ⁇ g, 4°C for 5 min, remove the supernatant, and rinse again.
  • On-board testing After resuspending the cells with 200 ⁇ l PBS, the flow cytometer can test on the computer.
  • the results of flow cytometry are shown in Figure 5.
  • the results of YK001 binding to Raji cells are shown in Figure 5 A, B and C, and the results of YK002 binding to Raji cells are shown in Figure 5 D, E and F
  • the results show that the bispecific antibodies YK001 and YK002 can specifically bind to Raji cells, that is, the bispecific antibody fusion protein retains the binding function of the monoclonal antibody Anti-CD19.
  • Collect T cells Collect 1 ⁇ 10 6 cells/tube.
  • Bs-antibody binding add bispecific antibodies YK001 and YK002 to 5 ⁇ g/ml respectively, and incubate on ice for 45min.
  • the results of flow cytometry are shown in Figure 6.
  • the results of YK001 binding to T cells are shown in Figure 6 A and B, and the results of YK002 binding to T cells are shown in Figure 6 C and D.
  • the results show that, Both the bispecific antibodies YK001 and YK002 can specifically bind to T cells, that is, the bispecific antibody fusion protein retains the binding function of the single-chain antibody Anti-CD3.
  • Raji-Luc was used as the target cell
  • PBMC was used as the immune effector cell to detect the killing effect of the bispecific antibody YK001 and YK002 mediated by the target cell
  • the anti-CD3 monoclonal antibody and anti-CD19 monoclonal antibody And 0527 ⁇ CD3 bispecific antibody as control.
  • Raji-Luc (Luciferase-labeled Raji cells) as target cells
  • the target cells were counted after being evenly blown, centrifuged at 1000 rpm for 5 min, and washed once with PBS. After centrifugation and washing of the target cells, the density is adjusted to 0.2 ⁇ 10 6 /ml with GT-T551 medium, and 50 ⁇ l is added to each well, so there are 10,000 cells in each well.
  • PBMC effector cells. Take out the PBMC frozen in the liquid nitrogen tank (refer to cell cryopreservation and recovery) and thaw it, add it to a 15ml centrifuge tube containing PBS or GT-T551 medium, centrifuge at 1000 rpm for 5 min, and wash twice with PBS or GT-T551 medium Count the number of cells, activity, and density, and adjust the density of viable cells to 2 ⁇ 10 6 /ml. Add 50 ⁇ l to each well, and there are 100,000 cells in each well.
  • the bispecific antibodies YK001 and YK002 were diluted with GT-T551 medium, and the initial concentration of antibodies YK001 and YK002 was adjusted to 10 nM. Sequentially dilute at a ratio of 1:5. Add 100 ⁇ l of the diluted antibody to the cells prepared above, mix well, put the 96-well plate back into the incubator, and test the killing effect after 18 hours.
  • steay-GLO promega
  • the calculation formula of the target cell killing ratio is as follows:
  • Target cell killing ratio 100 ⁇ (Only target-test well)/Only target;
  • bispecific antibodies YK001 and YK002 can effectively mediate PBMC to kill the tumor cell line Raji -Luc, YK001 and YK002 single molecules have the biological functions of Anti-CD19 and Anti-CD3 monoclonal antibodies at the same time, and YK001 is more effective in killing target cells than YK002.
  • the invention provides a bispecific antibody and its preparation method and application.
  • the bispecific antibody of the present invention includes a monoclonal antibody unit and a single-chain antibody unit; wherein, the monoclonal antibody unit is two complete light chain-heavy chain pairs that specifically bind to tumor cell surface antigen; the single-chain antibody unit includes 2 A single chain antibody, which includes a heavy chain variable region and a light chain variable region, specifically binds to immune cell surface antigens; the bispecific antibody provided by the present invention is a symmetrical structure connected by any of the following methods : (1) The C-terminus of the two single-chain antibodies are connected to the N-terminus of the two heavy chains of the monoclonal antibody through a connecting peptide; The C ends of the two heavy chains are connected.
  • the bispecific antibody of the present invention can simultaneously bind to immune cells and tumor cells, mediate a directed immune response, effectively kill tumor cells, and has good economic value and application prospects.

Abstract

本发明涉及一种双特异性抗体及其制备方法与应用。本发明的双特异性抗体包括单克隆抗体单元和单链抗体单元；其中，单克隆抗体单元为2个完整的轻链-重链对，特异性结合肿瘤细胞表面抗原；单链抗体单元包括2个单链抗体，单链抗体包括重链可变区和轻链可变区，特异性结合免疫细胞表面抗原；本发明提供的双特异性抗体为通过如下任意一种方式连接而成的对称结构：（1）2个单链抗体的C端分别通过连接肽与单克隆抗体的2条重链的N端连接；（2）2个单链抗体的N端分别通过连接肽与单克隆抗体的2条重链的C端连接。本发明的双特异性抗体能够同时结合免疫细胞和肿瘤细胞，介导导向性免疫反应，有效杀伤肿瘤细胞。

Description

一种双特异性抗体及其制备方法与应用
交叉引用
本申请要求2019年3月19日提交的专利名称为“一种双特异性抗体及其制备方法与应用”的第201910209330.4号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及生物技术及免疫学技术领域，具体涉及一种结合CD19和CD3的双特异性抗体及其制备方法与应用。
背景技术
抗体药物是以细胞工程技术和基因工程技术为主体的抗体工程技术制备的生物大分子药物，具有特异性高、性质均一、可针对特定靶点定向制备等优点。单克隆抗体在临床上主要应用于以下三个方面：肿瘤治疗、免疫性疾病治疗以及抗感染治疗。其中，肿瘤治疗是目前单克隆抗体应用最为广泛的领域，目前已经进入临床试验和上市的单克隆抗体产品中，用于肿瘤治疗的产品数量占比大概为50％。单克隆抗体***是一种针对病变细胞特异靶点刺激免疫***杀伤靶细胞的免疫疗法，为了增强抗体的效应功能，特别是杀伤肿瘤细胞的效果，人们尝试多种方法改造抗体分子，双特异性抗体是改善抗体治疗效果的发展方向之一，现已成为抗体工程研究领域的热点。
双特异性抗体(bispecific antibody，BsAb)是含有两种能够特异性识别并结合不同抗原或不同抗原位点的人工抗体。如果这两种抗原位于不同的细胞表面，则这种双特异性抗体能在这两种抗原分子之间架起桥梁，从而形成细胞之间的交联，介导细胞产生导向性的效应功能。BsAb在生物医学、特别是在肿瘤的免疫治疗中具有广阔的应用前景。用于免疫治疗的双特异性抗体(免疫双抗)是含有2种特异性细胞受体抗原结合位点的人工抗体，能在病变细胞(靶细胞)和功能细胞(免疫细胞)之间架起桥梁，激发具有导向性的免疫反应。通过BsAb介导免疫细胞(如T细胞，NK细胞等)杀死肿瘤细胞是目前免疫治疗应用研究的热点，其作用机理是BsAb能同时结合肿瘤相关抗原和免疫效应细胞上的靶分子，在活化免疫细胞的同时，直接导向免疫效应细胞对肿瘤细胞的特异性杀伤。
双特异性抗体可通过多种途径获得，其制备方法主要有：化学偶联法、杂交-杂交瘤法和基因工程抗体制备法。化学偶联法是将2个不同的单克隆抗体用化学偶联的 方式连接在一起，制备的双特异性单克隆抗体，这是最早的双特异性单克隆抗体。杂交-杂交瘤法是通过细胞杂交法或者三元杂交瘤的方式产生双特异性单克隆抗体，这些细胞杂交瘤或者三元杂交瘤是通过建成的杂交瘤融合，或者建立的杂交瘤和从小鼠的淋巴细胞融合而得到的，只能用于生产鼠源的双特异性抗体，因此，其应用受到了极大的限制。而随着分子生物学技术的迅速发展，出现了基因工程人源化或全人源的双特异性抗体的多种构建模式，主要包括双特异性微抗体、双链抗体、单链双价抗体、多价双特异性抗体四类。目前，国际上已有数种基因工程双特异性抗体药物进入临床试验阶段，并显示有较好的应用前景。
T细胞表面的CD3分子由4个亚基组成：δ、ε、γ、ζ，其分子质量分别为18.9k Da，23.1kDa，20.5kDa和18.7kDa，其长度分别为171、207、182、164个氨基酸残基。它们一起组成6条肽链，常与T细胞受体(T cell receptor，TCR)紧密结合形成含有8条肽链的TCR-CD3复合体，其结构示意图见图1。该复合体具有T细胞活化、信号转导以及稳定TCR结构的功能。CD3胞质段含有免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motif，ITAM)，TCR识别并结合由MHC(major histo-compatibility complex)分子提呈的抗原肽，导致CD3的ITAM的保守序列中的酪氨酸残基被T细胞内的酪氨酸蛋白激酶p56lck磷酸化，然后募集其它含有SH2(Scr homology 2)结构域的酪氨酸蛋白激酶(如ZAP-70)。ITAM的磷酸化以及与ZAP-70的结合是T细胞活化信号传导过程早期阶段的重要生化反应之一。因此，CD3分子的功能是转导TCR识别抗原所产生的活化信号。
CD19又名B4或Leu-12，属于免疫球蛋白(Ig)超家族成员，其分子量为95kDa，位于16号染色体的短臂上，含有15个外显子，编码556个氨基酸的I型跨膜糖蛋白，免疫球蛋白基因重组时，其最早在晚期祖B细胞和早期前B细胞中表达。CD19在整个B细胞的发育和成熟过程中均高表达，直至B细胞分化为浆细胞时，表达量下调，其在成熟B细胞中的表达是未成熟细胞的3倍。
CD19通过同时调节B细胞受体(B cell receptor，BCR)依赖和非依赖信号建立B细胞信号阈值，对B细胞的发育、增殖和分化起着重要的调控作用。CD19作为成熟B细胞的表面多分子复合物的主要组成部分，与受体CD21(CD2)，CD81(TAPA-1)以及CD225共同形成复合体，通过调节内源性和受体诱导信号减少触发B细胞***及分化所需抗原浓度的阈值。CD81作为伴侣蛋白，提供信号传导途径的分子对接位点，并且调节CD19的表达。CD19通过招募和放大Src家族蛋白酪氨酸激酶的活化，使蛋白酪氨酸激酶(PTK)激活，激活BCR信号。同时当BCR信号激活时，CD19也可 通过活化PI3K和下游的Akt激酶，增强BCR信号，促进B细胞的增殖。
CD19在正常及恶性B淋巴细胞中均有表达，被视为B细胞发育过程中一个涵盖阶段较长的最为可靠的表面标记物之一。在正常淋巴组织中，CD19在前B细胞、B细胞和滤泡树突状细胞、套细胞、滤泡间T细胞区的树突状大细胞中表达。此外，通过流式细胞学方法检测，CD19在人体组织分离得到的浆细胞中可以检测到。通常来说，CD19在B淋巴细胞瘤中表达，其中包括B淋巴细胞淋巴瘤、小淋巴细胞淋巴瘤、套细胞淋巴瘤、滤泡淋巴瘤、Burkitt淋巴瘤、边缘区淋巴。因此，CD19成为B细胞恶性肿瘤治疗的特异性分子靶点。近年来，靶向CD19的免疫治疗策略在临床前以及临床研究中广泛发展，包括单克隆抗体、双特异性抗体和嵌合抗原受体修饰T细胞(CAR-T)，并取得了显著优于常规小分子化疗方案的临床效果，推动了免疫治疗的进展。
肿瘤的过继免疫治疗是将自体或异体的免疫活性细胞经过体外扩增后输入患者体内，直接杀伤肿瘤细胞，调节和增强机体的免疫功能，主要包括LAK细胞、TIL细胞、激活的T淋巴细胞和CIK细胞的免疫治疗。而免疫疗法只能清除少量的、零散的肿瘤细胞，对于晚期的实体肿瘤疗效有限。故常将其作为一种辅助疗法与手术、化疗、放疗等常规方法联合应用：先用常规方法清扫大量的肿瘤细胞后，再用免疫疗法清除残存的肿瘤细胞，可提高肿瘤综合治疗的效果。过继免疫治疗作为肿瘤综合治疗中的一个新方法，已经与常规手术治疗、放疗、化疗及其他细胞和分子治疗广泛配合，在多种肿瘤的治疗中具有广泛的应用前景。然而，结合双特异性抗体，理想的肿瘤过继免疫治疗应为：双特异性抗体的一端结合免疫细胞的表面抗原(如CD3)，并随之一起输入体内，而双特异性抗体的另一端则能够很好地结合肿瘤细胞的表面抗原；这样，双特异性抗体就能在体内架起肿瘤细胞和免疫细胞之间的桥梁，使免疫细胞集中在肿瘤细胞周围，进而杀伤肿瘤细胞。通过这种方法可有效解决肿瘤细胞的转移和扩散，克服了手术、放化疗三大传统治疗方式后的“不彻底、易转移、副作用大”等弊端。因此，开发高效的、结合肿瘤细胞和免疫细胞的双特异性抗体具有重要意义。
发明内容
为解决现有技术中存在的技术问题，本发明的目的在于提供一种结合CD19和CD3、具有特异的靶向作用、能够高效激发导向性免疫反应的双特异性抗体及其制备方法与应用。
为实现上述目的，本发明的技术方案如下：本发明通过对结合CD19和CD3的双特异性抗体的分子结构进行设计和筛选，创造性地发现具有如下对称结构的双特异 性抗体与其对应的单克隆抗体和其它结构的双特异性抗体相比，能够更好地保留原抗体的特异性结合能力，同时具有两个单克隆抗体的生物学功能，在生产工艺和药用性能等方面具有明显优势：双特异性抗体包括(a)由2个完整的轻链-重链对组成的单克隆抗体单元，和(b)含有重链可变区和轻链可变区的2个完全相同的单链抗体的单链抗体单元，其中，单链抗体单元对免疫细胞的表面抗原CD3具有特异性结合能力，单克隆抗体单元对肿瘤细胞表面抗原CD19具有特异性结合能力；单链抗体单元通过连接肽连接于单克隆抗体单元的N端或C端。本发明开发了具有上述抗体分子结构的结合CD19和CD3的双特异性抗体，该双特异性抗体具有特异性的靶向作用，并能够高效激发具有导向性的免疫反应，杀伤肿瘤细胞。
具体地，首先，本发明提供一种双特异性抗体，所述双特异性抗体包括(a)单克隆抗体单元和(b)单链抗体单元；所述单克隆抗体单元为2个完整的轻链-重链对，能够特异性结合CD19；所述单链抗体单元包括2个单链抗体(ScFv)，所述单联抗体包括重链可变区和轻链可变区，能够特异性结合CD3；所述双特异性抗体为通过如下任意一种方式连接而成的对称结构：
(1)所述2个单链抗体的N端分别通过连接肽与所述单克隆抗体的2条重链的C端连接；
(2)所述2个单链抗体的C端分别通过连接肽与所述单克隆抗体的2条重链的N端连接。
作为优选，所述连接肽的氨基酸序列为(GGGGX)n，其中，X为Gly或Ser，n为1-4的自然数(即为1、2、3或4)。当采用上述序列的连接肽时，所述双特异性抗体能够更好的发挥抗原结合功能。
作为本发明的一种优选实施方式，所述连接肽的氨基酸序列如SEQ ID NO.13所示。
作为优选，所述单链抗体的轻链序列如SEQ ID NO.5所示或如SEQ ID NO.9所示。
所述单链抗体的重链序列如SEQ ID NO.6所示或如SEQ ID NO.10所示。
所述单链抗体的轻链和重链均能够特异性结合免疫细胞表面抗原CD3。
本发明采用融合肽的形式表达单链抗体，通过特定的抗体结构和序列设计，发现当单链抗体与单克隆抗体的连接方式不同时，分别采用特定的单链抗体融合肽序列能够更好地提升抗体结构的稳定性以及与两种抗原的结合。
对于所述双特异性抗体的单链抗体单元，作为优选，所述单链抗体的轻链和重链 组成融合肽，所述融合肽的序列为如下任意一种：
(1)当所述2个单链抗体的N端分别通过连接肽与所述单克隆抗体的2条重链的C端连接时，所述融合肽的序列如SEQ ID NO.17所示；
(2)当所述2个单链抗体的C端分别通过连接肽与所述单克隆抗体的2条重链的N端连接时，所述融合肽的序列如SEQ ID NO.16所示。
对于所述双特异性抗体的单克隆抗体单元，作为优选，所述单克隆抗体的轻链可变区的序列如SEQ ID NO.18所示或为如SEQ ID NO.18所示的氨基酸序列经一个或多个氨基酸的替换、缺失或***得到的具有相同功能多肽的氨基酸序列。
所述单克隆抗体的重链可变区的序列如SEQ ID NO.19所示或为如SEQ ID NO.19所示的氨基酸序列经一个或多个氨基酸的替换、缺失或***得到的具有相同功能多肽的氨基酸序列。
本发明中，所述双特异性抗体可以为鼠源抗体、人源化抗体、嵌合抗体或重组抗体。
作为本发明的一种实施方式，所述单克隆抗体的轻链和重链通过二硫键连接。所述单克隆抗体的Fc片段为人或人源化抗体的Fc片段。
作为优选，所述人或人源化抗体包括IgG1抗体、IgG2抗体、IgG3抗体、IgG4抗体中的一种。
作为本发明的一种优选实施方式，所述单克隆抗体的Fc片段为人或人源化IgG4抗体的Fc片段。
作为本发明的一种优选实施方式，所述单克隆抗体的轻链全长序列如SEQ ID NO.3所示；所述单克隆抗体的重链全长序列如SEQ ID NO.1或SEQ ID NO.20所示。
本发明中，上述“经一个或多个氨基酸的替换、缺失或***得到的具有相同功能的蛋白的氨基酸序列”是指在一个或多个氨基酸残基处不同于所示的序列但保留所得到的分子的生物学活性的序列，其可为“保守修饰的变体”或经“保守的氨基酸取代”改造得到的，“保守修饰的变体”或经“保守的氨基酸取代”是指本领域技术人员已知的氨基酸取代，进行这种取代通常不改变所得到的分子的生物学活性。一般而言，本领域技术人员公认在多肽非必需区的单个氨基酸取代基本上不改变生物学活性。示例性取代优选依照以下所示的取代进行：
表1 例示性保守氨基酸取代表
原残基 保守取代
Ala(A) Gly,Ser
Arg(R) Lys,His
Asn(N) Gln,His
Asp(D) Glu,Asn
Cys(C) Ser,Ala
Gln(Q) Asn
Glu(E) Asp,Gln
Gly(G) Ala
His(H) Asn,Gln
Ile(I) Leu,Val
Lys(K) Arg,His
Met(M) Leu,Ile,Tyr
Phe(F) Tyr,Met,Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr,Phe
Tyr(Y) Trp,Phe
Val(V) Ile,Leu
作为上述双特异性抗体的示例，本发明提供针对人的CD3和CD19的双特异性抗体，在具有上述单联抗体重链可变区、轻链可变区以及单克隆抗体的重链和轻链结构和序列中，本发明筛选得到2个能够最大程度地保留对应单克隆抗体的生物学功能，且在生产工艺及药用性能等方面具有明显优势的结合CD3和CD19的双特异性抗体，其结构和序列如下：
(1)单链抗体的轻链和重链融合肽的序列如SEQ ID NO.16所示，单克隆抗体的轻链序列如SEQ ID NO.3所示，单克隆抗体的重链序列如SEQ ID NO.1所示；抗体结构为2个单链抗体融合肽的C端分别通过序列如SEQ ID NO.13所示的连接肽连接于单克隆抗体2条重链的N端的对称结构(如图2的A所示)；
(2)单链抗体的轻链和重链融合肽的序列如SEQ ID NO.17所示，单克隆抗体的轻链序列如SEQ ID NO.3所示，单克隆抗体的重链序列如SEQ ID NO.20所示；抗体结构为2个单链抗体融合肽的N端分别通过序列如SEQ ID NO.13所示的连接肽连接于单克隆抗体两条重链的C端的对称结构(如图2的B所示)。
在上述双特异性抗体氨基酸序列的基础上，本发明还提供编码所述双特异性抗体的基因。
根据密码子编码规则以及密码子的简并性和偏好性，本领域技术人员可以根据上述双特异性抗体的氨基酸序列设计编码基因。
作为本发明的一种优选实施方式，所述单克隆抗体的轻链全长的编码基因序列如SEQ ID NO.4所示。
作为本发明的一种优选实施方式，所述单克隆抗体的重链全长的编码基因序列如SEQ ID NO.2所示或如SEQ ID NO.21所示。
作为本发明的一种优选实施方式，当所述2个单链抗体的C端分别通过连接肽与所述单克隆抗体的2条重链的N端连接时，所述单链抗体的编码基因序列如SEQ ID NO.14所示；当所述2个单链抗体的N端分别通过连接肽与所述单克隆抗体的2条重链的C端连接时，所述单链抗体的编码基因序列如SEQ ID NO.15所示。
上述基因序列可以组合用于表达所述双特异性抗体或分别与所述双特异性抗体的其余单元的其它编码基因序列组合用于表达所述双特异性抗体。
进一步地，本发明还提供包含所述基因的生物材料。
本发明中，所述生物材料包括重组DNA、表达盒、载体、宿主细胞、工程菌或细胞系。
本发明还提供所述双特异性抗体的制备方法，包括：构建含有所述单链抗体和所述单克隆抗体的编码基因的表达载体；将所述表达载体导入宿主细胞，获得稳定表达所述双特异性抗体的宿主细胞；培养宿主细胞，经分离纯化获得所述双特异性抗体。
在制备所述双特异性抗体时，本领域技术人员可根据需要选择本领域常规的宿主细胞、表达载体、将表达载体导入宿主细胞的方法以及抗体的分离纯化方法。
作为本发明的一种实施方式，所述的宿主细胞为CHO-K1细胞。
作为本发明的一种实施方式，所述表达载体为pG4HK。
所述表达载体的构建可以采用本领域常规方法，作为本发明的一种优选实施方式，所述表达载体的构建方法包括：将所述抗CD19单克隆抗体的轻链编码基因，通过SalI与BsiWI双酶切连接到表达载体pG4HK，获得抗CD19轻链的表达载体，质粒命名为pG4HK19VL；通过Hind III与BstEII双酶切将抗CD3单链抗体编码基因和抗CD19单克隆抗体的重链基因的融合片段连接到载体pG4HK19VL，获得双特异性抗体表达载体。
所述分离纯化可采用本领域常用的抗体分离纯化方法。
作为本发明的一种实施方式，所述的分离纯化包括如下步骤：
(1)通过重组rProtein A亲和层析柱从培养物上清中分离所有带Fc结构域的抗体；
(2)通过阴离子交换Q柱层析将双特异性抗体与副产物分离；
(3)通过分子筛层析纯化双特异性抗体。
在上述双特异性抗体的基础上，本发明还提供一种药物组合物，其包含本发明所述双特异性抗体。
作为优选，所述药物组合物还包括药学领域允许的其它有效成分或辅料。
进一步地，本发明提供所述双特异性抗体或所述双特异性抗体的编码基因或含有所述编码基因的生物材料的如下任一应用：
(1)在制备预防或治疗CD19表达的B细胞相关疾病的药物中的应用；
(2)在制备预防或治疗以CD19为靶标的疾病的药物中的应用；
(3)在制备用于杀伤CD19表达细胞的药物中的应用；
(4)在制备CD19和/或CD3的检测试剂中的应用。
本发明中，所述CD19表达的B细胞相关疾病包括但不限于B细胞相关肿瘤、B细胞引起的自身免疫病。
所述B细胞相关肿瘤但不限于B淋巴细胞瘤、B细胞性白血病。
本发明的有益效果如下：本发明利用基因工程和抗体工程方法构建包含单链抗体和完整单克隆抗体结构的结合CD19和CD3的双特异性抗体，该双特异性抗体融合蛋白保留了完整的单克隆抗体结构，而且具有高度稳定的对称结构，更好地保留了抗CD3单链抗体和抗CD19单克隆抗体的生物学功能，实现了一个双特异性抗体分子同时具有优异的抗CD19和抗CD3两个单克隆抗体的生物学功能，能够在肿瘤细胞和免疫效应细胞之间搭建桥梁，有效激活免疫效应细胞和导向性免疫反应，显著增强了免疫细胞杀伤肿瘤细胞的功效，同时最大程度地降低了ADCC效应，具有较高的安全性。此外，本发明提供的双特异性抗体由于具有结构完全对称的特点，在进行宿主表达时，不会产生其它结构的蛋白异构体，从而大大降低了提取和纯化工艺的难度，具有制备简单、产量高的优势，在肿瘤的免疫治疗中具有广阔的应用前景。
附图说明
图1为本发明背景技术中细胞表面抗原CD3分子的结构示意图。
图2为本发明实施例1中经筛选获得的2个双特异抗体YK001和YK002的分子结构示意图，其中，A为双特异抗体YK001；B为双特异抗体YK002。
图3为本发明实施例2中双特异抗体YK001和YK002的SDS-PAGE电泳图，其 中，A和C为还原SDS-PAGE电泳检测；B和D为非还原SDS-PAGE电泳检测；A和B为YK001双特异抗体SDS-PAGE电泳结果；C和D为YK002双特异抗体SDS-PAGE电泳结果；M代表蛋白分子量标记，泳道1为目的蛋白。
图4为本发明实施例2中双特异抗体YK001和YK002的HPLC-SEC纯度峰形图，其中，A为双特异抗体YK001；B为双特异抗体YK002。
图5为本发明实施例3中基于流式细胞分析方法测定的双特异抗体YK001和YK002与Raji细胞的结合效率，其中，A为阴性对照NC；B为YK001双特异抗体；C为阳性对照抗体(PC)Anti-CD19；D为阴性对照NC；E为YK002双特异抗体；F为阳性对照抗体(PC)Anti-CD19。
图6为本发明实施例3中基于流式细胞分析方法测定的双特异抗体YK001和YK002与T细胞的结合效率，其中，A为阴性对照NC；B为YK001双特异抗体；C为YK002双特异抗体，D为阳性对照(PC)Anti-CD3)。
图7为本发明实施例4中双特异抗体YK001和YK002有效介导PBMC细胞杀伤Raji肿瘤细胞的结果图，其中，(▼)表示YK001双特异抗体，
Figure PCTCN2020076516-appb-000001
表示YK002双特异抗体，(■)表示Anti-CD19单克隆抗体(◇)表示无关对照0527×CD3双特异性抗体(Her2×CD3双特异性抗体)，(●)表示Anti-CD3单克隆抗体。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用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 CD19×CD3双特异性抗体的结构和序列设计
本实施例以肿瘤细胞表面抗原CD19和免疫细胞表面抗原CD3作为靶点，设计双特异性抗体。
结合蛋白质结构设计软件和大量的人工实验筛选，本发明在多种结合CD19和CD3的双特异性抗体结构中筛选确定了包括单链抗体单元和单克隆抗体单元的具有对称结构的双特异性抗体结构。其中，抗-CD19单克隆抗体单元为IgG抗体，包括2个完整的轻链-重链对(即含有完整的Fab和Fc结构域，重链和轻链之间通过二硫键连接)，抗-CD3单链抗体单元包括2个单链抗体(ScFv)，每个单链抗体均包含重链可变区和轻链可变区 结构域，将重链可变区和轻链可变区经连接肽构建为融合肽。单链抗体和单克隆抗体之间采用连接肽连接，对于单链抗体和单克隆抗体的连接方式，设计2种不同的连接方式，由此获得两种不同对称结构的双特异性抗体：
(1)将抗-CD3单链抗体的C端与抗-CD19单克隆抗体的重链N端通过连接肽GGGGSGGGGSGGGGS(如SEQ ID NO.13所示)相连，获得双特异性抗体YK001(结构示意图如图2的A所示)；
(2)将抗-CD3单链抗体的N端与抗-CD19单克隆抗体的重链C端通过连接肽GGGGSGGGGSGGGGS(如SEQ ID NO.13所示)相连，获得双特异性抗体YK002(结构示意图如图2的B所示)。
上述双特异性抗体各结构域的氨基酸序列如下：
YK001的抗-CD19单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.19所示，重链全长氨基酸序列如SEQ ID NO.1所示；
YK002的抗-CD19单克隆抗体的重链可变区氨基酸序列如SEQ ID NO.19所示，重链全长氨基酸序列如SEQ ID NO.20所示；
抗-CD19单克隆抗体的轻链可变区的氨基酸序列如SEQ ID NO.18所示，轻链全长氨基酸序列如SEQ ID NO.3所示(YK001和YK002相同)；
YK001中抗-CD3单链抗体的氨基酸序列如SEQ ID NO.16所示；
YK002中抗-CD3单链抗体的氨基酸序列如SEQ ID NO.17所示。
实施例2 CD19×CD3双特异性抗体的制备
1、双特异性抗体编码基因设计与合成
根据实施例1中设计筛选得到的2个双特异性抗体YK001和YK002的氨基酸序列和宿主细胞的密码子偏好性，设计双特异性抗体的编码基因，具体序列如下：
YK001的抗-CD19单克隆抗体的重链编码核苷酸序列如SEQ ID NO.2所示；
YK002的抗-CD19单克隆抗体的重链编码核苷酸序列如SEQ ID NO.21所示；
抗-CD19单克隆抗体的轻链编码核苷酸序列如SEQ ID NO.4所示(YK001和YK002相同)；
YK001中抗-CD3单链抗体的编码核苷酸序列如SEQ ID NO.14所示；
YK002中抗-CD3单链抗体的编码核苷酸序列如SEQ ID NO.15所示。
为便于表达载体构建，合成抗-CD19单克隆抗体的轻链编码基因片段(YK001和YK002相同)以及抗CD3单链抗体编码基因-抗CD19单克隆抗体的重链编码基因的融合片段(YK001，即将抗-CD3单链抗体的C端与抗-CD19单克隆抗体的重链N端相连)和抗 CD19单克隆抗体的重链编码基因-抗CD3单链抗体编码基因的融合片段(YK002，即将抗-CD3单链抗体的N端与抗-CD19单克隆抗体的重链C端相连)。
2、双特异性抗体表达载体构建
(1)通过SalI与BsiWI双酶切将抗-CD19单克隆抗体的轻链编码基因连接到表达载体pG4HK，获得抗-CD19单克隆抗体轻链的表达载体，质粒命名为pG4HK19VL。
(2)通过Hind III与BstE II双酶切将抗CD3单链抗体编码基因-抗CD19单克隆抗体的重链编码基因的融合片段连接到载体pG4HK19VL，获得YK001双特异性抗体表达载体，质粒命名为pG4HK-YK001。
(3)通过Hind III与BstE II双酶切将抗CD19单克隆抗体的重链编码基因-抗CD3单链抗体编码基因的融合片段连接到载体pG4HK19VL，获得抗YK002双特异性抗体表达载体，质粒命名为pG4HK-YK002。
3、双特异性抗体的表达
(1)利用无内毒素大提试剂盒(Tiagen，4991083)进行质粒大提，具体操作按照试剂盒说明书进行。
(2)转染用细胞准备
①复苏CHO-K1细胞，接种6×10 6细胞到12ml CD-CHO培养基(含6mM GlutaMAX)中，密度为0.5×10 6/ml，于5％CO 2，37℃，135rpm摇床培养。
②转染前一天，调整细胞密度到0.5×10 6ml，于5％CO 2，37℃，135rpm摇床培养。
(3)电穿孔转染
①细胞计数测定细胞浓度，保证细胞活力95％以上。
②取1×10 7细胞，1000rpm离心5分钟，弃上清，用新鲜的CD-CHO培养基悬浮细胞，1000rpm离心5分钟，弃上清。再重复洗一次。
③用0.7ml CD-CHO培养基悬浮细胞，并加入40μg表达载体，混合均匀，转移至0.4cm电转杯中，电击。
④将电击后细胞迅速转移到CD-CHO培养基(without GlutaMAX)中，铺于96孔板中，于5％CO 2，37℃培养。
⑤转染后24小时每孔补加MSX至终浓度为50μM，5％CO 2，37℃培养。
⑥挑取高表达双特异性抗体的单克隆细胞株，进行分批补液发酵培养，培养14天后收集上清。
4、双特异性抗体的纯化
(1)料液预处理
发酵培养的上清液通过2000rpm离心，10min，然后用0.22uM滤膜过滤处理。
(2)亲和层析
用Mabselect SuRe亲和层析柱(购自GE公司，货号18-5438-02)捕获经过预处理的发酵液中的抗体，用平衡缓冲液(10mM PB，0.1M NaCl，pH7.0)充分平衡层析柱后，过亲和层析柱，用洗脱缓冲液(0.1M柠檬酸酸，pH 3.0)洗脱。
(3)阳离子交换层析
经亲和层析制备的样品，进一步通过SP阳离子交换层析纯化，阳离子交换柱购自GE公司(17-1014-01，17-1014-03)，用平衡缓冲液(50mM PBS，pH 5.5)平衡层析柱后，过SP柱子结合后，用洗脱缓冲液(50mM PBS，1.0M NaCl，pH 5.5)20个柱体积线性洗脱。
(4)阴离子交换层析
经SP阳离子交换层析纯化后，再进一步经过离子交换Q柱(购自GE公司，货号：17-1153-01，17-1154-01)，所用的缓冲液为50mM PBS，pH 5.5。
经上述纯化后的双特异性抗体YK001和YK002进行SDS-PAGE和HPLC-SEC检测。SDS-PAGE的结果如图3所示，YK001的还原SDS-PAGE电泳检测结果如图3的A所示，非还原SDS-PAGE电泳检测结果如图3的B所示；YK002的还原SDS-PAGE电泳检测结果如图3的C所示，非还原SDS-PAGE电泳检测结果如图3的D所示。HPLC-SEC检测结果如图4所示，其中，YK001的SEC检测结果如图4的A所示，YK002的SEC检测结果如图4的B所示。检测结果表明，经表达和纯化成功制备得到了双特异性抗体YK001和YK002，经纯化后的双特异性抗体的纯度在95％以上。
实施例3 双特异性抗体与肿瘤细胞和免疫细胞的结合活性测定
以Raji(购自ATCC，CCL-86)作为CD19阳性的细胞，T细胞作为CD3阳性的细胞，利用流式细胞分析法检测本发明的双特异性抗体与表达CD19的肿瘤细胞和表达CD3的免疫细胞的靶抗原的结合活性。
1、利用流式分析法检测双特异性抗体与Raji细胞的结合活性
(1)收集Raji细胞：收集1×10 6cells/tube。
(2)漂洗细胞：用1ml staining buffer(含有0.5％w/v BSA+2mM EDTA的PBS)漂洗细胞一次，350×g，4℃离心5min，离心后用200μl staining buffer重悬细胞。
(3)Bs-antibody binding:分别加入双特异抗体YK001和YK002至5μg/ml，冰上孵育45min。
(4)漂洗细胞：在细胞悬液中加入1ml staining buffer，混匀，350×g，4℃离心5min， 去上清，重新漂洗一次。离心后用100μl staining buffer重悬细胞。
(5)样品管加入biolegend抗体5μl(PE anti-human IgG Fc Antibody,Biolegend,409304)，同型对照管加入同型对照(PE Mouse IgG2a,κIsotype Ctrl(FC)Antibody，Biolegend,400213)，冰上避光孵育15min。
(6)漂洗细胞：在细胞悬液中加入1ml staining buffer，混匀，350×g，4℃离心5min，去上清，重新漂洗一次。
(7)上机检测：用200μl PBS重悬细胞后，流式细胞仪上机检测即可。
流式细胞检测结果如图5所示，其中，YK001结合Raji细胞的检测结果如图5的A、B和C所示，YK002结合Raji细胞的检测结果如图5的D、E和F所示，结果表明，双特异抗体YK001和YK002均能够特异性地结合Raji细胞，即双特异抗体融合蛋白保留了单克隆抗体Anti-CD19的结合功能。
2、利用流式分析法检测双特异性抗体与T细胞的结合活性.
(1)收集T细胞：收集1×10 6cells/tube。
(2)漂洗细胞：用1ml staining buffer(含有0.5％w/v BSA+2mM EDTA的PBS)漂洗细胞一次，350×g，4℃离心5min，离心后用200μl staining buffer重悬细胞。
(3)Bs-antibody binding:分别加入双特异抗体YK001和YK002至5μg/ml，冰上孵育45min。
(4)漂洗细胞：在细胞悬液中加入1ml staining buffer，混匀，350×g 4℃离心5min，去上清，重新漂洗一次。离心后用100μl staining buffer重悬细胞。
(5)样品管加入biolegend抗体5μl(PE anti-human IgG Fc Antibody,Biolegend,409304)，同型对照管加入同型对照(PE Mouse IgG2a,κIsotype Ctrl(FC)Antibody，Biolegend,400213)，冰上避光孵育15min。
(6)漂洗细胞：在细胞悬液中加入1ml staining buffer，混匀，350×g 4℃离心5min，去上清，重新漂洗一次。
(7)上机检测：用200μl PBS重悬细胞后，上机检测即可。
流式细胞检测结果如图6所示，其中，YK001结合T细胞的检测结果如图6的A和B所示，YK002结合T细胞的检测结果如图6的C和D所示，结果表明，双特异抗体YK001和YK002均能够特异性结合T细胞，即双特异抗体融合蛋白保留了单链抗体Anti-CD3的结合功能。
实施例4 双特异性抗体介导的体外细胞杀伤效率检测
本实施例以Raji-Luc为靶细胞，以PBMC为免疫效应细胞，检测双特异性抗体YK001 和YK002介导的靶细胞的杀伤作用，以抗-CD3单克隆抗体抗体和抗-CD19单克隆抗体以及0527×CD3双特异性抗体作为对照。
1、靶细胞准备
以Raji-Luc(荧光素酶标记的Raji细胞)为靶细胞，靶细胞吹匀后计数，1000rpm，离心5min，PBS洗涤一次。靶细胞离心洗涤后用GT-T551培养基调整密度为0.2×10 6/ml,每孔加入50μl，则每孔中细胞为10000个。
2、PBMC准备
以PBMC为效应细胞。将冻存在液氮罐中的PBMC取出(参考细胞冻存与复苏)解冻，加入含有PBS或GT-T551培养基的15ml离心管中，1000rpm，离心5min，用PBS或GT-T551培养基洗涤两次，计细胞数、活度与密度，并调整活细胞密度为2×10 6/ml，每孔加入50μl，则每孔中细胞为100000个。
3、抗体稀释
采用GT-T551培养基分别稀释双特异性抗体YK001和YK002，将抗体YK001和YK002的起始浓度调整到10nM。依次以1：5的比例稀释。将稀释好的抗体取100μl加入上述准备的细胞中，混匀，将96孔板放回培养箱，18小时后检测杀伤效果。
4、检测
由于Raji靶细胞携带Luciferase基因，故采用LUMINEX方法检测靶细胞杀伤效率。
以steay-GLO(promega公司)为底物，将试剂盒中的buffer解冻融化后加入底物粉末中，混匀，分装为每支5ml或10ml，完成steady-GLO底物重建。
将共培养的细胞吹匀后取100μl转移到不透明白板中，再加入100μl重建的steady-GLO底物，轻拍混匀，放置5min后读板，检测仪器为synergy HT。
5、数据处理
靶细胞杀伤比例的计算公式如下：
靶细胞杀伤比例＝100×(Only target-test well)/Only target；
将所有检测孔的靶细胞杀伤比例对应的抗体浓度转变为log10，以此作为横坐标、以杀伤比例作为纵坐标制作曲线图，结果如图7所示；通过软件Graphpad Prism 7.0分析结果，计算双特异性抗体的IC50，结果如表2所示。结果表明，与对照抗体(抗-CD3单克隆抗体和抗-CD19单克隆抗体以及0527×CD3双特异性抗体)相比，双特异抗体YK001和YK002均能够有效地介导PBMC杀伤肿瘤细胞系Raji-Luc，YK001和YK002单个分子都同时具有Anti-CD19和Anti-CD3单克隆抗体的生物学功能，YK001介导杀伤靶细胞的效力高于YK002。
表2 双特异性抗体YK001和YK002介导的靶细胞杀伤的IC50
  Anti-CD3 Anti-CD19 YK001 YK002 0527×CD3
IC50(nM) 0.02866 N/A 0.0004193 0.002445 ～0.4051
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种双特异性抗体及其制备方法与应用。本发明的双特异性抗体包括单克隆抗体单元和单链抗体单元；其中，单克隆抗体单元为2个完整的轻链-重链对，特异性结合肿瘤细胞表面抗原；单链抗体单元包括2个单链抗体，单链抗体包括重链可变区和轻链可变区，特异性结合免疫细胞表面抗原；本发明提供的双特异性抗体为通过如下任意一种方式连接而成的对称结构：(1)2个单链抗体的C端分别通过连接肽与单克隆抗体的2条重链的N端连接；(2)2个单链抗体的N端分别通过连接肽与单克隆抗体的2条重链的C端连接。本发明的双特异性抗体能够同时结合免疫细胞和肿瘤细胞，介导导向性免疫反应，有效杀伤肿瘤细胞，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种结合CD19和CD3的双特异性抗体，其特征在于，所述双特异性抗体包括(a)单克隆抗体单元和(b)单链抗体单元；所述单克隆抗体单元为2个完整的轻链-重链对，能够特异性结合CD19；所述单链抗体单元包括2个单链抗体；所述单链抗体包括重链可变区和轻链可变区，能够特异性结合CD3；所述双特异性抗体为通过如下任意一种方式连接而成的对称结构：
    (1)所述2个单链抗体的C端分别通过连接肽与所述单克隆抗体的2条重链的N端连接；
    (2)所述2个单链抗体的N端分别通过连接肽与所述单克隆抗体的2条重链的C端连接。
  2. 根据权利要求1所述的双特异性抗体，其特征在于，所述连接肽的氨基酸序列为(GGGGX)n，其中，X为Gly或Ser，n为1-4的自然数；
    优选地，所述连接肽的氨基酸序列如SEQ ID NO.13所示。
  3. 根据权利要求1或2所述的双特异性抗体，其特征在于，
    所述单链抗体的轻链序列如SEQ ID NO.5所示或如SEQ ID NO.9所示；
    所述单链抗体的重链序列如SEQ ID NO.6所示或如SEQ ID NO.10所示；
    优选地，所述单链抗体的轻链和重链组成融合肽，所述融合肽的序列为如下任意一种：
    (1)当所述2个单链抗体的C端分别通过连接肽与所述单克隆抗体的2条重链的N端连接时，所述融合肽的序列如SEQ ID NO.16所示。
    (2)当所述2个单链抗体的N端分别通过连接肽与所述单克隆抗体的2条重链的C端连接时，所述融合肽的序列如SEQ ID NO.17所示；
  4. 根据权利要求3所述的双特异性抗体，其特征在于，所述双特异性抗体为鼠源抗体、人源化抗体、嵌合抗体或重组抗体。
  5. 根据权利要求3或4所述的双特异性抗体，其特征在于，所述单克隆抗体的轻链和重链通过二硫键连接；
    所述单克隆抗体的Fc片段为人或人源化抗体的Fc片段，所述人或人源化抗体为IgG1、IgG2、IgG3或IgG4中的一种；
    优选地，所述单克隆抗体的Fc片段为人或人源化IgG4抗体的Fc片段；
    更优选地，所述单克隆抗体的轻链全长序列如SEQ ID NO.3所示；重链全长序列如SEQ ID NO.1或SEQ ID NO.20所示。
  6. 编码权利要求1～5任一项所述双特异性抗体的基因；
    优选地，所述单克隆抗体的轻链全长的编码基因序列如SEQ ID NO.4所示；
    和/或，
    所述单克隆抗体的重链全长的编码基因序列如SEQ ID NO.2所示或如SEQ ID NO.21所示；
    和/或，
    当所述2个单链抗体的C端分别通过连接肽与所述单克隆抗体的2条重链的N端连接时，所述单链抗体的编码基因序列如SEQ ID NO.14所示；当所述2个单链抗体的N端分别通过连接肽与所述单克隆抗体的2条重链的C端连接时，所述单链抗体的编码基因序列如SEQ ID NO.15所示。
  7. 包含权利要求6所述基因的生物材料，所述生物材料包括重组DNA、表达盒、载体、宿主细胞、工程菌或细胞系。
  8. 权利要求1～5任一项所述双特异性抗体的制备方法，其特征在于，包括：构建含有所述单链抗体和单克隆抗体的编码基因的表达载体；将所述表达载体导入宿主细胞，获得稳定表达所述双特异性抗体的宿主细胞；培养宿主细胞，经分离纯化获得所述双特异性抗体。
  9. 一种药物组合物，其特征在于，包含权利要求1～5任一项所述双特异性抗体。
  10. 权利要求1～5任一项所述双特异性抗体或权利要求6所述基因或权利要求7所述生物材料的如下任一应用：
    (1)在制备预防或治疗CD19表达的B细胞相关疾病的药物中的应用；
    (2)在制备预防或治疗以CD19为靶标的疾病的药物中的应用；
    (3)在制备用于杀伤CD19表达细胞的药物中的应用；
    (4)在制备CD19和/或CD3的检测试剂中的应用；
    优选地，所述CD19表达的B细胞相关疾病包括B细胞相关肿瘤、B细胞引起的自身免疫病。
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