CN1896229A - Production of anti-CD20 intrinsic antibody and its use - Google Patents

Abstract

Production of CD20 chimeric antibody and its use are disclosed. The process is based on preparation of anti-human CD20 single-cloning antibody hybridoma cell 1-28 and its antibody gene. It has cognitive epitope characteristic and mediated killer target cell functions.

Description

Preparation of a kind of anti-CD 20 intrinsic antibody and uses thereof

Technical field

The present invention relates to the monoclonal antibody (called after 1-28) of a kind of novel, selectively targeted CD20, this antibody has identification CD20 molecule, suppresses the growth of CD20 positive cell, by functions such as CDC effect specific killing target cells; Has good potential applicability in clinical practice.

The present invention relates to a kind of method of novel human-derivedization mouse monoclonal antibody: antibody variable region is ScFv; Constant region is the Fc section of IgG, the i.e. CH2domain of antibody and CH3domain; The variable region links to each other with the hinge area of constant region by antibody; Dimerization is the divalence structure by the halfcystine formation disulfide linkage of hinge area under field conditions (factors).The Humanized anti-CD 20 antibody TGS that realizes by present method has kept good identification antigenic activity and CDC function when reducing the mouse source property of monoclonal antibody 1-28 greatly, improved the potential applicability in clinical practice of mouse monoclonal antibody 1-28.

Background technology

(non-Hodgkin ' s lymphoma is the modal malignant tumour of immunity system NHL) to non-Hodgkin lymphoma, and the patient who newly was diagnosed as NHL in 2002 reaches 53,900 examples.Through behind the chemotherapy radiotherapy at initial stage, the illness of most of patients has obtained alleviation, but has only 25% patient to be cured.Therefore be badly in need of medicine and the application scheme that development makes new advances ^[1.2]

When using antibody that NHL patient is carried out immunotherapy, CD20 is the ideal target antigen.The CD20 molecule is made up of 297 amino acid, belongs to non-glycosylated phosphorprotein ^[3]CD20 molecule tetratransmembrane, aminoterminal and carboxyl terminal all are positioned at the cytoplasmic membrane inboard, stride film district and the 4th the 3rd and stride between the film district, by a ring district that is made up of 43 amino-acid residues, constitute its main epitope ^[4]But a large amount of experiments show that the anti-CD20 antibodies identified epitope but is diversified ^[5]

The research and the application development of anti-CD20 antibodies are rapid.1997,2002,2003, (Foodand Drug Administration FDA) ratified to be used at antibody Mabthera, Zevalin and the Bexxar of CD20 the treatment of NHL respectively, all demonstrates good effect in FDA Food and Drug Administration ^[6]Mabthera is a human mouse chimeric antibody, comprises the variable region of mouse source anti-CD-20 monoclonal antibody 2B8 (Ibritumomab) and the constant region of humanized IgG 1 heavy chain and κ chain.At present, Mabthera is mainly used in the treatment of intractable low differentiation or folliculus type B cell NHL clinically, can not bring out body basically and produce the HAMA reaction.Zevalin is by mouse IgG1-κ monoclonal antibody 2B8 and radionuclide ⁹⁰The Y coupling forms, and is used for the treatment of the NHL that Mabthera and other chemotherapeutics are failed to respond to any medical treatment, comprising intractable folliculus type NHL.Bexxar is the mouse resource monoclonal antibody---anti-B1 monoclonal antibody (Tositumomab, IgG2a-λ) and radionuclide ¹³¹The I covalent coupling forms, and being used for the treatment of has resistance to Mabthera, the CD20 that recurs again after the chemotherapy ⁺, folliculus type NHL.These three kinds of antibody of listing can be by CDC, ADCC, induce CD20 ⁺Cell generation apoptosis or the propagation that directly suppresses the Malignant B cell are brought into play its result of treatment.

Except that three kinds of antibody preparations of above-mentioned listing, also have some very potential anti-CD20 antibodies, as AME-133, IMMU-106 etc.AME-133 is based on Mabthera and uses the Humanized anti-CD 20 antibody that the method for molecular evolution constructs, and its immunogenicity is lower, avidity is higher, the ability of mediation ADCC is stronger, and potential applicability in clinical practice is better.IMMU-106 is a kind of reshaping antibody, further reduces its mouse source property, can bring into play its tumor killing effect by ADCC, CDC and mechanism of inducing apoptosis ^[7]Humans such as Teeling commentaries on classics human immunoglobulin gene's mouse has prepared the anti-CD-20 monoclonal antibody of a series of IgG1 κ type, and they are all strong in conjunction with CD20 ⁺Cell, and can raise monocyte cracking Malignant B cell.Wherein, the CDC function of 2F2 and 7D8 is very strong, can cracking Mabthera be had the target cell of resistance, as the low chronic lymphatic oncocyte of expressing of CD20 ^[8]

Summary of the invention

The present invention utilizes monoclonal technigue to obtain the hybridoma cell line 1-28 of the mouse resource monoclonal antibody (IgM/ κ hypotype) of strain secretion anti-humen CD 20.

Monoclonal antibody 1-28 has special combination activity to people B lymphoma cell clone (Daudi, Raji cell), the positive reaction rate is respectively 96.4% and positive rate 97.5%, human T lymphocyte leukemia cell system (Jurkat cell) is not had in conjunction with active, and the positive reaction rate is 0.98%.Mabthera combines experiment with the competition of 1-28 and shows: 1-28 antibody-FITC of 0.15 μ g is 34.8% ± 0.92 to Daudi cell positive reactivity, when adding various dose Mabthera antibody (0.15 μ g, 0.45 μ g, 1.35 μ g, 4.05 μ g, 12.1 μ g), Daudi cell positive reactivity is respectively 35.3% ± 0.65,35.9% ± 0.75,37.2% ± 2.65,38.8% ± 0.95,41.3% ± 0.34, and the result confirms that the epitope of other CD20 molecule of 1-28 antibody is different with Mabthera.

Target suppresses CD20 ⁺The growth result of cell shows: 1-28 antibody 33 μ g/ml are 35.2 ± 0.65 to the Daudi growth inhibition ratio, are 85.3 ± 3.65 to the growth inhibition ratio of Raji cell, and are 3.6 ± 2.75 to the growth inhibition ratio of Jurkat cell; Confirm that effect is specific to 1-28 antibody to the target cell growth-inhibiting; The 1-28 antibody of 50 μ g/ml is respectively 38.6 ± 0.8 and 59.2 ± 1.4 effects relatively to the growth inhibiting of Daudi and Raji cell, and Mabthera is respectively 9.9 ± 0.6 and 36.7 ± 2.8 to the growth inhibiting of Daudi and Raji cell when same dose, and prompting 1-28 antibody is better than Mabthera to Daudi and Raji cell growth inhibition.

The cellulotoxic experiment that the complement of target Raji cell relies on shows, 1-28 and Mabthera all can kill and wound CD20 male Raji cell by the CDC effect, the half casualty-producing concentrations is respectively 220ng/mL and 145ng/mL, and the Jurkat cell of CD20 feminine gender is not had the CDC effect.Prompting 1-28 and Mabthera are special to the lethal effect of Raji cell.

Antibody 1-28 energy specific recognition CD20 molecule, and different with Mabthera identification epi-position; Simultaneously can suppress the growth of CD20 positive cell, and can be by CDC effect specific killing target cell; Has good potential applicability in clinical practice.

Summary of the invention two

Mouse source property monoclonal antibody is used for the treatment of and has certain limitation.Mainly show: (1) as heterologous protein, and (human anti-mouse antibody HAMA), and then influences the result of treatment of monoclonal antibody, also may bring out anaphylaxis can to induce human body to produce the human anti-mouse antibody; (2) the Fc section of mouse monoclonal antibody can not mediate the performance of effector function effectively in human body; (3) the mouse monoclonal antibody is shorter in people intravital half life.Carrying out humanization modified to the mouse monoclonal antibody is to reduce its immunogenicity, increases the important channel of its effector function.

The present invention is directed to the limitation of mouse source property monoclonal antibody, propose to prepare the method for new small molecule antibody: antibody variable region is ScFv; Constant region is the Fc section of IgG, i.e. the CH2 domain of antibody and CH3 domain; The variable region links to each other with the hinge area of constant region by antibody; Halfcystine by hinge area forms disulfide linkage and dimerization under field conditions (factors).The antibody of this structure can reduce the mouse source property of monoclonal antibody greatly; This antibody is expressed by the gene structure of ScFv-Fc, is easy to express (expression of chimeric antibody need be considered the problem of the coupling of weight chain) than chimeric antibody; This structure antibody has than other small molecular antibodies (as single-chain antibody etc.) moderate, easy detection of molecular weight simultaneously, easily expression and purification (can affinity purification), good stability and antibody are advantages such as two valency structures.

Based on above-mentioned thinking, the present invention is spliced into single-chain antibody (scFv) with the variable region gene of 1-28 monoclonal antibody by Linker, and then by the CH of human IgG hinge region (hinge) with the human IgG1 ₂Domain and CH ₃Domain links to each other, and forms monomer structure (scFv-Fc), by the disulfide linkage formation dimeric structure of hinge region, is built into the humanized anti-CD20 antibodies (called after TGS) of novel texture.The TGS antibody capable is specific in conjunction with the CD20 positive cell, and can be by CDC effect specific killing target cell.Antibody TGS greatly reduces the mouse source property of monoclonal antibody 1-28 when keeping biologic activity, improved its potential applicability in clinical practice.This biomaterial is in preservation on June 24 in 2005, classification called after colon bacillus, and Escherichiacoli, deposit number are CGMCC No.1398, the address is common micro-organisms center, No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City.

Novel anti-CD20 antibodies TGS (ScFv-Fc) structural molecule formula

Embodiment

Below specifying with anti-CD-20 monoclonal antibody (1-28) gene is the process of fundamental construction TGS antibody, has verified the activity of this novel antibody simultaneously by biological experiment.Must be pointed out that present method not only is confined to the 1-28 antibody gene that preserve this chamber, other CD20 antibody genes and other hope reduce the antibody gene of mouse source property all can use the novel antibody that a process for preparing this structure.

The Design and Features of TGS is identified

1, material

The hybridoma 1-28 (IgM) of secretion anti-CD20 antibodies is stored in the Beijing Tian-Guang Biotechnology Co., Ltd. Is.

Daudi, Raji, 293T cell are used the RPMI-1640 of 10%FCS and the DMEM of 10%FCS (the Gibco BRL company) training of going down to posterity respectively

AMV reverse transcription test kit and pGEM ^-T Easy is available from promega company.

Be used to make up the carrier for expression of eukaryon pCMV-VH of chimeric antibody and pCMV-VL by Chinese Center for Disease Control virus fine strain of millet professor Mi Fang of institute be so kind as to give, E.coli XL1-Blue is this chamber preservation.Restriction enzyme is a Biolabs company product.

RTaq archaeal dna polymerase, T ₄Dna ligase, dNTP, dna molecular amount standard DL2000 are available from Takara company.

DNA glue reclaims test kit (OMEGA biotechnology company); Trizol and Lipofectamine ^TM2000 (Invitrogen); Hexadecyl trimethyl ammonium bromide (CTAB), OPD (Sigma company).

Goat anti-human igg (H+L) and horseradish peroxidase-labeled mountain sheep anti-mouse igg (CALTAG Laboratories); The goat-anti people Fc of FITC mark and ECL chemical luminous substrate detection kit (Pierce company); Foetal calf serum (Beijing Heng Shengma of unit biotechnology research institute).

People's complement is an AB serum of taking from the normal people;

Primer synthesizes in Beijing AudioCodes biotechnology limited liability company;

Determined dna sequence is rich inferior biotechnology limited liability company in Shanghai;

Mabthera (anti-humen CD 20 chimeric antibody) is available from PLA General Hospital.

2, method

2.1 primer design

Utilize universal primer to angle and get the 1-28 antibody variable gene, design of primers is as follows:

Be used for heavy chain gene and angle the upstream primer of getting: (5 ' → 3 ')

MuHC1：AGG?TCC?AGC?TGC?TCG?AGT?CAG?G

MuHC2：AGG?TCC?AGC?TGC?TCG?AGT?CAG?G

MuHC3：AGG?TCC?AGC?TTC?TCG?AGT?CTG?G

MuHC4：AGG?TCC?AGC?TTC?TCG?AGT?CAG?G

MuHC5：AGG?TCC?AAC?TGC?TCG?AGT?CTG?G

MuHC6：AGG?TCC?AAC?TGC?TCG?AGT?CAG?G

MuHC7：AGG?TCC?AAC?TTC?TCG?AGT?CTG?G

MuHC8：AGG?TCC?AAC?TTC?TCG?AGT?CAG?G

Be used for heavy chain gene and angle the downstream primer CH that gets ₂C is the CH according to the mouse source μ chain that checks in from GenBank ₂The design of aminoterminal nucleotide sequence.Therefore angle the gene of getting to comprise V _HAnd CH ₁And CH ₂Fragment gene.CH ₂C：GGA?CTA?GTC?TGGCAG?CAC?ATG?TG

Be used for light chain gene and angle the upstream primer of getting: (5 ' → 3 ')

MuLC1：CAA?GTT?CCG?AGC?TCG?TTG?TGA?CTC?AGG?AAT?CT

MuLC2：CCA?GTT?CCG?AGC?TCG?TGT?TGA?CGC?AGC?CGC?CC

MuLC3：CCA?GTT?CCG?AGC?TCG?TGC?TCA?CCC?AGT?CTC?CA

MuLC4：CCA?GTT?CCG?AGC?TCC?AGA?TGA?CCC?AGT?CTC?CA

MuLC5：CCA?GAT?GTG?AGC?TCG?TGA?TGA?CCC?AGA?CTC?CA

MuLC6：CCA?GAT?GTG?AGC?TCG?TCA?TGA?CCC?AGT?CTC?CA

MuLC7：CCA?GTT?CCG?AGC?TCG?TGA?TGA?CAC?AGT?CTC?CA

Be used for light chain gene and angle the downstream primer of getting: (5 ' → 3 ').MuCK：GCG?CCG?TCT?AGA?ATT?AAC?ACTCAT?TCC?TGT?TGA?A。This primer is according to C _LAmmonia terminal nucleotide sequence design, therefore angle the gene of getting to comprise the V of light chain _LAnd C _L

Be used for heavy chain variable region gene is cloned into the primer of carrier pCMV-VH:

LJ6H2-VHU(PvuII)：TGG CAGCTGACTCCGAGGTGCAGCTtCAGG

LJ6H2-VHD(BstEII)：AGAGAC GGTGACCAGAG

Be used for the Linker-chain variable region gene is cloned into the primer of carrier p1-28/H:

Linker-VLU(BstEII)：CTCC GATATCGTTCTCACCCAGTCTC，

Linker-VLD(Eco47III)：GTTTTAT CTCGAGCTTGGTC。

The line part is corresponding restriction enzyme site, and direction is 5 ' → 3 '.

2.2 total RNA extracts

According to the total RNA extraction agent of Trizol specification sheets, from the hybridoma of secretion IDEC-C2B8, extract total RNA.

2.3cDNA it is synthetic

With total RNA is template, oligo (dT) ₁₅Be primer, carry out reverse transcription according to AMV ThermoScript II specification sheets, total reaction volume is 20 μ L.

2.4 transferring of antibody variable gene

The cDNA that obtains with reverse transcription is a template, universal primer PCR amplification 1-28 antibody V _H-CH ₁-CH ₂And V _L-C _LGene; Respectively with pGEM ^-T Easy filters out recombinant plasmid after connecting, and finishes the structure of its sequencing vector; Order-checking is identified.

2.5 expression vector establishment

With the sequencing vector is template, with LJ6H2-VHU (PvuII) and LJ6H2-VHD (BstEII) is primer amplification 1-28 heavy chain variable region gene, behind Pvu II and BstE II double digestion, be connected, contain human IgG1's CH in this carrier with the carrier pCMV-VH that cuts through same enzyme ₁Domain, hinge domain, CH ₂Domain and CH ₃The domain gene.

Linked system is 15 μ L: 1 μ L carrier, 1 μ L enzyme cut back to close fragment (ratio that guarantees fragment and carrier is between 1: 3 to 1: 10), T ₄Dna ligase 0.5 μ L connects damping fluid 1.5 μ L, adds water and supplies 15 μ L, and 16 ℃ of connections are spent the night, and gets 7.5 μ L and connects product, transformed into escherichia coli XL1-Blue.

Operation is carried out according to molecular cloning method.The picking resistance is selected positive colony, and carries out enzyme and cut evaluation, selects dual evaluation to be male carrier p1-28/H and delivers to the order-checking of Bo Ya Bioisystech Co., Ltd.

With the sequencing vector is template, is primer amplification Linker-chain variable region gene with Linker-VLU (BstE II) and Linker-VLD (Eco47III); Carrier p1-28/H utilizes BstEII and Eco47III double digestion to remove CH ₁Be connected with the Linke-VL gene fragment of cutting behind the domain gene, obtain the expression vector p-TGS of TGS gene (ScFv-Fc) through same enzyme.

2.6 transfection

To contain the DMEM substratum of 10% FCS, at 37 ℃, 5%CO ₂Cultivate the 293T cell under the condition to logarithmic phase.Adjust cell concn to 2 * 10 with the DMEM substratum that does not contain antibiotic 10%FCS ⁵, in 24 orifice plates, add 500 μ L cell suspensions, continue to cultivate 24h, make before transfection cell reach the sheet that melts of 90%-95%.Change serum-free DMEM before the transfection into.The Lipofectamine of 3 μ g plasmid DNA and 2 μ L ^TM2000 respectively with the Opti-MEM of 70 μ L ^I Reduced Serum Medium mixes gently, hatches 5min.With the DNA of dilution and the Lipofectamine of dilution ^TM2000 mixings are gently hatched 30min.The DNA-Lipofectamine that adds 140 μ L in every hole ^TM2000 complex bodys.Cultivate after 4 hours in 37 ℃, the incubator of 5%CO2, remove rotaring redyeing system, add the DMEM of 10%FCS.Detect the expression of antibody after 72 hours.

2.7 the testing goal gene is transcribed rna level

Collect 293T cell after the transfection, PBS gives a baby a bath on the third day after its birth time, extracts total RNA by the working instructions of Trizol reagent.

OD260/OD280 measures total RNA purity, 1% agarose electrophoresis.With Oligo dT is synthetic the 1st chain cDNA of primer reverse transcription.So that LJ6H2-VHU (PvuII)/LJ6H2-VHD (BstEII), Linker-VLU (BstEII)/Linker-VLD (Eco47III) primer is to pcr amplification, the testing goal gene is transcribed rna level.

2.8ELISA

Adopt the sandwich ELISA method to detect:

Goat anti-human igg (H+L) (5ug/mL, 100uL/ hole) wraps by elisa plate, and 37 ℃, 1 hour; 1.5% casein sealing 1.5h; Add the expression supernatant and the empty carrier transfectional cell supernatant of TGS antibody respectively, (Perce company) makes typical curve with human IgG, hatches 1h for 37 ℃; PBST washes plate 3 times, each 5min; Add the anti-human IgG of HRP labelled goat (H+L) (1: 200), 37 ℃, 1h; PBST washes plate 3 times, each 5min; The OPD colour developing, room temperature lucifuge reaction 5min measures the A492 value with the enzyme linked immunological instrument.Determine expressing quantity with typical curve.

2.9Western?blot

12% SDS-PAGE separates the transfectional cell supernatant, and electricity forwards on the nitrocellulose filter (NC), 100V, 50min.Skim-milk with 5% adds goat anti-human igg (1: 5000) the room temperature oscillatory reaction 1h of HRP mark in room temperature vibration sealing 1 hour, and PBST washes film 3 times, each 10min.The DAB colour developing confirms to express the existence and the size thereof of target protein in the supernatant.

2.10 indirect immunofluorescence

The transfectional cell supernatant respectively with CD20 ⁺Bone-marrow-derived lymphocyte be the Daudi cell, Raji cell and T lymphoma cell line Jurkat cell are hatched jointly, with GAH-IgG-Fc-FITC (1: 1500) antibody test cell surface bonded antibody molecule, contrast of cell autofluorescence and two anti-contrasts are set simultaneously.By the method for observation and flow cytometry under inverted fluorescence microscope, judge chimeric antibody and Daudi cell bonded situation.

2.11 the cellulotoxic experiment that complement relies on

Get the Daudi, the Raji cell that are in logarithmic phase, wash 1 time, cell is resuspended in 10%FCS-RPMI 1640 substratum, adjust cell concn to 3 * 10 with 10%FCS-RPMI 1640 substratum ⁵Individual/ml, be seeded in 96 orifice plates with every hole 100 μ l.Add different concns antibody, hatched 20 minutes for 37 ℃ in 10 μ l/ holes.Add 37 ℃ in complement (1: 20) 10 μ l/ holes and hatch the cytotoxic activity that detects the complement dependence of antibody after 20 minutes with mtt assay.

3, result

3.1 1-28/V _HAnd 1-28/V _LThe clone of gene and sequential analysis

Utilize Trizol reagent to extract the total RNA of hybridoma cell line 1-28, identify through agarose gel electrophoresis 28s, 18s and 5s 3 bands clearly to occur.Behind synthetic the 1st chain cDNA, obtain V through the universal primer PCR amplification _H-CH ₁-CH ₂And V _L-C _LGene.Fig. 1 is the agarose gel electrophoresis analysis of total RNA and PCR product, and swimming lane 1 shows total RNA among the figure, and swimming lane 2 shows the 1-28 light chain gene, and swimming lane 3 shows 1-28 heavy chain gene (VH-CH1-CH2); Respectively with pGEM ^-T Easy filters out recombinant plasmid after connecting, and finishes the structure of its sequencing vector.Obtain angling the gene order information of getting through determined dna sequence, compare with the immunoglobulin gene sequence of having delivered, the gene of acquisition all meets mouse antibodies variable region skeleton construction.The nucleotide sequence of antibody 1-28 variable region gene:

Heavy chain amino acid sequence (Heavy chain):

Signal peptide CDR1

MMVLSLLYLLTALPGILSEVQLQESGPSLVKPSQTLSLTCSVT GDSITSGYWNWIRKFPGNKL

CDR2 CDR3

EYMG YISYSGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCAR DYDYPFAYW

GQGTLVTVS

The heavy chain nucleotide sequence:

ATGATGGTGTTAAGTCTTCTGTACTGTTGACAGCCCTTCCGGGTATCCTGTCAGAGGTG

CAGCTTCAGGAGTCAGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTG

TTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAG

GGAATAAACTTGAGTACATGGGGTACATAAGCTACAGTGGTAGCACTTACTACAATCCAT

CTCTCAAAAGTCGAATCTCCATCACCCGAGACACATCCAAGAACCAGTACTACCTGCAG

TTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGGGACTATGATTAC

CCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCT

Light-chain amino acid sequence (Light chain);

Signal peptide CDR1

MDFQVQIFSFLLISASVILSRGQIVLTQSPAIMSASPGEKVTMTC SASSSISYMHWYQQKPGT

CDR2 CDR3

SPKRWIY DISKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC HORSSYPYTFGGGTKL

EIK

The light chain nucleotide sequence:

ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATACTGTCC

AGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAA

GGTCACCATGACCTGCAGTGCCAGCTCAAGTATAAGTTACATGCACTGGTACCAGCAGA

AGCCAGGCACCTCCCCCAAAAGTGGATTTATGACACATCAAACTGGCTTCTGGAGTC

CCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCATG

GAGGCTGAAGATGCTGCCACTTATTACTGCCATCAGCGGAGTAGTTACCCGTACACGTTC

GGAGGGGGGACCAAGCTGGAAATAAAA

3.2TGS antibody Construction of eukaryotic

Anti-CD20 antibodies heavy chain variable region gene among the pcr amplification pGEM-1-28/H is building up on the pCMV-VH carrier, through PCR, enzyme cut and sequencing correct, finish the structure of p1-28/H.

Based on plasmid p1-28/H, CH1domain gene in the excision carrier substitutes and is cloned into the Linker-VL gene, makes up the carrier for expression of eukaryon pTGS of new small molecule anti-CD20 antibodies TGS (scFv-Fc).Fig. 2 is an antibody TGS expression vector establishment qualification result, and swimming lane 1 shows the BstEII/Eco47III double digestion evaluation of p1-28/H; Swimming lane 2 shows RCR amplification TGS variable region of light chain+Linker; Swimming lane 3 shows that the double digestion (PvuII/XholI) of expression vector pTGS obtains the qualification result of TGS variable region ScFv gene.

The nucleotide sequence of chimeric antibody TGS:

GAGGTGCAGCTGCAGGAGTCAGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCT

CACCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAAT

TCCCAGGGAATAAACTTGAGTACATGGGGTACATAAGCTACAGTGGTAGCACTTACTACA

ATCCATCTCTCAAAAGTCGAATCTCCATCACCCGAGACACATCCAAGAACCAGTTATAC

CTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGGGACTAT

GATTACCCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTACTCTGGCGGAGG

TGGAAGCGGTGGTGGCGGTTCCGGAGGCGGAGGATCAAATATCGTTCTCACCCAGTCTC

CAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCA

AGTATAAGTTACATGCACTGGTACCAGCAGAAGCCAGGCACCTCCCCCAAAAGATGGAT

TTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTG

GGACCTCTTATTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACT

GCCATCAGCGGAGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTCGAGATCAA

Agagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaa

cccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactgg

tacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccg

tcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaa

gccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctgg

tcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgga

ctccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgag

gctctgcacaaccactacacgcagaagagcctctccttaagtccgggaaaataa

The aminoacid sequence of chimeric antibody TGS:

E?V?Q?L?Q?E?S?G?P?S?L?V?K?P?S?Q?T?L?S?L?T?C?S?V?T?G?D?S?I?T?S?G?Y?W?N?W?I?R?K?F?P?G?N?K?L?E?Y?M?G?Y?I?S

Y?S?G?S?T?Y?Y?N?P?S?L?K?S?R?I?S?I?T?R?D?T?S?K?N?Q?Y?Y?L?Q?L?N?S?V?T?T?E?D?T?A?T?Y?Y?C?A?R?D?Y?D?Y?P?F?A?Y

W?G?Q?G?T?L?V?T?V?Y?S?G?G?G?G?S?G?G?G?G?S?G?G?G?G?S?N?I?V?L?T?Q?S?P?A?I?M?S?A?S?P?G?E?K?V?T?M?T?C?S?A

S?S?S?I?S?Y?M?H?W?Y?Q?Q?K?P?G?T?S?P?K?R?W?I?Y?D?T?S?K?L?A?S?G?V?P?A?R?F?S?G?S?G?S?G?T?S?Y?S?L?T?I?S?S?M

E?A?E?D?A?A?T?Y?Y?C?H?Q?R?S?S?Y?P?Y?T?F?G?G?G?T?K?L?E?I?K?E?P?K?S?C?D?K?T?H?T?C?P?P?C?P?A?P?E?L?L?G?G?P?S

V?F?L?F?P?P?K?P?K?D?T?L?M?I?S?R?T?P?E?V?T?C?V?V?V?D?V?S?H?E?D?P?E?V?K?F?N?W?Y?V?D?G?V?E?V?H?N?A?K?T?K

P?R?E?E?Q?Y?N?S?T?Y?R?V?V?S?V?L?T?V?L?H?Q?D?W?L?N?G?K?E?Y?K?C?K?V?S?N?K?A?L?P?A?P?I?E?K?T?I?S?K?A?K?G

Q?P?R?E?P?Q?V?Y?T?L?P?P?S?R?D?E?L?T?K?N?Q?V?S?L?T?C?L?V?K?G?F?Y?P?S?D?I?A?V?E?W?E?S?N?G?Q?P?E?N?N?Y?K?T

T?P?P?V?L?D?S?D?G?S?F?F?L?Y?S?K?L?T?V?D?K?S?R?W?Q?Q?G?N?V?F?S?C?S?V?M?H?E?A?L?H?N?H?Y?T?Q?K?S?L?S?L?S

P?G?K?Stop

3.3RT-PCR detect the TGS antibody expression

Liposome-mediated method transfection 293T cell, the Trizol method is extracted cell total rna, and OD260/OD280 is 2.0, and three bands of 28s, 18s and 5s appear in electrophoretic separation.With Oligo dT is synthetic the 1st chain cDNA of primer reverse transcription, and pcr amplification testing goal gene is in the expression of mRNA level.Fig. 3 is the result that transcribes that RT-PCR identifies chimeric antibody weight chain variable region gene: after swimming lane 1 shows the transfection of TGS antibody expression vector, transcribing of monoclonal antibody 1-28 heavy chain variable region gene arranged in the target cell, the transcribing of chain variable region gene of monoclonal antibody 1-28 arranged in 2 display target cells of swimming lane.

3.4TGS the expression amount of antibody and molecular weight determination

With the plasmid transfection 293T cell of chimeric antibody expression TGS, collected the transfectional cell culture supernatant in 72 hours, adopt sandwich ELISA method to measure chimeric antibody content in the supernatant, make typical curve with human IgG, calculate antibody TGS content in the cell expressing supernatant.The expression amount of TGS is 58 μ g/mL.Goat anti-human igg-the Fab of HRP mark and the TGS in supernatant reaction is negative, with predict the outcome consistent.

The result of SDS-PAGE shows that expressed proteins TGS exists with monomer (50kDa) and two kinds of forms of dimer (100kDa), does not have visible tripolymer and exists.The result of Western blot confirms that further TGS all can be discerned (GAIgG) by the goat anti-human immunoglobulin antibody with the monomer of 50kDa and the dimer of 100kDa, confirms that the TGS that obtains meets fusion rotein chimeric antibody feature.

3.5 the combined function of indirect immunofluorescence analysis antibody TGS

Respectively with expression supernatant and empty carrier transfectional cell supernatant and the CD20 of chimeric antibody TGS ⁺B leukemic lymphoblastoid clone Daudi or Raji cell hatch jointly, detect cell surface bonded chimeric antibody molecule with GAH-IgG-Fc-FITC, the contrast of cell autofluorescence is set simultaneously, two anti-contrasts and negative cells (Jurkat cell) contrast.By the method for observation and flow cytometry under inverted fluorescence microscope, judge chimeric antibody and Daudi cell bonded situation.Indirect immunofluorescence detect TGS to target cell be 97% in conjunction with result: Daudi and Raji cell positive rate, and TGS combines positive rate with the Jurkat cell and is lower than 2%; The result proves that TGS antibody can combine specifically with target cell.

3.6TGS the CDC effect of mediation

Activating the function that the rabbit complement kills and wounds target cell (CDC) in order to confirm that TGS has, is target cell with the Daudi cell, and the negative cell contrast of Jurkat cell is set simultaneously, detects the CDC function of TGS antibody.The result confirms: TGS antibody can kill and wound 28% Daudi cell when 5 μ g/mL, killing-efficiency was 65% when concentration was 9 μ g/mL, and killing-efficiency did not further improve when concentration continued to be elevated to 12.5 μ g/mL and 15.4 μ g/mL.Analyze by Origin5.0, drawing its half casualty-producing concentrations is 7.4 μ g/mL.And the TGS of 5 μ g/mL can not kill and wound the Jurkat cell by activating complement.Confirm that thus TGS antibody has the function of activating complement specific killing target cell.

In conjunction with the cellulotoxic experiment analysis that the combined function of indirect immunofluorescence analysis antibody TGS and complement rely on, change antibody behind the structure when reducing the mouse source property of monoclonal antibody 1-28 greatly, kept good identification antigenic activity and CDC function.Prompting, this novel antibody can be implemented in when keeping biologic activity, reduce the mouse source property of antibody, realize the humanization of antibody, improve the potential applicability in clinical practice of monoclonal antibody.

Reference

1.Fink-Bennett?DM.Thomas?K.90Y-ibritumomab?tiuxetan?in?the?treatment?of?relapsed?orrefractory?B-cell?non-Hodgkin′s?lymphoma.J?Nucl?Med?Technol.2003?Jun；31(2)：61-8；quiz69-70.

2.Kaminski?MS.Estes?J，Zasadny?KR，el?al.Ridioimmunotherapy?with?iodine?131I?tositumomabfor?relapsed?or?refractory?B-cell?non-Hodgkin?lymphoma：updated?results?and?long-term?followup?of?the?University?of?Michigan?experience.Blood.2000，96(15)：1259-1266.

3.Tedder?TF，McIntyre?G，Schlossman?SF.Heterogeneity?in?the?B1(CD20)cell?surface?moleculeexpressed?by?human?B-lymphocytes.Mol?Immunol.1988，25(12)：1321-1330.

4.Deans?JP，Li?H，Polyak?MJ.CD20-mediated?apoptosis：signalling?through?lipid?rafts.Immunology.2002，107(2)：176-182.

5.Polyak?MJ，Deans?JP.Alanine-170?and?proline-172?are?critical?determinants?for?extracelluarCD20?epitopes；heterogeneity?in?the?fine?specificity?of?CD20?monoclonal?antibodies?is?defined?byadditional?requirements?imposed?by?both?amino?acidsequence?and?quaternary?structure.Blood，2002.99(9)：3256-3262.

6. Wang Yu is firm, and .CD20 antigen and therapeutic anti-CD20 antibodies are doubly put forth energy in Shen. Chinese tumor biotherapy magazine .2005.12 (1): 76-79.

7.Stein?R，Qu?Z，Chen?S，et?al.Characterization?of?a?new?humanized?anti-CD20?monoclonalantibody，IMMU-106，and?Its?use?in?combination?with?the?humanized?anti-CD22?antibody，epratuzumab，for?the?therapy?of?non-Hodgkin′s?lymphoma.Clin?Cancer?Res，2004，10(8)：2868-2878.

8.Teeling?JL，French?RR，Cragg?MS，et?al.Characterization?of?new?human?CD20?monoclonalantibodies?with?potent?cytolyticactivity?against?non-Hodgkin?lymphomas.Blood.2004?Sep15；104(6)：1793-1800.

Claims (5)

1, the hybridoma 1-28 cell and the gene thereof of secretion anti-CD-20 monoclonal antibody.

2, sequence of a kind of anti-CD 20 intrinsic antibody and preparation method thereof.

3, claim 1,2 separation antibody or its antigen-binding portion thereof.

4, claim 1,2,3 has antibody or its antigen-binding portion thereof of following feature:

(A) have heavy chain CDR1, CDR2, CDR3 territory, this territory comprises the aminoacid sequence of SEQ ID NO:1, or by in the position 1,4,5,7 or 10 amino acid replace or replace by the amino acid of position 1,3,4,8,12;

(B) have light chain CDR1, CDR2, CDR3 territory, this territory comprises the aminoacid sequence of SEQ ID NO:2, or by in the position 2,3,4,6,7,9,10 amino-acid substitution or by in the position 3,6,10,11,12 amino acid replace.

5, claim 1-4 any one Antibody Preparation to non-Hodgkin lymphoma (non-Hodgkin ' s lymphoma, NHL), Mabthera treatment has after resistance, the chemotherapy CD20 of recurrence again ⁺The NHL clinical treatment of folliculus type.

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