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WO2021000530A1 - 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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WO2021000530A1
WO2021000530A1 PCT/CN2019/126066 CN2019126066W WO2021000530A1 WO 2021000530 A1 WO2021000530 A1 WO 2021000530A1 CN 2019126066 W CN2019126066 W CN 2019126066W WO 2021000530 A1 WO2021000530 A1 WO 2021000530A1
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region
bispecific antibody
seq
amino acid
antibody
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PCT/CN2019/126066
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English (en)
French (fr)
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张文军
李峰
华亚南
刘嘉熙
方春华
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广州爱思迈生物医药科技有限公司
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Priority to AU2019452936A priority Critical patent/AU2019452936A1/en
Priority to EP19936159.3A priority patent/EP3967713A4/en
Priority to JP2021561747A priority patent/JP7397882B2/ja
Priority to US17/616,043 priority patent/US20220324993A1/en
Publication of WO2021000530A1 publication Critical patent/WO2021000530A1/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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • 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
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • 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
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • 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 disclosure relates to an antibody and a preparation method and application thereof, in particular to a bispecific antibody and a preparation method and application thereof.
  • CD19, CD20, CD22, CD52, etc. are potential therapeutic targets for B cell-related diseases.
  • CD20 protein is also called B lymphocyte restricted differentiation antigen, Bp35 or B1, and is the human MS4A1 gene.
  • the encoded and expressed four-time transmembrane, highly hydrophobic, non-glycosylated phosphoprotein has a molecular weight of about 35kD.
  • CD20 is specifically expressed on the surface of pre-B cells and mature B cells, but not expressed in hematopoietic stem cells, progenitor cells, plasma cells and other normal somatic cells.
  • CD20 is also expressed on the cell surface of B-cell lymphomas such as non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL).
  • B-cell lymphomas such as non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL).
  • CD20 has no known natural ligands. Studies have shown that CD20 has calcium channel activity. It regulates the cell cycle by regulating the intracellular calcium ion concentration. It plays a special role in the differentiation and growth of B cells that are activated from the G0 phase to the G1 phase. , While regulating the cell cycle from S phase to mitosis phase, it can also induce cell apoptosis.
  • CD20 is specifically expressed on the surface of a variety of B-cell-related lymphoma cells, there is no CD20 expression on pre-B cells and other somatic cells, and CD20 is not easily shed or secreted, and does not undergo endocytosis after binding to antibodies, so it is It is an ideal target with great potential for treating B cell related diseases.
  • a variety of therapeutic monoclonal antibody drugs targeting CD20 have been marketed. According to the mechanism of action, they can be divided into two categories: Type I and Type II.
  • Type I antibodies mainly rely on complement-dependent cytotoxicity (CDC) and Antibody-dependent cytotoxicity (ADCC) functions.
  • Type II mainly functions through programmed cell death (PCD) and ADCC.
  • Rituximab Human-mouse chimeric antibody Rituximab (Rituximab, trade name: Rituxian/Rituxian) is the first-generation CD20 antibody (Type I). It was approved by the FDA in 1997 for the treatment of NHL, and was subsequently approved for the treatment of rheumatoid Diseases such as arthritis and CLL; the fully human monoclonal antibody Ofatumumab (trade name: Arzerra) is the second-generation CD20 antibody (Type I). Compared with Rituximab, Ofatumumab has a positive effect on CD20. The affinity is stronger, and the cell test shows that the CDC response is stronger. The ADCC response is similar to Rituxan.
  • humanized monoclonal antibody Obinutuzumab (Obinutuzumab, trade name: Gazyva) is the third-generation CD20 antibody (Type II) ), due to the glycosylation modification, which enhances the affinity of the antibody with NK cells, macrophages and dendritic cells, it has a stronger ADCC response than Rituximab.
  • Obinyutuzumab passes the neutrophil Cell-induced endocytosis and cell death can more effectively induce B cell apoptosis. In 2013, it was approved by the FDA in combination with chlorambucil to treat CLL that has not been treated in the past.
  • CD20 monoclonal antibody drugs targeting CD20 have shown better clinical advantages, but they often need to be combined with chemical drugs, and not all patients can respond to CD20 antibody therapy. This is because the curative effect of CD20 monoclonal antibody is related to the expression level of CD20 on tumor cells. Certain types, such as CLL cells, have a significantly lower CD20 expression level than other types of B-cell tumors, which limits the efficacy of CD20 monoclonal antibody.
  • monoclonal antibodies mainly kill tumor cells by combining ADCC, ADCP, CDC and other mechanisms of action produced by Fc ⁇ R, while Fc ⁇ R has polymorphisms in the population, resulting in significant differences in the affinity of Fc ⁇ R to Fc, and reducing the overall drug Effectiveness, leading to relapse and drug resistance. Once relapsed, treatment will be more difficult and the prognosis is extremely poor.
  • the median survival time is only 2-8 months. T cells with high tumor-killing effects cannot be effectively activated because they do not have Fc ⁇ R. Therefore, it is necessary to design more effective clinical antibody drugs to meet the current therapeutic situation, such as activating T cells to kill tumor cells more efficiently, or develop simultaneous Multifunctional antibodies that bind two or more antigen targets.
  • bispecific antibodies can simultaneously bind to two epitopes or two epitopes of one antigen, they can achieve higher potency through synergy; some bispecific antibodies can combine effector cells (such as T cells) Wait for recruitment to the target site, and directly kill the target cells by activating T cells.
  • effector cells such as T cells
  • T cells effector cells
  • the safety and effectiveness of bispecific antibodies have been greatly improved, and they are widely used in the treatment of tumors and autoimmune diseases. They are a research hotspot in tumor immunotherapy and have broad application prospects.
  • bispecific antibodies usually bind to tumor cell surface antigens and immune cell surface antigens at the same time, and kill tumor cells by activating the immune system.
  • CD3 is a homologous or heterodimeric protein complex and is an important component of T cell receptor (TCR). Its intracellular region contains immunoreceptor tyrosine activation motif (ITAM). When ITAM is phosphorylated It will then bind to the kinase ZAP70, thereby transducing T cell activation signals downstream.
  • Antibodies targeting CD3 can accumulate CD3 on the surface of T cells to activate T cells. This process mimics the process of TCR recognition of MHC-antigen peptides.
  • Bispecific antibodies with CD3 resistance and tumor-specific antigen resistance can simultaneously bind to T cells and tumor cells, activate and guide T cells to secrete granzymes, perforin, etc. to kill tumor cells. For example, dual antibodies that simultaneously bind CD20 and CD3 molecules Specific antibodies can recruit T cells to tumor cells expressing CD20 protein, causing CD20-positive tumor cells to be killed and eliminated.
  • bispecific antibodies were mostly produced by chemical cross-linking of purified monoclonal antibodies or the fusion of hybridoma cells expressing two different monoclonal antibodies.
  • the products produced by these methods have many problems such as unstable products, low yield, Improper antibody modification, immunogenicity, difficulty in production and purification, etc.
  • a large number of BsAbs have been produced through genetic engineering technology.
  • bispecific antibody forms which can be divided into two categories: those that do not contain Fc and those that contain Fc regions. Sex antibody.
  • Bispecific antibodies that do not contain Fc such as: BiTE, DART, TandAbs, Bi-Nanobody, etc.
  • BiTE Trigger-like peptide chain linking fragments or additional structures
  • the relative molecular weight and physical and chemical properties are quite different from those of natural IgG antibodies. It is easier to form multimers and produce immunogenicity.
  • Bispecific antibodies containing Fc can mediate ADCC and CDC, increase the half-life (FcRn-mediated), stability and solubility of antibodies in the blood, and are also the binding regions of protein A (Protein A) and protein G (Protein G) , To facilitate the purification of downstream products.
  • This type of bispecific antibody technology includes TrioMab, Knob-into-hole, DVD-Ig, DuoBody, SEEDbody, BEAT, etc.
  • the purpose of the present disclosure includes, for example, overcoming the above-mentioned shortcomings of the prior art to provide a stable and efficient bispecific antibody targeting CD20 and CD3, which can mediate T cell specific killing of tumor cells.
  • the present disclosure provides a bispecific antibody comprising a CD20-binding domain, a CD3-binding domain and a heterodimer Fc region, the CD20-binding domain and the The domains of CD3 are each independently selected from the Fab region, the ScFv region, or the sDab region.
  • bispecific antibody The most ideal kind of bispecific antibody is to maintain the natural structure of IgG antibody, which needs to overcome the problem of heavy chain and light chain mismatch during the antibody assembly process.
  • the present disclosure changes the antibody Fc and a few amino acids at the heavy chain-light chain interface. Based on the introduction of few site mutations without affecting the structure and function of the antibody, this artificially modified pairing method is used to prepare bispecific antibodies that simultaneously target leukocyte differentiation antigen 20 (CD20) and leukocyte differentiation antigen 3 (CD3), Compared with traditional monoclonal antibodies, the CD20 ⁇ CD3 bispecific antibody has the following advantages: (1) T cells that activate CD4 + and CD8 + specifically kill CD20-positive tumor cells, including tumor cells with low CD20 expression levels; (2) At a very low dosage, there is still a high-efficiency tumor killing effect; (3) It can cause low-level cytokine release and has higher safety; (4) The structure is similar to natural IgG antibodies, and has the same Anti-similar stability, half-life and physical and chemical properties.
  • the bispecific antibody of the present disclosure can specifically bind to CD20 epitope and CD3 epitope, and mediate T cell activation through antigen specificity. It is a new, stable and efficient target that can mediate T cell specific killing of tumor cells CD20 and CD3 bispecific antibody (ie CD20 ⁇ CD3 BsAb), which can cross-react with human and primate CD20 and CD3.
  • the bispecific antibody of the present disclosure is composed of three or four polypeptide chains, the three-chain structure includes a heavy chain, a light chain and the scFv-Fc chain, and the four-chain structure includes two heavy chains and two light chains.
  • the bispecific antibody forms an immunoglobulin domain that specifically binds to CD20, an immunoglobulin domain that specifically binds to CD3, and a heterodimer Fc region.
  • Two heavy chains (or one heavy chain and scFv-Fc chain) interact and combine to form a heterodimer form, forming the aforementioned heterodimer Fc region.
  • the four-chain bispecific antibody two light chains each bind to one of the heavy chains to form the above-mentioned Fab structure that specifically binds CD20 and the Fab structure that specifically binds CD3.
  • the CD20 ⁇ CD3 BsAb described in the present disclosure contains an Fc region, which can increase the half-life and stability of the antibody in the body.
  • the CD20-binding domain includes a first immunoglobulin Fab region, and the first immunoglobulin Fab region is mainly composed of mutually binding
  • the first heavy chain and the first light chain are formed, and the variable region of the first heavy chain has the amino acid sequence shown in SEQ ID No.: 2, and the variable region of the first light chain has the amino acid sequence shown in SEQ ID No.
  • the amino acid sequence shown in 4; and/or the CD3 binding domain includes a second immunoglobulin Fab region, the second immunoglobulin Fab region is mainly composed of a second heavy chain and a second The light chain is formed, the variable region of the second heavy chain has the amino acid sequence shown in SEQ ID No.: 6, and the variable region of the second light chain has the amino acid sequence shown in SEQ ID No.: 10 sequence;
  • the heterodimer Fc region is composed of two polypeptide chains, and each polypeptide chain contains asymmetric amino acid modifications with opposite charges.
  • both the CH1 portion of the first heavy chain and the CL portion of the first light chain include a non-half-half cysteine residue substituted for a natural cysteine residue. Cystine residues, and a cysteine residue that replaces the unnatural cysteine.
  • the non-cysteine residues in the CH1 portion of the first heavy chain that replace the natural cysteine residues and the first light chain form disulfide bonds.
  • the non-cysteine residue that replaces the natural cysteine residue is C220S, which is substituted
  • the cysteine residue of unnatural cysteine is L128C.
  • the non-cysteine residue that replaces the natural cysteine residue is C214S, which is substituted
  • the cysteine residue of unnatural cysteine is F118C.
  • the second immunoglobulin Fab region does not include non-cysteine residues that replace natural cysteine residues, and does not include Replace cysteine residues with non-natural cysteine residues.
  • the CD20-binding domain includes a first immunoglobulin Fab region
  • the CD3 binding domain includes a second immunoglobulin Fab region; the first heavy chain and the second heavy chain combine with each other to form a heterodimer Fc region.
  • the hinge region of the first heavy chain and the hinge region of the second heavy chain are covalently bonded through at least one disulfide bond.
  • the first heavy chain and the second heavy chain both comprise a CH2 domain and a modified CH3 domain, and the CH2 of the first heavy chain
  • the structural domain, the modified CH3 domain, the CH2 domain and the modified CH3 domain of the second heavy chain combine with each other to form a heterodimer Fc region.
  • At least one CH2 domain contains one, two or more mutations Site.
  • the mutation site is used to reduce the binding of Fc to its receptor Fc ⁇ R.
  • the mutation combination is L234A/L235A.
  • the modified CH3 domain in the first heavy chain and the modified CH3 domain in the second heavy chain comprise asymmetric amino acids with opposite charges Modification to form a heterodimer Fc region.
  • the inclusion of oppositely charged asymmetric amino acid modifications can promote the formation of heterodimers.
  • the modified CH3 domain in the first heavy chain contains at least one of P395K, P396K, and V397K mutations
  • the second heavy chain contains at least one of T394D, P395D, and P396D mutations; or,
  • the modified CH3 domain in the first heavy chain contains at least one of T394D, P395D, and P396D mutations
  • the modified CH3 domain in the second heavy chain contains at least one of the P395K, P396K, and V397K mutations .
  • the first heavy chain and the second heavy chain are derived from human antibodies IgG1, IgG2, IgG3 or IgG4. In one or more embodiments of the bispecific antibody of the present disclosure, the first heavy chain and the second heavy chain are derived from a human antibody IgG1.
  • the first heavy chain has an amino acid sequence as shown in SEQ ID No.: 1, and the first light chain has an amino acid sequence as shown in SEQ ID No.: The amino acid sequence shown in 3, the second heavy chain has the amino acid sequence shown in SEQ ID No.: 5 or SEQ ID No.: 7, and the second light chain has the amino acid sequence shown in SEQ ID No.: 9 sequence.
  • the CD20-binding domain includes the first immunoglobulin Fab region
  • the CD3 binding domain includes the scFv-Fc fusion protein Polypeptide chain
  • the heavy chain variable region of the scFv region in the scFv-Fc fusion protein polypeptide chain has the amino acid sequence shown in SEQ ID No.: 6
  • the light chain variable region has the amino acid sequence shown in SEQ ID No.: 10
  • the amino acid sequence of the first heavy chain and the scFv-Fc fusion protein polypeptide chain are combined to form a heterodimer Fc region; or,
  • the domain that can bind to CD20 includes the scFv-Fc fusion protein polypeptide chain, and the domain that can bind to CD3 includes the second immunoglobulin Fab region; the scFv-Fc fusion protein polypeptide chain has SEQ ID No.: 2
  • the amino acid sequence shown in the light chain variable region has the amino acid sequence shown in SEQ ID No.: 4.
  • the second heavy chain and the scFv-Fc fusion protein polypeptide chain combine to form a heterodimer Body Fc region.
  • the CD20-binding domain includes the first immunoglobulin Fab region
  • the CD3 binding domain includes the scFv-Fc fusion protein Polypeptide chain
  • the variable region of the first heavy chain has the amino acid sequence shown in SEQ ID No.: 8
  • the scFv region in the scFv-Fc fusion protein polypeptide chain has the amino acid sequence shown in SEQ ID No.: 11
  • the CDR3 of the heavy chain variable region in the polypeptide chain of the scFv-Fc fusion protein contains the N106T mutation, and the CDR3 sequence after the mutation is: HGNFGTSYVSWFA.
  • the scFv region in the polypeptide chain of the scFv-Fc fusion protein has a sequence such as SEQ ID No.: 12, SEQ ID No.: 13 or SEQ ID
  • the amino acid sequence shown in No.: 14, or its variable region has such as SEQ ID No.: 2, SEQ ID No.: 4, SEQ ID No.: 8, SEQ ID No.: 6 or SEQ ID No.: The amino acid sequence shown in 10.
  • the present disclosure provides an expression vector, which is obtained by linking the DNA sequence corresponding to the above amino acid sequence to the base.
  • the expression vector is a plasmid, a viral vector or a recombinant expression vector.
  • the basic carrier is pFUSE-hIgG 1-Fc2.
  • the present disclosure provides a host cell containing the above-mentioned expression vector.
  • the host cell is a mammalian cell. In one or more embodiments of the bispecific antibody of the present disclosure, the host cell is a CHO cell or a HEK293 cell.
  • the present disclosure provides a method for preparing the above-mentioned bispecific antibody, which includes the following steps:
  • step (b) Transfect or transform the expression vector constructed in step (a) into a host cell, and culture the host cell;
  • the method for separating and purifying the bispecific antibody is protein A affinity chromatography, cation exchange or anion exchange.
  • the present disclosure provides an antibody conjugate, which is formed by coupling a coupling substance with the above-mentioned bispecific antibody.
  • the coupling substance is a cytotoxin, a radioisotope, a fluorescent label, a luminescent substance, a chromogenic substance or an enzyme.
  • the present disclosure provides a pharmaceutical composition comprising the above-mentioned bispecific antibody.
  • the present disclosure provides the use of the above-mentioned bispecific antibodies in the preparation of drugs or pharmaceutical compositions for the treatment of tumors, rheumatoid arthritis, multiple sclerosis or systemic lupus erythematosus.
  • the tumor is a malignant tumor associated with CD20 expression.
  • the malignant tumor is acute B lymphocytic leukemia (B-ALL), diffuse large B cell lymphoma (DLBCL), chronic lymphocyte Leukemia (CLL), follicular lymphoma, non-Hodgkin's lymphoma (NHL), chronic myelogenous leukemia (CML), Burkitt lymphoma.
  • B-ALL acute B lymphocytic leukemia
  • DLBCL diffuse large B cell lymphoma
  • CLL chronic lymphocyte Leukemia
  • follicular lymphoma non-Hodgkin's lymphoma
  • NHL non-Hodgkin's lymphoma
  • CML chronic myelogenous leukemia
  • Burkitt lymphoma Burkitt lymphoma
  • the present disclosure provides the use of the above-mentioned bispecific antibodies in medicines or pharmaceutical compositions.
  • the present disclosure provides the use of the above-mentioned bispecific antibody in the treatment of tumors, rheumatoid arthritis, multiple sclerosis or systemic lupus erythematosus.
  • the CD20 ⁇ CD3 BsAb molecule of the present disclosure maintains the structure of a natural IgG antibody or a structure close to that of a natural IgG antibody, and has stability and physicochemical properties similar to monoclonal antibodies.
  • the present disclosure relates to the construction of bispecific antibodies.
  • a small number of amino acids on the heavy and light chains are mutated to make the two polypeptide chains have opposite charges, promote the formation of heavy chain heterodimers, and introduce unnatural
  • the disulfide bond overcomes light chain mismatches. This method involves very few mutations, has little effect on the function of the Fc region and does not affect the expression and yield of the protein in eukaryotic cells.
  • the CD20 ⁇ CD3 BsAb molecules described in this disclosure can not only bind to tumor cell surface antigens but also T cell surface CD3 molecules, relying on antigen-specific activation of TCR and increasing T cell targeting Tumor killing effect, using a smaller amount of antibody can play a significant effect of killing tumor cells.
  • the CD20 ⁇ CD3 BsAb molecule described in the present disclosure contains the Fc region of the antibody, which increases the half-life and stability of the antibody, and is easier to use the existing monoclonal antibody purification technology to simplify The production process of bispecific antibodies was introduced.
  • CD20-targeted chimeric antigen receptor T cell immunotherapy Compared with CD20-targeted chimeric antigen receptor T cell immunotherapy (CAR-T), the present disclosure does not involve operations such as exogenous virus, in vitro T cell culture and reinfusion, and has fewer toxic and side effects, and is safer. control.
  • the present disclosure relates to the modification of CD3 affinity weakening to improve the safety of CD3 class bispecific antibody molecules.
  • Figure 1 is a schematic diagram of an example of the structure of CD20 ⁇ CD3 BsAb described in the present disclosure
  • Figure 2 is the CD20 ⁇ CD3 BsAb purified by protein A affinity chromatography, where Figure 2A is the non-reducing and reducing SDS-PAGE gel image of CD20 ⁇ CD3 BsAb V1 protein A after one-step purification, and Figure 2B is the CD20 ⁇ CD3 BsAb V2 protein A after one-step purification, non-reducing and reducing SDS-PAGE electrophoresis gel image, where Figure 2C is CD20 ⁇ CD3 BsAb (three-chain bispecific antibody, anti-CD20 domain is scFv, anti-CD3 domain is Fab) protein A Non-reducing and reducing SDS-PAGE electrophoresis gel image after one-step purification;
  • FIG 3 shows the UV absorption peaks of CD20 ⁇ CD3 BsAb V1A280 purified by cation exchange chromatography (CEX); the purified protein A sample ( Figure 2A) is loaded onto the cation exchange column, eluted by NaCl gradient, and passed detection The eluted protein was collected by the absorbance at 280nm of ultraviolet light.
  • CD20 ⁇ CD3 BsAb V1 formed 1 main elution peak (peak D) and 4 small secondary elution peaks through CEX;
  • Figure 4 shows the elution curve of double antibody purification
  • (A) Take 1 mL of protein A purified sample ( Figure 2A) at a concentration of 1 mg/mL, and analyze the aggregate content of the sample after one-step protein purification by gel filtration chromatography (SEC). This figure shows the UV absorption peak of A280
  • (B) Take 1 mL of purified protein A sample ( Figure 2C, lane1) at a concentration of 1 mg/mL, and analyze the aggregate content of the sample after one-step protein purification by gel filtration chromatography (SEC) , The figure shows the UV absorption peak of A280;
  • Figure 5 is the result of the detection of the binding ability of CD20 ⁇ CD3 bispecific antibody to the antigen CD3; among them, Figure 5A compares the affinity of CD20 ⁇ CD3 BsAb V1 and V2 to CD3 antigen molecules by ELISA, CD20 ⁇ CD3 BsAb V1 and CD3 antigen The combined EC50 concentration is 0.8178 ⁇ g/mL, and the EC50 concentration of CD20 ⁇ CD3 BsAb V2 binding to CD3 antigen is 2.097 ⁇ g/ml;
  • Figure 5B shows the binding ability of three-chain bispecific antibodies to CD3 antigen molecules, 1: four-chain bispecific Antibodies, where the CD20-binding domain is Fab, and the CD3-binding domain Fab has an EC50 concentration of 4.9 ⁇ g/ml that binds to the CD3 antigen; 2: three-chain bispecific antibody, where the CD20-binding domain is scFv, which binds CD3 domain Fab, which binds to CD3 antigen with an
  • Figure 6 is the binding diagram of CD20 ⁇ CD3 BsAb V1 and CD20 ⁇ CD3 BsAb V2 with CD20 positive cells (Raji cells) detected by FACS;
  • Figure 7 is the ADCC cell killing experiment; among them, Figure 7A and B are the detection of CD20 ⁇ CD3 BsAb V1 and V2 mediated killing ability to target cells (Raji and Daudi cells), V1 and V2 killing tumor cells with high CD20 expression The effect is significant, the EC50 concentration is 0.2ng/ml; Figure 7C is to detect the killing ability of CD20 ⁇ CD3 triple-chain bispecific antibody to target cells (Daudi cells), 1: triple-chain bispecific antibody, which binds CD20 The domain of is scFv, which binds to CD3 domain Fab; 2: Three-chain bispecific antibody, where the CD20-binding domain is Fab, which binds CD3 domain scFv; 3: Four-chain bispecific antibody, which binds CD20 structure The domain is Fab, which binds to the CD3 domain Fab;
  • Figure 8 is a protein thermal stability test; where the protein sample is placed at 37°C and 40°C for different times: 0 days, 1 day, 4 days, and 7 days. SDS-PAGE electrophoresis diagrams of the protein samples;
  • Figure 9 shows the results of CD20 ⁇ CD3 BsAb cytokine release test
  • Figure 10 is a graph showing the detection results of CD20 ⁇ CD3 BsAb-mediated anti-tumor activity in mice.
  • BsAb bispecific antibody (bispecific antibody)
  • VH variable region of heavy chain (variable region of heavy chain)
  • VL variable region of light chain (variable region of light chain)
  • CH constant region of heavy chain (constant region of heavy chain)
  • CDR antigen complementarity determining region (complementarity determining region)
  • scFv single-chain variable fragment (single-chain variable fragment)
  • ADCC antibody-dependent cell-mediated cytotoxicity (antibody-dependent cellular cytotoxicity)
  • ELISA enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay)
  • FACS Fluorescence-activated cell sorting, that is, flow cytometry (Fluorescence-activated cell sorting)
  • the relevant terms used in the present disclosure have the same meaning as commonly understood in the technical field.
  • the operating steps of molecular cloning, cell culture, protein purification, immunological experiments, microbiology, animal models and other experiments described in the present disclosure are routine steps widely used in this field.
  • singular terms in the present disclosure include pluralities and plural meanings include singular meanings.
  • nucleotide sequences described in the present disclosure are arranged and written from left to right from the 5'end to the 3'end.
  • the amino acid sequences described in the present disclosure are arranged and written from left to right in the direction from the amino terminal (N-terminal) to the carboxy terminal (C-terminal).
  • amino acid refers to one of the 20 naturally-occurring amino acids or any non-natural analog that may be present in a specific defined position.
  • amino acid mutation in the present disclosure refers to amino acid substitutions, insertions, deletions and modifications in the polypeptide sequence, and any combination of amino acid substitutions, insertions, deletions and modifications.
  • the preferred amino acid modification herein is a substitution.
  • amino acid substitution or “substitution” refers to the substitution of an amino acid at a specific position in the parent polypeptide sequence with another amino acid.
  • substitution C220S refers to a variant polypeptide in which the aminocysteine at position 220 of the polypeptide has been replaced by the amino acid serine.
  • Amino acid mutations can be achieved through molecular cloning or chemical methods. Molecular cloning methods include PCR, site-directed mutagenesis, and full gene synthesis.
  • protein refers to molecules in which two or more amino acids are connected by peptide bonds, including natural proteins, artificial proteins, protein fragments, muteins, fusion proteins and the like.
  • domain refers to a specific structural region with independent functions in a biological macromolecule.
  • the domain has an independent tertiary structure and its function does not depend on the rest of the biological macromolecule.
  • the domain in this disclosure refers specifically to a protein In such regions, such as the VH domain of the heavy chain variable region and the VL domain of the light chain variable region, the combination of the domains can form a large domain.
  • CD3 refers to the cluster of differentiation-3 protein, which is a component of the T cell receptor complex (TCR) expressed on the surface of T cells. It is composed of CD3 ⁇ (human CD3 ⁇ UniProt Entry No.P0423), CD3 ⁇ (human CD3 ⁇ UniProt Entry No. .P07766), CD3 ⁇ (human CD3 ⁇ UniProt Entry No. P09693) and CD3 ⁇ (human CD3 ⁇ UniProt Entry No. P20963) homologous or heterodimer consisting of four peptide chains, unless explicitly specified, such as “cynomolgus CD3", " Mouse CD3", otherwise the "CD3" mentioned in this disclosure is human CD3.
  • CD3 ⁇ human CD3 ⁇ UniProt Entry No.P0423
  • CD3 ⁇ human CD3 ⁇ UniProt Entry No. .P07766
  • CD3 ⁇ human CD3 ⁇ UniProt Entry No. P09693
  • CD3 ⁇ human CD3 ⁇ UniProt Entry No. P20963
  • polypeptide chain that binds to CD3 or "antibody that binds to CD3” in the present disclosure includes antibodies or antibody fragments that specifically bind to CD3 single chain units, as well as those that specifically bind to CD3 homodimers or heterodimers. Antibody or antibody fragment.
  • the antibodies that specifically bind to CD3 described in the present disclosure can bind to CD3 protein molecules and can also bind to CD3 expressed on the cell surface.
  • CD20 refers to the B lymphocyte-restricted differentiation antigen (human CD20 UniProt Entry No. P11836) encoded and expressed by the human MS4A1 gene, including variants of human CD20 protein expressed naturally by cells and expressed by transfected foreign genes. Subtypes and homologous proteins of other species (such as cynomolgus CD20 protein).
  • antibody refers to an immunoglobulin molecule that contains at least one antigen recognition site and can specifically bind to an antigen.
  • antigen refers to a substance that can induce an immune response in the body and specifically bind to an antibody, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, hapten, or a combination of the foregoing .
  • the binding of antibody and antigen is mediated by the interaction between the two, including hydrogen bond, van der Waals force, ionic bond and hydrophobic bond.
  • the area on the surface of an antigen that binds to an antibody is an "antigenic determinant" or "epitope". Generally speaking, each antigen has multiple determinants.
  • antibody mentioned in the present disclosure includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, multispecific antibodies containing at least two different epitope binding domains (e.g., bispecific Antibodies), human antibodies, humanized antibodies, post-translationally modified antibodies, camelid antibodies, chimeric antibodies, fusion proteins containing antibody epitopes, and any other modified immunoglobulin molecules containing antigen recognition sites, as long as These antibodies exhibit the desired biological activity.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, that is, molecules that contain at least one antigen binding site.
  • the light chain of a natural human antibody is covalently connected to the heavy chain through two disulfide bonds, and the heavy chain-light chain dimer is then formed by the disulfide bond formed between the heavy chains to form an antibody molecule similar to the letter Y.
  • the number of disulfide bonds between the heavy chains of different types of human antibodies will vary.
  • the area between the Y-shaped arms and the trunk is the hinge area, which has a certain degree of flexibility.
  • Each antibody polypeptide chain includes a variable region and a constant region, which form different domain units through spatial folding.
  • Each light chain is composed of a variable region domain VL at the amino terminus and a constant region domain CL at the carboxy terminus.
  • Each heavy chain is composed of the amino-terminal variable region domain VH and the following three or four constant region domains (CH1, CH2, CH3, CH4).
  • variable region of an antibody is the antigen binding region, including the variable region of the heavy chain (VH) and the variable region of the light chain (VL).
  • the structure of VH and VL are similar.
  • the variable regions of the heavy and light chains are respectively composed of three complementary The determining region (complementarity determining region, CDR) and four framework regions (framework regions, FR) flanking the CDR.
  • the framework region supports CDRs and defines the spatial relationship between CDRs.
  • the CDR of the variable region is highly variable in sequence, so it is also called a hypervariable region (HVR).
  • CDR forms a specific loop structure and directly participates in antigen recognition.
  • Each variable region domain contains 3 segments of HVR, the HVRs on VH are H1, H2, H3, and the HVRs on VL are L1, L2, and L3.
  • the CDRs of the heavy chain or the light chain are denoted as CDR1, CDR2, and CDR3 from the amino terminus, respectively.
  • the variable regions of the heavy chain and the light chain are combined by non-covalent bonds.
  • the 3 CDRs of the heavy chain and the 3 CDRs of the light chain together constitute the antigen recognition site. This part of the amino acid residues is the main body of the antibody participating in the antigen binding. The specificity of the antibody to recognize the antigen.
  • Fab fragments
  • Fab region antigen-binding fragments that include the VH domain and CH1 domain of an immunoglobulin heavy chain, and the VL domain and CL domain of the light chain.
  • the first constant region domain CH1 of the heavy chain is combined with the constant region domain CL of the light chain, and the variable region domain VH of the heavy chain is combined with the variable region domain VL of the light chain.
  • Fc The terms “Fc”, “Fc region”, “Fc fragment” or “Fc molecule” are the effector regions of antibodies, which can cause, for example, CDC, ADCC, ADCP, cytokine release and the like.
  • the natural antibody Fc is usually composed of two identical protein fragments combined, which contains two or three immunoglobulin constant region domains.
  • the Fc described in the present disclosure includes natural Fc and mutant Fc. Although the boundaries of the Fc region can vary, the Fc region of a human IgG heavy chain is generally defined as containing residues starting from C226 or P230 to its carboxy terminus. Under the experimental conditions, the fragments of immunoglobulin monomers cleaved by papain are Fab and Fc, respectively.
  • the "hinge” or “hinge region” of an antibody refers to a flexible polypeptide comprising amino acids between the first and second constant domains (CH1 and CH2) of the antibody.
  • the Fc contained in the bispecific antibody of the present disclosure is a human immunoglobulin Fc.
  • the polypeptide sequence of the human immunoglobulin Fc region is derived from the wild-type human immunoglobulin Fc region.
  • Wild-type human immunoglobulin Fc refers to an amino acid sequence that is ubiquitous in the population, and it also contains polymorphisms in different positions of certain individuals in the Fc region.
  • the human immunoglobulin Fc described in the present disclosure also includes changes made to individual amino acids of wild-type human immunoglobulin Fc, for example, changing part of the interface amino acids in the Fc region to form a heavy chain heterodimer.
  • the amino acid numbering of the variable region of the antibody described in the present disclosure uses the coding scheme described by Kabat et al. in 1991, that is, the "Kabat index” or "Kabat numbering". Unless otherwise specified, the amino acid numbers of the antibody constant regions described in this disclosure use the EU index.
  • antigen-binding site refers to one or more amino acid residues that directly interact with an antigen-binding molecule.
  • the antigen-binding site of an antibody is composed of an antigen complementarity determining region (CDR).
  • CDR antigen complementarity determining region
  • Natural immunoglobulin molecules usually contain two An antigen binding site, Fab molecules usually contain an antigen binding site.
  • T cell activation refers to one or more immune responses of T lymphocytes, especially killer T lymphocytes, including: proliferation, differentiation, cytokine release, secretion of killer effector molecules, cell killing, etc.
  • EC50 refers to the half-maximal effective concentration, which refers to the antibody concentration corresponding to 50% of the maximum effect.
  • binding refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen.
  • an antibody that specifically binds to a certain antigen refers to an antibody that is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, The affinity (K D ) of 10 -8 M, 10 -9 M, or 10 -10 M or less binds the antigen.
  • the term "targeting" refers to specific binding.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen.
  • the antibody dissociates at an equilibrium of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less (K D ) Binding antigen.
  • single chain variable region antibody fragment refers to the fusion protein of immunoglobulin heavy chain variable region VH and light chain variable region VL, including different combinations of VH at the N-terminus and VL at the N-terminus, It can be prepared by conventional molecular cloning methods for constructing recombinant proteins (Sambrook JF, EFet al. Molecular cloning: a laboratory manual. 4th ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York: 2012).
  • humanized antibody refers to an antibody or antibody fragment obtained by replacing part or all of the CDR regions of a human immunoglobulin (recipient antibody) with a CDR region of a non-human antibody (donor antibody), wherein
  • the donor antibody may be a non-human (such as mouse, rat, or rabbit) antibody with the desired specificity, affinity, or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by corresponding non-human antibody amino acid residues, or by other antibody amino acid residues, to further improve or optimize one of the antibodies. Or multiple characteristics.
  • bispecific antibody refers to an antibody molecule capable of binding two independent antigens or having binding specificities for different epitopes within the same antigen.
  • one arm of the bispecific antibody molecule binds to a tumor-associated antigen, and the other arm binds to an immune cell-associated antigen (for example, a CD3 molecule), which can activate and initiate cellular immunity-related mechanisms at tumor cells.
  • an immune cell-associated antigen for example, a CD3 molecule
  • Bispecific antibodies can also be coupled to cytotoxins (e.g., radioisotopes, vinca alkaloids, methotrexate, etc.), radioisotopes, fluorescent labels, luminescent substances, chromogenic substances, or enzymes.
  • the moiety conjugated to an antibody or bispecific antibody of the present disclosure to form an antibody conjugate is a cytotoxin.
  • the cytotoxin refers to a substance that inhibits or prevents cell function and/or causes cell destruction, and includes small molecule cytotoxins.
  • the cytotoxin is selected from colchicine, emtansine, maytansinoid, auristatin, vindesine, tubulysin and the like.
  • the moiety that is conjugated to an antibody or bispecific antibody of the present disclosure to form an antibody conjugate is a radioisotope.
  • the radioisotopes include radioisotopes such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu.
  • the part conjugated with the antibody or bispecific antibody of the present disclosure to form an antibody conjugate is selected from fluorescent labels, luminescent substances and color-developing substances, for example: fluorescein isothiocyanate (FITC) , Luciferase (luciferase), horseradish peroxidase (HRP) and so on.
  • FITC fluorescein isothiocyanate
  • Luciferase Luciferase
  • HRP horseradish peroxidase
  • the part conjugated with the antibody or bispecific antibody of the present disclosure to form an antibody conjugate is an enzyme, such as an enzyme-active toxin of bacterial, fungal, plant or animal origin, including active fragments and/or Variants.
  • the present disclosure relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody or antibody fragment, bispecific antibody or antibody conjugate of the present disclosure, and optionally a pharmaceutically acceptable carrier, surfactant and/or diluent.
  • the pharmaceutical composition in addition to the antibodies, bispecific antibodies, or antibody conjugates of the present disclosure, further includes one or more additional therapeutic agents.
  • the additional therapeutic agents include, but are not limited to, chemotherapeutics, growth inhibitors, cytotoxic agents, agents for radiotherapy, anti-angiogenesis agents, apoptosis agents, anti-tubulin agents, and others Reagents for the treatment of cancer.
  • multispecific antibody refers to a bispecific, trispecific or tetraspecific antibody. Multispecific antibodies are composed of fragments of two or more different antibodies and are therefore capable of binding to two, three or four different antigens.
  • the bispecific antibodies of the present disclosure can be extracted and purified from host cells using standard experimental methods.
  • a bispecific antibody containing the Fc portion of an antibody can be purified using protein A or protein G affinity chromatography. Purification methods include, but are not limited to, affinity chromatography, ion exchange, size exclusion chromatography, and protein ultrafiltration.
  • the method for separation and purification of bispecific antibodies described in the present disclosure also includes a combination of the above methods.
  • Purification refers to the separation and/or recovery of target components from cells, cell cultures or other natural components. Unless otherwise specified, the antibodies described in this disclosure are all purified antibodies.
  • isolated antibody as used refers to an antibody that contains substantially no other molecules of different structure or antigen specificity, and a bispecific antibody molecule is an isolated antibody that contains substantially no other antibody molecules.
  • the term "host cell” instructs that the cell and its progeny into which the exogenous nucleic acid is introduced can be transformed or transfected with the nucleotide encoding the polypeptide to express the exogenous polypeptide.
  • the host cells described in this disclosure include but are not limited to CHO cells (Chinese hamster ovary cells), HEK293 cells (Human embryonic kidney cells 293), and BHK cells (Baby Hamster Kidney, baby hamsters). Kidney cells), myeloma cells, yeast, insect cells, or prokaryotic cells such as Escherichia coli.
  • the "host cell” described in this disclosure not only guides the cell into which the exogenous nucleic acid is introduced, but also includes the progeny of the cell. Because the progeny cells undergo mutations during cell division, they still belong to the description of the present disclosure. Term scope.
  • the present disclosure further includes nucleic acid sequences encoding these polypeptide chains.
  • the nucleic acid sequence is inserted into a suitable vector.
  • the vector includes but is not limited to: plasmid, phage expression vector, cosmid, artificial chromosome, phage and animal virus.
  • the expression vector contains elements for regulating expression, including but not limited to promoter, transcription initiation sequence, enhancer, signal peptide sequence, etc. Promoters include but are not limited to T7 promoter, T3 promoter, SP6 promoter, ⁇ -actin promoter, EF-1 ⁇ promoter, CMV promoter, and SV40 promoter.
  • the expression vector can be transferred into host cells using suitable methods known in the art, including but not limited to: calcium phosphate precipitation method, liposome transfection method, electroporation method, PEI (polyethylene imine) transfection method .
  • the bispecific antibody in the implementation of the present disclosure includes 3 or 4 polypeptide chains (Figure 1), respectively named the first heavy chain (the variable region of which has the amino acid sequence shown in SEQ ID No.: 2) and the first light chain.
  • Chain its variable region has the amino acid sequence shown in SEQ ID No.: 4
  • the second heavy chain its variable region has the amino acid sequence shown in SEQ ID No.: 6
  • the second light chain its variable region It has the amino acid sequence shown in SEQ ID No.: 10
  • the scFv-Fc chain has the amino acid sequence shown in SEQ ID No.: 11, SEQ ID No.: 12, SEQ ID No.: 13 or SEQ ID No.: 14 Sequence; or its variable region has SEQ ID No.: 2, SEQ ID No.: 4, SEQ ID No.: 6, SEQ ID No.: 8 or SEQ ID No.: 10
  • variable region of the bispecific antibody that specifically binds to the extracellular region of human CD20 consists of VH1 and VL1 ( Figure 1), and the sequence is derived from the fully human monoclonal antibody Ofatumumab (Patent No.: US2014093454A1).
  • variable region of the bispecific antibody that specifically binds to human CD3 is composed of VH2 and VL2 ( Figure 1), derived from a humanized sequence of the murine monoclonal antibody disclosed in the patent (Patent No.: CN201810263832.0) .
  • the deamination of the CDR region affects the stability of the antibody and the ratio of acid-base peaks.
  • the CDR3 of VH2 in this disclosure contains a potential deamination site (the original sequence of VH2-CDR3 is: HGNFGNSYVSWFA).
  • amino acid mutations are used to eliminate potential deamination.
  • VH2-CDR3 does not contain a deamination mutation (VH2-CDR3 sequence: HGNFG N TYVSWFA) CD20 ⁇ CD3 BsAb named V1, VH2-CDR3 contains N106T mutation (VH2-CDR3 sequence: HGNFG T SYVSWFA) CD20 ⁇ CD3 BsAb Named V2.
  • the Fc part of the antibody was modified according to the method in the PCT WO2017034770A1 patent published by the publisher.
  • the Fc part of one heavy chain was mutated as follows: P395K, P396K, V397K, and the mutations were marked as OA (SEQ ID No.: 1) ;
  • the Fc part of the other heavy chain has the following mutations: T394D, P395D, P396D, and the mutations are marked as OB (SEQ ID No.: 7) to form a heavy chain heterodimer.
  • the artificial synthesis of the first heavy chain nucleic acid sequence and the first light chain nucleic acid sequence uses the general molecular cloning method (Sambrook JF, EFet al. Molecular cloning: a laboratory manual. 4th ed.
  • the second light chain was cloned into the modified plasmid pCDNA3.1(+) (Invitrogen, catalog number V790-20)
  • the plasmid has been modified to add a human interleukin-2 (IL-2, Interleukin-2) signal peptide sequence to the N-terminus of the multiple cloning site to enable it to express and secrete antibodies in HEK293 cells.
  • IL-2 human interleukin-2
  • Interleukin-22 Interleukin-2
  • the resulting expression plasmid is labeled as pCDNA3.1-CD3-LC; clone the second heavy chain into the plasmid pFUSE-hIgG1-Fc2 (InvivoGene), the resulting expression plasmids are labeled pFUSE-CD3-HC-OA and pFUSE-CD3-HC-OA-N106T(VH2 -CDR3 contains the N106T mutation).
  • An endotoxin-free plasmid large-scale extraction kit (Endo-Free-Plasmid Maxi Kit (100), purchased from OMEGA, catalog number D6926-04) was used for large-scale plasmid extraction, and the operation steps were carried out according to the instructions provided by the kit.
  • Culture HEK293 cells to a cell density of 2.0 ⁇ 3.0 ⁇ 10 6 cells/mL, centrifuge the cell suspension for 5 min at 1000 rpm, discard the old culture supernatant, and use fresh medium (OPM-291CD05Medium, purchased from Shanghai OP Mai Biotechnology Co., Ltd.) resuspend the cells to a density of 1.0 ⁇ 106/mL. Perform co-transfection according to the plasmid combination provided in Table 1.
  • the transfected cell suspension was placed in a culture shaker at 37°C, 5% CO2, and 120 rpm for 5-7 days in the dark, and supplements were added on the fourth day. material.
  • the expression supernatant was collected by centrifugation, the cell supernatant was filtered with a 0.22 ⁇ m filter membrane, and the supernatant was captured with a protein A affinity chromatography medium (MabSelect SuRe TM , purchased from GE Healthcare, catalog number 17-5438-02).
  • a protein A affinity chromatography medium (MabSelect SuRe TM , purchased from GE Healthcare, catalog number 17-5438-02).
  • BsAb protein in the solution use equilibration buffer (137mM NaCl, 2.7mM KCl, 10mM Na 2 HPO 4 , 1.8mM KH 2 PO 4 ) to wash out non-specifically bound proteins (about 10 column volumes)
  • Buffer 100mM glycine, pH 3.0-pH 3.5
  • the eluted protein was analyzed by SDS-PAGE.
  • Figure 2-A and 2-B show that after the four-chain bispecific antibody is purified by protein A in one step, both V1 and V2 can reach high purity.
  • Reductive electrophoresis shows that the ratio of two heavy chains and two light chains is close to 1. :1:1:1.
  • Figure 2-C shows that the purity of the three-chain bispecific antibody is very high after one-step purification of protein A, and reduction electrophoresis shows that the ratio of heavy chain, light chain and scFv-Fc chain is close to 1:1:1.
  • AKTA pure 25L1 protein purification system uses the AKTA pure 25L1 protein purification system to further purify the protein obtained after the elution of protein A, and load the eluted sample onto a well-balanced cation exchange column (prepacked column Resource TM S, 1 mL, GE Healthcare, product Catalog No. 17-1178-01), equilibration buffer A (50mM sodium phosphate, pH 6.0) washes away non-specifically bound proteins until the UV absorption line is gentle, and elution buffer B (50mM sodium phosphate, 1M NaCl, pH 6.0) 15 column volumes were linearly eluted from 0%-50%, and the elution peaks were collected.
  • Figure 3 shows the elution curve.
  • the purified bispecific antibodies were tested for antigen CD3 binding by ELISA. Specific steps are as follows.
  • the CD3 antigen used in this experiment is a 1:1 mixture of human CD3e protein and human CD3d protein, purchased from Sino Biological Company.
  • Antigen coating Dilute the antigen to 100ng/mL with 0.1M sodium bicarbonate buffer (pH 9.5). Add the diluted antigen to the ELISA plate, 200 ⁇ L per well, seal the reaction well with a sealing film, and place it at 4°C for 16 hours. The plate was washed 5 times with 0.05% PBST.
  • Blocking Prepare a 3% M-PBS blocking solution with skimmed milk powder and PBS buffer, add 300 ⁇ L 3% M-PBS to each well, seal the reaction wells with a sealing film, and incubate at room temperature for 1 hour. The plate was washed 5 times with 0.05% PBST.
  • Color development Add 100 ⁇ L of TMB color development solution to each well, and incubate for 5-10 minutes at room temperature and dark.
  • Stop Add 50 ⁇ L of stop solution (1M HCl) to each well to stop the color reaction. After 3 minutes, read the OD value of the reaction solution in each well at 450nM on the microplate reader.
  • the EC50 concentration of CD20 ⁇ CD3 BsAb V1 binding to CD3 antigen is 0.8178 ⁇ g/mL
  • the EC50 concentration of CD20 ⁇ CD3 BsAb V2 binding to CD3 antigen is 2.097 ⁇ g/ml.
  • V2 has a higher binding capacity to CD3 antigen. Weak, which shows that the introduction of N106T weakened the antibody’s affinity for CD3. In practical applications, this feature will help avoid excessive T cell activation.
  • Raji cells are regarded as CD20 expression-positive and CD3 expression-negative cells (ie, CD20+/CD3-).
  • the Raji cells were cultured in a 37°C, 5% CO 2 incubator, and the cells were collected when the pooling degree was 80%-90%.
  • the CD20 ⁇ CD3 BsAb V1 and V2 samples were diluted to 20 ⁇ g/mL for use.
  • the cells were washed twice with PBS buffer containing 1% FBS, and the cells were resuspended to 1 ⁇ 10 7 cells/mL. Take a 1.5mL centrifuge tube, add 100 ⁇ L of the above cell suspension to each tube, and add 100 ⁇ L of the diluted sample. Mix well and incubate on ice for 1h.
  • the software Graphpad Prism 5.0 calculates and analyzes the binding activity of the double antibody sample and Raji cells.
  • CD20 ⁇ CD3 BsAb V1 and V2 have the same binding activity as the CD20 positive cell Raji.
  • the present disclosure performs ADCC toxicity detection on purified double antibody samples.
  • Raji cells are used as CD20-positive tumor cells.
  • 20 times the number of Raji cells was added to hPBMC, that is, 4.0 ⁇ 10 5 cells/well, 100 ⁇ L/well, so adjust the cell density to 4.0 ⁇ 10 6 /mL.
  • S spontaneously OD spontaneous release hole (target cell + effector cell)
  • Max OD maximum release hole (target cell);
  • CD20 ⁇ CD3 BsAb V1 and V2 bispecific antibodies effectively mediate hPBMC to kill CD20-positive tumor Raji cells.
  • the tumor cells have a significant killing effect, with EC50 concentrations of 0.21ng/mL and 0.24ng/ml, respectively.
  • CD20 ⁇ CD3 BsAb V2 has a weak affinity for CD3, it does not affect the killing ability of target cells Raji.
  • the CD20 ⁇ CD3 BsAb V1 samples purified by protein A were placed at 37°C and 40°C, and the storage time included: 0d, 1d, 4d, 7d.
  • the samples with different storage time were analyzed by SDS-PAGE to detect antibodies at different temperatures. Under the degradation and stability, the results show that the V1 sample can be stable for at least 7 days at 37°C and 40°C.
  • the present disclosure uses PBMC cells to determine the effect of bispecific antibodies on cytokine release. Wash the PBMC cells with PBS and adjust the cell density so that each well contains 2 ⁇ 10 5 cells. The antibody was diluted to 0.25 ⁇ g/mL with 1640 medium, diluted in gradients, and added to the above-mentioned wells. Place the 96-well plate in a 37°C, 5% CO 2 incubator for 41 hours. The concentration of cytokines in the sample was detected by double-antibody sandwich ELISA.
  • the specific method is: centrifuge the 96-well plate containing PBMC cells at 1000 rpm for 5 min, take the supernatant and add 80 ⁇ L/well to the corresponding well, and seal the reaction well with a sealing film , Incubate at room temperature for 120 minutes. Wash the plate 5 times, add 100 ⁇ L of biotinylated antibody per well, seal the reaction well with a parafilm, and incubate at room temperature for 60 minutes.
  • CD20 ⁇ CD3 BsAb V1, CD20 ⁇ CD3 BsAb V2, CD3 monoclonal antibody, and IgG to incubate overnight with hPBMC from different healthy donors.
  • CD20 ⁇ CD3 BsAb V1, CD20 ⁇ CD3 BsAb V2, CD3 monoclonal antibody, and IgG Take the supernatant to perform the above-mentioned ELISA sandwich method experiment to detect the cytokines IL-6 and IL- 2 to identify whether T cells are non-specifically activated and produce cytokine storms.
  • CD20 ⁇ CD3 BsAb The in vivo pharmacodynamic evaluation of CD20 ⁇ CD3 BsAb is based on the Raji subcutaneous xenograft model with positive expression of CD20.
  • Model establishment method Collect Raji cells in logarithmic growth phase, mix them with hPBMC, and inoculate them subcutaneously on the back of NOD-SCID mice. Randomly group them into control group and treatment group.
  • the bispecific antibody of the present disclosure is treated according to the grouping CD20 ⁇ CD3 BsAb V1 was delivered in low doses (0.01mg/kg and 0.05mg/mL), medium doses (0.1mg/kg and 0.5mg/mL) and high doses (2mg/kg) on day 1, day 3.
  • the drug was administered once on the 5th day and the 8th day.
  • the negative control group was given an equal volume of sterile saline.
  • the CD20 ⁇ CD3 BsAb V1 (2mg/kg, 0.5mg/kg, 0.1mg/kg, 0.05mg/kg, 0.01mg/kg) treatment group showed significant inhibition 21 days after starting treatment. Tumor effect. All treatment groups had no animal deaths caused by drug treatment, no obvious drug toxicity and severe weight loss, and they were well tolerated during the administration period.
  • the CD20 ⁇ CD3 BsAb molecules described in this disclosure can not only bind to tumor cell surface antigens but also T cell surface CD3 molecules, relying on antigen-specific activation of TCR and increasing T cell targeting Tumor killing effect, using a smaller amount of antibody can play a significant effect of killing tumor cells.
  • the CD20 ⁇ CD3 BsAb molecule described in the present disclosure contains the Fc region of the antibody, which increases the half-life and stability of the antibody, and is easier to use the existing monoclonal antibody purification technology to simplify The production process of bispecific antibodies was introduced.
  • CD20-targeted chimeric antigen receptor T cell immunotherapy CD20-targeted chimeric antigen receptor T cell immunotherapy (CAR-T)
  • the present disclosure does not involve operations such as foreign viruses, in vitro T cell culture and reinfusion, and has fewer side effects and is safer control.

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Abstract

提供一种双特异性抗体及其制备方法，所述抗体包含能结合CD20、CD3的结构域和异源二聚体Fc区域，所述能结合CD20和CD3的结构域各自独立地选自Fab区域、ScFv区域或sDab区域。所述双特异性抗体靶向CD20和CD3，介导T细胞特异性杀伤肿瘤细胞。

Description

一种双特异性抗体及其制备方法与应用
相关申请的交叉引用
本申请要求于2019年7月2日提交中国专利局的申请号为201910591110.2、名称为“一种双特异性抗体及其制备方法与应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及一种抗体及其制备方法与应用，具体涉及一种双特异性抗体及其制备方法与应用。
背景技术
B细胞表面的特异性抗原如CD19、CD20、CD22、CD52等是B细胞相关性疾病的潜在治疗靶点，其中CD20蛋白又被叫做B淋巴细胞限制性分化抗原、Bp35或B1，是人MS4A1基因所编码表达的四次跨膜、高度疏水的非糖基化磷蛋白，分子量为35kD左右。CD20特异性地表达于前B细胞以及成熟的B细胞表面，但在造血干细胞、祖细胞、浆细胞和其他正常体细胞中不表达。CD20还同时表达于B细胞淋巴瘤如：非霍奇金淋巴瘤(NHL)、慢性淋巴细胞白血病(CLL)等的细胞表面。CD20没有已知的天然配体，研究表明，CD20具有钙离子通道活性，通过调控细胞内钙离子浓度调节细胞周期，在B细胞激活即从G0期进入G1期的分化和生长过程中发挥特殊作用，同时调节细胞周期从S期进入有丝分裂期，也能诱导细胞凋亡。
由于CD20特异性地表达于多种B细胞相关的淋巴瘤细胞表面，前B细胞和其他体细胞上没有CD20的表达，并且CD20不轻易脱落或分泌，与抗体结合后不发生内吞，因此是治疗B细胞相关疾病非常具有潜力的理想靶点。目前针对CD20的治疗性单克隆抗体类药物有多种已上市，根据作用机理可将其分为两类：Type I和Type II，Type I类抗体主要通过补体依赖的细胞毒性作用(CDC)和抗体依赖的细胞毒性作用(ADCC)发挥功能，Type II类主要通过程序性细胞死亡(PCD)和ADCC发挥功能。人鼠嵌合抗体利妥昔单抗(Rituximab，商品名：Rituxian/美罗华)为第一代CD20抗体(Type I)，1997年被FDA批准用于治疗NHL，随后又被批准用于治疗类风湿性关节炎、CLL等疾病；全人源单抗奥法木单抗(Ofatumumab，商品名：Arzerra)为第二代CD20抗体(Type I)，与美罗华相比，奥法木单抗对CD20的亲和力更强，细胞试验显示CDC反应也更强，ADCC反应与美罗华类似，于2009年被FDA加速批准用于治疗氟达拉滨或阿仑单抗治疗无效的CLL，后又被批准用于治疗滤泡淋巴瘤、视神经脊髓炎、弥漫性和复发性多发性硬化症等疾病；人源化单抗奥滨尤妥珠单抗(Obinutuzumab，商品名：Gazyva)为第三代CD20抗体(Type II)，由于经过了糖基化改造，增强了抗体与NK细胞、巨噬细胞和树突状细胞的亲和力，因而比美罗华有更强的ADCC反应，同时奥滨尤妥珠单抗通过中性粒细胞导致的胞吞作用和细胞死亡能更有效地诱导B细胞凋亡，2013年被FDA批准与苯丁酸氮芥联合治疗过往未接受治疗的CLL。
与传统化疗方式相比，针对CD20靶点的单抗药物在临床上显示了更好的临床优势，但往往需要与化学药物联用，而且不是所有患者都能对CD20抗体疗法产生响应。这是由于CD20单抗的疗效与肿瘤细胞上CD20的表达水平相关，某些类别如CLL细胞上的CD20表达水平显著低于其他类别的B细胞肿瘤，限制了CD20单抗的疗效。另外，单抗主要是通过结合FcγR产生的ADCC、ADCP、CDC等作用机制杀伤肿瘤细胞，而FcγR在人群中具有多态性，导致了FcγR对Fc的亲和力有显著的差异，降低了药物的总体有效性，导致复发并产生耐药性，一旦复发，治疗难度会更大，预后极差，中位生存期只有2-8个月。具有高效肿瘤杀伤效果的T细胞因为没有FcγR而无法被有效激活，为此，需要设计更有效的临床抗体药物来满足目前的治疗形势，比如激活T细胞从而更加高效地杀伤肿瘤细胞，或者开发同时结合两个或多个抗原靶点的多功能抗体。
双特异性抗体由于能同时结合两种抗原表位或者一种抗原的两个表位，通过协同作用，可以达到更高的效能(potency)；有些双特异抗体可以将效应细胞(如T细胞)等招募至目标靶点，通过激活T细胞直接杀伤靶细胞。当前双特异抗体的安全性和有效性得到了很大的改善，被广泛应用于肿瘤、自身免疫疾病的治疗，是肿瘤免疫治疗的一个研发热点，具有广阔的应用前景。在治疗恶性肿瘤的实际应用中，双特异抗体通常同时结合肿瘤细胞表面抗原以及免疫细胞表面抗原，通过激活自身免疫系统杀伤肿瘤细胞。
CD3是同源或异源二聚体蛋白复合物，是构成T细胞受体(TCR)的重要组成成分，其胞内区域含有免疫受体酪氨酸激活基序(ITAM)，当ITAM磷酸化后会与激酶ZAP70结合，从而向下游转导T细胞激活信号。靶向CD3的抗体能够在T细胞表面聚集CD3，从而激活T细胞，这一过程模拟了TCR识别MHC-抗原肽的过程。具有CD3抗性和肿瘤特异性抗原抗性的双特异性抗体能够同时结合T细胞和肿瘤细胞，激活并引导T细胞分泌颗粒酶、穿孔素等杀伤肿瘤细胞，例如同时结合CD20和CD3分子的双特异性抗体，能够将T细胞招募至表达CD20蛋白的肿瘤细胞处，导致CD20阳性的肿瘤细胞被杀伤清除。
早期的双特异性抗体大多是通过已纯化好的单抗化学交联或表达两种不同单抗的杂交瘤细胞融合产生，但这些方法生产的产品会有许多问题如产物不稳定、产量低下、抗体修饰不当、免疫原性、生产纯化困难等。随着近些年基因工程技术的进步，BsAb大量通过基因工程技术制备，目前已有超过60种不同的双特异性抗体形式，总体可以分为两类：不包含Fc以及包含Fc区域的双特异性抗体。不包含Fc的双特异抗体，例如：BiTE、DART、TandAbs、Bi-Nanobody等，分子量小、可以在原核细胞中实现表达、生产复杂性降低且容易穿过组织和肿瘤细胞到达靶点，但是不能介导Fc相关的生物学功能，在体内会被快速清除，半衰期很短，若抗体中引入了非天然肽链连接片段或额外的结构，相对分子量和理化性质与天然IgG抗体差别较大，且更容易形成多聚体，产生免疫原性。包含Fc的双特异抗体能够介导ADCC以及CDC，增加抗体在血液中的半衰期(FcRn介导)、稳定性和溶解性，另外也是蛋白A(Protein A)、蛋白G(Protein G)的结合区域，方便下游产品的纯化，这一类的双特异抗体技术有TrioMab、Knob-into-hole、DVD-Ig、DuoBody、SEEDbody、BEAT等。
发明内容
本公开的目的包括，例如克服上述现有技术的不足之处而提供一种稳定、高效靶向CD20和CD3的双特异性抗体，该双特异性抗体能够介导T细胞特异性杀伤肿瘤细胞。
第一方面，本公开提供了一种双特异性抗体，其包含能结合CD20的结构域、能结合CD3的结构域和异源二聚体Fc区域，所述能结合CD20的结构域和能结合CD3的结构域各自独立地选自Fab区域、ScFv区域或sDab区域。
最理想的一种双特异抗体是保持IgG抗体的天然结构，这需要克服抗体装配过程中重链与轻链错配的问题，本公开通过改变抗体Fc以及重链-轻链交界面的少量氨基酸，在引入极少位点突变且不影响抗体结构和功能的基础上，利用该人工改造配对方法制备同时靶向白细胞分化抗原20(CD20)和白细胞分化抗原3(CD3)的双特异性抗体，与传统单抗相比，该CD20×CD3双特异抗体有如下优势：(1)激活CD4 +和CD8 +的T细胞特异地杀伤CD20表达阳性的肿瘤细胞，包括CD20表达水平很低的肿瘤细胞；(2)在很低的使用剂量下，仍有高效的肿瘤杀伤效果；(3)能够引起低水平的细胞因子释放，安全性更高；(4)与天然IgG抗体的结构类似，具有与单抗类似的稳定性、半衰期及理化性质。该双特异抗体可用于疾病治疗、诊断等多个应用领域。
本公开的双特异性抗体能特异性结合CD20表位和CD3表位，通过抗原特异性介导T细胞激活，是一种能够介导T细胞特异性杀伤肿瘤细胞的新型、稳定、高效靶向CD20和CD3的双特异性抗体(即CD20×CD3 BsAb)，它能够与人和灵长类动物的CD20和CD3交叉反应。
本公开的双特异抗体由三条或者四条多肽链组成，三链结构包括重链、轻链和scFv-Fc链，四链结构包括二条重链和二条轻链。所述双特异抗体形成一个特异性结合CD20的免疫球蛋白结构域、一个特异性结合CD3的免疫球蛋白结构域以及一个异源二聚体Fc区域。两条重链(或者一条重链与scFv-Fc链)相互作用并结合构成异源二聚体的形式，形成上述异源二聚体Fc区域。在四链双特异抗体中两条轻链各自与其中一条重链结合，形成上述特异性结合CD20的Fab结构以及特异性结合CD3的Fab结构。需要特别指出的是，本公开所述CD20×CD3 BsAb包含Fc区域，能够增加抗体在机体内的半衰期和稳定性。
在本公开所述双特异性抗体的一种或多种实施方式中，所述能结合CD20的结构域包括第一免疫球蛋白Fab区域，所述第一免疫球蛋白Fab区域主要由相互结合的第一重链与第一轻链形成，所述第一重链的可变区具有如SEQ ID No.:2所示的氨基酸序列，所述第一轻链的可变区具有如SEQ ID No.:4所示的氨基酸序列；和/或所述能结合CD3的结构域包括第二免疫球蛋白Fab区域，所述第二免疫球蛋白Fab 区域主要由相互结合的第二重链与第二轻链形成，所述第二重链的可变区具有如SEQ ID No.:6所示的氨基酸序列，所述第二轻链的可变区具有如SEQ ID No.:10所示的氨基酸序列；
所述异源二聚体Fc区域由两条多肽链构成，每条多肽链包含带相反电荷的不对称氨基酸修饰。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链的CH1部分以及第一轻链的CL部分均包含一个取代天然半胱氨酸残基的非半胱氨酸残基，以及一个取代非天然半胱氨酸的半胱氨酸残基。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链的CH1部分中取代天然半胱氨酸残基的非半胱氨酸残基与第一轻链的CL部分中取代非天然半胱氨酸的半胱氨酸残基形成二硫键。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链的CH1部分中，取代天然半胱氨酸残基的非半胱氨酸残基为C220S，取代非天然半胱氨酸的半胱氨酸残基为L128C。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一轻链的CL部分中，取代天然半胱氨酸残基的非半胱氨酸残基为C214S，取代非天然半胱氨酸的半胱氨酸残基为F118C。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第二免疫球蛋白Fab区域不包含取代天然半胱氨酸残基的非半胱氨酸残基，且不包含取代非天然半胱氨酸残基的半胱氨酸残基。
在本公开所述双特异性抗体的一种或多种实施方式中，所述能结合CD20的结构域包括第一免疫球蛋白Fab区域，所述能结合CD3的结构域包括第二免疫球蛋白Fab区域；所述第一重链与第二重链相互结合，形成异源二聚体Fc区域。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链的铰链区和所述第二重链的铰链区通过至少一个二硫键共价结合。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链和第二重链均包含CH2结构域和改造的CH3结构域，所述第一重链的CH2结构域、改造的CH3结构域与所述第二重链的CH2结构域、改造的CH3结构域相互结合形成异源二聚体Fc区域。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链和第二重链的CH2结构域中，至少一个CH2结构域包含一个、两个或多个突变位点。突变位点用以减少Fc与其受体FcγR的结合。在本公开所述双特异性抗体的一种或多种实施方式中，该突变组合为L234A/L235A。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链中改造的CH3结构域与第二重链中改造的CH3结构域包含带相反电荷的不对称氨基酸修饰，用以形成异源二聚体Fc区域。包含带相反电荷的不对称氨基酸修饰可促进异源二聚体的形成。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链中改造的CH3结构域含有P395K，P396K、V397K突变中的至少一种，且所述第二重链中改造的CH3结构域含有T394D，P395D、P396D突变中的至少一种；或者，
所述第一重链中改造的CH3结构域含有T394D，P395D、P396D突变中的至少一种，且所述第二重链中改造的CH3结构域含有P395K，P396K、V397K突变中的至少一种。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链和第二重链来自人抗体IgG1、IgG2、IgG3或IgG4。在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链和第二重链来自人抗体IgG1。
在本公开所述双特异性抗体的一种或多种实施方式中，所述第一重链具有如SEQ ID No.:1所示的氨基酸序列，第一轻链具有如SEQ ID No.:3所示的氨基酸序列，所述第二重链具有如SEQ ID No.:5或SEQ ID No.:7所示的氨基酸序列，第二轻链具有如SEQ ID No.:9所示的氨基酸序列。
在本公开所述双特异性抗体的一种或多种实施方式中，所述能结合CD20的结构域包括第一免疫球蛋白Fab区域，所述能结合CD3的结构域包括scFv-Fc融合蛋白多肽链；所述scFv-Fc融合蛋白多肽链中scFv区域的重链可变区具有如SEQ ID No.:6所示的氨基酸序列，轻链可变区具有如SEQ ID No.:10所示的氨基酸序列，所述第一重链与所述scFv-Fc融合蛋白多肽链相互结合，形成一个异源二聚体Fc区域；或者，
所述能结合CD20的结构域包括scFv-Fc融合蛋白多肽链，能结合CD3的结构域包括第二免疫球蛋白Fab区域；所述scFv-Fc融合蛋白多肽链中具有如SEQ ID No.:2所示的氨基酸序列，轻链可变区具有 如SEQ ID No.:4所示的氨基酸序列，所述第二重链与所述scFv-Fc融合蛋白多肽链相互结合，形成一个异源二聚体Fc区域。
在本公开所述双特异性抗体的一种或多种实施方式中，所述能结合CD20的结构域包括第一免疫球蛋白Fab区域，所述能结合CD3的结构域包括scFv-Fc融合蛋白多肽链；所述第一重链的可变区具有如SEQ ID No.:8所示的氨基酸序列；所述scFv-Fc融合蛋白多肽链中的scFv区域具有如SEQ ID No.:11所示的氨基酸序列，所述scFv-Fc融合蛋白多肽链中的重链可变区CDR3包含N106T突变,突变后的CDR3序列为：HGNFGTSYVSWFA。
在本公开所述双特异性抗体的一种或多种实施方式中，所述scFv-Fc融合蛋白多肽链中的scFv区域具有如SEQ ID No.:12、SEQ ID No.:13或SEQ ID No.:14所示的氨基酸序列，或者其可变区具有如SEQ ID No.:2、SEQ ID No.:4、SEQ ID No.:8、SEQ ID No.:6或SEQ ID No.:10所示的氨基酸序列。
第二方面，本公开提供了一种表达载体，所述表达载体是将上述氨基酸序列对应的DNA序列连接到基础体上得到的。
在本公开所述双特异性抗体的一种或多种实施方式中，所述表达载体为质粒、病毒载体或重组表达载体。
在本公开所述双特异性抗体的一种或多种实施方式中，所述基础载体为pFUSE-hIgG 1-Fc2。
第三方面，本公开提供了一种宿主细胞，所述宿主细胞含有上述表达载体。
在本公开所述双特异性抗体的一种或多种实施方式中，所述宿主细胞为哺乳动物细胞。在本公开所述双特异性抗体的一种或多种实施方式中，所述宿主细胞为CHO细胞或HEK293细胞。
第四方面，本公开提供了上述双特异性抗体的制备方法，其包括以下步骤：
(a)构建表达载体，所述表达载体是将编码如权利要求1～13任一项所述双特异性抗体的核苷酸连接到基础载体上得到的；
(b)将步骤(a)构建的表达载体转染或转化至宿主细胞中，并培养宿主细胞；
(c)分离、纯化双特异性抗体。
在本公开所述双特异性抗体的一种或多种实施方式中，所述分离、纯化双特异性抗体的方法为蛋白A亲和层析法、阳离子交换法或阴离子交换法。
第五方面，本公开提供了一种抗体偶联物，所述抗体偶联物是偶联物质与上述双特异性抗体偶联所形成。
在本公开所述双特异性抗体的一种或多种实施方式中，所述偶联物质为细胞毒素、放射性同位素、荧光标记物、发光物、显色物质或酶。
第六方面，本公开提供了一种药物组合物，其包含上述双特异性抗体。
第七方面，本公开提供了上述双特异性抗体在制备治疗肿瘤、风湿性关节炎、多发性硬化症或系统性红斑狼疮的药物或药物组合物中的用途。
在本公开所述双特异性抗体的一种或多种实施方式中，所述肿瘤为与CD20表达相关的恶性肿瘤。
在本公开所述双特异性抗体的一种或多种实施方式中，所述恶性肿瘤为急性B淋巴细胞性白血病(B-ALL)，弥漫性大B细胞淋巴瘤(DLBCL)，慢性淋巴细胞白血病(CLL)，滤泡淋巴瘤，非霍奇金氏淋巴瘤(NHL)，慢性骨髓细胞白血病(CML)，伯基特(Burkitt)淋巴瘤。
第八方面，本公开提供了上述双特异性抗体在药物或药物组合物中的用途。
第九方面，本公开提供了上述双特异性抗体在治疗肿瘤、风湿性关节炎、多发性硬化症或系统性红斑狼疮中的用途。
与现有技术相比，本公开的有益效果为：
(1)本公开所述CD20×CD3 BsAb分子保持了天然IgG抗体的结构或者接近天然IgG抗体的结构，具有与单抗类似的稳定性及理化性质。
(2)本公开涉及双特异性抗体的构建，对重链及轻链上少量氨基酸进行突变，使得两条多肽链带相反电荷，促进重链异源二聚体的形成，并通过引入非天然的二硫键克服轻链错配。该方法涉及突变极少，对Fc区域功能影响极小且不影响蛋白在真核细胞中的表达产量。
(3)与单克隆抗体相比，本公开所述CD20×CD3 BsAb分子能够在结合肿瘤细胞表面抗原的同时 也能结合T细胞表面CD3分子，依赖抗原特异性激活TCR进而增加了T细胞靶向肿瘤杀伤作用，用更少量的抗体就能发挥显著杀伤肿瘤细胞的效果。
(4)与BiTE等小分子双特异性抗体相比，本公开所述CD20×CD3 BsAb分子包含抗体Fc区域，增加了抗体的半衰期和稳定性，且更容易采用现有单抗纯化技术，简化了双特异抗体的生产工艺。
(5)与靶向CD20的嵌合抗原受体T细胞免疫疗法(CAR-T)相比，本公开不涉及外源病毒、体外T细胞培养回输等操作，毒副作用更小，更安全可控。
(6)本公开涉及CD3亲和力弱化改造，提高CD3类双特异抗体分子的安全性。
附图说明
图1为本公开所述CD20×CD3 BsAb结构实例示意图；
图2为蛋白A亲和层析纯化后的CD20×CD3 BsAb，其中图2A为CD20×CD3 BsAb V1蛋白A一步纯化后非还原和还原SDS-PAGE电泳胶图，其中图2B为CD20×CD3 BsAb V2蛋白A一步纯化后非还原和还原SDS-PAGE电泳胶图，其中图2C为CD20×CD3 BsAb(三链双特异抗体，anti-CD20结构域为scFv，anti-CD3结构域为Fab)蛋白A一步纯化后非还原和还原SDS-PAGE电泳胶图；
图3为通过阳离子交换层析(CEX)方法纯化的CD20×CD3 BsAb V1A280紫外吸收峰图；将蛋白A纯化后的样品(图2A)上样至阳离子交换柱，通过NaCl梯度洗脱，通过检测紫外光280nm处的吸收值收集洗脱的蛋白。CD20×CD3 BsAb V1通过CEX形成了1个主要洗脱峰(D峰)以及4个小的次要洗脱峰；
图4为双抗纯化的洗脱曲线；(A)取浓度为1mg/mL蛋白A纯化样品(图2A)1mL，通过凝胶过滤层析(SEC)分析蛋白一步纯化后样品的聚体含量，该图显示了A280紫外吸收峰图；(B)取浓度为1mg/mL蛋白A纯化样品(图2C，lane1)1mL，通过凝胶过滤层析(SEC)分析蛋白一步纯化后样品的聚体含量，该图显示了A280紫外吸收峰图；
图5为CD20×CD3双特异性抗体与抗原CD3的结合力的检测结果图；其中，图5A通过ELISA比较CD20×CD3 BsAb V1和V2对CD3抗原分子的亲和力，CD20×CD3 BsAb V1与CD3抗原结合的EC50浓度为0.8178μg/mL，CD20×CD3 BsAb V2与CD3抗原结合的EC50浓度为2.097μg/ml；图5B展示三链双特异抗体与CD3抗原分子的结合能力，1：四链双特异抗体，其中结合CD20的结构域为Fab，结合CD3的结构域Fab，其与CD3抗原结合的EC50浓度为4.9μg/ml；2：三链双特异抗体，其中结合CD20的结构域为scFv，结合CD3的结构域Fab，其与CD3抗原结合的EC50浓度为4.9μg/ml；3：三链双特异抗体，其中结合CD20的结构域为Fab，结合CD3的结构域scFv，其与CD3抗原结合的EC50浓度为0.6μg/ml；
图6为FACS检测CD20×CD3 BsAb V1和CD20×CD3 BsAb V2与CD20表达阳性的细胞(Raji细胞)的结合图；
图7为ADCC细胞杀伤实验；其中，图7A、B为检测CD20×CD3 BsAb V1和V2介导的对靶细胞(Raji和Daudi细胞)的杀伤能力，V1和V2对CD20高表达的肿瘤细胞杀伤效果显著，EC50浓度为均为0.2ng/ml；图7C为检测CD20×CD3三链双特异抗体介导的对靶细胞(Daudi细胞)的杀伤能力，1：三链双特异抗体，其中结合CD20的结构域为scFv，结合CD3的结构域Fab；2：三链双特异抗体，其中结合CD20的结构域为Fab，结合CD3的结构域scFv；3：四链双特异抗体，其中结合CD20的结构域为Fab，结合CD3的结构域Fab；
图8为蛋白热稳定性试验；其中，蛋白样品置于37℃和40℃不同时间：0天、1天、4天、7天时蛋白样品的SDS-PAGE电泳图；
图9为CD20×CD3 BsAb细胞因子释放试验结果图；
图10为CD20×CD3 BsAb介导的小鼠体内抗肿瘤活性检测结果图。
具体实施方式
为更好地说明本公开的目的、技术方案和优点，下面将结合附图和具体实施例对本公开作进一步说明。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
BsAb：双特异性抗体(bispecific antibody)
HC：重链(heavy chain)
LC：轻链(light chain)
VH：重链可变区(variable region of heavy chain)
VL：轻链可变区(variable region of light chain)
CH：重链恒定区(constant region of heavy chain)
CL：轻链恒定区(constant region of light chain)
CDR：抗原互补决定区(complementarity determining region)
scFv：单链可变区抗体片段(single-chain variable fragment)
ADCC：抗体依赖的细胞介导的细胞毒性作用(antibody dependent cellular cytotoxicity)
ELISA：酶联免疫吸附剂测定(enzyme-linked immunosorbent assay)
FACS：荧光激活细胞分选，即流式细胞术(Fluorescence-activated cell sorting)
EC50：半数效应浓度(half maximal effective concentration)
除非另外定义，否则在本公开中所使用的相关术语与该技术领域通常理解的含义相同。本公开所述分子克隆、细胞培养、蛋白纯化、免疫学实验、微生物学、动物模型等试验的操作步骤为该领域内被广泛应用的常规步骤。除非上下文另有说明，否则本公开单数术语包含复数且复数含义包括单数含义。除非另外指明，否则本公开所述核苷酸序列按照5’端至3’端的方向从左至右排列并书写。除非另外指明，否则本公开所述氨基酸序列按照从氨基端(N末端)到羧基端(C末端)的方向从左至右排列并书写。本公开所提及的氨基酸三字母缩写及核苷酸单字母缩写为该技术领域普遍接受的形式，氨基酸单字母缩写为IUPAC-IUB生物化学命名委员会(IUPAC-IUB Biochemical Nomenclature Commission)推荐的形式。
术语“氨基酸”是指20种天然存在的氨基酸之一或可能存在于特定的限定位置的任何非天然类似物。本公开所述“氨基酸突变”是指多肽序列中的氨基酸取代、插入、缺失和修饰，以及氨基酸取代、插入、缺失和修饰的任意组合。本文中优选的氨基酸修饰是取代。本公开中“氨基酸取代”或“取代”是指将亲本多肽序列中特定位置的氨基酸替代为另一种氨基酸。例如，取代C220S是指变体多肽，其中多肽的位置220处的氨基半胱氨酸已被氨基酸丝氨酸替代。氨基酸突变可以通过分子克隆或化学的方法实现，分子克隆的方法包括PCR、定点突变、全基因合成等。
术语“蛋白”、“肽链”、“多肽链”是指两个以上的氨基酸通过肽键连接的分子，包含天然蛋白、人工蛋白、蛋白片段、突变蛋白、融合蛋白等。
术语“结构域”是指生物大分子中具有独立功能的特异结构区域，结构域具有独立的三级结构，其功能不依赖于生物大分子中的其余部分，本公开中的结构域特指蛋白中这样的区域，如重链可变区VH结构域、轻链可变区VL结构域，结构域之间相互结合可以构成一个大的结构域。
术语“CD3”指分化抗原簇-3蛋白，是表达于T细胞表面构成T细胞受体复合物(TCR)的组成成分，是由CD3δ(人CD3δUniProt Entry No.P0423)、CD3ε(人CD3εUniProt Entry No.P07766)、CD3γ(人CD3γUniProt Entry No.P09693)和CD3ζ(人CD3ζUniProt Entry No.P20963)四条肽链构成的同源或异源二聚体，除非明确指明，如“食蟹猴CD3”、“小鼠CD3”，否则本公开中所提到的“CD3”为人CD3。
本公开所述“与CD3结合的多肽链”或“与CD3结合的抗体”包括与CD3单链单元特异性结合的抗体或抗体片段，以及与CD3同源或异源二聚体特异性结合的抗体或抗体片段。本公开所述特异性结合CD3的抗体能够与CD3蛋白分子结合也能够与表达于细胞表面的CD3结合。
术语“CD20”是指由人MS4A1基因编码表达的B淋巴细胞限制性分化抗原(人CD20UniProt Entry No.P11836)，包括细胞天然表达的以及经转染外源基因表达的人CD20蛋白的变异型、亚型和其他物种的同源蛋白(如食蟹候CD20蛋白)。
术语“抗体”指包含至少一个抗原识别位点并能特异性结合抗原的免疫球蛋白分子。在此，术语“抗原”是在机体内能诱发免疫应答且与抗体特异性结合的物质，如蛋白质、多肽、肽、碳水化合物、多聚核苷酸、脂质、半抗原或上述物质的组合。抗体与抗原的结合依靠二者间形成的相互作用来介导，包括氢键、范德华力、离子键以及疏水键。抗原表面与抗体结合的区域为“抗原决定簇”或“表位”，一般来说，每个抗原有多个决定簇。本公开所提及的术语“抗体”包含单克隆抗体(包括全长单克隆抗体)、多克隆抗体、抗体片段、包含至少两个不同表位结合结构域的多特异性抗体(例如，双特异性抗体)、人抗体、人源化抗体、翻译后修饰抗体、骆驼抗体、嵌合抗体、包含抗体抗原决定簇的融合蛋白、以及包含抗原 识别位点的任何其他修饰的免疫球蛋白分子，只要这些抗体展现出所期望的生物活性。具体来说，抗体包括免疫球蛋白分子和免疫球蛋白分子的免疫活性片段，即至少含有一个抗原结合位点的分子。
天然人抗体轻链通过两个二硫键与重链共价连接，重链-轻链二聚体再由重链间形成的二硫键构成类似字母Y的抗体分子。不同类别的人抗体重链间的二硫键数量会有差异。位于Y字形两臂与主干间的区域为铰链区，具有一定的柔性。每条抗体多肽链包括可变区以及恒定区，通过空间折叠形成不同的结构域单元。每条轻链由氨基端的可变区结构域VL以及羧基端的一个恒定区结构域CL构成。每条重链由氨基端的可变区结构域VH以及后面的三个或四个恒定区结构域(CH1、CH2、CH3、CH4)构成。
抗体的可变区为抗原结合区域，包括重链可变区(VH)和轻链可变区(VL)，VH和VL的结构类似，重链和轻链的可变区分别由三个互补决定区(complementarity determining region，CDR)以及位于CDR侧翼的四个框架区(framework region，FR)组成。框架区对CDR起到支撑作用，并限定了各个CDR间的空间关系。可变区的CDR在序列上高度可变，因而又被叫做高可变区(hypervariable region，HVR)，CDR形成特定的环状结构，直接参与抗原的识别。每个可变区结构域包含3段HVR，VH上的HVR分别为H1、H2、H3，VL上的HVR分别为L1、L2、L3。重链或轻链的CDR由氨基端开始分别表示为CDR1、CDR2、CDR3。重链和轻链可变区通过非共价键结合，重链的3个CDR和轻链的3个CDR共同构成了抗原识别位点，该部分氨基酸残基是抗体参与抗原结合的主体，构成抗体识别抗原的特异性。
术语“Fab”、“Fab区域”、“Fab片段”或“Fab分子”是抗原结合片段，包含免疫球蛋白重链的VH结构域、CH1结构域以及轻链的VL结构域、CL结构域，重链第一个恒定区结构域CH1与轻链的恒定区结构域CL结合，重链的可变区结构域VH和轻链的可变区结构域VL结合。
术语“Fc”、“Fc区域”、“Fc片段”或“Fc分子”是抗体的效应区，能够引起如CDC、ADCC、ADCP、细胞因子释放等。天然的抗体Fc通常是由两个相同的蛋白片段结合构成，该蛋白片段包含两个或三个免疫球蛋白恒定区结构域。本公开中所述Fc包含天然Fc以及突变的Fc。虽然Fc区的边界可以变化，但人IgG重链Fc区通常定义为包含从C226或P230开始到其羧基末端的残基。在实验条件下，免疫球蛋白单体经木瓜蛋白酶酶切生成的片段分别为Fab和Fc。抗体的“铰链”或“铰链区”是指包含抗体的第一和第二恒定结构域(CH1和CH2)之间的氨基酸的柔性多肽。
本公开所述双特异性抗体包含的Fc为人免疫球蛋白Fc。在通常情况下，人免疫球蛋白Fc区域的多肽序列来源于野生型的人免疫球蛋白Fc区域。野生型的人免疫球蛋白Fc指人群中普遍存在的氨基酸序列，其中也包含某些个体在Fc区域不同位置的多态性。本公开所述人免疫球蛋白Fc也包括对野生型人免疫球蛋白Fc个别氨基酸所作的改变，例如，为形成重链异源二聚体而在Fc区域改变部分界面氨基酸。
除非另有说明，否则本公开所述抗体可变区氨基酸编号使用Kabat等人在1991年所阐述的编码方案，即“Kabat索引”或“Kabat编号”。除非另有说明，否则本公开所述抗体恒定区氨基酸编号使用EU索引。
术语“抗原结合位点”指抗原结合分子与抗原产生直接相互作用的一个或多个氨基酸残基，抗体的抗原结合位点由抗原互补决定区(CDR)构成，天然免疫球蛋白分子通常包含两个抗原结合位点，Fab分子通常包含一个抗原结合位点。
术语“T细胞激活”指T淋巴细胞尤其是杀伤性T淋巴细胞的一种或多种免疫反应，包括：增值、分化、细胞因子的释放、杀伤性效应分子的分泌、细胞杀伤等。
术语“EC50”即半数效应浓度(half maximal effective concentration)，是指引起50％最大效应所对应的抗体浓度。
本文所用“特异性结合”是指，两种分子间的非随机的结合反应，如抗体及其所针对的抗原之间的反应。在某些实施方案中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M、或10 -10M或更小的亲和力(K D)结合该抗原。在本公开的一些实施方案中，术语“靶向”指特异性结合。
本文所用“K D”是指，特定抗体-抗原相互作用的解离平衡常数，用于描述抗体与抗原间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M、或10 -10M或更小的平衡解离常(K D)结合 抗原。
术语“单链可变区抗体片段”或“scFv”指免疫球蛋白重链可变区VH和轻链可变区VL的融合蛋白，包括N端为VH以及N端为VL的不同组合形式，利用构建重组蛋白的常规分子克隆方法即可制备(Sambrook JF,E.F.et al.Molecular cloning:a laboratory manual.4th ed.Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York:2012)。
术语“人源化抗体”是指人源免疫球蛋白(受体抗体)的部分或者全部CDR区被一非人源抗体(供体抗体)的CDR区替换后得到的抗体或者抗体片段，其中的供体抗体可以是具有预期特异性、亲和性或者反应性的非人源(如小鼠、大鼠或兔)抗体。此外，受体抗体的架构区(FR)的一些氨基酸残基也可被相应的非人源抗体的氨基酸残基替换，或者被其他抗体的氨基酸残基替换，以进一步改善或者优化抗体的一种或多种特性。
术语“双特异性抗体”是指能够结合两个独立抗原或对同一抗原内不同表位具有结合特异性的抗体分子。例如，在一些实施例中，双特异性抗体分子的一臂结合肿瘤相关抗原，另一臂结合免疫细胞相关抗原(例如，CD3分子)，这样可以在肿瘤细胞处激活并启动细胞免疫相关机制。
双特异性抗体还可以偶联细胞毒素(例如，放射性同位素、长春花生物碱、氨甲喋呤等)、放射性同位素、荧光标记物、发光物、显色物质或酶。在一些实施方案中，与本公开的抗体或双特异性抗体偶联以形成抗体偶联物的部分是细胞毒素。在一些实施方案中，所述细胞毒素是指抑制或阻止细胞功能和/或引起细胞破坏的物质，并包括小分子细胞毒素。在一些实施方案中，所述细胞毒素选自秋水仙素，emtansine，maytansinoid,auristatin，vindesine，tubulysin等。在一些实施方案中，与本公开的抗体或双特异性抗体偶联以形成抗体偶联物的部分是放射性同位素。在一些实施方案中，所述放射性同位素包括例如At 211，I 131，I 125，Y 90，Re 186，Re 188，Sm 153，Bi 212，P 32和Lu的放射性同位素。在一些实施方案中，与本公开的抗体或双特异性抗体偶联以形成抗体偶联物的部分选自荧光标记物、发光物和显色物质，例如：异硫氰酸荧光素(FITC)，荧光素酶(luciferase)，辣根过氧化物酶(HRP)等。在一些实施方案中，与本公开的抗体或双特异性抗体偶联以形成抗体偶联物的部分是酶，例如细菌、真菌、植物或动物起源的酶活性毒素，包括其活性片段和/或变体。
本公开涉及药物组合物，其包含本公开的抗体或抗体片段、双特异性抗体或抗体偶联物，和任选地药学上可接受的载体，表面活性剂和/或稀释剂。在一些实施方案中，除本公开的抗体、双特异性抗体或抗体偶联物外，所述药物组合物还包含一种或多种另外的治疗剂。在一些实施方案中，所述另外的治疗剂包括但不限于化疗剂、生长抑制剂、细胞毒性剂、用于放射疗法的试剂、抗血管生成剂、凋亡剂、抗微管蛋白剂和其它治疗癌症的试剂。
术语“多特异性抗体”是指双特异性，三特异性或四特异性抗体。多特异性抗体由两种或更多种不同抗体的片段组成，因此能够与两种，三种或四种不同的抗原结合。
本公开所述双特异性抗体可以用标准的实验方法从宿主细胞中提取纯化。例如，包含抗体Fc部分的双特异性抗体可以采用蛋白A或蛋白G亲和层析法纯化。纯化手段包括但不限于亲和层析色谱法、离子交换法、体积排阻色谱法以及蛋白超滤法。本公开所述双特异性抗体的分离纯化方法也包括上述方法的组合。在此使用的“纯化”是指从细胞、细胞培养物或其他天然组分中分离和/或回收目的组分。如无特别指明，本公开所述抗体均为纯化好的抗体。所使用术语“分离的抗体”是指基本不含有不同结构或抗原特异性的其他分子的抗体，双特异性抗体分子是基本不含有其他抗体分子的分离的抗体。
术语“宿主细胞”指导入外源核酸的细胞及其后代，可通过编码多肽的核苷酸转化或转染，从而表达外源多肽。本公开所述的宿主细胞包括但不限于CHO细胞(Chinese hamster ovary cells，中国仓鼠卵巢细胞)、HEK293细胞(Human embryonic kidney cells 293，人胚肾细胞293)、BHK细胞(Baby Hamster Kidney，幼仓鼠肾细胞)、骨髓瘤细胞、酵母、昆虫细胞或原核细胞如大肠杆菌(Escherichia coli)等。应当指出，本公开所述“宿主细胞”不仅指导入了外源核酸的细胞，同时还包括了该细胞的后代，由于在细胞分裂过程中后代细胞会发生突变，但仍属于本公开所述的术语范围。
本公开还进一步包含编码这些多肽链的核酸序列。在表达抗体的过程中，将核酸序列插入合适的载体中，载体包括但不限于：质粒、噬菌表达载体、柯斯质粒、人工染色体、噬菌体以及动物病毒。表达载体中包含用于调控表达的元件，包括但不限于启动子、转录起始序列、增强子、信号肽序等。启动子 包括但不限于T7启动子、T3启动子、SP6启动子、β-actin启动子、EF-1α启动子、CMV启动子以及SV40启动子。将表达载体转入宿主细胞中可使用本领域内已知的合适方法，包括但不限于：磷酸钙沉淀法、脂质体转染法、电穿孔法、PEI(聚乙烯亚胺)转染法。
实施例1：表达载体构建
(1)CD20×CD3 BsAb的序列设计
本公开实施中的双特异性抗体包括3条或者4条多肽链(图1)，分别命名为第一重链(其可变区具有SEQ ID No.:2所示氨基酸序列)、第一轻链(其可变区具有SEQ ID No.:4所示氨基酸序列)、第二重链(其可变区具有SEQ ID No.:6所示氨基酸序列)、第二轻链(其可变区具有SEQ ID No.:10所示氨基酸序列)和scFv-Fc链(具有如SEQ ID No.:11、SEQ ID No.:12、SEQ ID No.:13或SEQ ID No.:14所示氨基酸序列；或其可变区具有SEQ ID No.:2、SEQ ID No.:4、SEQ ID No.:6、SEQ ID No.:8或SEQ ID No.:10所示氨基酸序列)，形成一个特异性结合CD20的免疫球蛋白结构域、一个特异性结合CD3的免疫球蛋结构域以及一个异源二聚体Fc区域。两条重链相互作用或者一条重链与scFv-Fc链相互作用构成异源二聚体的形式，形成上述异源二聚体Fc区域。
特异性结合人CD20胞外区的双特异抗体可变区部分由VH1和VL1构成(图1)，序列来源于全人源单克隆抗体奥法木单抗(Ofatumumab)(专利号：US2014093454A1)。
特异性结合人CD3的双特异抗体可变区部分由VH2和VL2构成(图1)，来源于专利(专利号：CN201810263832.0)中披露的一种该鼠源单克隆抗体的人源化序列。
CDR区域脱氨影响抗体稳定性与酸碱峰比例，本公开中VH2的CDR3含有一个潜在的脱氨位点(VH2-CDR3原始序列为：HGNFGNSYVSWFA)。在某些实施例中，通过氨基酸突变来消除潜在的脱氨。VH2-CDR3不包含脱氨突变(VH2-CDR3序列为：HGNFG NTYVSWFA)的CD20×CD3 BsAb命名为V1，VH2-CDR3包含N106T突变(VH2-CDR3序列为：HGNFG TSYVSWFA)的CD20×CD3 BsAb命名为V2。在表达纯化后(实施例2和实施例3)，验证突变V1与V2的CD3抗原结合能力(实施例4)与肿瘤杀伤检测(实施例5)。
抗体Fc部分按照公开人已经公开的PCT WO2017034770A1专利中的方法进行部分序列的改造，其中一条重链的Fc部分做如下突变：P395K、P396K、V397K，突变标记为OA(SEQ ID No.:1)；另一条重链的Fc部分做如下突变：T394D、P395D、P396D，突变标记为OB(SEQ ID No.:7)，使其形成重链异源二聚体。
(2)表达质粒的分子克隆
人工合成第一重链核酸序列和第一轻链核酸序列利用通用的分子克隆的方法(Sambrook JF,E.F.et al.Molecular cloning:a laboratory manual.4th ed.Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York:2012)，将抗第一轻链克隆到改造过的质粒pCDNA3.1(+)(Invitrogen，货号V790-20)上，该质粒经过改造，在多克隆位点N端加入了一段人白介素-2(IL-2，Interleukin-2)信号肽序列，使其能够在HEK293细胞中表达分泌抗体，得到表达质粒标记为pCDNA3.1-CD20-LC；将第一重链重链克隆到质粒pFUSE-hIgG1-Fc2(InvivoGene)，得到表达质粒标记为pFUSE-CD20-HC-OB。
人工合成第二重链核酸序列和第二轻链核酸序列利用通用的分子克隆的方法，将第二轻链克隆到到改造过的质粒pCDNA3.1(+)(Invitrogen，货号V790-20)上，该质粒的经过改造在多克隆位点N端加入了一段人白介素-2(IL-2，Interleukin-2)信号肽序列，使其能够在HEK293细胞中表达分泌抗体，得到的表达质粒标记为pCDNA3.1-CD3-LC；将第二重链克隆到质粒pFUSE-hIgG1-Fc2(InvivoGene)，得到的表达质粒标记为pFUSE-CD3-HC-OA和pFUSE-CD3-HC-OA-N106T(VH2-CDR3包含N106T突变)。
实施例2：双特异抗体的瞬时转染与表达
采用无内毒素质粒大提试剂盒(Endo-Free-Plasmid Maxi Kit(100)，购自OMEGA公司，商品目录号为D6926-04)进行质粒大量提取，操作步骤按照试剂盒所提供的说明书进行。将HEK293细胞培养至细胞密度为2.0～3.0×10 6个/mL，将细胞悬液离心5min，转速为1000rpm，丢弃旧的培养上清，用新鲜的培养基(OPM-291CD05Medium，购自上海奥浦迈生物科技有限公司)重悬浮细胞，使其密度为1.0×106/mL。按照表1所提供的质粒组合进行共转染，转染后的细胞悬浮液放置在37℃、5％CO2、120rpm 的培养摇床中避光培养5-7天，并在第4天添加补料。
表1 CD20×CD3 BsAb的瞬时转染
Figure PCTCN2019126066-appb-000001
实施例3 双抗的纯化
离心收集表达上清，用0.22μm滤膜过滤细胞上清，用已经平衡好的蛋白A亲和层析填料(MabSelect SuRe TM，购自GE Healthcare公司，货号为17-5438-02)捕获上清液中的BsAb蛋白，使用平衡缓冲液(137mM NaCl，2.7mM KCl，10mM Na 2HPO 4，1.8mM KH 2PO 4)洗去非特异性结合的蛋白后(约10个柱体积)，用洗脱缓冲液(100mM甘氨酸，pH 3.0-pH 3.5)洗脱5-10个柱体积，收集洗脱液，并用中和缓冲液(1M Tris-HCl，pH9.0)调节pH至中性。洗脱蛋白经SDS-PAGE分析。图2-A、2-B显示四链双特异抗体经蛋白A一步纯化后，V1和V2均可以达到很高的纯度，还原性电泳显示两条重链和两条轻链的配比接近1:1:1:1。图2-C显示三链双特异抗体经蛋白A一步纯化后纯度非常高，且还原电泳显示重链、轻链和scFv-Fc链配比接近1:1:1。
接下来，用AKTA pure 25L1蛋白纯化系统将蛋白A洗脱后得到的蛋白进一步纯化，将洗脱样品上样至已经平衡好的阳离子交换柱(预装柱Resource TM S，1mL,GE Healthcare，产品目录号17-1178-01)，平衡缓冲液A(50mM磷酸钠，pH6.0)洗去非特异性结合的蛋白至紫外吸收线平缓，用洗脱缓冲液B(50mM磷酸钠，1M NaCl，pH 6.0)从0％-50％线性洗脱15个柱体积，收集洗脱峰，图3显示洗脱曲线。
取1mL蛋白A纯化后的样品(浓度为1mg/mL)，通过AKTA pure 25L1蛋白纯化系统的1mL上样环将样品上样至已经平衡好的分子筛预装柱(Superdex200 10/300GL increase，GE Healthcare，货号28-9909-44)，平衡缓冲液(1×PBS缓冲液：137mM NaCl,2.7mM KCl,10mM Na 2HPO 4,1.8mM KH 2PO 4)洗脱1.5柱体积，收集洗脱峰，图4显示洗脱曲线。SEC分析显示CD20×CD3 BsAb V1样品经一步纯化(蛋白A)后聚集体极少，单体含量超过99％
实施例4 抗体结合检测(ELISA)
(1)ELISA检测CD20×CD3双特异性抗体与抗原CD3的结合力
为了验证CD20×CD3 BsAb的抗原结合能力，纯化的双特异性抗体通过ELISA分别对抗原CD3结合检测。具体步骤如下。
1)本实验使用的CD3抗原，为人CD3e蛋白和人CD3d蛋白1:1的混合物，购自Sino Biological公司。
2)抗原包被：用0.1M碳酸氢钠缓冲液(pH 9.5)稀释抗原至100ng/mL。将稀释后的抗原加入酶标板中，每孔200μL，用封板膜封住反应孔，4℃放置16小时。0.05％PBST洗板5次。
3)封闭：用脱脂奶粉和PBS缓冲液配制3％M-PBS的封闭液，每孔加300μL 3％M-PBS，用封板膜封住反应孔，室温孵育1小时。0.05％PBST洗板5次。
4)加样：将双抗样品用PBS稀释至10μg/ml，然后4倍梯度稀释，共8个浓度。100μL/孔加入稀释后的样品，每个抗体浓度做2个复孔。用封板膜封住反应孔，室温孵育1.5小时。0.05％PBST洗板5次。
5)加入二抗：将HRP标记的羊抗人IgG(Goat Anti-Human IgG)(H+L)(购自proteintech公司，商品目录号为SA00001-17)用PBS按1:2000稀释。每孔加入100μL，用封板膜封住反应孔，室温孵育1小时。0.05％PBST洗板5次。
6)显色：每孔加入TMB显色液100μL，室温避光孵育5-10min。
7)终止：每孔加入终止液(1M HCl)50μL终止显色反应，3min后在酶标仪450nM处读取各孔反应液的OD值。
8)数据分析：应用软件GraphPad Prism 5进行数据处理后生成以log(样品浓度)为横坐标、OD450值为纵坐标的曲线，并得到EC50数据。
如图5所示，CD20×CD3 BsAb V1与CD3抗原结合的EC50浓度为0.8178μg/mL，CD20×CD3 BsAb V2与CD3抗原结合的EC50浓度为2.097μg/ml，V2对CD3抗原的结合力较弱，这说明N106T的引入减弱了抗体对CD3的亲和力，在实际应用中这一特性将有利于避免T细胞的过度激活。
(2)FACS检测双抗样品与CD20阳性细胞的亲和力
本公开中将Raji细胞作为CD20表达阳性、CD3表达阴性细胞(即CD20+/CD3-)。将Raji细胞置于37℃、5％CO 2培养箱中培养，汇集度80％-90％时收集细胞。同时将CD20×CD3 BsAb V1和V2样品稀释至20μg/mL，备用。用含1％FBS的PBS缓冲液将细胞洗涤2次，并将细胞重悬成1×10 7细胞/mL。取1.5mL离心管，每管中加入100μL上述细胞悬液，并加入100μL稀释好的样品。混匀，置于冰上孵育1h。离心(400g，5min)，弃上清，加入1mL 1％FBS/PBS缓冲液洗涤细胞，洗2次。用PE标记的羊抗人IgG Fc(Invitrogen，Cat.12-4998-82)重悬细胞，冰上避光孵育1h。再次离心(400g，5min)，弃上清，加入1mL 1％FBS/PBS缓冲液洗涤细胞，洗2次。最后用200μL1％FBS/PBS缓冲液重悬细胞，流式细胞仪检测样品与Raji细胞的结合活性。
根据平均荧光强度，软件Graphpad Prism 5.0计算分析双抗样品与Raji细胞的结合活性。
如图6所示，CD20×CD3 BsAb V1和V2同CD20阳性细胞Raji的结合活性相同。
实施例5 CD20×CD3双特异性抗体介导的体外细胞毒性检测
为进一步比较CD20×CD3 BsAb V1和V2在细胞水平对肿瘤细胞的杀伤活性，本公开对纯化的双抗样品进行ADCC毒性检测。
具体步骤如下：
本公开中将Raji细胞作为CD20阳性肿瘤细胞。制备Raji单细胞悬液。用无酚红的含10％FBS的RPMI1640培养基调整细胞密度为0.40×10 6/mL，50μL/孔加入96孔板，故2.0×10 4个细胞/孔。本实验加入20倍于Raji细胞数的hPBMC，即4.0×10 5个/孔，100μL/孔，故调整细胞密度为4.0×10 6/mL。用无酚红的10％FBS-RPMI 1640培养基将抗体稀释至0.1μg/mL，然后按1:4的比例进行倍比稀释，得到10个浓度分别为100ng/mL、25ng/mL、6.25ng/mL、1.56ng/mL、0.39ng/mL、0.10ng/mL、0.02ng/mL、0.006ng/mL的抗体。按实验设计加入相应抗体50μL/well，无需加入抗体的孔用相同体积的无酚红的含10％FBS的RPMI1640培养基补加。将细胞和抗体混匀后37℃，5％CO2培养箱培养过夜，约20h后用乳酸脱氢酶细胞毒性试剂盒(购自Beyotime公司)来检测细胞的毒性，进而检测CD20×CD3 BsAb的杀伤活性，计算公式：
杀伤率(％)＝(OD样品-S自发)/(Max-S自发)×100％
其中，S自发＝OD自发释放孔(靶细胞+效应细胞)，Max＝OD最大释放孔(靶细胞)；
如图7显示，纯化的CD20×CD3 BsAb V1和V2双特异性抗体有效介导hPBMC杀伤CD20阳性肿瘤Raji细胞。肿瘤细胞杀伤效果显著，EC50浓度分别为0.21ng/mL和0.24ng/ml，虽然CD20×CD3 BsAb V2对CD3的亲和力较弱，但并未影响对靶细胞Raji的杀伤力。
实施例6 抗体稳定性实验
将经蛋白A纯化后的CD20×CD3 BsAb V1样品分别置于37℃和40℃，放置时间包括：0d、1d、4d、7d，将不同放置时间的样品通过SDS-PAGE分析检测抗体在不同温度下的降解情况以及稳定性，结果显示V1样品在37℃和40℃能稳定至少7天。
实施例7 细胞因子释放检测
本公开采用PBMC细胞测定双特异性抗体对细胞因子释放的影响。PBS清洗PBMC细胞，调整细胞密度使得每个孔包含2×10 5个细胞。用1640培养基将抗体稀释至0.25μg/mL，梯度稀释，分别加入上述孔中。将96孔板置于37℃，5％CO 2培养箱中培养41小时。采用双抗体夹心ELISA法检测样品中细胞因子的浓度，具体方法为：将上述含有PBMC细胞的96孔板1000rpm离心5min，取上清以80μL/孔加入相应孔中，用封口膜封住反应孔，室温孵育120分钟。洗板5次，加入生物素化抗体100μL/孔，用封口膜封住反应孔，室温孵育60分钟。洗板5次，加入HRP-链霉亲和素100μL/孔，用封口膜封住反应孔，室温避光孵育10到20分钟，洗板5次，加入显色剂TMB溶液100μL/孔，用封口膜封住反应孔，室温避光孵育20分钟。加入终止液50μL/孔，混匀后立即用酶标仪检测450nm处的吸光度值。实验结果显示在图9A-9B中。
使用CD20×CD3 BsAb V1、CD20×CD3 BsAb V2、CD3单抗、IgG与来自不同健康供体的hPBMC共同孵育过夜，取其上清进行上述ELISA夹心法实验，检测细胞因子IL-6、IL-2的释放量，以此鉴定T细胞是否被非特异性活化并产生细胞因子风暴。实验结果显示，在不存在靶细胞的情况下，CD20×CD3 BsAb V1、CD20×CD3 BsAb V2抗体引起的IL-2、IL-6释放量均低于CD3单抗，引入N106T突变的V2产生的IL-2、IL-6更少(图9A)，说明本公开中的CD20×CD3 BsAb尤其是V2不会引起T细胞非特异性活化，若在临床上使用本公开的CD20×CD3双抗治疗CD20阳性肿瘤，将可以减少细胞因子风暴的发生，提高抗体使用的安全性。
实施例8 小鼠体内抗肿瘤活性检测
CD20×CD3 BsAb的体内药效学评价基于CD20阳性表达的Raji皮下移植瘤模型上完成。
模型建立方法：收集对数生长期的Raji细胞，与hPBMC混合后，接种于NOD-SCID小鼠背部皮下，随机分组，分为对照组和治疗组，治疗根据分组将本公开的双特异性抗体CD20×CD3 BsAb V1以低剂量(0.01mg/kg和0.05mg/mL)、中等剂量(0.1mg/kg和0.5mg/mL)和高剂量(2mg/kg)在第1天、第3天、第5天和第8天各给药1次。阴性对照组给予等体积的无菌生理盐水。分组时(即首次给药前)1次、给药后每周2次、安乐死前1次，测量并记录肿瘤长径、短径，计算肿瘤体积，并根据肿瘤体积绘制肿瘤生长曲线，比较各组间肿瘤生长曲线的差异。按照以下公式计算肿瘤体积：V＝1/2×长径×短径 2。图10显示了肿瘤体积随时间变化的曲线。每周两次测量肿瘤体积，记录小鼠体重变化。实验结束后，剥取肿瘤，称瘤重。
如图10所示，CD20×CD3 BsAb V1(2mg/kg、0.5mg/kg、0.1mg/kg、0.05mg/kg、0.01mg/kg)治疗组在开始治疗后21天均表现出显著的抑瘤作用。各治疗组均无药物治疗引起的动物死亡，无明显的药物毒性及严重的体重下降，给药期间耐受良好。
最后所应当说明的是，以上实施例仅用以说明本公开的技术方案而非对本公开保护范围的限制，尽管参照较佳实施例对本公开作了详细说明，本领域的普通技术人员应当理解，可以对本公开的技术方案进行修改或者等同替换，而不脱离本公开技术方案的实质和范围。
工业实用性
(1)与单克隆抗体相比，本公开所述CD20×CD3 BsAb分子能够在结合肿瘤细胞表面抗原的同时也能结合T细胞表面CD3分子，依赖抗原特异性激活TCR进而增加了T细胞靶向肿瘤杀伤作用，用更少量的抗体就能发挥显著杀伤肿瘤细胞的效果。
(2)与BiTE等小分子双特异性抗体相比，本公开所述CD20×CD3 BsAb分子包含抗体Fc区域，增加了抗体的半衰期和稳定性，且更容易采用现有单抗纯化技术，简化了双特异抗体的生产工艺。
(3)与靶向CD20的嵌合抗原受体T细胞免疫疗法(CAR-T)相比，本公开不涉及外源病毒、体外T细胞培养回输等操作，毒副作用更小，更安全可控。

Claims (21)

  1. 一种双特异性抗体，其特征在于，所述双特异性抗体包含能结合CD20的结构域、能结合CD3的结构域和异源二聚体Fc区域，所述能结合CD20的结构域和能结合CD3的结构域各自独立地选自Fab区域、ScFv区域或sDab区域。
  2. 如权利要求1所述的双特异性抗体，其特征在于，所述能结合CD20的结构域包括第一免疫球蛋白Fab区域，所述第一免疫球蛋白Fab区域主要由相互结合的第一重链与第一轻链形成，所述第一重链的可变区具有如SEQ ID No.:2所示的氨基酸序列，所述第一轻链的可变区具有如SEQ ID No.:4所示的氨基酸序列；和/或所述能结合CD3的结构域包括第二免疫球蛋白Fab区域，所述第二免疫球蛋白Fab区域主要由相互结合的第二重链与第二轻链形成，所述第二重链的可变区具有如SEQ ID No.:6所示的氨基酸序列，所述第二轻链的可变区具有如SEQ ID No.:10所示的氨基酸序列；
    所述异源二聚体Fc区域由两条多肽链构成，每条多肽链包含带相反电荷的不对称氨基酸修饰。
  3. 如权利要求2所述的双特异性抗体，其特征在于，所述第一重链的CH1部分以及第一轻链的CL部分均包含一个取代天然半胱氨酸残基的非半胱氨酸残基，以及一个取代非天然半胱氨酸的半胱氨酸残基。
  4. 如权利要求3所述的双特异性抗体，其特征在于，所述第一重链的CH1部分中取代天然半胱氨酸残基的非半胱氨酸残基与第一轻链的CL部分中取代非天然半胱氨酸的半胱氨酸残基形成二硫键。
  5. 如权利要求3或4所述的双特异性抗体，其特征在于，所述第一重链的CH1部分中，取代天然半胱氨酸残基的非半胱氨酸残基为C220S，取代非天然半胱氨酸的半胱氨酸残基为L128C。
  6. 如权利要求3-5中任一项所述的双特异性抗体，其特征在于，所述第一轻链的CL部分中，取代天然半胱氨酸残基的非半胱氨酸残基为C214S，取代非天然半胱氨酸的半胱氨酸残基为F118C。
  7. 如权利要求2-6中任一项所述的双特异性抗体，其特征在于，所述第二免疫球蛋白Fab区域不包含取代天然半胱氨酸残基的非半胱氨酸残基，且不包含取代非天然半胱氨酸残基的半胱氨酸残基。
  8. 如权利要求2-7中任一项所述的双特异性抗体，其特征在于，所述能结合CD20的结构域包括第一免疫球蛋白Fab区域，所述能结合CD3的结构域包括第二免疫球蛋白Fab区域；所述第一重链与第二重链相互结合，形成异源二聚体Fc区域。
  9. 如权利要求8所述的双特异性抗体，其特征在于，所述第一重链和第二重链来自人抗体IgG1、IgG2、IgG3或IgG4。
  10. 如权利要求8所述的双特异性抗体，其特征在于，所述第一重链具有如SEQ ID No.:1所示的氨基酸序列，第一轻链具有如SEQ ID No.:3所示的氨基酸序列，所述第二重链具有如SEQ ID No.:5或SEQ ID No.:7所示的氨基酸序列，第二轻链具有如SEQ ID No.:9所示的氨基酸序列。
  11. 如权利要求2所述的双特异性抗体，其特征在于，所述能结合CD20的结构域包括第一免疫球蛋白Fab区域，所述能结合CD3的结构域包括scFv-Fc融合蛋白多肽链；所述scFv-Fc融合蛋白多肽链中scFv区域的重链可变区具有如SEQ ID No.:6所示的氨基酸序列，轻链可变区具有如SEQ ID No.:10所示的氨基酸序列，所述第一重链与所述scFv-Fc融合蛋白多肽链相互结合，形成一个异源二聚体Fc区域；或者，
    所述能结合CD20的结构域包括scFv-Fc融合蛋白多肽链，能结合CD3的结构域包括第二免疫球蛋白Fab区域；所述scFv-Fc融合蛋白多肽链中具有如SEQ ID No.:2所示的氨基酸序列，轻链可变区具有如SEQ ID No.:4所示的氨基酸序列，所述第二重链与所述scFv-Fc融合蛋白多肽链相互结合，形成一个异源二聚体Fc区域。
  12. 如权利要求11所述的双特异性抗体，其特征在于，所述能结合CD20的结构域包括第一免疫球蛋白Fab区域，所述能结合CD3的结构域包括scFv-Fc融合蛋白多肽链；所述第一重链的可变区具有如SEQ ID No.:8所示的氨基酸序列；所述scFv-Fc融合蛋白多肽链中的scFv区域具有如SEQ ID No.:11所示的氨基酸序列，所述scFv-Fc融合蛋白多肽链中的重链可变区CDR3包含N106T突变,突变后的CDR3序列为：HGNFGTSYVSWFA。
  13. 如权利要求11所述的双特异性抗体，其特征在于，所述能结合CD20的结构域包括第一免疫球蛋白Fab区域，所述能结合CD3的结构域包括scFv-Fc融合蛋白多肽链时，所述scFv-Fc融合蛋白多肽链中的scFv区域的可变区具有如SEQ ID No.:6或SEQ ID No.:10所示的氨基酸序列；所述能结合CD20的结构域包括scFv-Fc融合蛋白多肽链，能结合CD3的结构域包括第二免疫球蛋白Fab区域时，所述scFv-Fc融合蛋白多肽链中的scFv区域具有如SEQ ID No.:12、SEQ ID No.:13、SEQ ID No.:14所示的氨基酸序列、或其可变区具有SEQ ID No.:4或SEQ ID No.:8所示的氨基酸序列。
  14. 一种抗体偶联物，其特征在于，所述抗体偶联物是偶联物质与权利要求1～13任一项所述的双特异性抗体偶联所形成。
  15. 如权利要求14所述的抗体偶联物，其特征在于，所述偶联物质为细胞毒素、放射性同位素、荧光标记物、发光物、显色物质或酶。
  16. 一种药物组合物，其特征在于，包含权利要求1-13任一项所述的双特异性抗体。
  17. 如权利要求1-13任一项所述的双特异性抗体在制备治疗肿瘤、风湿性关节炎、多发性硬化症或系统性红斑狼疮的药物或药物组合物中的用途。
  18. 如权利要求17所述的用途，其特征在于，所述肿瘤为与CD20表达相关的恶性肿瘤。
  19. 如权利要求18所述的用途，其特征在于，所述恶性肿瘤为急性B淋巴细胞性白血病、弥漫性大B细胞淋巴瘤、慢性淋巴细胞白血病、滤泡淋巴瘤，非霍奇金氏淋巴瘤、慢性骨髓细胞白血病或伯基特淋巴瘤。
  20. 一种用于制备如权利要求1-13任一项所述的双特异性抗体的方法，所述方法包括以下步骤：
    (a)构建表达载体，所述表达载体是将编码如权利要求1-13任一项所述的双特异性抗体的核苷酸连接到基础载体上得到的；
    (b)将步骤(a)构建的表达载体转染或转化至宿主细胞中，并培养宿主细胞；
    (c)分离、纯化所述双特异性抗体。
  21. 如权利要求20所述的方法，所述分离、纯化所述双特异性抗体的方法为蛋白A亲和层析法、阳离子交换法或阴离子交换法。
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