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WO2018188612A1 - 一种能与人cd19或cd20和人cd3结合的双特异性抗体及其应用 - Google Patents

一种能与人cd19或cd20和人cd3结合的双特异性抗体及其应用 Download PDF

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WO2018188612A1
WO2018188612A1 PCT/CN2018/082692 CN2018082692W WO2018188612A1 WO 2018188612 A1 WO2018188612 A1 WO 2018188612A1 CN 2018082692 W CN2018082692 W CN 2018082692W WO 2018188612 A1 WO2018188612 A1 WO 2018188612A1
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seq
domain
bispecific antibody
combination
antibody
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PCT/CN2018/082692
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French (fr)
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章华
张�成
范克索
汪笑峰
景书谦
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鸿运华宁（杭州）生物医药有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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
    • 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
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • 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
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • an antibody particularly a bispecific antibody capable of binding to both human CD19 or CD20 and human CD3, and a method for treating a disease associated with malignant or abnormal B cells.
  • B cells play an important role in humoral immune response and antigen presentation in humans.
  • the consequences of B-cell pathology are very serious, and the diseases caused by it are mainly two types.
  • the first type is malignant B-cell blood cancer, and the second type is auto-immune disease caused by abnormal B-cell recognition of self-antigen. (Janeway et al, 2001, Immunobiology: The Immune System in Health and Disease. 5th edition, New York: Garland Science).
  • CD19 is a 95kDa transmembrane glycoprotein involved in the regulation of B cell autoimmune response sensitivity (Tedder, 2009, Nat. Rev. Rheumatol. 5: 572-7; Stanciu-Herrera et al, 2008, Leuk. Res. :625-32), maintaining a balance between external immunity and autoimmune response.
  • CD19 is expressed on the surface of B cells in almost all types and stages, and also includes most malignant and abnormal B cells.
  • Antibody drugs against CD19 include Blinatumomab approved for B-ALL in 2014.
  • Blinatumomab is a T cell bispecific antibody formed by folding a single polypeptide chain, including two independent domains joined by a flexible peptide linker sequence, and a single-chain antibody variable region (scFv) structure that binds to B cell CD19.
  • the domain, and the other is a scFv that binds to the human T cell surface receptor CD3 (Nagorsen et al, 2012, Pharmacol Ther. 136: 334-42).
  • Lysocept which targets CD20
  • a traditional human-mouse chimeric antibody is used to treat non-Hodgkin's lymphoma, which causes immune damage and apoptosis to B cells through ADCC and CDC (Smith, 2003, Oncogene 22: 7359–68).
  • CD20 is associated with B cell responses to non-T cell-dependent antigens, and its distribution on the B cell surface is not as broad as CD19, mainly in mature B cell types, whereas pre-B cells, immature B cells, and most plasma cells are not.
  • CD20 is expressed or underexpressed, so drugs against CD20 are less effective in treating diseases caused by these specific types of B cells than drugs targeting CD19 (Johnson et al, 2009, Blood 113:3773-80), such as rheumatoid arthritis Such as autoimmune diseases.
  • the expression of CD20 was significantly greater than that of CD19 on the surface of B cell types expressing both CD19 and CD20, and the expression of CD20 was higher than that of normal B cells on the surface of malignant B cells of various types of chronic leukemia. The amount is down-regulated on these cells compared to normal B cells (Ginaldi et al., 1998, J. Clin. Pathol. 51: 364-9), thus treating CD28 may be a better choice for treating various types of chronic leukemia.
  • T cell bispecific antibodies have gradually become homogenized in the application of the principle of treatment of tumors, and more need to improve in antibody engineering and molecular design (Bano et al, 2016, Antibodies 5: 1-23), because such as Blinatumomab Although its molecular structure brings high activity, it also has shortcomings such as short half-life in vivo, low yield and complicated preparation process.
  • the novel bispecific antibodies referred to herein consist of a complete anti-human CD19 or human CD20 immunoglobulin G (IgG) molecule and two identical anti-CD3 scFv domains to overcome Blinatumomab stability and process The disadvantages of the aspect, while retaining a sufficiently high target cell killing activity.
  • T cell mediated to overcome the resistance of the CD20 monoclonal antibody rituximab (Rezvani et al, 2011, Best. Pract. Res. Clin. Haematol. 24: 203- 16).
  • the bispecific antibody herein has the activity of killing malignant B cells efficiently by T cell-mediated cytotoxicity, and has the advantages of structural stability, high expression amount, and simple purification process.
  • the bispecific antibodies herein can be used to treat all related diseases caused by malignant or abnormal B cells.
  • This article provides a novel bispecific antibody.
  • the antibody has the ability to specifically bind to human CD19 or human CD20 and human CD3.
  • This bispecific antibody can mediate T cell toxicity-dependent immunological killing responses against various types and stages of B cells.
  • the bispecific antibody can be used to prepare a medicament for treating a related disease caused by malignant or abnormal B cells.
  • a bispecific antibody provided herein is a bispecific antibody capable of binding to human CD19 or human CD20 and human CD3, the structure of which is characterized in that the bispecific antibody comprises a single chain antibody The domain (scFv domain), the immunoglobulin G domain (IgG) and the interdomain interpeptide linker sequence (Link 1 ), which are formed by fusion of one of the following:
  • N 'Representative amino terminus of the polypeptide chain C' represents a carboxyl terminus of the polypeptide chain
  • scFv represents a single-chain antibody domain
  • the representative IgG immunoglobulin G domain V L domain on behalf of IgG light chain variable region
  • C L domains representative of IgG light chain constant region
  • V H domain on behalf of IgG heavy chain variable region V H domain on behalf of IgG heavy chain variable region
  • C H2 domain on behalf of IgG heavy chain constant region 2 C H3 domains representative of IgG heavy chain constant region 3
  • the representative Link 1 and the interdomain linker peptide the representative Link 1 and the interdomain linker peptide.
  • the sequence of the interdomain peptide linker is selected from one of the following sequences: SEQ NO ID: 1, SEQ NO ID: 2. SEQ NO ID: 3, SEQ NO ID: 4, and SEQ NO ID: 5.
  • the scFv domain comprises a light chain variable region, a heavy chain variable region, and a domain internal peptide linker sequence, A combination to form a scFv domain:
  • N' represents the amino terminus of the polypeptide chain
  • C' represents the carboxy terminus of the polypeptide chain
  • VSL represents the light chain variable region of the scFv domain
  • VSH represents the heavy chain variable region of the scFv domain
  • Link 2 represents A peptide linker within the domain.
  • the sequence of the inter-domain endopeptide linker is selected from one of the following sequences: SEQ NO ID: 6, SEQ NO ID: 7, SEQ NO ID: 8, SEQ NO ID: 9, and SEQ NO ID: 10.
  • the scFv domain comprises an amino acid sequence of one of the following:
  • the light chain variable region sequence of the a.scFv domain is selected from one of the following sequences: SEQ NO ID: 11, SEQ NO ID: 13, and SEQ NO ID: 32;
  • the heavy chain variable region sequence of the scFv domain is selected from the group consisting of: SEQ NO ID: 12, SEQ NO ID: 14, SEQ NO ID: 33, and SEQ NO ID: 34;
  • the scFv domain of the antibody comprises an amino acid sequence combination of one of the following schemes: a combination of SEQ NO ID: 11 and SEQ NO ID: 12 (L1H1) a combination of SEQ NO ID: 13 and SEQ NO ID: 14 (L2H2), a combination of SEQ NO ID: 32 and SEQ NO ID: 33 (L9H9), and a combination of SEQ NO ID: 32 and SEQ NO ID: 34 (L9H10).
  • the IgG domain of the antibody comprises an amino acid sequence of one of the following:
  • the light chain variable region sequence of the IgG domain is selected from one of the following sequences: SEQ NO ID: 15, SEQ NO ID: 17, SEQ NO ID: 19, SEQ NO ID: 21, SEQ NO ID: 23, and SEQ NO ID: 25;
  • the heavy chain variable region sequence of the IgG domain is selected from one of the following sequences: SEQ NO ID: 16, SEQ NO ID: 18, SEQ NO ID: 20, SEQ NO ID: 22, SEQ NO ID: 24, and SEQ NO ID: 26; and
  • the IgG domain of the antibody comprises an amino acid sequence combination of one of the following: a combination of SEQ NO ID: 15 and SEQ NO ID: 16 (L3H3) a combination of SEQ NO ID: 17 and SEQ NO ID: 18 (L4H4), a combination of SEQ NO ID: 19 and SEQ NO ID: 20 (L5H5), SEQ NO ID: 21 and SEQ NO ID: 22 ( L6H6), a combination of SEQ NO ID: 23 and SEQ NO ID: 24 (L7H7), and a combination of SEQ NO ID: 25 and SEQ NO ID: 26 (L8H8).
  • the scFv domain comprises an amino acid sequence of one of the following:
  • the light chain variable region sequence of the a.scFv domain is selected from one of the following sequences: SEQ NO ID: 15, SEQ NO ID: 17, SEQ NO ID: 19, SEQ NO ID: 21, SEQ NO ID: 23, and SEQ NO ID: 25;
  • the heavy chain variable region sequence of the scFv domain is selected from one of the following sequences: SEQ NO ID: 16, SEQ NO ID: 18, SEQ NO ID: 20, SEQ NO ID: 22, SEQ NO ID: 24, and SEQ NO ID: 26; and
  • the scFv domain of the antibody comprises an amino acid sequence combination of one of the following schemes: a combination of SEQ NO ID: 15 and SEQ NO ID: 16 (L3H3) a combination of SEQ NO ID: 17 and SEQ NO ID: 18 (L4H4), a combination of SEQ NO ID: 19 and SEQ NO ID: 20 (L5H5), SEQ NO ID: 21 and SEQ NO ID: 22 ( L6H6), a combination of SEQ NO ID: 23 and SEQ NO ID: 24 (L7H7), and a combination of SEQ NO ID: 25 and SEQ NO ID: 26 (L8H8).
  • the IgG domain of the antibody comprises an amino acid sequence of one of the following:
  • the light chain variable region sequence of the IgG domain is selected from one of the following sequences: SEQ NO ID: 11, SEQ NO ID: 13, and SEQ NO ID: 32;
  • the heavy chain variable region sequence of the IgG domain is selected from the group consisting of SEQ NO ID: 12, SEQ NO ID: 14, SEQ NO ID: 33, and SEQ NO ID: 34;
  • the IgG domain of the antibody comprises an amino acid sequence combination of one of the following: a combination of SEQ NO ID: 11 and SEQ NO ID: L1H1), a combination of SEQ NO ID:13 and SEQ NO ID:14 (L2H2), a combination of SEQ NO ID:32 and SEQ NO ID:33 (L9H9), and SEQ NO ID:32 and SEQ NO ID:34 Combination (L9H10).
  • the IgG molecule of the antibody protein further comprises an amino acid sequence of one of the following schemes:
  • a light chain constant region amino acid sequence selected from one of the following sequences: SEQ NO ID: 27 and SEQ NO ID: 28;
  • a heavy chain constant region amino acid sequence selected from one of the following sequences: SEQ NO ID: 29, SEQ NO ID: 30, and SEQ NO ID: 31;
  • a bispecific antibody provided herein is a bispecific antibody capable of binding to human CD19 and human CD3.
  • a bispecific antibody provided herein is a bispecific antibody capable of binding to human CD20 and human CD3.
  • the bispecific antibody provided herein is a murine antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.
  • provided herein is a polynucleotide encoding a bispecific antibody provided herein.
  • a vector comprising a polynucleoside provided herein
  • provided herein is a host cell comprising a vector provided herein.
  • a pharmaceutical composition comprising a bispecific antibody provided herein in admixture with a pharmaceutically acceptable carrier.
  • a bispecific antibody described herein in the manufacture of a medicament for the prevention or treatment of B cell leukemia.
  • the B cell leukemia described herein is an acute B cell leukemia.
  • the B cell leukemia described herein is chronic B cell leukemia.
  • a bispecific antibody described herein in the manufacture of a medicament for the prevention or treatment of non-Hodgkin's lymphoma.
  • a bispecific antibody described herein in the manufacture of a medicament for preventing or treating an autoimmune disease caused by B cells.
  • the autoimmune disease caused by a B cell described herein is rheumatoid arthritis, multiple sclerosis, or systemic lupus erythematosus.
  • a bispecific antibody described herein in the manufacture of a medicament for preventing or treating a rejection reaction and associated symptoms associated with organ transplantation.
  • Figure 1 shows an SDS-PAGE of ⁇ CD19 (L6H6 comprising SEQ NO ID: 21 and SEQ NO ID: 22) / ⁇ CD3 (L2H2 comprising SEQ NO ID: 13 and SEQ NO ID: 14) bispecific antibodies .
  • the M lane represents the standard protein and the molecular weight is shown in the figure.
  • the #1 and #2 lanes were all ⁇ CD19/ ⁇ CD3 bispecific antibodies (different loading amounts), and the results showed that the molecular weight was about 55 Kd.
  • Figure 2 shows flow cytometry (FACS) detection of recombinantly expressed ⁇ CD19/ ⁇ CD3 bispecific antibody (solid double-peak and arrow indicated) specific binding to Raji tumor cells, positive control for ⁇ CD19 mAb (dashed doublet) And the arrow indicates), the negative control is the antibody solvent (single line with a single dotted line).
  • FACS flow cytometry
  • Figure 3 shows the killing curve of CIK against Raji tumor cells mediated by different concentrations of ⁇ CD19/ ⁇ CD3 bispecific antibody, E:T is 10:1.
  • the Y axis represents the number of remaining surviving Raji cells in the total amount of fixed cells, and the X axis represents the concentration of the antibody. This result suggests that in the case of E:T of 10:1, the lethality of CIK against Raji increases with increasing antibody concentration and is positively correlated. Its semi-killing concentration to Raji cells was 1 ng/mL.
  • Figure 4 shows the respective life cycles of NOD/SCID mice after in situ vaccination with Raji cells under different treatment conditions: PBS injection (solid line); CIK cell injection (dotted line); CIK cells plus 0.25 nmol of ⁇ CD19/ ⁇ CD3 bispecific antibody injection (dot solid line); CIK cells plus 2.5 nmol of ⁇ CD19/ ⁇ CD3 bispecific antibody injection (dashed line).
  • the injection cycle of each group was the first injection from the Raji cell inoculation day (Day 0), and then every two days, a total of ten times. The results showed that CIK cells plus different concentrations of ⁇ CD19/ ⁇ CD3 bispecific antibody injection significantly increased the survival cycle of mice.
  • polypeptide sequences are used herein to indicate polynucleotide and polypeptide sequences.
  • the amino terminus (N') of the polypeptide sequence is to the right at the left carboxy terminus (C'), and the 5' end of the upstream strand of the single-stranded nucleic acid sequence and the double-stranded nucleic acid sequence is to the left and their 3' end is to the right.
  • Particular portions of the polypeptide may be represented by amino acid residue numbers, such as amino acids 80 to 130, or by actual residues at the site, such as Lys80 to Lys130 or K80 to K130.
  • a particular polypeptide or polynucleotide sequence can also be described by interpreting its differences from the reference sequence.
  • peptide refers to a molecule comprising two or more amino acids joined together by a peptide. These terms encompass polypeptide analogs (eg, muteins, variants, and fusion proteins) such as native and artificial proteins, protein fragments, and protein sequences.
  • polypeptide analogs eg, muteins, variants, and fusion proteins
  • polypeptide fragment refers to a polypeptide having an amino terminal and/or carboxy terminal deletion compared to a corresponding full length protein.
  • the fragment length can be, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100, 150 or 200 amino acids.
  • the segment length can be, for example, up to 1000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11 or 10 amino acids.
  • the fragment may further comprise one or more additional amino acids at one or both ends thereof, for example, an amino acid sequence (eg, an Fc or leucine zipper domain) or an artificial amino acid sequence (eg, an artificial linker sequence) from a different native protein.
  • Polypeptides described herein include polypeptides modified for any reason and by any method, for example, to: (1) reduce proteolytic sensitivity, (2) reduce oxidation sensitivity, and (3) alter the affinity of protein-forming complexes. (4) altering the binding affinity and (5) imparting or modifying other physicochemical or functional properties.
  • Analogs comprise a mutein of a polypeptide. For example, single or multiple amino acid substitutions (eg, conservative amino acid substitutions) can be made in a native sequence (eg, a portion of a polypeptide that is outside of the domain that forms the intramolecular contact).
  • &quot Constant amino acid substitutions" are those that do not significantly alter the structural characteristics of the parent sequence (e.g., the replacement amino acid should not disrupt the helix present in the parent sequence or interfere with other secondary structure types that confer a maternal sequence property or are essential for its function).
  • domain-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company (1984)); Introduction to Protein Structure (Branden and Tooze, ed., Garland Publishing (1991)); And Thornton et al, 1991, Nature 354: 105-106, both incorporated herein by reference.
  • Non-peptide analogs of antibodies are commonly used to provide drugs with similar properties to the template peptide. These types of non-peptide chemicals are referred to as "peptide mimetics" or "peptidomimetics". Fauchere, 1986, Adv. Drug Res. 15: 29-69; Veber and Freidinger, 1985, Trends Neurosci. 8: 392-396; Evans et al, 1987, J. Med. Chem. 30: 1229-1239, both of which are incorporated by reference. The form is in this article. Peptidomimetics similar in structure to therapeutic peptides can be used to produce equal therapeutic or prophylactic effects.
  • polypeptide peptidomimetic structure i.e., a polypeptide having a desired biochemical property or pharmacological activity
  • a polypeptide having a desired biochemical property or pharmacological activity such as human antibody
  • one or more optional substituents selected from -CH 2 NH -, - CH 2 S
  • Substitution of one or more amino acids of a consensus sequence with the same type of D-amino acid system can also be used to generate more stable peptides.
  • intermolecular cyclization of the peptide can be formed by methods known in the art (Rizo and Gierasch, 1992, Annu. Rev. Biochem. 61: 387-418), in reference form and herein, for example by addition.
  • the internal cysteine residue of the sulfur bond generates constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variant.
  • a "variant" of a polypeptide comprises an amino acid sequence in which one or more amino acid residues have been inserted, deleted and/or replaced in the amino acid sequence relative to another polypeptide sequence.
  • a “derivative" of a polypeptide is a chemically modified polypeptide (eg, an antibody), for example by binding to other chemical moieties such as polyethylene glycol, albumin (eg, human serum albumin), phosphorylation, and glycosylation.
  • domain is a region having a specific structure and independent function in a biomacromolecule, which has considerable structural independence and has characteristics of being intact and repetitive in the same type of biomacromolecule.
  • polypeptide or protein regions such as domains, single chain antibodies (scFv) domains of the light chain variable region (V L).
  • Small domains can be combined to form larger domains, for example, a single-chain antibody domain is formed by a combination of a light chain variable region domain and a heavy chain variable region ( VH ) domain by a peptide linker sequence.
  • subunit is a domain or structural component of a protein or protein complex consisting of more than one polypeptide having independent and tertiary structures, eg, a complete human CD3 molecule comprising CD3 ⁇ , CD3 ⁇ and CD3 ⁇ Three subunits.
  • antibody is a protein comprising a scaffold or framework portion that binds to an antigen and optionally allows the antigen binding portion to adopt a conformation that promotes binding of the antibody to the antigen, ie comprises two full length heavy chains and two full length light Chain antibodies, as well as derivatives, variants, fragments, muteins, and fusion proteins, such as the bispecific antibodies herein.
  • antibodies include antibodies, antibody fragments (e.g., antigen binding portions of antibodies), antibody derivatives, and antibody analogs.
  • the antibody may comprise, for example, a selectable protein scaffold or a human scaffold with grafted CDRs or CDRs derivatives.
  • Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising, for example, a three-dimensional structure that stabilizes the antibody, and fully synthetic scaffolds comprising, for example, biocompatible multimers. See, for example, Korndorfer et al, 2003, Proteins: Structure, Function and Bioinformatics 53: 121-129; Roque et al, 2004, Biotechnol. Prog. 20: 639-654.
  • the antibody may be a peptidomimetic antibody ("PAMs") or a scaffold comprising a mock antibody that utilizes fibrin lignin as a scaffold.
  • PAMs peptidomimetic antibody
  • the antibody may have a structure such as a native immunoglobulin.
  • Immunoglobulin is a tetrameric molecule. In natural immunoglobulins, each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light" chain (about 25 kDa) and a “heavy” chain (about 50-70 kDa). The amino terminus of each chain includes a variable region domain of about 100 to 110 or more amino acids, primarily associated with antigen recognition. The carboxy terminal portion of each chain defines a constant region that is primarily associated with effector action. Human antibody light chains are classified into kappa and lambda light chains. The heavy chain was gamma and the antibody was determined to be IgG.
  • variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D” region of about 10 amino acids. See, Fundamental Immunology Ch. 7 (Paul Editor, 2nd ed., Raven Press, 1989).
  • the variable regions of each light/heavy chain pair form an antibody binding site such that one intact immunoglobulin has two binding sites.
  • Native immunoglobulin chains display the same basic structure of relatively conserved framework regions (FR) joined by three highly variable regions, also referred to as complementarity determining regions or CDRs. From the N-terminus to the C-terminus, the light and heavy chains each comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids for each domain is consistent with the definition of Kabat et al. in Sequences of Proteins of Immunological Interest, Fifth Edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991.
  • Single-chain antibody in which the V L and V H region by a peptide linker (synthetic amino acid residue sequence) linked to form antibodies continuous protein, wherein the peptide linker long enough to allow the protein chain folds back to itself and form Monovalent antigen binding sites (see, eg, Bird et al, 1988, Science 242: 423-26 and Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85: 5879-83).
  • peptide linker synthetic amino acid residue sequence
  • the complementarity determining region of a given antibody can be identified using the method described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991. (CDRs) and framework regions (FR).
  • CDRs can be incorporated into the molecule either covalently or non-covalently into an antibody.
  • Antibodies can incorporate CDR(s) into larger polypeptide chains.
  • the CDR(s) can be covalently linked to another township peptide chain, or non-covalently incorporated into the CDR(s).
  • CDRs allow antibodies to specifically bind to specific antigens of interest.
  • An antibody can have one or more binding sites. If there is more than one binding site, the binding site may have the same or different specificity as the other.
  • native human immunoglobulin G typically has two identical binding sites, while a "bispecific” or “bifunctional” antibody can have two or more binding sites, and these sites belong to two Different specific binding sites, such as the T cell bispecific antibody herein, have four binding sites, two of which recognize CD3 positive T cells, and the remaining two sites bind to CD19 positive or CD20 positive B cells.
  • T cell bispecific antibody or "bispecific antibody” is a bispecific antibody capable of recognizing T cells and an additional target.
  • murine antibody includes antibodies having one or more variable and constant regions derived from mouse immunoglobulin sequences.
  • humanized antibody is an antibody produced by grafting the complementarity determining region sequence of a mouse antibody molecule into a human antibody variable region framework.
  • an “antigen binding domain”, “antigen binding region” or “antigen binding site” is a portion of an antibody comprising an amino acid residue that interacts with an antigen and which contributes to the specificity and affinity of the antibody for the antigen. For an antibody that specifically binds to its antigen, this will include at least a portion of at least one of its CDR domains.
  • An “epitope” is a portion of a molecule that binds to an antibody (eg, by an antibody).
  • An epitope may comprise a non-contiguous portion of a molecule (eg, in a polypeptide, amino acid residues that are not contiguous in the primary sequence of the polypeptide are sufficiently close together in the tertiary and quaternary structure of the polypeptide that they are bound by one antibody) .
  • the "same percentage" of two polynucleotides or two polypeptide sequences was determined using the GAP computer program (GCG Wisconsin Package; version 10.3 (accelrys, San Diego, CA)) using its default parameter comparison sequence.
  • nucleic acid refers to DNA molecules (eg, cDNA or genomic DNA), RNA molecules (eg, mRNA), and nucleotides used. DNA or RNA analogs and hybrids thereof produced by analogs (eg, peptide nucleic acids and non-natural nucleotide analogs).
  • the nucleic acid molecule can be single stranded or double stranded.
  • a nucleic acid molecule herein comprises a contiguous open reading frame encoding an antibody, or a fragment, derivative, mutein or variant thereof thereof.
  • Two single-stranded polynucleotides are "complementary" to each other if their sequences are arranged in anti-parallel, such that each nucleotide in one polynucleotide is complementary to another polynucleotide In contrast to nucleotides, no gaps are introduced and there are no unpaired nucleotides at the 5' or 3' end of each sequence.
  • a polynucleotide is "complementary" to another polynucleotide if it can hybridize to each other under moderately stringent conditions. Thus, one polynucleotide may be complementary to another polynucleotide, but not its complement.
  • a “vector” is a nucleic acid that can be used to introduce another nucleic acid to which it is linked into a cell.
  • a "plasmid” which refers to a linear or circular double stranded DNA molecule to which additional nucleic acid segments can be ligated.
  • a viral vector e.g., replication defective retrovirus, adenovirus, and adenovirus companion virus
  • certain vectors can be autonomously replicated in the host cell into which they are introduced (e.g., bacterial vectors comprising bacterial origins of replication as well as episomal mammalian vectors).
  • vectors when introduced into a host cell, integrate into the genome of the host cell and are therefore replicated along with the host genome.
  • An "expression vector” is a type of vector that directs expression of a selected polynucleotide.
  • a nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression of the nucleotide sequence (eg, expression level, time or site).
  • a "regulatory sequence” is a nucleic acid that can affect the expression (eg, expression level, time or locus) of a nucleic acid to which it is operably linked.
  • a regulatory gene for example, acts directly on a regulated nucleic acid or through one or more other molecules (eg, a polynucleotide that binds to a regulatory sequence and/or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (eg, polyadenylation signals).
  • a “host cell” is a cell used to express a nucleic acid, such as a nucleic acid herein.
  • the host cell can be a prokaryote, such as E. coli, or it can be a eukaryote, such as a single cell eukaryote (eg, yeast or other fungus), a plant cell (eg, a tobacco or tomato plant cell), an animal cell (eg, Human cells, monkey cells, hamster cells, rat cells, mouse cells or insect cells) or hybridomas.
  • the host cell is a cultured cell transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in a host cell.
  • the phrase "recombinant host cell” can be used to describe a host cell transformed or transfected with a nucleic acid that is expected to be expressed.
  • a host cell can also be a cell that comprises the nucleic acid but is not expressed at a desired level unless a regulatory sequence is introduced into the host cell such that it is operably linked to the nucleic acid.
  • the term host cell refers not only to a particular subject cell but also to the progeny or progeny of that cell. Since certain modifications may occur to subsequent generations, such as mutations or environmental influences, the progeny may in fact be different from the parent cell but still fall within the scope of the term as used herein.
  • Differentiation cluster 3 on the surface of T cells is a co-receptor of T cell receptors and assists in the activation of cytotoxic T cells.
  • the CD3 molecule of mammalian T cells is composed of four subunits (one gamma subunit, one ⁇ subunit and two ⁇ subunits).
  • the T cell bispecific antibodies herein bind to CD3 molecule-positive T cells mainly by specificity against the epsilon subunit, but other subunits may also be involved in the formation of antibody epitopes.
  • Differentiation cluster 19 (CD19) on the surface of B cells can bind to B cell receptors, reduce the threshold of B cells in response to antigen-dependent activation, and participate in the maturation and differentiation of B cells.
  • Differentiation cluster 20 on the surface of B cells participates in the B cell immune response.
  • the T cell bispecific antibodies herein bind to B cells by specificity for CD19 or CD20.
  • the amino acid sequences of CD3 ⁇ , CD19, and CD20 of mouse, macaque, and human are listed in the Uniprot protein database (UniProt, Consortium. "UniProt: a hub for protein information.”. Nucleic acids research.
  • the antibodies referred to herein belong to a T cell-dependent bispecific antibody capable of specifically recognizing one of CD19 or CD20 on the surface of human B cells and CD3 on the surface of T cells. It can recruit T cells to attack CD19 or CD20-positive malignant or abnormal B cells at the time of administration, and play a role in treating related diseases caused by such B cells, including various B-cell leukemias and B-cell autoimmune diseases. Symptoms such as rejection caused by organ transplantation. Cell killing by such bispecific antibodies does not depend on the cytotoxic effects mediated by the Fc fragment of the IgG molecule and is faster, more active, and less potent than conventional antibodies.
  • Bispecific antibodies have a variety of different molecular configurations and have their own advantages and disadvantages.
  • Representative antibodies in the form of Triomab and BiTEs have been approved for marketing with Catumaxomab and Blinatumomab for the treatment of malignant ascites and B-ALL, respectively, which are also being tried for the treatment of NHL.
  • the former is highly immunogenic due to the structure of the rat/mouse chimeric antibody, and the preparation rate is very low, while the latter uses a single-chain antibody in a series of molecular forms, thereby having a short half-life in vivo, a low yield, and a complicated production process. And so on.
  • bispecific antibody proteins referred to herein are selected from novel constructs.
  • these bispecific antibodies are constructed from an intact anti-human CD19 or CD20 IgG molecule and two identical anti-CD3 scFv structures. Domain composition.
  • these bispecific antibodies consist of one intact anti-human CD3 IgG molecule and two identical anti-CD19 or CD20 scFv forms of the domain.
  • the two scFv domains are fused to the amino terminus (N') or carboxy terminus (C') of the light or heavy chain of the IgG molecule by a flexible peptide linker sequence.
  • Such molecular design is based on the desire to maximize retention of the excellent properties of IgG molecules.
  • IgG molecules As a fusion component of fusion proteins, IgG molecules have been shown to effectively increase the stability and in vivo half-life of the entire fusion protein while retaining its own affinity (CN 105854000 A). At the same time, the IgG preparation process based on the high affinity of Protein A for IgG molecules has been very mature and platformized. With IgG molecules, we can prepare these bispecific antibodies with a purification process similar to traditional antibodies, reducing the cost of the process. . Furthermore, thanks to its molecular structure and conserved sequences, IgG is a low immunogenic molecule, and the bispecific antibody proteins involved in this paper, although artificially engineered in molecular structure, are still largely based on IgG molecules. It is beneficial to reduce the stability and immunogenic uncertainty caused by excessive changes in molecular structure.
  • the bispecific antibody involved in this article a T cell bispecific antibody formed by scFv with IgG molecules.
  • the IgG molecular portion thereof may be selected from one of the following different antibody forms: mouse antibody, humanized antibody, chimeric antibody, monoclonal antibody, recombinant antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody, and the like. And may comprise any constant region known in the art.
  • the light chain constant region can be, for example, a kappa or lambda type light chain constant region, such as a human kappa or lambda light chain constant region (SEQ NO. 27 and SEQ NO. 27).
  • the heavy chain constant region is a gamma heavy chain constant region, such as the human gamma heavy chain constant region (SEQ NO. 29, SEQ NO. 30, and SEQ NO. 31).
  • the light or heavy chain constant region is a fragment, derivative, variant or mutein of the native constant region.
  • Recombinant DNA technology can also be applied.
  • a cloned DNA encoding a specific antibody, such as DNA encoding the constant domain of an antibody of the desired isotype, can be used in this procedure. See also Lanitto et al, 2002, Methods Mol. Biol. 178: 303-16.
  • the scFv domain which forms a bispecific antibody in combination with an IgG molecule does not comprise any constant region, which is composed of a light chain variable region domain and a heavy chain variable region domain linked by a peptide linker amino acid sequence, three
  • the arrangement is selected from one of the following:
  • Vs L is the light chain variable region amino acid sequence of the scFv domain
  • Vs H is the heavy chain variable region amino acid sequence of the scFv domain
  • N' represents the amino terminus of the polypeptide chain
  • C' represents the carboxy terminus of the polypeptide chain.
  • Link 2 is an intra-domain peptide linker amino acid sequence, the sequence being selected from the group consisting of: SEQ NO ID: 6, SEQ NO ID: 7, SEQ NO ID: 8, SEQ NO ID: 9, SEQ NO ID: 10.
  • the reason we chose the scFv domain to provide affinity for the second antigen is that it retains a structure similar to the IgG variable region (Fab), and the single-stranded construct is susceptible to fusion expression with IgG molecules.
  • the scFv domain is fused to IgG to form a symmetric structure, that is, two light chains or two heavy chains of an IgG molecule are linked to one scFv domain, and the linkage manner is carried out according to one of the following ways. :
  • scFv is a single-chain antibody variable region fragment
  • VH is an IgG domain heavy chain variable region
  • C L is an IgG domain light chain constant region domain
  • CH1 is an IgG domain heavy chain constant region domain 1
  • C H2 domain of an IgG heavy chain constant region domain 2 C H3 domain of an IgG heavy chain constant region domain 3
  • N ' represents an amino terminus of a polypeptide chain, C' on behalf of the carboxy terminus of the polypeptide chain.
  • Link 1 is an interdomain interpeptide linker sequence selected from the group consisting of: SEQ NO ID: 1, SEQ NO ID: 2, SEQ NO ID: 3, SEQ NO ID: 4, SEQ NO ID: 5.
  • bispecific antibodies herein as described above share four binding sites and two different specificities, derived from IgG molecules and scFv domains, respectively, and are distributed in one of the following ways:
  • IgG molecule recognizes CD19; scFv domain recognizes CD3;
  • IgG molecules recognize CD20; scFv domain recognizes CD3;
  • IgG molecules recognize CD3; scFv domain recognizes CD19;
  • the IgG molecule recognizes CD3; the scFv domain recognizes CD20.
  • the specificity against human CD19 or human CD20 may be derived from an antibody clone selected from the group consisting of 2H7, IDEC-C2B8, 4G7, B4, HD37, FMC63, etc., which has an affinity for human CD19 or human CD20 of 10 -9 /M or more, Higher affinity has better cell killing activity.
  • the specificity for human CD3 is derived from an antibody clone selected from the group consisting of OKT3 and UCHT-1, and its affinity for CD3 is required to be between 10 -5 /M and 10 -7 /M, and the scFv is lower or higher than this affinity. Not applicable to the bispecific antibodies described herein.
  • the IgG domain or scFv domain having different specificities described above can be represented by a combination of different light and heavy chain variable regions.
  • an IgG domain using a combination of L1H1 represents a light chain of L1 for the IgG domain.
  • the variable region is formed in combination with the heavy chain variable region of H1.
  • T cells Several antibodies against T cells, such as OKT3, TGN1412, and Rituximab against B cells, cause Cytokine release syndrome, and excessive release is called Cytokine storm.
  • An acute infusion reaction that can be life-threatening.
  • the T cell bispecific antibodies referred to herein are involved in simultaneous binding to both T cell and B cell immune cells, the probability of producing a Cytokine storm is higher. Therefore, it is particularly important to circumvent the cytokine storm when administering these bispecific antibodies. This risk can be reduced in two ways: first, because T cells are a major contributor to cytokine release, limiting the affinity of antibodies against CD3 is an important aspect of reducing cytokine release, the antibodies herein are directed against CD3.
  • Affinity must be maintained between 10 -5 /M and 10 -7 /M, maintaining a balance between recruiting T cell activity and stimulating cytokine release.
  • bispecific antibodies directed against human CD19 or human CD20 do not need to borrow Fc-mediated ADCC and CDC immune responses (eg, Blinatumomab does not contain Fc), and the effector function retained by the Fc portion of IgG will be introduced.
  • More T cells and immune cells other than B cells, such as NK cells, macrophages, neutrophils, eosinophils and basophils increase the risk of cytokine storms, so this article Part of the heavy chain constant region is mutated, eliminating its affinity for Fc receptors and has reduced the risk of cytokine storms.
  • the engineered IgG heavy chain constant region sequences are: SEQ NO ID: 29, SEQ NO ID: 30, SEQ NO ID: 31.
  • the immunogenicity of antibody drugs is a very important consideration in the development phase.
  • IgG molecules benefit from their structure and sequence conservation, and are a low immunogenic molecule.
  • the bispecific antibodies in this paper are structurally based on IgG.
  • the classical molecular structure is designed, the IgG part of which does not need to be described, and the two scFv domains also have a molecular structure close to the IgG variable region. This design reduces the introduction of structural new immune sites due to excessive artificialization of the molecular structure. risks of.
  • the peptide linker sequence is an artificial sequence portion that must be added, whether in constructing the scFv domain or joining the scFv and IgG domains.
  • the peptide linker portion is preferably rich in glycine and serine sequences. Because glycine and serine have smaller side chains, the peptide linker sequences are quite flexible, reducing the relative positional rigidity between the scFv and IgG domains, allowing them to freely bind to their respective targets. At the same time, the alternating presence of glycine and serine avoids excessive duplication of introduction of unnecessary immunogenicity into bispecific antibodies.
  • peptide linker length, balance structure flexibility and immunogenicity are important, so the following several peptide linker sequences are preferred for ligation of scFv and IgG domains: SEQ NO ID: 1, SEQ NO ID: 2, SEQ NO ID: 3, SEQ NO ID: 4, SEQ NO ID: 5; and construction of scFv: SEQ NO ID: 6, SEQ NO ID: 7, SEQ NO ID: 8, SEQ NO ID: 9, SEQ NO ID: 10.
  • the DNA encoding the scFv and the light or heavy chain DNA of the IgG domain are joined to form a fusion light chain or a fusion heavy chain DNA by DNA encoding a peptide linker sequence, and will also be introduced at the 5' end of the light chain DNA.
  • the DNA encoding the signal directs the peptide to form the gene, and the scFv and IgG sequences can be ligated on the basis of this gene.
  • the sequence of the scFv was obtained by gene synthesis and ligated to the DNA encoding the peptide linker sequence by PCR.
  • the light chain variable region and heavy chain variable region DNA sequences of the IgG domain are also obtained by gene synthesis, and are ligated with the constant region DNA of a specific antibody subtype to obtain a complete IgG light chain and heavy chain. DNA sequence.
  • the constant region DNA of a wild type antibody subtype can be obtained by cloning from a specific library and serving as a basis for sequence optimization.
  • the gene for expressing a bispecific antibody described herein is inserted into an expression vector by cloning for the production and expression of a bispecific antibody.
  • the DNA carrying the gene is introduced into the host cell by co-transfection or transformation, and the promoter is selected by optimizing adaptation, and the transformant or amplification coding station is selected.
  • the medium of the gene of the desired sequence is cultured at a suitable pH and temperature.
  • the DNA introduction method uses commonly used CaPO 4 , electroporation and PEI. These methods can be used to produce a large number of bispecific antibodies described herein after introduction of these vectors into E. coli, yeast or mammalian-based expression systems.
  • Suitable host cells suitable for expressing the nucleic acids in the vectors described herein include higher eukaryotic cells, and examples of mammalian host cell line expression include Chinese hamster ovary cell line (CHO) and human embryonic kidney cells (HEK293 or HEK293 in suspension culture).
  • the cell line a signal-directing peptide located at the N-terminus of the light chain, directs secretion of the bispecific antibody from the mammalian host cell line.
  • the vector for expression and cloning carries a selection marker capable of allowing the vector to be continuously replicated in the host cell for screening for cells capable of absorbing the nucleic acid encoding the bispecific antibody and having an operably linked to the bispecific antibody coding sequence And guide the promoter of mRNA synthesis.
  • a selection marker capable of allowing the vector to be continuously replicated in the host cell for screening for cells capable of absorbing the nucleic acid encoding the bispecific antibody and having an operably linked to the bispecific antibody coding sequence And guide the promoter of mRNA synthesis.
  • An example of this is the use of a vector bearing antibiotic resistance and a hepatitis B virus and a prion promoter (SV40), preferably a CHO host cell stably expressing a bispecific antibody.
  • proteins may undergo a variety of post-transcriptional modifications.
  • the type and extent of these modifications will depend on the host cell line used to express the protein as well as the culture conditions.
  • modifications include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation.
  • Frequent modification due to the action of carboxypeptidase results in the loss of a carboxy terminal basic residue such as lysine or arginine (as described in Harris, 1995, Journal of Chromatography 705: 129-134).
  • Monoclonal antibodies can be isolated and purified by a variety of established techniques. Such separation techniques include affinity chromatography using protein A-Sepharose, size exclusion chromatography, and high-sub-exchange chromatography, see, for example, Coligan, pages 2.7.1-2.7.12 and 2.9.1-2.9. .3 pages; Baines et al., "Purification of Immunoglobulin G (IgG),” Methods in Molecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc., 1992). Monoclonal antibodies can be purified by affinity chromatography using appropriate ligands based on specific properties of the antibody (eg, heavy or light chain isoforms, binding specificity, etc.).
  • Suitable ligands immobilized on a solid support include protein A, protein G, anti-constant (light or heavy chain) antibodies, anti-idiotypic antibodies, and TGF-p binding proteins or fragments or variants thereof.
  • the host cell line expresses the bispecific antibody protein, it is partially purified by affinity chromatography to be secreted in the cell culture supernatant.
  • affinity chromatography examples herein include capture of a bispecific antibody fused to a full length antibody using an affinity chromatography column of Protein A/Protein G, followed by elution from the chromatography column with low pH followed by collection. Mild elution conditions help prevent protein denaturation.
  • Antibodies with increased affinity can be isolated using molecular evolution of complementarity determining regions (CDRs) at the center of the antibody binding site, such as antibodies with increased affinity for c-erbB-2, such as Schier et al., 1996, J. Mol. Biol. .263: 551-567.
  • CDRs complementarity determining regions
  • the bispecific antibodies herein can be formulated with one or more excipients.
  • the bispecific antibodies herein can be combined with a pharmaceutically acceptable buffer, a pH adjusted to provide acceptable stability, and a pH that can be administered (e.g., parenterally administered).
  • one or more pharmaceutically acceptable antimicrobial agents can be added. Meta-cresol and phenol are preferred pharmaceutically acceptable antimicrobial agents.
  • One or more pharmaceutically acceptable salt solutions may be added to adjust the ionic strength or tonicity.
  • One or more excipients may be added to further adjust the isotonicity of the formulation. Glycerin is an example of an isotonicity regulating excipient.
  • Pharmaceutically acceptable means suitable for administration to humans or other animals and therefore does not contain toxic ingredients or undesirable contaminants and does not interfere with the activity of the active compounds therein.
  • the bispecific antibodies herein can be formulated as a solution formulation or as a lyophilized powder that can be reconstituted with a suitable diluent.
  • a lyophilized dosage form is one in which the bispecific antibody is stabilized, with or without the buffering ability of the reconstituted product to maintain pH over the intended shelf life.
  • the solution comprising the bispecific antibody discussed herein is preferably isotonic prior to lyophilization to allow it to form an isotonic solution upon reconstitution.
  • the pharmaceutically acceptable salt solution forms of the bispecific antibodies herein are within the scope of this document.
  • the acid commonly used to form acid addition salts is inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl. Sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
  • Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid.
  • Base addition salts include those derived from inorganic bases such as ammonium, alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salt solutions herein thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like. Nucleic acid and host cell
  • nucleic acid molecules comprising, for example, a polynucleotide encoding all or a portion of an antibody, such as one or both strands of a bispecific antibody described herein, or a fragment, derivative, mutation thereof Protein or variant; polynucleotide sufficient for use as a hybridization probe; PCR primer or sequencing primer for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide; for inhibiting polynucleoside Acid-expressed antisense nucleic acid and its complement.
  • the nucleic acid can be of any length.
  • nucleic acid can be single or double stranded and comprise RNA and/or DNA nucleotides as well as artificial variants thereof (eg, peptide nucleic acids).
  • each polypeptide sequence disclosed herein can be encoded by a greater number of other nucleic acid sequences.
  • nucleic acids that hybridize to other nucleic acids (eg, nucleic acids comprising any of the nucleotide sequences of A-1/A-2) under specific hybridization conditions.
  • Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Son (1989), 6.3.1-6.3.6.
  • moderately stringent conditions use a prewash solution containing 5X sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), about 50% formamide hybridization buffer, 6X
  • SSC sodium chloride/sodium citrate
  • the SSC is hybridized to a temperature of 55 ° C (or other similar hybridization solution, for example, containing 50% formamide, hybridized at 42 ° C), and eluted at 60 ° C using 0.5X SSC, 0.1% SDS.
  • Stringent hybridization conditions were hybridized in 6X SSC at 45 °C and then washed one or more times in 0.1X SSC, 0.2% SDS at 68 °C.
  • nucleosides comprising at least 65, 70, 75, 80, 85, 90, 95, 98 or 99% homology to each other are nucleosides.
  • the nucleic acids of the acid sequence can usually still hybridize to each other.
  • Mutations can be introduced using any technique known in the art.
  • one or more specific amino acid residues can be altered using, for example, a site-directed mutagenesis protocol.
  • one or more randomly selected residues can also be altered using, for example, a random mutagenesis protocol.
  • the mutant polypeptide can be expressed and screened for the desired properties.
  • This mutation can alter biological activity quantitatively or qualitatively. Examples of quantitative changes include increasing, decreasing or eliminating this activity. Examples of qualitative changes include altering the antigen specificity of the antibody. Mutations can also be introduced into the nucleic acid without significantly altering the biological activity of the polypeptide encoded thereby. For example, nucleotide substitutions that result in amino acid substitutions at non-essential amino acid residues can be made.
  • a vector comprising a nucleic acid encoding a polypeptide herein or a portion thereof.
  • vectors include, but are not limited to, plasmids, viral vectors, non-free gene mammalian vectors, and expression vectors, such as recombinant expression vectors.
  • a recombinant expression vector herein can comprise a nucleic acid encoding a bispecific antibody herein in a form suitable for expression of the nucleic acid in a host cell.
  • the recombinant expression vector comprises one or more regulatory sequences that are screened based on the host cell for expression operably linked to the pre-expressed nucleic acid sequence.
  • Regulatory sequences include constitutive expression of a guide nucleotide sequence in a plurality of species of host cells (eg, SV40 early gene enhancer, Rous sarcoma virus promoter, and cytomegalovirus promoter), directing only in certain hosts Expression of nucleotide sequences in cells (eg, tissue-specific regulatory sequences, see Voss et al, 1986, Trends Biochem. Sci. 11: 287, Maniatis et al, 1987, Science 236: 1237, the entire contents of which is incorporated herein by reference.
  • a guide nucleotide sequence in a plurality of species of host cells (eg, SV40 early gene enhancer, Rous sarcoma virus promoter, and cytomegalovirus promoter), directing only in certain hosts Expression of nucleotide sequences in cells (eg, tissue-specific regulatory sequences, see Voss et al, 1986, Trends Biochem. Sci. 11: 287, Maniatis e
  • directing inducible expression of the nucleotide sequence in response to a particular treatment or condition eg, a metal thiopurine promoter in a mammalian cell and a tetracycline reaction in both prokaryotic and eukaryotic systems (tet-sesponsive) Promoter and / or streptomycin reaction promoter (same as before)
  • a particular treatment or condition eg, a metal thiopurine promoter in a mammalian cell and a tetracycline reaction in both prokaryotic and eukaryotic systems (tet-sesponsive) Promoter and / or streptomycin reaction promoter (same as before)
  • tet-sesponsive tetracycline reaction in both prokaryotic and eukaryotic systems
  • streptomycin reaction promoter streptomycin reaction promoter
  • the host cell can be any prokaryotic or eukaryotic cell.
  • Prokaryotic host cells include Gram-negative or Gram-positive organisms such as E. coli or Bacillus. More advanced eukaryotic cells include insect cells, yeast cells, and established cell lines derived from mammalian sources. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al, 1998, Cytotechnology 28: 31) or CHO DXB-11, which lacks DHFR (see Urlaub et al, 1980, Proc. Natl.
  • CHO Chinese hamster ovary
  • CHO cell lines include CHO-K1 (ATCC#CCL-61), EM9 (ATCC#CRL-1861), and UV20 (ATCC#CRL-1862), and other host cells include the COS-7 line of monkey kidney cells (ATCC#).
  • CRL-1651) see Gluzman et al, 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL-163), AM-1/D cells (described in US Patent Serial No.
  • HeLa cells derived from African green monkey kidney cell line CV1 (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney Cells such as 293, 293EBNA or MSR 293, human epithelial A431 cells, human C010205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissues, primary transplants, HL- 60, U937, HaK or Jurkat cells. Suitable cloning and expression vectors for bacterial, fungal, yeast and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, 1985).
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques.
  • a gene encoding a screening marker eg, antibiotic resistance
  • Preferred screening markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate.
  • stable transfected cells containing the introduced nucleic acid can be identified by drug screening (e.g., cells incorporating the screened gene can survive while other cells die).
  • the transformed cells can be cultured under conditions that increase the expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification methods.
  • One such purification method is described in the examples below.
  • Polypeptides pre-administered herein include bispecific antibodies expressed by substantially homologous recombinant mammalian cells that are substantially free of contaminating endogenous materials. Indication
  • the bispecific antibodies herein can be used to treat a variety of diseases and conditions associated with abnormal and deteriorating B cells. Since the bispecific antibodies herein act by recruiting T cells to attack human CD19 or human CD20-positive B cells, various diseases induced by B cells are within their applicability, including but not limited to the following three categories.
  • the first category leukemia caused by malignant B cells, which are characterized by B cell proliferation, apoptosis, and uncontrolled differentiation, including B cell acute leukemia, B cell chronic leukemia, non-Hodgkin's lymphoma, hairy cell lymphoma. and many more. These diseases are potential bispecific antibody indications herein.
  • the second category autoimmune diseases caused by abnormal B cells.
  • Abnormal B cells in such diseases lose normal antigen tolerance, and abnormal expression is directed at autoantibodies, which can also induce human body by releasing pro-inflammatory cytokines and chemical hormones. Verify the reaction.
  • diseases of this type including the well-known multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, etc., which are also within the scope of the potential indications for bispecific antibodies herein.
  • the third category chronic rejection caused by organ transplantation, the disease is derived from the organ of the organ recipient's immune system to continue to reject the received organs, selective treatment of B cells depletion is beneficial to alleviate this rejection, and therefore this article Potential indications for bispecific antibodies.
  • a method of treating a subject comprises administering a therapeutic dose of a bispecific antibody provided herein.
  • the term "subject” refers to mammals, including humans, and can be used interchangeably with the term “patient.”
  • Murine antibodies or humanized antibodies thereof can be used to treat and control diseases or conditions associated with abnormal and deteriorating B cells in a patient.
  • the term “treating” includes alleviating or preventing other aspects of at least one symptom or condition, or reducing the severity of the disease, and the like.
  • the bispecific antibodies herein do not require a complete healing effect, or eradicate all symptoms or manifestations of the disease to constitute an effective therapeutic.
  • a drug as a therapeutic agent can reduce the severity of a given disease state, but can be considered as an effective therapeutic agent without eliminating all manifestations of the disease.
  • prophylactic administration can form an effective prophylactic agent without being completely effective in preventing the onset of symptoms. Only reduce the effects of the disease (for example, by reducing the number or severity of its symptoms, or by increasing another therapeutic effect, or by creating another effective effect), or by reducing the likelihood of disease occurrence or exacerbation in the subject enough.
  • One embodiment herein relates to a method of administering to a patient an antibody in an amount and for a time sufficient to induce a sustained improvement over a baseline level of an indicator sufficient to induce the severity of the response to a particular condition.
  • a pharmaceutical composition comprising an antibody herein is administered to a patient in a manner appropriate for the indication.
  • the pharmaceutical compositions may be administered by any suitable technique including, but not limited to, parenteral, topical or inhalation.
  • the pharmaceutical composition can be administered by rapid injection or continuous infusion by, for example, intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes. Administration may be considered, for example, for topical administration at the site of the disease or injury, such as transdermal administration and sustained release of the implant.
  • Administration by inhalation includes, for example, nasal or oral inhalation, administration with a spray, inhalation of the antibody in the form of an aerosol, and the like. Other options include oral formulations including tablets, syrups or lozenges.
  • an antibody herein in the form of a composition
  • a composition comprising one or more additional components, such as a physiologically acceptable carrier, adjuvant or diluent.
  • the composition may optionally additionally comprise one or more physiologically active agents as described below.
  • the composition comprises one, two, three, four, five or six physiologically active agents in addition to one or more of the T cell bispecific antibodies herein.
  • the pharmaceutical composition comprises a bispecific antibody herein and one or more materials selected from the group consisting of a buffer suitable for the antibody, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (eg, containing less than 10) Polypeptides of amino acids), proteins, amino acids, sugars such as dextrin, complexes such as EDTA, glutathione, stabilizers and adjuvants. Preservatives may also be added according to appropriate industry standards.
  • the composition can be formulated as a lyophilized powder using a suitable adjuvant solution as a diluent. The appropriate components are not toxic to the recipient at the dosages and concentrations employed.
  • kits for use by medical practitioners including one or more of the antibodies herein and Treatment The label or other description of any condition discussed herein.
  • the kit comprises a sterile preparation of one or more antibodies in one or more vials in the form of the above compositions.
  • the dosage and frequency of administration can vary depending on the route of administration, the particular antibody employed, the nature and severity of the condition being treated, whether the condition is acute or chronic, and the volume and overall symptoms of the patient. Suitable dosages can be determined by methods well known in the art, such as including dose escalation studies in clinical trials.
  • the bispecific antibodies described herein can be administered one or more times at regular intervals, for example, over a period of time.
  • long-term treatment is usually the most effective.
  • short-term administration is sufficient, for example, from one week to six weeks.
  • human antibodies are administered until the patient exhibits a selected medical condition or a medically relevant improvement in the indicator above baseline levels.
  • One example of a treatment regimen provided herein includes treating a B cell associated disorder with a subcutaneous injection of an antibody once a week at an appropriate dose.
  • the antibody can be administered weekly or monthly until the desired result is achieved, for example, the patient's symptoms subsided.
  • the treatment can be re-treated as needed or, alternatively, a maintenance dose can be administered.
  • compositions herein are directed to the use of, for example, one bispecific antibody herein, two or more bispecific antibodies herein, bispecific antibodies herein, and other non-incorporated antibodies, or bispecific herein.
  • Different conditions such as antibodies and various types of cells cultured in vitro (for example, CIK cells cultured in vitro).
  • the antibody is administered alone or in combination with other agents for treating the symptoms of pain in the patient, examples of which include proteins as well as non-protein drugs.
  • the dosages as known in the art should be adjusted accordingly.
  • “Co-administered" combination therapies are not limited to simultaneous administration, but also include treatment regimens that administer at least one antigen and protein in a course of treatment involving administration of at least one other therapeutic agent to the patient.
  • a method of making an agent for treating a B cell-associated disorder comprising administering a mixture of a bispecific antibody described herein and a pharmaceutically acceptable excipient.
  • the preparation method of the medicament is as described above.
  • the bispecific antibodies herein can be administered by any route known to physicians of ordinary skill to be effective.
  • Peripheral parenteral tract belongs to one of these methods.
  • Parenteral administration is generally understood in the medical literature as injecting a dosage form into the body by means of a sterile syringe or some other mechanical device such as a syringe pump.
  • Peripheral parenteral routes can include intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration.
  • the bispecific antibodies herein can also be administered by the oral, rectal, nasal or lower respiratory route, which are parenteral routes. Among these parenteral routes, the lower respiratory route and the oral route are preferred.
  • Step 1 Synthesis and subcloning of light and heavy chain genes of bispecific antibodies
  • variable region DNA sequence encoding the light heavy chain of the IgG molecule antibody and the DNA sequence encoding the scFv domain were synthesized by Nanjing Kingsray Biotechnology Co., Ltd. Using Overlapping PCR, the variable region of the IgG antibody light or heavy chain and the scFv domain DNA sequence were ligated through the polypeptide linker DNA sequence to form a complete DNA sequence encoding the fused light and heavy chain by designing the cleavage site. The points Nhe1 and Not1 are ligated into the human light chain or heavy chain constant region expression vector PTM5. After the integrity of the above DNA sequence is verified by sequencing, the PTM5 plasmid can be used in combination with the corresponding unfused light chain or heavy chain plasmid for transfection.
  • Step 2 Transient expression of bispecific antibodies in suspension HEK293 or CHO host cell lines
  • the suspension HEK293 or CHO expressing cell line was inoculated into a spinner flask and used for transfection after 24 hours at 37 ° C in spin culture.
  • Polyethylenimine (PEI) was used as a transfection medium during the transfection process, mixed with DNA and added to the cell culture.
  • the mixing ratio of PEI and DNA is preferably from 1:1 to 5:1.
  • the cells were further cultured at 37 ° C for more than 96 hours to express the antigen-binding protein, during which 0.5% tryptone was added to the cell culture as the amino acid source for expression, and finally the cell supernatant was collected for bispecificity. Purification of the antibody is separated.
  • Step 3 Purification and preparation of bispecific antibodies from cell culture supernatants
  • the cells are centrifuged to remove the cells, and the supernatant is subjected to an affinity chromatography column coupled with a protein A ligand, and the expressed bispecific antibody is eluted from the column with an eluent of pH 2.5-3.5. .
  • the preset neutralization buffer in the elution tube neutralizes the low pH of the eluent in time.
  • the protein solution collected after elution was dialyzed against PBS.
  • Step 4 Flow cytometry analysis (FACS) binding of bispecific antibodies to human B lymphoma Raji cells
  • Step 5 Detection of bispecific antibody binding to CIK cells by enzyme-linked immunosorbent assay Remove 10 5 cells/well of CIK cells from the cell culture supernatant, wash twice with PBS, and add to 96-well plates, 100% methanol (100 ⁇ L ) fixed at 4 ° C for 10 min. An additional 100 ⁇ l of freshly prepared 0.6% H 2 O 2 -PBS was added, treated at room temperature for 20 min, and washed twice with PBS.
  • T cell bispecific antibody (3.3 ⁇ g/well, 1.1 ⁇ g/well, 0.33 ⁇ g/well, 0.11 ⁇ g/well, 0.033 ⁇ g/well) was added and incubated at 37 ° C for 90 min. After multiple washings, 100 ⁇ l of a 1:5000 dilution of GxH-HRP secondary antibody (Sigma, commercially available) was added to each well, and a positive control group was added with a 1:5000 dilution of 100 ⁇ l of GxM-HRP secondary antibody (Sigma, commercially available). Incubate for 30 min at 37 °C.
  • TMB chromogenic substrate was added to each well, reacted at 37 ° C for 15 min, terminated by the addition of 50 ul of 2 M H 2 SO 4 , and the OD450 value was read.
  • the positive control was OKT3 (Abeam, commercially available); the negative control was PBS.
  • Step 6 Isolation of PBMC cells
  • the cell liquid was divided into 4 layers, and the white film layer was taken in a 50 mL centrifuge tube, PBS was added to 50 mL, mixed, centrifuged, and 1800 rpm for 6 minutes, the supernatant was removed, and the washing was repeated 2 times. Subsequent experiments such as counting can be performed after resuspending the cell pellet with appropriate PBS according to the experimental needs.
  • Step 7 CIK cell culture
  • CIK cell starter culture medium serum-free X-Vivo cell culture medium + 1000 U/mL IFN- ⁇
  • CIK cell starter culture medium serum-free X-Vivo cell culture medium + 1000 U/mL IFN- ⁇
  • CIK cell stimulating factor mixture serum-free X-Vivo cell culture medium + 50 ng/mL CD3 monoclonal antibody, 300 U/mL IL-2, 100 U/mL IL-1 ⁇
  • the next step is to determine the fluid replacement (free serum X-Vivo culture solution +300U/mL IL-2 ⁇ 2% autologous plasma) and the bottle separation according to the growth of CIK cells. Basically, the cells should be maintained at 1-2 ⁇ 10. The concentration of 6 grows around. Finally, the collected CIK cells were subjected to phenotypic detection by flow cytometry FC500, including: CD3, CD4, and the expression of these cell surface antigens in CIK cells was examined.
  • Step 8 Bispecific antibody-mediated detection of tumor cells by CIK or PBMC cells
  • the Raji single cell suspension was collected in a sterile 1.5 mL EP tube, centrifuged at 1000 rpm for 4 min, washed once with 1 mL of PBS, and 1 mL of Calcein AM (Cat. No. C1430, ThermoFisher) at a final concentration of 100 nM was added and incubated at 37 ° C in a cell culture incubator. After 30 min, the supernatant was removed by centrifugation, washed once with 1 mL of PBS, resuspended in 2.5 mL of complete medium, and added to a 96-well cell culture plate at 50 ⁇ L/well (i.e., 2 ⁇ 10 4 cells/well) overnight.
  • Calcein AM Cat. No. C1430, ThermoFisher
  • the experimentally designed effect-target ratio was added to the cultured CIK or PBMC cells at 100 ⁇ L/well, and control wells were set, and the same volume of medium was added without the addition of the wells of the CIK or PBMC cells.
  • the corresponding antibody was added according to the experimental design, 50 ⁇ L/well, and the wells without the addition of the antibody were supplemented with the same volume of the medium, and the co-culture was continued.
  • 96-well plates were taken out from 8h to 48h (time-dependent), and each well was transferred to 96-well U-shaped plate, centrifuged at 1200rpm for 3min, and added with 200 ⁇ L 2% FBS-PBS. Suspended and read data on a flow cytometer Guava 6HT. 50,000 cells were read per well, and the number of Calcein AM positive cells and their percentage to the negative control were calculated.
  • Step 9 In vivo pharmacodynamic study of bispecific antibody on human B lymphoma Raji cell in situ xenograft tumor model
  • CIK cells volume 0.2 mL, 1 ⁇ 10 7 CIK cells, 0.25 nmol ⁇ CD19(L6H6)/ ⁇ CD3(L2H2), or 2.5 nmol ⁇ CD19(L6H6)/ ⁇ CD3(L2H2) bispecific for each mouse in the experimental group.
  • Sex antibody volume 0.2 mL.
  • the animal's health status and death are monitored daily. Routine examinations include observing the effects of tumor cells on the daily behavior of the animal such as hair, behavioral activities ( ⁇ , etc.), food intake, body weight change (pre-weekly measurement of body weight 2 times per week, late Measured every other day or every day), appearance signs or other abnormal conditions.
  • the experimental indicator is to investigate the growth of tumor cells in animals.
  • the number of days after death from vaccination to the mouse is defined as its survival period.
  • the survival of each mouse was recorded, the Kaplan-Meier survival curve was calculated and the median Survival Time (MST) of each group was calculated, as well as the extended survival time of the drug-administered group relative to the control group (Increase in Life Span, ILS).
  • MST median Survival Time
  • the bispecific antibodies herein are ⁇ CD19 (L6H6, which comprises SEQ NO ID: 21 and SEQ NO ID: 22) / ⁇ CD3 (L2H2, which comprises SEQ NO ID: 13 and SEQ NO ID: 14)
  • L6H6 which comprises SEQ NO ID: 21 and SEQ NO ID: 22
  • ⁇ CD3 which comprises SEQ NO ID: 13 and SEQ NO ID: 14
  • Fig. 1 a mammalian cell expression system
  • These bispecific antibodies have the ability to bind to either human CD3 and either human CD19 or human CD20.
  • Figure 2 (Experimental step 4) also shows the bispecific antibody of this patent by using ⁇ CD19/ ⁇ CD3 as an example.
  • Raji cell-bound flow cytometry fluorescence color data Raji cells are a human B-cell lymphoma cell line with high expression of human CD19 molecules on the cell surface.
  • the data show that the surface binding ability of the ⁇ CD19/ ⁇ CD3 bispecific antibody and Raji cells is very similar to that of the control ⁇ CD19 mAb, and thus the molecular structure of the bispecific antibody does not affect the binding ability of the ⁇ CD19 antibody portion.
  • CIK cells are cytokine-induced killer T cells, and their induction and expansion are as described in the experimental step 6, and the surface thereof is highly expressed by human CD3 molecules.
  • the data showed that the binding value of 10 ⁇ g of ⁇ CD19/ ⁇ CD3 bispecific antibody to CIK cells was 0.47/0.06 (binding data/negative control), and the binding value of 0.4 ⁇ g of OKT3 in the positive control was 0.36/0.06 (binding data/negative control). .
  • the above binding results show that the bispecific antibody molecules of this patent are directed against two different antigen affinities.

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Abstract

提供了一种能与人CD19或CD20和人CD3结合的双特异性抗体，用于治疗与B细胞恶化相关的疾病。

Description

一种能与人CD19或CD20和人CD3结合的双特异性抗体及其应用
相关申请案的交叉参考
本申请案要求2017年4月12日提交的申请号为CN201710236421.8的中国专利申请的优先权，其披露内容以全文引用的方式并入本中。
技术领域
本文提供了一种抗体，特别是一种能够同时与人CD19或CD20和人CD3结合的双特异性抗体，还提供了它用于治疗由恶性或非正常B细胞所引起的相关疾病的方法。
背景技术
B细胞在人体的体液免疫反应和抗原递呈上发挥重要的作用。B细胞病变所带来的后果十分严重，其造成的疾病主要为两大类，第一类为恶性B细胞血液癌症，第二类则为非正常的B细胞识别自身抗原造成的自免疫类疾病(Janeway等，2001，Immunobiology:The Immune System in Health and Disease.5th edition，New York:Garland Science)。
CD19是一个95kDa大小的跨膜糖蛋白，参与B细胞自身免疫反应敏感性的调控(Tedder，2009，Nat.Rev.Rheumatol.5:572-7；Stanciu-Herrera等，2008，Leuk.Res.32:625-32)，维持对外免疫和自身免疫反应之间的平衡。CD19表达于几乎所有类型和阶段的B细胞表面，也包括大多数的恶性和非正常B细胞。针对CD19的抗体类药物(Katz等，2014，Leuk.Lymphoma55:999-1006)包括2014年获批以B-ALL为适应症的Blinatumomab。Blinatumomab是一个T细胞双特异性抗体，由单条多肽链折叠形成，其包括由柔性肽接头序列连接的两个独立的结构域，一个是结合B细胞CD19的单链抗体可变区(scFv)结构域，而另一个是结合人T细胞表面受体CD3的scFv(Nagorsen等，2012，Pharmacol Ther.136:334-42)。
以CD20为靶点的利妥昔于1997年上市，一个传统的人鼠嵌合型抗体用于治疗非霍奇金淋巴瘤，通过ADCC和CDC作用对B细胞造成免疫伤害和凋亡(Smith，2003，Oncogene 22:7359–68)。CD20与B细胞对非T细胞依赖的抗原的应答相关,其在B细胞表面的分布没有CD19广泛，主要为成熟B细胞类型，而pre-B细胞，非成熟B细胞和大多数的浆细胞不表达或低表达CD20，因此针对CD20的药物对这些特定类型的B细胞所引起的 疾病的治疗效果不如以CD19为靶标的药物(Johnson等，2009，Blood113:3773-80)，比如风湿性关节炎等自身免疫疾病。但在同时表达CD19和CD20的B细胞类型的表面，CD20的表达量明显大于CD19，且在各类型的慢性白血病的恶性B细胞表面，CD20的表达量高于正常的B细胞，而CD19的表达量在这些细胞上和正常B细胞相比均被下调(Ginaldi等，1998，J.Clin.Pathol.51:364-9)，因而治疗各类型的慢性白血病，CD20可能是更加好的选择。
发展至今，T细胞双特异性抗体在治疗肿瘤的原理应用上已经逐渐趋于同质化，更需要在抗体工程和分子设计上提高(Bano等，2016，Antibodies5:1-23)，因为如Blinatumomab，虽然其分子结构带来了高活性，但也使这个分子有体内半衰期短、产率低和制备工艺复杂等缺点。本文所涉及新型双特异性抗体由一个完整的抗人CD19或人CD20的免疫球蛋白G(IgG)分子和两个相同的抗CD3的scFv形式的结构域组成，以克服Blinatumomab在稳定性和工艺方面的缺点，并同时保留足够高的靶细胞杀伤活性。就CD20和CD3的双特异性抗体而言，借助T细胞介导，以克服CD20单抗药物利妥昔的抗药性(Rezvani等，2011，Best.Pract.Res.Clin.Haematol.24:203-16)。
本文的双特异性抗体具有高效地借助T细胞介导的细胞毒性杀死恶性B细胞的活性，并且还具有结构稳定，表达量高，纯化工艺简单等优势。本文的双特异抗体可被用于治疗所有由恶性或非正常B细胞所引起的相关疾病。
发明内容
本文提供了一种新式的双特异性抗体。该抗体具有能够与人CD19或人CD20和人CD3特异性结合的能力。该双特异性抗体可介导T细胞毒性依赖的针对各类型和阶段的B细胞的免疫杀伤反应。该双特异性抗体可用于制备治疗由恶性或非正常B细胞所引起的相关疾病的药物。
在一个实施方案中，本文所提供的双特异性抗体是一种能够与人CD19或人CD20和人CD3结合的双特异性抗体，其结构特征在于：所述的双特异性抗体包含单链抗体结构域(scFv结构域)、免疫球蛋白G结构域(IgG)和结构域间肽接头序列(Link 1)，这三者通过以下方式中的一种融合形成：
a.通过结构域间肽接头序列将scFv的羧基端和IgG轻链的氨基端连接：N’-scFv-Link 1-V L-C L-C’；
b.通过结构域间肽接头序列将scFv的氨基端和IgG轻链的羧基端连接：N’-V L-C L-Link 1-scFv-C’；
c.通过结构域间肽接头序列将scFv的羧基端和IgG重链的氨基端连接： N’-scFv-Link 1-V H-C H1-C H2-C H3-C’；及
d.通过结构域间肽接头序列将scFv的氨基端和IgG重链的羧基端连接：N’-V H-C H1-C H2-C H3-Link 1-scFv-C’；
其中:N’代表多肽链的氨基端,C’代表多肽链的羧基端,scFv代表单链抗体结构域，IgG代表免疫球蛋白G结构域,V L代表IgG结构域的轻链可变区,C L代表IgG结构域的轻链恒定区,V H代表IgG结构域的重链可变区,C H1代表IgG结构域的重链恒定区1,C H2代表IgG结构域的重链恒定区2,C H3代表IgG结构域的重链恒定区3,及Link 1代表结构域间肽接头。
在一个实施方案中，在本文所提供的双特异性抗体中，所述的结构域间肽接头(Link 1)的序列选自以下序列之一：SEQ NO ID:1、SEQ NO ID:2、SEQ NO ID:3、SEQ NO ID:4、及SEQ NO ID:5。
在一个实施方案中，在本文所提供的双特异性抗体中，所述的scFv结构域包含轻链可变区、重链可变区和结构域内肽接头序列，这三者通过以下方式中的一种组合形成scFv结构域：
a.通过结构域内肽接头序列将轻链可变区的羧基端和重链可变区的氨基端连接：N’-V SL-Link 2-V SH-C’顺序排列；及
b.通过结构域内肽接头序列将重链可变区的羧基端和轻链可变区的氨基端连接：N’-V SH-Link 2-V SL-C’顺序排列；
其中:N’代表多肽链的氨基端,C’代表多肽链的羧基端,V SL代表scFv结构域的轻链可变区,V SH代表scFv结构域的重链可变区,及Link 2代表结构域内肽接头。
在一个实施方案中，在本文所提供的双特异性抗体中,所述结构域间内肽接头(Link 2)的序列选自以下序列之一：SEQ NO ID:6、SEQ NO ID:7、SEQ NO ID:8、SEQ NO ID:9、及SEQ NO ID:10。
在一个实施方案中，在本文所提供的双特异性抗体中,所述scFv结构域包含以下方案之一的氨基酸序列：
a.scFv结构域的轻链可变区序列选自以下序列之一：SEQ NO ID:11、SEQ NO ID:13、及SEQ NO ID:32；
b.scFv结构域的重链可变区序列选自以下序列之一：SEQ NO ID:12、SEQ NO ID:14、SEQ NO ID:33、及SEQ NO ID:34；及
c.(a)的轻链可变区序列和(b)的重链可变区序列的组合。
在一个实施方案中，在本文所提供的双特异性抗体中,所述抗体的scFv结构域包含以下方案之一的氨基酸序列组合方式：SEQ NO ID:11与SEQ NO ID:12的组合(L1H1)、SEQ NO ID:13与SEQ NO ID:14的组合(L2H2)、 SEQ NO ID:32与SEQ NO ID:33的组合(L9H9)、及SEQ NO ID:32与SEQ NO ID:34的组合(L9H10)。
在一个实施方案中，在本文所提供的双特异性抗体中,所述的抗体的IgG结构域包含以下方案之一的氨基酸序列：
a.IgG结构域的轻链可变区序列选自以下序列之一：SEQ NO ID:15、SEQ NO ID:17、SEQ NO ID:19、SEQ NO ID:21、SEQ NO ID:23、及SEQ NO ID:25；
b.IgG结构域的重链可变区序列选自以下序列之一：SEQ NO ID:16、SEQ NO ID:18、SEQ NO ID:20、SEQ NO ID:22、SEQ NO ID:24、及SEQ NO ID:26；及
c.(a)的轻链可变区序列和(b)的重链可变区序列的组合。
在一个实施方案中，在本文所提供的双特异性抗体中,所述抗体的IgG结构域包含以下方案之一的氨基酸序列组合方式：SEQ NO ID:15与SEQ NO ID:16的组合(L3H3)、SEQ NO ID:17与SEQ NO ID:18的组合(L4H4)、SEQ NO ID:19与SEQ NO ID:20的组合(L5H5)、SEQ NO ID:21与SEQ NO ID:22的组合(L6H6)、SEQ NO ID:23与SEQ NO ID:24的组合(L7H7)、及SEQ NO ID:25与SEQ NO ID:26的组合(L8H8)。
在一个实施方案中，在本文所提供的双特异性抗体中,所述scFv结构域包含以下方案之一的氨基酸序列：
a.scFv结构域的轻链可变区序列选自以下序列之一：SEQ NO ID:15、SEQ NO ID:17、SEQ NO ID:19、SEQ NO ID:21、SEQ NO ID:23、及SEQ NO ID:25；
b.scFv结构域的重链可变区序列选自以下序列之一：SEQ NO ID:16、SEQ NO ID:18、SEQ NO ID:20、SEQ NO ID:22、SEQ NO ID:24、及SEQ NO ID:26；及
c.(a)的轻链可变区序列和(b)的重链可变区序列的组合。
在一个实施方案中，在本文所提供的双特异性抗体中,所述抗体的scFv结构域包含以下方案之一的氨基酸序列组合方式：SEQ NO ID:15与SEQ NO ID:16的组合(L3H3)、SEQ NO ID:17与SEQ NO ID:18的组合(L4H4)、SEQ NO ID:19与SEQ NO ID:20的组合(L5H5)、SEQ NO ID:21与SEQ NO ID:22的组合(L6H6)、SEQ NO ID:23与SEQ NO ID:24的组合(L7H7)、及SEQ NO ID:25与SEQ NO ID:26的组合(L8H8)。
在一个实施方案中，在本文所提供的双特异性抗体中,所述的抗体的IgG结构域包含以下方案之一的氨基酸序列：
a.IgG结构域的轻链可变区序列选自以下序列之一：SEQ NO ID:11、 SEQ NO ID:13、及SEQ NO ID:32；
b.IgG结构域的重链可变区序列选自以下序列之一：SEQ NO ID:12、SEQ NO ID:14、SEQ NO ID:33、及SEQ NO ID:34；及
c.(a)的轻链可变区序列和(b)的重链可变区序列的组合。
在一个实施方案中，在本文所提供的双特异性抗体中，其中所述抗体的IgG结构域包含以下方案之一的氨基酸序列组合方式：SEQ NO ID:11与SEQ NO ID:12的组合(L1H1)、SEQ NO ID:13与SEQ NO ID:14的组合(L2H2)、SEQ NO ID:32与SEQ NO ID:33的组合(L9H9)、及SEQ NO ID:32与SEQ NO ID:34的组合(L9H10)。
在一个实施方案中，在本文所提供的双特异性抗体中,所述的抗体蛋白质的IgG分子进一步包含以下方案之一的氨基酸序列：
a.选自以下序列之一的轻链恒定区氨基酸序列：SEQ NO ID:27及SEQ NO ID:28；
b.选自以下序列之一的重链恒定区氨基酸序列：SEQ NO ID:29、SEQ NO ID:30、及SEQ NO ID:31；
c.(a)的轻链恒定区序列和(b)的重链恒定区序列的组合。
在一个实施方案中，本文所提供的双特异性抗体是一种能够与人CD19和人CD3结合的双特异性抗体。
在一个实施方案中，本文所提供的双特异性抗体是一种能够与人CD20和人CD3结合的双特异性抗体。
在一个实施方案中，本文所提供的双特异性抗体是鼠源抗体、人源化抗体、嵌合抗体、单克隆抗体、重组抗体、IgGl抗体、IgG2抗体、IgG3抗体、或IgG4抗体。
在一个实施方案中，本文提供了一种多核苷酸，其编码本文所提供的双特异性抗体。
在一个实施方案中，本文提供了一种载体，其包含本文所提供的多核苷
酸。
在一个实施方案中，本文提供了一种宿主细胞，其包含本文所提供的载体。在一个实施方案中，本文提供了一种药用组合物，其包含与药用可接受载体混合的本文所提供的双特异性抗体。
在一个实施方案中，本文提供了本文所述的双特异性抗体在制备用于预防或治疗B细胞白血病的药物中的用途。在一个实施方案中，本文所述的B细胞白血病是急性B细胞白血病。在一个实施方案中，本文所述的B细胞白血病是慢性B细胞白血病。
在一个实施方案中，本文提供了本文所述的双特异性抗体在制备用于预 防或治疗非霍奇金淋巴瘤的药物中的用途。
在一个实施方案中，本文提供了本文所述的双特异性抗体在制备用于预防或治疗B细胞引起的自体免疫疾病的药物中的用途。在一个实施方案中，本文所述的B细胞引起的自体免疫疾病是风湿性关节炎、多发性硬化症、或系统性红斑狼疮。
在一个实施方案中，本文提供了本文所述的双特异性抗体在制备用于预防或治疗器官移植带来的排异反应及其相关症状的药物中的用途。
附图说明
图1显示了αCD19(L6H6，其包含SEQ NO ID:21与SEQ NO ID:22)/αCD3(L2H2，其包含SEQ NO ID:13与SEQ NO ID:14)双特异性抗体的SDS-PAGE图。M泳道代表标准蛋白，分子量如图所示。#1和#2泳道均为αCD19/αCD3双特异性抗体(不同上样量)，结果显示其分子量为55Kd左右。
图2显示了流式细胞术(FACS)检测重组表达的αCD19/αCD3双特异性抗体(实线双峰并箭头指示)与Raji肿瘤细胞的特异性结合，阳性对照为αCD19单抗(虚线双峰并箭头指示)，阴性对照为抗体溶剂(虚线单峰)。该结果显示该αCD19/αCD3双特异型抗体很好地保留αCD19针对CD19的亲和力。
图3显示了不同浓度的αCD19/αCD3双特异性抗体介导下的CIK对Raji肿瘤细胞的杀伤曲线，E:T为10：1。Y轴代表固定细胞总量中剩余存活的Raji细胞的数量，X轴代表了抗体的浓度。该结果提示了在E:T为10：1的情况下，CIK对Raji的杀伤力随着抗体浓度上升而提升，成正相关。其对Raji细胞的半杀伤浓度为1ng/mL。
图4显示了NOD/SCID小鼠在接受了Raji细胞原位接种后，在以下不同的治疗条件下的各自的生存周期：PBS注射(实线)；CIK细胞注射(点虚线)；CIK细胞加0.25nmol的αCD19/αCD3双特异性抗体注射(点实线)；CIK细胞加2.5nmol的αCD19/αCD3双特异性抗体注射(虚线)。各组的注射周期均为从Raji细胞接种日(第0天)进行第一次注射，之后每两天注射一次，共十次。结果显示了CIK细胞加不同浓度αCD19/αCD3双特异性抗体注射显著提高了小鼠的生存周期。
发明详述
定义
除非另外指明，本文使用标准的单字母或三字母缩写表明多聚核苷酸和多肽序列。多肽序列的氨基端(N’)在左羧基端(C’)在右，单链核酸序列和双链核酸序列的上游链的5’端在左而它们的3’端在右。多肽的具体部分可由氨基酸残基编号表示，例如氨基酸80至130，或由该位点的实际残基表示 例如Lys80至Lys130或K80至K130。也可通过解释其与参比序列的差异来描述具体的多肽或多聚核苷酸序列。
除非本文另外定义，与本文相关的科学和技术术语应具有本领域普通技术人员所理解的含义。进一步，除非上下文另有要求，单数术语应包括复数并且复数含义术语应包括单数含义。通常，与本文所述细胞和组织培养、分子生物学、免疫学、微生物学、遗传学和蛋白质核酸化学以及杂交相关的命名法和技术为本领域熟知和经常使用的。本文的方法和技术通常根据本领域已知的常规方法以及本说明书引用和讨论的各种普通和更具体的参考文献所描述的进行，除非另外说明。参见，例如，Sambrook等，Molecular Cloning:A Laboratory Manual，第2版,Cold Spring Harbor Laboratory Press(1989)和Ausubel等，Current Protocols in Molecular Biology,Greene Publishing Associates(1992)，以及Harlow and Lane Antibodies:A Laboratory Manual Cold Spring Harbor Laboratory Press(1990)，均以参考形式并于本文。酶反应和纯化技术根据操作说明进行，如本领域通常完成或本文所述。与本文描述的分析化学、合成有机化学和医学和药学化学相关的术语以及实验操作和技术均为本领域熟知和普遍使用者。标准技术可用于化学合成、化学分析、药物制备、制剂和给药以及患者的治疗。
术语“肽”、“多肽”和“蛋白”均指包含两个或多个通过肽健相互连接的氨基酸的分子。这些术语涵盖例如天然和人工蛋白、蛋白片段和蛋白序列的多肽类似物(例如突变蛋白、变异体和融合蛋白)。
如本文使用的术语“多肽片段”指与对应的全长蛋白相比具有氨基端和/或羧基端缺失的多肽。片段长度可为例如至少5、6、7、8、9、10、11、12、13、14、15、20、50、70、80、90、100、150或200个氨基酸。片段长度可为例如最多1000、750、500、250、200、175、150、125、100、90、80、70、60、50、40、30、20、15、14、13、12、11或10个氨基酸。片段可在其一端或两端进一步包含一个或多个附加氨基酸，例如，来自不同天然蛋白质的氨基酸序列(例如，Fc或亮氨酸拉链结构域)或人工氨基酸序列(例如，人工接头序列)。
本文所述的多肽包括以任何原因和经任何方法修饰的多肽，例如，以：(1)降低蛋白水解敏感性，(2)降低氧化敏感性，(3)改变形成蛋白复合物的亲和性，(4)改变结合亲和性以及(5)赋予或修饰其它物理化学或功能性质。类似物包含多肽的突变蛋白。例如，可在天然序列(例如在形成分子内接触的结构域之外的多肽部分)中进行单个或多个氨基酸替换(例如，保守氨基酸替换)。“保守氨基酸替换”为不显著改变母体序列结构特性者(例如，替换氨基酸不应破坏母体序列中出现的螺旋或干扰其它赋予母 体序列特性或对其功能是必须的二级结构类型)。领域公认的多肽二级和三级结构的实例描述于Proteins,Structures and Molecular Principles(Creighton编辑，W.H.Freeman and Company(1984))；Introduction to Protein Structure(Branden and Tooze编辑,Garland Publishing(1991))；和Thornton等，1991，Nature 354:105-106，均以参考形式并于本文。
本文也包含抗体的非肽类似物。非肽类似物普遍用于提供与模板肽具有相似性质的药物。这些非肽化学物类型被称作“模拟肽(peptide mimetics)”或“拟肽(peptidomimetics)”。Fauchere，1986，Adv.Drug Res.15:29-69；Veber和Freidinger，1985，Trends Neurosci.8:392-396；Evans等，1987，J.Med.Chem.30:1229-1239，均以参考形式并于本文。与治疗用肽结构相似的模拟肽可用于产生相等的治疗或预防效果。通常，拟肽与范例多肽的结构相似(即具有期望生物化学性质或药理学活性的多肽)，例如人类抗体，但是具有一个或多个任选被选自-CH 2NH-、-CH 2S-、-CH 2-CH 2-、-CH＝CH-(顺式和反式)、-COCH 2-、-CH(OH)CH 2-、和-CH 2SO-的连接通过本领域熟知的方法取代的肽连接。用相同类型的D-氨基酸系统取代一致序列的一个或多个氨基酸(例如，D-赖氨酸取代L-赖氨酸)也可用于生成更稳定的肽。此外，可通过本领域已知的方法(Rizo and Gierasch，1992，Annu.Rev.Biochem.61:387-418)，以参考形式并于本文)例如通过添加可形成将肽环化的分子间二硫键的内部半胱氨酸残基生成包含一致序列或基本相同的一致序列变异体的拘束肽(constrained peptides)。
多肽的“变异体”包含相对于另一多肽序列在氨基酸序列中插入、缺失和/或替换了一个或多个氨基酸残基的氨基酸序列。
多肽的“衍生物”为经化学修饰的多肽(例如，抗体)，例如通过与其它化学部分例如聚乙二醇、白蛋白(例如人血清白蛋白)结合、磷酸化和糖基化。
术语“结构域”为生物大分子中具有特异结构、独立功能的区域，其具有相当的结构独立性，有在同类型生物大分子中完整得、重复得出现的特性，本文中特别指肽、多肽或者蛋白中这样的区域，例如轻链可变区(V L)结构域，单链抗体(scFv)结构域。小的结构域可以组合形成更大的结构域，例如单链抗体结构域由轻链可变区结构域和重链可变区(V H)结构域通过肽接头序列组合形成。
术语“亚基”为一个由一条以上多肽组成的蛋白质或者蛋白复合物所包含的其中一个具有独立序列和三级结构的结构域或结构组分，例如完整的人CD3分子包含CD3γ，CD3ε和CD3δ三种亚基。
术语“抗体”为包含与抗原结合部分并任选为允许抗原结合部分采取促进 该抗体与该抗原结合的构象的支架或框架部分的蛋白，即包括两个全长重链和两个全长轻链的抗体，以及其衍生物，变异体、片段、突变蛋白和融合蛋白，例如本文的双特异性抗体。抗体的实例包括抗体、抗体片段(例如抗体的抗原结合部分)、抗体衍生物和抗体类似物。该抗体可包含例如可选择的蛋白支架或具有移植CDRs或CDRs衍生物的人工支架。该支架包括但不限于包含被引入的例如以稳定化该抗体的三维结构的抗体衍生支架以及包含例如生物相容性多聚体的全合成支架。参见，例如，Korndorfer等，2003，Proteins:Structure，Function and Bioinformatics 53:121-129；Roque等，2004,Biotechnol.Prog.20:639-654。此外，该抗体可以是模拟肽抗体(“PAMs”)或包含模拟抗体的支架，其如支架一样利用纤维蛋白连接素。
抗体可具有例如天然免疫球蛋白的结构。“免疫球蛋白”为四聚体分子。在天然的免疫球蛋白中，各四聚体由两个相同的多肽链对组成，各对具有一个“轻”链(约25kDa)和一个“重”链(约50-70kDa)。各链的氨基端包括约100至110或更多氨基酸的可变区结构域，主要与抗原识别相关。各链的羧基端部分确定了主要与效应器作用相关的恒定区。人的抗体轻链分为κ和λ轻链。重链为γ，并确定了抗体为IgG。在轻链和重链中，可变和恒定区由约12或更多个氨基酸的“J”区连接，重链也包括约10多个氨基酸的“D”区。参见，Fundamental Immunology Ch.7(Paul编辑，第2版，Raven Press，1989)。各轻/重链对的可变区形成抗体结合位点，这样一个完整的免疫球蛋白具有两个结合位点。
天然免疫球蛋白链显示出由三个高度可变区连接的相对保守骨架区(FR)的相同基本结构，也被称作互补决定区或CDRs。从N端到C端，轻和重链均包含结构域FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。各结构域氨基酸的分配与Kabat等在Sequences of Proteins of Immunological Interest，第5版，US Dept.of Health and Human Services,PHS，NIH，NIH Publication No.91-3242，1991中的定义一致。
单链抗体(scFv)为其中的V L和V H区由肽接头(合成的氨基酸残基序列)连接以形成连续蛋白质的抗体，其中该肽接头足够长以允许该蛋白链折叠回自身并形成单价抗原结合位点(参见，例如，Bird等，1988，Science242:423-26and Huston等，1988,Proc.Natl.Acad.Sci.USA85:5879-83)。
可使用Kabat等在Sequences of Proteins of Immunological Interest，第5版,US Dept.of Health and Human Services,PHS,NIH,NIH Publication No.91-3242，1991中描述的方法鉴定给定抗体的互补决定区(CDRs)和骨架区(FR)。可向分子中共价或非共价并入一个或多个CDRs使其成为抗体。抗体可以较大多肽链并入CDR(s)。可将CDR(s)共价连接至另一乡肽链，或 非共价并入CDR(s)。CDRs允许抗体与具体的相关抗原特异性结合。
抗体可有一个或多个结合位点。如果多于一个结合位点，该结合位点可与另一个具有相同或不同的特异性。例如，天然人免疫球蛋白G通常具有两个相同的结合位点，而“双特异性”或“双功能”抗体可以具有两个或者以上的结合位点，并且这些位点分属于两种具有不同特异性的结合位点，例如本文的T细胞双特异性抗体具有四个结合位点，其中两个位点识别CD3阳性的T细胞，剩余两个位点结合CD19阳性或者CD20阳性的B细胞。术语“T细胞双特异性抗体”或“双特异性抗体”为能够识别T细胞和一个额外靶点的双特异性抗体。
术语“鼠源抗体”包括具有一个或多个来源于小鼠免疫球蛋白序列的可变区和恒定区的抗体。
术语“人源化抗体”是将小鼠抗体分子的互补决定区序列移植到人抗体可变区框架中而制成的抗体。
“抗原结合结构域”、“抗原结合区”或“抗原结合位点”为包含与抗原相互作用的氨基酸残基并有助于抗体对抗原的特异性和亲和力的抗体的部分。对与其抗原特异性结合的抗体而言，这将包括至少部分的至少一个其CDR结构域。
“表位”为与抗体(例如，通过抗体)结合的分子部分。表位可包含分子的非连续部分(例如，在多肽中，在多肽的一级序列中不连续的氨基酸残基在该多肽的三级和四级结构中相互足够接近以致于被一个抗体结合)。
两个多聚核苷酸或两个多肽序列的“相同百分比”由使用GAP计算机程序(GCG Wisconsin Package；version 10.3(Accelrys，San Diego，CA)的一部分)使用其默认参数比较序列测定。
术语“多聚核苷酸”、“寡聚核苷酸”和“核酸”可在全文中交替使用并包括DNA分子(例如，cDNA或基因组DNA)、RNA分子(例如mRNA)、使用核苷酸类似物(例如，肽核酸和非天然核苷酸类似物)生成的DNA或RNA类似物及其杂交体。核酸分子可为单链或双链。在一个实施方案中，本文的核酸分子包含编码本文的抗体或其片段、衍生物、突变蛋白或变异体连续的开放阅读框。
如果它们的序列可反向平行排列则两个单链多聚核苷酸是相互“互补的”，这样一个多聚核苷酸中的各核苷酸与另一多聚核苷酸中的互补核苷酸相反，不会引入空隙并且各序列的5’或3’端没有未配对的核苷酸。如果两个多聚核苷酸可在中等严格条件下相互杂交那么一个多聚核苷酸与另一多聚核苷酸“互补”。因此，一个多聚核苷酸可与另一多聚核苷酸互补，但并不是它的互补序列。
“载体”为可用于将与其相连的另一核酸引入细胞的核酸。载体的一种类型为“质粒”，其指可连接附加核酸区段的线性或环状双链DNA分子。载体的另一类型为病毒载体(例如，复制缺陷逆转录病毒、腺病毒和腺病毒伴随病毒)，其中可将附加DNA区段引入病毒基因组。某些载体可在它们被引入的宿主细胞中自主复制(例如，包含细菌复制起点的细菌载体以及游离型哺乳动物载体)。其它载体(例如，非游离型哺乳动物载体)在引入宿主细胞时整合入宿主细胞的基因组中并因此与宿主基因组一起复制。“表达载体”为可引导所选多聚核苷酸表达的载体类型。
如果调控序列影响核苷酸序列的表达(例如，表达水平、时间或位点)那么核苷酸序列与调控序列“可操作地相连”。“调控序列”为可影响与其可操作相连的核酸的表达(例如，表达水平、时间或位点)的核酸。调控基因，例如，直接对受调控核酸发挥作用或通过一个或多个其它分子(例如，与调控序列和/或核酸结合的多聚核苷酸)的作用。调控序列的实例包括启动子、增强子和其它表达控制元件(例如，多腺苷酸化信号)。调控序列的进一步实例描述于例如Goeddel，1990,Gene Expression Technology:Methods in Enzymology,Volume 185,Academic Press,San Diego,CA and Baron等,1995,Nucleic Acids Res.23:3605-06。
“宿主细胞”为用于表达核酸例如本文的核酸的细胞。宿主细胞可为原核生物，例如大肠杆菌，或者其可为真核生物，例如单细胞真核生物(例如，酵母或其它真菌)、植物细胞(例如烟草或番茄植物细胞)、动物细胞(例如，人细胞、猴细胞、仓鼠细胞、大鼠细胞、小鼠细胞或昆虫细胞)或杂交瘤。通常，宿主细胞为可用多肽编码核酸转化或转染的培养细胞，其可接着在宿主细胞中表达。短语“重组宿主细胞”可用于表述用预期表达的核酸转化或转染的宿主细胞。宿主细胞也可为包含该核酸但是不以期望水平表达的细胞，除非向该宿主细胞引入了调控序列这样其与核酸可操作地相连。应理解的是术语宿主细胞不仅指具体的受试者细胞也指该细胞的子代或可能的子代。由于例如突变或环境影响后续世代会出现某些修饰，该子代事实上可能与母体细胞不同但是仍然属于本文使用的术语范围。CD3、CD19和CD20
T细胞表面的分化簇3(CD3)是T细胞受体的共受体，协助细胞毒性T细胞的激活。哺乳动物T细胞的CD3分子由四个亚基(一个γ亚基，一个δ亚基和两个ε亚基)共同组成。本文的T细胞双特异性抗体主要通过针对ε亚基的特异性来结合CD3分子阳性的T细胞，但其它亚基也有可能参与抗体表位的形成。B细胞表面的分化簇19(CD19)能够与B细胞受体结合，降低B细胞响应抗原依赖性激活的门槛，参与B细胞的成熟和分化。B细 胞表面的分化簇20(CD20)参与B细胞免疫应答。本文的T细胞双特异性抗体通过针对CD19或者CD20的特异性来结合B细胞。小鼠、猕猴和人的CD3ε、CD19、CD20的氨基酸序列在Uniprot蛋白数据库(UniProt,Consortium.“UniProt:a hub for protein information.”.Nucleic acids research.43)中的登录号如下：小鼠CD3ε登录号：P22646；猕猴CD3ε登录号：H9FCI9；人CD3ε登录号：P07766；小鼠CD19登录号：P25918；猕猴CD19登录号：F7F486；人CD19登录号：P15391；小鼠CD20登录号：P19437；猕猴CD20登录号：H9YXP1；人CD20登录号：P11836。双特异性抗体
本文所涉及的抗体属于T细胞依赖的双特异型抗体，能够特异性得识别人B细胞表面的CD19或CD20之一和T细胞表面的CD3。施用时能够招募T细胞攻击CD19或CD20阳性的恶性或者非正常B细胞，起到治疗由此类B细胞引起的相关疾病的作用，包括了各种B细胞性白血病、B细胞性自体免疫类疾病和器官移植引起的排异反应等症状。此类双特异性抗体所引起的细胞杀伤不依赖于IgG分子的Fc片段所介导的细胞毒性作用，并且比传统抗体起效更快，活性更高，用量更少。
双特异性抗体具有各种不同的分子构造，且具有各自的优缺点。具有代表性的Triomab和BiTEs形式的抗体已有Catumaxomab和Blinatumomab被批准上市，分别用于恶性腹水和B-ALL的治疗，后者还正尝试用于NHL的治疗。但前者因为使用大鼠/小鼠嵌合抗体的构造免疫源性很高，且制备得率很低，而后者使用单链抗体串联的分子形式因而具有体内半衰期短，产率低和生产工艺复杂等缺点。
本文所涉及的双特异性抗体蛋白质选用新的构造形式，在一个实施方案中，这些双特异性抗体由一个完整的抗人CD19或CD20的IgG分子和两个相同的抗CD3的scFv形式的结构域组成。在一个实施方案中，这些双特异性抗体由一个完整的抗人CD3IgG分子和两个相同的抗CD19或CD20的scFv形式的结构域组成。两个scFv结构域通过柔性肽接头序列与IgG分子的轻链或者重链的氨基端(N’)或者羧基端(C’)进行融合表达。这样的分子设计是基于对IgG分子的优秀性质加以最大保留的意愿。作为融合蛋白质的一个融合组件，IgG分子已经被证明可以在保留其自身的亲和力的同时有效得提高整个融合蛋白质的稳定性和体内半寿期(CN 105854000 A)。同时，基于Protein A对IgG分子的高亲和力而建立的IgG制备工艺流程已经非常成熟和平台化，借助IgG分子，我们可以用类似于传统抗体的纯化工艺来制备这些双特异性抗体，降低工艺成本。再者，得益与其分子构造和保守的序列，IgG是一个低免疫源性的分子，而本文涉及的双特异性抗体蛋白质虽 然进行了分子构造上的人为改造，却大体上仍然是基于IgG分子的，有利于减少因为分子构造过度变更带来的稳定性和免疫源性上的不确定性。
基于以上的考虑，我们设计了本文涉及的双特异型抗体，一种由scFv搭配IgG分子形成的T细胞双特异型抗体。其IgG分子部分可以选自以下不同的抗体形式之一：鼠源抗体、人源化抗体、嵌合抗体、单克隆抗体、重组抗体、IgGl抗体、IgG2抗体、IgG3抗体、IgG4抗体等的不同形式，并且可以包含本领域已知的任何恒定区。轻链恒定区可为例如κ或λ型轻链恒定区，例如人的κ或λ型轻链恒定区(SEQ NO.27和SEQ NO.27)。重链恒定区为γ型重链恒定区，例如人γ型重链恒定区(SEQ NO.29、SEQ NO.30和SEQ NO.31)。在一个实施方案中，该轻链或重链恒定区为天然恒定区的片段、衍生物、变异体或突变蛋白。也可应用重组DNA技术。可在该操作中应用编码具体抗体多的已克隆DNA，例如编码期望同种型的抗体恒定结构域的DNA。也可参见Lanitto等，2002，Methods Mol.Biol.178:303-16。而与IgG分子组合形成本文双特异性抗体的scFv结构域不包含有任何恒定区，其由轻链可变区结构域、重链可变区结构域由肽接头氨基酸序列连接而成，三者的排列方式选自以下的一种：
a.以N’-Vs L-Link 2-Vs H-C’顺序排列；及
b.以N’-Vs H-Link 2-Vs L-C’顺序排列；
其中:Vs L为scFv结构域的轻链可变区氨基酸序列，Vs H为scFv结构域的重链可变区氨基酸序列，N’代表多肽链的氨基端，C’代表多肽链的羧基端，Link 2为结构域内肽接头氨基酸序列，序列选自：SEQ NO ID:6、SEQ NO ID:7、SEQ NO ID:8、SEQ NO ID:9、SEQ NO ID:10。我们选择scFv结构域来提供针对第二个抗原的亲和力的原因是其保留了与IgG可变区(Fab)相类似的结构，并且单链的构造易于和IgG分子进行融合表达。该scFv结构域与IgG的进行融合表达后将形成对称的结构，即IgG分子的两条轻链或两条重链都连接一个scFv结构域，其链接方式按照以下的方式中的其中一种进行：
a.通过肽接头将scFv的羧基端和IgG轻链的氨基端链接：N’-scFv-Link 1-V L-C L-C’；
b.通过肽接头将scFv的氨基端和IgG轻链的羧基端链接：N’-V L-C L-Link 1-scFv-C’；
c.通过肽接头将scFv的羧基端和IgG重链的氨基端链接：N’-scFv-Link 1-V H-C H1-C H2-C H3-C’；及
d.通过肽接头将scFv的氨基端和IgG重链的羧基端链接：N’-V H-C H1-C H2-C H3-Link 1-scFv-C’；
其中:scFv为单链抗体可变区片段，V H为IgG结构域重链可变区，C L为IgG结构域轻链恒定区结构域，C H1为IgG结构域重链恒定区结构域1，C H2为IgG结构域重链恒定区结构域2，C H3为IgG结构域重链恒定区结构域3，N’代表多肽链的氨基端，C’代表多肽链的羧基端。Link 1为结构域间肽接头序列，序列选自：SEQ NO ID:1、SEQ NO ID:2、SEQ NO ID:3、SEQ NO ID:4、SEQ NO ID:5。
如上所述的本文的双特异性抗体共有四个结合位点和两种不同特异性，分别来自于IgG分子和scFv结构域，并按照以下的方式之一进行分配：
a.IgG分子识别CD19；scFv结构域识别CD3；
b.IgG分子识别CD20；scFv结构域识别CD3；
c.IgG分子识别CD3；scFv结构域识别CD19；及
d.IgG分子识别CD3；scFv结构域识别CD20。
针对人CD19或人CD20的特异性可以来自选自包括2H7、IDEC-C2B8、4G7、B4、HD37、FMC63等的抗体克隆，其对人CD19或人CD20的亲和力要求在10 -9/M以上，更高的亲和力具有更优秀的细胞杀伤活性。针对人CD3的特异性来自选自包括OKT3和UCHT-1等的抗体克隆，其对CD3的亲和力要求在10 -5/M到10 -7/M之间，低于或者高于此亲和力的scFv不适用于本文所述的双特异型抗体。以上所述的拥有不同特异性的IgG结构域或者scFv结构域可通过不同的轻重链可变区的组合方式来表示，例如选用L1H1组合的IgG结构域代表该IgG结构域选用L1的轻链可变区搭配H1的重链可变区组合形成。本文所涉及到的不同轻重链可变区氨基酸序列如下表总结：
Figure PCTCN2018082692-appb-000001
Figure PCTCN2018082692-appb-000002
Figure PCTCN2018082692-appb-000003
数个针对T细胞的抗体，如OKT3，TGN1412以及针对B细胞的Rituximab均会引起人体的细胞激素释放效应(Cytokine release syndrome)，而过度的释放被称作细胞激素风暴(Cytokine storm)，是一种可能危及生命的急性输液反应。因为本文所涉及的T细胞双特异性抗体牵涉到与T细胞和B细胞两种免疫细胞同时结合，产生Cytokine storm的几率更高。因此，规避施用这些双特异性抗体施用时的细胞激素风暴尤为重要。可以通过两个方面来减少该风险：第一，因为T细胞是细胞激素释放的主要贡献者，因此限制针对CD3的抗体的亲和力是降低细胞激素释放的一个重要方面，本文中的抗体针对CD3的亲和力必须保持在10 -5/M到10 -7/M之间，维持其招募T细胞活性和刺激细胞激素释放之间的平衡。第二，因为针对人CD19或者人CD20的双特异型抗体无需借用Fc介导的ADCC和CDC免疫反应来起作用(如Blinatumomab不含有Fc)，而且IgG的Fc部分保留的效应子功能将会引入更多的T细胞和B细胞以外的免疫细胞，如NK细胞，巨噬细胞，中性粒细胞，嗜酸性和嗜碱性粒细胞等的参与，增加细胞激素风暴的风险，所以本文通过对IgG部分的重链恒定区进行突变，消除其与Fc receptor的亲和力已减少细胞激素风暴的风险。经过改造的IgG重链恒定区序列为：SEQ NO ID:29、SEQ NO ID:30、SEQ NO ID:31。
抗体类药物的免疫源性是开发阶段非常重要的考虑项，IgG分子得益于其结构和序列的保守性，是一个低免疫源性的分子，本文的双特异性抗体在结构上基于IgG的经典分子结构设计而成，其IgG部分无需累述，其两个scFv结构域也拥有接近IgG可变区的分子结构，这样的设计减少了因为分子结构过度人为化引入结构性的新免疫位点的风险。但是，在构建这个分子时，无论是在组成scFv结构域或者连接scFv和IgG结构域时，肽接头序列是必须要添加的人为序列部分。为尽可能的减少引入的结构不确定性， 同时保持IgG和scFv两者的自由度，肽接头部分优选富含甘氨酸和丝氨酸的序列。因为甘氨酸和丝氨酸带有较小的侧链，使肽接头序列具有相当的灵活性，减少了scFv和IgG结构域之间相对位置的刚性，使两者可以自由得与各自的靶标结合。同时，甘氨酸和丝氨酸交替出现避免过度重复向双特异性抗体中引入不必要的免疫源性。优选肽接头长度，平衡结构柔度和免疫源性很重要，所以本文优选了以下的几个肽接头序列用于scFv和IgG结构域的连接：SEQ NO ID:1、SEQ NO ID:2、SEQ NO ID:3、SEQ NO ID:4、SEQ NO ID:5；以及scFv的构建：SEQ NO ID:6、SEQ NO ID:7、SEQ NO ID:8、SEQ NO ID:9、SEQ NO ID:10。
本文中，编码scFv的DNA与所述IgG结构域的轻链或重链DNA通过编码肽接头序列的DNA连接成融合轻链或融合重链DNA，同时在轻链DNA的5’末端还将引入编码信号引导肽的DNA来形成基因，并可以此基因基础连接scFv和IgG序列。本文通过基因合成的方法得到scFv的序列，并通过PCR的方法将其与编码肽接头序列的DNA相连接。本文也是通过基因合成的方法得所述IgG结构域的轻链可变区和重链可变区DNA序列，并与特定抗体亚型的恒定区DNA相连接组成得到完整的IgG轻链和重链的DNA序列。野生型抗体亚型的恒定区DNA可以通过从特定的文库克隆得到并作为序列优化的基础。在此所述的用于表达双特异性抗体的基因，通过克隆方式放入表达载体中用于双特异性抗体的产生和表达。在表达过程中，轻链和重链表达载体搭配后，用共转染或者转化的方式将带有其基因的DNA引入宿主细胞中，并用优化适应诱导启动子，选择转化子或者扩增编码所希望序列的基因的培养基在适宜的pH，温度中培养。DNA引入方式选用普遍使用的CaPO 4，电穿孔和PEI等方式。将这些载体引入大肠杆菌、酵母或哺乳动物为基础的表达系统后可使用这些方法生产大量本文所述的双特异性抗体。
适于表达此处所述载体中核酸的合适宿主细胞包括高等真核细胞，哺乳动物宿主细胞系表达的实例包括了中国仓鼠卵巢细胞系(CHO)和人胚胎肾脏细胞(HEK293或者悬浮培养的HEK293细胞系)，位于轻链N末端的信号引导肽指导双特异性抗体的从哺乳动物宿主细胞系中的分泌。用于表达和克隆的载体上带有能够使载体在宿主细胞中不断复制的选择标记，用于筛选有能力吸收编码双特异性抗体的核酸的细胞并且带有与双特异性抗体编码序列有效连接并指导mRNA合成的启动子。其中一个实例是用带有抗生素抗性以及乙型肝炎病毒和猿病毒启动子(SV40)的载体优选稳定表达双特异性抗体的CHO宿主细胞。
本领域技术人员应理解的是一些蛋白质，例如抗体，可能进行多种转录 后修饰。这些修饰的类型和程度取决于用于表达该蛋白的宿主细胞系以及培养条件。该类修饰包括糖基化作用、甲硫氨酸氧化、二酮哌嗪形成、天冬氨酸异构化和天冬酰胺脱酰胺作用的变化。由于羧肽酶的作用频繁修饰导致羧端碱性残基(例如赖氨酸或精氨酸)的丢失(如Harris，1995，Journal of Chromatography 705:129-134中所述)。
可通过多种已确立的技术分离和纯化单克隆抗体。该类分离技术包括使用蛋白A-琼脂糖的亲和色谱法、分子排阻色谱法和高子交换色谱法，参见，例如，Coligan第2.7.1-2.7.12页和第2.9.1-2.9.3页；Baines等，“Purification of Immunoglobulin G(IgG),”Methods in Molecular Biology，第10卷，第79-104页(The Humana Press，Inc.，1992)。可使用基于抗体的特殊性质(例如，重链或轻链同种型、结合特异性等)筛选的适当配基通过亲和色谱法纯化单克隆抗体。固定化于固体载体的适当配基的实例包括蛋白A、蛋白G、抗恒定区(轻链或重链)抗体、抗独特型抗体以及TGF-p结合蛋白或其片段或变异体。本文在宿主细胞系表达双特异型抗体蛋白质后，用亲和层析的方法将其分泌在细胞培养上清中的部分纯化。本文中的实例包括用蛋白A/蛋白G的亲和层析柱捕获融合有全长抗体的双特异性抗体，然后用低pH将其从层析柱料上洗脱后再收集。温和的洗脱条件有助于防止蛋白的变性。
可使用抗体结合位点中央的互补决定区(CDRs)的分子进化分离亲和性增加的抗体，例如对c-erbB-2亲和性增加的抗体，如Schier等，1996，J.Mol.Biol.263:551-567所述。当需要提高根据本文所述的双特异性抗体的针对其某一个靶点的亲和性时，可通过多种亲和成熟方案包括维持CDRs(Yang等，1995，J.Mol.Biol.254:392-403)、链替换(Marks等，1992，Bio/Technology10:779-783)、使用大肠杆菌的突变株(Low等，1996，J.Mol.Biol.250:350-368)DNA重排(Patten等，1997，Curr.Opin.Biotechnol.8:724-733)、噬菌体展示(Thompson等，1996，J.Mol.Biol.256:7-88)以及其它PCR技术(Crameri等，1998，Nature 391：288-291)。所有这些亲和力成熟方法讨论于Vaughan等，1998，Nature Biotechnology 16:535-539中。
可以用一种或多种赋形剂配制本文的双特异性抗体。本文的双特异性抗体可以与可药用的缓冲液、经调节提供可接受的稳定性的pH、以及可施用(例如胃肠外施用)的pH组合。任选地，可以添加一种或多种可药用的抗微生物剂。间甲酚和苯酚是优选的可药用抗微生物剂。可以添加一种或多种可药用盐溶液以调节离子强度或张力。可以添加一种或多种赋形剂以进一步调节制剂的等渗性。甘油是等渗性调节赋形剂的实例。可药用意味着适于施用于人类或其他动物，因此不含有毒性成分或不希望的污染物，并且不干扰其中活性化合物的活性。
可以以溶液制剂或能够用合适的稀释剂重构的冻干粉配制本文的双特异性抗体。冻干剂型是其中双特异性抗体稳定的一种剂型，具有或不具有重构产品在预期的使用货架期内保持pH的缓冲能力。包含在此讨论的双特异性抗体的溶液在冻干前优选是等渗的，使之重构后能够形成等渗溶液。
本文的双特异性抗体的可药用盐溶液形式在本文范围内。常用于形成酸加成盐的酸为无机酸，例如盐酸、氢溴酸、氢碘酸、硫酸、磷酸等，以及有机酸，例如对甲苯磺酸、甲磺酸、草酸、对溴苯基一磺酸、碳酸、琥珀酸、柠檬酸、苯甲酸、乙酸等。优选的酸加成盐是与无机酸，例如盐酸和氢溴酸形成的盐。
碱加成盐包括从无机碱，例如铵、碱或碱土金属氢氧化物衍生的那些盐、碳酸盐、碳酸氢盐等。在制备本文的盐溶液中有用的这类碱因此包括氢氧化钠、氢氧化钾、氢氧化铵、碳酸钾等。核酸和宿主细胞
本文也提供分离的核酸分子，该核酸分子包含例如编码全部或部分抗体的多聚核苷酸，例如本文所述的双特异性抗体的一条链或两条链，或其片段、衍生物、突变蛋白或变异体；足以用作杂交探针的多聚核苷酸；PCR引物或用于鉴定、分析、突变或扩增编码多肽的多聚核苷酸的测序引物；用于抑制多聚核苷酸表达的反义核酸以及其互补序列。该核酸可为任何长度。例如它们的长度可为5、10、15、20、25、30、35、40、45、50、75、100、125、150、175、200、250、300、350、400、450、500、750、1000、1500、3000、5000或更多个核苷酸，和/或包含一个或多个附加序列，例如调控序列，和/或是较大核酸例如载体的一部分。该核酸可为单链或双链并包含RNA和/或DNA核苷酸以及其人工变异体(例如，肽核酸)。熟练的技术人员可理解由于遗传密码的简并性，本文公开的各多肽序列可由更多数量的其他核酸序列编码。
本文进一步提供在具体杂交条件下与其他核酸(例如，包含任何A-1/A-2的核苷酸序列的核酸)杂交的核酸。杂交核酸的方法为本领域熟知。参见，例如，Current Protocols in Molecular Biology，John Wiley&Son(1989)，6.3.1-6.3.6。如本文定义，例如，中等严格条件使用包含5X氯化钠/柠檬酸钠(SSC)的预洗溶液、0.5％SDS、1.0mM EDTA(pH 8.0)、约50％甲酰胺的杂交缓冲液、6X SSC和55℃的杂交温度(或其他相似的杂交溶液，例如包含绚50％甲酰胺的，以42℃杂交)，并且洗脱条件为60℃，使用0.5X SSC、0.1％SDS。严格杂交条件在6X SSC中于45℃杂交，然后于68℃在0.1X SSC、0.2％SDS中洗涤一次或多次。此外，本领域技术人员可操作杂交和/或洗涤条件以增加或降低杂交严格度这样包含相互之间至少65、70、75、 80、85、90、95、98或99％同源的核苷酸序列的核酸通常仍可以相互杂交。影响杂交条件选择的基本参数和设计适当条件的指导列于例如Sambrook，Fritsch和Maniatis，1989，Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory Press，第9和11章；Current Protocols in Molecular Biology，1995，Ausubel等编辑，John Wiley&Sons，Inc.，第2.10和6.3-6.4节)并可由具有本领域普通技术的人员基于例如DNA的长度和/或碱基组成轻松确定。可通过突变在核酸中引入变化，藉此导致其编码的多肽(例如，抗原结舍蛋白)氨基酸序列的变化。可使用本领域已知的任何技术引入突变。在一个方面，可以使用例如定点诱变方案改变一个或多个具体氨基酸残基。在另一个方面，也可以使用例如随机诱变方案改变一个或多个随机选择的残基。无论其如何生成，可表达突变多肽并筛选期望性质。该突变可在数量上或性质上改变生物学活性。量变的实例包括增加、降低或消除该活性。质变的实例包括改变抗体的抗原特异性。也可将突变引入核酸而不显著改变其编码多肽的生物学活性。例如，可进行引起非必需氨基酸残基处氨基酸替换的核苷酸替换。
在另一方面本文提供包含编码本文多肽或其部分的核酸的载体。载体的实例包括但是不限于质粒、病毒载体、非游离基因哺乳动物载体和表达载体，例如重组表达载体。
本文的重组表达载体可包含适于该核酸在宿主细胞中表达的形式的本文编码双特异性抗体的核酸。该重组表达载体包括一个或多个调控序列，基于用于表达的宿主细胞进行筛选，其与该预表达的核酸序列可操作性相连。调控序列包括引导核苷酸序列在多个种类宿主细胞中组成型表达的(例如，SV40早期基因增强剂、劳斯氏肉瘤病毒启动子和细胞巨化病毒启动子)，引导仅在某些宿主细胞中核苷酸序列的表达的(例如，组织特异调控序列，参见Voss等，1986，Trends Biochem.Sci.11:287，Maniatis等，1987，Science 236:1237，其完整内容以参考形式并于本文)以及引导核苷酸序列响应具体处理或条件的诱导型表达的(例如，哺乳动物细胞中的金属硫堇启动子和原核和真核系统二者中的四环霉素反应(tet-sesponsive)启动子和/或链霉素反应启动子(同前))。本领域技术人员应理解表达载体的设计取决于例如用于转化的宿主细胞的选择、所需蛋白表达水平等因素。本文的表达载体可引入宿主细胞，藉此生产由本文所述核酸编码的蛋白或肽，包括融合蛋白或肽。
另一方面，本文提供可引入本文表达载体的宿主细胞。宿主细胞可为任何原核或真核细胞。原核宿主细胞包括革兰氏阴性或革兰氏阳性生物体，例如大肠杆菌或杆菌。更高级的真核细胞包括昆虫细胞、酵母细胞以及哺 乳动物源的确立细胞系。适当哺乳动物宿主细胞系的实例包括中国仓鼠卵巢(CHO)细胞或它们的衍生物例如Veggie CHO和在无血清培养基中生长的相关细胞系(参见Rasmussen等，1998，Cytotechnology 28:31)或CHO株DXB-11，其缺失DHFR(参见Urlaub等，1980，Proc.Natl.Acad,Sci.USA77:4216-20)。其它CHO细胞系包括CHO-K1(ATCC#CCL-61)、EM9(ATCC#CRL-1861)，和UV20(ATCC#CRL-1862)，其它宿主细胞包括猴肾细胞的COS-7系(ATCC#CRL-1651)(参见Gluzman等，1981，Cell 23:175)、L细胞、C127细胞、3T3细胞(ATCC CCL-163)，AM-1/D细胞(描述于美国专利序列号6210924)、HeLa细胞、BHK(ATCC CRL-10)细胞系、来源于非洲绿猴肾细胞系CV1的CV1/EBNA细胞系(ATCC CCL-70)(参见McMahan等，1991，EMBO J.10:2821)、人胚肾细胞例如293，293EBNA或MSR 293、人上皮A431细胞、人C010205细胞、其它经转化灵长动物细胞系、正常二倍体细胞、来源于初生组织体外培养物的细胞株、初移植体、HL-60、U937、HaK或Jurkat细胞。用于细菌、真菌、酵母和哺乳细胞宿主的适当克隆和表达载体描述于Pouwels等(Cloning Vectors:A Laboratory Manual,Elsevier,1985)。
可通过传统转化或转染技术将载体DNA引入原核或真核细胞中。对于稳定的哺乳动物转染而言，取决于使用的表达载体和转染技术，已知只有一小部分细胞可将外源DNA鏊合入它们的基因组中。为了鉴定和筛选这些整合子，通常将编码筛选标记(例如抗生素抗性)的基因与所关注基因一起引入宿主细胞。优选的筛选标记包括可赋予药物(如G418、潮霉素和甲氨喋呤)抗性的那些。在其它方法中可通过药物筛选鉴别包含被引入核酸的稳定转染细胞(例如，整合了筛选基因的细胞可存活，而其它细胞则死亡)。
可在提高多肽表达的条件下培养已转化细胞，可通过常规蛋白纯化方法回收多肽。一种该纯化方法描述于下文实施例。预用于本文的多肽包括基本同源的重组哺乳动物细胞表达的双特异性抗体，其基本不含污染性内源材料。适应症
本文的双特异性抗体可以用于治疗多种与非正常和恶化B细胞相关疾病和病症。因为本文的双特异性抗体通过招募T细胞攻击人CD19或人CD20阳性的B细胞起作用，所以由B细胞诱发的多种疾病均在其适用范围内，包括但不局限于以下三大类。第一类：恶性B细胞引起的白血病，此类疾病以B细胞增殖、凋亡、分化失控为特征，包括B细胞急性白血病、B细胞慢性白血病、非霍奇金淋巴瘤、毛细胞性淋巴瘤等等。这些疾病都 是潜在的本文的双特异性抗体适应症。第二类：非正常B细胞引起的自体免疫类疾病。此类疾病中非正常的B细胞丧失了正常的抗原耐受性，异常的表达针对自体分子的抗体(auto antibody),这些B细胞还可以通过释放促炎症的细胞激素和化学激素，诱发人体的验证反应。此类型的疾病有超过80种，包括了为人熟知的多发性硬化症，风湿性关节炎，系统性红斑狼疮等等，这些疾病也在本文的双特异性抗体潜在适应症的范围内。第三类：器官移植引起的慢性排异反应，此病症来源于器官接收者的免疫系统持续排斥接受的器官，选择性对B细胞进行耗竭治疗有利于缓解这种排异反应，因此也是本文的双特异性抗体的潜在适应症。治疗方法
一方面，治疗受试者的方法，包括给予治疗剂量的本文提供的双特异性抗体。本文中，术语“受试者”指哺乳动物，包括人类，可与术语“患者”交替使用。鼠源抗体或其人源化抗体可用于治疗和控制患者体内与非正常和恶化B细胞相关的疾病或症状。术语“治疗”包括减轻或预防至少一种症状或病症的其它方面，或者减轻疾病严重性，等等。本文的双特异性抗体不需要产生完全治愈的效果，或根除疾病的所有症状或表现，即可构成有效治疗剂。如相关领域所公认，作为治疗剂的药物可减少给定疾病状态的严重程度，但不需消除疾病的所有表现即可被认为是有效的治疗剂。相似地，预防给药治疗不需在预防症状出现上完全有效即可构成有效预防剂。只减少疾病的影响(例如，通过减少其症状的数量或严重度，或通过提高另一治疗效果，或通过产生另一有效作用)，或者减少受试者中疾病发生或加重的可能性就已经足够。本文的一个实施方案涉及包含以足以诱导反应具体病症严重性的指示剂高于基线水平的持续改善的量和时间给予患者抗体的方法。
如相关领域所理解，将包含本文的抗体的药用组合物以对适应症恰当的方式给予病人。药物组合物可采用任意适当技术包括但不限于肠道外、局部或吸入给药。如果是注射，可通过例如关节内、静脉内、肌肉内、损伤区内、腹膜内或皮下途径，以快速注射或连续输注给予药用组合物。可考虑例如在疾病或损伤部位局部给药，如透皮给药和埋植剂持续释放给药。吸入给药包括例如鼻腔或口腔吸入、采用喷雾剂、以气雾剂形式吸入抗体等等。其它选择包括口腔制剂包括片剂、糖浆剂或锭剂。
以包含一个或更多其它组分例如生理学可接受载体、辅料或稀释剂的组合物形式给予本文的抗体是有利的。组合物可任选额外包含一个或更多如下所述的生理学活性剂。在多个具体实施方案中，组合物包含除一个或更多本文的T细胞双特异性抗体之外的一个、两个、三个、四个、五个或六 个生理学活性剂。
在一个实施方案中，药物组合物包含本文的双特异性抗体以及一个或更多选自以下的物质：pH适合于抗体的缓冲液、抗氧化剂例如抗坏血酸、低分子量多肽(例如含少于10个氨基酸的多肽)、蛋白质、氨基酸、糖例如糊精、络合物例如EDTA、谷胱甘肽、稳定剂和辅料。根据适当工业标准，也可加入防腐剂。可使用适当辅料溶液作为稀释剂将组合物配制成冻干粉末。适当组分在所用剂量和浓度下对受者无毒。可用于药物处方组分的进一步实例见Remington's Pharmaceutical Sciences，第16版(1980)和20版(2000)，Mack Publishing Company提供医学从业者使用的试剂盒，其包括一种或更多本文的抗体以及治疗本文讨论任何病症的标签或其它说明。在一个实施方案中，试剂盒包括以上述组合物形式装在一个或多个管型瓶中的一种或多种抗体的无菌制剂。
给药剂量和频率可根据以下因素而改变：给药途径、所用具体抗体、所治疾病的性质和严重度、症状为急性还是慢性以及患者的体积和总体症状。可通过本领域熟知的方法确定适当剂量，例如在临床试验中包括剂量放大研究。
本文所述的双特异性抗体可在例如一段时间内按规律间隔给药一次或多次。对于治疗慢性症状，长期治疗通常最有效。但是，对于治疗急性症状，短期给药例如从一周至六周就已足够。通常，给予人类抗体直至患者表现出所选体征或指示剂高于基线水平的医学相关改善度为止。
本文提供的治疗方案的一个实例包括以适当剂量一周一次皮下注射抗体治疗B细胞相关病症。可持续每周或每月给予抗体直到达到所需结果例如病人症状消退。可按需要重新治疗，或者，可选择地，给予维持剂量。
本文方法和组合物的具体实施方案涉及使用例如一个本文的双特异性抗体、两个或更多本文双特异性抗体、本文的双特异性抗体和其它非本文的抗体、或者本文的双特异性抗体和体外培养的各类型细胞(例如体外培养的CIK细胞)等不同情形。在进一步的实施方案中，单独或与其它用于治疗使患者痛苦的症状的药剂组合给予抗体，这些药剂的实例包括蛋白质以及非蛋白质药物。当联合给予多种药物时，如本领域所熟知其剂量应相应调整。“联合给药”组合疗法不限于同时给药，也包括在涉及给予患者至少一种其它治疗剂的疗程中至少给予一次抗原和蛋白的治疗方案。
另一方面，本文提供制备治疗B细胞相关病症药剂的方法，其包含施用本文所说双特异性抗体与药学可接受辅料中的混合物。药剂制备方法如上所述。可以通过具有普通技能的内科医生已知有效的任意途径施用本文的双特异性抗体。外周肠胃外属于其中一种此类方法。医学文献中通常将肠 胃外施用理解为通过消毒注射器或一些其他机械装置例如注射泵向机体注射剂型。外周肠胃外可以包括静脉内、肌内、皮下和腹膜内施用途径。本文的双特异性抗体也可以进行经口、直肠、经鼻或下呼吸道途径施用，它们是非肠胃外途径。这些非肠胃外途径中，优选下呼吸道途径和经口途径。
具体实施方式
下面通过具体实施例，并结合附图，对本文的技术方案作进一步的具体说明。
本文中，若非特指，所采用的原料和设备等均可从市场购得或是本领域常用的。下述实施例中的方法，如无特别说明，均为本领域的常规方法。
步骤一：双特异性抗体的轻重链基因合成及亚克隆
编码IgG分子抗体轻重链的可变区DNA序列以及编码scFv结构域的DNA序列由南京金斯瑞生物科技有限公司合成。运用Overlapping PCR的方法，将IgG抗体轻链或者重链的可变区与scFv结构域DNA序列通过多肽接头DNA序列连接，形成的完整的编码融合后轻重链的DNA序列两端通过设计酶切位点Nhe1和Not1的，连接到已装入人轻链或者重链恒定区表达载体PTM5中。上述DNA序列的完整性通过测序验证后，该PTM5质粒可与对应的未融合轻链或者重链质粒组合用于转染。
步骤二：在悬浮HEK293或者CHO宿主细胞系中瞬时表达双特异性抗体
接种悬浮HEK293或者CHO表达细胞系至转瓶中，经过24小时,37℃旋转培养后被用于转染。转染过程中使用Polyethylenimine(PEI)作为转染介质，将其与DNA混合后加入到细胞培养中。PEI和DNA的混合优选配比为1:1至5:1。细胞在接受PEI与DNA混合物后继续37℃旋转培养96小时以上以表达抗原结合蛋白，其间向细胞培养中加入0.5％的胰蛋白胨作为表达需要的氨基酸源，最后收集细胞上清用于双特异性抗体的纯化分离。
步骤三：从细胞培养上清中的纯化和制备双特异性抗体
将细胞培养离心去除其中细胞，其上清经过偶联蛋白A配基的亲和层析柱后，用pH2.5-3.5的洗脱液将表达的双特异性抗体从层析柱上洗脱。洗脱管中预置中和缓冲液及时中和洗脱液的低pH值。洗脱后收集的蛋白质溶液对PBS透析。
步骤四：流式细胞仪分析鉴定(FACS)双特异性抗体与人B淋巴细胞瘤Raji细胞的结合
收集10 5个Raji细胞，加入1.5mL EP管，离心后弃上清，阴性对照样本用流式上样缓冲液(PBS，2％FBS)重悬。阳性处理组细胞每管加200μl， 共1μg抗体上清，室温孵育；1500rpm离心，弃上清，用流式上样缓冲液洗一次，再离心，重悬，每孔加入1:50稀释的FITC标记的羊抗人荧光抗体(BD Pharmingen)200μl，室温避光孵育30分钟(min)；离心，弃上清，再用流式上样缓冲液洗一次，离心，最后用流式上样缓冲液重悬，上机检测。
步骤五：通过酶联免疫反应检测双特异性抗体与CIK细胞的结合将10 5个/孔的CIK细胞除去细胞培养上清，PBS洗两遍，加入到96孔板中，100％甲醇(100μL)4℃固定10min。再加入100μl新鲜配制的0.6％H 2O 2-PBS，室温处理20min，PBS洗涤2遍。经过PBS-1％BSA封闭后，加入T细胞双特异性抗体100μl(3.3μg/孔，1.1μg/孔，0.33μg/孔，0.11μg/孔，0.033μg/孔)，37℃孵育90min。多次洗涤后，每孔加入1：5000稀释的GxH-HRP二抗(Sigma，市售)100μl，阳性对照组每孔加入1：5000稀释的GxM-HRP二抗(Sigma，市售)100μl，37℃孵育30min。洗涤5次后,每孔加入100μl TMB显色底物，37℃反应15min，加入50ul 2M H 2SO 4终止，读取OD450值。阳性对照为OKT3(Abcam，市售)；阴性对照为PBS。
步骤六：PBMC细胞的分离
取新鲜抗凝血，2000rpm离心10min，留上清自体血浆，56℃水浴45分钟后3000rmp 20分钟去沉淀置4℃待用。补加同等体积PBS于剩余细胞液中，轻轻吹打混匀后小心的加在淋巴细胞分离液上(不能将分离层打破，红细胞夜：分离液＝5:3)，600g离心20分钟。细胞液分为4层，吸取其中的白膜层置于50mL的离心管中，加入PBS至50mL,混匀后离心，1800rpm6分钟，去上清，重复洗涤2遍。根据实验需要用适当PBS重悬细胞沉淀后即可进行计数等后续实验。
步骤七：CIK细胞的培养
用CIK细胞启动培养液(无血清X-Vivo细胞培养液+1000U/mL IFN-γ)将每份新分离的PBMC细胞补满30mL，加到75cm 2培养瓶中，置于饱和湿度、37℃、5.0％CO 2培养箱培养。培养24小时后，加入CIK细胞刺激因子混合液1mL(无血清X-Vivo细胞培养液+50ng/mL CD3单克隆抗体、300U/mL IL-2、100U/mL IL-1α)，继续置于饱和湿度、37℃、5.0％CO 2培养箱内培养。接下来的步骤根据CIK细胞的生长情况决定补液(无血清X-Vivo培养液+300U/mL IL-2±2％自体血浆)、分瓶的事宜，基本上要维持细胞在1-2×10 6的浓度左右生长。最后用流式细胞仪FC500对收集的CIK细胞进行表型检测，包括：CD3，CD4，检测这些细胞表面抗原在CIK细胞的表达情况。
步骤八：双特异性抗体介导的CIK或者PBMC细胞杀伤肿瘤细胞的检 测
收集Raji单细胞悬液于无菌1.5mL EP管中，1000rpm，4min离心去上清，1mL PBS洗一遍，加终浓度为100nM的Calcein AM(货号C1430，ThermoFisher)1mL，37℃细胞培养箱孵育30min后，离心去上清，1mL PBS洗一遍，再加2.5mL完全培养基重悬，50μL/well(即2×10 4cell/well)加至96孔细胞培养板中培养过夜。按实验设计的效靶比加入培养的CIK或PBMC细胞，100μL/孔，设置对照孔，无需加入CIK或PBMC细胞的孔则用相同体积的培养基补入。加入杀伤细胞的同时按实验设计加入相应抗体，50μL/孔，无需加入抗体的孔则用相同体积的培养基补入，继续共培养。根据实验的选择，8h至48h(时间因需而定)内取出96孔板，将每孔细胞均转入96孔U型板，1200rpm，3min离心去上清，加200μL 2％FBS-PBS重悬，在流式细胞仪Guava 6HT上读取数据。每孔读取50000个细胞，计算Calcein AM阳性细胞的个数及其与阴性对照的百分比。
步骤九：双特异性抗体对人B淋巴细胞瘤Raji细胞原位异种移植肿瘤模型的体内药效学研究
将1×10 5个/0.2mL Raji细胞通过尾静脉给予每只NOD/SCID小鼠，共接种16只。待肿瘤细胞适应48h后开始分组给药。实验分为三个组开始，以每两天一次注射共给药10次，通过静脉注射，对照组一每只小鼠给予0.2mL的PBS，对照组二每只小鼠给予1×10 7个CIK细胞，体积0.2mL，实验组每只小鼠给予1×10 7个CIK细胞、0.25nmol的αCD19(L6H6)/αCD3(L2H2)、或者2.5nmol的αCD19(L6H6)/αCD3(L2H2)双特异性抗体，体积0.2mL。每天监测动物的健康状况及死亡情况，例行检查包括观察肿瘤细胞对动物日常行为表现的影响如毛发，行为活动(瘫痪等)，摄食摄水量，体重变化(前期每周测量2次体重，后期隔天或者每天测量)，外观体征或其它不正常情况。实验指标是考察肿瘤细胞在动物体内的生长情况。定义接种后到小鼠死亡的天数为其生存期。记录每只小鼠的生存期，做Kaplan-Meier生存曲线并算出每组的生存中位数(Median Survival Time,MST)，以及给药组相对对照组所延长的生存期(Increase in Life Span,ILS)。
以上所述的实施例只是本文的一种较佳的方案，并非对本文作任何形式上的限制，在不超出权利要求所记载的技术方案的前提下还有其它的变体及改型。
以上实施列的结果显示：本文的双特异性抗体，以αCD19(L6H6,其包含SEQ NO ID:21与SEQ NO ID:22)/αCD3(L2H2,其包含SEQ NO ID:13与SEQ NO ID:14)为例，能够顺利在哺乳细胞表达系统如HEK293或CHO中表达，分子量大小符合预期，无明显的降解(图1)。这些双特异性抗体具 有与人CD3和人CD19或人CD20两者之一结合的能力，图2(实验步骤四)同样通过以αCD19/αCD3为例，显示了本专利的双特异性抗体能与Raji细胞结合的流式细胞仪的荧光显色数据，Raji细胞是一个人B细胞淋巴瘤细胞株，其细胞表面高表达了人CD19分子。数据显示了αCD19/αCD3双特异性抗体和Raji细胞表面结合能力与对照αCD19单抗非常类似，因此该双特异性抗体的分子构造不会影响其αCD19抗体部分的结合能力。然后我们通过实验步骤五提供的方法，检测了αCD19/αCD3双特异性抗体与CIK细胞结合的酶联免疫反应的数据。CIK细胞为细胞激素诱导的杀伤型T细胞，其诱导和扩培如实验步骤六中所述，其表面高表达了人CD3分子。数据显示，10μg的αCD19/αCD3双特异性抗体与CIK细胞的结合数值为0.47/0.06(结合数据/阴性对照)，阳性对照0.4μg的OKT3的结合数值为0.36/0.06(结合数据/阴性对照)。以上结合结果显示了本专利的双特异性抗体分子针对两个不同抗原亲和力。然后通过针对Raji细胞的T细胞毒性实验(实验步骤八)，显示以不同E:T(效应细胞对靶细胞的比例)将CIK细胞与Raji细胞混合培养，加入不同浓度本文提供的αCD19/αCD3的双特异性抗体后CIK细胞将对Raji细胞造成杀伤效果。在E:T为10：1的情况下，双特异性抗体的半杀伤浓度约为1ng/mL(图3)。在接下来的实验中，在一个体内药效实验中(实验步骤十)，Raji细胞通过原位注射接种于NOD/SCID小鼠体内，以此小鼠模型检测αCD19/αCD3双特异性抗体的抗肿瘤生长的能力。生存周期数据(图4)显示同时注射不同浓度的αCD19/αCD3双特异性抗体和CIK细胞显著性得延长了小鼠的生存周期，超过了阴性对照组和单独CIK治疗组，显示了αCD19/αCD3双特异性抗体和CIK细胞的组合治疗能够显著抑制Raji细胞的在小鼠体内的生长速率。综上所述，本文的双特异型抗体分子构型稳定，并在体外和体内均显示了优越的T细胞依赖性的针对B细胞的细胞毒性。

Claims (27)

  1. 一种能够与人CD19或人CD20和人CD3结合的双特异性抗体，其结构特征在于：所述的双特异性抗体包含单链抗体结构域(scFv结构域)、免疫球蛋白G结构域(IgG)和结构域间肽接头序列(Link 1)，这三者通过以下方式中的一种融合形成所述的双特异性抗体：
    a.通过结构域间肽接头序列将scFv的羧基端和IgG轻链的氨基端连接：N’-scFv-Link 1-V L-C L-C’；
    b.通过结构域间肽接头序列将scFv的氨基端和IgG轻链的羧基端连接：N’-V L-C L-Link 1-scFv-C’；
    c.通过结构域间肽接头序列将scFv的羧基端和IgG重链的氨基端连接：N’-scFv-Link 1-V H-C H1-C H2-C H3-C’；及
    d.通过结构域间肽接头序列将scFv的氨基端和IgG重链的羧基端连接：N’-V H-C H1-C H2-C H3-Link 1-scFv-C’；
    其中:N’代表多肽链的氨基端,C’代表多肽链的羧基端,scFv代表单链抗体结构域，IgG代表免疫球蛋白G结构域,V L代表免疫球蛋白G结构域的轻链可变区,C L代表免疫球蛋白G结构域的轻链恒定区,V H代表免疫球蛋白G结构域的重链可变区,C H1代表免疫球蛋白G结构域的重链恒定区1,C H2代表免疫球蛋白G结构域的重链恒定区2,C H3代表免疫球蛋白G结构域的重链恒定区3,及Link 1代表结构域间肽接头。
  2. 根据权力要求1所述的双特异性抗体，其中所述的结构域间肽接头(Link 1)的序列选自以下序列之一：SEQ NO ID:1、SEQ NO ID:2、SEQ NO ID:3、SEQ NO ID:4、及SEQ NO ID:5。
  3. 根据权力要求1或2所述的双特异性抗体，其中所述的scFv结构域包含轻链可变区、重链可变区和结构域内肽接头序列，这三者通过以下方式中的一种组合形成scFv结构域：
    a.通过结构域内肽接头序列将轻链可变区的羧基端和重链可变区的氨基端连接：N’-V SL-Link 2-V SH-C’；及
    b.通过结构域内肽接头序列将重链可变区的羧基端和轻链可变区的氨基端连接：N’-V SH-Link 2-V SL-C’；
    其中:N’代表多肽链的氨基端,C’代表多肽链的羧基端,V SL代表scFv结构域的轻链可变区,V SH代表scFv结构域的重链可变区,及Link 2代表结构域内肽接头。
  4. 根据权力要求3所述的双特异性抗体，其中所述结构域间内肽接头(Link 2)的序列选自以下序列之一：SEQ NO ID:6、SEQ NO ID:7、SEQ NO ID:8、 SEQ NO ID:9、及SEQ NO ID:10。
  5. 根据权力要求1至4中任一项的双特异性抗体，其中所述scFv结构域包含以下方案之一的氨基酸序列：
    a.scFv结构域的轻链可变区序列选自以下序列之一：SEQ NO ID:11、SEQ NO ID:13、及SEQ NO ID:32；
    b.scFv结构域的重链可变区序列选自以下序列之一：SEQ NO ID:12、SEQ NO ID:14、SEQ NO ID:33、及SEQ NO ID:34；及
    c.(a)的轻链可变区序列和(b)的重链可变区序列的组合。
  6. 根据权力要求5所述的双特异性抗体，其中所述抗体的scFv结构域包含以下方案之一的氨基酸序列组合方式：SEQ NO ID:11与SEQ NO ID:12的组合(L1H1)、SEQ NO ID:13与SEQ NO ID:14的组合(L2H2)、SEQ NOID:32与SEQ NO ID:33的组合(L9H9)、及SEQ NO ID:32与SEQ NO ID:34的组合(L9H10)。
  7. 根据权力要求1至6中任一项所述的双特异性抗体，其中所述的抗体的IgG结构域包含以下方案之一的氨基酸序列：
    a.IgG结构域的轻链可变区序列选自以下序列之一：SEQ NO ID:15、SEQ NO ID:17、SEQ NO ID:19、SEQ NO ID:21、SEQ NO ID:23、及SEQ NO ID:25；
    b.IgG结构域的重链可变区序列选自以下序列之一：SEQ NO ID:16、SEQ NO ID:18、SEQ NO ID:20、SEQ NO ID:22、SEQ NO ID:24、及SEQ NO ID:26；及
    c.(a)的轻链可变区序列和(b)的重链可变区序列的组合。
  8. 根据权力要求7所述的双特异性抗体，其中所述抗体的IgG结构域包含以下方案之一的氨基酸序列组合方式：SEQ NO ID:15与SEQ NO ID:16的组合(L3H3)、SEQ NO ID:17与SEQ NO ID:18的组合(L4H4)、SEQ NOID:19与SEQ NO ID:20的组合(L5H5)、SEQ NO ID:21与SEQ NO ID:22的组合(L6H6)、SEQ NO ID:23与SEQ NO ID:24的组合(L7H7)、及SEQNO ID:25与SEQ NO ID:26的组合(L8H8)。
  9. 根据权力要求1至4中任一项的双特异性抗体，其中所述scFv结构域包含以下方案之一的氨基酸序列：
    a.scFv结构域的轻链可变区序列选自以下序列之一：SEQ NO ID:15、SEQ NO ID:17、SEQ NO ID:19、SEQ NO ID:21、SEQ NO ID:23、及SEQ NO ID:25；
    b.scFv结构域的重链可变区序列选自以下序列之一：SEQ NO ID:16、SEQ NO ID:18、SEQ NO ID:20、SEQ NO ID:22、SEQ NO ID:24、 及SEQ NO ID:26；及
    c.(a)的轻链可变区序列和(b)的重链可变区序列的组合。
  10. 根据权力要求9所述的双特异性抗体，其中所述抗体的scFv结构域包含以下方案之一的氨基酸序列组合方式：SEQ NO ID:15与SEQ NO ID:16的组合(L3H3)、SEQ NO ID:17与SEQ NO ID:18的组合(L4H4)、SEQ NOID:19与SEQ NO ID:20的组合(L5H5)、SEQ NO ID:21与SEQ NO ID:22的组合(L6H6)、SEQ NO ID:23与SEQ NO ID:24的组合(L7H7)、及SEQNO ID:25与SEQ NO ID:26的组合(L8H8)。
  11. 根据权力要求1至4,9,及10中任一项所述的双特异性抗体，其中所述的抗体的IgG结构域包含以下方案之一的氨基酸序列：
    a.IgG结构域的轻链可变区序列选自以下序列之一：SEQ NO ID:11、SEQ NO ID:13、及SEQ NO ID:32；
    b.IgG结构域的重链可变区序列选自以下序列之一：SEQ NO ID:12、SEQ NO ID:14、SEQ NO ID:33、及SEQ NO ID:34；及
    c.(a)的轻链可变区序列和(b)的重链可变区序列的组合。
  12. 根据权力要求11所述的双特异性抗体，其中所述抗体的IgG结构域包含以下方案之一的氨基酸序列组合方式：SEQ NO ID:11与SEQ NO ID:12的组合(L1H1)、SEQ NO ID:13与SEQ NO ID:14的组合(L2H2)、SEQ NOID:32与SEQ NO ID:33的组合(L9H9)、及SEQ NO ID:32与SEQ NO ID:34的组合(L9H10)。
  13. 根据权力要求7,8,11,及12中任一项所述的双特异性抗体，其中所述的抗体蛋白质的IgG分子进一步包含以下方案之一的氨基酸序列：
    a.选自以下序列之一的轻链恒定区氨基酸序列：SEQ NO ID:27及SEQNO ID:28；
    b.选自以下序列之一的重链恒定区氨基酸序列：SEQ NO ID:29、SEQNO ID:30、及SEQ NO ID:31；及
    c.(a)的轻链恒定区序列和(b)的重链恒定区序列的组合。
  14. 根据权力要求1至13中任一项所述的双特异性抗体，其中所述的双特异性抗体是一种能够与人CD19和人CD3结合的双特异性抗体。
  15. 根据权力要求1至13中任一项所述的双特异性抗体，其中所述的双特异性抗体是一种能够与人CD20和人CD3结合的双特异性抗体。
  16. 根据权力要求1至15中任一项所述的双特异性抗体，其中所述的双特异性抗体是鼠源抗体、人源化抗体、嵌合抗体、单克隆抗体、重组抗体、IgGl抗体、IgG2抗体、IgG3抗体、或IgG4抗体。
  17. 一种多核苷酸，其编码权力要求1至16中任一项所述的双特异性抗体。
  18. 一种载体，其包含权力要求17所述的多核苷酸。
  19. 一种宿主细胞，其包含权力要求18所述的载体。
  20. 一种药用组合物，其包含与药用可接受载体混合的权力要求1至16中任一项所述的双特异性抗体。
  21. 权力要求1至16中任一项所述的双特异性抗体在制备用于预防或治疗B细胞性白血病的药物中的用途。
  22. 根据权力要求21所述的用途,其中所述的白血病是急性白血病。
  23. 根据权力要求21所述的用途,其中所述的白血病是慢性白血病。
  24. 权力要求1至16中任一项所述的双特异性抗体在制备用于预防或治疗非霍奇金淋巴瘤的药物中的用途。
  25. 权力要求1至16中任一项所述的双特异性抗体在制备用于预防或治疗B细胞引起的自体免疫疾病的药物中的用途。
  26. 根据权力要求25所述的用途,其中所述的自体免疫疾病是风湿性关节炎、多发性硬化症、或系统性红斑狼疮。
  27. 权力要求1至16中任一项所述的双特异性抗体在制备用于预防或治疗器官移植带来的排异反应及其相关症状的药物中的用途。
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020135335A1 (en) * 2018-12-24 2020-07-02 Generon (Shanghai) Corporation Ltd. Multispecific antigen binding proteins capable of binding cd19 and cd3, and use thereof
US10870701B2 (en) 2016-03-15 2020-12-22 Generon (Shanghai) Corporation Ltd. Multispecific fab fusion proteins and use thereof
US11013800B2 (en) 2011-05-16 2021-05-25 Evive Biotech Ltd. Multi-specific Fab fusion proteins comprising a CD3-binding Fab fragment with N-terminal fusion to binding domains and methods of use
US20220002408A1 (en) * 2019-03-19 2022-01-06 Excytellc Bispecific antibody, preparation method thereof and application thereof
WO2022165171A1 (en) 2021-01-28 2022-08-04 Regeneron Pharmaceuticals, Inc. Compositions and methods for treating cytokine release syndrome
WO2023201226A1 (en) 2022-04-11 2023-10-19 Regeneron Pharmaceuticals, Inc. Compositions and methods for universal tumor cell killing
EP4061849A4 (en) * 2019-11-21 2024-05-29 Brown University BISPECIFIC ANTIBODIES AGAINST CHI3L1 AND CTLA4 WITH IMPROVED CYTOTOXIC EFFECTS ON TUMOR CELLS
WO2024173830A2 (en) 2023-02-17 2024-08-22 Regeneron Pharmaceuticals, Inc. Induced nk cells responsive to cd3/taa bispecific antibodies
US12221481B2 (en) 2019-05-21 2025-02-11 Novartis Ag CD19 binding molecules and uses thereof

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109836502B (zh) * 2018-11-12 2021-09-07 浙江大学 一种双特异性抗体及其应用
WO2020132574A1 (en) * 2018-12-21 2020-06-25 CentryMed Pharmaceutical Inc. Protease cleavable bispecific antibodies and uses thereof
CN112111012B (zh) * 2019-06-20 2023-07-04 成都恩沐生物科技有限公司 共价多特异性抗体
CN114685658B (zh) * 2020-12-30 2024-12-17 百力司康生物医药(杭州)有限公司 Ox40的靶向抗体及其制备方法和应用
CN115490770A (zh) * 2021-06-17 2022-12-20 鸿运华宁（杭州）生物医药有限公司 一种能与人eta和人cd3结合的双特异性抗体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1867586A (zh) * 2003-10-16 2006-11-22 麦克罗梅特股份公司 多特异性的去免疫化cd3结合物
CN104356241A (zh) * 2003-08-01 2015-02-18 美国政府（由卫生和人类服务部、国立卫生研究院的部长所代表） 免疫毒素的表达和纯化方法
US20150218217A1 (en) * 2014-01-31 2015-08-06 Cytomx Therapeutics, Inc. Matriptase and u-plasminogen activator substrates and other cleavable moieties and methods of use thereof
CN105530959A (zh) * 2013-03-15 2016-04-27 纪念斯隆-凯特琳癌症中心 多聚化技术

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5859205A (en) * 1989-12-21 1999-01-12 Celltech Limited Humanised antibodies
LU91067I2 (fr) * 1991-06-14 2004-04-02 Genentech Inc Trastuzumab et ses variantes et dérivés immuno chimiques y compris les immotoxines
US6136310A (en) * 1991-07-25 2000-10-24 Idec Pharmaceuticals Corporation Recombinant anti-CD4 antibodies for human therapy
WO1994012520A1 (en) * 1992-11-20 1994-06-09 Enzon, Inc. Linker for linked fusion polypeptides
ES2426468T3 (es) * 2005-12-30 2013-10-23 Merck Patent Gmbh Anticuerpos anti-CD19 con inmunogenicidad reducida
CA2693053C (en) * 2007-05-30 2021-01-05 Xencor, Inc. Methods and compositions for inhibiting cd32b expressing cells
CN102112494A (zh) * 2008-06-03 2011-06-29 雅培制药有限公司 双重可变结构域免疫球蛋白及其用途
EP2435476A4 (en) * 2009-05-27 2013-04-17 Synageva Biopharma Corp ANTIBODIES OBTAINED FROM BIRDS
US9458246B2 (en) * 2013-03-13 2016-10-04 Amgen Inc. Proteins specific for BAFF and B7RP1
US11161906B2 (en) * 2013-07-25 2021-11-02 Cytomx Therapeutics, Inc. Multispecific antibodies, multispecific activatable antibodies and methods of using the same
CN104558191B (zh) * 2015-01-21 2020-08-21 武汉友芝友生物制药有限公司 一种双特异性抗体cd20×cd3的构建及应用
CN104592393B (zh) * 2015-01-21 2018-09-28 武汉友芝友生物制药有限公司 一种双特异性抗体cd19×cd3的构建及应用
WO2016154621A1 (en) * 2015-03-26 2016-09-29 The California Institute For Biomedical Research SWITCHABLE NON-scFv CHIMERIC RECEPTORS, SWITCHES, AND USES THEREOF

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104356241A (zh) * 2003-08-01 2015-02-18 美国政府（由卫生和人类服务部、国立卫生研究院的部长所代表） 免疫毒素的表达和纯化方法
CN1867586A (zh) * 2003-10-16 2006-11-22 麦克罗梅特股份公司 多特异性的去免疫化cd3结合物
CN105530959A (zh) * 2013-03-15 2016-04-27 纪念斯隆-凯特琳癌症中心 多聚化技术
US20150218217A1 (en) * 2014-01-31 2015-08-06 Cytomx Therapeutics, Inc. Matriptase and u-plasminogen activator substrates and other cleavable moieties and methods of use thereof

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11013800B2 (en) 2011-05-16 2021-05-25 Evive Biotech Ltd. Multi-specific Fab fusion proteins comprising a CD3-binding Fab fragment with N-terminal fusion to binding domains and methods of use
US10870701B2 (en) 2016-03-15 2020-12-22 Generon (Shanghai) Corporation Ltd. Multispecific fab fusion proteins and use thereof
WO2020135335A1 (en) * 2018-12-24 2020-07-02 Generon (Shanghai) Corporation Ltd. Multispecific antigen binding proteins capable of binding cd19 and cd3, and use thereof
US20220002408A1 (en) * 2019-03-19 2022-01-06 Excytellc Bispecific antibody, preparation method thereof and application thereof
EP3882276A4 (en) * 2019-03-19 2022-03-23 Excyte LLC BISPECIFIC ANTIBODY, METHOD OF PRODUCTION THEREOF AND USE THEREOF
US12221481B2 (en) 2019-05-21 2025-02-11 Novartis Ag CD19 binding molecules and uses thereof
EP4061849A4 (en) * 2019-11-21 2024-05-29 Brown University BISPECIFIC ANTIBODIES AGAINST CHI3L1 AND CTLA4 WITH IMPROVED CYTOTOXIC EFFECTS ON TUMOR CELLS
WO2022165171A1 (en) 2021-01-28 2022-08-04 Regeneron Pharmaceuticals, Inc. Compositions and methods for treating cytokine release syndrome
US12274747B2 (en) 2021-01-28 2025-04-15 Regeneron Pharmaceuticals, Inc. Compositions and methods for treating cytokine release syndrome
EP4538292A2 (en) 2021-01-28 2025-04-16 Regeneron Pharmaceuticals, Inc. Compositions and methods for treating cytokine release syndrome
WO2023201226A1 (en) 2022-04-11 2023-10-19 Regeneron Pharmaceuticals, Inc. Compositions and methods for universal tumor cell killing
WO2024173830A2 (en) 2023-02-17 2024-08-22 Regeneron Pharmaceuticals, Inc. Induced nk cells responsive to cd3/taa bispecific antibodies

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