WO2017101863A1 - 双特异性偶联抗体及其制法和用途 - Google Patents

双特异性偶联抗体及其制法和用途 Download PDF

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WO2017101863A1
WO2017101863A1 PCT/CN2016/110438 CN2016110438W WO2017101863A1 WO 2017101863 A1 WO2017101863 A1 WO 2017101863A1 CN 2016110438 W CN2016110438 W CN 2016110438W WO 2017101863 A1 WO2017101863 A1 WO 2017101863A1
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antibody
protein
variable region
chain variable
light chain
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French (fr)
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蔡则玲
陈羿
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上海康岱生物医药技术股份有限公司
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Publication of WO2017101863A1 publication Critical patent/WO2017101863A1/zh
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    • A—HUMAN NECESSITIES
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    • 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
    • C07K16/2818—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 CD28 or CD152
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    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
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    • 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
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
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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
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70521—CD28, CD152

Definitions

  • the invention belongs to the field of biomedicine, in particular to the field of anti-tumor immunotherapy of dual-target monoclonal antibodies.
  • Her-2 is an epidermal growth factor receptor transmembrane protein encoded by the oncogene HER-2, and the intracellular domain of the receptor protein has tyrosine kinase activity.
  • the intracellular tyrosine kinase is activated and then activates multiple intracellular signaling pathways.
  • the most important signal channels have the function of stimulating cell growth and division, as well as anti-apoptosis and maintaining cell survival.
  • the HER-2 gene is found to be largely replicated or mutated, causing overexpression or sustained activation of Her-2 protein, resulting in cell canceration.
  • trastuzumab monoclonal antibody with Her-2 activity as a target for the inhibition of Her-2 activity was the first humanized monoclonal approved for the treatment of Her-2 positive breast cancer patients.
  • antibody. 11-26% of patients with malignant breast cancer have a curative effect on single trastuzumab. If combined with chemotherapy, the cure rate and survival rate of early and late breast cancer patients will be significantly improved. Although there is such a good effect, there are still a large number of Her-2 positive patients who do not respond to trastuzine, and most of those who respond to early treatment will produce trastuzumin at the later stage of treatment. Resistance to treatment.
  • trastuzumab The inhibition of tumor activity by trastuzumab depends not only on the inhibition of Her-2 activity, but also on the antibody-dependent cell-mediated cytotoxicity (ADCC) induced by trastuzumab. ).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Anti-Her-2 antibody that does not bind to the IgG Fc receptor (FcR) has been shown to have no anti-tumor effect.
  • trastuzumab In mice with FcR gene deletion, trastuzumab has proven to be ineffective and, in addition, trastuzumab
  • breast cancer tumor sections showed an increase in FcR-positive immune cells, mainly natural killer cells (NK).
  • acquired immunity also plays an important role in the inhibition of tumor effects by trastuzumab.
  • anti-Her-2 antibodies require CD8 ⁇ + cells, MyoD88 signaling pathway and RAG-dependent acquired immunological activity in order to achieve better anti-tumor effects.
  • Further studies have shown that traditional lymphocyte-mediated intercellular cytotoxicity of type I interferon (INF-I) and gamma interferon (INF ⁇ ) is important for the inhibition of trastuzumab in tumors, including CD8 ⁇ + T cells. The resulting INF ⁇ plays a key role.
  • INF ⁇ can directly inhibit the growth of Her-2 positive tumor cells in vitro, and can significantly enhance the ability of anti-Her-2 antibody to inhibit cell growth.
  • INF ⁇ significantly increased the expression of MHC class I on the surface of tumor cells. Combining these data explains the importance of INF ⁇ in the inhibition of tumors by trastuzumab.
  • trastuzumab Since acquired immunological activity is so important in the inhibition of tumor effect by trastuzumab, enhancing the activation of immune cells or maintaining the activation of immune cells, the effect of trastuzumab on tumor suppression is enhanced.
  • trastuzumab and anti-CD137 mAb enhances lymphocyte survival, particularly the viability of CD8+ T cells.
  • CD137 antibody also enhances the activation and function of NK cells and DC cells
  • the anti-tumor effect of the anti-Her-2 antibody can be enhanced by FcR+ cells and acquired immune cells.
  • blocking the PD-1 channel by the apoptosis-specific-1 (PD-1) antibody significantly increased the anti-tumor activity of the acquired immune T cells induced by trastuzumab.
  • T cells Activation of T cells requires not only stimulation of T cell receptors by MHC-polypeptide complexes on antigen presenting cells (APCs), but also a balance between costimulatory signals and suppression signals.
  • APCs antigen presenting cells
  • Multiple mechanisms interact to mediate activation of CD4+ and CD8+ T lymphocytes, including cell surface proteins that amplify or inhibit T cell responses.
  • Negative regulatory proteins on the surface of T cells such as CTLA-4, PD-1, B7 family of molecules B7-H4, T cell immune molecules and lymphocyte activating gene-3 (LAG-3), and their different types of cells Ligand binding results in a decrease in T cell growth and functional activity.
  • the CD28 molecule is a protein of the immunoglobulin family that is continuously expressed and mediates T cell costimulatory signals.
  • CD28 binds to the ligands B7-1 and B7-2 on APC, induces the production of interleukin-2 (IL-2) and anti-apoptotic factors, thereby stimulating the growth of T cells.
  • IL-2 interleukin-2
  • T cells When T cells are activated, they produce CTLA-4 on the cell surface, which is very similar to the CD28 molecule, and binds B7-1 and B7-2, and the binding strength and affinity are greater (especially in combination with B7-1). Therefore, a very small amount of CTLA-4 molecule can effectively compete with CD28 for its companion and reduce T cell response.
  • CTLA-4 has its intrinsic and extrinsic mechanisms of action, including the clearance of PKC-beta and CARMA1 from immune synaptosomes, limiting the presence of T cells and enhancing T-regulatory (Tregs) function. Collectively, these mechanisms inhibit cell growth, inhibit IL-2 production and cell survival pathways, and ultimately lead to the termination of the immune response.
  • the CTLA-4 knockout mouse experiment well demonstrates the importance of CTLA-4 as a negative regulatory molecule.
  • CTLA-4 deficient mice exhibit CD28-dependent large-scale autoreactive T cell expansion in lymph nodes, spleen, and several peripheral organs. These mice die from less than 4 weeks of birth due to diseases caused by diffuse lymph growth.
  • CTLA-4 a major immune checkpoint molecule in the T cell response, also has an effect on the memory formation of acquired immune responses. Recent studies have found that blocking CTLA-4 can reduce the number of professionally versatile CD4+ T memory cells, thereby regulating the quality of the overall memory cell pool. Another in vivo study found that injection of CTLA-4 antibody increased CD8+T memory cell expansion and then accumulated the number of immune cells that produced INFg and TNFa.
  • CTLA-4 monoclonal antibodies have demonstrated that selective blocking of CTLA-4 enhances internal or induced anti-tumor immune responses, which are developed for clinically targeted monoclonal antibodies targeting CTLA-4. Support is provided.
  • Two fully human antibodies, Ipilimumab and Tremelimumab bind to CTLA-4 and block its binding to the B7 chaperone, thereby enhancing T cell activation and proliferation.
  • Ipilimumab and Tremelimumab have conducted a large number of clinical studies on melanoma treatment in the past 10 years, and in 2011, Ipilimumab was approved by the US, EU and Australian Food and Drug Administration for the treatment of inoperable or metastatic melanoma. .
  • CTLA-4 blockade provides a new immune system regulation model that represents a milestone in the field of cancer therapy.
  • CTLA-4 monoclonal antibody therapy is being performed in clinical trials of multiple types of tumors, including melanoma, small cell lung cancer, non-small cell lung cancer, and prostate cancer.
  • a heavy chain variable region of an antibody comprising the following three complementarity determining region CDRs:
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:4.
  • a heavy chain of an antibody having the heavy chain variable region and the heavy chain constant region of the first aspect of the invention.
  • the heavy chain constant region is of human or murine origin.
  • the light chain variable region has the amino acid sequence set forth in SEQ ID NO: 10.
  • a light chain of an antibody having the light chain variable region and the light chain constant region of the third aspect of the invention.
  • the constant region of the light chain is of human or murine origin.
  • an antibody having:
  • the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the third aspect of the invention.
  • the antibody is an antibody specific for the CTLA-4 protein.
  • the antibody comprises: a single chain antibody, a double chain antibody, a monoclonal antibody, a chimeric antibody (such as a human mouse chimeric antibody), a murine antibody, a humanized antibody, and a bispecific antibody. (BiTE) and chimeric antigen receptor antibody (CAR).
  • a recombinant protein having:
  • the tag sequence comprises a 6His tag.
  • the recombinant protein specifically binds to the CTLA-4 protein.
  • a multispecific antigen binding molecule is provided, said multispecific antibody
  • the original binding molecule contains:
  • D2 a second antigen binding domain
  • D1 specifically binds to the target molecule Her-2 protein
  • D2 specifically binds to the target molecule CTLA-4 protein.
  • the multispecific antigen binding molecule has a higher ability to inhibit Her-2 protein-positive tumors than a single-specific antigen-binding molecule against Her-2 protein for Her-2 protein-positive tumors.
  • the multispecific antigen binding molecule is a polypeptide.
  • the multispecific antigen binding molecule is an antibody.
  • the multispecific antigen binding molecule is a bispecific antibody.
  • the D1 is an antibody or antibody fragment that specifically binds to the Her-2 protein.
  • the D2 is an antibody or antibody fragment that specifically binds to the CTLA-4 protein.
  • the D1 and/or the D2 is a single chain antibody (scFv).
  • D2 is a single chain antibody (scFv).
  • said D1 and said D2 are linked by a linker peptide comprising an antibody constant region sequence.
  • the D1 is an anti-Her-2 humanized antibody.
  • the D2 is an anti-CTLA-4 single chain antibody (scFv).
  • D1 is an anti-Her-2 humanized antibody
  • D2 is an anti-CTLA-4 single chain antibody (scFv)
  • scFv single chain antibody
  • the anti-Her-2 humanized antibody comprises a complementarity determining region CDR of at least one heavy chain variable region selected from the group consisting of:
  • the anti-Her-2 humanized antibody comprises the heavy chain set forth in SEQ ID NO.: 15.
  • the anti-Her-2 humanized antibody comprises a complementarity determining region CDR' of at least one light chain variable region selected from the group consisting of:
  • the anti-Her-2 humanized antibody comprises the light chain set forth in SEQ ID NO.: 17.
  • the anti-CTLA-4 single chain antibody comprises a complementarity determining region CDR of at least one heavy chain variable region selected from the group consisting of:
  • the anti-CTLA-4 single chain antibody comprises the heavy chain variable region set forth in SEQ ID NO.: 4.
  • the anti-CTLA-4 single chain antibody comprises a complementarity determining region CDR' of at least one light chain variable region selected from the group consisting of:
  • the anti-CTLA-4 single chain antibody comprises the light chain variable region set forth in SEQ ID NO.: 10.
  • the anti-CTLA-4 single chain antibody (scFv) sequence is set forth in SEQ ID NO.: 24.
  • a polynucleotide which encodes a polypeptide selected from the group consisting of:
  • the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the fourth aspect of the invention a light chain as described in the aspect, or an antibody according to the fifth aspect of the invention; or
  • a vector comprising the polynucleotide of the eighth aspect of the invention is provided.
  • the vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vector.
  • a genetically engineered host cell comprising the vector of the ninth aspect of the invention or the polynucleotide of the eighth aspect of the invention is integrated.
  • an immunoconjugate according to the eleventh aspect of the invention comprising:
  • a coupling moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.
  • the conjugate moiety is selected from the group consisting of: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent, or is capable of producing Detection of product enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, Prodrug activating enzymes (eg, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (eg, cisplatin) or any form of nanoparticles, and the like.
  • a fluorescent or luminescent label e.g., a radioactive label, an MRI (magnetic resonance imaging) or CT (computer tomography) contrast agent, or is capable of producing Detection of product enzymes, radio
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition is in the form of an injection.
  • the pharmaceutical composition is used for preparing a medicament for treating a tumor, the tumor being selected from the group consisting of gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, Colorectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, adrenal tumor, or bladder tumor.
  • the thirteenth aspect of the invention provides the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable according to the third aspect of the invention a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or a multi Use of a specific antigen binding molecule, or an immunoconjugate according to the eleventh aspect of the invention, for the preparation of a medicament, reagent, test plate or kit;
  • the reagent, the detection plate or the kit is used for: detecting CTLA-4 protein in the sample;
  • the agent is for treating or preventing a tumor expressing a CTLA-4 protein.
  • the tumor comprises: gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, or adrenal gland Tumor.
  • the tumor is selected from the group consisting of gastric cancer and follicular lymphoma.
  • the reagent comprises a chip, an immunoparticle coated with an antibody.
  • a fourteenth aspect of the invention there is provided a method of detecting CTLA-4 protein in a sample, the method comprising the steps of:
  • a method for preparing a recombinant polypeptide comprising:
  • Figure 1 shows a schematic diagram of the structure of a bispecifically coupled antibody, the upper part of which is trastuzumab, The variable regions of the heavy and light chains are represented by different colors.
  • the lower part of the structure is the scFv of human anti-CTLA-4 monoclonal antibody, the heavy chain variable region of the scFv antibody is linked to the C-terminus of the trastuzumab heavy chain, between the scFv heavy and light chain variable regions It is a 7 amino acid linker peptide.
  • Figure 2 shows the bispecifically coupled antibody nucleotide sequence and amino acid sequence.
  • Figure 2 (A) aa1-19 is a protein transmembrane signal peptide;
  • aa20-468 is a humanized anti-Her-2 monoclonal antibody (trastuzumab) heavy chain;
  • aa469-586 is a human anti-CTLA-4 scFv heavy chain Variable region;
  • aa587-593 is a linker peptide of human anti-CTLA-4 scFv heavy and light chain variable regions;
  • aa594-702 is a human anti-CTLA-4 scFv light chain variable region.
  • Figure 2 (B) aa1-20 is a protein transmembrane signal peptide;
  • aa21-234 is a humanized anti-Her-2 mono
  • Figure 3 shows SDS-PAGE electrophoresis analysis of bispecifically coupled antibodies, A: non-reduced 6% SDS-PAGE electrophoresis analysis, 3 ug/sample; B: reduction 10% SDS-PAGE electrophoresis analysis, 5 ug/sample.
  • FIG. 4 shows the purified bispecifically coupled antibody by HPLC-SEC analysis.
  • the bispecifically coupled antibody was purified by a Protein-A affinity chromatography column, passed through an anion chromatography column, and passed through a cation column.
  • Blue is the protein molecular weight standard
  • the red line is the trastuzumab peak map
  • the green peak is the bispecifically coupled antibody.
  • Figure 5 shows flow cytometry analysis of binding of bispecifically conjugated antibody to cell membrane Her-2.
  • Her-2 stably transfected mouse melanoma cells B16 were incubated with FITC-labeled sheep after incubation with different concentrations of antibodies.
  • the anti-human IgG1 Fc antibody detects the antibody bound to Her-2, and the FITC fluorescence intensity was detected by flow cytometry.
  • Mock is an antibody-free experimental group.
  • Figure 6 shows ELISA analysis of bispecifically coupled antibodies in vitro binding to CTLA-4, recombinant human CTLA-4-Fc fusion protein coated in 96-well ELISA plates, antibody anti-CTLA-4-Fc-conjugated goat anti-antibody
  • the human anti-human IgG1 Fab antibody is an alkaline phosphatase marker detected by human IgG1 Fab antibody.
  • Figure 7 shows the growth inhibition study of Her-2 positive tumor cells.
  • BT-474 cells were cultured for 4 days after co-culture with different concentrations of antibodies.
  • Figure 8 shows that enhanced activation of PBMCs expresses IL-2
  • PBMCs were isolated from 2 human donors, activated with PHA, and co-cultured with Raji cells and bispecific antibodies for 72 hours. The cells were pelleted by centrifugation, and the supernatant was collected, and the IL-2 content in the supernatant was measured by ELISA.
  • Figure 9 shows the amount of HBsAg antibody in plasma of HBsAg vaccine and bispecific antibody or control IgG1 protein monkey. Antibody content was determined by ELISA. The HBsAg protein was coated in a 96-well ELISA plate, and then incubated with 1000-fold diluted plasma, and antibodies bound to HBsAg were detected with an antibody against alkaline phosphatase-labeled goat anti-monkey IgG Fc.
  • the present inventors have obtained a human single-chain antibody (scFv) against CTLA-4 by extensive and intensive research, and the anti-CTLA-4 antibody has the advantages of high affinity and strong specificity.
  • the inventors have unexpectedly discovered that a recombinant bispecifically coupled antibody (humanized anti-Her-2 whole antibody (Trastuzumab) and anti-CTLA-4 human single-chain antibody was constructed using an anti-CTLA-4 antibody.
  • Bispecifically conjugated antibodies are not limited to the treatment of Her-2 positive breast cancer, but also include the treatment of other types of Her-2 positive malignancies.
  • the invention provides a multispecific antigen binding molecule comprising a first antigen binding domain (Dl) and a second antigen binding domain (D2), wherein the antigen binding domain D2 specifically binds to the CTLA-4 protein.
  • the amino acid sequence of the extracellular domain of the CTLA-4 protein is as follows:
  • the D1 component of the multispecific antigen binding molecule specifically binds to the extracellular ligand binding region of the target molecule Her-2 protein.
  • the Her-2 protein belongs to the epidermal growth factor receptor family (EGFR) and consists of an extracellular ligand binding domain, a single-stranded transmembrane domain, and an intracellular protein tyrosine kinase domain.
  • EGFR epidermal growth factor receptor family
  • the ligand binding region through its binding to the ligand, transmits extracellular signals to the cell, activates the EGFR signaling pathway, and promotes cell carcinogenesis.
  • amino acid sequence of the extracellular domain of the Her-2 protein is as follows:
  • the present invention provides an antibody against CTLA-4, the heavy chain variable region of which comprises the following three complementarity determining region CDRs:
  • the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:4:
  • the anti-CTLA-4 antibody light chain variable region has a complementarity determining region CDR selected from the group consisting of:
  • the anti-CTLA-4 antibody light chain variable region has the amino acid sequence set forth in SEQ ID NO: 10:
  • the anti-CTLA-4 antibody is a single-chain antibody, optionally having a linker peptide between the heavy chain variable region and the light chain variable region, and the length of the linker peptide is preferably It is 1-15 amino acids, more preferably 3-10 amino acids; preferably the amino acid sequence of the linker peptide is as follows:
  • a multispecific antigen-binding molecule comprising a binding domain that specifically binds to a target molecule Her-2 protein binding domain and specifically binds to a target molecule CTLA-4 protein is surprisingly constructed for Her -2 positive tumors have significant inhibitory effects, and can enhance the efficacy of trastuzumab against Her-2 positive tumors, as well as Her-2 positive tumors that are resistant to trastuzumab. Can be used as a sensitizer for trastuzumab.
  • the invention provides a multispecific antigen binding molecule comprising a first antigen binding domain (also referred to herein as "D1”) and a second antigen binding domain (also referred to herein as "D2”), wherein D1 specifically binds to the target molecule Her-2 protein; D2 specifically binds to the target molecule CTLA-4 protein.
  • D1 specifically binds to the target molecule Her-2 protein
  • D2 specifically binds to the target molecule CTLA-4 protein.
  • the multispecific antigen binding molecule may be a single multifunctional polypeptide, or it may be a multimeric complex of two or more polypeptides covalently or non-covalently bound to each other.
  • any antigen binding construct having the ability to simultaneously bind to Her-2 and CTLA-4 molecules is considered a multispecific antigen binding molecule.
  • any of the multispecific antigen binding molecules of the invention or variants thereof can be constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques), as will be appreciated by those of ordinary skill in the art.
  • the specific binding target molecule Her-2 protein binding domain comprises a complementarity determining region CDR of at least one heavy chain variable region selected from the group consisting of:
  • the specific binding target molecule Her-2 protein binding domain comprises the heavy chain set forth in SEQ ID NO.: 15:
  • the specific binding target molecule Her-2 protein binding domain comprises a complementarity determining region CDR' of at least one light chain variable region selected from the group consisting of:
  • the specific binding target molecule Her-2 protein binding domain comprises the light chain set forth in SEQ ID NO.: 17:
  • the binding domain that specifically binds to the target molecule CTLA-4 protein comprises a complementarity determining region CDR of at least one heavy chain variable region selected from the group consisting of:
  • the binding domain that specifically binds to the target molecule CTLA-4 protein comprises the heavy chain variable region set forth in SEQ ID NO.: 4.
  • the binding domain that specifically binds to the target molecule CTLA-4 protein comprises a complementarity determining region CDR' of at least one light chain variable region selected from the group consisting of:
  • the binding domain that specifically binds to the target molecule CTLA-4 protein comprises a light chain variable region comprising SEQ ID NO.: 10.
  • the binding domain that specifically binds to the target molecule CTLA-4 protein is an anti-CTLA-4 single chain antibody (scFv), the sequence of which is set forth in SEQ ID NO.: 24.
  • scFv anti-CTLA-4 single chain antibody
  • the anti-CTLA-4 single chain antibody is linked to the C-terminus of the trastuzumab heavy chain.
  • the multispecific antigen binding molecules of the invention comprise at least two independent antigen binding domains (D1 and D2).
  • the expression "antigen binding domain” means capable of specifically binding to a particular purpose Any peptide, polypeptide, nucleic acid molecule, scaffolding molecule, peptide display molecule or polypeptide-containing construct of an antigen.
  • the term "specifically binds" and the like means that the antigen binding domain forms a complex with a specific antigen characterized by a dissociation constant (KD) of 500 pM or less, and does not bind other unrelated under normal test conditions.
  • Antigen Preferably, "unrelated antigens" are proteins, peptides or polypeptides having less than 95% amino acid identity to each other.
  • Exemplary classifications of antigen binding domains that can be used in the context of the present invention include antibodies, antigen binding portions of antibodies, peptides that specifically interact with a particular antigen (eg, a peptibody), and specific antigen-specific interactions with each other.
  • Acting receptor molecule a protein comprising a ligand binding portion that specifically binds to a receptor of a particular antigen, an antigen binding scaffold (eg, DARPin, HEAT repeat protein, ARM repeat protein, triangular tetrapeptide repeat protein, and based on natural Other scaffolds for the presence of repeat proteins, etc. [see, for example, Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22: 849-857, and references cited therein) and aptamers or portions thereof.
  • an antigen binding scaffold eg, DARPin, HEAT repeat protein, ARM repeat protein, triangular tetrapeptide repeat protein, and based on natural Other scaffolds for
  • an antigen binding domain comprises less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about as measured in a surface plasmon resonance assay.
  • 90 pM less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5pM, less than about 0.2pM, less than about 0.1 pM, or less than about 0.05pM K D of binding to a specific antigen polypeptide or a portion thereof.
  • surface plasmon resonance refers to allow, for example using BIAcore TM system (GE Healthcare BiacoreLifeSciences the sector, Piscataway, NJ), by detecting alterations in protein concentrations biosensor matrix optical phenomena internal to analyze interactions in real time.
  • K D means that a particular protein - protein interactions (e.g., an antibody - antigen interaction) the equilibrium dissociation constant. Unless otherwise specified, K D disclosed herein refers to determining the value of K D values measured by surface plasmon resonance method at 25 °C.
  • an “antigen binding domain” may comprise or consist of an antigen binding fragment of an antibody or antibody.
  • antibody as used herein, means any antigen-binding molecule comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (eg, Her-2 protein or CTLA-4 protein) or Molecular complexes.
  • CDR complementarity determining region
  • antibody includes immunoglobulin molecules comprising four polypeptide chains (two heavy chains (H) and two light chains (L) interconnected by disulfide bonds) and multimers thereof (eg, IgM).
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH ) and a heavy chain constant region.
  • the heavy chain constant region comprises three domains: C H 1, C H 2 and C H 3.
  • Each light chain comprises a light chain variable domain (abbreviated herein as LCVR or V L) and a light chain constant region.
  • the light chain constant domain contains one domain (C L 1).
  • V L, V H regions regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs of), interposed between more conserved regions, called framework regions (FR).
  • each V H and V L made from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2 , FR3, composition CDR3, FR4 arranged three CDR's and four FR.
  • the FR of an antibody of the invention may be identical to a human germline sequence, or may be modified naturally or artificially.
  • Two or more CDR-defined amino acid consensus sequences can be defined based on side-by-side analysis.
  • the D1 and/or D2 component of the multispecific antigen binding molecule of the invention may comprise or consist of an antigen binding fragment of an intact antibody molecule.
  • the term "antigen-binding portion" of an antibody, "antigen-binding fragment” of an antibody, and the like includes any naturally occurring, enzymatically obtainable, synthetic or genetically modified polypeptide that specifically binds an antigen to form a complex or Glycoprotein. Any suitable standard technique, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of a DNA encoding an antibody variable domain and optionally an antibody constant domain, for example, an antigen-binding fragment of an antibody derived from an intact antibody molecule can be used.
  • DNA is known and/or readily available or can be synthesized, for example, from commercial sources, DNA libraries including, for example, phage antibody libraries.
  • the DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to align one or more variable domains and/or constant domains into a suitable layout, or to introduce codons, to generate cysteine residues. Base, modification, addition or deletion of amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab') 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) a dAb fragment; and (vii) a minimal recognition unit consisting of amino acid residues mimicking the hypervariable region of the antibody (eg, an independent complementarity determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 Peptide.
  • CDR independent complementarity determining region
  • the expression "antigen-binding fragment” also encompasses other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deficient antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-antibodies. , tetrabody antibodies, minibodies, nanobodies (eg, monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIP), and shark variable IgNAR domains.
  • SMIP small modular immunopharmaceuticals
  • variable domain can be of any size or amino acid composition and will typically comprise at least one CDR that is contiguous with one or more of the framework sequences or that conforms to the open reading frame.
  • binding fragment has an antigen domain L V V H domains of association, V H domains and V L, any suitable arrangement may be disposed opposite to each other.
  • the variable region may be dimeric and contain V H -V H, V H -V L or V L -V L dimer.
  • the antibody fragment may comprise the antigen binding monomeric V H or V L domains.
  • an antigen-binding fragment of an antibody can contain at least one variable domain covalently linked to at least one constant domain.
  • Non-limiting variable and constant domains may be stored in the internal binding fragment of an antibody antigen of the invention, an exemplary layout comprising: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L; (viii);) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L - C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3 ; and (xiv) V L -C H 1
  • variable domains and the constant domains may be directly connected to each other or may be connected by a full or partial hinge region or joint region.
  • the hinge region can be made up of at least 2 (eg, 5, 10, 15, 20, 40, respectively) that create a flexible or semi-flexible connection between adjacent variable domains and/or constant domains in a single polypeptide molecule. 60 or more amino acid compositions.
  • antigen-binding fragments can comprise any and / or with one or more monomers of the V H or V L domain of another variable and constant domains layout as set forth above (e.g., by disulfide bonds)) A homodimer or heterodimer (or other multimer) that is in a non-covalent association.
  • the multispecific antigen binding molecule of the invention may comprise or consist of a human antibody and/or a recombinant human antibody or fragment thereof.
  • the term "human antibody” includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • human antibodies can include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, in CDRs and, in particular, CDR3) (eg, by random mutagenesis or site-specific mutagenesis in vitro or by somatic mutation in vivo) Introduced mutation).
  • CDRs eg, in CDRs and, in particular, CDR3
  • the term "human antibody” is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the multispecific antigen binding molecule of the invention may comprise or consist of a recombinant human antibody or antigen binding fragment thereof.
  • recombinant human antibody is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into a host cell (described further below).
  • An antibody isolated from a recombinant human antibody combinatorial library (described further below), an antibody isolated from an animal (eg, a mouse) transgenic relative to a human immunoglobulin gene (see, eg, Taylor et al., (1992)) Nucl. Acids Res.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies undergo in vitro mutagenesis (or, when using a transgenic animal in terms of human Ig sequences, undergo in vivo somatic mutagenesis) and thus VH and V of the recombinant antibody L is amino acid sequence region derived from human germline although the V H and V L, and related sequences, may not naturally exist within the human antibody germline repertoire in vivo sequences.
  • the multispecific antigen binding molecule of the invention is a bispecific antibody; for example, a double comprising an antigen binding arm that specifically binds to the target molecule Her-2 and an antigen binding arm that specifically binds to the CTLA-4 protein Specific antibodies.
  • the multispecific antigen binding molecules of the invention can be constructed using methods known in the art for producing bispecific antibodies.
  • bispecific formats that can be used in the context of the present invention include, but are not limited to, for example, bispecific patterns based on scFv or diabodies, IgG-scFv fusions, dual variable domains (DVD)-Ig, cell hybridomas (Quadroma), knots, common light chain (for example, common light chain with knots, etc.), CrossMab, CrossFab, (SEED), leucine zipper, Duobody, IgG1/IgG2, dual-effect Fab (DAF) - IgG and Mab 2 bispecific patterns (for a review of the foregoing styles, see, for example, Klein et al, 2012, mAbs 4: 6, 1-11, and references cited therein).
  • the multispecific antigen binding molecules of the invention may also include one or more multimerization components.
  • the multimerization component can function to maintain association between the antigen binding domains (D1 and D2).
  • a "multimerization component" is any macromolecule, protein, polypeptide, peptide or amino acid that has the ability to associate with a second multimerization component having the same or similar structure or configuration.
  • the multimerization component can be a polypeptide comprising an immunoglobulin CH3 domain.
  • a non-limiting example of a multimerization component is the Fc portion of an immunoglobulin, for example, an Fc domain selected from the isotypes IgGl, IgG2, IgG3, and IgG4, and any allotype of IgG within each isoform group.
  • the multimerization component is an Fc fragment or amino acid sequence of from 1 to about 200 amino acids in length containing at least one cysteine residue.
  • the multimerization component is a cysteine residue or a short peptide comprising a cysteine.
  • Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.
  • the multispecific antigen binding molecule of the invention comprises two multimerization domains M1 and M2, wherein D1 is linked to M1 and D2 is linked to M2, and wherein association of M1 with M2 facilitates a single D1 and D2 in the multispecific antigen binding molecule are physically linked to each other.
  • M1 and M2 are identical to each other.
  • M1 may be an Fc domain having a specific amino acid sequence
  • M2 is an Fc domain having the same amino acid sequence as M1.
  • M1 and M2 may differ from each other at one or more amino acid positions.
  • M1 may comprise a first immunoglobulin (Ig) C H 3 domains and M2 may contain a second IgC H 3 domain, wherein the first and second differences to each other IgC H 3 domain of at least one amino acid, and wherein M1 having the same At least one amino acid difference reduces binding of the targeting construct to protein A compared to the reference construct of the M2 sequence.
  • the IgC H 3 domain of M1 binds to protein A and the IgC H 3 domain of M2 contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (according to the IMGT exon number; according to EU number H435R).
  • the CH3 of M2 may also contain a Y96F modification (according to the IMGT number; according to the EU number Y436F).
  • Other modifications that may be present in the CH3 of M2 include: D16E, L18M, N44S, K52N, V57M, and V82I in the case of the IgGl Fc domain (according to the IMGT number; according to EU numbers D356E, L358M, N384S, K392N, V397M And V422I); N44S, K52N and V82I in the case of the IgG2 Fc domain (numbered according to IMGT; N384S, K392N and V422I according to EU); and in the case of IgG4 Fc domain Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (according to IMGT number; according to EU numbers Q355R, N384S, K392N, V397M
  • a multispecific antigen binding molecule can be used to target tumor cells.
  • the multispecific antigen binding molecules of the invention can be conjugated to drugs, toxins, radioisotopes or other substances that are detrimental to cell viability.
  • the drug or toxin may be a substance that does not directly kill the cells but makes the cells more susceptible to attack by other foreign substances.
  • the multispecific antigen binding molecules of the invention are not themselves conjugated to a drug, toxin or radioisotope, but to other antigen binding molecules (referred to herein as "co-supplied molecules” ") Combination administration, such as other anti-tumor antibodies.
  • the multispecific antigen binding molecule can be conjugated to one or more cytotoxic drugs selected from the group consisting of: calicheamicin, espo Neomycin, methotrexate, doxorubicin, melphaxine, chlorambucil, ARA-C, vindesine, mitomycin C, cisplatin, etoposide, bleomycin, 5 -Fluorouracil, estramustine, vincristine, etoposide, doxorubicin, paclitaxel, larotaxel, tesitastat, orataxel, docetaxel, dolastatin 10, Australia Risstatin E, auristatin PHE and maytansin-based compounds (eg, DM1, DM4, etc.).
  • cytotoxic drugs selected from the group consisting of: calicheamicin, espo Neomycin, methotrexate, doxorubicin, melphaxine, chlorambucil, ARA-C, vinde
  • Multispecific antigen binding molecules may also or alternatively be conjugated to a toxin such as diphtheria toxin, Pseudomonasaeruginosa exotoxin A, ricin A chain, abrin A chain, eucalyptus root protein A chain, ⁇ - quercetin, oil twig (Aleuritesfordii) protein, carnation toxic protein, Phytolacaamericana protein, and the like.
  • a toxin such as diphtheria toxin, Pseudomonasaeruginosa exotoxin A, ricin A chain, abrin A chain, eucalyptus root protein A chain, ⁇ - quercetin, oil twig (Aleuritesfordii) protein, carnation toxic protein, Phytolacaamericana protein, and the like.
  • the multispecific antigen binding molecule may also or alternatively be conjugated to one or more radioisotopes selected from the group consisting of: 225 Ac, 211 At, 212 Bi, 213 Bi, 186 Rh, 188 Rh 177 Lu, 90 Y, 131 I, 67 Cu, 125 I, 123 I, 77 Br, 153 Sm, 166 Ho, 64 Cu, 121 Pb, 224 Ra and 223 Ra.
  • ADC antibody-drug conjugate
  • ARC antibody-radioisotope conjugate
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, It contains the above-described antibody or active fragment thereof or a fusion protein thereof, or the above-described multispecific antigen-binding molecule, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be used for the prevention and treatment of tumors.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the above specific binding molecule (or a conjugate thereof) of the present invention, and A pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • compositions such as injections and solutions are preferably prepared under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram body weight to about 5 milligrams per kilogram body weight per day.
  • the polypeptides of the invention may also be used with other therapeutic agents.
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the present invention has been subjected to extensive screening to obtain a human single-chain antibody (scFv) against CTLA-4, which has the advantages of high affinity and strong specificity;
  • the bispecific antibody of the present invention can deliver the anti-CTLA-4 antibody scFv to the Her-2 positive tumor region at a fixed point, so that the CTLA-4 antibody enhances the immune reaction in a local region without causing an excessive reaction of the entire immune system. Therefore, the side effects of the CTLA-4 antibody are reduced.
  • Single-chain variable region (scFv) antibodies to human anti-CTLA-4 were screened using the Phage Display Library technology.
  • PBMC peripheral blood mononuclear cell
  • the antibody heavy chain variable region gene (VH) and the light chain variable region gene (VL) were amplified by polymerase chain reaction (PCR) using the cDNA synthesized above as a template.
  • PCR primers (literature) for amplifying the VH and VL genes were designed and synthesized (GenScript). All PCR reactions were performed using a mixture of reverse primers and multiple forward primers, respectively. To amplify the VH gene, 12 different reverse primers (HVH) were used for PCR reaction with each of the four heavy chain J region forward primer (HJH) mixtures. The kappa and lamda-type VL genes were amplified in the same manner, that is, each HVkappa or HVlamda reverse primer was subjected to a PCR reaction to a corresponding HJkappa or HJlamda forward primer mixture.
  • Each PCR reaction volume was 100 ul and contained 2 ul of cDNA, 1 uM of forward or reverse primers, 200 uM of dNTPs, 5% of DMSO and 10X Pfu buffer. After the PCR reaction solution was heated at 94 ° C for 5 minutes, 5 units of Pfu (Stratagene) was added, followed by 30 cycles of reaction, each of which included denaturation at 94 ° C for 1 minute, withdrawal at 57 ° C for 1 minute, and extension at 72 ° C for 1 minute. After all the PCR reactions are completed, the PCR reaction mixture of the VH gene and the VL gene is separately mixed, and then subjected to agarose gel electrophoresis and gel purification ( Kit, Qiagen).
  • the purified VH gene and VL gene were ligated to the TA vector (Invitrogen), respectively, and the ligation product was transformed into competent XL-1 bacteria (Stratagene) by an electric shock method (GenePulser Xcell, Bio-Rad).
  • the transformed bacteria were collected in LB medium and then plated entirely into 10 150-mm LB/Agar dishes and incubated overnight at 37 °C.
  • the bacteria on the culture dish were scraped off and collected in 320 ml of LB medium, and dispensed into a 50-ml centrifuge tube, 20 ml per tube, and stored in a -80 ° C refrigerator.
  • the phage vector PIII (GenScript, USA) containing the phage coat protein PIII-encoding gene was artificially synthesized, and the multiple cloning site of this vector was located at the 5' end of the PIII gene for cloning the antibody variable region gene.
  • the 3' end of the antibody variable region gene is ligated with a His-Tag gene and an enterokinase cleavage site for purification of the scFV antibody.
  • the TA vector library of each 20 ml VH gene and VL gene was thawed, bacteria were collected by centrifugation, and a plasmid was prepared using HighPure Midi Plasmid Purification Kit (TianGen).
  • the purified VH-containing plasmid was digested with restriction endonucleases NcoI-HF and XhoI-HF, and the purified VL-containing gene plasmid was digested with restriction endonucleases NheI-HF and NotI-HF. All restriction enzymes were from New England Biolabs (NEB) and the digestion reaction was carried out overnight at 37 °C.
  • the digested plasmid was precipitated with ethanol, the plasmid was lysed in TE buffer, the plasmid was separated by agarose gel electrophoresis, and the agarose gel containing the VH or VL gene fragment was excised to purify the VH or VL gene ( Kit, Qiagen).
  • the VH gene and the VL gene were cloned into the phage vector PIII in two steps.
  • the phage vector PIII was digested with NheI-HF and NotI-HF restriction enzymes, and purified.
  • the purified vector and the purified VL gene were ligated with T4 ligase (NEB), which was ligated to the 5' end of the coat protein PIII gene, and they were in the same protein coding frame due to the design of the primers.
  • the ligation product was transformed into XL-1 competent bacteria (Stratagene) by electroporation, and the transformed bacteria were cultured on 40 150-mm LB/Agar dishes containing 2% glucose and 50 ug/ml carbenicillin.
  • the second step is to clone the VH gene into the above phage vector containing the VL gene.
  • the cloning sites NcoI and XhaoI are located at the 5' end of the VL gene, and there is a 7-15 amino acid linker coding between the VH gene and the VL gene. sequence.
  • the method of ligation and transformation was identical to the VL gene, and finally a human scFv phage display library was prepared.
  • the 5 ⁇ 1010 bacteria prepared above were inoculated into 1 liter of SB medium containing 2% glucose, 50 ug/ml carbenicillin and 20 ug/ml tetracycline, and cultured in a shaker incubator at 37 ° C until the cell density reached OD600. Between 0.5 and 0.7, 4x 1013 plaque forming units (PFU) of helper phage VCSM13 (Stratagene) and IPTG at a final concentration of 1 mM were then added. After incubating for 30 minutes at room temperature, it was diluted to 5 liters with SB medium, and incubation was continued for 2 hours at room temperature.
  • PFU plaque forming units
  • Kanamycin at a final concentration of 70 ug/ml was added and cultured overnight in a shaker incubator at 30 °C.
  • the bacteria were pelleted by centrifugation, the supernatant containing the phage was collected, transferred to a 500-ml clean centrifuge tube, and PEG 8000 (Sigma) was added to a final concentration of 4% (w/v) and NaCl to a final concentration of 3% (w/ v), precipitated phage.
  • the phage were suspended and stored in PBS (pH 7.4) containing 2% BSA.
  • the antigenic protein used for panning is the fusion protein of the extracellular domain (amino acids 37-162) of recombinant human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and the IgG constant region (R&D Systems, USA).
  • CTLA-4/Fc was dissolved in PBS, added to an immunoassay tube (Maxisorb, Nunc) at a concentration of 50 ug/ml, and incubated overnight at room temperature.
  • the immunoassay tubes were blocked with 4% semi-skimmed milk (PBS) for 1 hour at room temperature, and 1 x 1013 colony forming units of the above prepared scFv-expressing phage were added and incubated at room temperature for 2 hours (continuous shaking).
  • Unbound phages were washed away with PBS/0.1% Tween-20 and then washed with PBS. Wash 10 times each.
  • the specifically bound phage was eluted with 1 ml of eluent (100 mM NaCl, pH 2.2, 0.1% BSA) at room temperature, and then the eluate was neutralized with 60 ul of Tris (2M).
  • the above process was repeated twice with the selected phage, and the second and third rounds of panning were performed.
  • the concentrations of the fusion protein CTLA-4/Fc used in the second and third rounds of panning were 10 ug/ml and 5 ug/ml, respectively.
  • the XL-1 bacteria were cultured in SB medium to an OD600 of about 1, and the last round of selected positive phage was added and culture was continued for 1 hour in a 37 ° C shaker incubator.
  • the bacteria were pelleted by centrifugation, and the bacteria were cultured on a SB/Agar dish containing 2% glucose, 50 ug/ml carbenicillin and 20 ug/ml tetracycline, and cultured at 30 ° C overnight.
  • individual clones were selected and plasmid miniprep was prepared for scFv gene sequencing.
  • the scFv gene was subcloned from the obtained monoclonal plasmid into a bacterial expression vector (pET/Flag).
  • the scFv antibody was then expressed in BL21/DE3 bacteria and purified. Methods of expression and purification can be found in the published standard methods.
  • the in vitro binding ability of the scFv antibody and CTLA-4 was examined by ELISA (see Example 2 for specific procedures). After the ELISA plate was coated with the recombinant protein CTLA-4, different concentrations of scFV protein were added, and then the amount of scFV protein was detected by alkaline phosphatase-conjugated goat anti-human IgG Fab antibody.
  • scFv single-chain variable region
  • each FR and CDR is as follows:
  • each FR and CDR is as follows:
  • a biologically active recombinant bispecifically conjugated antibody is prepared which enhances the efficacy of Trastuzumab in the treatment of Her-2 positive breast cancer and in the treatment of Her-2 positive breast cancer resistant to Trastuzumab.
  • Figure 1 is a schematic view showing the structure of this antibody.
  • the gene sequence and protein sequence of the antibody heavy chain are shown in Figure 2A, and the gene sequence and protein sequence of the antibody light chain are shown in Figure 2B.
  • the heavy and light chain complete cDNA encoding trastuzumab was synthesized by GenScrip (USA), respectively, and the single-chain variable region (scFv) antibody sequence information of human anti-CTLA-4 was shown in Example 1, using phage display Library (PhageDisplayLibrary) technology screening obtained.
  • the present inventors have found that during the expression and preparation of trastuzumab, most of the heavy chain C-terminal lysine of the antibody is degraded, so when the bispecifically coupled antibody is constructed, the lysine is removed.
  • the acid is such that the bispecifically coupled antibody maintains integrity.
  • the Her-2 antibody heavy chain gene was amplified by polymerase chain reaction (PCR) using synthetic DNA as a substrate.
  • the Her-2 antibody sequence information was as described above, and the 5' primer was Her2-F (5'- ATTGAATTCCGCGGCCGCCACCATGGAG, SEQ ID NO.: 33), the primer at the 3' end is Her2-R (5'-TCCTGGGGACAGTGACAGTG, SEQ ID NO.: 34).
  • the scFv gene against CTLA-4 was also amplified by PCR, the 5' primer was CTLA-Fv-F (5'-CACTGTCACTGTCCCCAGGACAGGTCCAGCTGGTGCAGTC, SEQ ID NO.: 35), and the 3' primer was CTLA-Fv-R (5).
  • the two PCR fragments are ligated together.
  • the above two PCR fragments were purified by DNA gel and mixed with a NotI/XbaI double-digested mammalian cell expression vector (such as an expression plasmid containing a CMV promoter), and the Her-2 antibody heavy chain and CTLA- were in-fusion method.
  • the gene of the 4scFv antibody was ligated and cloned into an expression vector.
  • the antibody light chain cDNA was cloned into the same expression vector plasmid by subcloning.
  • the cloning enzymes are NotI and XbaI.
  • the host cell CHODG44 was obtained from Invitrogen, and the cell culture and passage methods were referred to the company's CHODG44 manual.
  • Non-transfected cells were suspension cultured in CDDG44 medium (Invitrogen) containing 8 mM L-glutamine and 5 ug/ml recombinant human insulin.
  • the method and steps for constructing an antibody protein stable expression cell line are briefly described below.
  • the antibody heavy chain and light chain expression vector plasmids were prepared using the TianGen plasmid bulking kit, and 100 ug of each plasmid DNA was digested with restriction endonuclease PuvI to linearize the plasmid.
  • DG44 cells are passed for at least three passages before the expression vector plasmid is transfected into cells.
  • the total number of DG44 cells was 1 ⁇ 10 7 , mixed with the digested plasmid in 0.8 ml of CDDG44 growth medium, transferred to a 0.4 cm electric shock cup (Bio-Rad), and electroporated with an electric transfection apparatus (Bio-Rad, GenePulserXcell). The cell/plasmid mixture was then incubated with the transfected cells in a T-75 cell culture flask and 20 ml of cell growth medium was added. T-75 square vials containing transfected cells were incubated for 24 hours at 37 ° C in an 8% CO 2 incubator.
  • Transfected cells were cultured for 24 hours, and the transfected cells were subjected to screening culture in a 96-well culture plate by limiting dilution.
  • the screening medium was OptiCHO containing 8 mM L-glutamine, 5 ug/ml recombinant human insulin and 100 nM methotrexate (MTX, Sigma).
  • the cells were cultured in an incubator at 37 ° C, 8% CO 2 .
  • the cell culture medium of each well with cell clones was analyzed by ELISA (alkaline phosphatase-conjugated goat anti-human IgG Fc antibody, Jackson ImmunoResearch Lab), and the clone with the highest expression was further amplified. Then, ELISA detection, re-amplification, and finally obtained the 12 stable cell lines with the highest expression.
  • a monoclonal cell strain with high antibody expression was selected and cultured to expand to 2 liters. The culture supernatant was used to purify the antibody. Purification methods include the Protein-A affinity layer (POROSMab Capture A, LifeTech), anion chromatography (Q-600C, TOSOH) and cationic chromatography (POROSXS, LifeTech).
  • Purification methods include the Protein-A affinity layer (POROSMab Capture A, LifeTech), anion chromatography (Q-600C, TOSOH) and cationic chromatography (POROSXS, LifeTech).
  • the purified antibody was analyzed by reduction and non-reduction SDS-PAGE electrophoresis, and subjected to HPLC-SEC (high pressure liquid phase-molecular sieve) (TSKgel G3000SWXL, TOSOH) analysis.
  • HPLC-SEC high pressure liquid phase-molecular sieve
  • Her-2 stably expressed B16 cells (mouse melanoma) were incubated with different concentrations of recombinant antibodies in vitro, and appropriate B16/Her-2 positive cells were used, and the cell density was adjusted to 3 ⁇ 10 6 with pre-cooled FACS working solution. /ml, dispense 100ul/tube, and block for 1 hour on ice. The antibody was then diluted to different concentrations with FACS working solution, and 10 ul of different concentrations of antibody was added to 100 ul of the cell suspension and incubated on ice for 30 minutes.
  • the blocking solution was discarded, and 50 ⁇ l/well of the serially diluted antibody protein was added and reacted in an incubator at 37 ° C for 1 hour.
  • the antibody protein solution was discarded, and the ELISA plate was washed three times with PBST, and 50 ul/well of a second antibody (alkaline phosphatase-conjugated goat anti-human IgG Fab antibody, Jackson ImmunoResearch Lab) was added and reacted in an incubator at 37 ° C for 1 hour.
  • the chromogenic antibody was discarded, and 200 ul/well PBST washing solution was added to the ELISA plate, and the ELISA plate was placed on a horizontal shaker for 5 minutes at a rotation speed of 100 rpm, and the washing solution was discarded. Repeat 5 times.
  • Add 50 ul/well of antibody chromogenic solution (PNPP) to the ELISA plate and place the plate in a 37 ° C incubator. The plate was read at a wavelength of 405 nm using
  • BT-474 cells (purchased from ATCC) were cultured in Dulbecco's MinimumEssential Medium (DMEM) containing 10% FBS. The cells were digested with trypsin, centrifuged, the supernatant was discarded, and the cells were suspended in a growth medium and counted. 150 ul per well was added to a 96-well cell culture plate containing 10,000 cells. Incubate overnight in a 5% CO 2 incubator at 37 °C. On the next day, recombinant antibodies or control antibodies were serially diluted with growth medium to prepare 10 different concentrations. Add 50 ul of diluted antibody or control antibody to each well in the cell plate.
  • DMEM Dulbecco's MinimumEssential Medium
  • CCK-8 cell proliferation and activity assay kit, DOJINDO
  • PBMC Peripheral blood mononuclear cells
  • RPMI1640 medium (10% FBS) at a cell density of 1 ⁇ 10 6 /ml.
  • the phytohemagglutinin (PHA) was added to a final concentration of 1 ug/ml, and the PBMC was cultured in a 5% CO 2 , 37 ° C incubator for 2 days.
  • the PBMCs were then washed once with PBS and the cells were suspended in RPMI 1640 growth medium at a density of 5 x 10 6 /ml.
  • miji cells treated with mitomycin C (Chinese cell bank) were prepared.
  • the suspended Raji cells were placed in RPMI1640 growth medium, and mitomycinC was added to a final concentration of 25 ug/ml.
  • the cells were washed 4 times with PBS, and the cells were suspended in RPMI 1640 growth medium at a density of 1 ⁇ 10 6 /ml.
  • an equal volume of PBMC and Raji cells 50 ul each cell was added to each well, and the antibody was added to a final concentration of 10 ug/ml or 30 ug/ml.
  • the cells were cultured in an incubator for 72 hours.
  • the cell culture supernatant was collected, and the IL-2 content in the supernatant was determined using an ELISA kit (Shanghai Yikesai Biological Products Co., Ltd.).
  • the scFv region of the bispecific antibody of the present invention can bind to CTLA-4, block the inhibitory effect of CTLA-4 on T cell CD28 co-stimulation, and thereby enhance the immune response of the immune system. Since the human anti-CTLA-4 antibody scFv can bind to CTLA-4 of cynomolgus monkeys, we used cynomolgus monkeys to detect the bispecific antibodies of the invention against recombinant hepatitis B vaccine HBsAg (GSK, Engerix B) in cynomolgus monkeys. The body causes an immune response.
  • cynomolgus monkeys (2-3 years old) were divided into 2 groups, each group containing 2 males and 2 females.
  • monkeys in the control group were intravenously injected with human IgG1
  • monkeys in the test group were intravenously injected with bispecific antibody at a dose of 10 mg of antibody per kilogram of monkey with an antibody protein concentration of 5 mg/ml.
  • each monkey was intramuscularly injected with 10 ug of recombinant hepatitis B vaccine.
  • day 35 and day 49 each monkey took blood to prepare plasma, and anti-HBsAg antibody titers in plasma were determined by ELISA.
  • HBsAg protein was diluted to 2 ug/ml with a coating solution (200 mM NaHCO3, pH 9.6), 50 ul per well was added to a 96-well ELISA plate, and stored in a refrigerator at 4 ° C overnight. On the next day, the ELISA plate was washed 3 times with PBST (PBS containing 0.05% Tween-20), 100 ul/well PBST blocking solution containing 3% BSA was added, and the ELISA plate was placed in an incubator at 37 ° C for 1 hour.
  • PBST PBS containing 0.05% Tween-20
  • the plasma of the same group was mixed in the same volume, and then the mixed plasma was diluted 100 times, 500 times, 2500 times and 12500 times with the binding solution (PBST containing 1% BSA).
  • the binding solution PBST containing 1% BSA.
  • the blocking solution was drained, and 50 ul/well of the diluted plasma mixture was added and incubated for 2 hours in a 37 ° C incubator.
  • the plasma solution was drained, and the ELISA plate was washed 3 times with PBST, and 50 ul/well 2000-fold diluted secondary antibody (alkaline phosphatase-conjugated goat anti-monkey IgGFc, Abcam, article number ab112765) was added and reacted in a 37 ° C incubator.
  • the second antibody was discarded, and 200 ul/well PBST washing solution was added to the ELISA plate, and the ELISA plate was placed on a horizontal shaker for 5 minutes at a rotation speed of 100 rpm, the washing solution was drained, and the washing process was repeated 5 times.
  • Add 50ul/well antibody color base The PNPP solution (Southern Biotech, China) was placed in an ELISA plate and placed in a 37 ° C incubator. The plate was read at a wavelength of 405 nm using a microplate reader (Bio-Rad).
  • the bispecific antibody was purified by Protein-A affinity chromatography column, anion column and cation column, and analyzed by reducing and non-reducing SDS-PAGE gel (Fig. 3).
  • Figure 3A shows that the molecular weight of the intact bispecific antibody is greater than about 200 kDa, very close to its theoretical value, 196 kDa.
  • the reduced SDS-PAGE gel showed that the heavy chain molecular weight of the bispecific antibody bispecific antibody was 75-80 kDa, which was also consistent with its theoretical molecular weight (74.4 kDa).
  • the affinity binding ability of the bispecific antibody to CTLA-4 was examined by ELISA.
  • the bispecific antibody specifically binds to CTLA-4 with an EC50 of 0.19 nM and a binding capacity to Ipilimumab (EC50 of 0.16 nM) ( Figure 6).
  • trastuzumab inhibited the growth of Her-2 positive breast cancer BT-474 cells in vitro, thus detecting the ability of bispecific antibodies to inhibit the growth of BT-474 cells (Fig. 7).
  • the results show that bispecific antibodies can inhibit the growth of BT-474 cells.
  • Its IC50 is 0.97 nM, which is similar to the IC50 (0.69 nM) of trastuzumab.
  • the purpose of this experiment was to demonstrate that the anti-Her-2 region of the bispecific antibody of the present invention has the ability to inhibit the growth of BT-474 cells.
  • bispecific antibody of the present invention is a coupling of two antibodies, they may interact with each other, such as affecting the formation of the correct spatial structure of the other party, or the binding of the other party to the corresponding antigen. This experiment demonstrates that each antigen-binding unit contained in the bispecific antibody of the present invention retains its respective activity.
  • anti-CTLA-4 antibody When anti-CTLA-4 antibody binds to CTLA-4, it blocks the negative regulatory signal of CTLA-4 on T cell activation, thus maintaining or enhancing the activation of T cells and increasing the expression of various intercellular substances, including IL-2. .
  • the present inventors tested the effect of bispecific antibodies on the expression of PHA-activated human PBMCIL-2, and found that bispecific antibodies can significantly increase IL-2 expression of PBMC compared to the experimental group without antibody (Fig. 8, with The control group increased by about 2 times). This result proves that bispecific antibodies can protect Hold or enhance the activation status of T cells.
  • Bispecific antibody enhances immune response of cynomolgus monkeys to HBsAg
  • the average titer of anti-HBsAg antibodies in plasma of cynomolgus monkeys after day 49 of vaccination was measured by ELISA, and the results showed that the plasma antibody titers of the vaccinated monkeys were nearly 10,000-fold (data not shown).
  • the anti-HBsAg antibody content of each monkey was detected by the same method. After the 7th week of vaccination, 2 of the 4 monkeys in the experimental group had an antibody content of 8000 units/ml, and 1 was close to 5000 units/ml. 1 is only 700 units / ml. In the control group of 4 monkeys, the antibody content of 3 was close to 1500 units/ml or less than 1000 units/ml, and only 1 monkey had an antibody content of 5000 units/ml (Fig. 9). Comparing the antibody contents of these two groups of monkeys, the results indicate that the bispecific antibody of the present invention has a function of enhancing the immune response caused by the vaccine in vivo.

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Abstract

提供一种双特异性偶联抗体及其制法和用途。具体地，提供了抗CTLA-4的人源单链抗体(scFv)，该抗CTLA-4抗体具有亲和力高、特异性强的优点。使用抗CTLA-4抗体构建重组双特异性偶联抗体(人源化抗Her-2全抗体(Trastuzumab)与抗CTLA-4的人源单链抗体(scFv，single-chain antibody fragment)融合)，能够显著增强机体的免疫反应，能够保持或增强T细胞的激活状态。

Description

双特异性偶联抗体及其制法和用途 技术领域
本发明属于是生物医药领域，具体地为双靶点单克隆抗体抗肿瘤免疫治疗领域。
背景技术
Her-2是由致癌基因HER-2编码的表皮生长因子受体跨膜蛋白，受体蛋白的胞内段具有酪氨酸激酶活性。在Her-2形成同源二聚体或与其家簇其它受体成员形成异源二聚体时，胞内段的酪氨酸激酶被激活，然后激活多条胞内信号通道。其中最主要的信号通道具有刺激细胞生长和分裂，以及抗自身凋亡和维持细胞生存的功能。在近三分之一的乳腺癌患者的癌细胞里发现HER-2基因被大量复制或突变，使Her-2蛋白过量表达或持续激活，造成细胞癌变。以Her-2为靶点，具有抑制Her-2活性的曲妥珠单克隆抗体(Trastuzumab)被开发出来，是第一个被批准用于治疗Her-2阳性乳腺癌患者的人源化单克隆抗体。11-26％的恶性乳腺癌患者对单一曲妥珠单抗药物治疗有疗效，如果和化疗联合使用，早期和晚期的乳腺癌患者的治愈率和生存率都会显著的提高。虽然有如此好的疗效，但仍有相当一部分Her-2阳性的病人对曲妥珠单克没有反应，而且那些早期治疗有反应的病人，其中大部分会在后期治疗时产生对曲妥珠单抗治疗的抗性。
曲妥珠单抗抑制肿瘤的活性，不仅取决于抑制Her-2的活性，还取决于曲妥珠单抗诱导的抗体依赖性细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity,ADCC)。不能结合IgG Fc受体(FcR)的抗Her-2抗体被证明没有抗肿瘤效果，在FcR基因缺失的小鼠里，曲妥珠单抗被证明也没有疗效，另外，在曲妥珠单抗治疗的病人乳腺肿瘤切片里发现FcR阳性免疫细胞增加，主要是自然杀伤细胞(NK)。这些数据说明了先天免疫系统ADCC的活性是曲妥珠单抗抑制肿瘤机制之一。不仅如此，获得性免疫在曲妥珠单抗抑制肿瘤效果中也起作很重要的作用。在具有免疫活性的小鼠Her-2阳性乳腺肿瘤模型里，为了达到更好的抗肿瘤效果，抗Her-2抗体需要CD8α+细胞，MyoD88信号通道和RAG依赖的获得性免疫活性。进一步的研究证明，传统的淋巴细胞介导的细胞间素毒性I型干扰素(INF-I)和gamma干扰素(INFγ)对曲妥珠单抗抑制肿瘤的效果很重要，其中CD8α+T细胞产生的INFγ起着关键的作用。重组INFγ能直接抑制Her-2阳性肿瘤细胞的体外生长，而且能显著增强抗Her-2抗体抑制细胞生长的能力。另外，INFγ显著增加I型MHC在肿瘤细胞表面的表达。综合这些数据，解释了INFγ在曲妥珠单抗抑制肿瘤效果中的重要性。
既然获得性免疫活性在曲妥珠单抗抑制肿瘤效果中有如此的重要性，增强免疫细胞的激活或者维持免疫细胞的激活，都会加强曲妥珠单抗抑制肿瘤的效果。事实上，有研究发现，曲妥珠单抗与抗CD137单抗联合使用，会加强淋巴细胞的存活，特别是CD8+T细胞的存活能力。CD137抗体还能增强NK细胞和DC细胞的激活和功 能，通过FcR+细胞和获得性免疫细胞加强抗Her-2抗体的抗肿瘤效果。另外，利用细胞程序死亡-1(PD-1)抗体阻断PD-1通道，能显著增加曲妥珠单抗诱导的获得性免疫T细胞抗肿瘤活性。
T细胞的激活不仅需要抗原呈现细胞(APCs)上的MHC-多肽复合物对T细胞受体的刺激，还需要共刺激信号和抑制信号之间的平衡。多个机制相互作用共同调解CD4+和CD8+T淋巴细胞的激活，包括扩增或抑制T细胞反应的细胞表面蛋白。T细胞表面上的负调节蛋白，比如CTLA-4，PD-1，B7家族分子B7-H4，T细胞免疫分子和淋巴细胞激活基因-3(LAG-3)，和在不同类型细胞上它们的配体结合，导致T细胞生长和功能活性的下降。为了有效的控制激活的免疫细胞和产生有效的免疫反应，机体有必要对这些免疫检查点负调节蛋白的表达进行严格的调控。在癌症和一些病毒感染中常见的慢性抗原暴露会使抗原特异性T淋巴细胞持续表达CTLA-4，PD-1和LAG-3这类分子，导致邻近细胞产生对抗原耐受性。因此，以免疫检查点负调节蛋白为靶点的药物开发正在受到极大的关注。
CD28分子是持续表达的属于免疫球蛋白家族的一个蛋白，它介导T细胞共刺激信号。CD28结合APC上的陪体B7-1和B7-2，诱导白介素-2(IL-2)和抗细胞凋亡因子的产生，从而刺激T细胞的生长。T细胞被激活后，在细胞表面产生CTLA-4，它和CD28分子非常类似，可以结合B7-1和B7-2，而且结合的力度和亲和力更大(特别是和B7-1的结合)。因此，很微量的CTLA-4分子就能有效的和CD28竞争结合其陪体，降低T细胞反应。另外，CTLA-4还有其内在和外在的作用机理，包括从免疫突触体清除PKC-beta和CARMA1的区域化，限制T细胞的存在时间和增强T调节细胞(Tregs)功能。总的来说，这些机制抑制细胞生长进程，抑制IL-2的产生和细胞生存途径，最终导致免疫反应终止。CTLA-4基因敲除小鼠实验很好地证明了CTLA-4作为负调节分子的重要性。CTLA-4缺陷型小鼠在淋巴结，脾脏和几个外周器官表现CD28依赖的大规模自身反应性T细胞扩张。这些小鼠由于扩散性的淋巴生长导致的疾病，在出生不到4周后死亡。CTLA-4作为T细胞反应的一个主要免疫检查点分子，它对获得性免疫反应的记忆形成也有影响。最近研究发现，阻断CTLA-4可以减少专业多功能的CD4+T记忆细胞数，进而调节总体记忆细胞池的质量。另外一个体内的研究发现，注射CTLA-4抗体可以增加CD8+T记忆细胞扩增，然后积累能产生INFg和TNFa的免疫细胞数。
利用不同的CTLA-4单克隆抗体研究已经证明，选择性的阻断CTLA-4能增强内部的或诱导的抗肿瘤免疫反应，这为在临床上以CTLA-4为靶点的单克隆抗体开发提供了支持。2个完全人源抗体，Ipilimumab和Tremelimumab，都可结合CTLA-4并阻断其与B7陪体的结合，进而增强T细胞的激活和增殖。Ipilimumab和Tremelimumab在过去的10年里，进行了大量的针对黑色素瘤治疗的临床研究，并在2011年，Ipilimumab被美国，欧盟和澳大利亚药监局批准用于治疗不能手术切除或转移性的黑色素瘤。CTLA-4阻断提供了一个新的免疫系统调节模式，它代表了肿瘤治疗领域中一个里程碑式的发现。目前，CTLA-4单克隆抗体治疗正在多个类型肿瘤的临床试验中进行，包括黑色素瘤，小细胞肺癌，非小细胞肺癌，以及前列腺癌。
发明内容
本发明的目的在于提供一种双特异性偶联抗体及其制法和用途。
本发明的第一方面，提供了一种抗体的重链可变区，所述的重链可变区包括以下三个互补决定区CDR：
SEQ ID NO:5所示的CDR1，
SEQ ID NO:6所示的CDR2，和
SEQ ID NO:7所示的CDR3。
在另一优选例中，所述重链可变区具有SEQ ID NO:4所示的氨基酸序列。
本发明的第二方面，提供了一种抗体的重链，所述的重链具有本发明第一方面所述的重链可变区和重链恒定区。
在另一优选例中，所述的重链恒定区为人源或鼠源的。
本发明的第三方面，提供了一种抗体的轻链可变区，所述轻链可变区具有选自下组的互补决定区CDR：
SEQ ID NO:11所示的CDR1’，
SEQ ID NO:12所示的CDR2’，和
SEQ ID NO:13所示的CDR3’。
在另一优选例中，所述的轻链可变区具有SEQ ID NO:10所示的氨基酸序列。
本发明的第四方面，提供了一种抗体的轻链，所述的轻链具有本发明第三方面所述的轻链可变区和轻链恒定区。
在另一优选例中，所述轻链的恒定区为人源或鼠源的。
本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明第一方面所述的重链可变区；和/或
(2)如本发明第三方面所述的轻链可变区。
在另一优选例中，所述抗体具有：如本发明第二方面所述的重链；和/或如本发明第三方面所述的轻链。
在另一优选例中，所述的抗体为特异性抗CTLA-4蛋白的抗体。
在另一优选例中，所述的抗体包括：单链抗体、双链抗体、单克隆抗体、嵌合抗体(如人鼠嵌合抗体)、鼠源抗体、人源化抗体、双特异性抗体(BiTE)以及嵌合抗原受体抗体(CAR)。
本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的的轻链、或如本发明第五方面所述的抗体的序列；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白特异性地与CTLA-4蛋白结合。
本发明的第七方面，提供了一种多特异性抗原结合分子，所述多特异性抗 原结合分子包含：
第一抗原结合结构域(D1)；和
第二抗原结合结构域(D2)；
其中，D1特异性结合靶分子Her-2蛋白；
D2特异性结合靶分子CTLA-4蛋白。
在另一优选例中，所述多特异性抗原结合分子对于Her-2蛋白阳性肿瘤的抑制能力高于针对Her-2蛋白的单特异性抗原结合分子对于Her-2蛋白阳性肿瘤的抑制能力。
在另一优选例中，所述多特异性抗原结合分子为多肽。
在另一优选例中，所述多特异性抗原结合分子为抗体。
在另一优选例中，所述多特异性抗原结合分子是双特异性抗体。
在另一优选例中，所述D1为特异性结合Her-2蛋白的抗体或抗体片段。
在另一优选例中，所述D2为特异性结合CTLA-4蛋白的抗体或抗体片段。
在另一优选例中，所述D1和/或所述D2为单链抗体(scFv)。
在另一优选例中，所述多特异性抗原结合分子中，仅D2为单链抗体(scFv)。
在另一优选例中，所述D1和所述D2通过连接肽相连，所述连接肽包括抗体恒定区序列。
在另一优选例中，所述D1为抗Her-2人源化抗体。
在另一优选例中，所述D2为抗CTLA-4单链抗体(scFv)。
在另一优选例中，D1为抗Her-2人源化抗体，D2为抗CTLA-4单链抗体(scFv)，D2连接在D1的重链恒定区末端。
在另一优选例中，所述抗Her-2人源化抗体包含选自下组的至少一个重链可变区的互补决定区CDR：
SEQ ID NO:18所示的CDRa，
SEQ ID NO:19所示的CDRb，和
SEQ ID NO:20所示的CDRc。
在另一优选例中，所述抗Her-2人源化抗体包含SEQ ID NO.:15所示的重链。
在另一优选例中，所述抗Her-2人源化抗体包含选自下组的至少一个轻链可变区的互补决定区CDR’：
SEQ ID NO:21所示的CDRa’，
SEQ ID NO:22所示的CDRb’，和
SEQ ID NO:23所示的CDRc’。
在另一优选例中，所述抗Her-2人源化抗体包含SEQ ID NO.:17所示的轻链。
在另一优选例中，所述抗CTLA-4单链抗体(scFv)包含选自下组的至少一个重链可变区的互补决定区CDR：
SEQ ID NO:5所示的CDR1，
SEQ ID NO:6所示的CDR2，和
SEQ ID NO:7所示的CDR3。
在另一优选例中，所述抗CTLA-4单链抗体(scFv)包含SEQ ID NO.:4所示的重链可变区。
在另一优选例中，所述抗CTLA-4单链抗体(scFv)包含选自下组的至少一个轻链可变区的互补决定区CDR’：
SEQ ID NO:11所示的CDR1’，
SEQ ID NO:12所示的CDR2’，和
SEQ ID NO:13所示的CDR3’。
在另一优选例中，所述抗CTLA-4单链抗体(scFv)包含SEQ ID NO.:10所示的轻链可变区。
在另一优选例中，所述抗CTLA-4单链抗体(scFv)序列如SEQ ID NO.:24所示。
本发明的第八方面，提供了一种多核苷酸，它编码选自下组的多肽：
(1)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的的轻链、或如本发明第五方面所述的抗体；或
(2)如本发明第六方面所述的重组蛋白、或如本发明第七方面所述的多特异性抗原结合分子。
本发明的第九方面，提供了一种载体，它含有本发明第八方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明的第十方面，提供了一种遗传工程化的宿主细胞，它含有本发明第九方面所述的载体或基因组中整合有本发明第八方面所述的多核苷酸。
本发明第十一方面所述的一种免疫偶联物，该免疫偶联物含有：
(a)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的的轻链、或如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、或如本发明第七方面所述的多特异性抗原结合分子；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
在另一优选例中，所述偶联物部分选自：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))、化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
本发明的第十二方面，提供了一种药物组合物，它含有：
(i)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的的轻链、或如 本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、或如本发明第七方面所述的多特异性抗原结合分子、或如本发明第十一方面所述的免疫偶联物；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为注射剂型。
在另一优选例中，所述的药物组合物用于制备治疗肿瘤的药物，所述的肿瘤选自下组：胃癌、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、肾上腺肿瘤、或膀胱肿瘤。
本发明的第十三方面，提供了如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的的轻链、或如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、或如本发明第七方面所述的多特异性抗原结合分子、或如本发明第十一方面所述的免疫偶联物的用途，用于制备药剂、试剂、检测板或试剂盒；
所述试剂、检测板或试剂盒用于：检测样品中CTLA-4蛋白；
所述药剂用于治疗或预防表达CTLA-4蛋白的肿瘤。
在另一优选例中，所述肿瘤包括：胃癌、淋巴瘤、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、或肾上腺肿瘤。
在另一优选例中，所述肿瘤选自：胃癌和滤泡性淋巴瘤。
在另一优选例中，所述的试剂包括芯片、包被抗体的免疫微粒。
本发明的第十四方面，提供了一种检测样品中CTLA-4蛋白的方法，所述方法包括步骤：
(1)将样品与本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CTLA-4蛋白。
本发明的第十五方面，提供了一种重组多肽的制备方法，该方法包含：
(a)在适合表达的条件下，培养本发明第十方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、或如本发明第七方面所述的多特异性抗原结合分子。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了双特异性偶联抗体结构示意图，结构图的上部分是曲妥珠单抗， 重链和轻链的可变区用不同的颜色表示。结构图的下部分是人源抗CTLA-4单克隆抗体的scFv，scFv抗体的重链可变区与曲妥珠单抗重链C端连接，在scFv重链和轻链可变区之间是一个7个氨基酸的连接肽。
图2显示了双特异性偶联抗体核苷酸序列和氨基酸序列。图2(A)重链核苷酸序列和氨基酸序列；图2(B)轻链核苷酸序列和氨基酸序列。图2(A)aa1-19是蛋白穿膜信号肽；aa20-468是人源化抗Her-2单克隆抗体(曲妥珠单抗)重链；aa469-586是人抗CTLA-4scFv重链可变区；aa587-593是人抗CTLA-4scFv重链和轻链可变区的连接肽；aa594-702是人抗CTLA-4scFv轻链可变区。图2(B)aa1-20是蛋白穿膜信号肽；aa21-234是人源化抗Her-2单克隆抗体(曲妥珠单抗)轻链。
图3显示了双特异性偶联抗体的SDS-PAGE电泳分析，A：非还原6％SDS-PAGE电泳分析，3ug/样品；B：还原10％SDS-PAGE电泳分析，5ug/样品。泳道1)人IgG1抗体；泳道2)双特异性偶联抗体。最左边的泳道是蛋白分子量标准(kDa)。
图4显示了HPLC-SEC分析纯化的双特异性偶联抗体，双特异性偶联抗体用Protein-A亲和层析柱纯化后，经过阴离子层析柱，流穿液再用阳离子柱分离。蓝色是蛋白分子量标准，红线是曲妥珠单抗峰图，绿色峰是双特异性偶联抗体。
图5显示了流式细胞技术分析双特异性偶联抗体与细胞膜Her-2的结合，Her-2稳定转染的小鼠黑色素瘤细胞(B16)于不同浓度抗体孵育后，用FITC标记的羊抗人IgG1Fc抗体检测结合Her-2的抗体，FITC萤光强度用流式细胞仪检测。Mock是无抗体的实验组。
图6显示了ELISA分析双特异性偶联抗体与CTLA-4体外结合，重组人CTLA-4-Fc融合蛋白包被于96-孔ELISA板里，与CTLA-4-Fc结合的抗体用羊抗人IgG1Fab抗体检测，羊抗人IgG1Fab抗体是碱性磷酸酶标记。
图7显示了Her-2阳性肿瘤细胞生长抑制研究，BT-474细胞与不同浓度的抗体共同培养4天后，进行检测。
图8显示了增强激活的PBMC表达IL-2，从2个人供体分离PBMC，用PHA激活后，与Raji细胞和双特异性抗体共同培养72个小时。离心沉淀细胞，收集上清，用ELISA法测上清中IL-2的含量。
图9显示注射HBsAg疫苗和双特异性抗体或对照IgG1蛋白猴子的血浆中HBsAg抗体的含量。抗体含量用ELISA方法检测。在96-孔ELISA板中包被HBsAg蛋白，然后与1000倍稀释的血浆孵育，用碱性磷酸酶标记的羊抗猴IgG Fc的抗体检测与HBsAg结合的抗体。
具体实施方式
本发明人通过广泛而深入的研究，获得一种抗CTLA-4的人源单链抗体(scFv)，该抗CTLA-4抗体，具有亲和力高、特异性强的优点。在此基础上，本发明人意外地发现，使用抗CTLA-4抗体构建重组双特异性偶联抗体(人源化抗Her-2全抗体(Trastuzumab)与抗CTLA-4的人源单链抗体(scFv，single-chainantibodyfragment)融合)，能够显著增强机体的免疫反应，能够保持或增强T细胞的激活状态，并且能够增强Trastuzumab治疗Her-2阳性乳腺癌的疗效，以及治疗对Trastuzumab起抗性的Her-2阳性乳腺癌。本发明 的双特异性偶联抗体不局限于Her-2阳性乳腺癌的治疗，也包括其它种类的Her-2阳性恶性肿瘤的治疗。
在具体描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
CTLA-4蛋白
本发明提供包含第一抗原结合结构域(D1)和第二抗原结合结构域(D2)的多特异性抗原结合分子，其中抗原结合结构域D2特异性结合CTLA-4蛋白。
在一优选的实施方式中，所述CTLA-4蛋白胞外段的氨基酸序列如下：
Figure PCTCN2016110438-appb-000001
Her-2蛋白
在本发明的上下文中，多特异性抗原结合分子的D1组分特异性结合靶分子Her-2蛋白的胞外配体结合区。
Her-2蛋白属于表皮生长因子受体家族(EGFR)，由胞外的配体结合区、单链跨膜区及胞内的蛋白酪氨酸激酶区三部分组成。配体结合区域通过与其配体结合，把细胞外的信号传递到细胞内，激活EGFR信号通道，促进细胞癌变的发生。
在一优选的实施方式中，所述Her-2蛋白胞外段的氨基酸序列如下：
Figure PCTCN2016110438-appb-000002
抗CTLA-4蛋白抗体
本发明提供了一种抗CTLA-4的抗体，该抗体的重链可变区包括以下三个互补决定区CDR：
Figure PCTCN2016110438-appb-000003
在另一优选例中，所述重链可变区具有SEQ ID NO:4所示的氨基酸序列：
Figure PCTCN2016110438-appb-000004
Figure PCTCN2016110438-appb-000005
其编码核苷酸序列为：
Figure PCTCN2016110438-appb-000006
在本发明的一个优选地实施方式中，所述抗CTLA-4抗体轻链可变区具有选自下组的互补决定区CDR：
Figure PCTCN2016110438-appb-000007
在另一优选例中，所述的抗CTLA-4抗体轻链可变区具有SEQ ID NO:10所示的氨基酸序列：
Figure PCTCN2016110438-appb-000008
其编码核苷酸序列为：
Figure PCTCN2016110438-appb-000009
在本发明的一个优选地实施方式中，所述抗CTLA-4抗体为单链抗体，其重链可变区和轻链可变区之间任选地具有连接肽，所述连接肽长度优选为1-15个氨基酸，更优地为3-10个氨基酸；较佳地所述连接肽的氨基酸序列如下：
Figure PCTCN2016110438-appb-000010
多特异性抗原结合分子
本发明人令人惊讶地发现，本发明构建的包含特异性结合靶分子Her-2蛋白结合结构域和特异性结合靶分子CTLA-4蛋白的结合结构域的多特异性抗原结合分子，对于Her-2阳性的肿瘤具有显著的抑制作用，而且能够增强曲妥珠单抗(Trastuzumab)对Her-2阳性的肿瘤的疗效，以及治疗对曲妥珠单抗起抗性的Her-2阳性肿瘤，能够用作曲妥珠单抗的增敏剂。
因此，本发明提供包含第一抗原结合结构域(在本文中也称作“D1”)和第二抗原结合结构域(在本文中也称作“D2”)的多特异性抗原结合分子，其中D1特异性结合靶分子Her-2蛋白；D2特异性结合靶分子CTLA-4蛋白。
根据本发明，多特异性抗原结合分子可以是单个多功能多肽，或它可以是彼此共价或非共价结合的两个或更多个多肽的多聚体复合物。如将因本公开而变得明显，将具有同时结合Her-2和CTLA-4分子的能力的任何抗原结合性构建体视为多特异性抗原结合分子。
本发明的任何多特异性抗原结合分子或其变体可以使用标准分子生物学技术(例如，重组DNA和蛋白质表达技术)构建，如本领域普通技术人员将知晓。
在本发明的一个优选地实施方式中，所述特异性结合靶分子Her-2蛋白结合结构域包含选自下组的至少一个重链可变区的互补决定区CDR：
Figure PCTCN2016110438-appb-000011
在另一优选例中，所述特异性结合靶分子Her-2蛋白结合结构域包含SEQ ID NO.:15所示的重链:
Figure PCTCN2016110438-appb-000012
其编码核苷酸序列为如SEQ ID NO.:14所示。
在另一优选例中，所述特异性结合靶分子Her-2蛋白结合结构域包含选自下组的至少一个轻链可变区的互补决定区CDR’：
Figure PCTCN2016110438-appb-000013
在另一优选例中，所述特异性结合靶分子Her-2蛋白结合结构域包含SEQ ID NO.:17所示的轻链：
Figure PCTCN2016110438-appb-000014
其编码核苷酸序列为如SEQ ID NO.:16所示。
在本发明的另一个优选地实施方式中，所述特异性结合靶分子CTLA-4蛋白的结合结构域包含选自下组的至少一个重链可变区的互补决定区CDR：
SEQ ID NO:5所示的CDR1，
SEQ ID NO:6所示的CDR2，和
SEQ ID NO:7所示的CDR3。
在另一优选例中，所述特异性结合靶分子CTLA-4蛋白的结合结构域包含SEQ ID NO.:4所示的重链可变区。
在另一优选例中，所述特异性结合靶分子CTLA-4蛋白的结合结构域包含选自下组的至少一个轻链可变区的互补决定区CDR’：
SEQ ID NO:11所示的CDR1’，
SEQ ID NO:12所示的CDR2’，和
SEQ ID NO:13所示的CDR3’。
在另一优选例中，所述特异性结合靶分子CTLA-4蛋白的结合结构域包含包含SEQ ID NO.:10所示的轻链可变区。
在另一优选例中，所述特异性结合靶分子CTLA-4蛋白的结合结构域为抗CTLA-4单链抗体(scFv)，其序列如SEQ ID NO.:24所示。
Figure PCTCN2016110438-appb-000015
在本发明的另一个优选地实施方式中，所述多特异性抗原结合分子中，所述抗CTLA-4单链抗体连接于曲妥珠单抗重链的C端。
抗原结合结构域
本发明的多特异性抗原结合分子包含至少两个独立的抗原结合结构域(D1和D2)。如本文所用，表述“抗原结合结构域”意指能够特异性结合特定目的 抗原的任何肽、多肽、核酸分子、支架型分子、肽展示分子或含多肽的构建体。
如本文所用，术语“特异性结合”等意指抗原结合结构域与以500pM或更小的解离常数(KD)为特征的特定抗原形成复合物，并且在普通测试条件下不结合其他不相关的抗原。优选地，“不相关的抗原”是彼此具有小于95％氨基酸同一性的蛋白质、肽或多肽。
可以在本发明上下文中使用的抗原结合结构域的示例性分类包括抗体、抗体的抗原结合部分、与特定抗原特异性相互作用的肽(例如，肽体(peptibody))、与特定抗原特异性相互作用的受体分子，包含特异性结合特定抗原的受体的配体结合部分的蛋白质、抗原结合支架(例如，DARPin，HEAT重复序列蛋白、ARM重复序列蛋白、三角形四肽重复序列蛋白和基于天然存在性重复序列蛋白的其他支架等[见，例如，Boersma和Pluckthun,2011,Curr.Opin.Biotechnol.22:849-857，和其中引用的参考文献])和适配体或其部分。
用于确定两个分子彼此是否特异性结合的方法是本领域熟知的并且例如包括平衡透析法、表面等离子体共振法等。例如，如本发明上下文中所用，抗原结合结构域包括如在表面等离子体共振测定法中所述测量，以小于约500pM、小于约400pM、小于约300pM、小于约200pM、小于约100pM、小于约90pM、小于约80pM、小于约70pM、小于约60pM、小于约50pM、小于约40pM、小于约30pM、小于约20pM、小于约10pM、小于约5pM、小于约4pM、小于约2pM、小于约1pM、小于约0.5pM、小于约0.2pM、小于约0.1pM，或小于约0.05pM的KD结合特定抗原或其部分的多肽。
如本文所用，术语“表面等离子体共振”指允许例如使用BIAcoreTM系统(GEHealthcare的BiacoreLifeSciences部门，Piscataway，NJ)，通过检测生物传感器基质内部蛋白质浓度的改变来分析实时相互作用的光学现象。
如本文所用，术语“KD”意指特定蛋白质-蛋白质相互作用(例如，抗体-抗原相互作用)的平衡解离常数。除非另外指明，否则本文中公开的KD值指通过表面等离子体共振测定法在25℃确定的KD值。
抗体和抗体的抗原结合片段
如上文所示，“抗原结合结构域”(D1和/或D2)可以包含抗体或抗体的抗原结合片段或由其组成。如本文所用，术语“抗体”意指包含与特定抗原(例如，Her-2蛋白或CTLA-4蛋白)特异性结合或与之相互作用的至少一个互补决定区(CDR)的任何抗原结合分子或分子复合物。术语“抗体”包括包含4条多肽链(由二硫键相互连接的两条重链(H)和两条轻链(L))的免疫球蛋白分子以及其多聚体(例如，IgM)。每条重链包含重链可变区(本文中缩写为HCVR或VH)和重链恒定区。重链恒定区包含3个结构域：CH1、CH2和CH3。每条轻链包含轻链可变域(本文中缩写为LCVR或VL)和轻链恒定区。轻链恒定域包含一个结构域(CL1)。VH区和VL区可以进一步再划分为超变区，名为互补性决定区(CDR)，其间插有更保守的区域，名为框架区(FR)。每个VH和VL由从氨基端至羧基端按以下顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4排列的3个CDR和4个FR的组成。在本发明的不同实施方案中，本发明抗体的(或其抗原结合部分)的FR可以与人种系序列相同，或可以经天然或人工修饰。可以基于并排分析两个或 更多个CDR限定氨基酸共有序列。
本发明的多特异性抗原结合分子的D1和/或D2组分可以包含完整抗体分子的抗原结合片段或由其组成。如本文所用，术语抗体的“抗原结合部分”、抗体的“抗原结合片段”等包括特异性结合抗原以形成复合物的任何天然存在的、酶促可获得的、合成性或基因修饰的多肽或糖蛋白。可以使用任何合适的标准技术如蛋白酶解消化或涉及操作并表达编码抗体可变域和任选地抗体恒定域的DNA的重组基因工程技术，例如，从完整抗体分子衍生抗体的抗原结合片段。这类DNA是已知的和/或从例如商业来源、DNA文库(包括例如，噬菌体抗体文库)轻易可获得或可以合成。可以对所述DNA测序并化学地或通过使用分子生物学技术操作，例如以将一个或多个可变域和/或恒定域排列成合适布局，或以引入密码子、产生半胱氨酸残基、修饰、添加或缺失氨基酸等。抗原结合片段的非限制性例子包括：(i)Fab片段；(ii)F(ab')2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段；和(vii)由模拟抗体高变区的氨基酸残基组成的最小识别单位(例如，独立的互补性决定区(CDR)如CDR3肽)或受约束的FR3-CDR3-FR4肽。如本文所用，表述“抗原结合片段”内部也涵盖其他工程化分子，如结构域特异性抗体，单结构域抗体，结构域缺失抗体，嵌合抗体，CDR移植抗体、双体抗体、三体抗体、四体抗体、微型抗体、纳米体(例如单价纳米体、双价纳米体等)、小模块免疫药物(SMIP)和鲨鱼可变IgNAR域。
抗体的抗原结合片段一般将包含至少一个可变域。可变域可以具有任何尺寸或氨基酸组成并且通常将包含与一个或多个框架序列毗邻或符合可读框的至少一个CDR。在具有与VL结构域缔合的VH结构域的抗原结合片段中，VH和VL结构域可以按任何合适的排列彼此相对设置。例如，可变区可以是二聚体并含有VH-VH、VH-VL或VL-VL二聚体。可选地，抗体的抗原结合片段可以含有单体性VH或VL结构域。
在某些实施方案中，抗体的抗原结合片段可以含有与至少一个恒定域共价连接的至少一个可变域。可以在本发明抗体的抗原结合片段内部存的可变域和恒定域的非限制、示例性布局包括：(i)VH-CH1；(ii)VH-CH2；(iii)VH-CH3；(iv)VH-CH1-CH2；(v)VH-CH1-CH2-CH3；(vi)VH-CH2-CH3；(vii)VH-CL；(viii)；)VL-CH1；(ix)VL-CH2；(x)VL-CH3；(xi)VL-CH1-CH2；(xii)VL-CH1-CH2-CH3；(xiii)VL-CH2-CH3；和(xiv)VL-CL。在可变域和恒定域的任何布局中，包括上文所列的任何示例性布局，可变域和恒定域可以彼此直接连接或可以由完整或部分的铰链区或接头区连接。铰链区可以由在单个多肽分子中相邻可变域和/或恒定域之间产生柔性连接或半柔性连接的至少2个(例如，5个、10个、15个、20个、40个、60个或更多个)氨基酸组成。另外，抗原结合片段可以包含具有上文所列的任何可变域和恒定域布局的彼此和/或与一个或多个单体性VH或VL结构域(例如，通过二硫键))处于非共价缔合的同型二聚体或异二聚体(或其他多聚体)。
本发明的多特异性抗原结合分子可以包含人抗体和/或重组人抗体或其片段或由其组成。如本文所用，术语“人抗体”包括具有源自人种系免疫球蛋白序列的可变区和恒定区的抗体。然而，人抗体可以包括不由人种系免疫球蛋白序列编码的(例如在CDR和尤其CDR3中)的氨基酸残基(例如，通过体外随机诱变或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，如本文所用， 术语“人抗体”不意在包括其中已经将从另一个哺乳动物物种(如小鼠)的种系衍生的CDR序列移植到人框架序列上的抗体。
本发明的多特异性抗原结合分子可以包含重组人抗体或其抗原结合片段或由其组成。如本文所用，术语“重组人抗体”意在包括通过重组手段所制备、表达、产生或分离的全部人抗体，如使用转染至宿主细胞(在下文进一步描述)中的重组表达载体表达的抗体、从重组人抗体组合文库(在下文进一步描述)分离的抗体、从相对于人免疫球蛋白基因而言转基因的动物(例如，小鼠)分离的抗体(见例如，Taylor等人,(1992)Nucl.AcidsRes.20:6287-6295)或通过涉及将人免疫球蛋白基因序列剪接至其他DNA序列的任何其他手段所制备、表达、产生或分离的抗体。此类重组人抗体具有源自人种系免疫球蛋白序列的可变区和恒定区。然而，在某些实施方案中，此类重组人抗体经历体外诱变(或，使用就人Ig序列而言为转基因的动物时，经历体内体细胞诱变)并且因此重组抗体的VH和VL区的氨基酸序列是尽管衍生自人种系VH和VL序列并且与之相关，但可能在体内人抗体种系库内部不天然存在的序列。
双特异性抗体
根据某些实施方案，本发明的多特异性抗原结合分子是双特异性抗体；例如，包含特异性结合靶分子Her-2的抗原结合臂和特异性结合CTLA-4蛋白的抗原结合臂的双特异性抗体。可以采用本领域已知的产生双特异性抗体的方法来构建本发明的多特异性抗原结合分子。可以在本发明上下文中使用的示例性双特异性样式包括而不限于例如基于scFv或双体抗体的双特异性样式、IgG-scFv融合物、双可变域(DVD)-Ig、细胞杂交瘤(Quadroma)、结入扣、共同轻链(例如，具有结入扣的共同轻链等)、CrossMab、CrossFab、(SEED)体、亮氨酸拉链，Duobody，IgG1/IgG2，双重作用Fab(DAF)-IgG和Mab2双特异性样式(关于前述样式的综述，见例如Klein等人,2012,mAbs4:6,1-11，和其中引用的参考文献)。
多聚化组分
在某些实施方案中，本发明的多特异性抗原结合分子还可以包括一种或多种多聚化组分。多聚化组分可以发挥维持抗原结合结构域(D1和D2)之间缔合的作用。如本文所用，“多聚化组分”是具有与具有相同或相似结构或构造的第二多聚化组分缔合的能力的任何大分子、蛋白质、多肽、肽或氨基酸。例如，多聚化组分可以是包含免疫球蛋白CH3域的多肽。多聚化组分的非限制性例子是免疫球蛋白的Fc部分，例如，选自同种型IgG1、IgG2、IgG3和IgG4以及每个同种型组内部任何同种异型的IgG的Fc结构域。在某些实施方案中，多聚化组分是含有至少一个半胱氨酸残基的1至约200个氨基酸长度的Fc片段或氨基酸序列。在其他实施方案中，多聚化组分是半胱氨酸残基或含有半胱氨酸的短肽。其他多聚化结构域包括：包含亮氨酸拉链、螺旋-环基序、或卷曲螺旋基序的肽或多肽或由其组成的肽或多肽。
在某些实施方案中，本发明的多特异性抗原结合分子包含两个多聚化结构域M1和M2，其中D1是与M1连接并且D2与M2连接，并且其中M1与M2的缔合促进单个多特异性抗原结合分子中D1和D2彼此物理连接。在某些实施方案 中，M1和M2彼此相同。例如，M1可以是具有特定氨基酸序列的Fc结构域，并且M2是具有与M1相同氨基酸序列的Fc结构域。可选地，M1和M2可以在一个或多个种氨基酸位置彼此不同。例如，M1可以包含第一免疫球蛋白(Ig)CH3域和M2可以包含第二IgCH3域，其中第一和第二IgCH3域彼此差异至少一个氨基酸，并且其中与具有相同M1和M2序列的参考构建体相比，至少一个氨基酸差异减少靶向构建体与蛋白A的结合。在一个实施方案中，M1的IgCH3域结合蛋白A并且M2的IgCH3域含有减少或消除蛋白A结合作用的突变如H95R修饰(按照IMGT外显子编号；按照EU编号为H435R)。M2的CH3还可以包含Y96F修饰(按照IMGT编号；按照EU编号为Y436F)。可以在M2的CH3中存在的其他修饰包括：在IgG1Fc结构域的情况下D16E、L18M、N44S、K52N、V57M和V82I(按照IMGT编号；按照EU编号为D356E、L358M、N384S、K392N、V397M和V422I)；在IgG2Fc结构域的情况下N44S、K52N和V82I(按照IMGT编号；按照EU编号为N384S、K392N和V422I)；和在IgG4Fc结构域的情况下Q15R、N44S、K52N、V57M、R69K、E79Q和V82I(按照IMGT编号；按照EU编号为Q355R、N384S、K392N、V397M、R409K、E419Q和V422I)。
肿瘤靶向作用
在本发明的另一个方面，多特异性抗原结合分子可用于靶向肿瘤细胞。
本发明的多特异性抗原结合分子可以与药物、毒素、放射性同位素或有损细胞生存力的其他物质缀合。可选地，药物或毒素可以是不直接杀伤细胞，但使细胞更易遭其他外部物质杀伤的物质。在涉及肿瘤靶向作用的另外的其他实施方案中，本发明的多特异性抗原结合分子本身不与药物、毒素或放射性同位素缀合，但是与其它抗原结合分子(本文中称作“协从分子”)组合施用，如其它的抗肿瘤抗体。
根据本发明的肿瘤靶向方面的某些实施方案，多特异性抗原结合分子(或协从抗体)可以与选自以下的一种或多种细胞毒药物缀合：刺孢霉素、埃斯波霉素、甲氨蝶呤、多柔比星、美法仓、苯丁酸氮芥、ARA-C、长春地辛、丝裂霉素C、顺铂、依托泊苷、博来霉素、5-氟尿嘧啶、雌氮芥、长春新碱、依托泊苷、多柔比星、紫杉醇、拉罗他赛、替司他赛、奥他赛(orataxel)、多西紫杉醇、多拉司他汀10、澳瑞司他汀E、澳瑞司他汀PHE和基于美坦辛的化合物(例如，DM1、DM4等)。多特异性抗原结合分子(或协从抗体)还可以或可选地与毒素缀合，如白喉毒素、铜绿假单胞菌(Pseudomonasaeruginosa)外毒素A、蓖麻毒蛋白A链、相思豆毒蛋白A链、蒴莲根毒蛋白A链、α-帚曲菌素、油桐(Aleuritesfordii)蛋白、香石竹毒蛋白、垂序商陆(Phytolacaamericana)蛋白等。多特异性抗原结合分子(或协从抗体)还可以或可选地与选自以下的一种或多种放射性同位素缀合：225Ac、211At、212Bi、213Bi、186Rh、188Rh、177Lu、90Y、131I、67Cu、125I、123I、77Br、153Sm、166Ho、64Cu、121Pb、224Ra和223Ra。因此，本发明的这个方面包括作为抗体-药物缀合物(ADC)或抗体-放射性同位素缀合物(ARC)的多特异性抗原结合分子。
药物组合物和施用方法
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物， 它含有上述的抗体或其活性片段或其融合蛋白，或者上述的多特异性抗原结合分子，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可用于预防和治疗肿瘤。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的特异性结合分子(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约8毫克/千克体重，较佳地该剂量是约10微克/千克体重-约1毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
本发明的主要优点在于：
(1)本发明经过大量筛选获得一种抗CTLA-4的人源单链抗体(scFv)，该抗CTLA-4抗体，具有亲和力高、特异性强的优点；
(2)使用本发明的抗CTLA-4单链抗体构建重组双特异性偶联抗体，能够增强Trastuzumab治疗Her-2阳性乳腺癌的疗效，以及治疗对Trastuzumab起抗性的Her-2阳性乳腺癌。
(3)本发明的双特异性抗体可以把抗CTLA-4抗体scFv定点输送到Her-2阳性肿瘤区域，使CTLA-4抗体在局部区域增强免疫反应，不会引起整个免疫系统的过度反应，因此降低CTLA-4抗体的副作用。
下面结合具体实施例，进一步详陈本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明详细条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(NewYork:ColdSpringHarborLaboratoryPress,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1抗CTLA-4单链可变区(scFv)抗体的制备
使用噬菌体展示库(Phage Display Library)技术筛选获得人抗CTLA-4的单链可变区(scFv)抗体。
具体实验步骤如下：
1.构建PIII噬菌体展示库
1.1人源cDNA模板的制备。
用血液RNA管(PAXgene)收集8个人供体的血液，并从中分离人外周血单核细胞PBMC(Ficoll Hypaque method)。用
Figure PCTCN2016110438-appb-000016
Midi Kit(Qiagen)从PBMC中抽提全RNA，利用全RNA做模板，oligo(dT)为引物，进行第一条cDNA链的合成(
Figure PCTCN2016110438-appb-000017
III First-Strand Synthesis kit，Invitrogen)。
1.2人源抗体可变区基因的扩增。
用上述合成的cDNA做模板，用聚合酶链式反应(PCR)方法，分别扩增抗体重链可变区基因(VH)和轻链可变区基因(VL)。
根据早前发表的资料和最新的V-base序列目录，设计出扩增VH和VL基因的PCR引物(文献)并合成(GenScript)。所有的PCR反应都是分别用一个反向引物和多个正向引物的混合液进行的。为了扩增VH基因，12个不同的反向引物(HVH)，每一个都和4个重链J区域的正向引物(HJH)混合液进行PCR反应。用同样的方法扩增kappa和lamda型的VL基因，即每一个HVkappa或HVlamda的反向引物都和向对应的HJkappa或HJlamda正向引物混合液进行PCR反应。每一个PCR反应体积为100ul，含有cDNA 2ul，正向引物或反向引物各1uM，dNTPs 200uM，DMSO 5％和10X Pfu缓冲液。PCR反应液在94℃加热5分钟后，加入5units的Pfu(Stratagene)，然后进行30个循环反应，每个循环反应包括94℃变性1分钟，57℃退伙1分钟和72℃延伸1分钟。所有PCR反应完成后，分别把VH基因和VL基因扩增的PCR反应液混合，然后进行琼脂糖凝胶电泳，并进行胶纯化(
Figure PCTCN2016110438-appb-000018
Kit，Qiagen)。
把纯化好的VH基因和VL基因分别和TA载体(Invitrogen)连接，然后用电击方法(GenePulser Xcell，Bio-Rad)把连接产物转化到感受态XL-1细菌中(Stratagene)。转化的细菌收集在LB培养基里，然后全部涂板到10个150-mm的LB/Agar培养皿，在37℃培养过夜。把培养皿上的细菌刮下收集到320ml的LB培养基中，分装到50-ml离心管里，每管20ml，保存在-80℃冰箱里。
1.3PIII噬菌体展示库的构建
人工合成含有噬菌体外壳蛋白PIII编码基因的噬菌体载体PIII(GenScript，USA)，此载体的多克隆位点位于PIII基因的5'端，用于克隆抗体可变区基因。抗体可变区基因的3’端连接一个His-Tag基因和肠激酶的酶切位点，用于纯化scFV抗体。
解冻各20mlVH基因和VL基因的TA载体库，离心收集细菌，用HighPure Midi质粒纯化试剂盒(TianGen)制备质粒。纯化的含VH基因质粒用限制性内切酶NcoI-HF和XhoI-HF进行双酶切，纯化的含VL基因质粒用限制性内切酶NheI-HF和NotI-HF进行双酶切。所有的限制性内切酶来自New England Biolabs(NEB)，酶切反应在37℃里进行过夜。用乙醇沉淀酶切的质粒，溶解质粒在TE缓冲液里，用琼脂糖凝胶电泳分离质粒，切下含VH或VL基因片段的琼脂糖胶，纯化出VH或VL基因(
Figure PCTCN2016110438-appb-000019
Kit，Qiagen)。
分2个步骤把VH基因和VL基因克隆到噬菌体载体PIII上。首先，用NheI-HF和NotI-HF限制性内切酶对噬菌体载体PIII进行双酶切，并纯化，把 纯化的载体和纯化的VL基因用T4连接酶(NEB)进行连接，VL基因连接在外壳蛋白PIII基因5'端，由于引物的设计，它们处于同一个蛋白编码框架内。把连接产物用电击方法转化进XL-1感受态细菌里(Stratagene)，转化的细菌培养在40个150-mm的LB/Agar培养皿上，培养皿含有2％葡萄糖，50ug/ml羧苄青霉素和20ug/ml四环素。在37℃里培养过夜后，用1600ml的SB(Super Broth)培养基把长出的细菌刮下，加入10％的甘油，分装保存在-80℃冰箱里。第二步是把VH基因克隆到上述含有VL基因的噬菌体载体里，克隆位点NcoI和XhaoI位于VL基因的5'端，VH基因和VL基因之间有一个7-15个氨基酸的连接肽编码序列。连接和转化的方法和VL基因相同，最后制备出人源scFv噬菌体展示库。
2.与CTLA-4结合的噬菌体的淘选
2.1scFv表达的噬菌体制备
把上述制备的5x 1010细菌接种进1升SB培养基里，培养基含有2％葡萄糖，50ug/ml羧苄青霉素和20ug/ml四环素，在37℃摇床培养箱里培养至细胞密度达到OD600在0.5和0.7之间，然后加入4x 1013空斑形成单位(PFU)的辅助噬菌体VCSM13(Stratagene)和终浓度为1mM的IPTG。在室温里培养30分钟后，用SB培养基稀释到5升，在室温下继续培养2小时。加入终浓度70ug/ml的卡那霉素，在30℃摇床培养箱里培养过夜。离心培养液沉淀细菌，收集含有噬菌体的上清，转入到500-ml干净的离心管里，加入PEG8000(Sigma)至终浓度4％(w/v)和NaCl至终浓度3％(w/v)，沉淀噬菌体。噬菌体悬浮并保存于含2％BSA的PBS(pH7.4)里。
2.2CTLA-4结合的噬菌体的淘选
用于淘选的抗原蛋白是重组人细胞毒T淋巴细胞相关抗原4(CTLA-4)的胞外段(氨基酸37-162)和IgG恒定区的融合蛋白(R&D Systems，USA)。融合蛋白CTLA-4/Fc溶解在PBS，以浓度50ug/ml的条件下加入到免疫测定试管(Maxisorb,Nunc)，在室温下孵育过夜。免疫测定试管用4％的半脱脂牛奶(PBS)在室温下封闭1个小时，加入1x 1013菌落形成单位的上述制备好的scFv表达的噬菌体，在室温下孵育2小时(不断摇晃)。没有结合的噬菌体用PBS/0.1％Tween-20清洗掉，然后用PBS清洗。各清洗10次。特异结合的噬菌体用1ml洗脱液(100mM NaCl，pH2.2，0.1％BSA)在室温下洗脱，然后用60ul的Tris(2M)中和洗脱液。用掏选出的噬菌体再重复2次上述过程，进行第2轮和第3轮的淘洗。第2轮和第3轮淘洗所用的融合蛋白CTLA-4/Fc的浓度分别是10ug/ml和5ug/ml。
2.3阳性噬菌体单克隆和基因测序
在SB培养基里培养XL-1细菌至OD600约为1，加入最后一轮掏选出的阳性噬菌体，继续在37℃摇床培养箱里培养1个小时。离心沉淀细菌，把细菌培养在SB/Agar培养皿上，培养皿含有2％葡萄糖，50ug/ml羧苄青霉素和20ug/ml四环素，培养在30℃里过夜。第2天，挑选单个克隆，做质粒微量制备，进行scFv基因测序。
3scFv抗体制备和分析
3.1scFv抗体的细菌表达和制备
把scFv基因从获得的单克隆质粒上亚克隆到细菌表达载体(pET/Flag)上， 然后在BL21/DE3细菌里表达scFv抗体并纯化。表达和纯化方法参见公开发表的标准方法。
3.2与CTLA-4体外结合的ELISA
用ELISA方法检测scFv抗体和CTLA-4体外结合能力(具体步骤参见实施例2)。ELISA板用重组蛋白CTLA-4包被后，加入不同浓度的scFV蛋白，然后用碱性磷酸酶偶联的羊抗人IgGFab抗体检测scFV蛋白量
本实施例中成功的获得了具有良好特异性和亲和力的人抗CTLA-4的单链可变区(scFv)抗体，经本领域常规方法测序，其序列信息如下：
人抗CTLA-4的单链可变区(scFv)抗体的重链可变区中，各FR和CDR如下：
  SEQ ID NO. 序列
FR1 25 QVQLVQSGGGVVQPGRSLRLSCAASGFTFS
CDR1 5 SYGMH
FR2 26 WVRQAPGKGLEWVA
CDR2 6 VIWYDGSRQYYADS
FR3 27 VKGRFTISRDDSKNTMYLQMNSLRAEDTAVYYCAR
CDR3 7 GGFWGAFDI
FR4 28 WGQGTMVTVSS
重量可变区序列：
Figure PCTCN2016110438-appb-000020
人抗CTLA-4的单链可变区(scFv)抗体的轻链可变区中，各FR和CDR如下：
  SEQ ID NO.:12中位置 序列
FR1' 29 DVVMTQSPGTLSLSPGEGATLSC
CDR1' 11 RASQHVISSYLA
FR2' 30 WYQQKPGQAPRLLVY
CDR2' 12 GASSRDT
FR3' 31 GVSDRFTGSGSGTDFTLTISRLEPEDSAVYFC
CDR3' 13 QQYGTSPWTF
FR4' 32 GQGTKLEIKR
轻链可变区序列：
Figure PCTCN2016110438-appb-000021
实施例2重组双特异性抗体的制备和检测
制备具有生物活性的重组双特异性偶联抗体，此抗体具有增强Trastuzumab治疗Her-2阳性乳腺癌的疗效，以及治疗对Trastuzumab起抗性的Her-2阳性乳腺癌。
图1是此抗体的结构示意图。抗体重链的基因序列和蛋白序列见图2A，抗体轻链的基因序列和蛋白序列见图2B。
1.技术方案
1.1双特异性偶联抗体蛋白表达质粒的构建
编码曲妥珠单抗的重链和轻链完整cDNA由GenScrip(USA)公司分别合成，人抗CTLA-4的单链可变区(scFv)抗体序列信息如实施例1所示，用噬菌体展示库(PhageDisplayLibrary)技术筛选获得。
本发明人发现，在曲妥珠单抗表达和制备过程中，绝大部分抗体的重链C-末端赖氨酸被降解掉，所以在构建双特异性偶联抗体时，去掉了这个赖氨酸，以使双特异性偶联抗体能保持完整性。
用聚合酶链式反应技术(PCR)以人工合成的DNA为底物扩增Her-2抗体重链基因，Her-2抗体序列信息如上所述，5’端的引物是Her2-F(5’-ATTGAATTCCGCGGCCGCCACCATGGAG，SEQ ID NO.:33)，3’端的引物是Her2-R(5’-TCCTGGGGACAGTGACAGTG，SEQ ID NO.:34)。抗CTLA-4的scFv基因也用PCR法扩增，5’端引物是CTLA-Fv-F(5’-CACTGTCACTGTCCCCAGGACAGGTCCAGCTGGTGCAGTC，SEQ ID NO.:35)，3’端引物是CTLA-Fv-R(5’-AGAATAGGGCCCTCTAGATTAACGTTTGATCTCCAGCTTGG，SEQ ID NO.:36)，其中引物CTLA-Fv-F的头20个核苷酸序列与引物Her2-R的核苷酸序列互补，这样在后面的In-fusion(ClontechLaboratories,Inc.)质粒克隆中，把这2个PCR片段连接起来。上面2个PCR片段经DNA胶纯化后，和NotI/XbaI双酶切的哺乳细胞表达载体(比如含CMV启动子的表达质粒)混合，用In-fusion方法把Her-2抗体重链和CTLA-4scFv抗体的基因连接和克隆到表达载体里。
用亚克隆方法把抗体轻链cDNA克隆到同样的表达载体质粒。克隆酶是NotI和XbaI。
1.2抗体蛋白表达细胞株的建立
宿主细胞CHODG44来源于Invitrogen公司，细胞培养和传代方法参照公司的CHODG44手册。无转染细胞悬浮培养在CDDG44培养基里(Invitrogen)，培养基含有8mML-glutamine和5ug/ml重组人胰岛素。
构建抗体蛋白稳定表达细胞系方法和步骤简述如下。用TianGen的质粒大抽试剂盒制备抗体重链和轻链表达载体质粒，并用限制性内切酶PuvI分别酶切各质粒DNA100ug，使质粒线形化。在表达载体质粒转染细胞前，DG44细胞至少要传三代。取DG44细胞总数1x107，与酶切的质粒混匀在0.8ml的CDDG44生长培养基里，转入0.4cm电击杯里(Bio-Rad)，用电转染仪(Bio-Rad，GenePulserXcell)电击细胞/质粒混合液，然后培养转染的细胞在一个T-75细胞培养方瓶里，加入20ml细胞生长培养基。把含有转染细胞的T-75方瓶置于37℃，8％CO2的培养箱里培养24个小时。
转染的细胞在培养24个小时后，用有限稀释法把转染的细胞在96孔培养板里进行筛选培养。筛选培养基是OptiCHO，含有8mML-glutamine，5ug/ml重组人胰岛素和100nM的氨甲碟呤(MTX，Sigma)。培养细胞在37℃，8％CO2的培养箱里。3个星期后，用ELISA方法(碱性磷酸酶偶联的羊抗人IgGFc抗体，Jackson ImmunoResearch Lab)对每个长有细胞克隆的孔的细胞培养液进行分析，把表达最高的克隆进一步扩增，再ELISA检测，再扩增，最后得到12个表达最高的稳定细胞株。
对这12个细胞克隆进行逐步提高MTX浓度的加压培养，以增加抗体的表 达量。共进行3轮的加压培养，每轮加压培养约3个星期。3轮加压后，对12个克隆进行抗体表达量检测，得到4-5个抗体高表达的细胞株。再对其中的一个细胞株用有限稀释法进行单克隆细胞株筛选，最后得到3-5个抗体高表达的单克隆细胞株。
1.3抗体的制备和理化性质的鉴定
选取一个抗体高表达的单克隆细胞株，培养扩增至2升。培养液上清用来纯化制备抗体。纯化方法包括Protein-A亲和层(POROSMabCaptureA，LifeTech)，阴离子层析(Q-600C，TOSOH)和阳离子层析(POROSXS，LifeTech)。
纯化的抗体用还原和非还原SDS-PAGE电泳分析，并进行HPLC-SEC(高压液相-分子筛)(TSKgelG3000SWXL，TOSOH)分析。
1.4流式细胞仪检测重组双特异性偶联抗体与细胞膜上Her-2的结合
Her-2稳定表达的B16细胞(小鼠黑色素瘤)与不同浓度的重组抗体在体外孵育，取适量B16/Her-2阳性细胞，用预冷的FACS工作液调整其细胞密度为3×106/ml，分装100ul/管，冰上封闭1小时。然后用FACS工作液稀释抗体到不同浓度，加10ul不同浓度抗体到100ul的细胞悬液中，冰上孵育30分钟。孵育完成后，向每管细胞悬液加1mlFACS工作液，涡旋混匀细胞，离心5分钟，1200rpm/min，弃去上清，重复洗涤一遍。用FACS工作液稀释FITC标记的羊抗人IgGFc抗体(JacksonImmunoResearchLab)，每管细胞悬液加10ul抗体，使其终浓度为10ug/ml，避光，冰上孵育30分钟。孵育完成后，向每管细胞悬液加1mlFACS工作液，涡旋混匀细胞，离心5分钟，1200rpm/min，弃去上清，重复洗涤一遍。用流式细胞仪C6(BDBiosciences)检测细胞。
1.5重组双特异性偶联抗体与CTLA-4结合的ELISA研究
25nM的重组人CTLA-4-Fc融合蛋白(R&DSystems)溶解在50mMNaHCO3(pH9.6)溶液了，在96-孔ELISA板里加入50ulCTLA-4蛋白，于4℃冰箱里过夜。第二天，用PBST(PBS含0.05％Tween-20)洗ELISA板3次，加入100ul/孔PBST含3％BSA的封闭溶液。放置ELISA板于37℃恒温箱里1小时。稀释抗体蛋白在PBST含1％BSA的结合溶液里，制备3倍系列稀释的抗体蛋白。倒掉封闭液，加入50ul/孔3倍系列稀释的抗体蛋白，在37℃恒温箱里反应1小时。倒掉抗体蛋白溶液，用PBST清洗ELISA板3次，加入50ul/孔的第二抗体(碱性磷酸酶偶联的羊抗人IgGFab抗体，JacksonImmunoResearchLab)，在37℃恒温箱里反应1小时。倒掉显色抗体，在ELISA板里加200ul/孔PBST清洗溶液，置ELISA板于水平摇床上5分钟，转速100转/分钟，倒掉清洗溶液。重复5次。加50ul/孔抗体显色液(PNPP)于ELISA板里，置板于37℃恒温箱里。用酶标仪在波长405nm下读板。
1.6抑制Her-2阳性乳腺癌细胞BT-474细胞体外生长
BT-474细胞(购自ATCC)培养在Dulbecco'sMinimumEssentialMedium(DMEM)里，含有10％的FBS。细胞用胰酶消化后离心，弃上清，悬浮细胞在生长培养基里，计数。在96-well细胞培养板里加入每孔150ul，含10000个 细胞。在5％CO2，37℃培养箱里培养过夜。第二天，用生长培养基系列稀释重组抗体或对照抗体，制备10个不同浓度。在细胞板里每孔加入50ul稀释的抗体或对照抗体。培养细胞4天后，每孔加20ul的CCK-8(细胞增殖和活性检测试剂盒，DOJINDO)，在细胞培养箱里培养4个小时后，用酶标仪在490nm/655nm下读板。
1.7增强PHA刺激的人PBMCIL-2的产生量
用Accuspin(Histopaque-1077，Sigma)方法，从2个人的新鲜血里制备外周血单个核细胞(PBMC)，悬浮PBMC在RPMI1640培养基里(10％FBS)，细胞密度1x106/ml。加入植物血凝素(PHA)至终浓度1ug/ml，在5％CO2，37℃培养箱里培养PBMC2天。然后用PBS洗PBMC一次，悬浮细胞在RPMI1640生长培养基里，密度为5x106/ml。同时准备丝裂霉素C(mitomycinC)处理的Raji细胞(中国细胞库)。悬浮Raji细胞在RPMI1640生长培养基里，加入mitomycinC至终浓度25ug/ml，在37℃里孵育1个小时后，用PBS清洗细胞4次，用RPMI1640生长培养基悬浮细胞，密度1x106/ml。在96-孔细胞培养板里，在每孔里加入等体积的PBMC和Raji细胞(50ul各细胞)，并加入抗体至终浓度10ug/ml或30ug/ml。在培养箱里培养细胞72个小时。收集细胞培养上清，用ELISA试剂盒(上海依科赛生物制品有限公司)测定上清里IL-2的含量。
1.8增强猴子对HBsAg抗原免疫反应
本发明双特异性抗体的scFv区域可以结合CTLA-4，阻断CTLA-4对T细胞CD28共刺激的抑制作用，进而增强免疫系统的免疫反应。由于人源抗CTLA-4抗体scFv可以和食蟹猴的CTLA-4结合，因此我们用食蟹猴检测本发明的双特异性抗体对重组乙型肝炎疫苗HBsAg(GSK，Engerix B)在食蟹猴体内引起免疫反应的影响。
8只食蟹猴(2-3岁)分2组，每组含雌雄各2只。在第1天和第29天，给对照组的猴子静脉注射人源IgG1，给试验组的猴子静脉注射双特异性抗体，注射剂量是每公斤猴子10mg抗体，抗体蛋白浓度为5mg/ml。在第2天和第30天，给每只猴子肌肉注射10ug的重组乙型肝炎疫苗。在第1天，第35天和第49天，每只猴子取血制备血浆，血浆中抗HBsAg抗体滴度用ELISA方法测定。
由于样品太多，所以先检测同组猴子血浆中抗体的平均滴度。方法简述如下：用包被液(200mM NaHCO3，pH9.6)稀释HBsAg蛋白至2ug/ml，在96-孔ELISA板里每孔加入50ul，于4℃冰箱里过夜。第二天，用PBST(PBS含0.05％Tween-20)洗ELISA板3次，加入100ul/孔PBST含3％BSA的封闭溶液，放置ELISA板于37℃恒温箱里1小时。取部分血浆，把同组同一天的血浆等体积混合，然后用结合溶液(PBST含1％BSA)稀释混合的血浆100倍，500倍，2500倍和12500倍。ELISA板封闭完成后，倒掉封闭液，加入50ul/孔稀释的血浆混合液，在37℃恒温箱里孵育2小时。倒掉血浆溶液，用PBST清洗ELISA板3次，加入50ul/孔2000倍稀释的第二抗体(碱性磷酸酶偶联的羊抗猴IgGFc，Abcam，货号ab112765)，在37℃恒温箱里反应2小时。倒掉第二抗体，在ELISA板里加200ul/孔PBST清洗溶液，置ELISA板于水平摇床上5分钟，转速100转/分钟，倒掉清洗溶液，重复清洗过程5次。加50ul/孔抗体显色底 物PNPP溶液(Southern Biotech,China)于ELISA板里，置板于37℃恒温箱里。用酶标仪(Bio-Rad)在波长405nm下读板。
从上述实验结果得出，1000倍稀释血浆的样品可以获得最好的ELISA结果。用同样的ELISA方法检测每个猴子的抗HBsAg抗体滴度，每个猴子的血浆稀释1000倍，其余的步骤同上述方法。为了方便比较每个猴子HBsAg抗体的含量，我们设定来自实验组猴子第49天混合血浆中的抗体单位是5000units/ml，用此混合血浆做标准，进行系列稀释，同时进行ELISA，然后计算每个猴子的HBsAg抗体的含量。每个样品做2个平行点。
2.实施效果
2.1双特异性抗体制备，纯化和鉴定
双特异性抗体经Protein-A亲和层析柱，阴离子柱和阳离子柱纯化后，用还原和非还原SDS-PAGE电泳胶分析(图3)。图3A显示完整双特异性抗体的分子量约大于200kDa，很接近其理论值，196kDa。还原SDS-PAGE电泳胶显示双特异性抗体双特异性抗体的重链分子量为75-80kDa，也与其理论分子量一直(74.4kDa)
HPLC-SEC分析纯化的双特异性抗体，表明40％纯化的抗体可能是二聚体，剩余的60％是单体(图4)。
2.2双特异性抗体结合细胞膜Her-2
用FlowCytometry检测双特异性抗体与细胞膜上Her-2结合的能力。用曲妥珠单抗和细胞膜上Her-2结合做对照，图5显示这2个抗体结合Her-2的萤光强度相似，说明双特异性抗体有和曲妥珠单抗相同的与细胞膜上Her-2的结合能力。
2.3双特异性抗体体外结合CTLA-4
用ELISA方法检测双特异性抗体与CTLA-4的亲和结合能力。双特异性抗体能特异性结合CTLA-4，EC50是0.19nM，与Ipilimumab的结合能力(EC50是0.16nM)相近(图6)。
2.4双特异性抗体抑制BT-474细胞生长
曲妥珠单抗可以抑制Her-2阳性乳腺癌BT-474细胞的体外生长，因此检测了双特异性抗体抑制BT-474细胞生长的能力(图7)。研究结果证明双特异性抗体可以很好的抑制BT-474细胞的生长。其IC50是0.97nM，和曲妥珠单抗的IC50(0.69nM)相近。本实验的目的是证明本发明的双特异性抗体的抗Her-2区域任具有抑制BT-474细胞的生长能力。由于本发明的双特异性抗体是2个抗体的偶联，它们之间有可能会互相影响，比如影响对方的正确空间结构的形成，或对方与相应抗原的结合。本实验证明了，本发明的双特异性抗体中包含的各抗原结合单元仍保留了各自的活性。
2.5双特异性抗体增强PBMC的IL-2表达
抗CTLA-4抗体与CTLA-4结合后，阻断CTLA-4对T细胞激活的负调节信号，因此保持或增强T细胞的激活状态，增加各类细胞间素的表达，其中包括IL-2。本发明人测试了双特异性抗体对PHA激活的人PBMCIL-2表达的影响，发现双特异性抗体与不加抗体的实验组相比，可以显著增加PBMC的IL-2表达(图8，与对照组相比增加了约2倍)。这个结果证明了双特异性抗体能够保 持或增强T细胞的激活状态。
2.6双特异抗体增强食蟹猴对HBsAg的免疫反应
用ELISA方法检测食蟹猴在注射疫苗第49天后血浆中的抗HBsAg抗体的平均滴度，结果显示注射疫苗猴子的血浆抗体滴度接近10000倍(数据没有显示)。用同样的方法检测每个猴子的抗HBsAg抗体的含量，在注射疫苗第7周后，试验组4只猴子中有2只的抗体含量在8000单位/毫升，1只接近5000单位/毫升，最后1只有700单位/毫升。对照组的4只猴子，3只的抗体含量接近1500单位/毫升或低于1000单位/毫升，只有1只猴子的抗体含量在5000单位/毫升(图9)。比较这2组猴子的抗体含量，结果表明本发明的双特异性抗体具有在体内增强疫苗引起的免疫反应的功能。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (16)

  1. 一种多特异性抗原结合分子，其特征在于，所述多特异性抗原结合分子包含：
    第一抗原结合结构域(D1)；和
    第二抗原结合结构域(D2)；
    其中，D1特异性结合靶分子Her-2蛋白；
    D2特异性结合靶分子CTLA-4蛋白；
    所述D1为特异性结合Her-2蛋白的抗体或抗体片段；和/或
    所述D2为特异性结合CTLA-4蛋白的抗体或抗体片段；
    其中，所述D1具有SEQ ID NO.:4所述的重量可变区，和SEQ ID NO.:10所述的轻链可变区。
  2. 一种抗体的重链可变区，其特征在于，所述的重链可变区包括以下三个互补决定区CDR：
    SEQ ID NO:5所示的CDR1，
    SEQ ID NO:6所示的CDR2，和
    SEQ ID NO:7所示的CDR3；
    优选地，所述重链可变区具有SEQ ID NO:4所示的氨基酸序列。
  3. 一种抗体的重链，其特征在于，所述的重链具有权利要求2所述的重链可变区和重链恒定区。
  4. 一种抗体的轻链可变区，其特征在于，所述轻链可变区具有选自下组的互补决定区CDR：
    SEQ ID NO:11所示的CDR1’，
    SEQ ID NO:12所示的CDR2’，和
    SEQ ID NO:13所示的CDR3’；
    优选地，所述的轻链可变区具有SEQ ID NO:10所示的氨基酸序列。
  5. 一种抗体的轻链，其特征在于，所述的轻链具有权利要求4所述的轻链可变区和轻链恒定区。
  6. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求2所述的重链可变区；和/或
    (2)如权利要求4所述的轻链可变区；
    优选地，所述抗体具有：如权利要求3所述的重链；和/或如权利要求5所述的轻链。
  7. 一种重组蛋白，所述的重组蛋白具有：
    (i)如权利要求2所述的重链可变区的序列、如权利要求3所述的重链的序列、如权利要求4所述的轻链可变区的序列、如权利要求5所述的轻链的序列、或如权利要求6所述的抗体的序列；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  8. 一种多特异性抗原结合分子，其特征在于，所述多特异性抗原结合分子包含：
    第一抗原结合结构域(D1)；和
    第二抗原结合结构域(D2)；
    其中，D1特异性结合靶分子Her-2蛋白；
    D2特异性结合靶分子CTLA-4蛋白；
    优选地，所述D1为特异性结合Her-2蛋白的抗体或抗体片段；和/或
    所述D2为特异性结合CTLA-4蛋白的抗体或抗体片段。
  9. 一种多核苷酸，其特征在于，它编码选自下组的多肽：
    (1)如权利要求2所述的重链可变区、如权利要求3所述的重链、如权利要求4所述的轻链可变区、如权利要求5所述的的轻链、或如权利要求6所述的抗体；或
    (2)如权利要求7所述的重组蛋白、或如权利要求8所述的多特异性抗原结合分子。
  10. 一种载体，其特征在于，它含有权利要求9所述的多核苷酸。
  11. 一种遗传工程化的宿主细胞，其特征在于，它含有权利要求10所述的载体或基因组中整合有权利要求8所述的多核苷酸。
  12. 一种免疫偶联物，其特征在于，该免疫偶联物含有：
    (a)如权利要求2所述的重链可变区、如权利要求3所述的重链、如权利要求4所述的轻链可变区、如权利要求5所述的轻链、如权利要求6所述的抗体、如权利要求7所述的重组蛋白、或如权利要求8所述的多特异性抗原结合分子；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  13. 一种药物组合物，其特征在于，它含有：
    (i)如权利要求2所述的重链可变区、如权利要求3所述的重链、如权利要求4所述的轻链可变区、如权利要求5所述的轻链、如权利要求6所述的抗体、如权利要求7所述的重组蛋白、如权利要求8所述的多特异性抗原结合分子、或如权利要求12所述的免疫偶联物；以及
    (ii)药学上可接受的载体。
  14. 如权利要求2所述的重链可变区、如权利要求3所述的重链、如权利要求4所述的轻链可变区、如权利要求5所述的轻链、如权利要求6所述的抗体、如权利要求7所述的重组蛋白、如权利要求8所述的多特异性抗原结合分子、或如权利要求12所述的免疫偶联物的用途，其特征在于，用于制备药剂、试剂、检测板或试剂盒；
    所述试剂、检测板或试剂盒用于：检测样品中CTLA-4蛋白；
    所述药剂用于治疗或预防表达CTLA-4蛋白的肿瘤。
  15. 一种检测样品中CTLA-4蛋白的方法，其特征在于，所述方法包括步骤：
    (1)将样品与权利要求6所述的抗体接触；
    (2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在CTLA-4蛋白。
  16. 一种重组多肽的制备方法，其特征在于，该方法包含：
    (a)在适合表达的条件下，培养权利要求11所述的宿主细胞；
    (b)从培养物中分离出重组多肽，所述的重组多肽是权利要求6所述的抗体、权利要求7述的重组蛋白、或如权利要求8所述的多特异性抗原结合分子。
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