CN108218987B - High-affinity, high specific, more antigen recognizing epitopes the anti-human CTLA4 antibody with higher function - Google Patents

High-affinity, high specific, more antigen recognizing epitopes the anti-human CTLA4 antibody with higher function Download PDF

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CN108218987B
CN108218987B CN201611193370.7A CN201611193370A CN108218987B CN 108218987 B CN108218987 B CN 108218987B CN 201611193370 A CN201611193370 A CN 201611193370A CN 108218987 B CN108218987 B CN 108218987B
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monoclonal antibody
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CN108218987A (en
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殷刘松
张贵斌
蒋忻坡
陈黎
徐飞
覃喜建
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Nanjing Jinsirui Science and Technology Biology Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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
    • C07K2317/90—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

Abstract

The present invention disclose a kind of high-affinity, high specific, more antigen recognizing epitopes the anti-human CTLA4 antibody with higher function.CTLA4 monoclonal antibody of the invention be capable of specificity with CTLA4 ining conjunction with, and the combination of CTLA4 and B7 albumen can be effectively blocked, specifically the immune negative regulator of releasing CTLA4, activation T cell secrete cytokines.Above-mentioned function meets or exceeds the level of unique CTLA4 targeted drug Yervoy currently on the market, and the epitope for having partial antibody to be different from Yervoy, has bigger diversity.

Description

High-affinity, high specific, more antigen recognizing epitopes it is anti-with higher function Human CTLA 4 antibody
Technical field
The invention belongs to tumour immunotherapies and molecular immunology field, and in particular to a kind of high-affinity, high specific, The anti-human CTLA4 antibody with higher function of more antigen recognizing epitopes.
Background technique
The immune system of vertebrate is one multi-level, by a variety of organs, tissue, cell and the functionality of molecular composition System, for protecting body to infect and safeguard inner equilibrium (Janeway et al., Immunology:The from external Immune System in Health and Disease.New York:Garland Science,2005).It includes natural Immune system and acquired immune system.Wherein, acquired immune system is by the T cell and B cell of height profession, Yi Jiduo Kind of effector molecule composition pathogen of the specific recognition including bacterium, fungi, virus etc. and can remove them.It obtains Property immune system is made of humoral immunity (B cell mediation) and cellular immunity (T cell mediation).Wherein, cellular immunity is to pass through T Cell receptor (TCR) identifies the antigen that major histocompatibility complex (MHC) presents on antigen presenting cell (APC) to cause 's.
T cell activation needs two-stage signal.First order signal, also referred to as main stimulus signal are special by TCR identification Property the MHC antigen that presents realize.This signal is antigentic specificity.The second level stimulus signal of T cell is activated, also referred to as It is by by expressing B7 family member B7.1 (also referred to as CD80) on APC and B7.2 (also referred to as costimulatory signal CD86), with expression in the CD28 in T cell in conjunction with when transmit and lead to t cell activation.CD28 is immunoglobulin (Ig) super Family, be made of two homologous polypeptides with extracellular variable region, expression on T cell surface, by with B7 protein binding Transmit the second signal of t cell activation.If without the second signal that CD28 is mediated, and the first signal that only TCR is mediated, T Cell will become incompetent (anergic).
Cytotoxic t lymphocyte-associated antigen 4 (CTLA4) (also known as CD152) is that a kind of cross-film in T cell is anti- Body is expressed in the T cell of activation, is found in 1987 (Brunet et al., Nature 328:267-270,1987). CTLA4 and CD28 are both Ig superfamily member, there is a single extracellular Ig structural domain, and CTLA4 is as CD28, also in relation with expression B7 albumen on the surface APC.But the effect of CTLA4 mainly passes through in conjunction with B7 albumen, so that t cell activation be inhibited to reach control Inner equilibrium carries out negative regulator to immune system.Also, due to the combination of the combination of CTLA4 and B7 albumen ratio CD28 and B7 albumen There is higher affinity, this mechanism can be after t cell activation, induction of T cell apoptosis, so that internal T cell stable state is maintained, Play immune negative regulation function.
CTLA4 monoclonal antibody is capable of the combination of specific inhibition CTLA4 and B7 albumen, to weaken or close CTLA4 Conduction to T cell negative regulator signal enhances immune response of the T cell to various antigens.In vitro and in vivo experiment all shows resistance T cell immune response (Walunas et al., Immunity 1:405-413,1994, and Kearney can be enhanced in disconnected CTLA4 Et al., J.Immunology 155:1032-1036,1995), antitumor immunity of organism power (Leach et can also be increased al.,Science 271:1734-1736,1996).It therefore, can with the negative regulator signal that monoclonal antibodies block CTLA4 conducts To provide the new treatment for the human diseases for benefiting from immunostimulation, such as the immunization therapy of cancer and communicable disease.At present CTLA4 monoclonal antibody is in clinical experimental stage for treating a variety of human cancers, including melanoma, prostate cancer, wing Guang cancer, colorectal cancer, malignant mesothelioma, gastrointestinal cancer, liver cancer, non-small cell lung cancer (Grosso et al., Cancer Immunology 13:5,2013).A kind of CTLA4 monoclonal antibody, Ipilimumab (trade name are had been successfully listed at present Yervoy) indicate that tumour immunotherapy is practical in clinical stage.Exempt from moreover, being verified with preclinical laboratory for difference Suppression is immunized from different in ability of the monoclonal antibody of epidemic disease regulatory factor in terms of synergistic treatment cancer, CTLA4 monoclonal antibody The monoclonal antibody or small molecule compound of molecule processed form combination treatment and are carrying out the clinical trial for various cancers.But The only a kind of CTLA4 monoclonal antibody listed at present, and CTLA4 monoclonal antibody is there is also different degrees of side reaction, Including inducing immunogenic, extra-inhibitory CTLA4 signal autoimmunity disease, and difference can be may cause in certain patients CTLA4 monoclonal antibody has different degrees of exploitability.Therefore, it is still necessary to develop new to block CTLA4 and B7 albumen In conjunction with functional antibodies, have higher affinity, specificity, functionality and diversity.
Summary of the invention
The object of the present invention is to provide a kind of human CTLA 4 monoclonal antibody and its applications.
Another object of the present invention is to provide the encoding genes of above-mentioned human CTLA 4 monoclonal antibody.
Another object of the present invention is to provide the preparation method of above-mentioned human CTLA 4 monoclonal antibody.
Another object of the present invention is to provide a kind of anti-tumor agent.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of human CTLA 4 monoclonal antibody, the protein sequence of the monoclonal antibody contain heavy chain variable region and light chain variable Area, the monoclonal antibody are selected from any one in following (1)~(5):
(1) heavy chain variable region described in has the amino acid sequence as shown in SEQ ID NO:1, the light chain variable region With the amino acid sequence as shown in SEQ ID NO:3；
(2) heavy chain variable region described in has the amino acid sequence as shown in SEQ ID NO:5, the light chain variable region With the amino acid sequence as shown in SEQ ID NO:7；
(3) heavy chain variable region described in has the amino acid sequence as shown in SEQ ID NO:9, the light chain variable region With the amino acid sequence as shown in SEQ ID NO:11；
(4) heavy chain variable region described in has the amino acid sequence as shown in SEQ ID NO:13, the light chain variable Area has the amino acid sequence as shown in SEQ ID NO:15；
(5) heavy chain variable region described in has the amino acid sequence as shown in SEQ ID NO:17, the light chain variable Area has the amino acid sequence as shown in SEQ ID NO:19.
The encoding gene of said monoclonal antibody.
Above-mentioned encoding gene is selected from any one in following (6)~(10):
(6) nucleotide sequence containing monoclonal antibody heavy variable region as described in the coding shown in SEQ ID NO:2, with And the nucleotide sequence of monoclonal antibody light chain variable region as described in the coding shown in SEQ IDNO:4；
(7) nucleotide sequence containing monoclonal antibody heavy variable region as described in the coding shown in SEQ ID NO:6, with And the nucleotide sequence of monoclonal antibody light chain variable region as described in the coding shown in SEQ IDNO:8；
(8) nucleotide sequence containing monoclonal antibody heavy variable region as described in the coding shown in SEQ ID NO:10, And the nucleotide sequence of monoclonal antibody light chain variable region as described in the coding shown in SEQ IDNO:12；
(9) nucleotide sequence containing monoclonal antibody heavy variable region as described in the coding shown in SEQ ID NO:14, And the nucleotide sequence of monoclonal antibody light chain variable region as described in the coding shown in SEQ IDNO:16；
(10) nucleotide sequence containing monoclonal antibody heavy variable region as described in the coding shown in SEQ ID NO:18, And the nucleotide sequence of monoclonal antibody light chain variable region as described in the coding shown in SEQ IDNO:20；
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned encoding gene.
Above-mentioned recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium is preparing human CTLA 4 monoclonal antibody In application.
A method of human CTLA 4 monoclonal antibody is prepared, will be transfected comprising the recombinant expression carrier of above-mentioned encoding gene Competent cell, and cultivated to obtain human CTLA 4 monoclonal antibody.
Invention technician has been prepared by the following clone 26A12E8,24H2C4B4,42B11G12D3, Unique V- Region Nucleotide/protein sequence of 41B6F9C8,42F8A6:
1) mouse is immunized with the human CTLA 4 extracellular region of recombinant expression, obtains the immune response for being directed to human CTLA 4；
2) it takes the spleen cell of mouse in step 1) to be merged, and the hybridoma of acquisition is screened, obtain Specific recognition human CTLA 4, and the protein bound positive female clone of CTLA4 and B7 can be blocked；
3) the female clone of the positive obtained in step 2) is subcloned, obtains stable hybridoma cell strain；
4) it is sequenced with the hybridoma cell strain obtained in step 3), obtains the variable region coding of antibody light chain and heavy chain Sequence.
It is anti-that the functional human CTLA 4 monoclonal of recombinant antibodies production is carried out with the variable region encoding sequences obtained in step 4) Body.
Monoclonal antibody of the present invention can specifically bind human CTLA 4, can block CTLA4 and B7 albumen knot It closes, and the immune negative regulator of CTLA4 can be released, activate T cell secrete cytokines.
Above-mentioned human CTLA 4 monoclonal antibody application in preparation of anti-tumor drugs.
A kind of anti-tumor agent, it includes above-mentioned human CTLA 4 monoclonal antibodies.
Beneficial effects of the present invention
CTLA4 monoclonal antibody of the invention be capable of specificity in conjunction with CTLA4, and can be effectively blocked The combination of CTLA4 and B7 albumen specifically releases the immune negative regulator of CTLA4, activates T cell secrete cytokines.Above-mentioned function The level of unique CTLA4 targeted drug Yervoy (Ipilimumab) currently on the market can be met or exceeded, and has portion The epitope for dividing antibody to be different from Yervoy has bigger diversity.
Detailed description of the invention
Fig. 1: the mouse serum titer detection after immune
Fig. 2: monoclonal antibody purification can specifically bind human CTLA 4 recombinant protein
Fig. 3: monoclonal antibody purification can specifically bind the cell line of expression human CTLA 4
Fig. 4: monoclonal antibody purification can block the combination of CTLA4 and B7 albumen
Fig. 5: monoclonal antibody purification can release the immune negative regulator of CTLA4, and stimulation T cell secretes interleukin-22
Fig. 6: the affinity determination of monoclonal antibody purification
Specific embodiment
The present invention relates to one kind to have functional human CTLA 4 antibody, below in conjunction with embodiment to implementation of the invention Scheme is described in detail.Unless otherwise indicated, the present invention used in technical and scientific term with it is of the art general The meaning that logical technician is generally understood is identical.Unless otherwise indicated, the method for embodiment as described below and material be can With the conventional products obtained by market purchase.Fields technician of the present invention will be understood that, method described below and material Material, is merely exemplary, and should not be taken as limiting the scope of the invention.
Embodiment 1: the acquisition of human CTLA 4 hybridoma cell strain
1) animal immune
Antigen uses the recombinant protein c TLA4-Fc for being fused to Fc sections of human IgG1 of human CTLA 4 extracellular domain (GenScript, Z03373).With the 200 μ l Freund's complete adjuvant (Sigma- for containing 50 μ g CTLA4-Fc fusion proteins Aldrich 1:1 lotion subcutaneous inoculation female Balb/c and C57bl/6 mouse).Then, every two weeks abdominal cavities/subcutaneous alternate injection The 1:1 lotion of incomplete Freund's adjuvant (Sigma-Aldrich) containing 25 μ g CTLA4-Fc is for up to 3 times, thus to mouse into Row booster immunization.Myeloma merges first 4 days, and a mouse for showing highest antibody titer (Fig. 1) receives 25ug CTLA4- The abdominal cavity booster immunization of Fc (being free of adjuvant).
2) hybridoma fusion and screening
It extracts spleen and homogenizes to generate single cell suspension, while it is unicellular to prepare myeloma cell (SP2/0) Suspension.Using electro' asion by 8.9 × 107A splenocyte and 4.1 × 107A SP2/0 murine myeloma cell is merged.It will melt The cell of conjunction is resuspended in the 100ml thymus nucleoside pyrimidine of selective agent containing hybridoma, the DMEM/ of hypoxanthine and aminopterin-induced syndrome In 10%FBS culture medium, and moved in 50 × 96 orifice plates with pipette with the volume of 100 μ l.By plate in 6%CO at 37 DEG C2 Middle incubation.After incubation in 7 days, ELISA described below is begun to use to combine, ELISA competition and FACS are in conjunction with carrying out testing needle To the antibody of CTLA4-Fc there are situations.
ELISA combination detection method: indirect ELISA is for assessing in supernatant antibody for the combination energy of CTLA4-Fc Power.Elisa plate (Nunc) is coated at 4 DEG C with the recombinant C TLA4-Fc of 0.5 μ g/ml or human IgG1 in the PBS in 100 holes μ l/ Overnight.Plate is washed with PBS-T (0.05% tween), and it is closed 0.5 at 37 DEG C with the PBST containing 1%BSA in 200 holes μ l/ Hour.Confining liquid then is discarded, 100 μ l Hybridoma Cell Culture supernatants are added to each plate, it is small to be then incubated at room temperature 1 When.Plate is washed three times with PBST, and (Fab- is special with the goat anti-mouse IgG of the conjugation horseradish peroxidase in 100 holes μ l/ It is anisotropic) 37 DEG C of (GenScript) incubation 0.5 hour.Plate is washed five times with PBST, TMB developing solution is then added (GenScript) and at room temperature it is incubated for 15 minutes in the dark.1MHCl terminate liquid (Sigma) by the way that 50 μ l are added terminates Reaction.Use microplate reader read plate at 450 nm.
ELISA race detection method: the antibody that competitive ELISA be used to assess supernatant kind matches CTLA4 with it The blocking ability of the combination of body B7 albumen.By 7 egg of recombinant human B of 0.5 μ g/ml in the PBS in 100 holes μ l/ of elisa plate (Nunc) It is white to be coated with overnight at 4 DEG C.Plate is washed with PBS-T (0.05% tween), and by it with the PBST containing 1%BSA in 200 holes μ l/ It is closed 0.5 hour at 37 DEG C.Confining liquid is then discarded, 50 μ l supernatants to be measured are added in each instrument connection, and 50 μ l are added in control wells Unrelated supernatant.Then the CTLA4-Fc (concentration is 0.15 μ g/ml) of 50 μ l biotin labelings is added in every hole, is incubated for 1 at 37 DEG C Hour.Plate is washed three times with PBST, and with the streptavidin HRP (SA-HRP, GenScript) in 100 holes μ l/ 37 DEG C are incubated for 10 minutes.Plate is finally washed five times with PBST, is then added TMB developing solution (GenScript) and at room temperature It is incubated for 15 minutes in the dark.1MHCl terminate liquid (Sigma) by the way that 50 μ l are added terminates reaction.Using microplate reader in 450nm Lower read plate.
FACS detection method: FACS Binding experiment be used to assess the antibody in supernatant for Chinese hamster ovary celI film surface table The binding ability of the CTLA4 reached.The Chinese hamster ovary celI of the expression CTLA4 for detection and the mother cell of negative control are collected, PBS is used Cleaning 3 times.2.5X10 is added in 96 orifice plates5A detection cell and supernatant 100ul to be measured, 4 DEG C are incubated for 1 hour.Then use PBS is cleaned cell 3 times, and the goat anti-mouse IgG of addition 100 μ liFluor label, 4 DEG C are incubated for 45 minutes.Finally cleaned with PBS Cell 3 times, signal is read with FACS BD Calibur.
3) Hybridoma Subclones
It is subcloned using limiting dilution assay.Using haemocytometer and in the thymus gland of selective agent containing hybridoma Cell is serially diluted in the DMEM/10%FBS culture medium of nucleoside pyrimidine, hypoxanthine and aminopterin-induced syndrome to determine cell Quantity, until cell density reaches 5-15 cell/ml.For each hybridoma, the cell solution of 200 μ l is moved with pipette Into 96 holes, density is 1-3 cells/well.After culture is cultivated 1 week at 37 DEG C in 5%CO2, supernatant is carried out Above-mentioned ELISA is combined, ELISA competition and FACS binding test, come assess for CTLA4-Fc antibody there are situations.
Embodiment 2: the variable region sequencing of monoclonal antibody and antibody recombinant production
Using Rapid ELISA mouse antibodies subtype identification kit (Clonotyping System-HRP, SouthernBiotech it after) carrying out subtype identification to the antibody in Hybridoma Cell Culture supernatant, uses TRIzol (Ambion) From 3 × 106-5×106A hybridoma extracts total serum IgE, and utilizes antibody subtype specific primer and universal primer (PrimeScriptTM1stStrand cDNA Synthesis Kit, Takara) by its reverse transcription be cDNA.Then pass through RACE PCR (GenScript) expands rat immune globulin heavy chain and light chain V- region segments, and by sub- gram of resulting PCR fragment It is grand that Insert Fragment is sequenced into pMD18-T carrier system (Takara), and using vector-specific primers.It is final to obtain Clone 26A12E8,24H2C4B4,42B11G12D3, unique V- Region Nucleotide/protein sequence of 41B6F9C8,42F8A6.
26A12E8 heavy chain variable amino acid sequence: SEQ ID NO:1
26A12E8 heavy chain variable region DNA sequence dna: SEQ ID NO:2
26A12E8 chain variable region amino acid sequence: SEQ ID NO:3
26A12E8 light chain variable region DNA sequence dna: SEQ ID NO:4
24H2C4B4 heavy chain variable amino acid sequence: SEQ ID NO:5
24H2C4B4 heavy chain variable region DNA sequence dna: SEQ ID NO:6
24H2C4B4 chain variable region amino acid sequence: SEQ ID NO:7
24H2C4B4 light chain variable region DNA sequence dna: SEQ ID NO:8
42B11G12D3 heavy chain variable amino acid sequence: SEQ ID NO:9
42B11G12D3 heavy chain variable region DNA sequence dna: SEQ ID NO:10
42B11G12D3 chain variable region amino acid sequence: SEQ ID NO:11
42B11G12D3 light chain variable region DNA sequence dna: SEQ ID NO:12
41B6F9C8 heavy chain variable amino acid sequence: SEQ ID NO:13
41B6F9C8 heavy chain variable region DNA sequence dna: SEQ ID NO:14
41B6F9C8 chain variable region amino acid sequence: SEQ ID NO:15
41B6F9C8 light chain variable region DNA sequence dna: SEQ ID NO:16
42F8A6 heavy chain variable amino acid sequence: SEQ ID NO:17
42F8A6 heavy chain variable region DNA sequence dna: SEQ ID NO:18
42F8A6 chain variable region amino acid sequence: SEQ ID NO:19
42F8A6 light chain variable region DNA sequence dna: SEQ ID NO:20
It is respectively synthesized comprising light chain variable region+constant region and heavy chain variable region+constant region DNA fragmentation, it is inserted respectively Enter in pTT5 expression vector, forms expression plasmid.
By above-mentioned plasmid co-transfection HEK293-6E cell, and after cultivating 10 days in 37 DEG C of shaking flasks, supernatant is collected for resisting Body purifying.Before purifying, by pipeline and albumin A column 0.2M NaOH pyrogen removal.Column is used and contains 0.05M Tris and 1.5M The buffer of NaCl (pH8.0) rebalances.Then by the cell culture supernatant of harvest, using 2 × above-mentioned buffer 1:1 Dilute simultaneously filtration sterilization.The supernatant of filtering and albumin A column are incubated at room temperature 2 hours, with and 1 × above-mentioned buffer column scrubber Afterwards, IgG is eluted using sterile 0.1M sodium citrate (pH3.5), has collected eluent and the sterile 1M with 1/9th volumes Tris-HCl (pH9) is neutralized.It aseptically, is PBS (pH7.4) to remove any wash by the product buffer-exchanged Simultaneously the sample is concentrated in de- buffer.After concentration, using 1.43 extinction coefficient Ec (0.1%) by OD280nm to antibody It is quantified.
The antibody of purifying is divided with 10% pre-prepared colloid (GenScript) by SDS-PAGE by BioRad electrophoresis system Analysis.The gel is dyed with Estain2.0 (GenScript) and by comparing colored zone and Protein Ladder (GenScript) estimate molecular size and purity.
Embodiment 3: combination of the monoclonal antibody to human CTLA 4 recombinant protein
Indirect ELISA is for assessing antibody purification for the binding ability of CTLA4-Fc.By elisa plate (Nunc) with 100 μ The recombinant C TLA4-Fc of 0.5 μ g/ml or human IgG1 are coated with overnight at 4 DEG C in the PBS in the hole l/.With PBS-T (0.05% tween) Plate is washed, and it is closed 0.5 hour with the PBST containing 1%BSA in 200 holes μ l/ at 37 DEG C.Then discard confining liquid, Xiang Shou The 100 μ l of antibody purification of 10 μ g/ml is added in hole, and according to 3 times of gradient dilutions, amounts to 11 test concentrations gradients.Then in room Temperature is lower to be incubated for 1 hour.Plate is washed three times with PBST, and the goat anti-mouse of the conjugation horseradish peroxidase with 100 holes μ l/ 37 DEG C of IgG (Fab- specificity) (GenScript) is incubated for 0.5 hour.Plate is washed five times with PBST, TMB colour developing is then added Liquid (GenScript) is simultaneously incubated for 15 minutes in the dark at room temperature.By the way that the 1MHCl terminate liquid (Sigma) of 50 μ l is added eventually Only react.Use microplate reader read plate at 450 nm.26A12E8,24H2C4B4,42B11G12D3,41B6F9C8 are cloned, The binding ability such as Fig. 2 of 42F8A6 for recombinant protein c TLA4-Fc.Compared with Yervoy, 24H2C4B4,41B6F9C8 and 42F8A6 meets or exceeds its antigen binding capacity.
Embodiment 4: combination of the monoclonal antibody to the cell line of expression human CTLA 4
The Chinese hamster ovary celI of the expression CTLA4 for detection and the mother cell of negative control are collected, is cleaned 3 times with PBS.96 2.5X10 is added in orifice plate5A detection cell and 5 μ g/ml antibody purification 100ul, 4 DEG C are incubated for 1 hour.Then cleaned carefully with PBS Born of the same parents 3 times, the goat anti-mouse IgG of addition 100 μ liFluor label, 4 DEG C are incubated for 45 minutes.Finally cleaned cell 3 times with PBS, Signal is read with FACS BD Calibur.Clone 26A12E8,24H2C4B4,42B11G12D3,41B6F9C8,42F8A6 for Express combination such as Fig. 3 of the CHO cell line of CTLA4.Compared with Yervoy, above 5 clones meet or exceed it and combine carefully The binding ability of the antigen of after birth expression.
Embodiment 5: the combination of monoclonal antibodies block CTLA4 and B7 albumen
Elisa plate (Nunc) was coated at 4 DEG C with 7 albumen of recombinant human B of 0.5 μ g/ml in the PBS in 100 holes μ l/ Night.Plate is washed with PBS-T (0.05% tween), and it is small in 37 DEG C of closings 0.5 with the PBST containing 1%BSA in 200 holes μ l/ When.Confining liquid is then discarded, 50 μ g/ml, 50 μ l of antibody purification to be measured is added in first instrument connection, and according to 3 times of gradient dilutions, altogether Count 11 test concentrations gradients.Then the CTLA4-Fc (concentration is 0.15 μ g/ml) of 50 μ l biotin labelings is added in every hole, 37 DEG C be incubated for 1 hour.Plate is washed three times with PBST, and with the streptavidin HRP in 100 holes μ l/ (SA-HRP, GenScript it) is incubated for 10 minutes for 37 DEG C.Plate is washed five times with PBST finally, TMB developing solution (GenScript) then is added And it is incubated for 15 minutes in the dark at room temperature.1MHCl terminate liquid (Sigma) by the way that 50 μ l are added terminates reaction.Use enzyme Mark instrument read plate at 450 nm.26A12E8,24H2C4B4,42B11G12D3 are cloned, 41B6F9C8,42F8A6 are for blocking The combination of CTLA4 and B7 recombinant protein such as Fig. 4.Compared with Yervoy, above 5 clones are for CTLA4 and B7 recombinant protein In conjunction with blocking ability be superior to the former.
Embodiment 6: monoclonal antibody Epitope Identification
Competitive ELISA is used to assess the epitope of antibody purification.It will be in PBS of the elisa plate (Nunc) with 100 holes μ l/ The recombinant C TLA4-Fc of 0.5 μ g/ml is coated with overnight at 4 DEG C.Plate is washed with PBS-T (0.05% tween), and by it with 200 μ The PBST containing 1%BSA in the hole l/ is closed 0.5 hour at 37 DEG C.Then discard confining liquid, every hole is separately added into a pair of (wherein one A marked biotin) it is used for the test antibodies of competitive assay, each 100 μ l of antibody purification (10 μ g/ml).Then at 37 DEG C It is incubated for 1 hour.Plate is washed three times with PBST, and with the streptavidin HRP in 100 holes μ l/ (SA-HRP, GenScript it) is incubated for 10 minutes for 37 DEG C.Plate is washed five times with PBST, be then added TMB developing solution (GenScript) and It is incubated for 15 minutes in the dark at room temperature.1MHCl terminate liquid (Sigma) by the way that 50 μ l are added terminates reaction.Use microplate reader Read plate at 450 nm.26A12E8 is cloned, 42B11G12D3,41B6F9C8 identify same epitope (epitope #1), clone 24H2C4B4 identifies that epitope #2, clone 42F8A6 identify epitope #3.The above epitope is different from the identification epitope of Yervoy, wherein Epitope #2 is Chong Die with Yervoy epitope moiety.Therefore, the antibody that the present invention screens has bigger diversity.
Embodiment 7: the functional detection of monoclonal antibody
Separation CD4+T cell (MiltenylBiotec) from PBMC, the CHO-K1 cell being overexpressed with source of people CD80 ( The stable cell lines of source of people CD80 are overexpressed on CHO-K) it co-cultures, the monoclonal antibody of anti-human CTLA4 is added by concentration gradient In the CHO-K1 cell co-culture system that CD4+T cell and source of people CD80 are overexpressed, humanized IgG 1 is used as negative control, Positive control of the Ipilimumab (Yervoy, Bristol-Myers Squibb Company) as experiment, the PBS of no antibody As ground control, CTLA4-Fc (GenScript, Z03373-50) albumen is added in cultivating system, in 37 DEG C/5%CO2 Incubator be incubated for 24 hours after, take out the supernatant of 100ul, detect the content (Cisbio) of IL-2.26A12E8 is cloned, The activation capability such as Fig. 5 of 24H2C4B4,42B11G12D3,41B6F9C8,42F8A6 for T cell.It is above compared with Yervoy The cell function level of clone meets or exceeds the former.
Embodiment 8: the affinity determination of monoclonal antibody
Chip surface 5min is balanced with the HBS-EP buffer of 10 μ l/min flow velocitys, is then injected with the flow velocity of 10 μ l/min The 1:1 mixed liquor 7mim of " NHS+EDC " activates chip, the capture antibody (Goat that will be diluted in 10mM sodium-acetate buffer Anti-mouse IgG) it is coupled with 10 μ l/min flow velocitys injection about 7min, ethyl alcohol is finally injected with the flow velocity of 10 μ l/min Amine 7min carries out surface-closed.
Three pre- circulations are carried out using HBS-EP buffer as sample makes baseline stability, 10 μ l/min flow velocitys to balance chip 0~the 5min of antibody being diluted in HBS-EP buffer is injected (to believe by adjusting capture time to control antibody and antigen binding Number in~100RU), buffer balances 1min.Low concentration antigen 0.33nMPD-L1-Fc 5min is injected with 30 μ l/min flow velocitys, Antigen is in conjunction with antibody, and the buffer 15min of 30 μ l/min flow velocitys injection later is dissociated, the injection of 100 μ l/min flow velocitys 50mMHCl totally four times, each 10s is regenerated, and one cycle terminates.Change antigen concentration (such as 1nMPD-L1-Fc) to carry out down The circulation measurement of one gradient concentration until all gradient concentrations (0.33nM, 1nM, 3nM, 9nM, 27nMPD-L1-Fc) and repeats Concentration (such as 9nMPD-L1-Fc) measurement terminates.
Experimental data by it is double reduce (control channel and zero-dose) after, in Biacore T200evaluation The fitting of " 1:1Binding " model is carried out in software.26A12E8,24H2C4B4,42B11G12D3 are cloned, The affinity of 41B6F9C8,42F8A6 such as Fig. 6.Compared with Yervoy (KD (M)=2.5E-10), the affinity of above 5 clones Reach at or above the former level.
SEQUENCE LISTING
<110>Nanjing Genscript Biotechnology Co., Ltd.
<120>high-affinity, high specific, more antigen recognizing epitopes the anti-human CTLA4 antibody with higher function
<130> 2016
<160> 20
<170> PatentIn version 3.3
<210> 1
<211> 134
<212> PRT
<213>artificial sequence
<221>26A12E8 heavy chain variable amino acid sequence
<400> 1
Met Asp Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Val Asn Glu Gly
1 5 10 15
Val Tyr Cys Gln Val His Leu Gln Gln Ser Gly Asp Val Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Asn Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Ser Tyr Trp Ile Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu
50 55 60
Glu Trp Ile Gly Arg Ile Ala Pro Gly Ser Gly Thr Thr Tyr Tyr Asn
65 70 75 80
Glu Met Phe Thr Gly Lys Ala Thr Leu Thr Val Val Thr Ser Ser Thr
85 90 95
Thr Ala Tyr Ile His Leu Ser Ser Leu Ser Phe Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Asp Tyr Phe Asp Tyr Trp Asp Gln Gly Thr
115 120 125
Thr Leu Thr Val Ser Ser
130
<210> 2
<211> 402
<212> DNA
<213>artificial sequence
<221>26A12E8 heavy chain variable region DNA sequence dna
<400> 2
atggactgga gttgggtctt tctcttcctc ctgtcagtaa atgaaggtgt ctactgtcag 60
gtccacctgc agcagtctgg agatgttttg gtaaagcctg gggcctcagt gaacttgtcc 120
tgcaaggctt ctggctacac cttcaccagc tactggatta actggataaa acagaggcct 180
ggacagggcc ttgagtggat aggacgtatt gctcctggaa gtggtaccac ttactacaat 240
gaaatgttca cgggcaaggc aacactgact gtggtcacat cctccaccac agcctacatt 300
cacctcagca gcctgtcatt tgaggactct gctgtctatt tctgtgcacg gggggactac 360
tttgactact gggaccaagg caccactctc acagtctcct ca 402
<210> 3
<211> 128
<212> PRT
<213>artificial sequence
<221>26A12E8 chain variable region amino acid sequence
<400> 3
Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Ser Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
20 25 30
Met Ser Ala Ser Pro Gly Gly Lys Val Thr Ile Thr Cys Ser Ala Ser
35 40 45
Lys Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Thr Ser
50 55 60
Pro Lys Leu Leu Ile Tyr Asp Thr Ser Thr Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Pro Ser Tyr Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg
100 105 110
Thr Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Arg
115 120 125
<210> 4
<211> 384
<212> DNA
<213>artificial sequence
<221>26A12E8 light chain variable region DNA sequence dna
<400> 4
atgcattttc aagtgcagat tttcagcttc ctgctaatca gtgcctccgt cataatgtcc 60
agcggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctcc cggggggaag 120
gtcaccatca cctgcagtgc cagcaaaagt gtaagttaca ttcactggtt ccagcagaag 180
ccaggcactt ctcccaaact cttgatttat gacacatcca ccctggcttc tggggtccct 240
gctcgcttca gtggcagtgg atctgggccc tcttactctc tcacaatcag ccgagtggag 300
gctgaagatg ctgccactta ttactgccag caaaggacta cttacccgct cacgttcggt 360
ggtgggacca aactggaggt gaga 384
<210> 5
<211> 142
<212> PRT
<213>artificial sequence
<221>24H2C4B4 heavy chain variable amino acid sequence
<400> 5
Met Asn Phe Gly Phe Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Val Lys Leu Val Glu Ser Gly Gly Gly Ser Val Lys
20 25 30
Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu
35 40 45
Ser Asn Phe Asp Met Ser Trp Ile Arg Gln Thr Pro Glu Lys Arg Leu
50 55 60
Glu Trp Val Ser Ser Phe Thr Pro Asp Gly Asn Thr Tyr Phe Pro Asp
65 70 75 80
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile
85 90 95
Leu Tyr Leu Gln Met Asn Ser Val Arg Ser Glu Asp Thr Ala Met Tyr
100 105 110
Phe Cys Ala Arg Lys Gly Pro Tyr Tyr Tyr Gly Pro Arg Gly Trp Phe
115 120 125
Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
130 135 140
<210> 6
<211> 426
<212> DNA
<213>artificial sequence
<221>24H2C4B4 heavy chain variable region DNA sequence dna
<400> 6
atgaacttcg ggttcagctt gattttcctt gtccttgttt taaaaggtgt ccagtgtgaa 60
gtgaagctgg tggagtcggg gggaggctca gtgaaacctg gagggtccct gaaactctcc 120
tgtgcagcct ctggattcac tctcagtaat tttgacatgt cttggattcg ccagactcca 180
gagaagaggc tggagtgggt ctcatccttt actcctgatg gtaacaccta ctttccagac 240
agtgtgaagg gccgattcac catctccaga gataacgcca ggaacatctt gtacctgcaa 300
atgaacagtg tgaggtctga ggacacggcc atgtatttct gtgcaagaaa ggggccttat 360
tactacggtc ctagaggctg gttcttcgat gtctggggcg cagggaccac ggtcaccgtc 420
tcctca 426
<210> 7
<211> 131
<212> PRT
<213>artificial sequence
<221>24H2C4B4 chain variable region amino acid sequence
<400> 7
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
20 25 30
Val Ser Leu Gly Gln Arg Ala Thr Met Ser Cys Arg Ala Ser Glu Ser
35 40 45
Val Glu Gly Tyr Gly Thr Ser Phe Ile Tyr Trp Tyr Gln Gln Lys Ser
50 55 60
Gly Gln Pro Pro Asn Leu Leu Ile Tyr Leu Thr Ser His Leu Lys Pro
65 70 75 80
Gly Val Pro Ala Arg Phe Thr Gly Ser Gly Ser Arg Thr Asp Phe Thr
85 90 95
Leu Thr Ile Asp Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys
100 105 110
Gln Gln Asn Asn Glu Asp Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
115 120 125
Glu Leu Lys
130
<210> 8
<211> 393
<212> DNA
<213>artificial sequence
<221>24H2C4B4 light chain variable region DNA sequence dna
<400> 8
atggagacag acacactcct gctatgggtg ctgctgctct gggttccagg ttccacaggt 60
aatattgtgt tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 120
atgtcctgca gagccagtga aagtgttgaa ggttatggca ctagttttat atactggtac 180
caacagaaat caggacagcc acccaatctc ctcatctatc ttacatccca cctgaaacct 240
ggggtccctg ccaggttcac tggcagtggg tctaggacag acttcaccct caccattgat 300
cctgtggagg ctgatgatgc tgcaacctat tactgtcagc aaaataatga ggatcctctc 360
acgttcggtg ctgggaccaa gctggaactg aaa 393
<210> 9
<211> 134
<212> PRT
<213>artificial sequence
<221>42B11G12D3 heavy chain variable amino acid sequence
<400> 9
Met Asp Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Val Asn Glu Gly
1 5 10 15
Val Tyr Cys Gln Val His Leu Gln Gln Ser Gly Asp Val Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Asn Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Ser Tyr Trp Ile Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu
50 55 60
Glu Trp Ile Gly Arg Ile Ala Pro Gly Ser Gly Thr Thr Tyr Tyr Asn
65 70 75 80
Glu Met Phe Thr Gly Lys Ala Thr Leu Thr Val Val Thr Ser Ser Thr
85 90 95
Thr Ala Tyr Ile Gln Leu Ser Ser Leu Ser Phe Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Asp Tyr Phe Asp Tyr Trp Asp Gln Gly Thr
115 120 125
Thr Leu Thr Val Ser Ser
130
<210> 10
<211> 402
<212> DNA
<213>artificial sequence
<221>42B11G12D3 heavy chain variable region DNA sequence dna
<400> 10
atggactgga gttgggtctt tctcttcctc ctgtcagtaa atgaaggtgt ctactgtcag 60
gtccacctgc agcagtctgg agatgttttg gtaaagcctg gggcctcagt gaacttgtcc 120
tgcaaggctt ctggctacac cttcaccagc tactggatta actggataaa acagaggcct 180
ggacagggcc ttgagtggat aggacgtatt gctcctggaa gtggtaccac ttactacaat 240
gaaatgttca cgggcaaggc aacactgact gtggtcacat cctccaccac agcctacatt 300
caactcagca gcctgtcatt tgaggactct gctgtctatt tctgtgcacg gggggactac 360
tttgactact gggaccaagg caccactctc acagtctcct ca 402
<210> 11
<211> 128
<212> PRT
<213>artificial sequence
<221>42B11G12D3 chain variable region amino acid sequence
<400> 11
Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Ser Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
20 25 30
Met Ser Ala Ser Pro Gly Gly Lys Val Thr Ile Thr Cys Ser Ala Ser
35 40 45
Lys Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Thr Ser
50 55 60
Pro Lys Leu Leu Ile Tyr Asp Thr Ser Thr Leu Ala Ser Gly Val Pro
65 70 75 80
Pro Arg Phe Ser Gly Ser Gly Ser Gly Pro Ser Tyr Ser Leu Thr Ile
85 90 95
Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg
100 105 110
Thr Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Arg
115 120 125
<210> 12
<211> 384
<212> DNA
<213>artificial sequence
<221>42B11G12D3 light chain variable region DNA sequence dna
<400> 12
atgcattttc aagtgcagat tttcagcttc ctgctaatca gtgcctccgt cataatgtcc 60
agcggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctcc cggggggaag 120
gtcaccatca cctgcagtgc cagcaaaagt gtaagttaca ttcactggtt ccagcagaag 180
ccaggcactt ctcccaaact cttgatttat gacacatcca ccctggcttc tggagtccct 240
cctcgcttca gtggcagtgg atctgggccc tcttactctc tcacaatcag ccgaatggag 300
gctgaagatg ctgccactta ttactgtcag caaaggacta cttacccgct cacgttcggt 360
ggtgggacca aactggaggt gaga 384
<210> 13
<211> 134
<212> PRT
<213>artificial sequence
<221>41B6F9C8 heavy chain variable amino acid sequence
<400> 13
Met Asp Trp Ser Trp Val Phe Leu Phe Leu Leu Ser Val Asn Glu Gly
1 5 10 15
Val Tyr Cys Gln Val His Leu Gln Gln Ser Gly Asp Val Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Asn Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Ser Tyr Trp Ile Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu
50 55 60
Glu Trp Ile Gly Arg Ile Ala Pro Gly Ser Gly Thr Thr Tyr Tyr Asn
65 70 75 80
Glu Met Phe Thr Gly Lys Ala Thr Leu Thr Val Val Thr Ser Ser Thr
85 90 95
Thr Ala Tyr Ile His Leu Asn Ser Leu Ser Phe Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Asp Tyr Phe Asp Tyr Trp Asp Gln Gly Thr
115 120 125
Thr Leu Thr Val Ser Ser
130
<210> 14
<211> 402
<212> DNA
<213>artificial sequence
<221>41B6F9C8 heavy chain variable region DNA sequence dna
<400> 14
atggactgga gttgggtctt tctcttcctc ctgtcagtaa atgaaggtgt ctactgtcag 60
gtccacctgc agcagtctgg agatgttttg gtaaagcctg gggcctcagt gaacttgtcc 120
tgcaaggctt ctggctacac cttcaccagc tactggatta actggataaa acagaggcct 180
ggacagggcc ttgagtggat aggacgtatt gctcctggaa gtggtaccac ttactacaat 240
gaaatgttca cgggcaaggc aacactgact gtggtcacat cctccaccac agcctacatt 300
cacctcaaca gcctgtcatt tgaggactct gctgtctatt tctgtgcacg gggggactac 360
tttgattact gggaccaagg caccactctc acagtctcct ca 402
<210> 15
<211> 128
<212> PRT
<213>artificial sequence
<221>41B6F9C8 chain variable region amino acid sequence
<400> 15
Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Met Met Ser Ser Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
20 25 30
Met Ser Ala Ser Pro Gly Gly Lys Val Thr Ile Thr Cys Ser Ala Ser
35 40 45
Lys Ser Val Ser Tyr Ile His Trp Phe Gln Gln Lys Pro Gly Thr Ser
50 55 60
Pro Lys Leu Leu Ile Tyr Asp Thr Ser Thr Leu Ala Ser Gly Val Pro
65 70 75 80
Ala Arg Phe Ser Gly Ser Gly Ser Gly Pro Ser Tyr Ser Leu Thr Ile
85 90 95
Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg
100 105 110
Thr Thr Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Val Arg
115 120 125
<210> 16
<211> 384
<212> DNA
<213>artificial sequence
<221>41B6F9C8 light chain variable region DNA sequence dna
<400> 16
atgcattttc aagtgcagat tttcagcttc ctgctaatca gtgcctccgt catgatgtcc 60
agcggacaaa ttgttctcac ccagtctcca gcaatcatgt ctgcatctcc cggggggaag 120
gtcaccatca cctgcagtgc cagcaaaagt gtaagttaca ttcactggtt ccagcagaag 180
ccaggcactt ctcccaaact cttgatttat gacacatcca ccctggcttc tggagtccct 240
gctcgcttca gtggcagtgg atctgggccc tcttactctc tcacaatcag ccgagtggag 300
gctgaagatg ctgccactta ttactgccag caaaggacta cttacccgct cacgttcggt 360
ggtgggacca aactggaggt gaga 384
<210> 17
<211> 137
<212> PRT
<213>artificial sequence
<221>42F8A6 heavy chain variable amino acid sequence
<400> 17
Met Gly Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
20 25 30
Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Ser Tyr Asp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
50 55 60
Glu Trp Ile Gly Trp Ile His Pro Arg Asp Gly Ser Thr Glu Tyr Asn
65 70 75 80
Glu Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser
85 90 95
Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Arg Gly Leu Leu Gly Pro Leu Asp Tyr Trp Gly
115 120 125
Gln Gly Thr Thr Leu Thr Val Ser Ser
130 135
<210> 18
<211> 411
<212> DNA
<213>artificial sequence
<221>42F8A6 heavy chain variable region DNA sequence dna
<400> 18
atgggatgga gctggatctt tctcttcctc ctgtcaggaa ctgcaggtgt ccactcccag 60
gttcagctgc agcagtctgg acctgagctg gtgaagcctg gggcttcagt gaagttgtcc 120
tgcaaggctt ctggctacac cttcacaagc tacgatataa actgggtgaa gcagaggcct 180
ggacagggac ttgagtggat tggatggatt catcctagag atggtagtac tgagtacaat 240
gagaagttca agggcaaggc cacatttact gtagacacat cctccagcac agcgtacatg 300
gagctccaca gcctgacatc tgaggactct gcggtctatt tctgtgcaag aaggggtctc 360
ctgggacctc ttgactactg gggccaaggc accactctca cagtctcctc a 411
<210> 19
<211> 130
<212> PRT
<213>artificial sequence
<221>42F8A6 chain variable region amino acid sequence
<400> 19
Met Gly Ile Lys Met Glu Thr His Ser Gln Val Phe Val Tyr Met Leu
1 5 10 15
Leu Trp Leu Ser Gly Val Glu Gly Asp Ile Val Met Thr Gln Ser His
20 25 30
Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys
35 40 45
Ala Ser Gln Asp Val Gly Thr Leu Val Ala Trp Tyr Gln Gln Lys Pro
50 55 60
Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg His Thr
65 70 75 80
Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Thr Ile Ser Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys
100 105 110
Gln Gln Tyr Ser Ser Tyr Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu
115 120 125
Leu Lys
130
<210> 20
<211> 390
<212> DNA
<213>artificial sequence
<221>42F8A6 light chain variable region DNA sequence dna
<400> 20
atgggcatca agatggagac acattctcag gtctttgtat acatgttgct gtggttgtct 60
ggtgttgaag gagacattgt gatgacccag tctcacaaat tcatgtccac atcagtagga 120
gacagggtca gcatcacctg caaggccagt caggatgtgg gtactcttgt agcctggtat 180
caacagaaac cagggcaatc tcctaaatta ttgatttact gggcatccac ccggcacact 240
ggagtccctg atcgcttcac aggcagtgga tctgggacag atttcactct caccattagc 300
aatgtgcagt ctgaagactt ggcagattat ttctgtcagc aatatagcag ctatcccacg 360
ttcggtgctg ggaccaagct ggagctgaaa 390

Claims (8)

1. a kind of human CTLA 4 monoclonal antibody, it is characterised in that: the protein sequence of the monoclonal antibody contain heavy chain variable region and Light chain variable region, the heavy chain variable region have the amino acid sequence as shown in SEQ ID NO:9, the light chain variable region With the amino acid sequence as shown in SEQ ID NO:11.
2. the encoding gene of monoclonal antibody described in claim 1.
3. encoding gene according to claim 2, it is characterised in that the encoding gene contains as shown in SEQ ID NO:10 The coding monoclonal antibody heavy variable region nucleotide sequence, and as described in the coding such as SEQ IDNO:12 shown in singly The nucleotide sequence of monoclonal antibody light variable region.
4. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing encoding gene described in claim 3.
5. recombinant expression carrier as claimed in claim 4, expression cassette, transgenic cell line or recombinant bacterium are preparing human CTLA 4 list Application in clonal antibody.
6. a kind of method for preparing human CTLA 4 monoclonal antibody, it is characterised in that will be comprising encoding base described in Claims 2 or 3 The recombinant expression carrier of cause transfects competent cell, and is cultivated to obtain human CTLA 4 monoclonal antibody.
7. human CTLA 4 monoclonal antibody application in preparation of anti-tumor drugs described in claim 1.
8. a kind of anti-tumor agent, it is characterised in that: it includes human CTLA 4 monoclonal antibodies described in claim 1.
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Denomination of invention: Anti human CTLA4 antibody with high affinity, high specificity and multi antigen recognition epitope

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