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WO2018188612A1 - Anticorps bispécifique pouvant être combiné avec cd19 humain ou cd20 humain et cd3 humain, et applications associées - Google Patents

Anticorps bispécifique pouvant être combiné avec cd19 humain ou cd20 humain et cd3 humain, et applications associées 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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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

L'invention concerne un anticorps bispécifique pouvant être combiné avec CD19 humain ou CD20 humain et CD3 humain, destiné à être utilisé dans le traitement de maladies associées à la détérioration de cellules B.
PCT/CN2018/082692 2017-04-12 2018-04-11 Anticorps bispécifique pouvant être combiné avec cd19 humain ou cd20 humain et cd3 humain, et applications associées WO2018188612A1 (fr)

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