CN1618812A - Human Source antibody for anti CD20 and epithelial cell adhesion molecule - Google Patents
Human Source antibody for anti CD20 and epithelial cell adhesion molecule Download PDFInfo
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Abstract
A humanized antibody to CD20 and epithelial cell adhesion molecule (EpCAM), and its preparing process and application are disclosed. Said antibody has high affinity and specificity to CD20 and EpCAM.
Description
Technical field
The invention belongs to field of immunology, be specifically related to a kind of anti-CD20 and anti-epithelial cell adhesion molecule (epithelial cell adhesion molecule, human antibody EpCAM) and method for making thereof and purposes.
Background technology
Antibody is as the existing developing history that goes up a century of the preparation of disease prevention, diagnosis and treatment.The early stage method for preparing antibody be with certain natural antigen through various approach immune animals, after sophisticated B cell clone is subjected to antigenic stimulation, with antibody-secreting in serum and body fluid.
In fact the antibody in the serum is the mixture of multiple monoclonal antibody, therefore is referred to as polyclonal antibody.Polyclonal antibody is that the mankind have purpose to utilize the antibody the first step.The unhomogeneity of polyclonal antibody has limited the further research and the application of antagonist 26S Proteasome Structure and Function.
The birth of hybridoma technology is considered to the qualitative leap first time of antibody engineering development, also is a milestone of modern biotechnology development.Utilize the monoclonal antibody of this technology preparation in medical diagnosis on disease, treatment and scientific research, to be widely used.Mostly this monoclonal antibody is by mouse B cell and rat bone marrow tumour cell to have mouse source property through the hybridoma excretory that cytogamy forms, and enters the people and knows from experience the rejection that causes body; The molecular weight of complete antibody molecule is bigger, and the ability that penetrates blood vessel in vivo is relatively poor; Production cost is too high, is not suitable for large-scale industrial production.At the beginning of the eighties, the achievement in research of antibody gene 26S Proteasome Structure and Function combines with recombinant DNA technology, has produced the genetic engineering antibody technology.
The development of antibody engineering technology is about to the mouse resource monoclonal antibody and transform humanized antibody as, perhaps directly produces human antibody, significantly reduces side effect that mouse source antibody brings such as human antimouse antibody reaction etc., helps clinical application.
At present, three kinds of methods are mainly adopted in the generation of humanization or human antibody:
(1) mouse source antibody is transformed: Fc section and part Fab with human antibodies replace mouse source antibody fragment, reduce the fragment in mouse source, to alleviate the untoward reaction of human antagonist;
(2) transgenic method: human antibody gene is transferred to mouse, produces human antibodies behind the immune animal;
(3) display technique of bacteriophage: the novel method that is developed recently, utilize the characteristic screening antibody of phage can directly screen human antibody, this method utilizes human immunoglobulin gene to make up the combinatorial library that contains VH and VL, with phage surface indicating system screening antigen-specific human antibody.It has replaced the B cell clone with bacterial clone, is described as the revolutionary character progress of antibody technique.
Above-mentioned three kinds of methods cut both ways.The first two kind method can be screened high-affinity antibody, but expense is big, and is not easy to operate, and the third method is easy and simple to handle, cost is few, but avidity is lower sometimes.
Adopt phage antibody library technique screening antibody needn't carry out animal immune, be easy to prepare antibody, screening total man's endogenous antibody and the high-affinity antibody of private antigen.Phage antibody library technique is one of breakthrough of life science, also the research of antibody engineering has been guessed a new climax simultaneously.
The phage system of selection is all unrestricted for the separation of antibody or small peptide, and be applicable to the molecular studies of other biologically active, conjugated protein as cytokine, acceptor, enzyme substrates, enzyme inhibitors, abzyme, DNA, make up receptor domain with specific binding molecules and cellulose binding domain etc.It is reported, the support difference of antibody can be formed for the suitable binding partner of all kinds molecule, and many examples can illustrate, host's support can hold many some suitable metathetical zones of effectively can regulating, (can put the storehouse of preparation localized variation according to this), these supports comprise: beta sheet albumen, alpha-helix bundle protein, these two proteic combinations and green fluorescent protein (GFP) and cellulose binding domain etc.
CD20 is that a kind of cytolemma of non-saccharification is striden the film phosphorylated protein, and molecular weight is 35kD, and distribution density is 50,000～200, a 000 site/cell, its function is not really clear, may have regulating effect to the propagation and the differentiation of striding film conduction, bone-marrow-derived lymphocyte of Ca2+.Studies show that, the CD20 wide expression reaches in pre-B lymphocyte, prematurity and ripe bone-marrow-derived lymphocyte, activation bone-marrow-derived lymphocyte and surpasses among 90% the B cellularity NHL (general B cell antigen), all do not have expression and organize, also do not have the existence of free CD20 in the human serum at hemopoietic stem cell, plasmocyte, lymph progenitor cell and other.CD20 can not come off from cytolemma easily, can internalization not take place because of with combining of antibody antigenic modulation taking place yet.CD20 is different from tumour specific antigen, in organ that lymphoma is invaded such as spleen, lymphoglandula, there is a large amount of normal lymphocytes, also can with the CD20 antibodies of labelled nuclide, thereby can amplify the effect that kills and wounds the tumour cell of invading these organs, so CD20 can be used as the best target spot of bone-marrow-derived lymphocyte knurl treatment.The visible accession number HS.438040 of concrete sequence of CD20.
EpCAM is a kind of cell surface glycoprotein, is distributed in minority and normally reaches the superfluous epithelize cell of great majority.Studies show that, have the expression of EpCAM at the tumor cell surface of most of colon cancer patients.Anti-EpCAM antibody has been proved and can have caused a series of immune response, comprises the cytotoxic reaction (ADCC) of antibody dependent cellular mediation and complementary reaction etc., thus the inhibition growth of tumor.Therefore EpCAM is considered to an important colorectal carcinoma oncotherapy target spot.The visible accession number HS.23582 of concrete sequence of EpCAM.
At present, also lack effectively antibody at CD20 and epithelial cell adhesion molecule (EpCAM), therefore, to press for exploitation new for the high specific of CD20 and EpCAM and the monoclonal antibody of counteragent, the monoclonal antibody of especially humanized high-affinity in this area.
Summary of the invention
Purpose of the present invention just provides specific antibody, the especially Humanized monoclonal antibodies of a kind of anti-CD20 and EpCAM.
In a first aspect of the present invention, a kind of antibody is provided, the variable region of heavy chain of this antibody contains the aminoacid sequence of SEQ IDNO:2 or 6.
In another preference, the variable region of light chain of this antibody contains the aminoacid sequence of SEQ ID NO:4 or 8.
More preferably, described anti-CD20 antibodies has the light chain shown in heavy chain shown in the SEQ ID NO:2 and the SEQ ID NO:4.A kind of anti-CD20 antibodies has the aminoacid sequence shown in the SEQ ID NO:10.
More preferably, described anti-EpCAM antibody has the light chain shown in heavy chain shown in the SEQ ID NO:6 and the SEQ ID NO:8.A kind of anti-EpCAM antibody has the aminoacid sequence shown in the SEQ ID NO:14.
In another preference, described antibody is humanized monoclonal antibody.It more preferably is single-chain antibody.
In a second aspect of the present invention, provide a kind of isolated nucleic acid molecule, its encode specific antibody of anti-CD20 of the present invention and anti-EpCAM.
In another preference, described nucleic acid molecule contains the nucleotide sequence of the encoding antibody variable region of heavy chain shown in SEQ ID NO:1 or 5.
In another preference, described nucleic acid molecule also contains the nucleotide sequence of the encoding antibody variable region of light chain shown in SEQ ID NO:3 or 7.
More preferably, the nucleic acid molecule of described coding anti-CD20 antibodies has the light chain encoding sequence shown in heavy chain encoding sequence shown in the SEQ ID NO:1 and the SEQ ID NO:3.
More preferably, the nucleic acid molecule of described coding anti-EpCAM antibody has the light chain encoding sequence shown in heavy chain encoding sequence shown in the SEQ ID NO:5 and the SEQ ID NO:7.
In a third aspect of the present invention, a kind of carrier is provided, it contains the nucleotide sequence of the specific antibody of coding anti-CD20 of the present invention and anti-EpCAM, and the expression regulation sequence that links to each other with the operability of described nucleotide sequence.
In fourth aspect present invention, a kind of host cell is provided, it contains the above-mentioned carrier of the present invention.
In a fifth aspect of the present invention, a kind of method for preparing antibody is provided, this method comprises:
A) cultivate the above-mentioned transformed host cells of the present invention, thus expressing antibodies; With
D) the described antibody of separation and purification, thus the specific antibody of anti-CD20 of the present invention or anti-EpCAM obtained.
In a sixth aspect of the present invention, a kind of composition is provided, it contains the of the present invention anti-CD20 of safe and effective amount and/or the specific antibody and the pharmaceutically acceptable carrier of anti-EpCAM.
Description of drawings
Fig. 1 has shown the evaluation of antagonism CD20 single-chain antibody, shows that anti-CD20 antibodies has high-affinity and specificity.
Fig. 2 has shown the evaluation to the anti-EpCAM single-chain antibody, shows that anti-EpCAM antibody has high-affinity and specificity.
Embodiment
The inventor utilizes the phage display system, and filtering out first with CD20 and EpCAM has good affinity and specific antibody, after the mensuration sequence.Synthetic full gene has carried out recombinant expressedly, has obtained the anti-CD20 of reorganization and the monoclonal antibody of anti-EpCAM.
The invention provides a kind of reorganization Humanized anti-CD 20 monoclonal antibody, it comprises people source Heng Qu (as permanent district, people source IgG1-Fc), and its variable region of heavy chain and variable region of light chain have unique prior art constructions that is different from.
The invention provides a kind of reorganization anti-EpCAM Humanized monoclonal antibodies, it comprises people source Heng Qu (as permanent district, people source IgG1-Fc), and its variable region of heavy chain and variable region of light chain have unique prior art constructions that is different from.
The present invention also provides aminoacid sequence and its variable region chain thereof of anti-CD20 or EpCAM monoclonal antibody, and other protein or fusion expressed product with these chains.Particularly, the present invention includes and have the hypervariable region of containing (complementary determining region, any protein of light chain CDR) and heavy chain or protein conjugate and fusion expressed product (being immune conjugate and fusion expressed product), as long as identical or at least 90% homology of hypervariable region of this hypervariable region and light chain of the present invention and heavy chain, preferably at least 95% homology.
The antigen binding characteristic of antibody can be described by 3 specific zones that are positioned at heavy chain and variable region of light chain, be called hypermutation zone (CDR), should intersegmentally be divided into 4 frame areas (FR), the aminoacid sequence of 4 FR is relatively conservative, does not participate in association reaction directly.These CDR form ring texturees, and the βZhe Die that the FR by therebetween forms is close mutually on space structure, and the CDR on CDR on the heavy chain and the corresponding light chain has constituted the antigen binding site of antibody.Can use ordinary method, determine that by the aminoacid sequence of antibody more of the same type which amino acid has constituted FR or CDR zone.
In addition, also find the dependency structure that is made of variable region of light chain recently, compare with corresponding variable region of heavy chain that its bonded kinetics is smaller, isolating weight chain variable zone self has antigen-binding activity.
Herein the hypervariable region of the V chain of Jian Dinging or complementary determining region (complementarity determiningregion, CDR) interesting especially because relate to conjugated antigen to small part in them.Therefore, the present invention includes those the monoclonal antibody light chains and the molecule of weight chain variable chain, as long as its CDR has the homology of (preferably more than 95%) more than 90% with the CDR that identifies herein with band CDR.
As used herein, term " antibody of the present invention " refers to CD20 monoclonal antibody and/or chimeric CD20 antibody; And EpCAM monoclonal antibody and/or chimeric EpCAM antibody.This term also comprises the fusion rotein that forms with GST etc., if in this fusion rotein the antibody moiety activity that combines of reservation and CD20 or EpCAM still.
CD20 monoclonal antibody of the present invention contains the heavy chain of aminoacid sequence shown in the SEQ ID NO:2, and contains the light chain of the aminoacid sequence shown in the SEQ ID NO:4.
EpCAM monoclonal antibody of the present invention contains the heavy chain of aminoacid sequence shown in the SEQ ID NO:6, and contains the light chain of the aminoacid sequence shown in the SEQ ID NO:8.
The present invention not only comprises complete monoclonal antibody, also comprises having immunocompetent antibody fragment, as Fab or (Fab ')
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody, as have the murine antibody binding specificity but keep antibody from people's antibody moiety.The technology that can be used for formation chimeric antibody of the present invention is as known in the art.
The present invention also provides coding said monoclonal antibody or its segmental dna molecular.The Nucleotide full length sequence of monoclonal antibody of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.A kind of feasible method is to synthesize relevant sequence, especially fragment length more in short-term with artificial synthetic method.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.In addition, also the encoding sequence of light chain and heavy chain can be merged, form single-chain antibody.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
Among the present invention, " nucleic acid molecule " refers to polynucleotide, and for example thymus nucleic acid (DNA) and Yeast Nucleic Acid (RNA) also comprise the DNA that formed by nucleotide analog or equivalent, the analogue of RNA, strand (sense strand or antisense strand) and double stranded polynucleotide.This term also comprises homologous sequence.For a person skilled in the art, obviously, the nucleic acid molecule of any one above-mentioned form has all been contained all above-mentioned equivalents of this " nucleic acid molecule " natch.
In a preference of the present invention, the nucleotide sequence of the described CD20 monoclonal antibody variable region of heavy chain of encoding comprises sequence shown in the SEQ ID NO:1, and the nucleotide sequence of the described monoclonal antibody variable region of light chain of encoding comprises sequence shown in the SEQ ID NO:3.
In a preference of the present invention, the nucleotide sequence of the described EpCAM monoclonal antibody variable region of heavy chain of encoding comprises sequence shown in the SEQ ID NO:5, and the nucleotide sequence of the described monoclonal antibody variable region of light chain of encoding comprises sequence shown in the SEQ ID NO:7.
In addition, at present can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The invention still further relates to the carrier that comprises above-mentioned suitable dna sequence dna and suitable promotor or control sequence.These carriers can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS7,293 cells etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
The present invention also provides a kind of pharmaceutical composition for the treatment of transplant rejection, and it contains above-mentioned monoclonal antibody or immune conjugate, and pharmaceutically acceptable carrier.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intraperitoneal, intravenously or topical.
Pharmaceutical composition of the present invention contains above-mentioned monoclonal antibody and the pharmaceutically acceptable carrier or the vehicle of the present invention of safe and effective amount.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as injection, solution should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition, be that immune conjugate with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Major advantage of the present invention is: monoclonal antibody of the present invention has high-affinity and specificity to CD20 and EpCAM, and monoclonal antibody of the present invention is humanized.The monoclonal antibody of this high-affinity has significant values clinically.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The variable region gene of anti-CD20 of screening and anti-EpCAM antibody from antibody library
The present invention utilize phage antibody combinatorial library technology with human antibody light chain gene and heavy chain gene vitro recombination and by phage display technique (phage display) screening system at the antigenic specific antibody gene of purpose.This technology is walked around the hybridoma approach, just can prepare and produces the monoclonal antibody that can be used for the testing goal antigen presentation without immunity.Method is as follows:
According to the Chinese patent application 01126858.1 (applying date: 2001.09.25; Publication number: 1408874 open days: 2003.04.09; Denomination of invention: the screening of prokaryotic cell prokaryocyte internal antibody library construction, antibody and optimization and purposes) method of describing in makes up human antibody library.In brief, the gene for preparing heavy chain immunoglobulin and light chain from lymphocyte, carry out amplification in vitro with PCR method, and it is cloned into phage vector, the characteristics of utilizing the antibody molecule fragment to present at phage surface, with CD20 albumen or EpCAM is antigen, screens corresponding specific antibody.
As antigen three-wheel is eluriated in phage single-chain antibody library, people source with CD20 Prokaryotic Expression purified product, specific enrichment occurred, obtain to produce the positive colony of anti-CD antibody.Mammals two-hybrid system with Clontech company is identified the specificity and the avidity of this anti-CD20 single-chain antibody.Through identifying that the CD20 single-chain antibody has specificity and avidity (Fig. 1) preferably.
As antigen three-wheel is eluriated in phage single-chain antibody library, people source with EpCAM Prokaryotic Expression purified product, specific enrichment occurred, obtain to produce the positive colony of anti-CD antibody.Mammals two-hybrid system with Clontech company is identified the specificity and the avidity of this anti-EpCAM single-chain antibody.Through identifying that the EpCAM single-chain antibody has specificity and avidity (Fig. 2) preferably.
Anti-CD20 antigen that is obtained and the antigenic scFv gene of anti-EpCAM carry out sequencing with ordinary method, carry out the Blast comparative analysis with the known array in the NCBI human normal immunoglobulin database, the result shows that its heavy chain gene sequence and light chain gene all meet human normal immunoglobulin variable region skeleton construction feature.
The VH of anti-CD20 antibodies forms (SEQ ID NO:1) by 333bp, 111 the amino acid whose polypeptide (SEQ ID NO:2) of encoding; VL forms (SEQ ID NO:3) by 339bp, 113 the amino acid whose polypeptide (SEQID NO:4) of encoding.
The VH of anti-EpCAM antibody forms (SEQ ID NO:5) by 333bp, 111 the amino acid whose polypeptide (SEQ ID NO:6) of encoding; VL forms (SEQ ID NO:7) by 339bp, 113 the amino acid whose polypeptide (SEQID NO:8) of encoding.
Embodiment 2
Anti-CD20 single-chain antibody recombinant expressed
According to heavy chain of antibody and the sequence of light chain shown in SEQ ID NO:1 and 3, and the catenation sequence that is used to connect heavy chain and light chain of design, with the dna fragmentation (sequence is shown in SEQ ID NO:9) of the complete synthesis single-chain antibody of artificial synthetic method.
With this dna fragmentation is that template is carried out the PCR reaction with following primer,
Forward primer: CGG AAT TCC CAG GAG TCG GGC (SEQ ID NO:11)
Reverse primer: CCGCTCGAGACGTTTGATTTCCACCTT (SEQ ID NO:12)
With the PCR product after EcoR I and Xho I enzyme are cut, the pGEX 5X-2 expression vector (available from Amersham company) of packing into, transformed into escherichia coli.This single-chain antibody gene is expressed through protokaryon GST emerging system, and affinity purification obtains purity and reaches 90% solubility expression product, i.e. the fusion rotein of GST-single-chain antibody (GST merges anti-CD20 single-chain antibody), and molecular weight is about 52KD.
The specific reaction of the fusion rotein (antibody) that obtains is active to be assessed in the proteic ability of external precipitation people CD20 with it.Found that this fusion rotein (SEQ ID NO:10) can combine with CD20 albumen specifically.
Embodiment 3
Anti-EpCAM single-chain antibody recombinant expressed
According to heavy chain of antibody and the sequence of light chain shown in SEQ ID NO:5 and 7, and the catenation sequence that is used to connect heavy chain and light chain of design, with the dna fragmentation (sequence is shown in SEQ ID NO:13) of the complete synthesis single-chain antibody of artificial synthetic method.
With this dna fragmentation is that template is carried out the PCR reaction with following primer,
Forward primer: CGG AAT TCC GTG CAG TCT GGG CCT (SEQ ID NO:15)
Reverse primer: CCGCTCGAGACGTTTGATTTCCAGCTTGG (SEQ ID NO:16)
With the PCR product after EcoR I and Not I enzyme are cut, the pGEX 5X-2 expression vector (available from Amersham company) of packing into, transformed into escherichia coli.This single-chain antibody gene is expressed through protokaryon GST emerging system, and affinity purification obtains purity and reaches 87% solubility expression product, i.e. the fusion rotein of GST-single-chain antibody (GST merges the anti-EpCAM single-chain antibody), and molecular weight is about 52KD.
The specific reaction of the fusion rotein (antibody) that obtains is active to be assessed in the proteic ability of external precipitation people EpCAM with it.Found that this fusion rotein (SEQ ID NO:14) can combine with EpCAM albumen specifically.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Research Center of Shanghai Human Genome
＜120〉human antibody of anti-CD20 and epithelial cell adhesion molecule
<130>036974
<160>16
<170>PatentIn?version?3.1
<210>1
<211>333
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)..(333)
＜223〉heavy chain of anti-CD20 human antibody
<400>1
cag?gag?tcg?ggc?cca?gga?ctg?gtg?aag?cct?tcg?gag?acc?ctg?tcc?ctc 48
Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu?Thr?Leu?Ser?Leu
1 5 10 15
acc?tgc?gtt?gtc?tct?ggt?ggc?tcc?atc?agc?agt?agt?aac?tgg?tgg?agc 96
Thr?Cys?Val?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Ser?Asn?Trp?Trp?Ser
20 25 30
tgg?gtc?cgc?cag?ccc?cca?ggg?aag?ggg?ctg?gag?tgg?att?ggg?gaa?atc 144
Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Glu?Ile
35 40 45
tat?cat?agt?ggg?agc?ccc?aac?tac?aac?ccg?tcc?ctc?aag?agt?cga?gtc 192
Tyr?His?Ser?Gly?Ser?Pro?Asn?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val
50 55 60
acc?ata?tca?gta?gac?aag?tcc?aag?aac?cag?ttc?tcc?ctg?aag?ctg?agc 240
Thr?Ile?Ser?Val?Asp?Lys?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser
65 70 75 80
tct?gtg?acc?gcc?gcg?gac?acg?gcc?gtg?tat?tac?tgt?gca?aga?att?att 288
Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Ile?Ile
85 90 95
cgg?att?cat?agg?acg?ggg?tgg?ggc?caa?ggt?acc?ctg?gtc?acc?gtc 333
Arg?Ile?His?Arg?Thr?Gly?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val
100 105 110
<210>2
<211>111
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉heavy chain of anti-CD20 human antibody
<400>2
Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu?Thr?Leu?Ser?Leu
1 5 10 15
Thr?Cys?Val?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Ser?Asn?Trp?Trp?Ser
20 25 30
Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Glu?Ile
35 40 45
Tyr?His?Ser?Gly?Ser?Pro?Asn?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val
50 55 60
Thr?Ile?Ser?Val?Asp?Lys?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser
65 70 75 80
Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Ile?Ile
85 90 95
Arg?Ile?His?Arg?Thr?Gly?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val
100 105 110
<210>3
<211>339
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)..(339)
＜223〉light chain of anti-CD20 human antibody
<400>3
ctt?gag?att?gtg?atg?acc?cag?act?cca?ctc?tcc?tcg?cct?gtc?acc?ctt 48
Leu?Glu?Ile?Val?Met?Thr?Gln?Thr?Pro?Leu?Ser?Ser?Pro?Val?Thr?Leu
1 5 10 15
gga?cag?ccg?gcc?tcc?atc?tcc?ttc?agg?tct?agt?caa?agc?ctc?gta?cac 96
Gly?Gln?Pro?Ala?Ser?Ile?Ser?Phe?Arg?Ser?Ser?Gln?Ser?Leu?Val?His
20 25 30
agt?gat?gga?aac?acc?tac?ttg?aat?tgg?ttt?cag?cag?agg?cca?ggc?caa 144
Ser?Asp?Gly?Asn?Thr?Tyr?Leu?Asn?Trp?Phe?Gln?Gln?Arg?Pro?Gly?Gln
35 40 45
tct?cca?agg?cgc?cta?att?tat?aag?gtt?tct?aac?tgg?gac?tct?ggg?gtc 192
Ser?Pro?Arg?Arg?Leu?Ile?Tyr?Lys?Val?Ser?Asn?Trp?Asp?Ser?Gly?Val
50 55 60
cca?gac?aga?ttc?agc?ggc?agt?ggg?tca?ggc?act?gat?ttc?aca?ctg?aaa 240
Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys
65 70 75 80
atc?agc?ggg?gcg?gag?gct?gag?gat?gtt?ggg?gtt?tat?tac?tgc?atg?caa 288
Ile?Ser?Gly?Ala?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Met?Gln
85 90 95
ggt?aca?cac?tgg?ctt?acg?ttc?ggc?caa?ggg?acc?aag?gtg?gaa?atc?aaa 336
Gly?Thr?His?Trp?Leu?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys
100 105 110
cgt 339
Arg
<210>4
<211>113
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉light chain of anti-CD20 human antibody
<400>4
Leu?Glu?Ile?Val?Met?Thr?Gln?Thr?Pro?Leu?Ser?Ser?Pro?Val?Thr?Leu
1 5 10 15
Gly?Gln?Pro?Ala?Ser?Ile?Ser?Phe?Arg?Ser?Ser?Gln?Ser?Leu?Val?His
20 25 30
Ser?Asp?Gly?Asn?Thr?Tyr?Leu?Asn?Trp?Phe?Gln?Gln?Arg?Pro?Gly?Gln
35 40 45
Ser?Pro?Arg?Arg?Leu?Ile?Tyr?Lys?Val?Ser?Asn?Trp?Asp?Ser?Gly?Val
50 55 60
Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys
65 70 75 80
Ile?Ser?Gly?Ala?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Met?Gln
85 90 95
Gly?Thr?His?Trp?Leu?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys
100 105 110
Arg
<210>5
<211>333
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)..(333)
＜223〉heavy chain of antibody of anti-EpCAM
<400>5
gtg?cag?tct?ggg?cct?gag?gtg?aag?aag?cct?ggg?acc?tca?gtg?aag?gtc 48
Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr?Ser?Val?Lys?Val
1 5 10 15
tcc?tgc?aag?gct?tct?gga?ttc?acc?ttt?act?agc?tct?gct?gtg?cag?tgg 96
Ser?Cys?Lys?Ala?Ser?Gly?Phe?Thr?Phe?Thr?Ser?Ser?Ala?Val?Gln?Trp
20 25 30
gtg?cga?cag?gct?cgt?gga?caa?cgc?ctt?gag?tgg?ata?gga?tgg?atc?gtc 144
Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Ile?Gly?Trp?Ile?Val
35 40 45
gtt?ggc?agt?ggt?aac?aca?aac?tac?gca?cag?aag?ttc?cag?gaa?aga?gtc 192
Val?Gly?Ser?Gly?Asn?Thr?Asn?Tyr?Ala?Gln?Lys?Phe?Gln?Glu?Arg?Val
50 55 60
acc?att?acc?agg?gac?atg?tcc?aca?agc?aca?gcc?tac?atg?gag?ctg?agc 240
Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr?Ala?Tyr?Met?Glu?Leu?Ser
65 70 75 80
agc?ctg?aga?tcc?gag?gac?acg?gcc?gtg?tat?tac?tgt?gca?aga?cgt?agt 288
Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?Ser
85 90 95
atg?act?act?ttt?gac?tat?tgg?ggc?caa?ggt?acc?ctg?gtc?acc?gtc 333
Met?Thr?Thr?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val
100 105 110
<210>6
<211>111
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉heavy chain of antibody of anti-EpCAM
<400>6
Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr?Ser?Val?Lys?Val
1 5 10 15
Ser?Cys?Lys?Ala?Ser?Gly?Phe?Thr?Phe?Thr?Ser?Ser?Ala?Val?Gln?Trp
20 25 30
Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Ile?Gly?Trp?Ile?Val
35 40 45
Val?Gly?Ser?Gly?Asn?Thr?Asn?Tyr?Ala?Gln?Lys?Phe?Gln?Glu?Arg?Val
50 55 60
Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr?Ala?Tyr?Met?Glu?Leu?Ser
65 70 75 80
Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?Ser
85 90 95
Met?Thr?Thr?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val
100 105 110
<210>7
<211>339
<212>DNA
＜213〉artificial sequence
<220>
<221>CDS
<222>(1)..(339)
＜223〉light chain of anti-EpCAM antibody
<400>7
ctt?gag?att?gtg?atg?acc?cag?act?cca?ctc?tct?ctg?tcc?gtc?acc?cct 48
Leu?Glu?Ile?Val?Met?Thr?Gln?Thr?Pro?Leu?Ser?Leu?Ser?Val?Thr?Pro
1 5 10 15
gga?cag?ccg?gcc?tcc?atc?tcc?tgc?aag?tct?agt?cag?agc?ctc?ctg?cat 96
Gly?Gln?Pro?Ala?Ser?Ile?Ser?Cys?Lys?Ser?Ser?Gln?Ser?Leu?Leu?His
20 25 30
agt?gat?gga?aag?acc?tat?ttg?tat?tgg?tac?ctg?cag?aag?cca?ggc?cag 144
Ser?Asp?Gly?Lys?Thr?Tyr?Leu?Tyr?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln
35 40 45
cct?cca?cag?ctc?ctg?atc?tat?gaa?gtt?tcc?aac?cgg?ttc?tct?gga?gtg 192
Pro?Pro?Gln?Leu?Leu?Ile?Tyr?Glu?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val
50 55 60
cca?gat?agg?ttc?agt?ggc?agc?ggg?tca?ggg?aca?gat?ttc?aca?ctg?aaa 240
Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys
65 70 75 80
atc?agc?cgg?gtg?gag?gct?gag?gat?gtt?ggg?gtt?tat?tac?tgc?atg?caa 288
Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Met?Gln
85 90 95
agt?ata?cag?ctt?ctg?acg?ttc?ggc?caa?ggg?acc?aag?ctg?gaa?atc?aaa 336
Ser?Ile?Gln?Leu?Leu?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys
100 105 110
cgt 339
Arg
<210>8
<211>113
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉light chain of anti-EpCAM antibody
<400>8
Leu?Glu?Ile?Val?Met?Thr?Gln?Thr?Pro?Leu?Ser?Leu?Ser?Val?Thr?Pro
1 5 10 15
Gly?Gln?Pro?Ala?Ser?Ile?Ser?Cys?Lys?Ser?Ser?Gln?Ser?Leu?Leu?His
20 25 30
Ser?Asp?Gly?Lys?Thr?Tyr?Leu?Tyr?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln
35 40 45
Pro?Pro?Gln?Leu?Leu?Ile?Tyr?Glu?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val
50 55 60
Pro?Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys
65 70 75 80
Ile?Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Met?Gln
85 90 95
Ser?Ile?Gln?Leu?Leu?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys
100 105 110
Arg
<210>9
<211>720
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉anti-CD20 single-chain antibody
<220>
<221>CDS
<222>(1)..(720)
<223>
<400>9
cag?gag?tcg?ggc?cca?gga?ctg?gtg?aag?cct?tcg?gag?acc?ctg?tcc?ctc 48
Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu?Thr?Leu?Ser?Leu
1 5 10 15
acc?tgc?gtt?gtc?tct?ggt?ggc?tcc?atc?agc?agt?agt?aac?tgg?tgg?agc 96
Thr?Cys?Val?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Ser?Asn?Trp?Trp?Ser
20 25 30
tgg?gtc?cgc?cag?ccc?cca?ggg?aag?ggg?ctg?gag?tgg?att?ggg?gaa?atc 144
Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Glu?Ile
35 40 45
tat?cat?agt?ggg?agc?ccc?aac?tac?aac?ccg?tcc?ctc?aag?agt?cga?gtc 192
Tyr?His?Ser?Gly?Ser?Pro?Asn?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val
50 55 60
acc?ata?tca?gta?gac?aag?tcc?aag?aac?cag?ttc?tcc?ctg?aag?ctg?agc 240
Thr?Ile?Ser?Val?Asp?Lys?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser
65 70 75 80
tct?gtg?acc?gcc?gcg?gac?acg?gcc?gtg?tat?tac?tgt?gca?aga?att?att 288
Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Ile?Ile
85 90 95
cgg?att?cat?agg?acg?ggg?tgg?ggc?caa?ggt?acc?ctg?gtc?acc?gtc?tcg 336
Arg?Ile?His?Arg?Thr?Gly?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser
100 105 110
agt?ggt?gga?ggc?ggt?tca?ggc?gga?ggt?ggc?tct?ggc?ggt?agt?gca?ctt 384
Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Ser?Ala?Leu
115 120 125
gag?att?gtg?atg?acc?cag?act?cca?ctc?tcc?tcg?cct?gtc?acc?ctt?gga 432
Glu?Ile?Val?Met?Thr?Gln?Thr?Pro?Leu?Ser?Ser?Pro?Val?Thr?Leu?Gly
130 135 140
cag?ccg?gcc?tcc?atc?tcc?ttc?agg?tct?agt?caa?agc?ctc?gta?cac?agt 480
Gln?Pro?Ala?Ser?Ile?Ser?Phe?Arg?Ser?Ser?Gln?Ser?Leu?Val?His?Ser
145 150 155 160
gat?gga?aac?acc?tac?ttg?aat?tgg?ttt?cag?cag?agg?cca?ggc?caa?tct 528
Asp?Gly?Asn?Thr?Tyr?Leu?Asn?Trp?Phe?Gln?Gln?Arg?Pro?Gly?Gln?Ser
165 170 175
cca?agg?cgc?cta?att?tat?aag?gtt?tct?aac?tgg?gac?tct?ggg?gtc?cca 576
Pro?Arg?Arg?Leu?Ile?Tyr?Lys?Val?Ser?Asn?Trp?Asp?Ser?Gly?Val?Pro
180 185 190
gac?aga?ttc?agc?ggc?agt?ggg?tca?ggc?act?gat?ttc?aca?ctg?aaa?atc 624
Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile
195 200 205
agc?ggg?gcg?gag?gct?gag?gat?gtt?ggg?gtt?tat?tac?tgc?atg?caa?ggt 672
Ser?Gly?Ala?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Met?Gln?Gly
210 215 220
aca?cac?tgg?ctt?acg?ttc?ggc?caa?ggg?acc?aag?gtg?gaa?atc?aaa?cgt 720
Thr?His?Trp?Leu?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
225 230 235 240
<210>10
<211>240
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉anti-CD20 single-chain antibody
<400>10
Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu?Thr?Leu?Ser?Leu
1 5 10 15
Thr?Cys?Val?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Ser?Asn?Trp?Trp?Ser
20 25 30
Trp?Val?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Glu?Ile
35 40 45
Tyr?His?Ser?Gly?Ser?Pro?Asn?Tyr?Asn?Pro?Ser?Leu?Lys?Ser?Arg?Val
50 55 60
Thr?Ile?Ser?Val?Asp?Lys?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser
65 70 75 80
Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Ile?Ile
85 90 95
Arg?Ile?His?Arg?Thr?Gly?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser
100 105 110
Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Ser?Ala?Leu
115 120 125
Glu?Ile?Val?Met?Thr?Gln?Thr?Pro?Leu?Ser?Ser?Pro?Val?Thr?Leu?Gly
130 135 140
Gln?Pro?Ala?Ser?Ile?Ser?Phe?Arg?Ser?Ser?Gln?Ser?Leu?Val?His?Ser
145 150 155 160
Asp?Gly?Asn?Thr?Tyr?Leu?Asn?Trp?Phe?Gln?Gln?Arg?Pro?Gly?Gln?Ser
165 170 175
Pro?Arg?Arg?Leu?Ile?Tyr?Lys?Val?Ser?Asn?Trp?Asp?Ser?Gly?Val?Pro
180 185 190
Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile
195 200 205
Ser?Gly?Ala?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Met?Gln?Gly
210 215 220
Thr?His?Trp?Leu?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
225 230 235 240
<210>11
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>11
cggaattccc?aggagtcggg?c 21
<210>12
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>12
ccgctcgaga?cgtttgattt?ccacctt 27
<210>13
<211>720
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉anti-EpCAM single-chain antibody
<220>
<221>CDS
<222>(1)..(720)
<223>
<400>13
gtg?cag?tct?ggg?cct?gag?gtg?aag?aag?cct?ggg?acc?tca?gtg?aag?gtc 48
Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr?Ser?Val?Lys?Val
1 5 10 15
tcc?tgc?aag?gct?tct?gga?ttc?acc?ttt?act?agc?tct?gct?gtg?cag?tgg 96
Ser?Cys?Lys?Ala?Ser?Gly?Phe?Thr?Phe?Thr?Ser?Ser?Ala?Val?Gln?Trp
20 25 30
gtg?cga?cag?gct?cgt?gga?caa?cgc?ctt?gag?tgg?ata?gga?tgg?atc?gtc 144
Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Ile?Gly?Trp?Ile?Val
35 40 45
gtt?ggc?agt?ggt?aac?aca?aac?tac?gca?cag?aag?ttc?cag?gaa?aga?gtc 192
Val?Gly?Ser?Gly?Asn?Thr?Asn?Tyr?Ala?Gln?Lys?Phe?Gln?Glu?Arg?Val
50 55 60
acc?att?acc?agg?gac?atg?tcc?aca?agc?aca?gcc?tac?atg?gag?ctg?agc 240
Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr?Ala?Tyr?Met?Glu?Leu?Ser
65 70 75 80
agc?ctg?aga?tcc?gag?gac?acg?gcc?gtg?tat?tac?tgt?gca?aga?cgt?agt 288
Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?Ser
85 90 95
atg?act?act?ttt?gac?tat?tgg?ggc?caa?ggt?acc?ctg?gtc?acc?gtc?tcg 336
Met?Thr?Thr?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser
100 105 110
agt?ggt?gga?ggc?ggt?tca?ggc?gga?ggt?ggc?tct?ggc?ggt?agt?gca?ctt 384
Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Ser?Ala?Leu
115 120 125
gag?att?gtg?atg?acc?cag?act?cca?ctc?tct?ctg?tcc?gtc?acc?cct?gga 432
Glu?Ile?Val?Met?Thr?Gln?Thr?Pro?Leu?Ser?Leu?Ser?Val?Thr?Pro?Gly
130 135 140
cag?ccg?gcc?tcc?atc?tcc?tgc?aag?tct?agt?cag?agc?ctc?ctg?cat?agt 480
Gln?Pro?Ala?Ser?Ile?Ser?Cys?Lys?Ser?Ser?Gln?Ser?Leu?Leu?His?Ser
145 150 155 160
gat?gga?aag?acc?tat?ttg?tat?tgg?tac?ctg?cag?aag?cca?ggc?cag?cct 528
Asp?Gly?Lys?Thr?Tyr?Leu?Tyr?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Pro
165 170 175
cca?cag?ctc?ctg?atc?tat?gaa?gtt?tcc?aac?cgg?ttc?tct?gga?gtg?cca 576
Pro?Gln?Leu?Leu?Ile?Tyr?Glu?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro
180 185 190
gat?agg?ttc?agt?ggc?agc?ggg?tca?ggg?aca?gat?ttc?aca?ctg?aaa?atc 624
Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile
195 200 205
agc?cgg?gtg?gag?gct?gag?gat?gtt?ggg?gtt?tat?tac?tgc?atg?caa?agt 672
Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Met?Gln?Ser
210 215 220
ata?cag?ctt?ctg?acg?ttc?ggc?caa?ggg?acc?aag?ctg?gaa?atc?aaa?cgt 720
Ile?Gln?Leu?Leu?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg
225 230 235 240
<210>14
<211>240
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉anti-EPCAM single-chain antibody
<400>14
Val?Gln?Ser?Gly?Pro?Glu?Val?Lys?Lys?Pro?Gly?Thr?Ser?Val?Lys?Val
1 5 10 15
Ser?Cys?Lys?Ala?Ser?Gly?Phe?Thr?Phe?Thr?Ser?Ser?Ala?Val?Gln?Trp
20 25 30
Val?Arg?Gln?Ala?Arg?Gly?Gln?Arg?Leu?Glu?Trp?Ile?Gly?Trp?Ile?Val
35 40 45
Val?Gly?Ser?Gly?Asn?Thr?Asn?Tyr?Ala?Gln?Lys?Phe?Gln?Glu?Arg?Val
50 55 60
Thr?Ile?Thr?Arg?Asp?Met?Ser?Thr?Ser?Thr?Ala?Tyr?Met?Glu?Leu?Ser
65 70 75 80
Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Arg?Ser
85 90 95
Met?Thr?Thr?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser
100 105 110
Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Ser?Ala?Leu
115 120 125
Glu?Ile?Val?Met?Thr?Gln?Thr?Pro?Leu?Ser?Leu?Ser?Val?Thr?Pro?Gly
130 135 140
Gln?Pro?Ala?Ser?Ile?Ser?Cys?Lys?Ser?Ser?Gln?Ser?Leu?Leu?His?Ser
145 150 155 160
Asp?Gly?Lys?Thr?Tyr?Leu?Tyr?Trp?Tyr?Leu?Gln?Lys?Pro?Gly?Gln?Pro
165 170 175
Pro?Gln?Leu?Leu?Ile?Tyr?Glu?Val?Ser?Asn?Arg?Phe?Ser?Gly?Val?Pro
180 185 190
Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile
195 200 205
Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Met?Gln?Ser
210 215 220
Ile?Gln?Leu?Leu?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg
225 230 235 240
<210>15
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>15
cggaattccg?tgcagtctgg?gcct 24
<210>16
<211>29
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>16
ccgctcgaga?cgtttgattt?ccagcttgg 29
Claims (10)
1. an antibody is characterized in that, the variable region of heavy chain of this antibody contains the aminoacid sequence of SEQ ID NO:2 or 6.
2. antibody as claimed in claim 1 is characterized in that the variable region of light chain of this antibody contains the aminoacid sequence of SEQ IDNO:4 or 8.
3. antibody as claimed in claim 1 is characterized in that, it is humanized monoclonal antibody.
4. an isolated nucleic acid molecule is characterized in that, the described antibody of its coding claim 1.
5. nucleic acid molecule according to claim 4 is characterized in that, it contains the nucleotide sequence of the encoding antibody variable region of heavy chain shown in SEQ ID NO:1 or 5.
6. nucleic acid molecule according to claim 4 is characterized in that, it also contains the nucleotide sequence of the encoding antibody variable region of light chain shown in SEQ ID NO:3 or 7.
7. a carrier is characterized in that, it contains the nucleotide sequence of the described antibody of coding claim 1, and the expression regulation sequence that links to each other with the operability of described nucleotide sequence.
8. a host cell is characterized in that, it contains the described carrier of claim 7.
9. a method for preparing antibody is characterized in that, this method comprises:
A) cultivate the described host cell of claim 7, thus expressing antibodies; With
D) the described antibody of separation and purification, thus described antibody obtained.
10. a composition is characterized in that, it contains the described antibody of claim 1 and the pharmaceutically acceptable carrier of safe and effective amount.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310108756 CN1618812A (en) | 2003-11-21 | 2003-11-21 | Human Source antibody for anti CD20 and epithelial cell adhesion molecule |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310108756 CN1618812A (en) | 2003-11-21 | 2003-11-21 | Human Source antibody for anti CD20 and epithelial cell adhesion molecule |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1618812A true CN1618812A (en) | 2005-05-25 |
Family
ID=34758704
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200310108756 Pending CN1618812A (en) | 2003-11-21 | 2003-11-21 | Human Source antibody for anti CD20 and epithelial cell adhesion molecule |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1618812A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100455598C (en) * | 2006-11-29 | 2009-01-28 | 中国抗体制药有限公司 | Antibody of anti human CD20 from human resources functionally, and application |
| CN102174465A (en) * | 2011-01-12 | 2011-09-07 | 武汉格蓝丽富科技有限公司 | Method for separating enriched target cells from tissues |
| CN110950959A (en) * | 2020-02-25 | 2020-04-03 | 和铂医药(上海)有限责任公司 | EpCAM-targeted antibody and preparation and application thereof |
-
2003
- 2003-11-21 CN CN 200310108756 patent/CN1618812A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100455598C (en) * | 2006-11-29 | 2009-01-28 | 中国抗体制药有限公司 | Antibody of anti human CD20 from human resources functionally, and application |
| CN102174465A (en) * | 2011-01-12 | 2011-09-07 | 武汉格蓝丽富科技有限公司 | Method for separating enriched target cells from tissues |
| CN110950959A (en) * | 2020-02-25 | 2020-04-03 | 和铂医药(上海)有限责任公司 | EpCAM-targeted antibody and preparation and application thereof |
| CN110950959B (en) * | 2020-02-25 | 2020-07-03 | 和铂医药(上海)有限责任公司 | EpCAM-targeted antibody and preparation and application thereof |
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