WO2020047818A1 - 双特异性抗体及其使用方法 - Google Patents

双特异性抗体及其使用方法 Download PDF

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WO2020047818A1
WO2020047818A1 PCT/CN2018/104518 CN2018104518W WO2020047818A1 WO 2020047818 A1 WO2020047818 A1 WO 2020047818A1 CN 2018104518 W CN2018104518 W CN 2018104518W WO 2020047818 A1 WO2020047818 A1 WO 2020047818A1
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monoclonal antibody
cancer
bispecific antibody
antibody
variable region
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PCT/CN2018/104518
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English (en)
French (fr)
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季匡华
王愈善
江欣倩
黄逸君
周詹姆斯
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广西慧宝源健康产业有限公司
强普生技股份有限公司
桂林商源植物制品有限公司
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Publication of WO2020047818A1 publication Critical patent/WO2020047818A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention belongs to the field of antibody engineering, and in particular, the present invention relates to a bispecific antibody and application thereof.
  • the bispecific antibody is excellent in effect, and in particular, it is excellent in the effect of persistently maintaining the affinity of T cells.
  • Antibodies are also called immunoglobulins (Ig).
  • Natural antibodies contain four heterologous polypeptide chains, of which two chains with larger molecular weights are called heavy chains (HC) and two chains with smaller molecular weights are called light chains (LC).
  • the regions in the light and heavy chains that have large changes in the amino acid sequence near the N-terminus are called variable regions, that is, the light chain variable region (VL) and heavy chain variable region (VH), which account for 1 / 4 and 1/2, VL and VH each include three complementary determining regions (CDRs), namely VL-CDR1, VL-CDR2 and VL-CDR3, and VH-CDR1, VH-CDR2 and VH-CDR3; they will be close to C
  • the region where the amino acid sequence at the end is relatively stable is called the constant region, in which the heavy chain constant distinguishes three segments, which are the heavy chain constant region 1 (CH1), the heavy chain constant region 2 (CH2), and the heavy chain constant region 3 (CH
  • bispecific antibodies Natural monoclonal antibodies specifically bind to one antigen target, while bispecific antibodies (BsAb) are antibody fusions that can specifically bind to two different antigen targets.
  • BsAb bispecific antibodies
  • Chinese patent application CN104892765A discloses a bispecific antibody against CD3 antigen and Her-2 antigen, including two identical light chain sequences and two different heavy chain sequences, which can respectively recognize Her on the surface of breast cancer cells.
  • -2 antigen and CD3 antigen on the surface of T cells can significantly enhance the activity of antibody-mediated T cells to kill tumor target cells;
  • Chinese patent application CN106831996A provides a novel bispecific antibody comprising an antigen-binding portion against human CD3E and / or an antigen-binding portion against HER2;
  • Cisoka HER2XCD3 which is composed of a monovalent unit and a single chain unit, wherein the monovalent unit has specific binding ability to the surface antigen CD3 of immune cells, and the single chain unit has the tumor surface antigen HER2. Specific binding ability, and the single-stranded unit comprises a single-chain variable fragment fused to an Fc fragment;
  • Chinese patent application CN106632681A discloses anti-EGFR and anti-CD3 bispecific antibodies, which can specifically bind tumor cell surface antigen epidermal growth factor receptor and immune cell surface antigen differentiation cluster 3, wherein the single-chain antibody ScFv of the anti-CD3 antibody is located in the anti-EGFR The C-terminus of the constant region of the antibody;
  • Cisoka patent application CN104774268A discloses a bispecific antibody EGFR ⁇ CD3, which is composed of a monovalent unit and a single chain unit, wherein the monovalent unit has specific binding ability against the surface antigen CD3 of immune cells, and the single chain unit against the surface antigen of tumor cells EGFR has specific binding ability, and the single-stranded unit includes a single-stranded variable fragment fused to an Fc fragment;
  • Chinese patent application CN104829729A discloses an anti-Her2 / CD3 bispecific antibody, which includes a first antibody segment and a second antibody segment, the first antibody segment is an anti-tumor antigen antibody segment, and the second antibody segment is an anti-human CD3
  • the molecular antibody segment utilizes adoptively transferred T cells to carry and continuously express the antibody protein in the body, so that the bispecific antibody exerts a killing effect in the body, while accompanied by a sufficient number of T effector cells, which optimizes the effect of the effect. effectiveness.
  • the prior art does not suggest obtaining a bispecific antibody of the present invention that is specifically tandemly linked including a transmembrane domain.
  • the bispecific antibodies of the present invention designed by the inventors have excellent effects, especially the effect of being able to maintain adhesion to T cells for a long time, which is more beneficial to practical applications.
  • the invention provides novel bispecific antibodies and intermediate products of the recombinant DNA technology, preparation methods, uses in treatment and the like.
  • the present invention provides a bispecific antibody having a structure of the following formula,
  • VH is the heavy chain variable region of the first monoclonal antibody
  • VL is the light chain variable region of the first monoclonal antibody
  • CH1 is the heavy chain constant region 1 of the first monoclonal antibody
  • CK is the light chain constant region of the first monoclonal antibody, in which CH1 and CK are connected by a disulfide bond (
  • VH is the heavy chain variable region of the second monoclonal antibody
  • VL ' is the light chain variable region of a second monoclonal antibody
  • TM is a transmembrane protein domain
  • the antigenic targets targeted by the first monoclonal antibody and the second monoclonal antibody are different.
  • the bispecific antibody of the first aspect of the present invention preferably, one of the first monoclonal antibody and the second monoclonal antibody specifically binds to T cells.
  • a CD3 antigen that specifically binds to T cells is more preferable.
  • the other of the first monoclonal antibody and the second monoclonal antibody specifically binds to a pathogen. More preferably, the other of the first monoclonal antibody and the second monoclonal antibody specifically bind to a tumor or cancer cell, such as a specific antigen or an overexpressing antigen of the tumor or cancer cell, such as an epidermal growth factor receptor, a carcinoembryonic antigen Or prostate-specific membrane antigen.
  • a tumor or cancer cell such as a specific antigen or an overexpressing antigen of the tumor or cancer cell, such as an epidermal growth factor receptor, a carcinoembryonic antigen Or prostate-specific membrane antigen.
  • the transmembrane protein domain is a transmembrane domain of a leukocyte differentiation antigen, and may be, for example, a transmembrane domain of a cell adhesion molecule-1, CD48, or CD80. More preferably, the amino acid sequence of the transmembrane protein domain is as shown in SEQ ID NO: 1, 2 or 3.
  • VL or VL ' comprises an amino acid sequence such as the light chain variable region or amino acid sequence in a fusion peptide shown in SEQ ID NO: 4, 5, 6, or 11 ID NO: VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region in the fusion peptide shown in 10, 13, 14, or 15.
  • VH or VH comprises an amino acid sequence such as the heavy chain variable region or amino acid sequence in a fusion peptide shown in SEQ ID NO: 7, 8, 9, or 12 SEQ ID NO: VH-CDR1, VH-CDR2, and VH-CDR3 in the heavy chain variable region in the fusion peptide shown in 10, 13, 14, or 15.
  • VL-CK amino acid sequence of VL-CK is shown in SEQ ID NO: 4, 5, 6, or 11, wherein the first three are specifically binding epidermal growth factor receptor, carcinoembryonic antigen, and prostate-specific VL-CK of a monoclonal antibody to a membrane antigen; the latter is a VL-CK of a monoclonal antibody that specifically binds to the CD3 antigen of T cells.
  • VH-CH1 is shown in SEQ ID NO: 7, 8, 9 or 12, wherein the first three are specifically binding epidermal growth factor receptor, carcinoembryonic antigen and prostate specific VH-CH1 of a monoclonal antibody against a membrane antigen; the latter is VH-CH1 of a monoclonal antibody that specifically binds to the CD3 antigen of T cells.
  • VH'-VL ' is shown as SEQ ID NO: 10, 13, 14 or 15, wherein the former is a VH'- of a monoclonal antibody that specifically binds to the CD3 antigen of T cells.
  • VL '; the latter three are VH'-VL' of monoclonal antibodies that specifically bind epidermal growth factor receptor, carcinoembryonic antigen, and prostate specific membrane antigen, respectively.
  • the invention provides an isolated nucleic acid encoding a bispecific antibody of the first aspect of the invention.
  • the invention provides a vector comprising a nucleic acid of the second aspect of the invention.
  • the vector of the third aspect of the invention is a plasmid.
  • the invention provides a host cell comprising the vector of the third aspect of the invention or transfected with a vector of the third aspect of the invention.
  • the host cell of the fourth aspect of the invention is a mammalian cell.
  • the present invention provides a method for producing a bispecific antibody of the first aspect of the present invention, which comprises the following steps:
  • step (b) collecting the bispecific antibody of the first aspect of the present invention from the culture obtained in step (a).
  • the invention provides a pharmaceutical composition comprising the bispecific antibody of the first aspect of the invention and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition of the sixth aspect of the present invention is used to treat a disease treated by the first monoclonal antibody or the second monoclonal antibody, such as a tumor or cancer.
  • the present invention provides the use of the bispecific antibody of the first aspect of the present invention in the manufacture of a medicament for treating a disease treated by a first monoclonal antibody or a second monoclonal antibody; accordingly, the present invention also provides A method of treating a disease treated by a first monoclonal antibody or a second monoclonal antibody includes administering to a subject in need thereof an effective amount of a bispecific antibody of the first aspect of the invention.
  • the disease treated by the first monoclonal antibody or the second monoclonal antibody is a tumor or cancer.
  • the tumor or cancer is selected from the group consisting of colorectal cancer, rectal cancer, throat cancer, head and neck cancer, lung cancer, gastric cancer, breast cancer, pancreatic cancer, cervical cancer, ovarian cancer, prostate cancer, and prostate cancer.
  • FIG. 1 is a schematic diagram of the construct of the bispecific antibody A of Example 1.
  • FIG. 1 is a schematic diagram of the construct of the bispecific antibody A of Example 1.
  • FIG. 2 is a schematic structural diagram of the bispecific antibody A of Example 1.
  • FIG. 2 is a schematic structural diagram of the bispecific antibody A of Example 1.
  • FIG. 3 is a schematic diagram of a construct of the bispecific antibody B of Example 1.
  • FIG. 3 is a schematic diagram of a construct of the bispecific antibody B of Example 1.
  • FIG. 4 is a graph of an experimental result of a T-cell adhesion time test in Example 1.
  • FIG. 5 is a schematic diagram of the construct of the bispecific antibody C of Example 2.
  • FIG. 6 is a schematic structural diagram of a bispecific antibody C of Example 2.
  • FIG. 7 is a schematic diagram of a construct of the bispecific antibody D of Example 2.
  • FIG. 8 is a graph showing an experimental result of a T cell adhesion time test in Example 2.
  • the bispecific antibody of the present invention is composed of two peptide chains, including a first peptide chain and a second peptide chain, wherein the structure of the first peptide chain is shown as VL-CK-TM, and the structure of the second peptide chain is shown as VH- CH1-VH'-VL ', where VH is the heavy chain variable region of the first monoclonal antibody, VL is the light chain variable region of the first monoclonal antibody, and CH1 is the heavy chain of the first monoclonal antibody Constant region 1, CK is the light chain constant region of the first monoclonal antibody, VH 'is the heavy chain variable region of the second monoclonal antibody, VL' is the light chain variable region of the second monoclonal antibody, and TM is a cross Membrane protein domain, and-indicates a direct peptide bond or a linker peptide.
  • the purpose is to distinguish the modified product, that is, the first product and the second product are different, but not to the structure of the product itself and / or Composition composition is limited.
  • the first monoclonal antibody and the second monoclonal antibody are different monoclonal antibodies.
  • a monoclonal antibody consists of a pair of light chains and a pair of heavy chains, and the four chains are connected to each other by disulfide bonds.
  • each heavy chain usually includes a heavy chain variable region and a heavy chain constant region.
  • the heavy chain constant region typically contains three domains, namely CH1, CH2, and CH3; each light chain typically contains a light chain variable region and a light chain constant region.
  • Each variable region typically contains three complementary determining regions, which are highly formally variable regions of sequence and / or structurally defined loops that are separated by four more conserved framework regions (FR), namely Each variable region is arranged from amino terminal to carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the CDR sequences in this paper can be determined according to IMGT rules (Brochet X., Nucleic Acids Research, 2008, 36: W503-508 and Lefranc MP., Nucleic Acids Research, 1999, 27: 209-212), or according to Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD. 1991). The Kabat numbering of residues can be determined for a given antibody variable region.
  • one of the first monoclonal antibody and the second monoclonal antibody specifically binds to T cells, such as a CD3 antigen that specifically binds to T cells; and the first monoclonal antibody and the first Another specific binding agent in the two monoclonal antibodies, preferably specifically binding to a tumor or cancer cell, such as a tumor or cancer cell specific antigen or an overexpressing antigen, such as epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA) Or prostate specific membrane antigen (PSMA).
  • EGFR epidermal growth factor receptor
  • CEA carcinoembryonic antigen
  • PSMA prostate specific membrane antigen
  • the medicinal use of the bispecific antibody of the first aspect of the present invention is mainly determined by a monoclonal antibody fragment that specifically binds to a pathogen, and another monoclonal antibody fragment that specifically binds to T cells has the effect of persistently maintaining adhesion to T cells, thereby promoting Corresponding medicinal effect.
  • the VL comprises a light chain variable region or an amino acid sequence in a fusion peptide shown in SEQ ID NO: 4, 5 or 6 as shown in SEQ ID NO: 13, 14 or 15
  • the amino acid sequence is VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region in the fusion peptide shown in SEQ ID NO: 10; and vice versa
  • VL contains the amino acid sequence as shown in SEQ ID NO: 11
  • the light chain variable region or amino acid sequence of the fusion peptide shown is as follows: VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region of the fusion peptide shown in SEQ ID NO: 10, then VLL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region of the fusion peptide shown in SEQ ID NO: 10, then VL
  • VH comprises the heavy chain variable region or amino acid sequence in the fusion peptide shown in SEQ ID NO: 7, 8 or 9 as shown in SEQ ID NO: 13, 14 or 15.
  • VH contains the amino acid sequence shown in SEQ ID NO: 12
  • the variable region or amino acid sequence of the heavy chain in the fusion peptide is as shown in SEQ ID NO: 10
  • a transmembrane protein domain or transmembrane domain refers to a region on a polypeptide or protein that has a structure that is thermodynamically stable in the membrane and typically includes a single transmembrane alpha helix of a transmembrane protein, mainly composed of hydrophobic amino acids composition.
  • the preferred transmembrane protein domain of the present invention is a transmembrane domain (CD) of a leukocyte differentiation antigen, and may be, for example, a transmembrane domain of cell adhesion molecule-1 (ICAM-1, also called CD54), CD48, or CD80.
  • CD transmembrane domain of a leukocyte differentiation antigen
  • linker peptides refer to those that are inserted between the variable, constant, and transmembrane protein domains of a bispecific antibody to provide sufficient mobility for each region or domain to provide the bispecific antibody binding function.
  • the linker peptide usually includes a flexible amino acid residue, such as Gly.
  • each linker peptide is independent, and may have the same sequence and / or length, or may be different from each other.
  • the nucleic acid of the second aspect of the present invention is generally two, namely a first nucleic acid and a second nucleic acid, which encode the first and second peptide chains in the bispecific antibody of the present invention, respectively.
  • the first nucleic acid and the second nucleic acid are preferably constructed on the same construct and expressed by the same vector.
  • the first nucleic acid and the second nucleic acid can also be constructed on different vectors, expressed separately, and then combined together.
  • the vector is preferably an expression vector, including derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmid, vectors derived from a combination of plasmid and phage DNA, and viral nucleic acid (RNA or DNA) vectors, etc. .
  • Vectors suitable for expression in bacterial cells include commercially available BlueScript (Stratagene), pIN vectors (VanHeeke, J.
  • the vector expressed in the yeast system is a vector containing a constitutive or inducible promoter; a preferred expression vector is suitable for expression in mammalian cells, such as the expression vector used in a specific embodiment of the present invention.
  • An expression vector may contain or be associated with any suitable promoter, enhancer, and other elements that promote expression.
  • suitable promoter, enhancer, and other elements that promote expression include strong expression promoters (e.g., human CMV IE promoters / enhancers and RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), polyA termination sequences, A replication origin region of a plasmid product, an antibiotic resistance gene as a selection marker, and / or a convenient cloning site.
  • the expression vector can be located in a host cell and / or the nucleic acid of the second aspect of the invention can be delivered (eg, transfected) into the host cell via the vector.
  • host cells include yeast, bacteria, and mammalian cells, such as CHO or HEK cells.
  • a nucleic acid encoding a bispecific antibody of the invention is transfected into Expi293F cells.
  • the bispecific antibody of the first aspect of the present invention can be obtained by culturing the host cell of the fourth aspect of the present invention, and isolating and purifying the culture obtained from the culture.
  • Methods for separation and purification include chromatography, magnetic bead separation, or colloidal filtration.
  • pharmaceutically acceptable excipients refer to pharmaceutical excipients that do not produce side effects, allergies or other adverse reactions when used to mammals (especially humans) as appropriate, and include non-toxic solid or liquid fillers, diluents, packages Sealing materials or preparation materials.
  • suitable fillers, solvents, and / or excipients include one or more of water, amino acids, saline, phosphate buffered saline, glucose, glycerol, ethanol, and the like, and combinations thereof. It is also preferred to include isotonic agents, such as sugars, polyols or sodium chloride, in the pharmaceutical compositions of the invention.
  • the pharmaceutical composition of the present invention may further include an antioxidant (eg, tryptamine) and a stabilizer (eg, Tween-20).
  • the form, route of administration, dosage, and schedule of the pharmaceutical composition will naturally depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, and the like.
  • the pharmaceutical composition of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.
  • the pharmaceutical composition of the present invention is an injection, such as an isotonic, sterile aqueous solution, or a dry (especially freeze-dried) composition thereof.
  • the dry composition can be formulated as an injectable solution when sterile water or physiological saline is added.
  • the bispecific antibody of the present invention may exist as a salt in the pharmaceutical composition of the present invention.
  • the pharmaceutically acceptable salts include acid addition salts, the corresponding inorganic acids include hydrochloric acid or phosphoric acid, and the corresponding organic acids include acetic acid, oxalic acid, tartaric acid, and the like.
  • Pharmaceutically acceptable salts can also include salts formed with free carboxyl groups on proteins.
  • Corresponding inorganic bases include hydroxides of sodium, potassium, ammonium, calcium or iron, and corresponding organic bases include isopropylamine, trimethylamine, Glycine, Histidine, etc.
  • an individual refers to a human or non-human mammal, preferably a human, including men, women, and children.
  • the non-human mammal is preferably a drug for testing animals, such as rats, mice, rabbits, cats, dogs, monkeys and horses.
  • treatment refers to reversing, alleviating, or inhibiting the progression of one or more disorders or conditions of a given disease to a subject having a given disease.
  • treatment refers not only to treatment that results in a complete cure of the disease, but also to treatment that slows the progression of the disease and / or prolongs the survival rate of the subject.
  • an effective amount refers to the amount of drug required to treat a disease with a reasonable benefit / risk ratio sufficient to treat the corresponding disease.
  • the total daily dosage of the bispecific antibody of the present invention or the pharmaceutical composition of the present invention will be determined by the physician within the scope of safe medical judgment.
  • the specific therapeutically effective amount for any particular patient will depend on a number of factors, including the severity of the disorder and condition to be treated; the activity of the particular polypeptide employed; the particular composition employed; the age, weight, Basic health, gender and diet; time of administration; route of administration and excretion rate of the polypeptide used; duration of treatment; drugs known in combination with or in combination with the polypeptide used.
  • the present invention refers to public documents, which are used to describe the present invention more clearly, and the entire contents thereof are incorporated herein by reference, as if their entire texts have been repeatedly described herein.
  • a plasmid (pLNCX, commercially available from Clontech) containing the construct shown in FIG. 1 is first constructed, in which: the leader sequence (leaderseq) and internal The light chain variable region and the light chain constant region ( ⁇ -TumorVL-CK) of the anti-epidermal growth factor receptor antibody are sequentially inserted between the ribosome entry site (IRES) and its amino acid sequence is as shown in SEQ ID (NO: 4), linker (amino acid sequence: GGGGSGGGGSGGGGS) and Intercellular Adhesion Molecule1 (ICAM-1) domain D1-D2-D3 (ICAM1-D1-3, its amino acid sequence As shown in SEQ ID ID NO: 1), the second leader sequence (Leader seq) of the plasmid is inserted into the heavy chain variable region encoding the anti-epidermal growth factor receptor antibody and CH1 ( ⁇ -Tumor VH) in sequence.
  • the leader sequence leaderseq
  • linker linker (amino acid sequence: GGGSGGG)
  • heavy chain variable region ⁇ -CD3 VH
  • ⁇ -CD3 VH heavy chain variable region
  • ScFv linker anti-CD3 antigen
  • anti-CD3 light chain The DNA of the variable region ( ⁇ -CD3) VL and histidine tag (6His).
  • the complete amino acid sequence of ⁇ -CD3VH, scFvlinker and ⁇ -CD3VL is shown in SEQ ID NO: 10.
  • the constructed positive plastids were transfected into Expi293F cells (commercially available from Thermo Fisher Scientific), and the cell culture solution Expi293 TM Expression Medium (commercially available from Thermo Fisher Scientific) was used at 37 ° C and 5% carbon dioxide concentration. Suspension culture was performed under the conditions, and the number of Expi293F cells in the Expi293F cell culture medium was maintained at 3-5 ⁇ 10 6 cells / ml by subculture.
  • the bispecific antibody B was constructed and prepared as a control according to the method basically the same as that described above. Compared with the bispecific antibody A, the only difference was that it did not carry the cell adhesion molecule-1 (Intercellular Adhesion Molecule 1, ICAM-1) domain D1-D2-D3 (ICAM1-D1-3).
  • ICAM1-D1-3 Intercellular Adhesion Molecule 1, ICAM-1) domain D1-D2-D3
  • Bispecific antibodies A and B were tested for T cell adhesion time. Specifically, first, six 250 ml cell culture flasks were prepared, and 20 ml of serum-free cell culture solution AIM V (commercially available) was added to each cell culture flask. (From ThermoFisher Scientific Co., Ltd.), and then add bispecific antibody A at a concentration of 5mg / mL to the cell culture fluid in three of the cell culture flasks as the experimental group sample, and the cells in the other three cell culture flasks A bispecific antibody B at a concentration of 5 mg / mL was added to the culture medium as a control sample.
  • AIM V serum-free cell culture solution
  • the above 6 cell culture flasks were placed in a cell culture incubator, and cultured at 37 ° C and 5% carbon dioxide concentration, and 0 hours after the cultivation, 24 hours after the cultivation, 48 hours after the cultivation, and 72 hours after the cultivation.
  • the analysis samples were taken from the cytosol samples of the experimental group and the cytosol samples of the control group, and the analysis samples of the experimental group and the control group were analyzed with the fluorescent secondary antibody anti-human IgG fluorescent isothiocyanate.
  • Anti-human IgG-FITC for staining, and put the stained analysis sample in the flow cytometer, because anti-human IgG-FITC will bind with bispecific antibody A and bispecific antibody B Therefore, by detecting T cells and T cells labeled with anti-human IgG-FITC, it can be known whether the T cells in the experimental group and the control group adhere to their corresponding bispecific antibodies.
  • plasmid containing the construct shown in FIG. 5 is first constructed, in which: the leader sequence (Leaderseq) and internal The light chain variable region and the light chain constant region ( ⁇ -CD3) of the light chain constant region ( ⁇ -CD3, VL-CK, amino acids) of the ribosome entry site (internal, ribosome, entry site, IRES) are inserted in sequence.
  • the sequence is shown in SEQ ID No.
  • linker (amino acid sequence is GGGGSGGGGSGGGGS) and Intercellular Adhesion Molecule1 (ICAM-1) domain D1-D2-D3 (ICAM1-D1-3 DNA whose amino acid sequence is shown in SEQ ID NO: 1), and the second leader sequence (Leader seq) carried by the plasmid is inserted into the heavy chain encoding the anti-CD3 antigen (anti-CD3) variable in order Region and CH1 ( ⁇ -CD3 VH-CH1, whose amino acid sequence is shown in SEQ ID NO: 12), linker (amino acid sequence: GGGSGGG), heavy chain variable region of anti-epidermal growth factor receptor antibody ( ⁇ -EGFR (VH), linker peptide (scFv linker), light chain of anti-epidermal growth factor receptor antibody Region ( ⁇ -EGFR VL) and histidine tag (6His) of the DNA, wherein the complete amino acid sequence of ⁇ -EGFR VH, scFv linker, and ⁇ -
  • a bispecific antibody C was prepared by Expi293F cells and nickel affinity chromatography.
  • a bispecific antibody D was constructed and prepared as a control according to a method basically the same as that described above. Compared with the bispecific antibody C, the only difference is that it does not carry the cell adhesion molecule-1 (Intercellular Adhesion Molecule 1, ICAM-1) domain D1-D2-D3 (ICAM1-D1-3).
  • ICAM1-D1-3 Intercellular Adhesion Molecule 1, ICAM-1) domain D1-D2-D3
  • the T cell adhesion time test was performed on the bispecific antibodies A and B according to the test method described in Example 1. The experimental results are shown in Figure 8. Although the affinity of the bispecific antibody C (EGFR_CD3 + ICAM1) and the bispecific antibody D (EGFR_CD3) with T cells decreased with time, after 24 hours, At the three time points of 48 hours and 72 hours after culture, the degree of affinity of bispecific antibody C and T cells was significantly higher than that of bispecific antibody D and T cells.

Abstract

本发明提供了一种双特异性抗体，其包括第一肽链VL-CK-TM和第二肽链VH-CH1-VH'-VL'，其中，VH是第一单克隆抗体的重链可变区，VL是第一单克隆抗体的轻链可变区，CH1是第一单克隆抗体的重链恒定区1，CK是第一单克隆抗体的轻链恒定区，VH'是第二单克隆抗体的重链可变区，VL'是第二单克隆抗体的轻链可变区，而且TM是跨膜蛋白结构域。

Description

双特异性抗体及其使用方法 技术领域
本发明属于抗体工程领域，具体而言，本发明涉及一种双特异性抗体及其应用，该双特异性抗体效果优异，尤其对T细胞的亲和性的持久保持的效果优异。
背景技术
抗体又称免疫球蛋白(Ig)。天然抗体含有四条异源性多肽链，其中分子量较大的两条链称为重链(HC)，而分子量较小的两条链称为轻链(LC)。轻链和重链中靠近N端氨基酸序列变化较大的区域称为可变区，即轻链可变区(VL)和重链可变区(VH)，分别占重链和轻链的1/4和1/2，VL和VH各自包括了三个互补决定区(CDR)，即VL-CDR1、VL-CDR2和VL-CDR3以及VH-CDR1、VH-CDR2和VH-CDR3；将靠近C端的氨基酸序列相对稳定的区域，称为恒定区，其中重链恒定区分三段，依次分别是重链恒定区1(CH1)、重链恒定区2(CH2)和重链恒定区3(CH3)，CH1与轻链恒定区(CL，也称CK)通过二硫键连接。
天然的单克隆抗体特异性结合一个抗原靶点，而双特异性抗体(BsAb)是可以与两个不同抗原靶点特异性结合的抗体融合体。双特异性抗体目前研究较多的有两类，分别为串联scFv与双特异性四价抗体(Tand-Abs)。
例如，中国专利申请CN104892765A公开了一种抗CD3抗原和Her-2抗原的双特异性抗体，包括2条相同的轻链序列和2条不同的重链序列，可以分别识别乳腺癌细胞表面的Her-2抗原和T细胞表面的CD3抗原，能够显著增强抗体介导的T细胞杀伤肿瘤靶细胞的活性；
中国专利申请CN106831996A提供了新型双特异性抗体，其包含针对人CD3E的抗原结合部和/或针对HER2的抗原结合部；
中国专利申请CN104829728A公开了一种双特异性抗体HER2XCD3，其由单价单元和单链单元组成，其中单价单元针对免疫细胞的表面抗原CD3具有特异性结合能力，单链单元针对肿瘤细胞表面抗原HER2具有特异性结合能力，并且单链单元包含与Fc片段融合的单链可变片段；
中国专利申请CN106632681A公开了抗EGFR和抗CD3双特异抗体，其能 够特异性结合肿瘤细胞表面抗原表皮生长因子受体和免疫细胞表面抗原分化簇3，其中抗CD3抗体的单链抗体ScFv位于抗EGFR抗体恒定区的C末端；
中国专利申请CN104774268A公开了一种双特异性抗体EGFR×CD3，其由单价单元和单链单元组成，其中单价单元针对免疫细胞的表面抗原CD3具有特异性结合能力，单链单元针对肿瘤细胞表面抗原EGFR具有特异性结合能力，并且单链单元包含与Fc片段融合的单链可变片段；
中国专利申请CN104829729A公开了一种携带抗Her2/CD3双特异抗体，其包括第一抗体段和第二抗体段，所述第一抗体段为抗肿瘤抗原抗体段，第二抗体段为抗人CD3分子抗体段，利用过继转输的T细胞携带并在体内持续表达该抗体蛋白，从而使该双特异性抗体在体内发挥杀伤作用的同时，伴随足量的T效应细胞，更优化了效应发挥的效率。
然而，现有技术没有启示获得本发明的包括跨膜结构域的特定串联方式连接的双特异抗体。令人意外的是，本发明人设计的本发明的双特异抗体的效果优异，尤其是能够持久保持与T细胞黏附的效果，更有利于实际应用。
发明简述
本发明提供了新的双特异性抗体及其重组DNA技术的中间产物、制备方法和治疗方面的用途等。
具体而言，在第一方面，本发明提供了具有下式结构的双特异性抗体，
VL-CK-TM
    |
VH-CH1-VH’-VL’
其中，VH是第一单克隆抗体的重链可变区，
VL是第一单克隆抗体的轻链可变区，
CH1是第一单克隆抗体的重链恒定区1，
CK是第一单克隆抗体的轻链恒定区，其中CH1和CK通过二硫键(|)连接，
VH’是第二单克隆抗体的重链可变区，
VL’是第二单克隆抗体的轻链可变区，
TM是跨膜蛋白结构域，和，
-表示直接肽键连接或通过连接肽连接。
第一单克隆抗体和第二单克隆抗体所针对的抗原靶点是不相同的。优选在本 发明第一方面的双特异性抗体中，第一单克隆抗体和第二单克隆抗体中的一个特异结合T细胞。更优选其中，特异结合T细胞的CD3抗原。
也优选在本发明第一方面的双特异性抗体中，第一单克隆抗体和第二单克隆抗体中的另一个特异结合病原。更优选其中，第一单克隆抗体和第二单克隆抗体中的另一个特异结合肿瘤或癌细胞，例如肿瘤或癌细胞的特异性抗原或过表达抗原，例如表皮生长因子受体、癌胚抗原或前列腺特定膜抗原。
优选在本发明第一方面的双特异性抗体中，跨膜蛋白结构域是白细胞分化抗原的跨膜结构域，例如可以是细胞黏附分子-1、CD48或CD80的跨膜结构域。更优选其中，跨膜蛋白结构域的氨基酸序列如SEQ ID NO：1、2或3所示。
优选在本发明第一方面的双特异性抗体中，VL或VL’包含氨基酸序列如SEQ ID NO：4、5、6或11所示的融合肽中的轻链可变区或氨基酸序列如SEQ ID NO：10、13、14或15所示的融合肽中的轻链可变区中的VL-CDR1、VL-CDR2和VL-CDR3。
也优选在本发明第一方面的双特异性抗体中，VH或VH’包含氨基酸序列如SEQ ID NO：7、8、9或12所示的融合肽中的重链可变区或氨基酸序列如SEQ ID NO：10、13、14或15所示的融合肽中的重链可变区中的VH-CDR1、VH-CDR2和VH-CDR3。
在本发明的具体实施方式中，VL-CK的氨基酸序列如SEQ ID NO：4、5、6或11所示，其中前三者分别是特异结合表皮生长因子受体、癌胚抗原和前列腺特定膜抗原的单克隆抗体的VL-CK；后者是特异结合T细胞的CD3抗原的单克隆抗体的VL-CK。
在本发明的具体实施方式中，VH-CH1的氨基酸序列如SEQ ID NO：7、8、9或12所示，其中前三者分别是特异结合表皮生长因子受体、癌胚抗原和前列腺特定膜抗原的单克隆抗体的VH-CH1；后者是特异结合T细胞的CD3抗原的单克隆抗体的VH-CH1。
在本发明的具体实施方式中，VH’-VL’的氨基酸序列如SEQ ID NO：10、13、14或15所示，其中前者是特异结合T细胞的CD3抗原的单克隆抗体的VH’-VL’；后三者分别是特异结合表皮生长因子受体、癌胚抗原和前列腺特定膜抗原的单克隆抗体的VH’-VL’。
在本发明的具体实施方式中，连接肽可以是GGGSGGG、VEGGSGGSGGSGGSGGVD或(GGGGS)n，其中n＝1～5。
在第二方面，本发明提供了分离的核酸，其编码本发明第一方面的双特异性抗体。
在第三方面，本发明提供了载体，其包含本发明第二方面的核酸。优选本发明第三方面的载体是质粒。
在第四方面，本发明提供了宿主细胞，其包含本发明第三方面的载体，或者被本发明第三方面的载体转染。优选本发明第四方面的宿主细胞是哺乳动物细胞。
在第五方面，本发明提供了生产本发明第一方面的双特异性抗体的方法，其包括以下步骤：
(a)培养本发明第四方面的宿主细胞；和。
(b)从步骤(a)获得的培养物中收集本发明第一方面的双特异性抗体。
在第六方面，本发明提供了药物组合物，其包括本发明第一方面的双特异性抗体和药学上可接受的辅料。本发明第六方面的药物组合物用于治疗第一单克隆抗体或第二单克隆抗体所治疗的疾病，例如肿瘤或癌症。
在第七方面，本发明提供了本发明第一方面的双特异性抗体在制备治疗第一单克隆抗体或第二单克隆抗体所治疗的疾病的药物中的用途；相应地，本发明也提供了治疗第一单克隆抗体或第二单克隆抗体所治疗的疾病的方法，其包括向有需要的个体施用有效量的本发明第一方面的双特异性抗体。
优选在本发明第七方面的用途或方法中，第一单克隆抗体或第二单克隆抗体所治疗的疾病是肿瘤或癌症。更优选其中肿瘤或癌症选自大肠癌、直肠癌、咽喉癌、头颈部癌、肺癌、胃癌、乳癌、胰脏癌、子宫颈癌、卵巢癌、摄护腺癌及前列腺癌。
附图说明
图1为实施例1的双特异性抗体A的构建体示意图。
图2为实施例1的双特异性抗体A的结构示意图。
图3为实施例1的双特异性抗体B的构建体示意图。
图4为实施例1的T细胞黏合时间试验的实验结果图。
图5为实施例2的双特异性抗体C的构建体示意图。
图6为实施例2的双特异性抗体C的结构示意图。
图7为实施例2的双特异性抗体D的构建体示意图。
图8为实施例2的T细胞黏合时间试验的实验结果图。
发明详述
本发明的双特异性抗体由两条肽链组成，包括第一肽链和第二肽链，其中第一肽链的结构如VL-CK-TM所示，第二肽链的结构如VH-CH1-VH’-VL’所示，其中，VH是第一单克隆抗体的重链可变区，VL是第一单克隆抗体的轻链可变区，CH1是第一单克隆抗体的重链恒定区1，CK是第一单克隆抗体的轻链恒定区，VH’是第二单克隆抗体的重链可变区，VL’是第二单克隆抗体的轻链可变区，TM是跨膜蛋白结构域，而且-表示直接肽键连接或通过连接肽连接。
在本文中，“第一”和“第二”在修饰相同的产品的时候，是为了区分所修饰的产品，即第一产品和第二产品是不同的，但是不对产品本身的结构和/或组成构成限定。例如，第一单克隆抗体和第二单克隆抗体是不同的单克隆抗体。
在本文中，单克隆抗体由一对轻链和一对重链组成，该四条链是通过二硫键相互连接。其中，每条重链通常包含重链可变区和重链恒定区。重链恒定区通常包含三个域，即CH1、CH2、和CH3；每条轻链通常包含轻链可变区和轻链恒定区。每个可变区通常包含三个互补决定区，其是序列上和/或结构上确定的环的形式上高度可变的区域，其被更加保守的四个框架区(FR)隔开，即每个可变区从氨基端到羧基端以以下的顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
本文中的CDR序列可以根据IMGT规则确定(Brochet X.,Nucleic Acids Research，2008，36:W503-508和Lefranc MP.,Nucleic Acids Research，1999，27:209-212)，也可以根据由Kabat等人(Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD.1991)描述的方法进行。对于给定的抗体可变区可以确定残基的Kabat编号。
优选在本发明第一方面的双特异性抗体中，第一单克隆抗体和第二单克隆抗体中的一个特异结合T细胞，如特异结合T细胞的CD3抗原；而第一单克隆抗体和第二单克隆抗体中的另一个特异结合病原，优选特异结合肿瘤或癌细胞，例如肿瘤或癌细胞的特异性抗原或过表达抗原，例如表皮生长因子受体(EGFR)、 癌胚抗原(CEA)或前列腺特定膜抗原(PSMA)。本发明第一方面的双特异性抗体的药用用途主要由特异结合病原的单克隆抗体片段确定，另一个特异结合T细胞的单克隆抗体片段起到持久保持与T细胞黏附的效果，从而促进相应的药用效果。
在本发明的具体实施方式中，VL包含氨基酸序列如SEQ ID NO：4、5或6所示的融合肽中的轻链可变区或氨基酸序列如SEQ ID NO：13、14或15所示的融合肽中的轻链可变区中的VL-CDR1、VL-CDR2和VL-CDR3的时候，则VL’包含氨基酸序列如SEQ ID NO：11所示的融合肽中的轻链可变区或氨基酸序列如SEQ ID NO：10所示的融合肽中的轻链可变区中的VL-CDR1、VL-CDR2和VL-CDR3；反之亦然，VL包含氨基酸序列如SEQ ID NO：11所示的融合肽中的轻链可变区或氨基酸序列如SEQ ID NO：10所示的融合肽中的轻链可变区中的VL-CDR1、VL-CDR2和VL-CDR3的时候，则VL’包含氨基酸序列如SEQ ID NO：4、5或6所示的融合肽中的轻链可变区或氨基酸序列如SEQ ID NO：13、14或15所示的融合肽中的轻链可变区中的VL-CDR1、VL-CDR2和VL-CDR3。
在本发明的具体实施方式中，VH包含氨基酸序列如SEQ ID NO：7、8或9所示的融合肽中的重链可变区或氨基酸序列如SEQ ID NO：13、14或15所示的融合肽中的重链可变区中的VH-CDR1、VH-CDR2和VH-CDR3的时候，VH’包含氨基酸序列如SEQ ID NO：12所示的融合肽中的重链可变区或氨基酸序列如SEQ ID NO：10所示的融合肽中的重链可变区中的VH-CDR1、VH-CDR2和VH-CDR3；反之亦然，VH包含氨基酸序列如SEQ ID NO：12所示的融合肽中的重链可变区或氨基酸序列如SEQ ID NO：10所示的融合肽中的重链可变区中的VH-CDR1、VH-CDR2和VH-CDR3的时候，VH’包含氨基酸序列如SEQ ID NO：7、8或9所示的融合肽中的重链可变区或氨基酸序列如SEQ ID NO：13、14或15所示的融合肽中的重链可变区中的VH-CDR1、VH-CDR2和VH-CDR3。
在本文中，跨膜蛋白结构域或跨膜结构域指的是多肽或蛋白质上的区域，该区域结构在膜中热力学稳定，并且通常包括跨膜蛋白的单一跨膜α螺旋，主要由疏水氨基酸组成。本发明优选的跨膜蛋白结构域是是白细胞分化抗原的跨膜结构域(CD)，例如可以是细胞黏附分子-1(ICAM-1，也称CD54)、CD48或CD80的跨膜结构域。
在本文中，连接肽指的是插入双特异性抗体各可变区、恒定区和跨膜蛋白结构域之间为各区域或结构域提供足够的可动性以提供双特异性抗体结合功能的一个或多个氨基酸残基。连接肽通常包括柔性氨基酸残基，如Gly，在本发明的双特异性抗体中，各连接肽是独立的，可以具有相同的序列和/或长度，也可以互不相同。
本发明第二方面的核酸通常是两条，即第一核酸和第二核酸，分别编码本发明的双特异性抗体中的第一肽链和第二肽链。第一核酸和第二核酸优选构建在同一构建体上，由同一载体表达。当然，尽管不优选，第一核酸和第二核酸也可以分别构建在不同载体上，分别表达，然后再组合在一起。
在本文中，载体优选是表达载体，包括SV40的衍生物、细菌质粒、噬菌体DNA、杆状病毒、酵母质粒、衍生自质粒和噬菌体DNA的组合的载体、和病毒核酸(RNA或DNA)载体等。适于在细菌细胞中表达的载体包括已经商用的BlueScript(Stratagene)、pIN载体(Van Heeke,J Biol Chem，264,5503-5509(1989))和pET载体(Novagen,Madison WI)等；适于在酵母系统中表达的载体是包含组成型或诱导型启动子的载体；优选的表达载体适于在哺乳动物细胞中表达，例如本发明具体实施方式中所采用的表达载体。
表达载体可以包含或相关于任何合适的启动子、增强子和其它促进表达的元件。这类元件的实例包括强表达启动子(例如，人CMV IE启动子/增强子以及RSV、SV40、SL3-3、MMTV、和HIV LTR启动子)，polyA终止序列、用于在大肠杆菌中的质粒产物的复制起始区、作为选择标志物的抗生素抗性基因、和/或方便的克隆位点。
表达载体可以位于宿主细胞中和/或经由载体将本发明第二方面的核酸递送(如，转染)至宿主细胞中。宿主细胞的实例包括酵母、细菌、和哺乳动物细胞，如CHO或HEK细胞。在本发明的具体实施方式中，编码本发明的双特异性抗体的核酸转染入Expi293F细胞中。
培养本发明第四方面的宿主细胞，并分离、纯化培养获得的培养物，可以获得本发明第一方面的双特异性抗体。分离纯化的方法包括层析法、磁珠分离法或胶体过滤法。
在本文中，药学上可接受的辅料指指酌情使用至哺乳动物(特别是人)时不 产生副作用、过敏或其他不良反应的药用辅料，包括非毒性固体或液体填充剂、稀释剂、包封材料或制剂辅料等。合适的填充剂、溶剂和/或赋形剂的实例包括水、氨基酸、盐水、磷酸盐缓冲盐水、葡萄糖、甘油、乙醇等的一种或多种，以及其组合。也优选本发明的药物组合物中包括等渗剂，如糖、多元醇或氯化钠。另外优选本发明的药物组合物还可包含抗氧化剂(如，色胺)和稳定剂(如，吐温-20)。
药物组合物的形式、施用途径、剂量和方案自然取决于待治疗的病况、疾病的严重性、患者的年龄、重量和性别等。本发明的药物组合物可配制用于局部、口服、肠胃外、鼻内、静脉内、肌肉内、皮下或眼内施用等。优选本发明的药物组合物是注射剂，如等渗、无菌的水溶液，或其干燥(特别是冷冻干燥)的组合物。干燥的组合物在添加无菌水或生理盐水时可构成可注射溶液。
本发明的双特异性抗体在本发明的药物组合物中可以以盐形式存在。药学上可接受的盐包括酸加成盐，相应的无机酸包括盐酸或磷酸，相应的机酸包括乙酸、草酸、酒石酸等。药学上可接受的盐还可以包括与蛋白质上游离羧基基团形成的盐，相应的无机碱包括钠，钾、铵、钙或铁的氢氧化物，相应的有机碱包括异丙胺、三甲胺、甘氨酸、组氨酸等。
在本文中，个体指的是人或非人哺乳动物，优选是人，包括男人、女人和儿童。非人哺乳动物优选是药物试验用动物，如大鼠、小鼠、兔、猫、狗、猴和马等。
在本文中，治疗指对具有给定疾病的受试者给药后逆转、减轻、抑制该疾病得一种或多种病症或病况的进展。因此，治疗不仅指导致疾病完全治愈的治疗，还指减缓疾病进展和/或延长受试者生存率的治疗。
在本文中，有效量指以合理的收益/风险比治疗达到足以治疗相应疾病的所需的药物量。本发明的双特异性抗体或本发明的药物组合物的每日总用量将由医师在安全医学判断范围内决定。用于任何特定患者的特定的治疗上的有效量将取决于多种因素，包括待治疗的病症和病症的严重性；采用的特定多肽的活性；采用的特定组合物；患者的年龄、体重、基本健康、性别和饮食；施用时间；施用途径和采用的多肽的排泄率；治疗持续时间；与采用的多肽组合或同时使用的药物等医学领域已知的因素。
本发明引用了公开文献，这些文献是为了更清楚地描述本发明，它们的全文内容均纳入本文进行参考，就好像它们的全文已经在本文中重复叙述过一样。
为了便于理解，以下将通过具体的实施例和附图对本发明进行详细地描述。需要特别指出的是，这些描述仅仅是示例性的描述，并不构成对本发明范围的限制。依据本说明书的论述，本发明的许多变化、改变对所属领域技术人员来说都是显而易见了。
具体实施方式
以下通过实施例进一步说明本发明的内容。如未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段和市售的常用仪器、试剂，可参见《抗体工程(第二版)》(北京大学医学出版社)、《分子克隆实验指南(第3版)》(科学出版社)、《微生物学实验(第4版)》(高等教育出版社)以及相应仪器和试剂的厂商说明书等参考。
实施例1 本发明的双特异性抗体A的制备及其效果实验
按照常规重组DNA技术制备，简而言之，首先构建含有如图1所示之构建体的质粒(pLNCX，可购自Clontech公司)，其中：在质粒带有的先导序列(Leader seq)和内部核糖体进入位点(internal ribosome entry site,IRES)之间依次插入编码抗表皮生长因子受体抗体的轻链可变区和轻链恒定区(α-Tumor VL-CK，其氨基酸序列如SEQ ID NO：4所示)、连接肽(linker，氨基酸序列为GGGGSGGGGSGGGGS)和细胞黏附分子-1(Intercellular Adhesion Molecule 1,ICAM-1)结构域D1-D2-D3(ICAM1-D1-3，其氨基酸序列如SEQ ID NO：1所示)的DNA，在质粒带有的第二个先导序列(Leader seq)后依次插入编码抗表皮生长因子受体抗体的重链可变区和CH1(α-Tumor VH-CH1，其氨基酸序列如SEQ ID NO：7所示)、连接肽(linker，氨基酸序列为GGGSGGG)、抗T细胞的CD3抗原(anti-CD3)的重链可变区(α-CD3 VH)、连接肽(scFv linker)、抗T细胞的CD3抗原(anti-CD3)的轻链可变区(α-CD3 VL)和组氨酸标签(6His)的DNA，其中α-CD3 VH、scFv linker和α-CD3 VL的完整的氨基酸序列如SEQ ID NO：10所示。
接著，将上述构建的阳性质体转染入Expi293F细胞(可购自Thermo Fisher Scientific公司)，用细胞培养液Expi293 TM Expression Medium(可购自Thermo  Fisher Scientific公司)于37℃、二氧化碳浓度5％的条件下进行悬浮培养，并透过继代培养(subculture)方式来使Expi293F细胞培养液中的Expi293F细胞个数维持在3–5×10 6cells/ml。经培养7天后，用超声波破碎细胞，以1000rpm离心10分钟收集细胞上清液，以镍亲和性层析法(Nickel Affinity Chromatography)从前述之上清液中纯化出Expi293F细胞所表达的双特异性抗体A。
经鉴定，α-Tumor VL-CK的轻链恒定区与α-Tumor VH-CH1的CH1之间通过二硫键共价相连成完整的双特异性抗体。该双特异性抗体A表达后的结构如图2所示。
另外，如图3所示，按照基本相同于上述的方法构建并制备作为对照的双特异性抗体B，与双特异性抗体A相比，所不同仅在于不带有细胞黏附分子-1(Intercellular Adhesion Molecule 1,ICAM-1)结构域D1-D2-D3(ICAM1-D1-3)。
对双特异性抗体A和B进行针对T细胞的黏合时间试验，具体而言，首先，准备6个250ml细胞培养瓶，各个细胞培养瓶中分别加入20ml的无血清细胞培养液AIM V(可购自ThermoFisher Scientific公司)，接著，在其中三个细胞培养瓶内的细胞培养液中加上浓度为5mg/mL的双特异性抗体A作为实验组样本，并在另外三个细胞培养瓶内的细胞培养液中加上浓度为5mg/mL的双特异性抗体B作为对照组样本。上述的6个细胞培养瓶放入细胞培养箱中，于37℃、二氧化碳浓度5％的条件下进行培养，并且在培养后0小时、培养后24小时、培养后48小时以及培养后72小时这4个时间点，分别从实验组的细胞液样本及对照组的细胞液样本中取出分析样本，将实验组的分析样本及对照组的分析样本以萤光二级抗体抗人类IgG萤光异硫氰酸盐(anti-human IgG-FITC)进行染色，并将经染色后的分析样本放入流式细胞仪中，由于anti-human IgG-FITC会与双特异性抗体A及双特异性抗体B结合，因此透过检测T细胞以及被anti-human IgG-FITC标记的T细胞，可以得知实验组及对照组中的T细胞是否与其对应的双特异性抗体製备黏合。
实验结果如图4所示，双特异性抗体A(EGFR_CD3+ICAM1)及双特异性抗体B(EGFR_CD3)与T细胞的亲和程度虽然都随时间而下降，但是在培养后24小时、培养后48小时以及培养后72小时这3个时间点，双特异性抗体A与 T细胞的亲和程度都显著高于双特异性抗体B与T细胞的亲和程度。由上述实施例1之双特异性抗体A对T细胞黏合时间试验的实验结果可知，能够稳定免疫突触结构的ICAM-1之结构域D1-D2-D3有助于双特异性抗体A提高对于T细胞的亲和性。
实施例2 本发明的双特异性抗体C的制备及其效果实验
按照常规重组DNA技术制备，简而言之，首先构建含有如图5所示之构建体的质粒(pLNCX，可购自Clontech公司)，其中：在质粒带有的先导序列(Leader seq)和内部核糖体进入位点(internal ribosome entry site,IRES)之间依次插入编码抗T细胞的CD3抗原(anti-CD3)的轻链可变区和轻链恒定区(α-CD3 VL-CK，其氨基酸序列如SEQ ID NO：11所示)、连接肽(linker，氨基酸序列为GGGGSGGGGSGGGGS)和细胞黏附分子-1(Intercellular Adhesion Molecule 1,ICAM-1)结构域D1-D2-D3(ICAM1-D1-3，其氨基酸序列如SEQ ID NO：1所示)的DNA，在质粒带有的第二个先导序列(Leader seq)后依次插入编码抗T细胞的CD3抗原(anti-CD3)的重链可变区和CH1(α-CD3 VH-CH1，其氨基酸序列如SEQ ID NO：12所示)、连接肽(linker，氨基酸序列为GGGSGGG)、抗表皮生长因子受体抗体的重链可变区(α-EGFR VH)、连接肽(scFv linker)、抗表皮生长因子受体抗体的轻链可变区(α-EGFR VL)和组氨酸标签(6His)的DNA，其中α-EGFR VH、scFv linker和α-EGFR VL的完整的氨基酸序列如SEQ ID NO：13所示。
接著，按照与实施例1相同的方式通过Expi293F细胞和镍亲和性层析法制备出双特异性抗体C。
经鉴定，α-CD3 VL-CK的轻链恒定区与α-CD3 VH-CH1的CH1之间通过二硫键共价相连成完整的双特异性抗体。该双特异性抗体C表达后的结构如图6所示。
另外，如图7所示，按照基本相同于上述的方法构建并制备作为对照的双特异性抗体D，与双特异性抗体C相比，所不同仅在于不带有细胞黏附分子-1(Intercellular Adhesion Molecule 1,ICAM-1)结构域D1-D2-D3(ICAM1-D1-3)。
对双特异性抗体A和B进行针对T细胞的黏合时间试验按照实施例1所述的试验方法进行。实验结果如图8所示，双特异性抗体C(EGFR_CD3+ICAM1) 及双特异性抗体D(EGFR_CD3)与T细胞的亲和程度虽然都随时间而下降，但是在培养后24小时、培养后48小时以及培养后72小时这3个时间点，双特异性抗体C与T细胞的亲和程度都显著高于双特异性抗体D与T细胞的亲和程度。由上述实施例2之双特异性抗体C对T细胞黏合时间试验的实验结果并结合实施例1的结果，可知，不论是使VL-CK及VH-CH对目标细胞具有结合特异性，且使VH-VL对T细胞具有结合特异性，或是使VL-CK及VH-CH对T细胞具有结合特异性，且使VH-VL对目标细胞具有结合特异性，皆不影响具有跨膜蛋白的双特异性抗体对于T细胞的亲和性之提高。
Figure PCTCN2018104518-appb-000001
Figure PCTCN2018104518-appb-000002
Figure PCTCN2018104518-appb-000003
Figure PCTCN2018104518-appb-000004
Figure PCTCN2018104518-appb-000005
Figure PCTCN2018104518-appb-000006
Figure PCTCN2018104518-appb-000007
Figure PCTCN2018104518-appb-000008
Figure PCTCN2018104518-appb-000009
Figure PCTCN2018104518-appb-000010
Figure PCTCN2018104518-appb-000011

Claims (10)

  1. 具有下式结构的双特异性抗体，
    Figure PCTCN2018104518-appb-100001
    其中，VH是第一单克隆抗体的重链可变区，
    VL是第一单克隆抗体的轻链可变区，
    CH1是第一单克隆抗体的重链恒定区1，
    CK是第一单克隆抗体的轻链恒定区，其中CH1和CK通过二硫键(|)连接，
    VH’是第二单克隆抗体的重链可变区，
    VL’是第二单克隆抗体的轻链可变区，
    TM是跨膜蛋白结构域，和
    -表示直接肽键连接或通过连接肽连接。
  2. 权利要求1所述的双特异性抗体，其中，第一单克隆抗体和第二单克隆抗体中的一个特异结合T细胞，优选特异结合T细胞的CD3抗原；第一单克隆抗体和第二单克隆抗体中的另一个特异结合病原，优选特异结合肿瘤或癌细胞，更优选特异结合表皮生长因子受体、癌胚抗原或前列腺特定膜抗原。
  3. 权利要求1所述的双特异性抗体，其中，跨膜蛋白结构域是白细胞分化抗原的跨膜结构域，优选是细胞黏附分子-1、CD48或CD80的跨膜结构域，更优选跨膜蛋白结构域的氨基酸序列如SEQ ID NO：1、2或3所示。
  4. 权利要求1所述的双特异性抗体，其中，VL-CK的氨基酸序列如SEQ ID NO：4、5、6或11所示。
  5. 权利要求1所述的双特异性抗体，其中，VH-CH1的氨基酸序列如SEQ ID NO：7、8、9或12所示。
  6. 权利要求1所述的双特异性抗体，其中，VH’-VL’的氨基酸序列如SEQ ID NO：10、13、14或15所示。
  7. 重组DNA技术的中间产物，其为：
    (1)分离的核酸，其编码权利要求1-6中任一项所述的双特异性抗体；
    (2)载体(优选为质粒)，其包含上述(1)所述的核酸；或，
    (3)宿主细胞(优选为哺乳动物细胞)，其包含上述(2)所述的载体，或者被上述(2)所述的载体转染。
  8. 生产权利要求1-6中任一项所述的双特异性抗体的方法，其包括以下步骤：
    (a)培养权利要求所述7宿主细胞；和。
    (b)从步骤(a)获得的培养物中收集权利要求1-6中任一项所述的双特异性抗体。
  9. 药物组合物，其包括权利要求1-6中任一项所述的双特异性抗体和药学上可接受的辅料。
  10. 权利要求1-6中任一项所述的双特异性抗体在制备治疗第一单克隆抗体或第二单克隆抗体所治疗的疾病的药物中的用途，优选其中第一单克隆抗体或第二单克隆抗体所治疗的疾病是肿瘤或癌症，更优选其中肿瘤或癌症选自大肠癌、直肠癌、咽喉癌、头颈部癌、肺癌、胃癌、乳癌、胰脏癌、子宫颈癌、卵巢癌、摄护腺癌及前列腺癌。
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