WO2017070943A1 - 双特异性抗体、其制备方法和用途 - Google Patents

双特异性抗体、其制备方法和用途 Download PDF

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WO2017070943A1
WO2017070943A1 PCT/CN2015/093383 CN2015093383W WO2017070943A1 WO 2017070943 A1 WO2017070943 A1 WO 2017070943A1 CN 2015093383 W CN2015093383 W CN 2015093383W WO 2017070943 A1 WO2017070943 A1 WO 2017070943A1
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variable region
antibody
cells
seq
human
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PCT/CN2015/093383
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English (en)
French (fr)
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马永
曹锫沛
王安良
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江苏众红生物工程创药研究院有限公司
常州京森生物医药研究所有限公司
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Priority to CN201580008856.6A priority Critical patent/CN107207608B/zh
Priority to US15/554,476 priority patent/US10752686B2/en
Priority to PCT/CN2015/093383 priority patent/WO2017070943A1/zh
Publication of WO2017070943A1 publication Critical patent/WO2017070943A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a bispecific antibody, and more particularly to a single chain antibody which specifically binds to both human CD26 and human CD3, a preparation method thereof and a novel use of a medicament for treating a cell which expresses a CD26 type tumor in a cell.
  • CD26 is a ubiquitous multifunctional type II transmembrane glycoprotein with multiple biological functions and can also be present in plasma in dissolved form.
  • CD26 is often present as a homodimer with a monomer of 766 amino acids and a relative molecular mass of about 110 kDa.
  • the amino acid residues are divided into five parts from the inside to the outside: intracellular regions (1 to 6), transmembrane regions (7 to 28), highly glycosylated regions (29 to 323), and cysteine-rich regions (324). ⁇ 551) and the C-terminal catalytic domain (552-766), the three-dimensional structure and function of CD26 molecule are closely related.
  • the role of CD26 in immune regulation has been extensively studied.
  • CD26 is a molecular marker of T cell activation and also acts as a costimulatory molecule in T cell signaling, and involves a variety of T cell functions, including T cell maturation and migration. , cytokine secretion, T cell-dependent antibody production, B cell immunoglobulin transformation, etc. (Ohnuma et al. (2011) Adv Clin Chem, 53, 51-84).
  • CD26 can interact with a variety of proteins, such as ADA, CD45, FAP-alpha, etc., and can also bind to ECM, resulting in increased or decreased expression of CD26 cell infiltration activity, showing that CD26 plays an important role in tumor biology.
  • the expression of CD26 on a variety of neoplastic cell surfaces is significantly elevated.
  • CD26 is highly expressed in certain offensive T cell malignancies, malignant mesothelioma, nephroma, and certain colon cancer (Havre et al. (2008). ) Front Biosci, 13, 1634-1645).
  • Certain CD26 + colon cancer cell subsets, CD26 + malignant mesothelioma cells have distinct tumor stem cell characteristics (Ghani et al. (2011) Biochem Biophys Res Commun, 404, 735-742 and Pang et al. (2010) Cell Stem Cell, 6,603-615), so CD26 can be used as a molecular marker for a variety of tumors.
  • targeted anticancer drugs targeting human CD26 are mainly monoclonal antibodies, and their immunogenicity and therapeutic effects still need further improvement.
  • Y's Therapeutics has entered the clinical stage of the anti-CD26 murine humanized monoclonal antibody drug YS110, which specifically binds to the CD26 epitope on the surface of target cells, followed by the Fc segment of CD26 and the expression of IgG Fc receptor. The binding of NK cells, macrophages and neutrophils then acts through the ADCC effect of the antibodies.
  • YS110 the anti-CD26 murine humanized monoclonal antibody drug
  • YS110 specifically binds to the CD26 epitope on the surface of target cells
  • the binding of NK cells, macrophages and neutrophils then acts through the ADCC effect of the antibodies.
  • the mouse still contains the mouse component after anti-humanization, and the HAMA effect cannot be completely excluded.
  • the Fc segment of the antibody has a fucose modification phenomenon during the expression process, inhibits the AD
  • Human CD3 is only present on the surface of T cells and consists of three different chain CD3 ⁇ chains, CD3 ⁇ chains, and CD3 ⁇ chains, often tightly bound to TCR to form TCR-CD3 complex.
  • CD3 molecules are linked to T cell antigen receptors via salt bridges and participate in T cell signaling divert. Clustering of CD3 on T cells (eg, by immobilized anti-CD3 antibodies) can result in T cell activation similar to T cell receptor binding.
  • the OKT3 antibody used clinically is a classic representative of anti-CD3 antibodies for the treatment of allograft rejection.
  • the bispecific antibody provided by the present invention comprises a variable region fragment of an antibody that specifically binds to human CD26, and a variable region fragment of an antibody that specifically binds to human CD3.
  • the bispecific antibody wherein the heavy chain variable region in the variable region fragment of the antibody that specifically binds to human CD26 and the heavy chain fragment in the variable region fragment of the antibody that specifically binds to human CD3
  • the light chain variable region in the variable region fragment of the antibody adjacent to the variable region or specifically binding to human CD26 is adjacent to the light chain variable region in the variable region fragment of the antibody that specifically binds to human CD3.
  • the N-terminal to C-terminus of the above bispecific antibody is arranged in the following order: a light chain variable region, a heavy chain variable region of an antibody that specifically binds to human CD26, and an antibody that specifically binds to human CD3 a heavy chain variable region, a light chain variable region; or a heavy chain variable region, a light chain variable region of an antibody that specifically binds to human CD26, a light chain variable region of an antibody that specifically binds to human CD3, Heavy chain variable region.
  • variable region fragment of the antibody that specifically binds to human CD26 comprises a heavy chain variable region as set forth in SEQ ID NO: 1 and a light chain variable region as set forth in SEQ ID NO: 2;
  • the variable region fragment of the antibody that specifically binds to human CD3 includes a heavy chain variable region as set forth in SEQ ID NO: 4 and a light chain variable region as set forth in SEQ ID NO: 5.
  • variable region of the CD26 antibody is obtained by codon-optimizing the variable region gene sequence of the monoclonal antibody YS110, or from other well-known CD26 antibody variable regions.
  • variable region of the CD3 antibody is derived from OKT-3, TR-66, X35-3, VIT3, CLB-T3/3, CLB-T3.4.2, F111-409, WT31, WT32, CRIS7, F101.01, BMA030 (BW264/56), YTH12.5, SPv-T3b, 11D8, CD3-specific antibodies to XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2.
  • CD3-specific antibodies are well known in the art.
  • the amino acid sequence of the bispecific antibody of the invention is as set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.
  • the nucleotide sequence encoding the above antibody provided by the present invention is represented by SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23.
  • the present invention also provides an expression vector comprising the above nucleotide sequence.
  • the vector is preferably pCHO1.0.
  • the invention also provides a recombinant host cell comprising the above expression vector.
  • the cell is preferably a CHO-S cell.
  • the invention also provides a preparation method of the above antibody, comprising the following steps:
  • Step 1 the above recombinant host cells are cultured under suitable conditions to express the protein of interest;
  • Step 2 Purification of the above target protein by chromatography.
  • the invention also provides the use of the bispecific antibody in the preparation of a medicament for treating cells with high expression of CD26 type tumors.
  • the invention also provides the use of the bispecific antibody for the preparation of a T cell mediated tumor cell immunotherapy drug activated by CD3.
  • the above bispecific antibody is used in the preparation of a medicament for treating kidney cancer, prostate cancer, colon cancer, mesothelioma.
  • the invention successfully expresses anti-CD26 and anti-CD3 bispecific antibodies by eukaryotic cell system, and is suitable for subsequent large-scale production, and has specificity for binding to CD26 protein, affinity reaches 10 -9 M, and cells with high expression of CD26
  • the strain has a strong cytotoxic effect and can be used to treat solid tumors associated with high expression of CD26.
  • the bispecific antibody mediates T cell killing of CD26 high expression tumor cells, has better curative effect than anti-CD26 monoclonal antibody alone, and has no fucose modification and no complicated glycoform distribution due to deletion of Fc fragment. Compared with humanized IgG antibody, it has the advantages of low immunogenicity and easier quality control.
  • Figure 1 is a gel electrophoresis map of the ZHB BsAb series of genes.
  • Lane 1 is a 500 bp DNA Ladder; Lane 2 is a ZHB BsAb-1 gene containing AvrII and BstZ17I restriction sites at both ends, and Lane 3 is a ZHB BsAb-2 gene containing AvrII and BstZ17I restriction sites at both ends, lanes 4 is the ZHB BsAb-3 gene containing the AvrII and BstZ17I cleavage sites at both ends, and Lane 5 is the ZHB BsAb-4 gene containing the AvrII and BstZ17I cleavage sites at both ends.
  • Figure 2-a, Figure 2-b, Figure 2-c, and Figure 2-d show the construction process of the ZHB BsAb series gene expression plasmid.
  • Figure 2-a shows the process of constructing the expression plasmid pCHO1.0 by ZHB BsAb-1;
  • Figure 2-b shows the process of constructing the expression plasmid pCHO1.0 by ZHB BsAb-2;
  • Figure 2-c shows the construction of ZHB BsAb-3 To the expression plasmid pCHO1.0 process map;
  • Figure 2-d is ZHB BsAb-4 was constructed into a map of the expression plasmid pCHO1.0.
  • Figure 3-a, Figure 3-b, Figure 3-c, and Figure 3-d show the SDS-PAGE electrophoresis pattern of the ZHB BsAb series polyclonal cell line fed-batch flow plus daily expression.
  • Figure 3-a shows the SDS-PAGE electrophoresis pattern of the ZHB BsAb-1 polyclonal cell line fed-batch and daily expression;
  • the lane 1 is the CHO-S empty cell supernatant culture control, and the lane 2 is 10-
  • the pre-stained protein in the 250KD range was loaded with Marker, and the lanes 3-10 were the ZEB BsAb-1 polyclonal cell line fed with the 3-10 day expression amount; the arrow indicated ZHB BsAb-1;
  • Figure 3-b shows the SDS-PAGE electrophoresis pattern of the ZHB BsAb-2 polyclonal cell line fed-batch flow plus daily expression; lane 1 is the CHO-S empty cell supernatant culture control, and lane 2 is the 10-250KD range.
  • the pre-stained protein was loaded with Marker, and lanes 3-10 were the ZHB BsAb-2 polyclonal cell line fed batch plus the 3-10 day expression amount; the arrow indicated ZHB BsAb-2;
  • Figure 3-c shows the SDS-PAGE electrophoresis pattern of the ZHB BsAb-3 polyclonal cell line fed-batch flow plus daily expression; lane 1 is the CHO-S empty cell supernatant culture control, and lane 2 is the 10-250KD range.
  • the pre-stained protein was loaded with Marker, and lanes 3-10 were for the ZHB BsAb-3 polyclonal cell line fed batch and the expression amount was 3-10 days; the arrow indicated ZHB BsAb-3;
  • Figure 3-d shows the SDS-PAGE electrophoresis pattern of the ZHB BsAb-4 polyclonal cell line fed-batch flow plus daily expression; lane 1 is the CHO-S empty cell supernatant culture control, and lane 2 is the 10-250KD range. The pre-stained protein was loaded with Marker. Lanes 3-10 were for the ZHB BsAb-4 polyclonal cell line fed batch plus the 3-10 day expression; the arrow indicated ZHB BsAb-4.
  • Figure 4-a, Figure 4-b, Figure 4-c, and Figure 4-d show the SDS-PAGE electrophoresis pattern of the ZHB BsAb series polyclonal cell line fed-batch flow-wise culture supernatant.
  • Figure 4-a shows the SDS-PAGE electrophoresis pattern of the ZHB BsAb-1 polyclonal cell line fed-batch and culture supernatant cultured through column chromatography.
  • Lane 1 is a pre-stained protein-loaded Marker in the range of 10-250 KD, and lanes 2-10 are purified samples of ZHB BsAb-1 collected in different collection tubes; the arrow indicates ZHB BsAb-1.
  • Figure 4-b shows the SDS-PAGE electrophoresis pattern of the ZHB BsAb-2 polyclonal cell line fed-batch flow-wise culture supernatant.
  • Lane 1 is a pre-stained protein-loaded Marker in the range of 10-250 KD, and lanes 2-10 are purified samples of ZHB BsAb-2 collected in different collection tubes; the arrow indicates ZHB BsAb-2.
  • Figure 4-c shows the SDS-PAGE electrophoresis pattern of the ZHB BsAb-3 polyclonal cell line fed-batch flow-wise culture supernatant.
  • Lane 1 is a pre-stained protein-loaded Marker in the range of 10-250 KD, and lanes 2-10 are purified samples of ZHB BsAb-3 collected in different collection tubes; the arrow indicates ZHB BsAb-3.
  • Figure 4-d shows the SDS-PAGE electrophoresis pattern of the ZHB BsAb-4 polyclonal cell line fed-batch flow-wise culture supernatant.
  • Lane 1 is a pre-stained protein-loaded Marker in the range of 10-250 KD, and lanes 2-10 are purified samples of ZHB BsAb-4 collected in different collection tubes; the arrow indicates ZHB BsAb-4.
  • Figure 5-a Figure 5-b are electrophoresis patterns of anti-CD26-scFv and anti-CD3-scFv genes on agarose gel.
  • Figure 5-a shows the anti-CD26-scFv gene agarose gel electrophoresis.
  • Lane 1 is a 200 bp DNA ladder;
  • Lane 2 is an anti-CD26-scFv gene containing XhoI and XbaI restriction sites at both ends.
  • Figure 5-b is an electrophoresis map of the anti-CD3-scFv gene agarose gel
  • Lane 1 is a 500 bp DNA ladder
  • Lane 2 is an anti-CD3-scFv gene containing XhoI and XbaI restriction sites at both ends.
  • Figure 6-a and Figure 6-b are diagrams showing the construction process of anti-CD26-scFv and anti-CD3-scFv gene expression plasmids.
  • Fig. 6-a is a process diagram of constructing the anti-CD26-scFv gene into the expression plasmid pPICZ ⁇
  • Fig. 6-b is a process diagram of constructing the anti-CD3-scFv gene into the expression plasmid pPICZ ⁇ .
  • Figure 7-a, Figure 7-b are small-scale induction maps of anti-CD26-scFv and anti-CD3-scFv recombinant Pichia pastoris strains.
  • Figure 7-a is a small-scale induction map of anti-CD26-scFv recombinant Pichia pastoris strain.
  • Lane 1 is a pre-stained protein-loading Marker in the range of 10-250 KD
  • lanes 2-9 are the anti-CD26-scFv supernatant cultures induced by methanol in different strains obtained by antibiotic Zeocin compression screening; the arrow indicates anti-CD26 -scFv.
  • Figure 7-b is a diagram showing the minimization induction of anti-CD3-scFv recombinant Pichia pastoris strains.
  • Lane 1 is a pre-stained protein-loading Marker in the range of 10-250 KD, and lanes 2-9 are anti-CD3-scFv supernatant cultures induced by different clones obtained by antibiotic Zeocin pressure screening; the arrow indicates anti-CD3- scFv.
  • Figure 8-a shows SDS-PAGE electrophoresis of anti-CD26-scFv and anti-CD3-scFv samples collected by IMAC affinity purification
  • Fig. 8-a is a SDS-PAGE electrophoresis pattern of anti-CD26-scFv collected by IMAC affinity purification.
  • Lane 1 is a pre-stained protein-loaded Marker in the range of 10-250 KD, and lanes 2-8 are purified samples of anti-CD26-scFv collected in different collection tubes; the arrow indicates anti-CD26-scFv.
  • Figure 8-b is a SDS-PAGE electropherogram of anti-CD3-scFv collected by IMAC affinity purification.
  • Lane 1 is a pre-stained protein-loaded Marker in the range of 10-250 KD, and lanes 2-7 are anti-CD3-scFv purified samples collected in different collection tubes; the arrow indicates anti-CD3-scFv.
  • Figure 9-a and Figure 9-b are electrophoresis plots of the anti-CD26 humanized antibody heavy and light chain genes on agarose gel.
  • Figure 9-a is an electrophoresis map of the anti-CD26 humanized antibody heavy chain gene agarose gel.
  • Lane 1 is a 500 bp DNA ladder;
  • Lane 2 is an anti-CD26 humanized antibody heavy chain gene containing AvrII and BstZ17I restriction sites at both ends.
  • Figure 9-b is an electrophoresis map of the anti-CD26 humanized antibody light chain gene agarose gel.
  • Lane 1 is a 500 bp DNA ladder;
  • Lane 2 is an anti-CD26 humanized antibody light chain gene containing EcoRV and PacI restriction sites at both ends.
  • Figure 10 is a diagram showing the construction process of an anti-CD26 humanized antibody gene expression plasmid.
  • Figure 11 is a diagram showing the SDS-PAGE electrophoresis of the anti-CD26 humanized antibody polyclonal cell line fed-batch flow plus daily expression.
  • Lane 1 is a pre-stained protein-loaded Marker in the range of 10-250 KD, and lanes 2-9 are anti-CD26 humanized antibodies.
  • the cell line was fed in batches with the expression of the 2nd-9th day; the lane 10 was the CHO-S empty cell supernatant culture control, and the arrows indicated the anti-CD26 humanized antibody heavy and light chains, respectively;
  • Figure 12 is an SDS-PAGE electrophoresis pattern of anti-CD26 humanized antibody polyclonal cell line fed-batch flow-wise culture supernatant culture solution pre-packed with proteinA affinity column.
  • Lane 1 is a pre-stained protein-loaded Marker in the range of 10-250 KD
  • Lane 2 is an anti-CD26 humanized antibody
  • the arrows indicate anti-CD26 humanized antibody heavy and light chains, respectively.
  • Figure 13 is a graph showing the binding and dissociation of affinity detection results.
  • A is the binding and dissociation curve of BsAb1
  • B is the binding and dissociation curve of BsAb4
  • C is the binding and dissociation curve of BsAb3
  • D is the binding and dissociation curve of BsAb2
  • E is the binding of anti-CD26 single-chain antibody With the dissociation curve.
  • Figure 14 is a cytotoxic effect of PBMC on 786-0 cells and A375 cells mediated by parental single-chain antibody and bispecific antibody BsAb1, BsAb2, BsAb3, BsAb4.
  • Figure 15 shows the cytotoxic effect of BsAb4-mediated PBMC cells on 786-0 cells under a microscope.
  • Figure 15-a shows 786-0 cells without BsAb4 added; 15-b is 786-0 cells with apoptosis added with BsAb4.
  • Figure 16 is a bispecific antibody BsAb2, BsAb4-mediated cytotoxic effect of T cells on 786-0 cells.
  • 16-a is a cytotoxic effect of BsAb2-mediated T cells on 786-0 cells
  • 16-b is a cytotoxic effect of BsAb4-mediated T cells on 786-0 cells.
  • the protein domain that targets the first antigen CD26 is operably linked to the protein domain that targets the second antigen CD3, maintaining their respective spatial structures and exerting their respective physiological activities.
  • the protein functional region targeting the first antigen CD26 and the protein functional region targeting the second antigen CD3 can be directly fused together without affecting their respective functions, and the protein functional region targeting the second antigen CD3 can be
  • the N-terminus of the functional domain of the protein targeting the first antigen CD26 may also be ligated to the C-terminus of the functional domain of the protein targeting the first antigen CD26, or other spacer sequences such as ligation fragments may be added therebetween.
  • the heavy chain variable region and the light chain variable region of the protein functional region targeting the first antigen CD26 can be at the N-terminus of the protein functional region targeting the first antigen CD26; the protein function targeting the second antigen CD3 Both the heavy chain variable region and the light chain variable region in the region are at the N-terminus of the protein functional region that targets the second antigen CD3.
  • bispecific in a “bispecific antibody” refers to the simultaneous specific targeting of two different antigens, which in the present invention are human CD26 and human CD3, respectively.
  • antibody in addition to the specific immunoglobulin, also refers to a fragment of an immunoglobulin (eg, at least an immunologically active portion of an immunoglobulin molecule), such as Fab, Fab', F. (ab') 2, Fv fragment, single-chain antibody molecule or A multispecific antibody formed by any fragment of an immunoglobulin molecule of one or more CDR regions.
  • single-chain antibody or: scFv in the present invention refers to an engineered antibody in which a light chain variable region is directly linked to a heavy chain variable region or linked by a short peptide.
  • the bispecific antibody consists of an anti-CD26-scFv-anti-CD3-scFv, or an anti-CD3-scFv-anti-CD26-scFv, or an anti-CD26 IgG-linked short peptide-
  • the anti-CD3-scFv composition may also consist of an anti-CD3 scFv-linked short peptide-anti-CD26 IgG.
  • the invention relates to a method of preventing or treating a disease caused by high expression of CD26 with said bispecific antibody, comprising administering a therapeutically effective amount of a bispecific antibody or pharmaceutical composition as described above.
  • the treated tumor is selected from the group consisting of an offensive T cell malignancy, a malignant mesothelioma, a renal tumor, and a colon cancer.
  • variable regions are joined in a different order to form four bispecific antibodies, designated ZHB BsAb-1 (shown as SEQ ID NO. 7), ZHB BsAb-2 (shown as SEQ ID NO. 8), ZHB BsAb. -3 (as shown in SEQ ID NO. 9), ZHB BsAb-4 (shown as SEQ ID NO. 10), wherein ZHB BsAb-1 is selected as a sandfly yellow-associated protein (eg SEQ ID NO.
  • ZHB BsAb-2 uses a silkworm Fibroin-related protein (as shown in SEQ ID NO. 12) as a secretory expression signal peptide
  • ZHB BsAb-3 is a cypridina Noctiluca.
  • Related White (as shown in SEQ ID NO. 13) as a secretory expression signal peptide
  • ZHB BsAb-4 uses a pinemoth-associated protein (as shown in SEQ ID NO. 14) as a secretory expression signal peptide to obtain an optimized ZHB BsAb-1 gene (as shown in SEQ ID NO. 16), ZHB BsAb-2 gene (shown as SEQ ID NO.
  • ZHB BsAb-3 gene shown as SEQ ID NO. 18
  • ZHB BsAb- 4 genes as shown in SEQ ID NO. 19
  • the above four genes are optimized according to mammalian cell CHO codon bias, respectively, to obtain optimized ZHB BsAb-1 (as shown in SEQ ID No. 20), ZHB BsAb-2 (shown as SEQ ID No. 21), ZHB BsAb-3 (shown as SEQ ID No. 22), ZHB BsAb-4 (shown as SEQ ID No. 23)
  • CAI Codon Adaptation Index
  • CAI CHO codon adaptation index
  • ZHB BsAb-1, ZHB BsAb-2, ZHB BsAb-3, and ZHB BsAb-4 genes in mammalian cells were 0.68, 0.68, 0.68, 0.68, respectively.
  • Codon optimization and calculation showed that the optimized ZHB BsAb-1, ZHB BsAb-2, ZHB BsAb-3, and ZHB BsAb-4 genes had a CAI index of 0.86, 0.86, and 0.87 in the mammalian cell CHO expression system. 0.87. It can be seen that the gene sequence obtained by codon optimization can increase the expression level of ZHB BsAb-1, ZHB BsAb-2, ZHB BsAb-3 and ZHB BsAb-4 genes in mammalian cell CHO expression system.
  • the ideal distribution of GC content is 30%-70%, and any peak outside this region will affect transcription and translation efficiency to varying degrees.
  • the average content of unquantified gene GC bases of ZHB BsAb-1, ZHB BsAb-2, ZHB BsAb-3, and ZHB BsAb-4 is 51.83%, 51.90%, 51.89%, 51.87%, and is optimized after removal.
  • the GC content outside the 30%-70% region was finally optimized.
  • the average GC base content of ZHB BsAb-1, ZHB BsAb-2, ZHB BsAb-3, and ZHB BsAb-4 genes was 54.15%, 54.41%, 54.75. %, 54.54%.
  • Example 2 Anti-CD26 and anti-CD3 bispecific antibody expression plasmid construction, stable expression and purification
  • Optimized ZHB BsAb-1 (shown as SEQ ID No. 20), ZHB BsAb-2 (shown as SEQ ID No. 21), ZHB BsAb-3 (shown as SEQ ID No. 22), The ZHB BsAb-4 (shown as SEQ ID No. 23) gene is introduced upstream of the AvrII cleavage site (as shown in SEQ ID NO. 24) and the kozak sequence (shown as SEQ ID NO. 25), and the BstZ17I enzyme is introduced downstream.
  • the cleavage site (as shown in SEQ ID NO.
  • the above plasmid was used as a template to amplify the target gene, and 1% agarose electrophoresis was used to recover the PCR product (Fig. 1)) and use AvrII and BstZ17I
  • the product was digested with PCR and the pCHO1.0 vector (purchased from Invitrogen).
  • the T4 ligase was ligated into the pCHO1.0 vector backbone and transformed into Top10 competent cells and coated with kanamycin.
  • the resistant LB plates were cultured overnight at 37 °C.
  • the expression plasmids of the fusion protein of different forms of anti-CD26 single-chain antibody and anti-CD3 single-chain antibody were obtained, respectively, which were recorded as pCHO1.0- ZHB BsAb-1, pCHO1.0-ZHB BsAb-2, pCHO1.0-ZHB BsAb-3, pCHO1.0-ZHB BsAb-4 (The plasmid construction flow chart is shown in Figure 2-a, 2-b, 2-c, 2-d)).
  • Anti-CD26 and anti-CD3 bispecific antibodies stably transfected and expressed different genes
  • the pCHO1.0-ZHB BsAb-1, pCHO1.0-ZHB BsAb-2, pCHO1.0-ZHB BsAb-3, pCHO1.0-ZHB BsAb-4 plasmid was digested overnight by NruI (R0192S, purchased from NEB). After linearization and electroporation into CHO-S cells, penicillin and MTX were added for pressure screening, and cell viability was calculated after one week. When the cell viability was greater than 30%, the cells were transferred to a CO 2 shaker.
  • Anti-CD26 and anti-CD3 bispecific antibody polyclonal cell lines expanded culture and purification
  • the polyclonal cell lines obtained by the pressure screening in step 2 were subjected to fed-batch fed-batch culture to obtain ZHB BsAb-1, ZHB BsAb-2, ZHB BsAb-3, ZHB BsAb-4 supernatant culture solution, 12000 rpm, The supernatant was collected by cryogenic centrifugation at 15 min, and filtered through a 0.45 ⁇ m filter.
  • the pre-treated ZHB BsAb-1, ZHB BsAb-2, ZHB BsAb-3, and ZHB BsAb-4 were purified using a fully automated intelligent protein purification system (AKTA york150, purchased from GE healcare).
  • the purification effects of ZHB BsAb-1, ZHB BsAb-2, ZHB BsAb-3 and ZHB BsAb-4 were analyzed by SDS-PAGE. The results are shown in Figure 4-a, Figure 4-b, Figure 4-c and Figure 4-d. It is shown that each collection tube that meets the requirements is combined, concentrated by ultrafiltration, replaced into PBS buffer, sterilized by filtration, and stored at 4 ° C for use as a backup.
  • the anti-CD26 heavy chain variable region (shown as SEQ ID NO. 1) and the light chain variable region (shown as SEQ ID NO. 2) are fused directly to the short peptide (shown as SEQ ID NO. 3) , constituting an anti-CD26-scFv (as shown in SEQ ID NO. 29); an anti-CD3 heavy chain variable region (as shown in SEQ ID NO. 4) and a light chain variable region (as shown in SEQ ID NO. 5) Direct fusion with a short peptide (shown as SEQ ID NO. 6) to form an anti-CD3-scFv (as set forth in SEQ ID NO. 30).
  • codon-optimized anti-CD26-scFv and anti-CD3-scFv genes have a CAI index of 0.84 and 0.83, respectively, in the Pichia pastoris expression system; codon-optimized anti-CD26-scFv and anti-CD3
  • the frequency of the scFv gene showing a low utilization codon in the Pichia pastoris system was 0; the average GC base content of the anti-CD26-scFv and anti-CD3-scFv genes after optimization was 44.99%, 44.10%.
  • the optimized anti-CD26-scFv and anti-CD3-scFv whole genes were introduced into the XhoI (as shown in SEQ ID NO. 35) and XbaI (as shown in SEQ ID NO. 36) double-cleavage sites and carried out. Gene synthesis. The synthesized fragments were separately constructed into pUC57 plasmid to obtain a long-term preservation plasmid, which was sequentially designated as pUC57-CD26-scFv, pUC57-CD3-scFv plasmid.
  • the above plasmid was used as a template to amplify the target gene, and 1% agarose electrophoresis was used to recover the PCR product (Fig. 5-a, 5-b), and the XhoI and XbaI double-digested PCR to recover the product and the pPICZ ⁇ 1 vector (purchased from Invitrogen), and the T4 ligase ligated the double-cut product into the pPICZ ⁇ 1 vector backbone and transformed into the Top10 receptor.
  • the cells were cultured overnight at 37 ° C in LB plates containing Zeocin.
  • YPDS solid medium preparation Invitrogen EasySelect Pichia Expression Kit specification provided, wherein yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agarose 15g / L, D - sorbitol 182g / L.
  • X-33 strain (C18000, purchased from Invitrogen) was prepared into electrocompetent cells according to the method provided by the EasySelect Pichia Expression Kit.
  • the pPICZ ⁇ -CD26-scFv and pPICZ ⁇ -CD3-scFv plasmids were linearized by SacI restriction enzyme digestion, and the linearized vector was electrotransformed into X-33 competent yeast cells after ethanol precipitation, and coated to contain 0.5. -2.0 mg/mL Zeocin (R250-01, available from Invitrogen) in YPDS solid medium, incubated at 30 ° C for 3-5 d.
  • BMGY medium Invitrogen Multi-Copy Pichia Expression Kit instructions, including yeast extract 10g / L, peptone 20g / L, K 2 HPO 4 3g / L, KH 2 PO 4 11.8g / L, YNB 13.4g / L, biotin 4 ⁇ 10 -4 g / L, glycerol 10 g / L.
  • BMMY medium preparation Invitrogen Multi-Copy Pichia Expression Kit instructions, including yeast extract 10g / L, peptone 20g / L, K 2 HPO 4 3g / L, KH 2 PO 4 11.8g / L, YNB13.4g / L, biotin 4 ⁇ 10 -4 g / L, methanol 5 mL / L.
  • Fig. 7-a, 7-b are the identification expressions of the recombinant strains containing the optimized codon anti-CD26-scFv and anti-CD3-scFv, respectively.
  • the supernatants of the anti-CD26-scFv and anti-CD3-scFv recombinant strains containing the optimized codon were respectively obtained, and the supernatant was collected by centrifugation at 12000 rpm for 15 minutes, and the binding buffer was added to make it in the supernatant.
  • the final concentration was 300 mM NaCl, 20 mM NaH 2 PO 4 , 10 mM Imidazole, adjusted to pH 7.5, and filtered through a 0.45 ⁇ m filter.
  • the anti-CD26-scFv and anti-CD3-scFv fermentation broth obtained by pretreatment were purified by HisTrap FF crude affinity column using a fully automated intelligent protein purification system.
  • the binding buffer was 300 mM NaCl, 20 mM NaH 2 PO 4 . 20 mM Imidazole, pH 7.5; elution buffer was 300 mM NaCl, 20 mM Na 2 HPO 4 , 20 mM Imidazole, pH 7.5.
  • Each elution peak was collected when the elution buffer was eluted, and the purity was confirmed by SDS-PAGE electrophoresis.
  • 8-b are electrophoresis patterns of the collection tubes after anti-CD26-scFv and anti-CD3-scFv elution, respectively.
  • Each collection tube was combined with ultrafiltration to concentrate, the replacement buffer was PBS solution, and sterilized by filtration, and stored at 4 ° C for use as a backup.
  • CD26 heavy chain variable region shown as SEQ ID NO. 1
  • human IgG1 heavy chain constant region shown herein as anti-CD26-HC
  • anti-CD26-HC Directly fused to the CD26 light chain variable region (shown as SEQ ID NO. 2) and the human IgGl light chain constant region, as shown in SEQ ID NO. 37, to form an anti-CD26 humanized antibody light chain region
  • the present invention is named as anti-CD26-LC, as shown in SEQ ID NO. 38, and mouse (Mus musculus) IgG k (as shown in SEQ ID NO. 39) is selected as a secretory expression signal peptide to obtain anti-CD26-HC optimization.
  • the pre-gene sequence (shown as SEQ ID NO. 40) and the anti-CD26-LC optimized pre-gene sequence (shown as SEQ ID NO. 41) are optimized for codon bias according to the mammalian cell expression system to obtain anti-CD26 - HC-optimized gene (as set forth in SEQ ID NO. 42) and anti-CD26-LC optimized gene (shown as SEQ ID NO. 43). Furthermore, it was calculated that the CAI indices of codon-optimized anti-CD26-HC and anti-CD26-LC genes in mammalian cell expression systems were 0.86 and 0.86, respectively; anti-CD26-HC and anti-CD26 were optimized by codon.
  • the frequency of the low-utilization codon in the mammalian cell expression system of the -LC gene is 0; the average GC base content of the anti-CD26-HC and anti-CD26-LC genes after optimization is 53.89%, 53.10%.
  • the optimized anti-CD26-HC gene is introduced upstream into the AvrII cleavage site (as shown in SEQ ID NO. 24) and the kozak sequence (as shown in SEQ ID NO. 25), and the BstZ17I cleavage site (such as SEQ) is introduced downstream.
  • ID NO.26 The whole gene was synthesized, and the synthesized fragment was constructed into pUC57 plasmid to obtain a long-preserved plasmid, which was recorded as pUC57-CD26-HC plasmid; and the optimized anti-CD26-LC gene was upstream.
  • the EcoRV (as shown in SEQ ID NO.
  • cleavage site and the kozak sequence are introduced, and the PacI cleavage site (shown as SEQ ID NO. 45) is introduced downstream for the whole gene.
  • the synthesized fragment was constructed into pUC57 plasmid to obtain a long-term preservation plasmid, which was designated as pUC57-CD26-LC plasmid.
  • pUC57-CD26-HC plasmid was used as a template to amplify the target gene, and 1% agarose electrophoresis was used to recover the PCR product.
  • the product and pCHO1.0 vector were double-digested with AvrII and BstZ17I, and the T4 ligase was ligated into the pCHO1.0 vector backbone and transformed into Top10 competent cells. Incubate overnight at 37 ° C in LB plates containing kanamycin resistance. The positive clones were screened the next day, and sequenced and aligned, which was identical to the expected sequence, and the expression plasmid of anti-CD26-HC was obtained, which was designated as pCHO1.0-CD26-HC.
  • the M13 F (as shown in SEQ ID NO. 27) and M13 R (as shown in SEQ ID NO. 28) were used as primers, the pUC57-CD26-LC plasmid was used as a template to amplify the target gene, and the PCR product was recovered by 1% agarose electrophoresis. (As shown in 9-b), the product was recovered by EcoRV and PacI double enzyme PCR and pCHO1.0-CD26-HC, and the T4 ligase was ligated into the pCHO1.0-CD26-HC vector backbone and transformed into Top10. In competent cells, culture was carried out overnight at 37 ° C in LB plates containing kanamycin resistance. On the second day, positive clones were screened and sequenced, which was identical to the expected sequence, and the expression plasmid of anti-CD26 humanized antibody was obtained, which was recorded as pCHO1.0-CD26(H+L). Shown).
  • FIG. 11 shows the anti-CD26 humanized antibody polyclonal cell line for the expression of glucose fed-batch expression.
  • the polyclonal cell strain obtained by the pressure screening in the step 2 was subjected to fed-batch fed-batch culture to obtain an anti-CD26 humanized antibody supernatant culture solution, and the supernatant was collected by centrifugation at 15 000 min for 15 min, and subjected to 20 mM NaH 2 PO 4 .
  • the tangential flow ultrafiltration system was changed to buffer, and the pH was adjusted to 7.0, and the 0.45 ⁇ m filter was filtered.
  • the anti-CD26 humanized antibody obtained by pretreatment was purified by HiTrap proteinA FF prepacked column using a fully automated intelligent protein purification system.
  • the binding buffer was 20 mM NaH 2 PO 4 , pH 7.0; the elution buffer was 20 mM NaH 2 PO 4 , 0.1 M citric acid, pH 3.0.
  • Each elution peak was collected by elution buffer, and the purification effect was confirmed by SDS-PAGE electrophoresis. The results are shown in Fig. 12.
  • the respective collection tubes which meet the requirements were combined and concentrated by ultrafiltration.
  • the replacement buffer was PBS solution and filtered.
  • the bacteria were stored at 4 ° C for use as a spare.
  • Human renal clear cell adenocarcinoma cell line 786-0 cells in logarithmic growth phase ( CRL-1932), human mesothelioma cell NCI-H2452 cells ( CRL-5946), renal cancer cell Caki-1 cells ( HTB-46), human colon cancer cell line COLO 205 cells ( CCL-222), human prostate cancer cell line PC-3 ( CRL-1435), human malignant melanoma cell A375 cells ( CRL-1619) was digested with trypsin, centrifuged at 1000 rpm for 5 min, and washed twice with PBS; 1.0 ⁇ 10 6 cells each were resuspended in 250 ⁇ L PBS, and added with anti-CD26 antibody at a final concentration of 10 ug/mL at room temperature.
  • Jurkat cells in logarithmic growth phase (TIB-152) was collected, centrifuged at 1000 rpm for 5 min, and washed twice with PBS; 1.0 ⁇ 10 6 cells each were resuspended in 250 ⁇ L PBS, and added with anti-CD3 single-chain antibody at a final concentration of 30 ug/mL.
  • 1h after the end of the incubation, wash twice with PBS, add 250 ⁇ L of Anti-His-tag-Alexa Fluor 488 antibody diluted 1:2000, incubate for 1 h at room temperature, after the incubation, wash three times with PBS, resuspend in 300 ⁇ L PBS After that, the test was carried out by flow cytometry.
  • the bispecific antibodies BsAb1, BsAb2, BsAb3 and BsAb4 can specifically bind to CD26 molecules on the surface of tumor cells, especially BsAb2 and BsAb4 have higher binding rate to CD26 on the surface of tumor cells.
  • Jurkat cells in logarithmic growth phase were trypsinized, and the cells were collected by centrifugation at 1000 rpm for 5 min, and washed twice with PBS; 1.0 ⁇ 10 6 cells each were resuspended in 250 ⁇ L of PBS, and the final concentration was 10 ug. /mL of BsAb1, BsAb2, BsAb3, and BsAb4 were incubated for 1 h at room temperature. After the incubation, wash twice with PBS, add 250 ⁇ L of Anti-His-tag-Alexa Fluor 488 antibody diluted 1:2000, and incubate for 1 h at room temperature. After the incubation, the cells were washed three times with PBS, resuspended in 300 ⁇ L of PBS, and detected by flow cytometry.
  • the binding rate of BsAb1 to Jurkat cells was 0.1% by FACS analysis; the binding rate of BsAb2 to Jurkat cells was 92.0%; the binding rate of BsAb3 to Jurkat cells was 0.2%; the binding rate of BsAb4 to Jurkat cells was 56.5%. .
  • the bispecific antibodies BsAb1, BsAb2, BsAb3 and BsAb4 can specifically bind to CD3 molecules on the surface of Jurkat cells, especially the binding ratio of BsAb2 and BsAb4 to the surface of Jurkat cells.
  • KD (BsAb1) 9.29 ⁇ 10 -9 M
  • KD (BsAb2) 3.44 ⁇ 10 -9 M
  • KD (BsAb3) 1.0 ⁇ 10 -8 M
  • KD (BsAb4) 1.97 ⁇ 10 -9 M
  • KD (anti-CD26-scFv) 1.00 ⁇ 10 -9 M.
  • bispecific antibodies BsAb1, BsAb2, BsAb3, BsAb4 and CD26 have certain affinity; and compared with the parental single-chain antibody, BsAb2 and BsAb4 are close to their affinity constants.
  • mice in logarithmic growth phase were trypsinized, centrifuged at 1000 rpm for 5 min, and washed twice with PBS; 1.0 ⁇ 10 6 cells were resuspended in 1 mL PBS, and the final concentration was 2.5.
  • the cells were cultured at 37 ° C for 30 min in ⁇ M Calcein-AM solution; after the incubation, the cells were washed three times with PBS; the cells were diluted to 6.0 ⁇ 10 5 with RPMI-1640 medium (GIBCO, Cat# 31800022), 50 uL per well. Add U-type 96-well cell culture plate, and add 50 ⁇ L of the experimental group.
  • the blank control group is added with 50 ⁇ L of RPMI-1640 medium, the positive control group is added with 50 ⁇ L of 3% Triton-100, and the experimental group is added with 50 ⁇ L of BsAb1 (10 ng/mL).
  • 50 ⁇ L BsAb2 (10 ng/mL)
  • 50 ⁇ L BsAb3 (10 ng/mL)
  • 50 ⁇ L BsAb4 (10 ng/mL)
  • 50 ⁇ L Anti-CD3-scFv (10 ng/mL
  • 50 ⁇ L Anti-CD26-scFv 10 ng/mL
  • Incubate at 37 °C for 30 min. After the incubation, add PBMC cells at a ratio of 786-0:PBMC of 1:15.
  • A375 cells were used as a negative control, and the operation was the same as above.
  • the cytotoxic effects of bispecific antibodies and parental single chain antibody mediated PBMC on 786-0 cells and A375 cells are shown in Table 3, Figure 14.
  • the cleavage ratio of the bispecific antibody BsAb1 to 786-0 cells at 10 ng/mL was 15.9%; the cleavage ratio of BsAb2 to 786-0 cells at 10 ng/mL was 27.4%; BsAb3 at 10 ng/mL for 786-0 cells.
  • the lysis rate was 4.3%; the cleavage ratio of BsAb4 to 786-0 cells at 10 ng/mL was 25.5%; the cleavage ratio of single-chain antibody anti-CD26-scFv to 786-0 cells at 10 ng/mL was 4.1%; single-chain antibody The cleavage ratio of anti-CD3-scFv to 786-0 cells at 10 ng/mL was 4.1%.
  • the ratio of cleavage of the bispecific antibody BsAb1 to the negative control A375 cells at 10 ng/mL was 0.2%; the ratio of BsAb2 cleavage to A375 cells at 10 ng/mL was 2.5%; the ratio of BsAb3 at 10 ng/mL to A375 cells was 1.5. %; BsAb4 cleavage ratio of A375 cells at 10 ng/mL was 2.4%; single-chain antibody anti-CD26-scFv cleavage ratio of A375 cells at 10 ng/mL was 0.3%; single-chain antibody anti-CD3-scFv at 10 ng/ The ratio of lysis of mL to A375 cells was 0.4%.
  • Parental single-chain antibody anti-CD26-scFv, anti-CD3-scFv and bispecific antibodies BsAb1, BsAb2, BsAb3, BsAb4 have no cytotoxic effect on negative control A375 cells; parental single-chain antibody anti-CD26-scFv and anti-CD3-scFv and bispecific antibodies BsAb1, BsAb2, BsAb3, and BsAb4 have certain cytotoxic effects on 786-0 cells.
  • the bispecific antibodies BsAb1, BsAb2 and BsAb4 were more cytotoxic to 786-0 cells than the parental single-chain antibodies anti-CD26-scFv and anti-CD3-scFv.
  • mice in logarithmic growth phase were trypsinized, centrifuged at 1000 rpm for 5 min, and washed twice with PBS; 1.0 ⁇ 10 6 cells were resuspended in 1 mL PBS, and the final concentration was 2.5.
  • the cells were cultured at 37 ° C for 30 min in ⁇ M Calcein-AM solution; after the incubation, the cells were washed three times with PBS; after the cells were diluted to 6.0 ⁇ 10 5 with RPMI-1640 medium, U-type 96-well cells were added at 50 ⁇ L per well.
  • the culture plate 50 ⁇ L of the experimental group was added, in which 50 ⁇ L of RPMI-1640 medium was added to the blank control group, 50 ⁇ L of 3% Triton-100 was added to the positive control group, and 50 ⁇ L of different concentrations of anti-CD26 humanized antibody (10 ng/mL) were added to the experimental group. , 1ng/mL, 0.01ng/mL) was incubated at 37 ° C for 30 min. After the incubation, PBMC cells were added at a ratio of 786-0:PBMC of 1:15, and incubation was continued at 37 ° C for 3 h. After the incubation, appropriate amount of cell culture was taken. The supernatant was detected by a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 515 nm.
  • DMEM medium (GIBCO, Cat#12800-082).
  • Anti-CD26 humanized antibody has no obvious cytotoxic effect on negative control A375 cells. Compared with it, anti-CD26 humanized antibody showed a concentration-dependent cytotoxic effect on 786-0 cells. However, the cytotoxic effects of the bispecific antibodies BsAb1, BsAb2, and BsAb4 of the present invention on 786-0 cells were significantly enhanced compared to the anti-CD26 humanized antibodies.
  • PBMC cells were isolated from human blood according to the instructions of Lymphocyte Separation Medium (MP, Cat# 50494). The isolated PBMC cells were subjected to Human Erythrocyte Lying Kit (R&D SYSTEM, Cat#). WL1000) instructions for lysing red blood cells, operating according to the instructions of Human T Cell Enrichment Columns (R&D SYSTEM, Cat# HTCC-10), enriching T cells, and enriching T cells, 1.0 ⁇ each 10 6 cells were resuspended in 250 ⁇ L PBS, washed twice with PBS, and added with anti-CD3 single-chain antibody at a final concentration of 30 ug/mL for 1 h at room temperature.
  • the 786-0 cells in the logarithmic growth phase were trypsinized, and the cells were collected by centrifugation at 1000 rpm for 5 min, and washed twice with PBS; 1.0 ⁇ 10 6 cells were resuspended in 1 mL of PBS, and Calcein was added at a final concentration of 2.5 ⁇ M.
  • -AM solution the cells were cultured at 37 ° C for 30 min; after the incubation, the cells were washed three times with PBS; after the cells were diluted to 5.0 ⁇ 10 5 with RPMI-1640 medium, 50 uL per well was added to the U-type 96-well cell culture plate.
  • 50 ⁇ L of the experimental group was added, in which 50 ⁇ L of RPMI-1640 medium was added to the blank control group, 50 ⁇ L of 3% Triton-100 was added to the positive control group, and 50 ⁇ L of anti-CD26-scFv (10 1 pg/mL, 10 3 pg/ was added to the experimental group.
  • BsAb 2 (10 -1 pg/mL, 10 0 pg/mL, 10 1 pg/mL, 10 2 pg/mL, 10 3 pg/mL, 10 4 pg/mL, 10 5 pg/mL, 10 6 pg/mL), 50 ⁇ L BsAb4 (10 -1 pg/mL, 10 0 pg/mL, 10 1 pg/mL, 10 2 pg/mL, 10 3 pg/ mL, 10 4 pg/mL, 10 5 pg/mL, 10 6 pg/mL), incubate at 37 ° C for 30 min, after the end of the incubation, add T cells according to the ratio of 786-0: T cells 1:5, continue Incubate at 37 ° C for 7 h, after the end of the incubation, take appropriate amount of cell
  • the A375 cells in the logarithmic growth phase were trypsinized and washed twice with PBS; 1.0 ⁇ 10 6 cells were resuspended in 1 mL PBS, and a final concentration of 2.5 ⁇ M Calcein-AM solution was added, and the cells were cultured at 37 ° C for 30 min.
  • the cells were washed three times with PBS; after diluting the cells with DMEM medium to 5.0 ⁇ 10 5 , 50 ⁇ L per well was added to the U-type 96-well cell culture plate, and 50 ⁇ L of the experimental group sample was added, and the blank control group was added with 50 ⁇ L of RPMI-1640.
  • Anti-CD26-scFv, BsAb2 and BsAb4 have cytotoxic effects on 786-0 cells compared with the negative control, but the cytotoxic effect of the parental single-chain antibody anti-CD26-scFv on 786-0 cells has no significant concentration.
  • Gradient-dependent, BsAb2 and BsAb4 showed a significant concentration-dependent killing effect on 786-0 cells compared to the parental single-chain antibody anti-CD26-scFv (Fig. 16).
  • NCI-H2452 cells, PC-3 cells, Caki-1 cells, and COLO 205 cells in logarithmic growth phase were trypsinized, centrifuged at 1000 rpm for 5 min, and washed twice with PBS; 1.0 ⁇ 10 6 cells were resuspended in 1 mL PBS, and a final concentration of 2.5 ⁇ M Calcein-AM solution was added, and the cells were cultured at 37 ° C for 30 min. After the incubation, the cells were washed three times with PBS; the cells were diluted to 4.5 with RPMI-1640 medium.
  • bispecific antibodies BsAb1, BsAb2, BsAb3 and BsAb4 could mediate the cytotoxic effect of PBMC on tumor cell lines with high CD26 expression on the cell surface at a concentration of 10 ng/mL.
  • the human CD3 and human CD26 bispecific antibodies provided by the present invention can effectively mediate the cytotoxic effect of PBMC on tumor cells with high CD26 expression on the cell surface; and the difference in bispecific antibody structure has certain biological activities.
  • BsAb2 and BsAb4 have higher biological activities in the different structures of human CD3 and human CD26 bispecific antibodies studied.

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Abstract

提供了一种双特异性抗体，及其制备方法和其在制备治疗细胞高表达CD26型肿瘤的药物中的应用，所述抗体同时与人CD26和人CD3特异性结合。

Description

双特异性抗体、其制备方法和用途 技术领域
本发明涉及一种双特异性抗体，尤其涉及同时与人CD26和人CD3的特异性结合的单链抗体，及其制备方法和在治疗细胞高表达CD26型肿瘤的药物的新用途。
背景技术
CD26是一种普遍存在的多功能Ⅱ型跨膜糖蛋白，具有多种生物学功能，也可以以溶解形式存在于血浆中。CD26常以同源二聚体形式存在，其单体含766个氨基酸，相对分子质量约110kDa。氨基酸残基从内向外分为5个部分：胞内区(1～6)、跨膜区(7～28)、高度糖基化区(29～323)、富含半胱氨酸区(324～551)和C端催化结构域(552～766)，CD26分子三维结构与功能密切相关。CD26在免疫调节中的作用已被广泛研究，CD26是T细胞活化的分子标志，也在T细胞信号转导过程中作为共刺激分子，还涉及多种T细胞功能，包括T细胞发生成熟及迁移，细胞因子分泌，T细胞依赖的抗体产生，B细胞免疫球蛋白转型等(Ohnuma et al.(2011)Adv Clin Chem,53,51-84)。
CD26可以和多种蛋白相互作用，如ADA、CD45、FAP-alpha等，还可以结合ECM，导致表达CD26细胞浸润活性的增加或降低，可见CD26在肿瘤生物学发挥重要作用。CD26在多种的新生瘤细胞表面的表达量明显升高，例如，CD26高表达于某些进攻性T细胞恶性肿瘤，恶性间皮瘤，肾瘤，某些结肠癌(Havre et al.(2008)Front Biosci,13,1634-1645)。某些CD26+结肠癌细胞亚群，CD26+恶性间皮瘤细胞都有明显肿瘤干细胞特征(Ghani et al.(2011)Biochem Biophys Res Commun,404,735-742and Pang et al.(2010)Cell Stem Cell,6,603-615)，因此CD26可作为多种肿瘤的分子标志。
目前，以人CD26为靶点的靶向性抗癌药物还主要是一些单抗药物，而其免疫原性、治疗效果等方面仍需进一步改善。例如Y’s Therapeutics公司已进入临床阶段的抗CD26鼠源人源化单克隆抗体药物YS110，可与靶细胞表面的CD26抗原决定簇特异性地结合，之后CD26的IgG的Fc段与表达IgG Fc受体的NK细胞、巨噬细胞和中性粒细胞结合进而通过抗体的ADCC效应发挥作用。然而Fc受体中可能存在激动型和抑制型两种类型，从而影响抗体的作用效果。且鼠抗人源化以后仍然含有鼠源成分，不能完全排除HAMA效应。抗体的Fc段在表达过程中存在着岩藻糖修饰现象，抑制ADCC效应，且重链糖型分布复杂，增加分离纯化和质量控制的难度。
人CD3仅存在于T细胞表面，由三种不同链CD3γ链，CD3δ链，和CD3ε链组成，常与TCR紧密结合形成TCR-CD3复合体。CD3分子通过盐桥与T细胞抗原受体相连，参与T细胞的信号 转导。CD3在T细胞上成簇(如通过固定的抗CD3抗体)可以导致类似T细胞受体结合引起的T细胞活化。临床上使用的OKT3抗体是抗CD3抗体的经典代表，用于治疗同种异体移植排斥。
定向T细胞攻击癌细胞的双特异性抗体的研究已有二十多年，在抗体药物前期开发出了大量分化抗原用于肿瘤的靶向治疗，现今在新技术平台下，大部分均被研究用于构建与CD3靶向偶联的双特异性抗体来提高疗效，像CD19、CD33、CEA、EpCAM、HER-2/neu、PSMA或EGF受体等。(S.R.Frankel,P.A.Baeuerle(2013)Curr Opin Chem Biol 17,385-392)。截止目前，还没有靶向CD26&CD3组合形式的双特异性抗体结构出现，CD26及CD3分子重链及轻链可变区在双特异性抗体中的的排列顺序对CD26及CD3双特异性抗体的生物学活性及亲和力也全然未知。
发明内容
本发明的目的在于提供具有杀伤细胞高表达CD26型肿瘤活性的CD26、CD3双特异性抗体。
本发明提供的双特异性抗体，包含与人CD26特异性结合的抗体的可变区片段，以及与人CD3特异性结合的抗体的可变区片段。
优选的，所述双特异性抗体，其中与人CD26特异性结合的抗体的可变区片段中的重链可变区与与人CD3特异性结合的抗体的可变区片段中的重链可变区相邻，或者与人CD26特异性结合的抗体的可变区片段中的轻链可变区与与人CD3特异性结合的抗体的可变区片段中的轻链可变区相邻。
作为优选，上述双特异性抗体N-末端到C-末端按如下顺序排列：与人CD26特异性结合的抗体的轻链可变区、重链可变区，与人CD3特异性结合的抗体的重链可变区、轻链可变区；或者，与人CD26特异性结合的抗体的重链可变区、轻链可变区，与人CD3特异性结合的抗体的轻链可变区、重链可变区。
优选的，所述与人CD26特异性结合的抗体的可变区片段，包括如SEQ ID NO：1所示的重链可变区以及如SEQ ID NO：2所示的轻链可变区；所述与人CD3特异性结合的抗体的可变区片段，包括如SEQ ID NO：4所示的重链可变区以及如SEQ ID NO：5所示的轻链可变区)。
所述CD26抗体的可变区来源于单克隆抗体YS110的可变区基因序列经密码子优化后所得，或来源于其他公知的CD26抗体可变区。
所述CD3抗体的可变区来源于OKT-3、TR-66、X35-3、VIT3、CLB-T3/3、CLB-T3.4.2、F111-409、WT31、WT32、CRIS7、F101.01、BMA030(BW264/56)、YTH12.5、SPv-T3b、11D8、 XⅢ-141、XⅢ-46、XⅢ-87、12F6、T3/RW2-8C8、T3/RW2-4B6、OKT3D、M-T301、SMC2的CD3特异性抗体。这些CD3特异性抗体在本领域中是公知的。
优选地，本发明的双特异性抗体的氨基酸序列如SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9或SEQ ID NO：10所示。优选地，本发明提供的编码上述抗体的核苷酸序列如SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22或SEQ ID NO：23所示。
本发明还提供了含有上述核苷酸序列的表达载体。所述载体优选为pCHO1.0。
本发明还提供了含有上述表达载体的重组宿主细胞。所述细胞优选为CHO-S细胞。
本发明还提供了上述抗体的制备方法，包括如下步骤：
步骤一：将上述重组宿主细胞在合适的条件下培养，表达目的蛋白；
步骤二：以层析法对上述目的蛋白进行纯化。
本发明还提供了所述双特异性抗体在制备治疗细胞高表达CD26型肿瘤的药物中的应用。
本发明还提供了所述双特异性抗体在制备通过CD3活化的T细胞介导的肿瘤细胞免疫治疗药物中的应用。
优选的，上述双特异性抗体在制备治疗肾癌、前列腺癌、结肠癌、间皮瘤的药物中的应用。
本发明用真核细胞系统成功表达了抗CD26、抗CD3双特异性抗体，适用于后续的大规模生产，其对CD26蛋白的结合具有特异性，亲和力达到10-9M，对CD26高表达细胞株有较强的细胞毒效应，可用于治疗CD26高表达相关实体肿瘤。该双特异性抗体介导T细胞杀伤CD26高表达的肿瘤细胞，比单纯抗CD26单克隆抗体有更好的疗效，且由于缺失Fc片段，无岩藻糖修饰，也无复杂的糖型分布，与人源化的IgG抗体相比具有免疫原性低、更易质控等优点。
附图说明
图1为ZHB BsAb系列基因琼脂糖凝胶电泳图。
其中，泳道1为500bp DNA Ladder；泳道2为两端含有AvrII和BstZ17I酶切位点的ZHB BsAb-1基因，泳道3为两端含有AvrII和BstZ17I酶切位点的ZHB BsAb-2基因，泳道4为两端含有AvrII和BstZ17I酶切位点的ZHB BsAb-3基因，泳道5为两端含有AvrII和BstZ17I酶切位点的ZHB BsAb-4基因。
图2-a、图2-b、图2-c、图2-d为ZHB BsAb系列基因表达质粒构建过程图。
其中，图2-a为ZHB BsAb-1构建到表达质粒pCHO1.0过程图；图2-b为ZHB BsAb-2构建到表达质粒pCHO1.0过程图；图2-c为ZHB BsAb-3构建到表达质粒pCHO1.0过程图；图2-d为ZHB  BsAb-4构建到表达质粒pCHO1.0过程图。
图3-a、图3-b、图3-c、图3-d为ZHB BsAb系列多克隆细胞株补料分批流加每天表达量鉴定SDS-PAGE电泳图。
其中，图3-a为ZHB BsAb-1多克隆细胞株补料分批流加每天表达量鉴定SDS-PAGE电泳图；泳道1为CHO-S空细胞上清培养液对照，泳道2为10-250KD范围的预染蛋白上样Marker，泳道3-10为ZHB BsAb-1多克隆细胞株补料分批流加第3-10天表达量；箭头所指即为ZHB BsAb-1；
图3-b为ZHB BsAb-2多克隆细胞株补料分批流加每天表达量鉴定SDS-PAGE电泳图；泳道1为CHO-S空细胞上清培养液对照，泳道2为10-250KD范围的预染蛋白上样Marker，泳道3-10为ZHB BsAb-2多克隆细胞株补料分批流加第3-10天表达量；箭头所指即为ZHB BsAb-2；
图3-c为ZHB BsAb-3多克隆细胞株补料分批流加每天表达量鉴定SDS-PAGE电泳图；泳道1为CHO-S空细胞上清培养液对照，泳道2为10-250KD范围的预染蛋白上样Marker，泳道3-10为ZHB BsAb-3多克隆细胞株补料分批流加第3-10天表达量；箭头所指即为ZHB BsAb-3；
图3-d为ZHB BsAb-4多克隆细胞株补料分批流加每天表达量鉴定SDS-PAGE电泳图；泳道1为CHO-S空细胞上清培养液对照，泳道2为10-250KD范围的预染蛋白上样Marker，泳道3-10为ZHB BsAb-4多克隆细胞株补料分批流加第3-10天表达量；箭头所指即为ZHB BsAb-4。
图4-a、图4-b、图4-c、图4-d为ZHB BsAb系列多克隆细胞株补料分批流加培养上清培养液经过色谱柱纯化收集样品SDS-PAGE电泳图
其中，图4-a为ZHB BsAb-1多克隆细胞株补料分批流加培养上清培养液经过色谱柱纯化收集样品SDS-PAGE电泳图。泳道1为10-250KD范围的预染蛋白上样Marker，泳道2-10为不同收集管收集的ZHB BsAb-1纯化样品；箭头所指即为ZHB BsAb-1。
图4-b为ZHB BsAb-2多克隆细胞株补料分批流加培养上清培养液经过色谱柱纯化收集样品SDS-PAGE电泳图。泳道1为10-250KD范围的预染蛋白上样Marker，泳道2-10为不同收集管收集的ZHB BsAb-2纯化样品；箭头所指即为ZHB BsAb-2。
图4-c为ZHB BsAb-3多克隆细胞株补料分批流加培养上清培养液经过色谱柱纯化收集样品SDS-PAGE电泳图。泳道1为10-250KD范围的预染蛋白上样Marker，泳道2-10为不同收集管收集的ZHB BsAb-3纯化样品；箭头所指即为ZHB BsAb-3。
图4-d为ZHB BsAb-4多克隆细胞株补料分批流加培养上清培养液经过色谱柱纯化收集样品SDS-PAGE电泳图。泳道1为10-250KD范围的预染蛋白上样Marker，泳道2-10为不同收集管收集的ZHB BsAb-4纯化样品；箭头所指即为ZHB BsAb-4。
图5-a、图5-b为抗CD26-scFv和抗CD3-scFv基因琼脂糖凝胶电泳图。
其中，图5-a为抗CD26-scFv基因琼脂糖凝胶电泳图，泳道1为200bp DNA Ladder；泳道2为两端含有XhoI和XbaI酶切位点的抗CD26-scFv基因。
图5-b为抗CD3-scFv基因琼脂糖凝胶电泳图，泳道1为500bp DNA Ladder；泳道2为两端含有XhoI和XbaI酶切位点的抗CD3-scFv基因。
图6-a、图6-b为抗CD26-scFv和抗CD3-scFv基因表达质粒构建过程图。
其中，图6-a为抗CD26-scFv基因构建到表达质粒pPICZα过程图；图6-b为抗CD3-scFv基因构建到表达质粒pPICZα过程图。
图7-a、图7-b为抗CD26-scFv和抗CD3-scFv重组毕赤酵母菌株小量诱导鉴定图。
其中，图7-a为抗CD26-scFv重组毕赤酵母菌株小量诱导鉴定图。泳道1为10-250KD范围的预染蛋白上样Marker，泳道2-9为抗生素Zeocin加压筛选筛选获得的不同克隆通过甲醇诱导表达抗CD26-scFv上清培养液；箭头所指即为抗CD26-scFv。
图7-b为抗CD3-scFv体重组毕赤酵母菌株小量诱导鉴定图。泳道1为10-250KD范围的预染蛋白上样Marker，泳道2-9为抗生素Zeocin加压筛选获得的不同克隆通过甲醇诱导表达抗CD3-scFv上清培养液；箭头所指即为抗CD3-scFv。
图8-a、图8-b为抗CD26-scFv和抗CD3-scFv经过IMAC亲和纯化收集样品SDS-PAGE电泳图
其中，图8-a为抗CD26-scFv经过IMAC亲和纯化收集样品SDS-PAGE电泳图。泳道1为10-250KD范围的预染蛋白上样Marker，泳道2-8为不同收集管收集的抗CD26-scFv纯化样品；箭头所指即为抗CD26-scFv。
图8-b为抗CD3-scFv经过IMAC亲和纯化收集样品SDS-PAGE电泳图。泳道1为10-250KD范围的预染蛋白上样Marker，泳道2-7为不同收集管收集的抗CD3-scFv纯化样品；箭头所指即为抗CD3-scFv。
图9-a和图9-b为抗CD26人源化抗体重链和轻链基因琼脂糖凝胶电泳图。
其中，图9-a为抗CD26人源化抗体重链基因琼脂糖凝胶电泳图。泳道1为500bp DNA Ladder；泳道2为两端含有AvrII和BstZ17I酶切位点的抗CD26人源化抗体重链基因。
图9-b为抗CD26人源化抗体轻链基因琼脂糖凝胶电泳图。泳道1为500bp DNA Ladder；泳道2为两端含有EcoRV和PacI酶切位点的抗CD26人源化抗体轻链基因。
图10为抗CD26人源化抗体基因表达质粒构建过程图。
图11为抗CD26人源化抗体多克隆细胞株补料分批流加每天表达量鉴定SDS-PAGE电泳图。
其中，泳道1为10-250KD范围的预染蛋白上样Marker，泳道2-9为抗CD26人源化抗体多克 隆细胞株补料分批流加第2-9天表达量；泳道10为CHO-S空细胞上清培养液对照，箭头所指分别为抗CD26人源化抗体重链和轻链；
图12为抗CD26人源化抗体多克隆细胞株补料分批流加培养上清培养液经过预装proteinA亲和柱纯化收集样品SDS-PAGE电泳图
其中，泳道1为10-250KD范围的预染蛋白上样Marker，泳道2为抗CD26人源化抗体，箭头所指分别为抗CD26人源化抗体重链和轻链。
图13为亲和力检测结果结合和解离图。其中A是BsAb1的结合与解离曲线，B是BsAb4的结合与解离曲线，C是BsAb3的结合与解离曲线，D是BsAb2的结合与解离曲线，E是抗CD26单链抗体的结合与解离曲线。
图14为亲本单链抗体及双特异性抗体BsAb1，BsAb2，BsAb3，BsAb4介导的PBMC对786-0细胞和A375细胞的细胞毒作用。
图15显微镜下观察BsAb4介导的PBMC细胞对786-0细胞的细胞毒效应。
其中图15-a是未添加BsAb4的786-0细胞；15-b是添加BsAb4的发生细胞凋亡的786-0细胞。
图16为双特异性抗体BsAb2，BsAb4介导的T细胞对786-0细胞的细胞毒作用。
其中16-a是BsAb2介导的T细胞对786-0细胞的细胞毒作用；16-b是BsAb4介导的T细胞对786-0细胞的细胞毒作用。
具体实施方式
本发明的一个目的在于提供一种双特异抗体，其包括一种靶向第一抗原CD26的蛋白功能区和一种靶向第二抗原CD3的蛋白功能区。靶向第一抗原CD26的蛋白功能区与靶向第二抗原CD3的蛋白功能区有效连接，保持其各自的空间结构并发挥其各自的生理活性。所述靶向第一抗原CD26的蛋白功能区与靶向第二抗原CD3的蛋白功能区可以在不影响其各自功能的情况下直接融合在一起，且靶向第二抗原CD3的蛋白功能区可以连接在靶向第一抗原CD26的蛋白功能区的N端，也可以连接在靶向第一抗原CD26的蛋白功能区的C端，也可以在其间加入其它间隔序列，如连接片段。此外，靶向第一抗原CD26的蛋白功能区的重链可变区、轻链可变区均可在靶向第一抗原CD26的蛋白功能区的N端；靶向第二抗原CD3的蛋白功能区中的重链可变区、轻链可变区均可在靶向第二抗原CD3的蛋白功能区的N端。
本发明所用的术语”双特异抗体”中的:双特异”是指同时特异性靶向两种不同的抗原，在本发明中所述两种不同的抗原分别是人CD26和人CD3。
本发明中的:抗体”一词，除特指完整的免疫球蛋白外，也指免疫球蛋白的片段(如至少是免疫球蛋白分子的一个免疫活性区段)，如Fab、Fab′、F(ab′)2、Fv片段、单链抗体分子或由含 有一个或多个CDR区的免疫球蛋白分子的任意片段形成的多特异性抗体。
本发明中的”单链抗体”或:scFv”指的是由轻链可变区与重链可变区直接相连或通过一个连接短肽连接而成的工程抗体。
在某些实施方案中，所述双特异抗体是由抗CD26-scFv-抗CD3-scFv组成，也可以由抗CD3-scFv-抗CD26-scFv组成，也可以由抗CD26 IgG-连接短肽-抗CD3-scFv组成，还可以由抗CD3 scFv-连接短肽-抗CD26 IgG组成。
在一个实施方案中，本发明涉及用所述的双特异抗体预防或治疗由CD26高表达而引起的疾病的方法，包含给予治疗有效剂量的如上所述的双特异抗体或药物组合物。在某些实施方案中，所述治疗的肿瘤选自进攻性T细胞恶性肿瘤，恶性间皮瘤，肾瘤，结肠癌。
下面将通过下述非限制性实施例进一步说明本发明，本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。下述实验方法如无特别说明，均为常规方法，所使用的实验材料如无特别说明，均可容易地从商业公司获取。
实施例1抗CD26和抗CD3双特异抗体序列设计
靶向第一抗原CD26重链可变区(如SEQ ID NO.1所示)和轻链可变区(如SEQ ID NO.2所示)以连接短肽(如SEQ ID NO.3所示)进行融合组成抗CD26蛋白功能区；靶向第二抗原CD3重链可变区(如SEQ ID NO.4所示)和轻链可变区(如SEQ ID NO.5所示)，以连接短肽(如SEQ ID NO.6所示)进行融合组成抗CD3蛋白功能区；上述靶向第一抗原CD26蛋白功能区和靶向第二抗原CD3蛋白功能区中重链可变区和轻链可变区以不同的顺序连接形成四种双特异抗体，分别命名为ZHB BsAb-1(如SEQ ID NO.7所示)，ZHB BsAb-2(如SEQ ID NO.8所示)，ZHB BsAb-3(如SEQ ID NO.9所示)，ZHB BsAb-4(如SEQ ID NO.10所示)，其中ZHB BsAb-1选择以黄白蛉(sandfly yellow)相关蛋白(如SEQ ID NO.11所示)作为分泌表达信号肽，ZHB BsAb-2以家蚕蚕丝(Silkworm Fibroin)相关蛋白(如SEQ ID NO.12所示)作为分泌表达信号肽，ZHB BsAb-3以海萤夜光虫(cypridina Noctiluca)相关蛋白(如SEQ ID NO.13所示)作为分泌表达信号肽，ZHB BsAb-4以蜂鸟窝(pinemoth)相关蛋白(如SEQ ID NO.14所示)作为分泌表达信号肽，得到优化前的ZHB BsAb-1基因(如SEQ ID NO.16所示)，ZHB BsAb-2基因(如SEQ ID NO.17所示)，ZHB BsAb-3基因(如SEQ ID NO.18所示)，ZHB BsAb-4基因(如SEQ ID NO.19所示)，将上述四种基因按照哺乳动物细胞CHO密码子偏爱性进行优化，分别得到优化后的ZHB BsAb-1(如SEQ ID No.20所示)、ZHB BsAb-2(如SEQ ID No.21所示)、ZHB BsAb-3(如SEQ ID No.22所示)、ZHB BsAb-4(如SEQ ID No.23所示)下面分别对该四种融合蛋白进行密码子优化前后各参数对比说明：
1)密码子适应指数(CAI)
通常CAI＝1时被认为该基因在该表达系统中是最理想的高效表达状态，CAI指数越低表明该基因在该宿主中表达水平越差。通过计算，密码子没有优化前，ZHB BsAb-1，ZHB BsAb-2，ZHB BsAb-3，ZHB BsAb-4基因在哺乳动物细胞CHO密码子适应指数(CAI)分别为0.68，0.68，0.68，0.68。通过密码子优化，并经过计算可知优化后的ZHB BsAb-1，ZHB BsAb-2，ZHB BsAb-3，ZHB BsAb-4基因在哺乳动物细胞CHO表达系统中CAI指数依次为0.86，0.86，0.87，0.87。可以看出经过密码子优化后得到的基因序列可以提高ZHB BsAb-1，ZHB BsAb-2，ZHB BsAb-3，ZHB BsAb-4基因在哺乳动物细胞CHO表达系统中的表达水平。
2)最优密码子使用频率(FOP)
经过计算可知，没有基于哺乳动物细胞CHO表达系统密码子优化前，ZHB BsAb-1，ZHB BsAb-2，ZHB BsAb-3，ZHB BsAb-4基因序列的低利用率密码子(利用率低于40％的密码子)出现百分比为15％，17％，17％，16％。这四条未进行优化的基因采用串联稀有密码子，导致降低翻译效率，解散翻译装配物。通过密码子优化后，ZHB BsAb-1，ZHB BsAb-2，ZHB BsAb-3，ZHB BsAb-4基因在哺乳动物细胞CHO表达系统中出现低利用率密码子的频率为0。
3).GC碱基含量(GC curve)
GC含量理想分布区域为30％-70％，在这个区域外的出现任何峰都会不同程度地影响转录和翻译效率。经过计算可知，ZHB BsAb-1，ZHB BsAb-2，ZHB BsAb-3，ZHB BsAb-4未经优化的基因GC碱基平均含量为51.83％，51.90％，51.89％，51.87％，优化后去除在30％-70％区域外出现的GC含量，最终得到优化后ZHB BsAb-1，ZHB BsAb-2，ZHB BsAb-3，ZHB BsAb-4基因的GC碱基平均含量为54.15％，54.41％，54.75％，54.54％。
实施例2：抗CD26和抗CD3双特异抗体表达质粒构建、稳定表达以及纯化
1.抗CD26和抗CD3双特异抗体表达质粒构建
分别将优化后的ZHB BsAb-1(如SEQ ID No.20所示)、ZHB BsAb-2(如SEQ ID No.21所示)、ZHB BsAb-3(如SEQ ID No.22所示)、ZHB BsAb-4(如SEQ ID No.23所示)基因上游引入AvrII酶切位点(如SEQ ID NO.24所示)和kozak序列(如SEQ ID NO.25所示)，下游引入BstZ17I酶切位点(如SEQ ID NO.26所示)，并将合成的片段，分别构建到pUC57质粒(购自南京金斯瑞科技有限公司)中，得到一种长期保存质粒，依次记为pUC57-ZHB BsAb-1，pUC57-ZHB BsAb-2，pUC57-ZHB BsAb-3，pUC57-ZHB BsAb-4质粒。
分别以M13 F(如SEQ ID NO.27所示)和M13 R(如SEQ ID NO.28所示)为引物，上述质粒为模板扩增目的基因，1％琼脂糖电泳回收PCR产物(如图1所示)，并用AvrII和BstZ17I 双酶切PCR回收产物和pCHO1.0载体(购自Invitrogen公司)，T4连接酶连接双酶切产物至pCHO1.0载体骨架中，并转化到Top10感受态细胞中，涂于含有卡那霉素抗性的LB平板中37℃过夜培养。第二天筛选阳性克隆菌，并测序比对，与预期序列完全一致，即分别得到了抗CD26单链抗体与抗CD3单链抗体不同形式的融合蛋白的表达质粒，依次记为pCHO1.0-ZHB BsAb-1，pCHO1.0-ZHB BsAb-2，pCHO1.0-ZHB BsAb-3，pCHO1.0-ZHB BsAb-4(质粒构建流程图如图2-a，2-b，2-c，2-d所示)。
2.抗CD26和抗CD3双特异抗体不同基因稳定转染及表达
将pCHO1.0-ZHB BsAb-1，pCHO1.0-ZHB BsAb-2，pCHO1.0-ZHB BsAb-3，pCHO1.0-ZHB BsAb-4质粒通过NruI(R0192S，购于NEB公司)过夜酶切进行线性化，电转染到CHO-S细胞中后，分别加入嘌呤霉素和MTX进行加压筛选，一周后计算细胞活率，当细胞活率大于30％以上转移到CO2摇床中继续悬浮加压筛选，37℃，8％CO2，130rpm培养，通过不断增加嘌呤霉素和MTX浓度来加压筛选多克隆细胞株直到上述四种融合蛋白有较高的表达量，图3-a、图3-b、图3-c、图3-d分别为ZHB BsAb-1、ZHB BsAb-2、ZHB BsAb-3、ZHB BsAb-4多克隆细胞株进行葡萄糖补料流加表达量鉴定。
3.抗CD26和抗CD3双特异抗体多克隆细胞株扩大培养及纯化
按步骤2中加压筛选获得的多克隆细胞株进行中补料分批流加培养得到ZHB BsAb-1，ZHB BsAb-2，ZHB BsAb-3，ZHB BsAb-4各上清培养液，12000rpm，15min低温离心收集上清，0.45μm滤膜过滤。
运用全自动智能蛋白纯化系统(AKTA avant150，购自GE healcare)对预处理获得的ZHB BsAb-1，ZHB BsAb-2，ZHB BsAb-3，ZHB BsAb-4各培养液进行纯化。SDS-PAGE电泳分析ZHB BsAb-1、ZHB BsAb-2、ZHB BsAb-3、ZHB BsAb-4纯化效果，结果依次如图4-a、图4-b、图4-c、图4-d所示，合并符合要求的各个收集管，并超滤浓缩，置换至PBS缓冲液中，过滤除菌，4℃保存以作备用。
实施例3抗CD26-scFv和抗CD3-scFv分子构建、表达与纯化
1.抗CD26-scFv和抗CD3-scFv分子构建
抗CD26重链可变区(如SEQ ID NO.1所示)和轻链可变区(如SEQ ID NO.2所示)以连接短肽(如SEQ ID NO.3所示)进行直接融合，组成抗CD26-scFv(如SEQ ID NO.29所示)；抗CD3重链可变区(如SEQ ID NO.4所示)和轻链可变区(如SEQ ID NO.5所示)，以连接短肽(如SEQ ID NO.6所示)进行直接融合，组成抗CD3-scFv(如SEQ ID NO.30所示)。
分别对抗CD26-scFv和抗CD3-scFv蛋白C端融合组氨酸标签(如SEQ ID NO.15所示)， 得到优化前抗CD26-scFv基因(如SEQ ID NO.31所示)和优化前抗CD3-scFv基因(如SEQ ID NO.32所示)并按照毕赤酵母密码子偏爱性进行优化，得到优化后抗CD26-scFv基因(如SEQ ID NO.33所示)和优化后抗CD3-scFv基因(如SEQ ID NO.34所示)。并进一步经计算可得到，密码子优化后的抗CD26-scFv和抗CD3-scFv基因在毕赤酵母表达系统中CAI指数分别为0.84，0.83；通过密码子优化后的抗CD26-scFv和抗CD3-scFv基因在毕赤酵母系统中出现低利用率密码子的频率为0；优化后抗CD26-scFv和抗CD3-scFv基因的GC碱基平均含量为44.99％，44.10％。
分别将优化后的抗CD26-scFv和抗CD3-scFv全基因上下游引入XhoI(如SEQ ID NO.35所示)和XbaI(如SEQ ID NO.36所示)双酶切位点并进行全基因合成。将合成的片段，分别构建到pUC57质粒中，得到一种长期保存质粒，依次记为pUC57-CD26-scFv，pUC57-CD3-scFv质粒。
分别以M13 F(如SEQ ID NO.21所示)和M13 R(如SEQ ID NO.22所示)为引物，上述质粒为模板扩增目的基因，1％琼脂糖电泳回收PCR产物(如图5-a，5-b所示)，并用XhoI和XbaI双酶切PCR回收产物和pPICZα1载体(购自Invitrogen公司)，T4连接酶连接双酶切产物至pPICZα1载体骨架中，并转化到Top10感受态细胞中，在含有Zeocin的LB平板中37℃过夜培养。第二天筛选阳性克隆菌，并测序比对，与预期序列完全一致，即得到抗CD26-scFv和抗CD3-scFv的表达质粒，分别记为pPICZα-CD26-scFv和pPICZα-CD3-scFv(质粒构建流程如图6-a、6-b所示)。
2.抗CD26-scFv和抗CD3-scFv重组菌株筛选
YPDS固体培养基配制：Invitrogen公司EasySelect Pichia Expression Kit说明书提供，其中酵母提取物10g/L，蛋白胨20g/L，葡萄糖20g/L，琼脂糖15g/L，D-山梨醇182g/L。
按EasySelect Pichia Expression Kit提供的方法将X-33菌株(C18000，购自Invitrogen)制备成电感受态细胞。将pPICZα-CD26-scFv和pPICZα-CD3-scFv质粒，用SacI限制性内切酶酶切线性化，乙醇沉淀后将线性化载体电转化进入到X-33感受态酵母细胞，涂布到含有0.5-2.0mg/mL Zeocin(R250-01，购自Invitrogen公司)的YPDS固体培养基，30℃培养3-5d。
3.抗CD26-scFv和抗CD3-scFv重组工程菌株诱导表达及鉴定
BMGY培养基配制：Invitrogen公司Multi-Copy Pichia Expression Kit说明书提供，其中酵母提取物10g/L，蛋白胨20g/L，K2HPO43g/L，KH2PO411.8g/L，YNB 13.4g/L，生物素4×10-4g/L，甘油10g/L。
BMMY培养基配制：Invitrogen公司Multi-Copy Pichia Expression Kit说明书提供，其中酵母提取物10g/L，蛋白胨20g/L，K2HPO43g/L，KH2PO411.8g/L，YNB13.4g/L，生物素4×10-4g/L， 甲醇5mL/L。
挑取步骤2中获得的单克隆于5mL BMGY培养基中，于50mL无菌离心管中30℃，220rpm培养，至OD600＝2.0-6.0时，取1mL保存菌种，并将剩余菌液重悬后转移到BMMY中小量诱导表达，每隔24h补加甲醇至终浓度为1％。一周后，离心收集菌液上清，通过SDS-PAGE凝胶电泳分析，图7-a，7-b分别为含有优化后密码子抗CD26-scFv和抗CD3-scFv重组菌株诱导表达鉴定图。
4.抗CD26-scFv和抗CD3-scFv的纯化
按步骤3中表达方法分别得到含有优化后密码子的抗CD26-scFv和抗CD3-scFv重组菌株发酵液上清，12000rpm，15min低温离心收集上清，加入结合缓冲液，使其在上清中终浓度分别为300mM NaCl，20mM NaH2PO4，10mM Imidazole，调pH7.5，0.45μm滤膜过滤。
运用全自动智能蛋白纯化系统对预处理获得的抗CD26-scFv和抗CD3-scFv毕赤酵母发酵液分别用HisTrap FF crude亲和柱进行纯化，结合缓冲液为300mM NaCl，20mM NaH2PO4，20mM Imidazole，pH7.5；洗脱缓冲液为300mM NaCl，20mM Na2HPO4，20mM Imidazole，pH7.5。洗脱缓冲液洗脱时收集各个洗脱峰，SDS-PAGE电泳鉴定纯度，图8-a，图8-b分别为抗CD26-scFv和抗CD3-scFv洗脱后各收集管电泳图。将各收集管合并超滤浓缩，置换缓冲液为PBS溶液，并过滤除菌，4℃保存以作备用。
实施例4抗CD26人源化抗体分子构建、表达与纯化
1.抗CD26人源化抗体重链和轻链分子构建
分别对抗CD26重链可变区(如SEQ ID NO.1所示)和人源IgG1重链恒定区进行直接融合，形成抗CD26人源化抗体重链区(本发明命名为抗CD26-HC，如SEQ ID NO.37所示)，对抗CD26轻链可变区(如SEQ ID NO.2所示)和人源IgG1轻链恒定区进行直接融合，形成抗CD26人源化抗体轻链区(本发明命名为抗CD26-LC，如SEQ ID NO.38所示)，并选择鼠(Mus musculus)IgG k(如SEQ ID NO.39所示)作为分泌表达信号肽，得到抗CD26-HC优化前基因序列(如SEQ ID NO.40所示)和抗CD26-LC优化前基因序列(如SEQ ID NO.41所示)，按照哺乳动物细胞表达系统进行密码子偏爱性进行优化，得到抗CD26-HC优化后基因(如SEQ ID NO.42所示)和抗CD26-LC优化后基因(如SEQ ID NO.43所示)。并进一步经计算可得到，密码子优化后的抗CD26-HC和抗CD26-LC基因在哺乳动物细胞表达系统中CAI指数分别为0.86，0.86；通过密码子优化后的抗CD26-HC和抗CD26-LC基因在哺乳动物细胞表达系统中出现低利用率密码子的频率为0；优化后抗CD26-HC和抗CD26-LC基因的GC碱基平均含量为53.89％，53.10％。
将优化后的抗CD26-HC基因上游引入AvrII酶切位点(如SEQ ID NO.24所示)和kozak序列(如SEQ ID NO.25所示)，下游引入BstZ17I酶切位点(如SEQ ID NO.26所示)进行全基因合成，将合成的片段构建到pUC57质粒中，得到一种长期保存的质粒，记为pUC57-CD26-HC质粒；同时将优化后的抗CD26-LC基因上游引入EcoRV(如SEQ ID NO.44所示)酶切位点和kozak序列(如SEQ ID NO.14所示)，下游引入PacI酶切位点(如SEQ ID NO.45所示)进行全基因合成，将合成的片段，构建到pUC57质粒中，得到一种长期保存质粒，记为pUC57-CD26-LC质粒。
以M13 F(如SEQ ID NO.27所示)和M13 R(如SEQ ID NO.28所示)为引物，pUC57-CD26-HC质粒为模板扩增目的基因，1％琼脂糖电泳回收PCR产物(如图9-a所示)，用AvrII和BstZ17I双酶切PCR回收产物和pCHO1.0载体，T4连接酶连接双酶切产物至pCHO1.0载体骨架中，转化到Top10感受态细胞中，在含有卡那霉素抗性的LB平板中37℃培养过夜。第二天筛选阳性克隆菌，并测序比对，与预期序列完全一致，即得到抗CD26-HC的表达质粒，记为pCHO1.0-CD26-HC。
以M13 F(如SEQ ID NO.27所示)和M13 R(如SEQ ID NO.28所示)为引物，pUC57-CD26-LC质粒为模板扩增目的基因，1％琼脂糖电泳回收PCR产物(如9-b所示)，用EcoRV和PacI双酶PCR回收产物和pCHO1.0-CD26-HC，T4连接酶连接双酶切产物至pCHO1.0-CD26-HC载体骨架中，转化到Top10感受态细胞中，在含有卡那霉素抗性的LB平板中37℃过夜培养。第二天筛选阳性克隆菌，并测序比对，与预期序列完全一致，即得到抗CD26人源化抗体的表达质粒，记为pCHO1.0-CD26(H+L)(质粒构建流程如图10所示)。
2.抗CD26人源化抗体稳定转染及表达
将pCHO1.0-CD26(H+L)质粒通过NruI过夜酶切进行线性化，电转染到CHO-S细胞中后，分别加入嘌呤霉素和MTX进行加压筛选，一周后计算细胞活率，当细胞活率大于30％以上转移到CO2摇床中继续悬浮加压筛选，37℃，8％CO2，130rpm培养，通过不断增加嘌呤霉素和MTX浓度来加压筛选多克隆细胞株直到抗CD26人源化抗体有较高的表达量，图11为抗CD26人源化抗体多克隆细胞株进行葡萄糖补料流加表达量鉴定。
3.抗CD26人源化抗体多克隆细胞株扩大培养及纯化
按步骤2中加压筛选获得的多克隆细胞株进行中补料分批流加培养得到抗CD26人源化抗体上清培养液，12000rpm，15min低温离心收集上清，并用20mM NaH2PO4进行切向流超滤系统更换缓冲液，调pH＝7.0，0.45μm滤膜过滤。
运用全自动智能蛋白纯化系统对预处理获得的抗CD26人源化抗体通过HiTrap proteinA FF预装柱进行纯化，结合缓冲液为20mM NaH2PO4，pH7.0；洗脱缓冲液为20mM NaH2PO4， 0.1M citric acid，pH3.0。洗脱缓冲液洗脱时收集各个洗脱峰，SDS-PAGE电泳鉴定纯化效果，结果如图12所示，合并符合要求的各个收集管，并超滤浓缩，置换缓冲液为PBS溶液，过滤除菌，4℃保存以作备用。
实施例5检测六种肿瘤细胞系表面CD26的阳性率
实验方法：将处于对数生长期的人肾透明细胞腺癌细胞786-0细胞(
Figure PCTCN2015093383-appb-000001
CRL-1932)，人间皮瘤细胞NCI-H2452细胞(
Figure PCTCN2015093383-appb-000002
CRL-5946)，肾癌细胞Caki-1细胞(
Figure PCTCN2015093383-appb-000003
HTB-46)，人结肠癌细胞COLO 205细胞(
Figure PCTCN2015093383-appb-000004
CCL-222)，人前列腺癌细胞PC-3细胞(
Figure PCTCN2015093383-appb-000005
CRL-1435)，人恶性黑色素瘤细胞A375细胞(
Figure PCTCN2015093383-appb-000006
CRL-1619)用胰酶消化，于1000rpm，离心5min收集细胞，PBS清洗2次；每份取1.0×106个细胞用250μLPBS重悬，分别加入终浓度为10ug/mL的抗CD26抗体于室温孵育1h，孵育结束后，用PBS清洗两次，再加入按1:2000稀释后的Anti-His-tag-Alexa Fluor 488抗体(MBL，Cat#D291-A48)250μL，于室温孵育1h，孵育结束后，用PBS清洗三次，300μLPBS重悬后，用流式细胞仪(BD，Accuri C6)进行检测。
实验结果：实验结果见表1。
表1 不同肿瘤细胞系表面CD26蛋白的阳性率
Figure PCTCN2015093383-appb-000007
结论：786-0细胞，NCI-H2452细胞，Caki-1细胞，COLO 205细胞，PC-3细胞表面均高表达CD26；A375细胞基本不表达CD26，可作为本实验的阴性对照。
实施例6检测Jurkat细胞表面CD3的阳性率
实验方法：将处于对数生长期的Jurkat细胞(
Figure PCTCN2015093383-appb-000008
TIB-152)收集，于1000rpm，离心5min收集细胞，PBS清洗2次；每份取1.0×106个细胞用250μL PBS重悬，加入终浓度为30ug/mL的抗CD3单链抗体于室温孵育1h，孵育结束后，用PBS清洗两次，再加入按1:2000稀释后的Anti-His-tag-Alexa Fluor 488抗体250μL，于室温孵育1h，孵育结束后，用PBS清洗三次，300μLPBS重悬后，用流式细胞仪进行检测。
实验结果：经FACS分析，Jurkat细胞表面CD3的阳性率为46.6％。
结论：Jurkat细胞表面有CD3表达。
实施例7检测双特异性抗体与CD26高表达的肿瘤细胞的结合率
实验方法：将处于对数生长期的786-0细胞、NCI-H2452细胞用胰酶消化，于1000rpm，离心5min收集细胞，PBS清洗2次；每份取1.0×106个细胞用250μL PBS重悬，分别加入终浓度为10ug/mL的BsAb1，BsAb2，BsAb3，BsAb4于室温孵育1h，孵育结束后，用PBS清洗两次，再加入按1:2000稀释后的Anti-His-tag-Alexa Fluor 488抗体250μL，于室温孵育1h，孵育结束后，用PBS清洗三次，300μLPBS重悬后，用流式细胞仪进行检测。
实验结果：见表2
表2 双特异性抗体与CD26高表达的肿瘤细胞的结合率
Figure PCTCN2015093383-appb-000009
结论：双特异性抗体BsAb1，BsAb2，BsAb3，BsAb4均可与肿瘤细胞表面的CD26分子发生特异性结合，特别是BsAb2及BsAb4与肿瘤细胞表面的CD26分子结合率较高。
实施例8检测双特异性抗体与Jurkat(CD3+)细胞的结合率
实验方法：将处于对数生长期的Jurkat细胞用胰酶消化，于1000rpm，离心5min收集细胞，PBS清洗2次；每份取1.0×106个细胞用250μLPBS重悬，分别加入终浓度为10ug/mL的BsAb1，BsAb2，BsAb3，BsAb4于室温孵育1h，孵育结束后，用PBS清洗两次，再加入按1:2000稀释后的Anti-His-tag-Alexa Fluor 488抗体250μL，于室温孵育1h，孵育结束后，用PBS清洗三次，300μLPBS重悬后，用流式细胞仪进行检测。
实验结果：经FACS分析，BsAb1与Jurkat细胞的结合率为0.1％；BsAb2与Jurkat细胞的结合率为92.0％；BsAb3与Jurkat细胞的结合率为0.2％；BsAb4与Jurkat细胞的结合率为56.5％。
结论：双特异性抗体BsAb1，BsAb2，BsAb3，BsAb4均可与Jurkat细胞表面的CD3分子发生特异性结合，特别是BsAb2及BsAb4与Jurkat细胞表面的CD3分子结合率较高。
实施例9双特异性抗体对CD26蛋白的亲和力鉴定
实验方法：用分子相互作用仪Fortebio Qke检测双特异性抗体与CD26蛋白的亲和力，按照浸入即读氨基反应第二代生物传感器(Fortebio，AR2G)说明书操作。
实验结果：见图13。亲和力常数为：KD(BsAb1)＝9.29×10-9M；KD(BsAb2)＝3.44×10-9M；KD(BsAb3)＝1.0×10-8M；KD(BsAb4)＝1.97×10-9M；KD(anti-CD26-scFv)＝1.00×10-9M。
结论：双特异性抗体BsAb1，BsAb2，BsAb3，BsAb4与CD26蛋白均具备一定亲和力；并且，与亲本单链抗体相比，BsAb2，BsAb4与其亲和力常数较为接近。
实施例10检测双特异性抗体和亲本单链抗体介导的PBMC对786-0细胞和A375细胞的细 胞毒效应
实验方法：将处于对数生长期的786-0细胞用胰酶消化，于1000rpm，离心5min收集细胞，PBS清洗2次；取1.0×106个细胞重悬于1mLPBS中，加入终浓度为2.5μM的Calcein-AM溶液，在37℃培养细胞30min；孵育结束后，用PBS洗涤细胞三次；将细胞用RPMI-1640培养基(GIBCO，Cat#31800022)稀释至6.0×105后，每孔50uL加入U-型96孔细胞培养板中，再加入50μL实验组样品，其中空白对照组加入50μLRPMI-1640培养基，阳性对照组加入50μL3％Triton-100，实验组分别加入50μL BsAb1(10ng/mL)，50μL BsAb2(10ng/mL)，50μL BsAb3(10ng/mL)，50μL BsAb4(10ng/mL)，50μL Anti-CD3-scFv(10ng/mL)，50μL Anti-CD26-scFv(10ng/mL)，于37℃孵育30min，孵育结束后，按786-0：PBMC为1:15的比例加入PBMC细胞，继续于37℃孵育3h，孵育结束后取适量细胞培养上清，用酶标仪于激发波长485nm，发射波长515nm条件下进行检测。
同时，以A375细胞为阴性对照，操作同上。
实验结果：显微镜下观察BsAb4介导的PBMC细胞对786-0细胞的细胞毒效应。图15-a为将786-0细胞与PBMC细胞孵育3h后在显微镜下拍摄的照片，白色虚线里是生长正常的癌细胞，15-b为将786-0细胞用BsAb4(10ng/ml)孵育30min后，再与PBMC细胞孵育3h后在显微镜下拍摄的照片，箭头标记的是T细胞的位置，白色圆圈里是发生凋亡的癌细胞。
双特异性抗体和亲本单链抗体介导的PBMC对786-0细胞和A375细胞的细胞毒效应见表3，图14。双特异性抗体BsAb1在10ng/mL对786-0细胞的裂解比例为15.9％，；BsAb2在10ng/mL对786-0细胞的裂解比例为27.4％；BsAb3在10ng/mL对786-0细胞的裂解比例为4.3％；BsAb4在10ng/mL对786-0细胞的裂解比例为25.5％；单链抗体anti-CD26-scFv在10ng/mL对786-0细胞的裂解比例为4.1％；单链抗体anti-CD3-scFv在10ng/mL对786-0细胞的裂解比例为4.1％。
双特异性抗体BsAb1在10ng/mL对阴性对照A375细胞的裂解比例为0.2％，；BsAb2在10ng/mL对A375细胞的裂解比例为2.5％；BsAb3在10ng/mL对A375细胞的裂解比例为1.5％；BsAb4在10ng/mL对A375细胞的裂解比例为2.4％；单链抗体anti-CD26-scFv在10ng/mL对A375细胞的裂解比例为0.3％；单链抗体anti-CD3-scFv在10ng/mL对A375细胞的裂解比例为0.4％。
表3 双特异性抗体和亲本单链抗体对786-0细胞和A375细胞的细胞毒效应
Figure PCTCN2015093383-appb-000010
结论：亲本单链抗体anti-CD26-scFv，anti-CD3-scFv和双特异性抗体BsAb1，BsAb2，BsAb3，BsAb4对阴性对照A375细胞均无细胞毒作用；亲本单链抗体anti-CD26-scFv和anti-CD3-scFv以及双特异性抗体BsAb1，BsAb2，BsAb3，BsAb4对786-0细胞均有一定的细胞毒作用。并且，双特异性抗体BsAb1，BsAb2和BsAb4对786-0细胞的细胞毒作用效果优于亲本单链抗体anti-CD26-scFv和anti-CD3-scFv。
实施例11检测抗CD26人源化抗体介导的PBMC对786-0细胞和A375细胞的细胞毒 效应
实验方法：将处于对数生长期的786-0细胞用胰酶消化，于1000rpm，离心5min收集细胞，PBS清洗2次；取1.0×106个细胞重悬于1mLPBS中，加入终浓度为2.5μM的Calcein-AM溶液，在37℃培养细胞30min；孵育结束后，用PBS洗涤细胞三次；将细胞用RPMI-1640培养基稀释至6.0×105后，每孔50uL加入U-型96孔细胞培养板中，再加入50μL实验组样品，其中空白对照组加入50μLRPMI-1640培养基，阳性对照组加入50μL3％Triton-100，实验组分别加入50μL不同浓度的抗CD26人源化抗体(10ng/mL，1ng/mL，0.01ng/mL)于37℃孵育30min，孵育结束后，按786-0：PBMC为1:15的比例加入PBMC细胞，继续于37℃孵育3h，孵育结束后取适量细胞培养上清，用酶标仪于激发波长485nm，发射波长515nm条件下进行检测。
同时，以A375细胞为阴性对照，操作同上，所用培养基为DMEM培养基(GIBCO，Cat#12800-082)。
实验结果：见表4。抗CD26人源化抗体在10ng/mL，1ng/mL，0.01ng/mL条件下对786-0细胞的裂解比例依次为9.6％，1.5％，0.4％；抗CD26人源化抗体在10ng/mL，1ng/mL，0.01 ng/mL条件下对A375细胞的裂解比例依次为0.2％，0.1％，0.4％。
表4 抗CD26人源化抗体对786-0细胞和A375细胞的细胞毒效应
Figure PCTCN2015093383-appb-000011
结论：抗CD26人源化抗体对阴性对照A375细胞无明显细胞毒作用，与其相比，抗CD26人源化抗体对786-0细胞呈现出浓度依赖性的细胞毒作用。但是，相比抗CD26人源化抗体，本发明的双特异性抗体BsAb1、BsAb2、BsAb4对786-0细胞的细胞毒作用显著增强。
实施例12检测双特异性抗体介导的T细胞对786-0细胞和A375细胞的细胞毒效应
实验方法：
1.分离T细胞
按照淋巴细胞分离液(Lymphocyte Separation Medium，MP，Cat#50494)说明书操作，从人的血液中分离PBMC细胞，分离后的PBMC细胞按人红细胞裂解试剂盒(Human Erythrocyte Lying Kit，R&D SYSTEM，Cat#WL1000)说明书操作裂解红细胞，按人T细胞富集柱(Human T Cell Enrichment Columns，R&D SYSTEM，Cat#HTCC-10)说明书操作，富集T细胞，对富集的T细胞，每份取1.0×106个细胞用250μL PBS重悬，用PBS清洗两次，加入终浓度为30ug/mL的抗CD3单链抗体于室温孵育1h，孵育结束后，用PBS清洗两次，再加入按1:2000稀释后的Anti-His-tag-Alexa Fluor 488抗体250μL，于室温孵育1h，孵育结束后，用PBS清洗三次，300μLPBS重悬后，用流式细胞仪进行检测富集到的T细胞中，CD3+的比例为93.5％。
2.检测T细胞介导的双特异性抗体对786-0细胞和A375细胞的细胞毒效应
将处于对数生长期的786-0细胞用胰酶消化，于1000rpm，离心5min收集细胞，PBS清洗2次；取1.0×106个细胞重悬于1mLPBS中，加入终浓度为2.5μM的Calcein-AM溶液，在37℃培养细胞30min；孵育结束后，用PBS洗涤细胞三次；将细胞用RPMI-1640培养基稀释至5.0×105后，每孔50uL加入U-型96孔细胞培养板中，再加入50μL实验组样品，其中空白对照组加入50μLRPMI-1640培养基，阳性对照组加入50μL3％Triton-100，实验组分别加入50μL anti-CD26-scFv(101pg/mL，103pg/mL，104pg/mL，105pg/mL)，50μL BsAb2(10-1pg/mL，100pg/mL，101pg/mL，102pg/mL，103pg/mL，104pg/mL，105pg/mL，106pg/mL)，50μL BsAb4(10-1pg/mL，100pg/mL，101pg/mL，102pg/mL，103pg/mL，104pg/mL，105pg/mL，106pg/mL)， 于37℃孵育30min，孵育结束后，按786-0：T细胞为1:5的比例加入T细胞，继续于37℃孵育7h，孵育结束后取适量细胞培养上清，用酶标仪于激发波长485nm，发射波长515nm条件下进行检测。
将处于对数生长期的A375细胞用胰酶消化，PBS清洗2次；取1.0×106个细胞重悬于1mLPBS中，加入终浓度为2.5μM的Calcein-AM溶液，在37℃培养细胞30min；用PBS洗涤细胞三次；将细胞用DMEM培养基稀释至5.0×105后，每孔50uL加入U-型96孔细胞培养板中，再加入50μL实验组样品，其中空白对照组加入50μLRPMI-1640培养基，阳性对照组加入50μL3％Triton-100，实验组分别加入50μL anti-CD26-scFv(104pg/mL)，50μL BsAb2(104pg/mL)，50μL BsAb4(104pg/mL)，于37℃孵育30min，孵育结束后，按A375：T细胞为1:5的比例加入T细胞，继续于37℃孵育7h，孵育结束后取适量细胞培养上清，用酶标仪于激发波长485nm，发射波长515nm条件下进行检测。
实验结果：见表5，图16。亲本单链抗体anti-CD26-scFv、BsAb2、BsAb4在104pg/mL浓度条件下对阴性对照A375的裂解率分别为1.2％，2.0％，1.5％。BsAb2、BsAb4在不同浓度时对786-0细胞的细胞毒效应如表4所示。
表5 不同浓度双特异性抗体对786-0细胞的细胞毒效应
Figure PCTCN2015093383-appb-000012
结论：与阴性对照相比，anti-CD26-scFv、BsAb2、BsAb4对786-0细胞均有细胞毒作用，但是亲本单链抗体anti-CD26-scFv对786-0细胞的细胞毒效应无明显浓度梯度依赖，与亲本单链抗体anti-CD26-scFv相比，BsAb2、BsAb4对786-0细胞的杀伤作用呈现明显的浓度依赖性(图16)。由GraphPad Prism 5.0软件计算得BsAb2的IC50为1.388ng/mL，BsAb4的IC50为3.096ng/mL。
实施例13检测双特异性抗体介导的PBMC对其他CD26高表达肿瘤细胞的细胞毒效应
实验方法：分别将处于对数生长期的NCI-H2452细胞、PC-3细胞、Caki-1细胞、COLO 205细胞用胰酶消化，于1000rpm，离心5min收集细胞，PBS清洗2次；取1.0×106个细胞重悬于1mLPBS中，加入终浓度为2.5μM的Calcein-AM溶液，在37℃培养细胞30min；孵育结束后，用PBS洗涤细胞三次；将细胞用RPMI-1640培养基稀释至4.5×105后，每孔50uL加入U-型96孔细胞培养板中，再加入50μL实验组样品，其中空白对照组加入50μLRPMI-1640 培养基，阳性对照组加入50μL3％Triton-100，实验组分别加入50μL10 ng/mL 的BsAb1，BsAb2，BsAb3，BsAb4，于37℃孵育30min，孵育结束后，分别按NCI-H2452/PC-3/Caki-1/COLO 205：PBMC为1:15的比例加入PBMC细胞，继续于37℃孵育5h，孵育结束后取适量细胞培养上清，用酶标仪于激发波长485nm，发射波长515nm条件下进行检测。
实验结果：见表6。
表6 双特异性抗体介导的PBMC对其他CD26高表达肿瘤细胞的细胞毒效应
Figure PCTCN2015093383-appb-000013
实验结果表明，在10ng/mL的浓度条件下，双特异性抗体BsAb1，BsAb2，BsAb3，BsAb4均可以介导PBMC对细胞表面高表达CD26的肿瘤细胞株产生细胞毒作用。
结论：本发明提供的人CD3和人CD26双特异性抗体可有效介导PBMC对细胞表面高表达CD26的肿瘤细胞产生细胞毒作用；并且，双特异性抗体结构的不同对其生物学活性有一定的影响，在所研究的不同结构的人CD3和人CD26双特异性抗体中，BsAb2及BsAb4具有更高的生物学活性。

Claims (10)

  1. 一种双特异性抗体，包括与人CD26特异性结合的抗体的可变区片段，以及与人CD3特异性结合的抗体的可变区片段。
  2. 如权利要求1所述的双特异性抗体，其中与人CD26特异性结合的抗体的可变区片段中的重链可变区与与人CD3特异性结合的抗体的可变区片段中的重链可变区相邻，或者与人CD26特异性结合的抗体的可变区片段中的轻链可变区与与人CD3特异性结合的抗体的可变区片段中的轻链可变区相邻。
  3. 如权利要求2所述的双特异性抗体，其N-末端到C-末端按如下顺序排列：
    与人CD26特异性结合的抗体的轻链可变区、重链可变区，与人CD3特异性结合的抗体的重链可变区、轻链可变区；或者，
    与人CD26特异性结合的抗体的重链可变区、轻链可变区，与人CD3特异性结合的抗体的轻链可变区、重链可变区。
  4. 如权利要求1至3中任一项所述的双特异性抗体，所述与人CD26特异性结合的抗体的可变区片段，包括如SEQ ID NO：1所示的重链可变区以及如SEQ ID NO：2所示的轻链可变区；所述与人CD3特异性结合的抗体的可变区片段，包括如SEQ ID NO：4所示的重链可变区以及如SEQ ID NO：5所示的轻链可变区。
  5. 如权利要求4所述的双特异性抗体，氨基酸序列如SEQ ID NO：7、SEQ ID NO：8、SEQ ID NO：9或SEQ ID NO：10所示。
  6. 一种权利要求5所述的双特异性抗体的编码基因，其核苷酸序列如SEQ ID NO：20、SEQ ID NO：21、SEQ ID NO：22或SEQ ID NO：23所示。
  7. 一种双特异性抗体的制备方法，包含如下步骤：
    步骤一：将权利要求6所述的编码基因构建到pCHO1.0载体中；
    步骤二：将上述载体线性化后电转染到CHO-S细胞中；
    步骤三：将上述重组宿主CHO-S细胞在合适的条件下培养，表达目的蛋白；
    步骤四：以层析法对上述目的蛋白进行纯化。
  8. 权利要求1至5中任一项所述的双特异性抗体在制备治疗细胞表面高表达CD26型肿瘤的药物中的应用。
  9. 权利要求1至5中任一项所述的双特异性抗体在制备治疗肾癌、前列腺癌、结肠癌、间皮瘤的药物中的应用。
  10. 权利要求1至5中任一项所述的双特异性抗体在制备通过CD3活化的T细胞介导的 肿瘤细胞免疫治疗药物中的应用。
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