CN102134276A

CN102134276A - Chimeric antibody resisting CTLA-4 (Cytotoxic T Lymphocyte Antigen)

Info

Publication number: CN102134276A
Application number: CN2010100231582A
Authority: CN
Inventors: 谈珉; 何进秋; 张大鹏; 钱卫珠; 李彩辉
Original assignee: ANTIBODIES NATIONAL ENGINEERING RESEARCH CENTER; Shanghai CP Guojian Pharmaceutical Co Ltd
Current assignee: ANTIBODIES NATIONAL ENGINEERING RESEARCH CENTER; Shanghai CP Guojian Pharmaceutical Co Ltd; Shanghai National Engineering Research Center of Antibody Medicine Co
Priority date: 2010-01-22
Filing date: 2010-01-22
Publication date: 2011-07-27

Abstract

The invention discloses a chimeric antibody resisting CTLA-4 (Cytotoxic T Lymphocyte Antigen) belonging to the technical field of biology. CDR amino acid sequences of a heavy chain variable region of the chimeric antibody are respectively selected from GYTFTKY, DIYPGGDYTNYNE and FRQLGLRG; and CDR amino acid sequences of a light chain variable region are respectively selected from RASESVGSYGNSFML and RASNLES or NNEDPWT. The chimeric antibody resisting the CTLA-4, disclosed by the invention, has high activity compared with a commercialized antibody BNI3 resisting human CTLA-4.

Description

A kind of anti-CTLA-4 chimeric antibody

Technical field

The invention belongs to biomedicine field, particularly, the invention discloses a kind of chimeric antibody, Preparation Method And The Use.

Background technology

CTLA-4 is made up of 237 amino acid, is I type transmembrane glycoprotein, and the relative molecular weight of monomer whose is about 43KD, mainly exist at the T cell surface with dimeric forms, activated T cells high expression level CTLA-4 combines the B7 molecule with CD28 competitiveness, suppressor T cell activation then.And CTLA-4 antibody can stop combining of CTLA-4 and its native ligand, thereby sealing CTLA-4 is to the conduction of T cell negativity conditioning signal, strengthen the T cell to various antigenic reactivities, the immunotherapy that development CTLA-4 antibody is used for tumour is the focus of present therapy of tumor.Because mouse source antibody may bring out human antimouse antibody immunne response (HAMA) after entering human body, thereby people have developed human mouse chimeric antibody and have reduced the human antimouse antibody immune response.Utilize biotechnology to obtain behind the mouse source CTLA-4 monoclonal anti of high-affinity it is transformed that to prepare anti-CTLA-4 human mouse chimeric antibody be the problem that those skilled in the art put forth effort to solve always.

Summary of the invention

The invention discloses a kind of anti-CTLA-4 chimeric antibody, its variable region of heavy chain cdr amino acid sequence is selected from GYTFTKY, DIYPGGDYTNYNE, FRQLGLRG respectively, and light chain variable cdr amino acid sequence is selected from RASESVGSYGNSFML, RASNLES or NNEDPWT respectively; Particularly, its weight chain variable region amino acid sequence shown in SEQ ID NO:10, the light chain variable region amino acid sequence shown in SEQ ID NO:12, constant region behaviour antibody constant region; Further, the aminoacid sequence of its CH is shown in SEQ ID NO:2, and the aminoacid sequence of light chain chain constant region is shown in SEQ ID NO:4; Further, its heavy chain amino acid sequence is shown in SEQ ID NO:6, and light-chain amino acid sequence is shown in SEQ ID NO:8.

The invention also discloses a kind of nucleic acid molecule, the anti-CTLA-4 chimeric antibody that its coding is above-mentioned, wherein the nucleotide sequence of encoding heavy chain variable region is shown in SEQ ID NO:9, and the nucleotide sequence of encoded light chain variable region is shown in SEQ ID NO:11; Further, the nucleotide sequence of encoding heavy chain is shown in SEQID NO:5, and the nucleotide sequence of coding light chain is shown in SEQ ID NO:7.

The invention also discloses the expression vector that contains above-mentioned nucleic acid molecule, be pcDNA3.1/ZEO (+) or pcDNA3.1 (+).

In addition, the invention also discloses a kind of host cell, it is transformed by above-mentioned expression vector, further, is the CHO-K1 cell.

More specifically, it is antigen that the present inventor gives expression to people CTLA-4 extracellular region protein with e. coli bl21 (DE3) (available from Merck KGaA company), adopts cell-fusion techniques preparation secretion anti-CTLA-4 mono-clonal (IgG1/ κ hypotype) hybridoma cell line 16C11.Cell proliferation experiment shows that 16C11 has the function of impelling the Jurkat T cell proliferation that stimulates through PHA.For overcoming the limitation of mouse source antibody 16C11 in clinical application, from hybridoma, clone its weight chain variable region gene by the method for RT-PCR, link to each other with people's constant region gene respectively, made up anti-CTLA-4 chimeric antibody.Experiment in vitro shows that this inosculating antibody physical efficiency combines with the Jurkat cell well; With corresponding mouse source antibody competition conjugated antigen, and their IC ₅₀) value similar, illustrate that this chimeric antibody has kept avidity and the specificity similar to mouse source antibody.

Description of drawings

3. 2. 1. the CTLA-4 extracellular region protein figure that Figure 115 %SDS-PAGE proteins gel electrophoresis analysis purifying obtains be that 6. 5. 4. albumen Marker be molecular sieving peak (reduction) for anionresin pillar elution peak (reduction) for molecular sieving peak M for anionresin pillar elution peak

Figure 26 C11 heavy chain of antibody and light chain variable region nucleotide sequence and aminoacid sequence, what represent in the parantheses is the CDR district;

The antigen-binding activity experimental result of Fig. 3 mouse source antibody 16C11 and chimeric antibody c16C11;

Fig. 4 competes the inhibition experimental result

Embodiment

Following examples, experimental example only further specify the present invention, should not be construed as limitation of the present invention.

Jurkat cell U.S. ATCC TIB-152

PGEM-T carrier U.S. Promega company

Pet22b (+) American I nvitrogen company

T4DNA ligase enzyme American I nvitrogen company

BL21 (DE3) Merck KGaA company

BNI3 U.S. GeneTex company

Embodiment 1: people's antibody is light, the clone of weight chain constant area gene

Separate healthy human lymphocyte with lymphocyte separation medium (ancient cooking vessel state biotech development company product), extract total RNA with Trizol reagent (Invitrogen company product), according to document (Cloned humanand mouse kappa immunoglobulin constant and J region genes conservehomology in functional segments.Hieter PA, Max EE, Seidman JG, MaizelJV Jr, Leder P.Cell.1980Nov; 22 (1 Pt 1): 197-207.) and document (Thenucleotide sequence of a human immunoglobulin C gammal gene.EllisonJW, Berson BJ, Hood LE.Nucleic Acids Res.1982 Jul10; 10 (13): 4071-4079) Bao Dao sequence designs primer respectively and adopts RT-PCR reaction amplification heavy chain of antibody and constant region of light chain gene.The PCR product reclaims and is cloned in the pGEM-T carrier through the agarose gel electrophoresis purifying, confirms to have obtained correct clone after the sequence verification.SEQ ID NO:1 and SEQ ID NO:2 have shown the nucleotide sequence and the aminoacid sequence of CH (CH) respectively.SEQ ID NO:3 and SEQID NO:4 have shown the nucleotide sequence and the aminoacid sequence of constant region of light chain (CL) respectively.Correct clone's note in this example is made pGEM-T/CH and pGEM-T/CL.

Embodiment 2: the segmental expression of people CTLA-4 extracellular region

Extract 5 * 10 by " Trizol Reagent " test kit (U.S. Gibco BRL company product) specification sheets ⁶Total RNA of Jurkat cell.Design primer CTLA-4 antisense carries out reverse transcription amplification, obtains by CTLA-4 justice and CTLA-4 antisense CTLA-4 antisense extracellular region gene being increased behind the cDNA again.Primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized, and sequence is as follows:

CTLA-4 justice:

5’-CCCCATATGGCAATGCAGTGGCCCAGCCTGCT-3’

The CTLA-4 antisense:

5’-CCCAAGCTTGGTACCTTATCAGTCAGAATCTGGGCACGGTTCTGG-3’

The PCR product reclaims purifying purpose segment and is cloned in the pGEM-T carrier after 1% agarose gel electrophoresis separates, the screening positive clone order-checking is analyzed sequencing result.Then with the correct pGEM-T/CTLA-4 plasmid of NdeI HindIII double digestion order-checking, 1% agarose gel electrophoresis separates back recovery purifying purpose segment and is connected with the T4DNA ligase enzyme with the plasmid pet22b (+) that cuts with enzyme, is built into expression vector pet22b (+) (CTLA-4).And transformed into escherichia coli BL21 (DE3), identify correct clone, induced 4 hours with 37 ℃ of 1mM IPTG, can obtain CTLA-4 extracellular region fragment albumen, mainly the form with inclusion body exists.

Embodiment 3: segmental renaturation of people CTLA-4 extracellular region and purifying

The inclusion body that escherichia coli expression obtains dissolves the sex change inclusion body with 4M urea 20mMTris 100mM beta-mercaptoethanol PH8.5,4 ℃ are stirred 2h, and beta-mercaptoethanol is removed in the process desalination, desalting column Hiprep26/10 Desalting G25 prepacked column, Buffer:2M urea 20mMTris 8mM beta-mercaptoethanol PH 8.5.Demineralised liquid is splashed into renaturation solution with the speed of 1ml/min, and renaturation buffer:50mMTris 5mM Nacl 0.05g/l PEG8000 5% glycerol PH8.5 constantly stirs.Drip off and be placed on 16 ℃ and spend the night.Use Millipore 3KD nitrocellulose filter bag that renaturation solution is carried out ultrafiltration and concentration, concentrated solution carries out purifying with anion-exchange column DEAE, balance liquid: 20mMTris PH 8.5, eluting liquid: 20mMTris 1M Nacl PH 8.5,0-60B%20 times of column volume wash-out of flow velocity 6ml/min collected target protein.Elutriant is further purified with molecular sieve SuperdexG75Hiload26/60 320ml prepacked column.Balance liquid 20mM PB 150mM NaCl PH 6.8, flow velocity 3.6ml/min is in charge of and collects albumen and analyze with 15%SDS-PAGE, obtains purity greater than 90% CTLA-4 extracellular region protein (as shown in Figure 1).

Embodiment 4: preparation-cytogamy hybridoma of anti-people CTLA-4 monoclonal antibody 16C11 prepares monoclonal antibody

With the Jurkat cellular immunization BALB/c mouse (available from available from the Shanghai Experimental Animal Center) that stimulates back high expression level CTLA-4 through PHA, make bone-marrow-derived lymphocyte in its spleen can produce the antibody of anti-people CTLA-4, the splenocyte and the NS-1 (BALB/c mouse myeloma cell) that get immunity back mouse merge, cultivate through the HAT selectivity, through cultivating, people CTLA-4 protein extracellular protein screening behind the renaturation purifying that obtains with embodiment 3 goes out anti-people CTLA-4 positive colony, after cloning, filter out subclone again, to guarantee that antibody is to be produced by single clone cell, collect single clone cell culture supernatant then, behind Protein A column purification, just obtain the monoclonal antibody 16C11 of anti-people CTLA-4.

Embodiment 5: the clone of anti-people CTLA-4 monoclonal antibody 16C11 variable region gene

Extract total RNA of the hybridoma 16C11 of the anti-people CTLA-4 monoclonal antibody of 2 * 106 secretions by " Trizol Reagent " test kit (U.S. Gibco BRL company product) specification sheets.Select antibody (IgG2a, κ) appropriate location of heavy chain and constant region of light chain is designed 3 gene-specific primer GSP1, GSP2, GSP3 respectively, and wherein GSP1 distance variable district gene is used for reverse transcription reaction farthest, GSP2 is used for first run pcr amplification, and GSP3 is used for the nido amplification.Primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized, and sequence is as follows: GSP1-H, 5 '-GTA GAG GTC AGA CTG CAG GAC-3 '; GSP2-H, 5 '-CTC AGGGAA ATA GCC CTT GAC-3 '; GSP3-H, 5 '-AGA TCC AGG GGC CAG TGG ATA GAC-3 ' .GSP1-L, 5 '-TTG CTG TCC TGA TCA GTC CAA CT-3 '; GSP2-L, 5 '-TGT CGTTCA CTG CCA TCA ATC TT-3 '; GSP3-L, 5 '-TTG TTC AAG AAG CAC ACG ACTGA-3 '.

According to 5 ' RACE test kit (U.S. Gibco BRL company product) specification sheets is that primer becomes cDNA with total RNA reverse transcription with GSP1, add poly (C) tail for then 3 ' the end of the first chain cDNA, being that primer carries out pcr amplification with GSP2 and AAP behind the tailing, is that primer carries out the nest-type PRC amplification with AUAP and GSP3 for 100 times with the amplified production dilution again.Warm start, reaction conditions are all adopted in twice PCR reaction: 94 ℃ 5 minutes; 94 ℃ 45 seconds, 60 ℃ 45 seconds, 72 ℃ 1 minute 10 seconds, 30 circulations; 72 ℃ 7 minutes.The nest-type PRC product reclaims purifying purpose segment and is cloned in the pGEM-T easy carrier after 1% agarose gel electrophoresis separates, the screening positive clone order-checking is analyzed sequencing result.Be template with the correct pGEM-T/VH that checks order then, design primer H justice AAG CTT GCC GCCACC ATG GAA TGTAAC TGG ATA C and H antisense GCT AGC TGA GGA GAC GGT GAC TG, adopt Onestep RT-PCR reaction amplification VH chain variable region gene and make its 5 ' end contain restriction enzyme sites HindIII, 3 ' end contain restriction enzyme sites Nhe I, reaction conditions is: 50 ℃ 30 minutes; 95 ℃ 15 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 50 seconds, 30 circulations; 72 ℃ 10 minutes.Reclaim the PCR product and be cloned in the pGEM-T carrier through the agarose gel electrophoresis purifying, screening positive clone sequence verification, result prove that the sequence of this sequence and 5 ' RACE is in full accord.SEQ ID NO:9 and SEQ ID NO:10 have shown the nucleotide sequence and the aminoacid sequence of 16C11 variable region of heavy chain respectively.Correct clone's note in this example is made pGEM-T/VH.

With the correct pGEM-T/VL that checks order is template, design primer L justice AAG CTT GCC GCC ACCATG GAG ACA GAC ACA CTC CT and L antisense TGG TGC AGC CAC AGT CCG TTT TAT TTCCAG adopt Onestep RT-PCR reaction amplification VL gene and make its 5 ' end contain restriction enzyme sites HindIII, 3 ' end contains the complementary sequence of human antibody light chain constant region 5 ' end, and reaction conditions is: 94 ℃ 5 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 1 minute, 30 circulations; 72 ℃ 10 minutes.Reclaim the PCR product and be cloned in the pGEM-T carrier through the agarose gel electrophoresis purifying, screening positive clone sequence verification, result prove that the sequence of this sequence and 5 ' RACE is in full accord.SEQ ID NO:11 and SEQ ID NO:12 have shown the nucleotide sequence and the aminoacid sequence of 16C11 variable region of light chain respectively.Correct clone's note in this example is made pGEM-T/VL.

Embodiment 6: make up chimeric antibody c16C11

Plasmid pGEM-T/VH HindIII and Nhe I double digestion that above-mentioned Onestep RT-PCR order-checking is correct, reclaiming the enzyme section that obtains about 440bp through the agarose gel electrophoresis purifying breaks, be connected with the plasmid pGEM-T/CH that cuts with enzyme, selecting correct clone back cuts with HindIII and EcoR I enzyme, reclaim the purpose fragment through the agarose gel electrophoresis purifying, be connected with T4DNA ligase enzyme (Invitrogen company product) with the plasmid pcDNA3.1 (+) that cuts with enzyme (American I nvitrogen company product), be built into carrier for expression of eukaryon pcDNA3.1 (+) (VHCH).

Adopt the Overlapping PCR clone pGEM-T/VL that above-mentioned Onestep RT-PCR order-checking is correct directly with the correct clone pGEM-T/CL fusion of constant region of light chain, reaction conditions is: 50 ℃ 30 minutes; 95 ℃ 15 minutes; 94 ℃ 50 seconds, 58 ℃ 50 seconds, 72 ℃ 50 seconds, 30 circulations; 72 ℃ 10 minutes, obtain PCR product VLCL, its 5 ' end contains restriction enzyme sites HindIII, 3 ' end contains restriction enzyme sites EcoR I.Reclaim the PCR product and be cloned in the pGEM-T carrier (Promega company product) the screening positive clone order-checking through the agarose gel electrophoresis purifying.The VLCL gene that order-checking is correct downcuts from the pGEM-T carrier with HindIII and the two enzymic digestions of EcoR I, be cloned in pcDNA3.1/ZEO (+) carrier (American I nvitrogen company product), be built into carrier for expression of eukaryon pcDNA3.1/ZEO (+) (VLCL).

In 3.5cm tissue culture ware, inoculate 3 * 105 CHO-K1 cells, cell cultures is carried out transfection when 90%-95% merges: (plasmid pcDNA3.1 (+) is 4 μ g (VHCH) to get plasmid 10 μ g, plasmid pcDNA3.1/ZEO (+) is 6 μ g (VLCL)) and 20 μ l Lipofectamine2000 Reagent (Invitrogen company product) be dissolved in 500 μ l serum-free DMEM substratum respectively, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, therebetween with the blood serum medium that contains in the DMEM substratum replacement culture dish of 3ml serum-free, then the DNA-liposome complex that forms is joined in the plate, the CO2 incubator is cultivated after 4 hours and is added the DMEM perfect medium that 2ml contains 10% serum, places the CO2 incubator to continue to cultivate.Cell changed the selection substratum screening resistance clone that contains 600 μ g/ml G418 and 250 μ g/ml Zeocin after 24h was carried out in transfection.Get cells and supernatant and detect the screening high-expression clone with ELISA: goat anti-human igg (Fc) bag quilt is in elisa plate, 4 ℃ are spent the night, seal 2h with 2%BSA-PBS in 37 ℃, add resistance clone culture supernatant to be measured or standard substance (Human myeloma IgG1, κ), 37 ℃ of incubation 2h, add HRP-goat anti-human igg (κ) and carry out association reaction, 37 ℃ of incubation 1h add TMB in 37 ℃ of effect 5min, use H at last ₂SO ₄Termination reaction is surveyed the A450 value.With the high-expression clone serum free medium enlarged culturing that screening obtains, use ProteinA affinity column (GE company product) separation and purification chimeric antibody c16C11.Antibody purification is dialysed with PBS, at last with the uv-absorbing standard measure, antibody purification is through order-checking, its heavy chain nucleotide sequence and aminoacid sequence are respectively as SEQ ID NO:5, shown in the SEQ ID NO:6, light chain nucleotide sequence and aminoacid sequence are respectively as SEQ ID NO:7, shown in the SEQ ID NO:8.

Experimental example 1: antigen is in conjunction with experiment

With the Jurkat cell resuspended one-tenth 1 * 106cells/ml of 1%FCS-PBS, add different dilution inosculating antibody people CTLA-4 monoclonal antibody c16C11 and mouse source anti-people CTLA-4 monoclonal antibody 16C11 BNI3 (available from GeneTex company) respectively, place 4 ℃ to hatch 60min, wash cell 2 times with 1%FCS-PBS, add FITC-goat anti-mouse igg (H+L) (available from ZYMED company) again and hatch 60min in 4 ℃, wash cell after flow cytometer detect and use the Cellquest software analysis.

Experimental result is seen Fig. 3, and chimeric antibody c16C11 has similar binding curve with mouse source antibody 16C11, all is the concentration gradient dependency, illustrates that the two can both combine with the Jurkat cell-specific well.But the EC of c16C11 ₅₀Value (1.493 ± 0.183) is significantly less than 16C11 (5.465 ± 0.873), illustrates that c16C11 has stronger combination active (p＜0.01) than 16C11.

Experimental example 2: competition suppresses experiment

(preparation method A liquid: 20mg16C11 antibody is dissolved in the 1ml pure water with the fluorescent-labeled antibody FITC-16C11 of fixed sub-saturated concentration, B liquid: 10mg FITC (AMRESCO company) is dissolved in 100ml 25mMNaHCO3,25mM Na2CO3, in the solution of PH 9.6, A liquid and B liquid mix, after the lucifuge dialysis in 24 hours, changed dialyzate (PBS) once in per 8 hours, totally 6 times, unconjugated FITC is removed with the G25 desalting column in the back) and the unmarked antibody purification of serial dilution join target cell Jurkat (in 1 * 106/ml) after mixing respectively, hatch 60min for 4 ℃, 1%FCS-PBS washes cell 2 times, and flow cytometer detects and use the Cellquest software analysis.(preparation method is referring to Bohua Li for anti-people CD3 chimeric antibody c 12F6, HaoWang, Construction and characterization of a humanizedanti-human CD3monoclonal antibody 12F6 with effective immunoregulationfunctions.Immunology.116:4 such as Jianxin Dai are 487-498) as negative control.Each concentration of competition antibody is established 3 multiple pipes, calculation of half inhibitory concentration IC ₅₀Value, maximum fluorescence intensity is illustrated in the average fluorescent strength that obtains when not competing antibody.

Experimental result is seen Fig. 4, and what antibody 16C11 and c 16C11 can block fluorescent-labeled antibody FITC-16C11 and Jurkat cell fully combines their IC ₅₀Be worth approachingly, show that chimeric antibody has and similar specificity and the avidity of former mouse source antibody, and control antibodies can not be blocked fluorescent-labeled antibody FITC-16C11 and combines with the Jurkat cell, competition inhibition analysis experimental result sees Table 1.

Table 1. competition inhibition analysis

^aStudent ' s unpaired t checks demonstration: the IC of chimeric antibody ₅₀There was no significant difference between value and the corresponding mouse source antibody.

SEQUENCE?LISTING

＜110〉Antibodies National Engineering Research Center

Shanghai CP Guojian Pharmaceutical Co.,Ltd

＜120〉a kind of anti-CTLA-4 chimeric antibody

<130>77

<160>12

<170>PatentIn?version?3.2

<210>1

<211>990

<212>DNA

＜213〉nucleotide sequence of human antibody heavy chain's constant region (CH)

<400>1

gctagcacca?agggcccatc?ggtcttcccc?ctggcaccct?cctccaagag?cacctctggg 60

ggcacagcgg?ccctgggctg?cctggtcaag?gactacttcc?ccgaaccggt?gacggtgtcg 120

tggaactcag?gcgccctgac?cagcggcgtg?cacaccttcc?cggctgtcct?acagtcctca 180

ggactctact?ccctcagcag?cgtggtgacc?gtgccctcca?gcagcttggg?cacccagacc 240

tacatctgca?acgtgaatca?caagcccagc?aacaccaagg?tggacaagaa?agttgagccc 300

aaatcttgtg?acaaaactca?cacatgccca?ccgtgcccag?cacctgaact?cctgggggga 360

ccgtcagtct?tcctcttccc?cccaaaaccc?aaggacaccc?tcatgatctc?ccggacccct 420

gaggtcacat?gcgtggtggt?ggacgtgagc?cacgaagacc?ctgaggtcaa?gttcaactgg 480

tacgtggacg?gcgtggaggt?gcataatgcc?aagacaaagc?cgcgggaaga?gcagtacaac 540

agcacgtacc?gtgtggtcag?cgtcctcacc?gtcctgcacc?aggactggct?gaatggcaag 600

gagtacaagt?gcaaggtctc?caacaaagcc?ctcccagccc?ccatcgagaa?aaccatctcc 660

aaagccaaag?ggcagccccg?agaaccacag?gtgtacaccc?tgcccccatc?ccgggatgag 720

ctgaccaaga?accaggtcag?cctgacctgc?ctggtcaaag?gcttctatcc?cagcgacatc 780

gccgtggagt?gggagagcaa?tgggcagccg?gagaacaact?acaagaccac?gcctcccgtg 840

ctggactccg?acggctcctt?cttcctctac?agcaagctca?ccgtggacaa?gagcaggtgg 900

cagcagggga?acgtcttctc?atgctccgtg?atgcatgagg?ctctgcacaa?ccactacacg 960

cagaagagcc?tctccctgtc?tcccggtaaa 990

<210>2

<211>330

<212>PRT

＜213〉aminoacid sequence of human antibody heavy chain's constant region (CH)

<400>2

Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys

1 5 10 15

Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr

20 25 30

Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser

35 40 45

Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser

50 55 60

Leu?Ser?Ser?Val Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr

65 70 75 80

Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys

85 90 95

Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys

100 105 110

Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro

115 120 125

Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys

130 135 140

Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp

145 150 155 160

Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu

165 170 175

Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu

180 185 190

His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn

195 200 205

Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly

210 215 220

Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu

225 230 235 240

Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr

245 250 255

Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn

260 265 270

Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe

275 280 285

Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn

290 295 300

Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr

305 310 315 320

Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

325 330

<210>3

<211>318

<212>DNA

＜213〉nucleotide sequence of human antibody light chain constant region (CL)

<400>3

actgtggctg?caccatctgt?cttcatcttc?ccgccatctg?atgagcagtt?gaaatctgga 60

actgcctctg?ttgtgtgcct?gctgaataac?ttctatccca?gagaggccaa?agtacagtgg 120

aaggtggata?acgccctcca?atcgggtaac?tcccaggaga?gtgtcacaga?gcaggacagc 180

aaggacagca?cctacagcct?cagcagcacc?ctgacgctga?gcaaagcaga?ctacgagaaa 240

cacaaagtct?acgcctgcga?agtcacccat?cagggcctga?gctcgcccgt?cacaaagagc 300

ttcaacaggg?gagagtgt 318

<210>4

<211>106

<212>PRT

＜213〉aminoacid sequence of human antibody light chain constant region (CL)

<400>4

Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln

1 5 10 15

Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr

20 25 30

Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser

35 40 45

Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr

50 55 60

Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys

65 70 75 80

His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro

85 90 95

Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys

100 105

<210>5

<211>1353

<212>DNA

＜213〉embedding and antibody c16C11 heavy chain nucleotide sequence

<400>5

caggtccagc?tgcagcagtc?tggagctgag?atggtaaggc?ctgggacttc?agtgaagatg 60

tcctgcaagg?ctgctggata?caccttcact?aaatactgga?taggttgggt?aaagcagagg 120

cctggacatg?gccttgagtg?gattggagat?atttaccctg?gaggtgatta?tactaactac 180

aatgagaagt?tcaagggcaa?ggccacactg?actgcagaca?catcctccag?cacagcctac 240

atgcagctca?gcagcctgac?ctctgaagac?tctgccctct?actactgtgc?aagattcaga 300

caactcgggc?tacgtgggtg?gtttgcttac?tggggccaag?ggactctggt?cactgtctct 360

gcagctagca?ccaagggccc?atcggtcttc?cccctggcac?cctcctccaa?gagcacctct 420

gggggcacag?cggccctggg?ctgcctggtc?aaggactact?tccccgaacc?ggtgacggtg 480

tcgtggaact?caggcgccct?gaccagcggc?gtgcacacct?tcccggctgt?cctacagtcc 540

tcaggactct?actccctcag?cagcgtggtg?accgtgccct?ccagcagctt?gggcacccag 600

acctacatct?gcaacgtgaa?tcacaagccc?agcaacacca?aggtggacaa?gaaagttgag 660

cccaaatctt?gtgacaaaac?tcacacatgc?ccaccgtgcc?cagcacctga?actcctgggg 720

ggaccgtcag?tcttcctctt?ccccccaaaa?cccaaggaca?ccctcatgat?ctcccggacc 780

cctgaggtca?catgcgtggt?ggtggacgtg?agccacgaag?accctgaggt?caagttcaac 840

tggtacgtgg?acggcgtgga?ggtgcataat?gccaagacaa?agccgcggga?agagcagtac 900

aacagcacgt?accgtgtggt?cagcgtcctc?accgtcctgc?accaggactg?gctgaatggc 960

aaggagtaca?agtgcaaggt?ctccaacaaa?gccctcccag?cccccatcga?gaaaaccatc 1020

tccaaagcca?aagggcagcc?ccgagaacca?caggtgtaca?ccctgccccc?atcccgggat 1080

gagctgacca?agaaccaggt?cagcctgacc?tgcctggtca?aaggcttcta?tcccagcgac 1140

atcgccgtgg?agtgggagag?caatgggcag?ccggagaaca?actacaagac?cacgcctccc 1200

gtgctggact?ccgacggctc?cttcttcctc?tacagcaagc?tcaccgtgga?caagagcagg 1260

tggcagcagg?ggaacgtctt?ctcatgctcc?gtgatgcatg?aggctctgca?caaccactac 1320

acgcagaaga?gcctctccct?gtctcccggt?aaa 1353

<210>6

<211>451

<212>PRT

＜213〉embedding and antibody c16C11 heavy chain amino acid sequence

<400>6

Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Ala?Glu?Met?Val?Arg?Pro?Gly?Thr

1 5 10 15

Ser?Val?Lys?Met?Ser?Cys?Lys?Ala?Ala?Gly?Tyr?Thr?Phe?Thr?Lys?Tyr

20 25 30

Trp?Ile?Gly?Trp?Val?Lys?Gln?Arg?Pro?Gly?His?Gly?Leu?Glu?Trp?Ile

35 40 45

Gly?Asp?Ile?Tyr?Pro?Gly?Gly?Asp?Tyr?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

50 55 60

Lys?Gly?Lys?Ala?Thr?Leu?Thr?Als?Asp?Thr?Ser?Ser?Ser?Thr?Ala?Tyr

65 70 75 80

Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Leu?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Phe?Arg?Gln?Leu?Gly?Leu?Arg?Gly?Trp?Phe?Ala?Tyr?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ala?Ala?Ser?Thr?Lys?Gly?Pro?Ser

115 120 125

Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala

130 135 140

Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val

145 150 155 160

Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala

165 170 175

Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val

180 185 190

Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His

195 200 205

Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys

210 215 220

Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly

225 230 235 240

Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met

245 250 255

Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His

260 265 270

Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val

275 280 285

His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr

290 295 300

Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly

305 310 315 320

Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile

325 330 335

Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val

340 345 350

Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser

355 360 365

Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu

370 375 380

Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro

385 390 395 400

Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val

405 410 415

Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met

420 425 430

His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser

435 440 445

Pro?Gly?Lys

450

<210>7

<211>657

<212>DNA

＜213〉embedding and antibody c16C11 light chain nucleotide sequence

<400>7

ggtaaaattg?tgctgaccca?atctccagct?tctttggctg?tgtctctaag?gcagagggcc 60

gccatatcct?gcagagccag?tgaaagtgtt?ggtagttatg?gcaatagttt?tatgctctgg 120

taccagcaga?aaccaggaca?gccacccaaa?ctcctcatct?atcgtgcatc?caacctagaa 180

tctggggtcc?ctgccaggtt?cagtggcagt?gggtctagga?cagacttcac?cctcaccatt 240

gatcctgtgg?aggctgatga?tgctgcaacc?tattactgtc?agcaaaataa?tgaggatccg 300

tggacgttcg?gtggaggcac?caagctggaa?atcaaacgga?ctgtggctgc?accatctgtc 360

ttcatcttcc?cgccatctga?tgagcagttg?aaatctggaa?ctgcctctgt?tgtgtgcctg 420

ctgaataact?tctatcccag?agaggccaaa?gtacagtgga?aggtggataa?cgccctccaa 480

tcgggtaact?cccaggagag?tgtcacagag?caggacagca?aggacagcac?ctacagcctc 540

agcagcaccc?tgacgctgag?caaagcagac?tacgagaaac?acaaagtcta?cgcctgcgaa 600

gtcacccatc?agggcctgag?ctcgcccgtc?acaaagagct?tcaacagggg?agagtgt 657

<210>8

<211>219

<212>PRT

＜213〉embedding and antibody c16C11 light-chain amino acid sequence

<400>8

Gly?Lys?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Ser?Leu?Ala?Val?Ser?Leu

1 5 10 15

Arg?Gln?Arg?Ala?Ala?Ile?Ser?Cys?Arg?Ala?Ser?Glu?Ser?Val?Gly?Ser

20 25 30

Tyr?Gly?Asn?Ser?Phe?Met?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Pro

35 40 45

Pro?Lys?Leu?Leu?Ile?Tyr?Arg?Ala?Ser?Asn?Leu?Glu?Ser?Gly?Val?Pro

50 55 60

Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Arg?Thr?Asp?Phe?Thr?Leu?Thr?Ile

65 70 75 80

Asp?Pro?Val?Glu?Ala?Asp?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Asn

85 90 95

Asn?Glu?Asp?Pro?Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys

100 105 110

Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu

115 120 125

Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe

130 135 140

Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln

145 150 155 160

Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser

165 170 175

Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu

180 185 190

Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser

195 200 205

Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys

210 215

<210>9

<211>363

<212>DNA

＜213〉embedding and antibody c16C11 weight chain variable region nucleotide sequence

<400>9

caggtccagc?tgcagcagtc?tggagctgag?atggtaaggc?ctgggacttc?agtgaagatg 60

tcctgcaagg?ctgctggata?caccttcact?aaatactgga?taggttgggt?aaagcagagg 120

cctggacatg?gccttgagtg?gattggagat?atttaccctg?gaggtgatta?tactaactac 180

aatgagaagt?tcaagggcaa?ggccacactg?actgcagaca?catcctccag?cacagcctac 240

atgcagctca?gcagcctgac?ctctgaagac?tctgccctct?actactgtgc?aagattcaga 300

caactcgggc?tacgtgggtg?gtttgcttac?tggggccaag?ggactctggt?cactgtctct 360

gca 363

<210>10

<211>121

<212>PRT

＜213〉embedding and antibody c16C11 weight chain variable region amino acid sequence

<400>10

Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Ala?Glu?Met?Val?Arg?Pro?Gly?Thr

1 5 10 15

Ser?Val?Lys?Met?Ser?Cys?Lys?Ala?Ala?Gly?Tyr?Thr?Phe?Thr?Lys?Tyr

20 25 30

Trp?Ile?Gly?Trp?Val?Lys?Gln?Arg?Pro?Gly?His?Gly?Leu?Glu?Trp?Ile

35 40 45

Gly?Asp?Ile?Tyr?Pro?Gly?Gly?Asp?Tyr?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

50 55 60

Lys?Gly?Lys?Ala?Thr?Leu?Thr?Ala?Asp?Thr?Ser?Ser?Ser?Thr?Ala?Tyr

65 70 75 80

Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Leu?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Phe?Arg?Gln?Leu?Gly?Leu?Arg?Gly?Trp?Phe?Ala?Tyr?Trp?Gly

100 105 110

Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ala

115 120

<210>11

<211>324

<212>DNA

＜213〉embedding and antibody c16C11 light chain variable region nucleotide sequence

<400>11

gacatcgtac?ttacccaaag?tcccagtagc?ctcagcgcga?gtgtaggtga?ccgcgtgacc 60

atcacctgcc?gcgcgagtca?aagcatcaga?aacaatctcc?attggttcca?acagaagcca 120

ggtaaggctc?caaaactcct?cataaaatat?gccagccaga?gcatatctgg?cgtgccgtct 180

agattctctg?gctccggctc?cggcacagac?ttcacactaa?cgatatcctc?cctacagcct 240

gaggactttg?ctacgtatta?ttgccaacaa?tcaaatacgt?ggcctctgac?ttttggccag 300

ggcactaagg?tggagattaa?gagg 324

<210>12

<211>113

<212>PRT

＜213〉embedding and antibody c16C11 light chain variable region amino acid sequence

<400>12

Gly?Lys?Ile?Val?Leu?Thr?Gln?Ser?Pro?Ala?Ser?Leu?Ala?Val?Ser?Leu

1 5 10 15

Arg?Gln?Arg?Ala?Ala?Ile?Ser?Cys?Arg?Ala?Ser?Glu?Ser?Val?Gly?Ser

20 25 30

Tyr?Gly?Asn?Ser?Phe?Met?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Pro

35 40 45

Pro?Lys?Leu?Leu?Ile?Tyr?Arg?Ala?Ser?Asn?Leu?Glu?Ser?Gly?Val?Pro

50 55 60

Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Arg?Thr?Asp?Phe?Thr?Leu?Thr?Ile

65 70 75 80

Asp?Pro?Val?Glu?Ala?Asp?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Asn

85 90 95

Asn?Glu?Asp?Pro?Trp?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys

100 105 110

Arg

Claims

1. anti-CTLA-4 chimeric antibody, its variable region of heavy chain cdr amino acid sequence is selected from GYTFTKY, DIYPGGDYTNYNE, FRQLGLRG respectively, and variable region of light chain cdr amino acid sequence is selected from RASESVGSYGNSFML, RASNLES or NNEDPWT respectively.

2. the described anti-CTLA-4 chimeric antibody of claim 1, its weight chain variable region amino acid sequence shown in SEQ IDNO:10, the light chain variable region amino acid sequence shown in SEQ ID NO:12, constant region behaviour antibody constant region.

3. the described anti-CTLA-4 chimeric antibody of claim 2, the aminoacid sequence of CH is shown in SEQ IDNO:2, and the aminoacid sequence of light chain chain constant region is shown in SEQ ID NO:4.

4. the described anti-CTLA-4 chimeric antibody of claim 2, its heavy chain amino acid sequence is shown in SEQ ID NO:6, and light-chain amino acid sequence is shown in SEQ ID NO:8.

5. nucleic acid molecule, the arbitrary described anti-CTLA-4 chimeric antibody of its coding claim 2 ~ 4, wherein the nucleotide sequence of encoding heavy chain variable region is shown in SEQ ID NO:9, and the nucleotide sequence of encoded light chain variable region is shown in SEQ ID NO:11.

6. the described nucleic acid molecule of claim 5, wherein the nucleotide sequence of encoding heavy chain is shown in SEQ ID NO:5, and the nucleotide sequence of coding light chain is shown in SEQ ID NO:7.

7. an expression vector contains claim 5 or 6 described nucleic acid molecules, is pcDNA3.1/ZEO (+) or pcDNA3.1 (+).

8. a host cell is transformed by the expression vector of claim 7.

9. the described host cell of claim 8 is the CHO-K1 cell.