CN107556386A - Anti- EGFRvIII and CD3 specificity double targeting antibodies, the minicircle dna containing double targeting antibodies expression cassettes and applications - Google Patents

Abstract

The invention provides the double targeting antibodies of a species specificity and its methods and applications.The first antigen-binding domains and specificity and the second antigen-binding domains of EGFRvIII antigen bindings that there is the double targeting antibodies of specificity specificity to be combined with mankind CD3, the tumour cell that can specifically identify and be overexpressed with reference to EGFRvIII antigens and EGFRvIII, including positive tumor disease of liver cancer and other EGFRvIII etc., while it can also combine φt cell receptor；And mediate T cell plays lethal effect, so as to remove EGFRvIII infection, there is very big application value in diagnosis and treatment, prevention, detection EGFRvIII relevant diseases.Present invention also offers a kind of minicircle dna for the nucleotide sequence for having and encoding the double targeting antibodies of the specificity.The invention provides the double targeting antibodies of specificity and minicircle dna can be used for preparing antibody pharmaceutical compositions or EGFRvIII positive tumor cell diagnostic reagents.

Description

The double targeting antibodies of anti-EGFRvIII and CD3 specificity, containing double targeting antibodies expression cassettes Minicircle dna and application

Technical field

The present invention relates to biological medicine and diagnosis and treatment field, and in particular to a kind of double targetings of anti-EGFRvIII and CD3 specificity Antibody, the minicircle dna containing double targeting antibodies expression cassettes and application.

Background technology

EGF-R ELISA type III mutant (EGFRvIII) is EGFR extracellular fragment 2-7 Exon deletions (801bp, 267aa) causes exons 1 and 8 to merge, and a new glycine is generated at fusion, therefore is formd outside film One new antigenic determinant, in malignant tumours such as glioma, breast cancer, non-small cell type lung cancer, prostate cancer, oophoromas Middle expression rate is very high, and this mutant is the product of neoplastic process, only occurs in tumour cell.EGFRvIII is lacked Most of ligand binding region of EGFR extracellular fragments, autophosphorylation can occur, under constitutive activation EGFR-TK induces A series of signal transduction is swum, causing cell, it improves cell propagation, wetting capacity, suppresses apoptotic to vicious transformation Death, generation, development with tumour have substantial connection, and it is not expressed in the normal tissue, therefore are diagnosing tumor and targeting Treat preferable molecular target.

In recent years, in immunotherapy of tumors field, occur using double targeting antibodies (Bispecific antibody, BsAb) mediate T cell kills the new treatment of cancer cell.In human immune system, cytotoxic T lymphocyte (Cytotoxic T Lymphocyte, CTL) important role is play, there is φt cell receptor (T cell receptor, TCR), can specificity knowledge Not and target antigens expressed cell is combined, and secrete perforin and granzyme killing target cell.Tumour cell is exempted from due to lacking The reason such as epidemic disease adhesion molecule and costimulatory molecules so that immunity of organism effectively can not be identified and activated, so that CTL can not be clear Except cancer cell.BsAb can be simultaneously with T cell and tumor cell specific antigen binding, can be between T cell and tumour cell Immunological synapse (immune synapse) is formed, the function of φt cell receptor (TCR) equally is played, so as to activate T cell immunization machine System, mediate T cell kill target cell.

Several EGFRvIII antibody are reported at present, can specific recognition and combination EGFRvIII antigens and EGFRvIII sun Property tumour cell, this several antibody include：

(1) the monoclonal antibody mAb 806 (anti-EGFRvIII) in mouse source, earliest anti-EGFRvIII resist Body, come by expressing EGFRvIII NR6 l cells to mouse immune.The antibody can only resist with reference to EGFRvIII The positive cell of former and EGFRvIII expression, it is impossible to, can not mediate T cell killing with reference to T cell；ADCC can only be passed through (antibody-dependent cell-mediated cytotoxicity) or CDC (complement-dependent Cytotoxicity) EGFRvIII antigen positive tumour cells are killed in targeting, for can also there is host's anti-shifting during human treatment Plant reaction, main correlative theses (Cancer Res.2001Jul 15；61(14):5349-54.Cancer Res.2001Jul15；61(14):5355-61.).

(2) recombinant monoclonal antibodies recombinant antibody anti-EGFRvIII (Rab DMvIII), by Stanford universities are researched and developed, and antibody variable region is connected with amino acid chain, is then connected with the IgG of people Fc sections, and lead to Knobs-into-holes transformations are crossed, humanization is improved while increasing affinity, eliminates most of mouse source sequence, the antibody EGFRvIII antigens and the cell of the EGFRvIII expression positives can be combined, but T cell can not be combined, can not mediate T cell Killing；It can only can be targetted by ADCC or CDC and kill EGFRvIII antigen positive tumour cells, clinical efficacy is still not clear, main Want correlative theses (BMC Biotechnol.2010Oct 7；10:72.).

(3) the monoclonal antibody CH12 (anti-EGFRvIII) in mouse source, the antibody of China research and development, by Shanghai Cancer Inst, Shanghai Yijie Biotech companies research and develop, currently the clinical II phases test, can only With reference to the positive cell of EGFRvIII antigens and EGFRvIII expression, it is impossible to, can not mediate T cell killing with reference to T cell；Can only EGFRvIII antigen positive tumour cells are killed by ADCC or CDC targetings.Patent resources US8506963B2, main theory of correlation Text (Oncotarget.2015Nov 17；6(36):38840-53.FASEB J.2012Jan；26(1):73-80.J Biol Chem.2011Feb 18；286(7):5913-20.).

The double target antibodies of EGFRvIII × CD3 that there is no examination ＆ approval to pass through in the market.The present invention is intended to provide one Efficiently, inexpensive, anti-double targeting antibodies of EGFRvIII and CD3 specificity and its preparation method and application.

The content of the invention

In a first aspect, the invention provides the double targeting antibodies of a species specificity, the double targeting antibodies of specificity have spy The first antigen-binding domains and specificity and the second antigen binding of EGFRvIII antigen bindings that the opposite sex is combined with mankind CD3 Domain.

The invention provides the double targeting antibodies (Bispecific antibody, BsAb) of specificity, can simultaneously with CD3 and EGFRvIII antigen bindings；Specifically identify and combine EGFRvIII antigen-positive cells, while also in relation with expression CD3 T cell, can form immunological synapse (immune synapse) between T cell and tumour cell, play φt cell receptor (T Cell receptor, TCR) the same function, so as to activate T cell immunologic mechanism, mediate T cell plays killing EGFRvIII The effect of positive tumor cell, so as to remove EGFRvIII positive tumor cells.

" double targetings " expression of the present invention " can simultaneously with CD3 and EGFRvIII antigen bindings ".

In an embodiment of the invention, the invention provides the double targeting antibodies of specificity, can be with CD3 and EGFRvIII With reference to；The double targeting antibodies of the specificity are also known as " the anti-double targeting antibodies of CD3/ anti-EGFRs vIII specificity " in the text.It is " anti- The anti-EGFR vIII parts of the double targeting antibodies of CD3/ anti-EGFRs vIII specificity " are available for the thin of targeted expression EGFRvIII Born of the same parents, as the anti-CD3 parts of SMMC-7721 liver cancer cell lines, and the double targeting antibodies molecules of specificity can be used for activating T cell.It is logical Cross and combined while the CD3 on the EGFRvIII and T cell on EGFRvIII positive cells, promote the T cell orientation of activation Kill the EGFRvIII positive cells that (cell cracking) is targeted.

The double targeting antibodies of anti-CD3/ anti-EGFRs vIII specificity of the present invention, especially, for diagnosing, treating EGFRvIII positive tumor cells, and treatment, prevention and/or control with EGFRvIII unconventionality expression and/or by The method and composition for the physiologic derangement that EGFRvIII unconventionality expression triggers.Such disorder includes but is not limited to:It is cancer, non- Cancer hyperproliferative cell is disorderly.

The double targeting antibodies of exemplary specificity of the present invention are listed in the Tables 1 and 2 in text.Specificity provided by the invention is double In targeting antibodies, weight chain variable district (HCVR) that the first antigen-binding domains and the second antigen-binding domains include and light Chain variable region (LCVR)).The table 2 of table 1 lists HCVR, LCVR of the double targeting antibodies of specificity provided by the invention amino acid sequence Row, and its sequence number in sequence table.Table 2 is listed corresponding to coding HCVR, LCVR amino acid sequence of the present invention Nucleotide sequence, and its sequence number in sequence table.

The amino acid sequence of the double targeting antibodies of specificity provided by the invention includes：At least one the first antigen binding structures It is the HCVR amino acid sequences in domain, the LCVR amino acid sequences of at least one the first antigen-binding domains, at least one the second anti- The LCVR amino acid sequences of the HCVR amino acid sequences of former binding structural domain and at least one the second antigen-binding domains； Specific HCVR and LCVR is preferably any amino acid sequence selected from table 1.

Encoding the nucleotide sequence of the double targeting antibodies of specificity provided by the invention includes：At least one the first antigen bindings The HCVR nucleotide sequences of domain, the LCVR nucleotide sequences of at least one the first antigen-binding domains, at least one the The LCVR nucleotides of the HCVR nucleotide sequences of two antigen-binding domains and at least one the second antigen-binding domains Sequence；Specific HCVR and LCVR is preferably any nucleotide sequence selected from table 2.

Amino acid sequence in the sequence table of table 1

In an embodiment of the present invention, 3 not homotactic anti-CD3 × double targeting antibodies of anti-EGFR vIII specificity, Numbering is respectively BsAb-1, BsAb-2, BsAb-3 in table 1, and wherein BsAb-1 is by the special of the optimum efficiency that filters out Property double targeting antibodies.

In some embodiments of the invention, 1 each sequence of table is specially：

In SEQUENCE NO.1,

CD3.HCVR

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGRVNPNRRGTTYNQKFEGRVTMTTDTSTS TAYMELRSLRSDDTAVYYCARANWLDYWGQGTTVTVSS；

In SEQUENCE NO.2,

CD3.LCVR

DIQMTQSPSSLSASVGDRVTITCSVSSSVSSIYLHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQVYSGYPLTFGGGTKVEIK；

In SEQUENCE NO.3,

EGFRVIII-HCVR

EIQLVQSGAEVKKPGESLRISCKGSGFNIEDYYIHWVRQMPGKGLEWMGRIDPENDETKYGPIFQGHVTISADTSIN TVYLQWSSLKASDTAMYYCAFRGGVYWGQGTTVTVSS；

In SEQUENCE NO.4,

EGFRVIII-LCVR

DVVMTQSPDSLAVSLGERATINCKSSQSLLDSDGKTYLNWLQQKPGQPPKRLISLVSKLDSGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCWQGTHFPGTFGGGTKVEIK；

In SEQUENCE NO.5,

EGFRVIII-HCVR

EIQLVQSGAEVKRPGESLRISCKSSGFNIEDYYIHWVRQMPGKGLEWMGKIDPENDETKYGPIFQGHVTISADTSIN TVYLQWGSLKASDTAMYYCAFRGSVYWGQGTTVTVSS；

In SEQUENCE NO.6,

EGFRVIII-LCVR

DVVMTQSPDSLAVSIGERATINCKSSQSLIDSDGRTYLNWLQQKPGQPPKRLISLVSKLDSGVPDRFSGSGSGTDFT LTIGSLQAEDVAVYYCWQGTHFPGTFGGGTKVEIK；

In SEQUENCE NO.7,

EGFRVIII-HCVR

EIQLVQSGAEVRKPGESLRIGCKGSGFNIEDYYIHWVRQMPGKGLEWMGKLDPENDETKYGPIFQGHVTISADTGIN TVYLQWSSLKAGDTAMYYCAFRGSVYWGQGTTVTVSS；

In SEQUENCE NO.8,

EGFRVIII-LCVR

DVVMTQSPDSLAVSIGEKATINCKGSQSLLDSDGKTYLNWLQQKPGQPPKRLISLVGKIDSGVPDRFSGSGSSTDFT LTIGSLQAEDVAVYYCWQGTHFPGTFGSGTKVEIK；

Nucleotide sequence in the sequence table of table 2

In some embodiments of the invention, 2 each sequence of table is specially：

SEQUENCE NO.9,

CD3.HCVR

CAGGTTCAGCTGGTGCAGTCTGGTGCTGAGGTGAAGAAGCCTGGTGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGG TTACACATTCACTGACTACTACATGCACTGGGTGAGACAGGCCCCTGGTCAAGGTCTTGAGTGGATGGGTAGAGTTA ATCCTAACCGGAGGGGTACTACCTACAACCAGAAATTCGAGGGCCGTGTCACCATGACCACAGACACATCCACCAGC ACAGCCTACATGGAGCTGCGTAGCCTGCGTTCTGACGACACCGCCGTGTATTACTGCGCCCGTGCTAACTGGCTTGA CTACTGGGGCCAGGGCACCACCGTCACCGTCTCCTCC；

SEQUENCE NO.10,

CD3.LCVR

CAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGCAGTGTCAGCTCAAG TGTTTCCTCCATTTACTTGCACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGCACATCCA ACTTGGCTTCTGGAGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTG CAACCTGAAGATTTTGCAACTTACTACTGCCAAGTCTACAGTGGTTACCCTCTCACCTTCGGCGGAGGGACCAAGGT GGAGATCAAA；

In SEQUENCE NO.11,

EGFRVIII-HCVR

GAGATCCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGCGCATCAGCTGCAAGGGCAGCGG CTTCAACATCGAGGACTACTACATCCACTGGGTGCGCCAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCCGCATCG ACCCCGAGAACGACGAGACCAAGTACGGCCCCATCTTCCAGGGCCACGTGACCATCAGCGCCGACACCAGCATCAAC ACCGTGTACCTGCAGTGGAGCAGCCTGAAGGCCAGCGACACCGCCATGTACTACTGCGCCTTCCGCGGCGGCGTGTA CTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC；

In SEQUENCE NO.12,

EGFRVIII-LCVR

GACGTGGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGCGCCACCATCAACTGCAAGAGCAG CCAGAGCCTGCTGGACAGCGACGGCAAGACCTACCTGAACTGGCTGCAGCAGAAGCCCGGCCAGCCCCCCAAGCGCC TGATCAGCCTGGTGAGCAAGCTGGACAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACC CTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCTGGCAGGGCACCCACTTCCCCGGCACCTT CGGCGGCGGCACCAAGGTGGAGATCAAG；

In SEQUENCE NO.13,

EGFRVIII-HCVR

GAGATCCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGCGCCCCGGCGAGAGCCTGCGCATCAGCTGCAAGAGCAGCGG CTTCAACATCGAGGACTACTACATCCACTGGGTGCGCCAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCAAGATCG ACCCCGAGAACGACGAGACCAAGTACGGCCCCATCTTCCAGGGCCACGTGACCATCAGCGCCGACACCAGCATCAAC ACCGTGTACCTGCAGTGGGGCAGCCTGAAGGCCAGCGACACCGCCATGTACTACTGCGCCTTCCGCGGCAGCGTGTA CTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC；

In SEQUENCE NO.14,

EGFRVIII-LCVR

GACGTGGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCATCGGCGAGCGCGCCACCATCAACTGCAAGAGCAG CCAGAGCCTGATCGACAGCGACGGCCGCACCTACCTGAACTGGCTGCAGCAGAAGCCCGGCCAGCCCCCCAAGCGCC TGATCAGCCTGGTGAGCAAGCTGGACAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCGGCACCGACTTCACC CTGACCATCGGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCTGGCAGGGCACCCACTTCCCCGGCACCTT CGGCGGCGGCACCAAGGTGGAGATCAAG；

In SEQUENCE NO.15,

EGFRVIII-HCVR

GAGATCCAGCTGGTGCAGAGCGGCGCCGAGGTGCGCAAGCCCGGCGAGAGCCTGCGCATCGGCTGCAAGGGCAGCGG CTTCAACATCGAGGACTACTACATCCACTGGGTGCGCCAGATGCCCGGCAAGGGCCTGGAGTGGATGGGCAAGCTGG ACCCCGAGAACGACGAGACCAAGTACGGCCCCATCTTCCAGGGCCACGTGACCATCAGCGCCGACACCGGCATCAAC ACCGTGTACCTGCAGTGGAGCAGCCTGAAGGCCGGCGACACCGCCATGTACTACTGCGCCTTCCGCGGCAGCGTGTA CTGGGGCCAGGGCACCACCGTGACCGTGAGCAGC；

In SEQUENCE NO.16,

EGFRVIII-LCVR

GACGTGGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCATCGGCGAGAAGGCCACCATCAACTGCAAGGGCAG CCAGAGCCTGCTGGACAGCGACGGCAAGACCTACCTGAACTGGCTGCAGCAGAAGCCCGGCCAGCCCCCCAAGCGCC TGATCAGCCTGGTGGGCAAGATCGACAGCGGCGTGCCCGACCGCTTCAGCGGCAGCGGCAGCAGCACCGACTTCACC CTGACCATCGGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCTGGCAGGGCACCCACTTCCCCGGCACCTT CGGCAGCGGCACCAAGGTGGAGATCAAG；

Specifically, SEQUENCE NO.9-16 correspond to coding SEQUENCE NO.1-8 amino acid sequence respectively.

In view of sequence homology, it will be appreciated by persons skilled in the art that scheme, which is implemented as follows, should include guarantor of the present invention Protect scope：

In some embodiments of the invention, the HCVR of the first described antigen-binding domains is and the shown in table 1 Any HCVR amino acid sequences have at least 90%, at least 95%, at least 98% or at least 99% in one antigen-binding domains The amino acid sequence of homology.

In some embodiments of the invention, the LCVR of the first described antigen-binding domains is and the shown in table 1 Any LCVR amino acid sequences have at least 90%, at least 95%, at least 98% or at least 99% in one antigen-binding domains The amino acid sequence of homology.

In some embodiments of the invention, the HCVR of the second described antigen-binding domains is and the shown in table 1 Any HCVR amino acid sequences have at least 90%, at least 95%, at least 98% or at least 99% in one antigen-binding domains The amino acid sequence of homology.

In some embodiments of the invention, the LCVR of the second described antigen-binding domains is and the shown in table 1 Any LCVR amino acid sequences have at least 90%, at least 95%, at least 98% or at least 99% in one antigen-binding domains The amino acid sequence of homology.

In some embodiments of the invention, described the first antigen-binding domains of coding HCVR sequence be with table 2 In the first shown antigen-binding domains any HCVR nucleotide sequences have at least 90%, at least 95%, at least 98% or At least nucleotide sequence of 99% homology.

In some embodiments of the invention, described the first antigen-binding domains of coding LCVR sequence be with table 2 In the first shown antigen-binding domains any LCVR nucleotide sequences have at least 90%, at least 95%, at least 98% or At least nucleotide sequence of 99% homology.

In some embodiments of the invention, described the second antigen-binding domains of coding HCVR sequence be with table 2 In the first shown antigen-binding domains any HCVR nucleotide sequences have at least 90%, at least 95%, at least 98% or At least nucleotide sequence of 99% homology.

In some embodiments of the invention, described the second antigen-binding domains of coding LCVR sequence be with table 2 In the first shown antigen-binding domains any LCVR nucleotide sequences have at least 90%, at least 95%, at least 98% or At least nucleotide sequence of 99% homology.

It should be understood that there is base degeneracy because of situations such as " nucleotide sequence ", be mutated, those of ordinary skill in industry The species of some bases is can adjust, although described base change result in the change of codon, but not causes codon to turn over The change for the amino acid translated, very common, the codon of leucine just has a variety of codons, such as：UUA、UUG、CUU.

In addition, EpCAM antibody amino acids sequences of the present invention come from human antibody amino acid sequence, by messenger HVGR (host versus graft reaction, HVGR) can be reduced in body application.Nucleotide sequence passes through Codon optimization is crossed, is expressed beneficial in mammalian cell.

In some embodiments of the invention, the amino acid sequence of the double targeting antibodies of specificity is：Optionally from table 1 Sequence, it is a kind of combination gained (" V in (a)~(h) by following type of attachment_LCD3 " represents the of the double targeting antibodies of specificity The LCVR of one antigen-binding domains；“V_HCD3 " represents the HCVR of the first antigen-binding domains of the double targeting antibodies of specificity； “V_LEGFRvIII " represents the LCVR of the second antigen-binding domains of the double targeting antibodies of specificity；“V_HEGFRvIII " represents special The HCVR of second antigen-binding domains of different in nature double targeting antibodies)：

(a)V_LEGFRvIII-linker1-V_HEGFRvIII-linker2-V_LCD3-linker3-V_HCD3,

(b)V_HEGFRvIII-linker1-V_LEGFRvIII-linker2-V_LCD3-linker3-V_HCD3,

(c)V_LEGFRvIII-linker1-V_HEGFRvIII-linker2-V_HCD3-linker3-V_LCD3,

(d)V_HEGFRvIII-linker1-V_LEGFRvIII-linker2-V_HCD3-linker3-V_LCD3,

(e)V_LCD3-linker1-V_HCD3-linker2-V_LEGFRvIII-linker3-V_HEGFRvIII,

(f)V_LCD3-linker1-V_HCD3-linker2-V_HEGFRvIII-linker3-V_LEGFRvIII,

(g)V_HCD3-linker1-V_LCD3-linker2-V_LEGFRvIII-linker3-V_HEGFRvIII,

(h)V_HCD3-linker1-V_LCD3-linker2-V_HEGFRvIII-linker3-V_LEGFRvIII,

Wherein, whippletree "-" represents each amino acid sequence order and arranged.

In an embodiment of the invention, the HCVR (V of the first antigen-binding domains or the second antigen-binding domains_H) With LCVR (V_L) between Linker (Linker1, Linker3) amino acid sequence be (GGGGS) n (n=1-4), (GGS) 4 Or (Gly) n (n=6-8), but not limited to this.

In an embodiment of the invention, the amino acid sequence of the Linker (Linker2) between each antigen-binding domains It is classified as (GGGGS) n (n=1-4), but not limited to this.

In a preferred embodiment, bispecific single-chain antibody amino acid sequence is with any with (a)~(h) The homology of shown sequence is not less than 90%, 95%, 98% or 99% amino acid sequence.

In some embodiments of the invention, the HCVR (V of each antigen-binding domains_H) and LCVR (V_L) between, each antigen knot It is connected between conjunction domain by linker, Linker is connection peptide, and the connection peptide is mainly employed with glycine and serine Based on the peptide sequence that forms, wherein, glycine is the amino acid that molecular weight is minimum, side chain is most short, can increase the flexible of side chain Property；Serine is the most strong amino acid of hydrophily, can increase the hydrophily of peptide chain.

In some embodiments of the invention, the N sections of the double targeting antibodies of specificity have immunoglobulin kappa chain signal peptide Amino acid sequence.

The N-terminal of the double targeting antibodies of the specificity provided by the invention adds the secretion letter of light chain immunoglobulin κ chains Number peptide, so as to ensure the expression of antibody and be secreted into outside host cell.

Second aspect, present invention also offers a kind of side of the double targeting antibodies of the specificity prepared as described in relation to the first aspect Method, this method include：The specificity that the double targeting antibodies sequences of specificity described in first aspect are used for described in construction expression is double The recombinant expression carrier of targeting antibodies；Import host cell；Under expression condition, host cell is cultivated, expression, is isolated and purified, Obtain the double targeting antibodies of the specificity.

The third aspect, present invention also offers the double targeting antibodies of specificity as described in relation to the first aspect by following a kind of or more Kind method is applied：

(1) the double targeting antibodies of the specificity are individually applied；

(2) by the double targeting antibodies of specificity and one kind in chemotherapy, radiotherapy, operation, biological therapy, immunization therapy or Several use in conjunction；

(3) the double targeting antibodies of the specificity are directly delivered into patient's body by the way of delivering in vivo to be controlled Treat；

(4) first pass through in-vitro transfection technology to mix the double targeting antibodies of the specificity with immune effector cell, then will Described defeated time patient's body of immune effector cell for being mixed with specific double targeting antibodies implements treatment.

In some embodiments of the invention, in the application (3) and (4) as described in the third aspect, as above institute is applied to patient The antibody stated, it can be applied more than once to patient.

In some embodiments of the invention, in the application (3) as described in the third aspect, the mode of described delivery is targeting Deliver, including but not limited to using can the industry common carrier such as liposome liposomes (or its polymer) of targeting Delivery carry out Deliver.

By applying the antibody of first aspect present invention offer or the medicine of fourth aspect offer to the mammal including people Compositions, it can prevent or treat liver cancer or other EGFRvIII relevant disease, such as EGFRvIII abnormal expressions or disorder Related disease.

Fourth aspect, the invention provides a kind of pharmaceutical composition, including at least one as described in relation to the first aspect special Property double targeting antibodies, and pharmaceutically useful supporting agent, excipient or diluent.

Pharmaceutical preparation can be made according to conventional methods for pharmaceutical composition provided by the invention., preferably will be anti-in production process Body is mixed with carrier or diluted with carrier, or is fitted into the carrier of vessel form.When the carrier serves as a diluent, it can be solid Body, semisolid or liquid, as the vesica, excipient or culture medium for antibody.Therefore, preparation can be tablet, pill, powder Agent, sachet, capsule, elixir, supensoid agent, emulsion, solution, syrup, aerosol, soft hard gelatin capsule, injection without The forms such as bacterium solution, sterile powder.The example of suitable carrier, excipient or diluent includes lactose, glucose, sucrose, mountain Pears alcohol, mannitol, calcium silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, hydroxybenzoic acid Methyl esters, nipasol, talcum powder, magnesium stearate and mineral oil.Preparation can also include filler, anticoagulant, profit Lubrication prescription, wetting agent, flavor enhancement, emulsifying agent, preservative etc..

In some embodiments of the invention, described pharmaceutical composition also includes second therapeutic agent, and second therapeutic agent is to appoint The activating agent what is advantageously combined with the double targeting antibodies of described specificity.Including but not limited to combine and/or activate CD3 signals Other activating agents (including other antibody or its antigen-binding fragment etc.) transmitted, and/or it is not direct combined with CD3, but live Change or stimulate the activating agent of activated immune cell.

In some embodiments of the invention, described second therapeutic agent includes and the agent interfering of antibody synergy, anti- EGFRvIII monoclonal antibodies, anti-EGFRvIII polyclonal antibodies, nucleoside analog, archaeal dna polymerase inhibitor, siRNA preparations, Treatment vaccine, cell factor, toxin, chemotherapeutant, radiotherapy dose, hormone, antibody Fc fragment as antivirotic, TLR activators, molecule or co-stimulators containing CpG.

5th aspect, the invention provides a kind of diagnostic reagent, including at least one specificity as described in relation to the first aspect Double targeting antibodies, and diagnosticum, described diagnosticum include but is not limited to fluorogen, chromophore, dyestuff, radio isotope, Chemiluminescent molecule, paramagnetic ion or spin trapping reagent.

6th aspect, the invention provides a kind of antibody drug, for the double targetings of any specificity as described in relation to the first aspect Antibody, it is incorporated in external evoked mankind's T cell with mankind CD3 knots and breeds.

7th aspect, the invention provides a kind of antibody drug, for the double targetings of any specificity as described in relation to the first aspect Antibody, being killed to EGFRvIII positive cells for external evoked T cell mediation is incorporated in mankind CD3 knots.

Eighth aspect, the invention provides a kind of nucleic acid molecules, the double targetings of specificity encoded as described in relation to the first aspect are anti- The amino acid sequence of body；In some embodiments, the amino acid sequence of described nucleic acid molecule encoding, it is and such as first aspect The amino acid sequence of the double targeting antibodies of specificity has at least 90%, at least 95%, at least 98% or at least 99% homologous The amino acid sequence of property.

9th aspect, the invention provides a kind of restructuring load of the double targeting antibodies of the specificity expressed as described in relation to the first aspect Body；In some embodiments, described recombinant vector has any sequence of nucleic acid molecules that eighth aspect present invention provides.

In some embodiments of the invention, described carrier is that minicircle dna recombinates matrix grain or minicircle dna.

In some embodiments of the invention, the minicircle dna restructuring matrix grain is in the polyclonal position of minicircle dna empty plasmid Any sequence of nucleic acid molecules that eighth aspect present invention provides is inserted in point to combine.Specific insertion method can use conventional Gene cloning, it can also use seamless clone (Seamless cloning).

In some embodiments of the invention, the minicircle dna empty plasmid is preferably p2 Ф C31 plasmids or pMC.BESPX matter Grain.Specifically, the empty plasmid p2 Ф C31 construction methods are with reference to Chen ZY etc., Molecular Therapy, 8 (3), 495- 500 (2003), Chen ZY etc., Human Gene Therapy, 16 (1), 126-131 (2005) and United States Patent (USP) US7897380B2.Specifically, described empty plasmid pMC.BESPX construction methods and complete genome sequence reference Chen ZY etc., Nature Biotechnology,28,(12),1289-1291(2010)。

The p2 Ф C31 weights that p2 Ф C31 plasmids, the pMC.BESPX plasmids used in exemplary embodiments of the present invention obtains respectively Group matrix grain, pMC.BESPX restructuring matrix grains, difference are：There is coding Ф C31 recombinases and I- on p2 Ф C31 carriers The nucleotide sequence of Sce1 restriction endonucleases, and there is no the core for encoding Ф C31 recombinases and I-Sce1 restriction endonucleases on pMC.BESPX carriers Nucleotide sequence, thus using pMC.BESPX carriers prepare minicircle dna matrix grain it is more high-quality, greatly reduce recombinase and The pollution of the nucleotide sequence of restriction endonuclease；But pMC.BESPX need to support the use E. coli ZYCY10P3S2T Engineering bacteria, ZYCY10P3S2T engineering bacterias have coding Ф C31 recombinases and I-Sce1 restriction endonuclease functions, no Ф C31 recombinases The pMC.BESPX of (i.e. phiC31 groups enzyme) and I-Sce1 restriction endonuclease nucleotide coding sequences restructuring matrix grains need to support the use ZYCY10P3S2T engineering bacterias could produce internal locus specificity restructuring (and E. coli TOP 10 is without this function) And final production goes out minicircle dna；Accordingly, p2 Ф C31 restructuring matrix grain, which can support the use TOP 10, can produce internal site Specificity restructuring, and final production goes out minicircle dna.

In some embodiments of the invention, described minicircle dna is that minicircle dna provided by the invention restructuring matrix grain passes through The locus specificity restructuring of specific recombination site produces.Minicircle dna restructuring matrix grain provided by the invention has specificity weight Group site, described " minicircle dna restructuring matrix grain " produce minicircle dna by the locus specificity restructuring of specific recombination site With skeleton DNA sequence dna.The method of p2 Ф C31 restructuring matrix grain production minicircle dnas is with reference to Chen ZY etc., Molecular Therapy, 8 (3), 495-500 (2003), Chen ZY etc., Human Gene Therapy, 16 (1), 126-131 (2005) and United States Patent (USP) US7897380B2.The method of pMC.BESPX restructuring matrix grain production minicircle dnas is with reference to Chen ZY etc., Nature Biotechnology,28,(12),1289-1291(2010)。

As used herein, " skeleton DNA sequence dna " there is the duplication for being responsible for bacterial plasmid in standard plasmid or screening to contain plasmid The function such as host DNA sequence dna, such as bacterium replication sequence, resistant gene, unmethylated CpG motifs etc..

In some embodiments of the invention, described recombinant vector (is special in the present invention also comprising expression desired polypeptides Property double targeting antibodies) needed for all necessary elements gene expression system, generally it include elements below：Promoter, coding The gene order of polypeptide, terminator；Additionally alternative is including signal coding sequence etc.；These elements are to be operatively connected 's.

As used herein, described " being operably connected " refers to two or more nucleic acid regions or the function of nucleotide sequence The space arrangement of property.Such as：Promoter region is placed in the ad-hoc location relative to target gene nucleotide sequence so that nucleotide sequence Transcription guided by the promoter region, so as to, promoter region is " operably connected " on the nucleotide sequence. Preferably, the signal peptide is immunoglobulin kappa chain signal peptide.Preferably, the label is His labels, GST labels, c-myc At least one of label and Flag labels.Signal peptide above and the preferred embodiment that tag class is the present inventor, this area Technical staff can select suitable signal peptide and label according to specific needs.

Tenth aspect, the invention provides a kind of host cell (preferably eucaryon with the carrier as described in terms of the 9th Cell or mammalian cell including people)；Additionally provide in the vector introduction host cell as described in terms of the 9th Method；Additionally provide by host cell of the culture with the carrier under conditions of generation antibody is allowed, and separating force institute The method of caused antibody.

Tenth on the one hand, and the invention provides a kind of detection kit, including solid detection holder, the inspection of described solid Survey holder and include the double targeting antibodies of at least one specificity as described in relation to the first aspect.In some embodiments, it is described solid Body holder is the surface of solids that can connect macromolecular such as antibody, protein, polypeptide, peptide, polynucleotides, such as magnetic beads, latex Pearl, micro titer plate well, glass plate, nylon, agarose, polyacrylamide, silica dioxide granule, nitrocellulose filter etc..

12nd aspect, the invention provides a kind of method that abnormal cell is detected in testing sample, it includes making institute Sample is stated to be in contact with the double targeting antibodies of at least one specificity as described in relation to the first aspect.In some embodiments, it is described Tumor bed of the sample from liver or through excision.

As described herein, " abnormal cell " be have for the cell type be atypical characteristics (including atypia give birth to The long, typical growth of atypia opening position or the typical effect for atypia target) any cell.Such cell includes Cancer cell, hyperplasia of prostate cell or dysplasia cell, inflammatory cell or autoimmunity cell.

In some embodiments of the invention, using the method as described in terms of the 12nd to the tissue or device donated before transplanting Official is screened, to provide the tissue or organ that are substantially free of EGFRvIII.

In some embodiments of the invention, blood supply product are screened using the method as described in terms of the 12nd, To provide the blood supply product for being substantially free of EGFRvIII.

13rd aspect, double targeting antibodies of specificity as described in relation to the first aspect or the recombinant vector as described in terms of the 9th Preparing diagnosis, preventing, the application in the reagent or medicine for the treatment of EGFRvIII relevant diseases.

As described in the present invention, described " EGFRvIII diseases " includes but is not limited to EGFRvIII abnormal expressions or disorder Relevant disease, such as, the liver cancer of EGFRvIII antigen positives.

Fourteenth aspect, present invention also offers using the double targeting antibodies treating cancers of specificity as described in relation to the first aspect Or the method for tumour, include following methods any one：

(1) genetic engineering antibody is individually used for treating cancer or tumour；

(2) by the one or more in the genetic engineering antibody and chemotherapy, radiotherapy, operation, biological therapy, immunization therapy Combine and be used for treating cancer or tumour；

(3) genetic engineering antibody is directly delivered to patient's body by the way of internal targeting Delivery and carries out cancer Or oncotherapy；

(4) first pass through in-vitro transfection technology to mix the genetic engineering antibody with immune effector cell, then by described in The defeated time patient's body of immune effector cell for being mixed with genetic engineering antibody implements cancer or oncotherapy.

In some embodiments of the invention, in methods described (4), the immune effector cell is that CIK cell or D-CIK are thin Born of the same parents.

In some embodiments of the invention, in methods described (4), the double targeting antibodies of specificity of described first aspect with Immune effector cell mixing ratio be：0.001~1ug/ul double the targeting antibodies and 1 × 10 of specificity⁴~1.6 × 10⁵It is immune Effector cell mixes.

Technical scheme provided by the invention, by designing the double targeting antibodies of a group-specific, and by antibody expression frame (DNA Sequence) insertion minicircle dna carrier (preferably Kay MA*, He CY and Chen ZY* (* communicates author) .A Robust system for production of minicircle DNA vecrtors.Nat Biotechnology,28:1287, 2010) in.The antibody expressed by minicircle dna has following characteristic：1) EGFRvIII positive antigens are specifically bound；2) it is specific With reference to T cell；3) mediate T cell plays lethal effect to EGFRvIII antigen-positive cells.In addition, in certain embodiments, Because antibody expression frame carries the coded sequence of secreting signal peptide, can be secreted after antibody expression to extracellular, holding bioactivity.

Brief description of the drawings

Fig. 1 is the plasmid map of micro-loop matrix grain prepared by the embodiment of the present invention 1；

Fig. 2 is the plasmid map of minicircle dna prepared by the embodiment of the present invention 2；

Fig. 3 is that the Western Blot that the embodiment of the present invention 3 provides detect BsAb antibody expressions in culture medium supernatant Result；

Fig. 4-5 is EGFRvIII × CD3 BsAb cell binding experiments flow cytometer inspection that the embodiment of the present invention 4 provides Survey result；Wherein, a-c represents BsAb-1, BsAb-2, BsAb-3 result respectively；B is BsAb result, and C is control (control)；

The result for EGFRvIII × CD3 BsAb joint DCIK cell killing experiments that Fig. 6-7 embodiment of the present invention 5 provides.

Embodiment

As described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art For, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as Protection scope of the present invention.

Outer without special instruction in the embodiment of the present invention, agents useful for same and consumptive material are commercial goods.

Embodiment 1

The structure of micro-loop matrix grain with coding EGFRvIII × CD3 BsAb antibody nucleotide sequences, including following step Suddenly：

1) BsAb antibody gene expression cassettes are designed

The BsAb antibody genes expression cassette of the embodiment of the present invention 1 also wraps in addition to the nucleotide sequence containing coding BsAb Include promoter, secreting signal peptide, protein purification label, the coded sequence of polyA tailing signals.

In an embodiment, the BsAb antibody genes expression cassette of the embodiment of the present invention 1 includes what is be sequentially connected：

Nucleotide sequence (the Murine Ig kapa-chain signal of encoding immune globulin κ chain signal peptides Peptide, SEQ ID NO:17) gene order (V of-BsAb antibody_LEGFRvIII-linker1-V_HEGFRvIII- linker2-V_HCD3-linker3-V_LCD3；EGFRvIII LCVR-linker1- in specific nucleotide sequence corresponding table 2 The nucleotides sequence of EGFRvIII HCVR-linker2-CD3 HCVR-linker3-CD3 LCVR- encoding histidine 6-His labels Arrange (SEQ ID NO:18)-dual terminator codon (TAGTGA)；Wherein,

The nucleotide sequence of encoding immune globulin κ chain signal peptides, such as SEQ ID NO:Shown in 17.

The nucleotide sequence of encoding histidine 6-His labels, such as SEQ ID NO:Shown in 18.

The BsAb antibody genes expression cassette of the embodiment of the present invention 1 also includes the nucleotides sequence of cytomegalovirus CMV promoter Row, positioned at the N sections of Signal peptide secreting signal peptides；Sequence is SEQ ID NO:19.

The BsAb antibody genes expression cassette of the embodiment of the present invention 1 also includes bovine growth hormone polynucleotide poly A (BGH Poly A) nucleotide sequence, positioned at terminator codon (TGATAG) C-terminal；Sequence is SEQ ID NO:20.

In order to strengthen the expression efficiency of minicircle dna carrier, element chimeric intron are with the addition of (between CMV promoter Between Signal peptide secreting signal peptides；Document report, 10-20 times can be strengthened in various kinds of cell), sequence SEQ ID NO:21。

In the BsAb antibody gene expression cassettes of the embodiment of the present invention 1, linker1, linker2 amino acid sequence are identical, It is (GGGGS)₃, nucleotide sequence is different, nucleosides shown in respectively SEQUENCE NO.22 and SEQUENCE NO.23 Acid sequence.

Linker3 amino acid sequences are VEGGSGGSGGSGGSGGVD, and nucleotides sequence is classified as shown in SEQUENCE NO.24 Nucleotide sequence.

Specifically, each sequence is as follows：

In SEQUENCE NO.17,

Murine Ig kapa-chain signal peptide

ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT；

In SEQUENCE NO.18,

6-His

CATCATCACCATCATCAT；

In SEQUENCE NO.19,

CMV promoter

GTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACG TTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGAC TAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGG TAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACG CCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA TCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCT TACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTAC ACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTT TTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTG TACGGTGGGAGGTCTATATAAGCAGAGGTCGTTTAGTGAACCGTCAGATCACTAGTAGCTTTATTGCGGTAGTTTAT CACAGTTAAATTGCT；

In SEQUENCE NO.20,

BGH poly A

GGTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTG GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGG GCTCTATGGAACCAGCTG；

In SEQUENCE NO.21,

chimeric intron

AACGCAGTCAGTGCTCGACTGATCACAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGGCCAATAGAAACTGG GCTTGTCGAGACAGAGAAGATTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTC TCCACAGGGGTACCGAAGCCGCTAGCGCTACCGGTC；

In SEQUENCE NO.22,

Linker1

GGCGGCGGAGGCAGCGGCGGCGGAGGCAGCGGCGGCGGAGGCAGCGGCGGCGGAGGCAGC；

In SEQUENCE NO.23,

Linker2

GGCGGCGGAGGCAGCGGCGGCGGAGGCAGCGGCGGAGGCGGATCC；

In SEQUENCE NO.24,

Linker3

GTCGAAGGTGGAAGTGGAGGTTCTGGTGGAAGTGGAGGTTCAGGTGGAGTTGAC；

2) described BsAb antibody genes expression cassette is inserted to the attB of p2 Ф C31 empty carriers or pMC.BESXP empty carriers Between attP sites, specific steps include A) or B) in it is any：

A)

PCT application (the application number that specific steps were submitted with reference to inventor in 2014：PCT/CN2014/083741) explanation Book：

Embodiment 1 be " a kind of minicircle dna restructuring matrix grain p2 Ф C31.Bab's of expression casette containing genetic engineering antibody Construction method ", to the p2 Ф with coding EGFRvIII × CD3 BsAb antibody nucleotide sequences for obtaining the embodiment of the present invention 2 C31 micro-loop matrix grains；And

A kind of " the minicircle dna restructuring matrix grain pMC.Bab of expression casette containing the genetic engineering antibody structure of embodiment 6 The step of method "；EGFRvIII × CD3 BsAb antibody nucleotide sequences are encoded to having for the embodiment of the present invention 2 is obtained PMC.BESXP micro-loop matrix grains.

Step 1-2 of the present invention) place different from above-mentioned PCT application be only that, the step 1-2 of the embodiment of the present invention 2) will be upper State " EGFRvIII × CD3 that " BiTE gene order " in PCT application embodiment 1 or 6 replaces with the embodiment of the present invention 2 BsAb gene order ".

B)

The method for directly using digestion-connection, two digestions of SmaI-ApaI are designed at BsAb antibody gene expression cassettes both ends Site and pMC.BESXP empty carriers or p2 Ф C31 empty carriers, pass through digestion-connection by the BsAb antibody genes expression cassette of synthesis Mode insert between p2 Ф C31 empty carriers or attB the and attP sites of pMC.BESXP empty carriers；It is real that the present invention is obtained respectively Apply the p2 Ф C31 micro-loop matrix grains or pMC.BESXP micro-loop matrix grains with coding BsAb antibody nucleotide sequences of example 1.

The plasmid map of pMC.BESXP micro-loop matrix grains prepared by the embodiment of the present invention 1 (is named as p- as shown in Figure 1 EGFRvIII×CD3)。

Embodiment 2

The structure of minicircle dna with coding EGFRvIII × CD3 BsAb antibody nucleotide sequences

PCT application (the application number submitted with reference to inventor in 2014：PCT/CN2014/083741) specification：

Embodiment 2 and embodiment 7 " a kind of preparation method of the minicircle dna of expression casette containing genetic engineering antibody ", point Not to the minicircle dna with coding EGFRvIII × CD3 BsAb antibody nucleotide sequences for obtaining the embodiment of the present invention 2.

The place different from above-mentioned PCT application embodiment 2 and embodiment 7 of the invention is only that the embodiment of the present invention 3 will be upper State the micro-loop matrix grain that the micro-loop matrix grain in PCT application embodiment 2 or 7 replaces with the offer of the embodiment of the present invention 1.

Micro-loop collection of illustrative plates prepared by the embodiment of the present invention 2 is as shown in Figure 2.

Embodiment 3

The expression and purifying of BsAb antibody, comprise the following steps：

(1) expression of EGFRvIII × CD3 BsAb antibody in HEK 293T cells

Using X-treme GENEHP DNA Transfection Reagent plasmid transfections kits (Roche companies) By minicircle dna (minicircle dna prepared by the grain of micro-loop matrix shown in Fig. 2) transfection HEK293T cells of embodiment 3, HEK293T cells EGFRvIII BsAb micro-loops carrier is transfected after 72 hours, in Western Blot detection culture medium supernatant cytoplasm EGFRvIII BsAb antibody expressions, as a result as shown in figure 3, BsAb-1, BsAb-2 and BsAb-3 have expression, but BsAb- 1 in the relative highest of culture medium supernatant secreting, expressing.

(2) purifying of EGFRvIII × CD3 BsAb antibody

Gauge mould transfects HEK 293T cells in the antibody BsAb that will have secreting, expressing, collects culture supernatant, then use His-Tag affine resins purify (Omplete His-Tag Purification Resin, Roche), and antibody after purification is adopted With ELISA method detection protein concentration；Protein concentration reaches 1.0mg/ml after purification, is placed in -80 DEG C long-term preservation, is ready for use on stream Formula cell art is detected and external knurl of killing is tested.

Embodiment 4

The binding activity of Flow cytometry EGFRvIII × CD3 BsAb antibody and Jurkat, SMMC-7721 cell, Comprise the following steps：

A) cell culture：Jurkat cell (1640medium；Suspension growth, the cell line of CD3 expression)；SMMC-7721 (DMEM medium；The liver cancer cell lines of EGFRvIII expression),

B) pancreatin digestion SMMC-7721, add serum-containing media neutralize after centrifugation abandon supernatant and obtain cell；Jurkat is suspension Cell is directly collected by centrifugation in cell；

C) tears are washed 2 times with precooling PBS, 200g centrifugation 5min, collects cell, count respectively；

D) mean allocation is per experimental group 1 × 10⁵Individual/group sample, is grouped as follows：

Jurkat cell, 2 groups:Blank group (the supernatant after Blank, pMC.BESXP empty carrier transfection HEK 293T cells； Prepare in advance)；EGFRvIII × CD3 BsAb groups (the antibody of the gained of embodiment 3 after purification；Prepare in advance)

SMMC-7721 cells, 2 groups:As above, blank group (being same as above) and EGFRvIII BsAb groups are (obtained by embodiment 3 for packet Antibody after purification；Prepare in advance) Jurkat, 2 groups:It is grouped as above, blank group (being same as above) and EGFRvIII × CD3 BsAb groups (the antibody of the gained of embodiment 3 after purification；Prepare in advance)

E) the above-mentioned μ l/ groups of supernatant 100 are added and is incubated 30min on ice；

F) 1ml precoolings PBS washings are added, 200g centrifugation 5min, cell is collected, adds prior precooling and the APC- diluted The μ l (1 of PBS 100 of mouse anti-6His antibody:1000 dilutions), 30min is incubated on ice,

G) 1ml precoolings PBS washings are added, 200g centrifugation 5min, collect cell, then be resuspended with 200 μ l PBS.Up flow type is thin Born of the same parents' instrument (BD C6) observation combines situation.

Fig. 4 is the flow cytomery result of BsAb combination CD3 positive cells (Jurkat)；Fig. 5 combines for BsAb The flow cytomery result of EGFRvIII antigen-positive cells (SMMC-7721).It is left in Fig. 4 and Fig. 5 each streaming figure Side curve is control group；The right curve is BsAb experimental groups.Experimental result Fig. 4 shows that only BsAb-1 and CD3 antigens have stronger Binding function, BsAb-2 and BsAb-3 combinations are very weak.Experimental result Fig. 5 shows that only BsAb-1 has weaker with tumour cell Specific binding, BsAb-2 and BsAb-3 are without binding ability.

Embodiment 5

The method of the antibody combined DCIK cell killings SMMC-7721 cells of EGFRvIII × CD3 BsAb-1

Target cell (T) is SMMC-7721 cells in this example；Effector cell (E) is (the BMDC regulation of DCIK cells Cytokine induced kill cell, dendritic cell activated and cytokine induced killer Cell) kill knurl experiment and use commercial kit CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega companies, cat.G1780)

Step reference reagent box specification, it is specific as follows：

A) trypsin digestion collects cultured target cell (SMMC-7721), after counting, is resuspended in the DMEM containing 5%FBS In culture medium, according to 2 × 10⁴It is grouped, is inoculated into 96 orifice plates in/hole and Fig. 6.

B) centrifugal process is collected effector cell DCIK (E) and counted, and is resuspended in the DMEM culture mediums containing 5%FBS, presses respectively According to E:T=4:1 and E:T=8:1 ratio, added according to packet in middle Fig. 6 in corresponding hole.

C) each BsAb (refined solution is diluted to 1ug/ml, i.e. 1ng/ul) is added according to Fig. 6, addition is 10ng/ holes, 10 μ l, one group is set to compare (DCIK groups) without BsAb, every group of 4 multiple holes；

D) 37 DEG C of cell culture incubators be incubated 3 hours 15 points；

E) maximum release lactic dehydrogenase (LDH) hole of target cell adds 10 μ l lysates, mixes；

F) 37 DEG C of cell culture incubators continue after being incubated 45 minutes, and 96 hole 250g are centrifuged 4 minutes, shift each hole supernatants of 10 μ l Into 96 new orifice plates, then each hole adds 50 μ l LDH substrate solutions, is incubated at room temperature 30 minutes；

G) each hole adds 50 μ l terminate liquid, and absorbance is determined at 490nm.

H) according to absorbance, calculated according to formula below and kill knurl efficiency：

Kill knurl efficiency (%)=[(E_BsAb-E_spon-T_spon)/(T_max-T_spon)]×100

Wherein, E_BsAbRepresent each experimental group release lactic dehydrogenase (LDH)；E_sponRepresent the spontaneous release LDH of effector cell； T_sponRepresent target cell spontaneous release LDH, T_maxRepresent the maximum release LDH of target cell.(according to practical illustration book, the porose number of institute Culture medium ground control is subtracted according to before processing, experimental group subtracts volume correction value).

The result of joint DCIK cell killing SMMC-7721 cell experiments is as shown in Figure 7.

In the figure 7, the longitudinal axis represents to kill the relative value of ratio of outflow；Abscissa represents different groups and effect target ratio (E:T).

As shown in Figure 7, compared with independent DCIK groups (no BsAb groups), EGFRvIII × CD3 BsAb is added and kill ratio of outflow Improve, it is best that wherein EGFRvIII × CD3 BsAb-1 kill knurl effect, in effect target ratio (E:T it is) 4:1 and 8:It is reachable respectively when 1 43.26%th, 68.40%, illustrate that EGFRvIII × CD3 BsAb specific can mediate DCIK (primary T cell) to kill target cell Wound.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention All any modification, equivalent and improvement made within refreshing and principle etc., should be included in the scope of the protection.

Claims (10)

1. the double targeting antibodies of a species specificity, it is characterised in that the double targeting antibodies of specificity have specificity and mankind CD3 With reference to the first antigen-binding domains and specificity and the second antigen-binding domains of EGFRvIII antigen bindings.

2. the double targeting antibodies of specificity as claimed in claim 1, it is characterised in that the double targeting antibodies of specificity are included extremely The LCVR amino of the HCVR amino acid sequences of few first antigen-binding domains, at least one the first antigen-binding domains Acid sequence, the HCVR amino acid sequences of at least one the second antigen-binding domains and at least one the second antigen binding knots The LCVR amino acid sequences in structure domain；Wherein, the HCVR and LCVR are respectively HCVR and LCVR amino acid sequences optional in table 1 Row.

3. the double targeting antibodies of a species specificity are applied by following one or more methods：

(1) the double targeting antibodies of the specificity are individually applied；

(2) by the double targeting antibodies of the specificity and the one or more in chemotherapy, radiotherapy, operation, biological therapy, immunization therapy Use in conjunction；

(3) the double targeting antibodies of the specificity are directly delivered into patient's body by the way of delivering in vivo to be treated；

(4) first pass through in-vitro transfection technology to mix the double targeting antibodies of the specificity with immune effector cell, then by described in The defeated time patient's body of immune effector cell for being mixed with specific double targeting antibodies implements treatment.

4. a kind of pharmaceutical composition, including at least one double targeting antibodies of specificity as claimed in claim 1, and diagnosticum, Pharmaceutically useful supporting agent, excipient or diluent.

A kind of 5. nucleic acid molecules, it is characterised in that the amino acid sequence of the double targeting antibodies of coding specificity as claimed in claim 1 Row.

6. a kind of recombinant vector, it is characterised in that there is any sequence of nucleic acid molecules as claimed in claim 7.

7. recombinant vector as claimed in claim 6, it is characterised in that the recombinant vector is expression vector or minicircle dna.

8. a kind of host cell, it is characterised in that include recombinant vector as claimed in claim 6.

9. a kind of antibody drug, it is characterised in that be the double targeting antibodies of any specificity as claimed in claim 1, with the mankind CD3 is combined, and in the killing carried out to EGFRvIII positive cells of external evoked T cell mediation.

10. specificity pair targeting antibodies as claimed in claim 1 or such as recombinant vector as claimed in claim 6 are examined in preparation Break, prevent, the application in the reagent or medicine for the treatment of EGFRvIII diseases.