CN1124501A - Anti-EGFR single-chain Fvs and anti-EGFR antibodies - Google Patents

Abstract

This invention relates to new anti-EGFR antibodies and single-chain Fvs (scFvs) thereof which can be obtained from phage-antibody libraries constructed from cells of an immunized mammalian, preferably a mouse. Two of the single-chain Fvs isolated from the phage-antibody libraries were engineered to create partially humanized whole antibody molecules. These chimeric anti-EGFR antibodies contain constant regions of human immunoglobulins, and can be used as well as the single-chain Fvs as agents for the diagnosis and therapy of human tumors.

Description

The single chain variable fragment of anti-epidermal growth factor receptor and antibody

The present invention relates to new anti-egfr antibodies and antibody fragment, preferably the single chain variable fragment (scFv) that can obtain from phage antibody library by immune Mammals (preferred mouse) cell construction.The antibody fragment that is separated to from phage antibody library can produce the humanized complete antibody molecule of part with gene engineering method.These chimeric anti-egfr antibodies contain the constant region of human normal immunoglobulin, and they and fragment thereof can be used as the reagent of diagnosis and treatment people tumour.

In addition, the present invention proves that phage antibody library compares with the standard hybridoma technology, is a kind of more general alternative method from immune Mammals separation antibody.

The invention still further relates to be used for the treatment of and contain described antibody or segmental pharmaceutical composition such as the tumour of melanoma, neurospongioma or cancer.Described antibody or fragment also can be used for the interior location of external or body of described tumour and the diagnostic use of assessment.

Several technical terms that this specification sheets relates to are defined as follows:

" FR " (framework region) refers to support four subprovinces of light chain or the variable region of heavy chain of three CDR.

" CDR " (complementarity-determining region) refers to the light chain that has hypervariable sequence and form ring structure or three subprovinces of variable region of heavy chain, and main being responsible for directly contacts with antigen.

" chimeric " or part humanized antibody refer to contain from people's constant region with from the antibody of the variable region (comprising CDR) of inhuman (as mouse).

" humanization " or full-length human antibody refer to contain constant region and the FR from the people, and contain the antibody of the CDR in inhuman source.

" EGF " and " EGFR " refers to Urogastron and its acceptor.

" PCR " refers to polymerase chain reaction.

" scFv " refers to the single chain variable fragment as a kind of antibody fragment.

" V _L" refer to variable region of light chain.

" V _K" refer to the K variable region of light chain.

" V _H" refer to variable region of heavy chain.

PBS refers to the salt solution of phosphate buffered

FCS refers to foetal calf serum

HBSS refers to the Hanks balanced salt solution

FITC refers to fluorescein isothiocyanate

MTC refers to the cell mixing cultivation

Urogastron (EGF) is a kind of short epidermis and the mitotic polypeptide hormone of epithelial cell.Do the time spent when EGF and sensitive cells, it combines with membrane receptor (EGFR).EGFR is the transmembrane glycoprotein of a kind of about 170KD, is the gene product of the former oncogene of C-erb-B.

MAb 425 is the mouse monoclonal antibodies that produce at the people A431 cancerous cell line of knowing (ATCC CRL 1555), and its polypeptide epitope (antigenic determinant) with people EGFR external function district combines, and suppresses the combination of EGF.Find the external mediation cytotoxicity of MAb 425 (ATCC HB9629), and the colorectal carcinoma deutero-clone tumor cell in vitro growth of inhibition skins (Rodeck etc., Cancer Res.1987,47:3692).The humanization of MAb 425 and the form of chimeric antibody are disclosed among the WO92/15683.

In recent years, described the segmental method of functional antibodies that can in prokaryotic host cell such as intestinal bacteria, produce (Skerra and Pluckthun, Science 1988,240:1038; Better etc., Science 1988.240:1041).This comprises Fv fragment and Fab fragment, and wherein the Fv fragment is meaningful especially.Strand Fv (V has wherein also been described _LAnd V _HChain links together) (Bird etc., Science 1988,242:423; Huston etc., Proc Natl, Acad Sci USA 1988,85:5879).

Phage antibody library provides a kind of substitute technology to hybridoma technology from the immune animal separation antibody.Hybridoma technology is to make the cell immortalization that produces antibody.The phage antibody technology is that (Nature 1991,349:293) for Winter, G. and Milstein C. for the gene immortalization that makes encoding antibody.In the phage antibody technology, pcr amplification antibody heavy chain variable region (V _H) and variable region of light chain (V _L) gene, the surface expression that the variable region random groups is incorporated in phage particle is an antibody fragment, then from phage antibody library screening and target antigen bonded antibody.

In the time may causing strong immune response at the spleen of animal, it is very successful that hybridoma technology is separated mouse monoclonal antibody.For example, personnel selection A431 tumour cell intraperitoneal immune mouse, from its spleen, be separated to anti-Human epidermal growth factor receptor (EGFR) mouse MAb (Marthy etc., Arch, Biochem, Biopbys, 1987,252:549).The phage antibody technology is to use almost that the antibodies from any source express cell can filter out a large amount of different antibodies rapidly as parent material than the potential advantages of hybridoma technology.Another advantage of phage antibody technology is the encoding gene of having cloned the target antibody variable region, can carry out further genetic manipulation at any time.

Once report will be converted into the whole antibody molecule from the isolating anti-tetanus toxoid of phage antibody library Fab fragment (Berder etc., Hum.Antibod.Hybridomas 1993,4:74).

In nearly ten years, used external immunity as a kind of substitute technology of active immunity produce people and mouse system a large amount of different antigenic monoclonal antibodies (mAb) (as Vanx, D.J, T; Helenius, A, and Mellman, I; Nature, 1988,336:36; Gathu-ru, J.K etc.; J.Immunol.Methods, 1991,137:95; Borrebaeck, C.A.K; Immunol, Today, 1988,9:355).The advantage of this method is only to need a spot of antigen, and this method can be used for producing hybridoma.But external immunity back produces weak avidity IgM antibody and be difficult to make the human lymphocyte immortalization to become the problem that always exists of technology for this reason.

A kind of new way that obtains antibody is: pcr amplification heavy chain (V _H) and light chain (V _L) variable region gene whole, then it being recombinated at random and being expressed as phage shows library (7-9).The clonal antibody variable region gene also merges with parcel clothing albumen (gene 3) and to become strand Fv fragment (scFv) (10).Phage particle is at its surperficial displaying antibody fragment, and the elutriation of available antibodies binding characteristic is selected.The advantage of this technology is that the reorganization at random of V gene can produce the new pairing with new specificity and affinity, and this specific specificity and affinity possibly can't be selected in natural process.In addition, this method makes and may use the natural or external immune lymphocyte of mouse or people source.

The cross reacting antibody of low-affinity has been had in the effort that produces the mAb of anti-EGFR with mouse B cells in vitro immunity and hybridoma technology in the past.In order to overcome these obstacles, carried out external immunity back in conjunction with the PCR clone technology.

Therefore, the purpose of this invention is to provide the antibody and the antibody fragment that have high-affinity with the EGF acceptor, it can be by above obtaining with favorable method described below.

The mouse-anti EGFR antibody that the present invention will be separated to from three kinds of different bacteriophages antibody libraries with by the isolating mouse mAb of standard hybridoma technology (425) (Marthy etc., Arch, Biochem, Biophys, 1987,252:549; Kettleborough etc., Protein Eng, 1991,4:773) compare.Not only from the spleen of immunized mice but also from the draining lymph node and the external immunized mice cell preparation library of immunized mice.Isolating two strand Fv (scFv) carry out genetic manipulation from the library, and the mouse variable region is connected in human constant region to produce chimeric complete antibody molecule.

Say that in detail the present invention relates to can be from the anti-FGFR strand Fv of phage antibody library acquisition, described library is from the mammiferous spleen of preferred immunity or draining lymph node cell or external immunocyte structure.Say in principle, the invention is not restricted to scFv but also prolong and other anti-egfr antibodies fragment such as Fab or F (ab ') ₂

DNA and the aminoacid sequence of scFv more of the present invention are determined.Therefore, another object of the present invention is a kind of strand Fv fragment, and wherein the variable region of heavy chain and light chain is contained and is selected among the SEQ ID NO.1-32, the DNA and/or the aminoacid sequence of one of the heavy chain that provides among preferred Fig. 5-8 and sequence of light chain.

Because have only under many situations function completely whole antibody just can be used for the diagnosis and therapeutic purpose, so interest of the present invention also is the variable region from strand Fv is connected with the constant region of human normal immunoglobulin, forms the humanized anti-egfr antibodies of part completely.

Therefore, one of purpose of the present invention provides from as mentioned, hereinafter define or as the dna sequence dna of defined antibody fragment in the claim, the anti-EGFR complete antibody that makes up with the dna sequence dna of human normal immunoglobulin constant region, wherein as a preferred embodiment, heavy chain comprises the aminoacid sequence of γ-1 chain, and light chain comprises the aminoacid sequence of K-chain.

Anti-EGFR scFv of the present invention is isolating with the phage antibody library technology.Therefore, the present invention relates to prepare the method for the strand Fv of anti-EGFR, comprise the following steps: (i) from immune mammalian cell, preferred mouse cellular segregation RNA, (ii) synthetic first gang of cDNA (iii) increases from V among the cDNA of immunocyte _HAnd V _KGene, (iv) described gene is cloned in the phagemid carrier with suitable restriction site together, (v) connect mixture and transform prokaryotic cell prokaryocyte with this, (vi) usefulness purifying EGFR screens the phage antibody at EGFR in phage library, and (vii) produces required strand Fv in prokaryotic host cell such as intestinal bacteria.

In addition, one of purpose of the present invention provides a kind of method for preparing anti-EGFR complete antibody, comprise and to be cloned at least a carrier for expression of eukaryon as mentioned above or as the segmental variable region of the anti-egfr antibodies coding DNA that defined method in the claim produces, this carrier contains the genomic dna of coding human normal immunoglobulin constant region, with described carrier transformed eukaryotic karyocyte, express and separation antibody.

Anti-EGFR scFv and primary anti-EGFR complete antibody can be used for diagnosis and treatment people tumour.Therefore the present invention relates to contain as defined above or as the pharmaceutical composition of defined anti-EGFR single-chain Fv or anti-EGFR complete antibody in the claim.

Result of the present invention and advantage are comprehensively as follows:

Separated new mouse-anti EGFR antibody from phage antibody library.This new antibodies has four kinds of different V at least _HSubgroup and four kinds of different V _KSubgroup (Kabat etc., Sequences of proteins of immunological interest, 5th Eds., U.S.Dept, of Health and Human Services, Bethesda 1991).Used pairing and the sequence of the pairing that their show and sequence and the isolating mouse MAb of usefulness hybridoma technology is different.Mouse 425MAb has a V _H2b and V _K4 pairings, and in phage antibody, do not observe.The V of scFvL311D _HWith 425V _HHas the highest concordance rate (84.9%).Main difference is in CDR.ScFv S42D and 425V _KHas the highest consistence (83.2%).Main difference also in CDR, CDR3 particularly.Among the present invention, separated many different new anti-egfr antibodies from phage antibody library, these antibody are all different with 425MAb, and at least two scFv discern the different EGFR epi-position of being discerned with 425MAb.These are different with former report, the latter report from combinatorial library isolated antibody and hybridoma technology isolated antibody closely similar (Caton and Koprowski, Proc Natl Acad Sci USA1990,87:6450).

In three phage antibody libraries, from obtaining needed selection number of steps of high-affinity antibody and the diversity that is separated to high-affinity antibody, best library is the library that produces from the shunting lymphoglandula.Select lymphoglandula two reasons to be arranged as the RNA source that makes up phage antibody.At first, work has in the past illustrated that Hou foot pad immunization Cong lymphonodi poplitei can obtain the B cell (Venn and Dresser, J.Immunol.Methods 1987.102:95) of more a high proportion of generation high-affinity IgG antibody from spleen than after the peritonaeum immunization.The second, the shunting lymphoglandula is considered to the good source of separation of human anti-tumour antibody.Therefore, separating mouse-anti EGFR antibody from the lymphonodi poplitei of immunized mice is model from mammary cancer patient axillary gland separation of human anti-egfr antibodies.Proved the feasibility of from the library, separating high-affinity antibody again from the suitably big or small library of a small amount of lymphoglandula material preparation.

Though from three phage antibody libraries, all separated mouse-anti EGFR antibody, do not known that the avidity of which new isolated antibody is higher than with the isolating mouse 425MAb of hybridoma technology.In first kind of analysis, the scFv that phage antibody produces it seems with combining of EGFR and is better than the scFv (Fig. 2) that makes up from 425MAb.In other experiments with chimeric whole antibody molecule, a kind of chimeric antibody (S4 2D) shows that EGFR avidity equates with the avidity of chimeric 425 antibody.The avidity of second chimeric antibody (L3 11D) is lower than 4 times of the avidity (Fig. 4) of 425 chimeric antibody.The binding data that obtains with scFv makes us misreading, may be because the scFv preparation can contain monomer and dimeric mixture (Grif-fiths etc., EMBO J.1993,12:725).And chimeric IgG antibody can not form dimer according to reason, has proved that chimeric L3 11D and S4 2D antibody have the suitable size of divalence monomer I gG chimeric antibody.But to 425, the analysis revealed of L3 11D and S4 2D scFv affinity purification preparation, these preparations contain monomer, dimer and other polymer form really.In addition, the monomer of each scFv is different with the relative proportion of polymer form.The per-cent of the dimeric forms of 425scFv is minimum.Such as expected, dimer and particularly higher polymer form show with the binding ratio monomeric form of purifying EGFR strong.As if 425scFv a little less than the dimerization tendency than new isolating scFv.

Though the expression of antibody fragment on the phage particle surface formed the basis of strong method that rapid screening has the antibody of target property, phage antibody and antibody fragment itself (scFv or Fab) all can not be the target finished product.Next illustrate from the isolating mouse scFv of phage library and how to change into antibody molecule completely rapidly.This can combine the variable region of mouse with people's constant region, to form the humanized chimeric antibody of part.

These results show and may utilize the phage antibody technology to be separated to various anti-egfr antibodies fragments from immune mouse.Can make up the complete antibody molecule that has suitable constant region from these antibody fragments then.Under some situation, hybridoma technology is still a kind of selective method of separating monoclonal antibody from mouse.If there is high immunogenicity antigen to utilize, and if produce the just enough usefulness of minority hybridoma cell line of one or more different anti-antigen-antibodies, may just there be any reason to consider the phage antibody technology.If but specifc immunity inoculation method such as foot pad injection are favourable to producing high-affinity antibody, if or the multiple antibody of anti-certain the antigenic various different epi-positions of requirement, or require the antibody of epi-position that may be more weak at very hidden epi-position and immunogenicity, then can select to bite the body antibody technique.In addition, carry out further genetic manipulation if wish antagonist, then the phage antibody technology is favourable, because cloned antibody gene.

This with the external immunity of particulate antigen and PCR-clone technology bonded approach produced with EGFR reaction and not with the scFv fragment of other antigenic cross-reaction.Here Bao Dao immune programme for children depends on antigen presentation (it be not soluble and be film vesicle preparation) and substratum self (not containing FCS).According to reports these two kinds of methods all be by antigen offered antigen presenting cell increase the method for external immune efficacy (as Brams, P etc.; J.Im-munol, Methods, 1987,98:11).

The result who obtains with MTC consistent with the report that has earlier (as Borrebacck, C.A.K and M ller, S.A; J.Immunol., 1986,136:3710; M ller S, A and Borrebaeck, C.A.K, at Borrebaeck, the In Vitro Immunization in Hybridoma Tcchnology of C.A.K (volume), Elsevier Science Pub-lishers B.V., Amsterdam 1988, described in the P3), the source of MTC supernatant as the lymphokine of improving external immunization method used in these document suggestions.Film vesicle preparation should be counted as many antigens, because there are many different antigenic determinants in this vesicle.Therefore, as if they should induce the polyclone of certain level to activate.We get rid of this, because anti-EGFR specific reaction is different with the significant reaction that standard polyclone activator obtains.

We are not with the B cell immortalization for external immunity backs, and have been to use the molecule strategy with antibody V _HAnd V _LThe gene immortalization.These monoclonal antibody fragments are expressed in bacterium and are produced.The phage indicating system is to separate the favorable method of the antibody fragment of anti-specific antigen.Exist terminator codon to make between antibody fragment and g3p coating protein to show and changing (Hoogenboon etc., Mucl Acids Res 1991 19:4133) with containing suppressor gene or not conforming between the bacterial strain secretion solubility scFv fragment of suppressor gene on the surface.

Because the raising of mRNA level in the B cell that specific reaction and exo-antigen stimulate, external immunity has contribution to separating antigenic antibody with high specificity fragment.After two-wheeled was selected, 100% clone was combined into the positive to EGFR.On the contrary, the clone that methods of vaccination produces in the body only after four-wheel is selected just be 100% positive (Kettleporough etc., EP94104160 and Eur.J.Immunol 1994,24:952).

Utilizing the phage of natural antibody gene to show that the library can make is not carrying out immunization or making the specific human antibodies fragment after external immunity.Antibody fragment can directly originate in the bacterium, is a kind of simple, quick and economic method therefore.Biomaterial and general method

Other fragment of the microorganism of mentioning among the application, clone, plasmid, phagemid, promotor, resistance marker, ori and carrier has supply of commodities or can be public's gained with other approach.As long as do not have out of Memory in this application, they are only as for example, and are optional to the present invention, can replace with other instrument and biomaterial that is fit to respectively.

Preferred use host bacterium clone scFv also produces scFv albumen.These hosts' example is intestinal bacteria or bacillus.

According to the present invention,, preferably use eucaryon host such as COS, CHO or yeast in order to produce anti-egfr antibodies completely.

In specification sheets, describe technology required in this invention in detail.Other technology that does not have to describe in detail is corresponding to standard method well known to those skilled in the art, or is method in greater detail in the document of being quoted from and patent application and normative document.

Brief description of drawings

Fig. 1. the aminoacid sequence of isolating scFv from phage antibody library.(A) from the scFv in lymphoglandula library.(B) from the scFv in spleen library.Complementary determining region (CDR) and framework region (FR) have been indicated.

Fig. 2. scFv combines with EGFR's.Estimated the concentration of scFv in the bacterium supernatant liquor, measured combining of scFv and purifying EGFR with ELISA.(A). from the scFv in lymphoglandula library.(B). from the scFv in spleen library.P1 (positive control) is the scFv from MAb425.L1 and S1 (negative control) are from the lymphoglandula before selecting and the non-binding property scFv in spleen library.

Fig. 3. be used for making up the intermediate carrier that the mammalian cell of variable region is expressed.(A) V _HCarrier.(B) V _KCarrier.

Fig. 4. chimeric whole antibody combines with EGFR's.Measured antibody concentration in the COS cell conditioned medium liquid with ELISA, tested combining of antibody and purifying EGFR with ELISA.

Fig. 5. DNA and the aminoacid sequence of scFv No.L2 11C.

(A): light chain; (B): heavy chain.

Amino acid position:

(A)FR-1：1-23，CDR-1：24-34，

FR-2：35-49，CDR-2：50-56，

FR-3：57-88，CDR-3：89-97，

FR-4：98-109.

(B)FR-1：1-30，CDR-1：31-35，

FR-2：36-49，CDR-2：50-66，

FR-3：67-98，CDR-3：99-108，

FR-4:109-119. DNA and the aminoacid sequence of Fig. 6 .scFv No.L2 12B.(A): light chain; (B) heavy chain.Amino acid position:

(A)FR-1：1-23，CDR-1：24-38，

FR-2：39-49，CDR-2：50-56，

FR-3：57-88，CDR-3：89-97，

FR-4：98-109.

(B)FR-1：1-30，CDR-1：31-35，

FR-2：36-49，CDR-2：50-66，

FR-3：67-98，CDR-3：99-108，

FR-4：109-119.

Fig. 7. DNA and the aminoacid sequence of scFv NO.L3 11D.(A) light chain; (B): heavy chain.

The amino acid position of FR and CDR corresponding to provide among Fig. 6 those.

Fig. 8. DNA and the aminoacid sequence of scFv NO.S4 2D.(A): light chain; (B): heavy chain.Amino acid position:

(A)FR-1：1-23，CDR-1：24-35，

FR-2：36-50，CDR-2：51-57，

FR-3：58-89，CDR-3：90-98，

FR-4：99-110

(B)FR-1：1-30，CDR-1：31-35，

FR-2：36-49，CDR-2：50-66，

FR-3：67-98，CDR-3：99-107，

FR-4：108-118.

The sequence of Fig. 5-8 also provides in appended sequence table, and this sequence table is a part disclosed by the invention.(1) structure of phage antibody library and screening

Made up three phage libraries, one from using human carcinoma cell line A431 (8.8 * 10 ⁵Individual) immune mouse spleen, the purifying EGFR (6.5 * 10 that uses by oneself ⁶Individual) the mouse lymphonodi poplitei of foot pad immunity, the A431 vesicle (1.1 * 10 of using by oneself ⁵Individual) mouse lymphotactin (embodiment 1,2 is seen in the detailed structure and the external immunity of A431 vesicle) of external immunity.Before the selection, at least 46 clones all do the fingerprinting analysis with the BstNI enzyme (Clackson etc., Nature 1991,352:624) to determine the diversity of repertoire (repertoires) in each library.Observed multiple different digestion pattern.Before selection, also 96 clones' scFv and combining of EGFR in each library have been tested with the ELISA method.ScFv none from spleen and lymphoglandula library combines with EGFR.A scFv in external immune library combines with EGFR.After carrying out taking turns selection with the immunity pipe (im-munotabes) of EGFR bag quilt, observe that EGFR associativity scFv has obvious enrichment in lymphoglandula library and the external immune library.Needs second are taken turns selection and could detect any EGFR associativity scFv from the gland libraries.After third round was selected, the EGFR of most of scFv was combined into the positive in lymphoglandula library and the external immune library.The 4th take turns selection after, the EGFR of most of scFv is combined into the positive (table 1) in the spleen library.

Table 1: every EGFR associativity clone number (%) of selecting the back to exist of taking turns

	The lymphoglandula library	The spleen library	External immunocyte library
				Before the selection	????0	????0	????1
The 1st takes turns	????77	????0	????84
				The 2nd takes turns	????86	????26	????100
The 3rd takes turns	????90	????77	????100
				The 4th takes turns	Undetermined	????97	Undetermined

(2) EGFR associativity clone's sequential analysis

Every take turns selection after, (Clackson etc., Nature 1991,352:624) analyzed the scFv that inserts among the EGFR associativity clone with the BstNI fingerprinting.Shown the enrichment of some digestion pattern.For V _HAnd V _KOrder-checking, select and third and fourth the taking turns and selected clone of spleen library from second and the third round in lymphoglandula library with different B stNI fingerprint.The clone who analyzes the later several rounds selection is because of expecting that the antibody than high-affinity is that (Clackson etc., Natare 1991,352:624) in the later several rounds selection.

16 clones to the lymphoglandula library check order, and obtain six kinds of different scFv (Fig. 1).Wherein five kinds is to have unique V _HAnd V _KPairing.The 6th kind is the V that exists in the past _HVarient, wherein have 6 amino acid to change, 5 is in framework region (FR) 1.Two application that are attributable to the VH1BACKSFI primer of degeneracy in this variation (Hoogenboon etc., Nucl.Acids Res.1991,19:4133).Other may be the result of PCR mistake.V _HBe divided into two subgroup: V _H2b and V _H3d, and V _KBe divided into four subgroup: V _K3, V _K4, V _K5 and V _K6 (Kabat etc., Sequences of proteins of immunological interest, 5th Eds., U.S.Dept.of Health and Hu-man Services, Bethesda 1991).10 monospecific polyclonals to the spleen library check order, and find 4 kinds of different scFv.Wherein three kinds is unique V _HAnd V _KPairing, and the 4th kind with existing one of match similar, only at V _HTwo amino acid differences are arranged, and one of them is at complementary determining region (CDR) 2; At V _KIn two amino acid differences are arranged.V _HSubgroup is categorized as V _H2a, V _H2c and V _H3d, V _KBe divided into V _K3 and V _K4 subgroups.Relatively the scFv that obtains from lymphoglandula and spleen library finds to have only a kind of scFv to have in two libraries: scFvL3 10A/scFv S4 10H (Fig. 1).When detecting, should clone's performance combine with EGFR is strong with ELISA.Although extreme care to eliminate the crossed contamination in two kinds of libraries, is difficult to get rid of a small amount of strong EGFR associativity clone's pollution.But consider the inbreeding of Balb/c mouse, it is possible producing same scFv independently from two different libraries.(3) with EGFR bonded affinity and specific analysis

Based on diversity, select further to analyze from several scFv in lymphoglandula and spleen library to antigenic good combination and dna sequence dna.With combining of the EGFR of these scFv of elisa assay and purifying, with uncorrelated antigenic combine and with the combining of the tumor cell line of not expressing EGFR.ScFv as positive control makes (P1) from mouse 425 MAb.As the scFv of negative control from making (being respectively L1 and S1) by the isolating phage antibody of lymphoglandula and spleen library before selecting.The extent of dilution of the purifying scFv of concentration known in the extent of dilution of the scFv that will detect and the Western trace is relatively determined the concentration of scFv.

Measure combining of scFv and purifying EGFR with ELISA, and draw result (Fig. 2).According to they with EGFR to combine the scFv classification be possible.These classifications are repeatedly having repeatability in the experiment.Combine the strongest scFv with EGFR and be from the L2 1C in lymphoglandula library and L3 10A with from the S4 10H in spleen library.As previously mentioned, scFv L3 10A has identical dna sequence dna with S4 10H.At V _HAnd V _KIt is more rudimentary than S4 10H all the time with the closely similar a kind of scFv (S4 5A) of scFv S4 10H that the district has respectively that two amino acid change.On the contrary, observed sequence difference does not then show the bonded remarkably influenced between L2 12B and the L3 11D.Among the isolating scFv of institute, have only four kinds, L2 8C and L2 11C show that associativity is poorer than scFv 425.

Tested combining of these scFv uncorrelated with series of standards albumen with plastics (Protalbinic acid, egg lytic enzyme, cytochrome C, Glycerose-3-phosphate dehydrogenase, CBA albumin and BSA) with ELISA.There is not a kind of scFv to provide the above signal of baseline.

Tested combining of these scFv and three kinds of tumor cell lines with ELISA.Clone A431 and MDA MB 468 are the tumour cells that contain EGFR that are separated to from vaginal orifice and breast respectively.Clone SK-MEL-the 23rd, a kind of melanoma cell series that contains Sphingolipids,sialo, at this as negative control.In ten kinds of scFv of test, only there are four kinds both also to combine (L2 12B, L3 11D, L2 11C and S4 2D, Fig. 5-8) with the tumour cell of band EGFR with purifying EGFR.Do not detect and the combining of SK-MEL-23.This afterclap there are several possible explanations.A kind of is that the EGFR that is used for immunization, selection and ELISA is that (Weber etc., Science 1984,224:294) for excretory EGFR associated protein.This albumen C-end have more 17 amino acid (Gunther etc., J.Biol.Chem.1990,265:22082).Test combining of scFv and this 17 amino acid whose peptides with ELISA, do not observed any combination.Excretory EGFR associated protein may have difference with the EGFR of tumor cell surface in conformation or glycosylation.

For combining of further research and tumour cell, purifying three kinds of scFv (L2 11A, L3 11D and S4 2D), and with flow cytometry analysis with the combining of A431 tumour cell.425 scFv are as positive control.Among three kinds of scFv that tried, have only L3 11D and S4 2D to combine with the A431 cell.These two kinds of scFv have similar binding characteristic to scFv425.

From with EGFR and the tumour cell (L3 11D and S4 2D) that contains EGFR purifying scFv of two kinds of isolates preparations of bonded all, combine in the experiment in competition and to test with mouse 425 MAb.The scFv425 of purifying can suppress combining of mouse 425MAb and EGFR in finite concentration, and scFv L3 11D and S4 2D can not suppress combining of mouse 425 MAb and EGFR under these concentration.Epi-position on these two kinds of EGFR that scFv discerned as if with mouse 425 MAb discerned different.(4) the chimeric complete antibody that obtains from scFv

Selected two kinds of scFv (L3 11D and S4 2D) to change the complete antibody molecule into.Mouse V will encode _HAnd V _KDna clone in intermediate carrier, this intermediate carrier contains the dna sequence dna and the donor splicing site signal (Fig. 3) of coding immunoglobulin (Ig) leader.V _HThe location of cloning site means V in the intermediate carrier _HFirst residue become L-glutamic acid by no winter propylhomoserin.From then on intermediate carrier contains V _HAnd V _KAnd the dna fragmentation that is connected with leader and donor splicing site sequence is cloned into, and (Antibod.Hybridomas 1991,2:124) for Maeda etc., Hum in the mammalian cell expression vector of the DNA that contains coding people γ-1 constant regional people K constant region.To every kind of chimeric antibody, with heavy chain and light chain expression vector cotransfection COS cell.As positive control, also with the coding heavy chain of 425 chimeric antibodies and light chain expression vector cotransfection cell (Kettleporough etc., Protein Eng.1991,4:773).The collecting cell nutrient solution, the antibody concentration and antibody and the EGFR bonded ability (Fig. 4) that exist with mensuration with elisa assay.When relatively reaching with the needed antibody concentration of antigen bonded half maximum value, chimeric S4 2D antibody is the same with the combination of EGFR with 425 chimeric antibodies good.But binding ratio 425 chimeric antibodies of L3 11D chimeric antibody and GEFR is low 4 times.The avidity that records 425 chimeric antibodies with the competition inhibition analysis is 1.9 * 10 ⁸M ^-1These results are unexpected, because former scFv analytical data has shown combining all than scFv425 good (Fig. 2) of scFv S4 2D and L3 11D and EGFR.L3 11D and S4 2D chimeric antibody sample through albumen-A purifying carry out SDS-PAGE analysis under reduction and non-reduced condition.Also tested combining of L311D and S4 2D chimeric antibody and A431 and SK-MEL-23 cell with flow cytometer.Two kinds of chimeric antibodies all combine well with EGFR expressivity A431 cell, and do not combine with SK-MEL-23 cell of EGFR feminine gender.(5) treatment and diagnostic use

Antibody fragment of the present invention and complete antibody can give patient and treat.Therefore, an object of the present invention is to provide contain at least a above with claim in defined antibody or antibody fragment as the pharmaceutical composition of activeconstituents, wherein be combined with one or more pharmaceutically acceptable carriers, vehicle or thinner.

Antibody of the present invention should make intravenously or intestines are injected outward, and generally speaking, the dosage scope of antibody should be enough big to produce the effect that desirable tumor suppression and tumour are dissolved.Dosage will depend on the degree of patient's age, sex and disease, variation range be 0.1mg/kg to 200mg/kg, preferred 0.1mg/kg is to 100mg/kg/ time, once a day or repeatedly, lasting a couple of days.

Intestines external administration preparation comprises aseptic aqueous solution or non-aqueous solution, suspension and emulsion.Examples of non-aqueous is known other solvent that is suitable for these purposes in propylene glycol, polyoxyethylene glycol, vegetables oil and sweet oil, injectable organic ester such as ethyl oleate and this area.Antibody of the present invention can be used for containing physiology and can accept in the composition of carrier.The example of this suitable carrier has salt solution, PBS, Ringers solution or lactic acid Ringers solution.Also can there be sanitas and other additive such as microbiotic, oxidation inhibitor and sequestrant in the pharmaceutical preparation.

This antibody (fragment) can also be by currently known methods and cytokine such as IL-2 coupling to support its cytotoxicity.

Pharmaceutical preparation of the present invention is suitable for various tumours, comprises melanoma, neurospongioma and cancer, and the tumour of the recycle system and solid tumor.

Be diagnostic purpose, antibody can or be done radio-labeled with for example radiopaque dye-coupling.Preferred marking method is the Lodogen method.Antibody is preferably with F (ab ') ₂Or the scFv pieces is administered for diagnostic purpose.This provides better result, makes to there is no need subtracting background.Embodiment 1:A431 vesicle

Method (J.Biol Chem, 1982,257:1523 to descriptions such as Cohen; Yeaton etc., J.Biol, Chem, 1983,258:9254) carry out some and revise, produce the vesicle preparation of demoulding.Cover with of the PBC washing of the flask of A431 cell with calcic and magnesium.Added hypotonic PBS and shaking flask 15 minutes, then with vesicle damping fluid (100mM NaCl, 50mMNa ₂HPO ₄, 5mM KCl, 0.5mM MgSO ₄, pH8.5) washed cell.Add the vesicle damping fluid, in room temperature and 37 ℃ of stirrings down.By wire netting damping fluid is strained in the 50ml pipe in the ice then, 150Xg, 4 ℃ are centrifugal 5 minutes.Discard precipitation, supernatant liquor was with 39000rpm ultracentrifugation 90 minutes.Final precipitation is resuspended in the 10mM Hepes damping fluid (pH7.4).For analyzing the EGFR from vesicle, sample precipitates with 9 volume ethanol, and is resuspended with 0.08M Tris (pH6.8), carries out SDS-PAGE with MAb425 as standard and analyzes.

Carry out quantitatively making standard with BSA, with the protein content of improved Coomassie blue method at the 595nm reading to these prepared products.For analyzing the EGFR from vesicle, sample precipitates (4 ℃ are spent the night) with 9 volume ethanol.To precipitate with Tris (0.08M, pH6.8) resuspended, carry out SDS-PAGE electrophoresis (5% stacking gel then; 1 hour, 35mA; 10% running gel; 2.5 hour; 40mA).Sample and standard substance are double.One of them uses Coomassie blue stain, another transfer printing (12V on nitro-cellulose film; 4 ℃ 16 hours) and with mouse mAb 425 (anti-EGFR) and with the anti-mouse IgG antibody treatment of alkaline phosphatase coupling.

Three kinds of substratum are used for external immunization.Substratum-1 (M1), substratum-2 (M2) and mixing thymocyte substratum (MTC).M1 is had HL1 (Ventrex Laboratories, USA) composition of 50mM 2-mercaptoethanol and 2mM L-glutamine (Gibco) by benefit.M2 has 50mM 2-mercaptoethanol by benefit; 40U/ml IL-2 (Gen-zyme); 20mg/ml adjuvant peptide (sigma); The 2mML-glutamine; The blue or green toxin (Gibco) of 100U/ml; The HL1 of 100mg/ml Streptomycin sulphate (Gibco) forms.The FCS (Biological Industries) of adding 4% or 20% in M2.The method of describing as Uaux (1) prepares MTC.Main points are: the single cell suspension of thymus gland being pressed aseptic 50 mesh sieves preparation Balb/c in three ages in week and C57/BL-1 mouse thymus.The collecting cell suspension washes twice with HBSS, expects the number of blue exclusion mensuration viable cell with tongue.Then with every milliliter 2.5 * 10 ⁶The density of every strain thymocyte is cultivated thymocyte in the HL1 substratum that contains 4%FCS, 2mM L-glutamine, 100U/ml penicillin and 100mg./ml Streptomycin sulphate.After 48 hours, reclaim supernatant liquor, filter the filter of a 0.22mm, preserve down in-70 ℃.

The preparation method of thymocyte as described, from non-immunity eight ages in week BALB/C mice obtain splenocyte suspensions.Expect blue exclusion method mensuration viability with tongue.Embodiment 2: external immunity and screening

Three kinds of substratum are used for external immunization.Substratum-1 (M1), substratum-2 (M2) and mixing thymocyte substratum (MTC).M1 is had HL1 (Ventrex Laboratories, USA) composition of 50mM 2-mercaptoethanol and 2mM L-glutamine (Gibco) by benefit.M2 has 50mM 2-mercaptoethanol by benefit; 40U/ml IL-2 (Gen-zyme); 20mg/ml adjuvant peptide (sigma); The 2mML glutamine; The blue or green toxin (Gibco) of 100U/ml; The HL1 of 100mg/ml Streptomycin sulphate (Gibco) forms.The FCS (Biological Industries) of adding 4% or 20% in M2.The method of describing as Uaux (1) prepares MTC.Main points are: the single cell suspension of thymus gland being pressed aseptic 50 mesh sieves preparation Balb/c in three ages in week and C57/BL-1 mouse thymus.The collecting cell suspension washes twice with HBSS, expects the number of blue exclusion mensuration viable cell with tongue.Then with every milliliter 2.5 * 10 ⁶The density of every strain thymocyte is cultivated thymocyte in the HL1 substratum that contains 4%FCS, 2mM L-glutamine, 100U/ml penicillin and 100mg./ml Streptomycin sulphate.After 48 hours, reclaim supernatant liquor, filter the filter of a 0.22mm, preserve down in-70 ℃.

The preparation preparation method of thymocyte as described, from non-immunity eight ages in week BALB/c mouse obtain splenocyte suspensions.Expect blue exclusion method mensuration viability with tongue.

Thymus gland was pressed the single cell suspension of aseptic 50 mesh sieves preparation Balb/c in three ages in week and C57/BL-1 mouse thymus.The collecting cell suspension washes twice with HBSS, expects the number of blue exclusion mensuration viable cell with tongue.Then with every milliliter 2.5 * 10 ⁶The density of every strain thymocyte is cultivated at the HL1 that contains 4%FCS, 2mML-glutamine, 100U/ml penicillin and 100mg/ml Streptomycin sulphate and is wherein cultivated thymocyte.After 48 hours, reclaim supernatant liquor, filter and preserve.The method of thymocyte as described, from non-immunity eight ages in week BALB/c mouse obtain splenocyte suspensions.Expect blue exclusion method mensuration viability with tongue.

On one 6 orifice plates (Costar), carry out external immunity.Contain 10 in 3.5ml M1-substratum (having the HL1-substratum of 50 μ m 2-mercaptoethanols and 2mM L-glutamine (Gibco) to form) by benefit ⁷The hole of splenocyte with the vesicle of the band EGFR of proper concn hatch (37 ℃, 5%CO ₂).Adding is from the vesicle of the cell of not expressing EGFR or PBS hole in contrast.After several hours, in every hole, add 3.5ml and contain 4% or M2-substratum (having the HL1 of 50 μ M2-mercaptoethanols, 40U/ml IL-2 (Genzyme), 20 μ g/ml adjuvant peptides (Sigma), 2mML-glutamine, 100U/ml penicillin (Gibco), 100 μ g/ml Streptomycin sulphates (Gibeo) to form) of 10%FCS (Biologocal Lndusrties) by benefit.In some experiments, there is the MTC substratum of adjuvant peptide (20 μ g/ml) and IL-2 (40U/ml) (to mix thymocyte substratum (Vaax etc. with benefit, Nature 1988,336:36)) replacement M2 (note FCS in the culture, the final concentration of IL-2 and adjuvant peptide has lowered 50%).Culturing cell is 72,96,120 or 144 hours under the same conditions, detects cell at last and whether has the specific immune sphaeroprotein or carry out the RNA separation.

Screen with purifying antigen or A431 immobilized cell.(Carroll etc., Hybridoma 1990,9:81), but some modification substantially as previously mentioned for operation steps.In brief, (Nunc is Maxisorb) with EGFR (2.5 μ g/ml), GD among the PBS for 96 aseptic orifice plates ₃Sphingolipids,sialo (2 μ g/ml) or RNase (10 μ g/ml) bag are spent the night.When the A431 cell was used as antigen, culturing cell was fixed with 0.1% glutaraldehyde to confluent growth again in 96 orifice plates.The lymphocyte flushing of external immunity also is resuspended in the HL1 substratum of benefit with 2%FCS and 2mM L-glycosamine, and density is 5 * 10 ⁵Cell/ml adds 1 * 10 in every hole ⁵Individual cell, hatch 48 hours (37 ℃, 5%CO ₂).Do 16 parts for every group.Lymphocyte is washed 5 removals in the PBS that contains 0.1%Tween-20.Detect specific immune sphaeroprotein (1 hour, 37 ℃) with the anti-rat immune globulin of the rabbit of peroxidase labelling (Dako).In citric acid-phosphoric acid buffer 2,2 '-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid-di-ammonium salts (ABTS) is (Sigma) as substrate for azino-two.Embodiment 3: library construction

From mouse spleen (Murthy etc., Arch, Biochem by intraperitoneal A431 cellular immunization, Biophys, 1987,252:549), by with purifying EGFR foot pad immunity De Shu lymphonodi poplitei with by the RNA that makes with the outer immune mouse cell of A431 vesicular body, made up three kinds of libraries.Synthesized first gang of cDNA.Pcr amplification is also assembled V _HAnd V _K(Clackson etc., Natare 1991,352:624) for gene.By PCR, added Notl and Sfil restriction site, scFv is cloned among the phagemid carrier pHEN1 (Hoogen-boom etc., Nucl.Acids Res.1991,19:4133).To connect the mixture electroporation to Bacillus coli cells, the bacterium colony of formation scrape in the substratum with produce the library original seed (Marks etc., J.Mol.Biol, 1991,222:581).Embodiment 4: library screening

(Promega, Madison WI) save phage antibody (Marks etc., J.Mol.Biol 1991.222:581) from the library with the M13KO7 helper phage.(Paisley's immunity pipe UK) is spent the night with the bag of 2.5 μ g/mlEGFR among the 4ml PBS for Nunc, Life Sciences.After PBS washing three times, this pipe in the PBS that contains 2% milk powder (PBSM) 37 ℃ hatched at least 1 hour.With phage (10 ¹²-10 ¹³) be resuspended among the 4ml PBSM, incubated at room is 1 hour in the pipe of EGFR bag quilt.Use PBS, 0.1%Tween washes pipe 20 times, washes 20 times with PBS again.In 1ml 0.1M triethylamine, be inverted mixed culture after 10 minutes, the phage of elution of bound.Add 0.5ml 1MTris-HCl in the phage of wash-out, the pH7.5 neutralization is used for ehec infection TG1 cell.With the cells infected coated plate, choose individual bacterium colony and be used for inducing in a small amount ScFv.All the other bacterium colonies are scraped in the substratum, and portion is used to prepare the phage of screening next time.The generation of embodiment 5:scFv and analysis

(, I.C.) in intestinal bacteria HB2151, produce solubility scFv by existing description as, Kettleleborough etc.As standard, assessed the concentration of scFv in the bacterium supernatant liquor with the purifying scFv preparation of concentration known.Filtering supernatant adds sodiumazide to 0.1%.With the serial dilutions of this supernatant liquor and standard substance with 96 hole menifold points Immobilon-PVDF filter membrane (Millipore, Watford, UK) on.(Towbin etc., Proc.Natl.Acad.Sci.USA 1979,76:4350) handle this filter membrane as the Western blotting.Use antibody (9E10) (Munro and Pelham at C-terminal tail, Cell 1986,46:291) and peroxidase link coupled sheep anti-mouse igg and IgM antibody (Jackson Immuno Research Lab Inc, West Grove PA) detects scFv.With the ECL system carry out these reactions (Amersham, Aytesbury, UK).Autoradiogram(ARGM) optical density meter scanning before the flash of light.Do typical curve and be used for evaluating the scFv concentration of supernatant liquor.

Carry out the ELISA detection of antigen-binding with the plate (2.5 μ g/ml) of EGFRyy bag quilt.The supernatant liquor that contains scFv dilutes with PBSM, and adds on the plate.With aforesaid 9E10 antibody test bonded scFv.Also tested combining of uncorrelated albumen of supernatant liquor and plastics with series of standards.Elisa plate is spent the night with 100 μ g/ml bag with Protalbinic acid, egg lytic enzyme, cytochrome C, Glycerose-3-phosphate dehydrogenase, mouse albumin (CBA kind) and BSA.In wrapping, add the undiluted supernatant liquor (two covers) that contains 2% milk powder, and detect bonded scFv as stated above by plate.

The ELISA that carries out the cell associativity with tumor cell line A431 (ATCC CRL 1555), MDA MB 468 (ATCC HTB 132) and SK-MEL-23 (negative control) detects.Cell grows in 96-hole tissue culture ware (Nunc) that poly--D-Methionin is handled and converges.These cells washed with DMEM, with 37 ℃ of sealings of the PBS that contains 2.5%BSA 2 hours.After the sucking-off, in every hole, add supernatant liquor and isopyknic 2XYT substratum that contains 4% milk powder, cultivated 1 hour for 4 ℃.Carry out detection as mentioned above in conjunction with scFv.

Being at war with property ELISA as follows; With the elisa plate and the pre-cultivation of 50 μ l purifying scFv (100 μ g/ml) of EGFR bag quilt, counted 10 minutes, add the concentration that mouse MAb425 (50 μ l) reaches 3.13-200ng/ml then.After hatching and washing, with peroxidase link coupled sheep anti-mouse igg and IgM antibody test bonded mouse MAb425.Embodiment 6:DNA analyzes

In order to carry out the BstNI fingerprinting, will carry out pcr amplification from single clone's scFv insertion sequence, (Clackson etc., Nature 1991,352:624) with BstNI digestion for product.(United States Biochemical, Cleveland OH) carries out dna sequencing with the Sequenase test kit.The purifying of embodiment 7:scFv

Centrifugal and filter 0.2 μ m filter membrane and make bacterium supernatant liquor clarification, application of sample is equipped with the EGFR (5mg) of purifying in a 1ml (Pharmacia, Uppsala is on post Sweden) with the Sepharose 4B coupling of cyanogen bromide-activated then.This post is used 5ml 0.2M glycine (pH5.0) flushing again with 30ml PBS.With 0.2M glycine/HCl (pH2.8) wash-out scFv.Elutriant 10X PBS neutralization.Collect protein-contg stream part, (Am-icon, Stonehouse UK) change damping fluid into the PBS that contains 1%BSA and 0.05% sodiumazide through ultrafiltration.Embodiment 8: the facs analysis of purifying scFv

The A431 cell is cultivated with tryptic digestion and in containing the DMEM of 10%FCS.Cell washes twice with cold DMEM, filters the sieve of one 45 μ m.On ice, cell (106) was hatched 30 minutes with purifying scFv in 50 μ l contain the PBS of 1%BSA.After twice of cold PBS flushing, with 9E10 antibody (100 μ g/ml) the detection bonded scFv of 50 μ lFITC couplings.After 30 minutes, cell with the PBS flushing once, and is fixing in the PBS that contains 1% formaldehyde on ice, and (Becton-Dickinson, Cow-ley UK) analyze with FACSCAN.Embodiment 9: the structure of chimeric complete antibody, analysis and expression

Utilize PstI and BstEII site, with selected scFv V _HCoding DNA subclone in the middle of V _HIn the carrier, this carrier contains that (Rechavi etc., Proc.Natl.Acad.Sci.USA 1983,80:855) and donor splicing site (Fig. 3) from the eucaryon leader of people's antibody HG3CL.Utilization can be 5 ' and the 3 ' terminal PCR primer in XhoI and SstI site of introducing with V _KCoding DNA insert in the middle of V _KIn the carrier

(VkFor：5′-CCG?TTT?CAG?CTC?GAG?CTT?GGT?CCC-3′，

VkBack:5 '-GAC ATT GAG CTC ACC CAG TCT CCA-3 ') with Sstl-XhoI fragment cloning in the middle VK carrier that contains eucaryon leader and donor splicing site, wherein the eucaryon leader is from reconstruct people CAMPATH-1 light chain (Riechmann etc., Nature 1988,332:21).The DNA that the coding variable region is added the eucaryon flanking region is with HindIII-Bam HI fragment cloning (Maeda etc., Hum.Antibod.Hyridomas 1991 2:124) in the mammalian cell expression vector of the genomic dna that contains encode people's γ-1 constant region or people K constant region.With heavy chain and light chain expression vector electroporation in the COS cell.After 72 hours, collect nutrient solution, and with the chimeric anti-egfr antibodies of elisa assay (Kettleborough etc., Protein Eng1991,4:773).Embodiment 10: produce scFv from external immunocyte

The method of following discloses is only modified a little to aforesaid method.Described in immunity, library construction and screening such as the embodiment 1-4.Describe following step below in detail:

Screen the clone after initial library and the three-wheel elutriation, selected some monoamine penicillin G resistance bacterium colonies.Alkaline bleach liquor cleavage prepares phagemid dna, and in order to heat shock transfection E.Coli HB2151, and the latter one does not contain the suppressor gene bacterial strain.Colony inoculation is arrived among 2xTY-Amp-Glu 30 ℃ of grow overnight.The 5ml equal portions are inoculated in the 2xTY nutrient solution that contains 100mg penbritin/ml and 0.1% glucose, and 1 hour (to logarithmic phase) cultivated in 30 ℃ of joltings.Collecting cell adds isopropyl ss-D-sulfo-gala pyranoside (IPTG) and reaches expression (Be Bellis, D. and Schwartz, the I. of final concentration 1mM to lure different solubility scFv; Nucleic Acids Res; 1990,18:1311).Culture is 30 ℃ of jolting grow overnight.Taking-up contains the supernatant liquor of scFv, and filter membrane centrifugal and filtration 0.22mm makes clarification, detects then.By existing description (Kettleborough etc., EP94104160 and Eur.J.Immunol, 1994,24:952), with combining of ELISA bacteria tested supernatant liquor and EGFR.Utilization is checked the segmental specificity of selecting of scFv with culture plate and other antigen and the plastics of various relevant with EGFR and incoherent albumen bag quilts with ECISA.Used antigen is: RNase, BSA, OVA, GD ₃Sphingolipids,sialo, Vitronectic receptor (VNR), platelet glycoprotein IIbIIIa (GPIIbIIIa) and disialyl-lacto-N-tetrose (DSLNT).Under every kind of antigenic optimum concn, wrap and spent the night.The ELISA culture plate of bag quilt sealed 1 hour for 37 ℃ with the skim-milk (w/v) among the 1.5%PBS.After the flushing, in the microtitre hole, add 100ml scFv supernatant liquor, cultivated 2 hours for 37 ℃.Rabbit anti-mouse antibody (Dako) with anti--C-myc antibody 9E10 (MyC 1-9E10.2 crossbred nutrient solution) and alkaline phosphatase coupling detects bonded scFv.

Three kinds of tumor cell line A431, MDA MB231 human breast carcinoma (ATCC that contain EGFR, HTB26) and HT29 human colon adenocarcinoma (ATCC, HTB38), the binding ability that is used to test EGFR on scFv and the cell with a kind of clone WM164 that does not express EGFR, with facs analysis and not the immunofluorescence of fixed cell test.For carrying out indirect immunofluorescence analysis, cell is layered on the Terasaki plate (2 * 10 ⁴Cells/well) and cultivated 24 hours.Then cell with contain the segmental 20ml thickness of scFv bacterium supernatant liquor incubated at room temperature 90 minutes.First antibody is (anti--C-as myc) to carry out 60 minutes with the room temperature that is incubated in of second antibody.The rabbit anti-mouse antibody (DaKo) of second antibody-FICT coupling was by dilution in 1: 20.

For carrying out facs analysis, 5 * 10 ⁵(PBS-BSA) wash three times cultivated 20 minutes for 4 ℃ with the bacterium supernatant liquor that 50ml is thick individual cell with the PBS that contains 1%BSA and 0.1% sodiumazide.After washing twice with cold PBS-BSA, detect bonded scFv with being diluted in the anti--C-myc antibody among PBS-BSA and the sheep anti-mouse antibody (Becton Pickinson) of FITC coupling at 1: 25.Add iodate third ingot (PI) and reach final concentration 5mg/ml.In having the EPICS ProfILe II of air cooled argon laser, carry out the flow cytometry analysis.Light (15mV) with 488nm excites.The bandpass filter of 530nm is used to collect FITC emission light, and the bandpass filter of 625nm is used to collect PI emission light.One number converter is set on the scatterer of front side and gets rid of the PI staining cell and select viable cell.

By the segmental pcr amplification of cloning (G  ssow, D, Clackson, T; Nu-cleic Acids Res, 1989,17:4000) and the assay determination of BstNI digestion pattern (8) original library and select the diversity in library, back.With Sequenase (Sequenase) test kit (USB) with dideoxy chain termination to some cloning and sequencings (Sanger, F etc.; Proc, Nat, Acad, Sci, U.S.A 1977,74:5463).

Thickness bacterium supernatant liquor (10ml) carries out SDS-PAGE with 12.5% gel.Basic press the described method of Towbin (Towbin etc., J.Proc.Nat.Acad.Sci., U.S.A, 1979,76:4350) carry out the Western trace.Albumen is transferred to that Immobilon-P (Millipore) goes up or Nitrocellulose (on the Bio-Rad) by electroblotting.With the PBS sealing trace that contains 2% skimmed milk (w/v).Anti-mouse antibody (Jackson) and enhanced chemiluminescence system (ECL, Amersham) detection scFv fragment with anti--C-myc antibody (9E10), peroxidase coupling.

The quantitative analysis of demoulding vesicle shows that the albumen total concn is 2.5mg/ml, wherein has only 10-14%, is equivalent to EGFR (Sato etc., J.Natl.Cancer Inst, 1989,21:1601; Yeaton, R etc., J.Biol Chem., 1983,258:9254) content is about 250-350ng/ml.With PAGE-SDS electrophoretic analysis Coomassie blue stain then, show that vesicle contains quite complicated egg white mixture.Do not detect proteolytic degradation.The Western engram analysis shows under our experiment condition, has the complete molecule of EGF acceptor in film vesicle preparation.

In order to determine whether FCS and lymphokine are needed, compared to contain 2% or MTC and the M2 of 4%FCS.The vesicle and the PBS that contain EGFR are used separately as antigen and contrast.Splenocyte was hatched 3 hours in M1 (serum-free) having or do not have on antigenic 16 orifice plates.Add MTC or M2 then, screen with the A431 immobilized cell after 72,96,120 or 1 44 hours.In all experiments, the viable count that is write down is between 20-40%, and (Gavilondo-Cowley, J. etc. conform to the document result; In Vitro Immuniza-tion in Hybridoma Technology, Elsevier Science Publishers B.V., Amsterdam, P131).Obtaining maximum specificity at the 4th day during with MTC replys; And with containing 4% or during the M2 of 20%FCS (final concentration is 2% or 10%), maximum answering delay to the 6 days (table 2).But MTC and 10%FCS may evoke nonspecific response by polyclone activation, and this can be from non-ly specially replying the result that ratio represents and find out with special.So we determine to use is to cultivate 6 days with the M2 that 4%FCS replenishes in later experiment.

Exist EGFR to strengthen greatly to this antigenic replying on the vesicle surface.Press and above-mentioned similar experimental technique, compared from the vesicle of expressing or not expressing the clone of EGFR.Lymphocyte and vesicle were cultivated in M1 3 hours, added the M2 that contains 4%FCS then.Every group lymphocyte was cultivated 48 hours in 96 orifice plates with EGFR, A431-fixed cell, RNase or GD3 bag quilt after 6 days.As desired, these result of experiment show the polyspecific pattern (table 3) of replying.When the vesicle of expressing EGFR was used as antigen, what optical density was represented obviously improved with the reactive of EGFR.

In sum, these results suggest (though is that all right ripe): exist measurable antigen dependency to reply after the external immunity, this external immunity produces the several groups of anti-EGFR immunity lymphocytes that are suitable for variable region PCR clone.

To after the scFv fragment cloning of external immunity is in the pHEN1 phagemid, obtain 1.1 * 10 ⁵Individual clone's library.This library be with other two carry out in the body the parallel generation in library that immunity produces.The structure of these phage libraries was existing in the past to be described (Kettleborough etc., EP 94104160 and Eur.J.Immund, 1994,24:952).

In order to select fragment, with the immunity pipe elutriation phage of EGFR bag quilt with EGFR bonded scFv.The phage of wash-out is used for ehec infection SupE strain again.Carrying out three-wheel altogether selects.There is a no antigenic test tube to carry out parallel laboratory test in every the wheel to calculate background.In the elutriation first time, in immune pipe, add 1.5 * 10 ¹⁰Individual phage particle has gone out 6.6 * 10 from the immunity pipe wash-out that wraps quilt ⁴Individual; And from the background phage-infest, only obtain 200 clones.After the elutriation, add 1 * 10 for the third time ¹¹Individual phage, wash-out goes out 5.6 * 10 ¹⁰Individual.

In order further to identify the scFv fragment, before selection and every take turns selection after, we select 22 clones altogether from phage-infest.Analyze the diversity in library with the segmental BstNI restriction enzyme mapping of cloning.Library performance difference before selecting is very big.It is identical that the fingerprint in conjunction with the clone after the first round is selected shows several groups restriction map.

Based on their restriction enzyme mapping, from selecting, different rounds select the clone.Dna sequencing shows in the selected clone of major part and has different sequences.The length of the complementary determining region (CDR) of clone 10D2,5D3,10E2,1B3,4B3 and 5E2 is different with composition.Maximum variation sees V _HAnd V _LThe CDR3 district of sequence.Clone 5D3 and 1E3 select from third round.ELISA and flow cytometry analysis show that they are extremely strong with combining of EGFR, and have identical sequence.

Grow in the presence of IPTG by the coli strain HB2151 that does not contain suppressor gene, obtain solubility scFv fragment.

In order to confirm the generation of scFv, the individual clone's of gel electrophoresis analysis inoculum.The Western engram analysis shows one 35, the clearly band about 000KD.

Identify to have EGFR by ELISA in conjunction with active clone.In order to check selected clone's cross reactivity, carry out elisa assay with synantigen not.Antigen (EGFR, RNase, BSA, KLH, OVA, GD3 Sphingolipids,sialo, Vitronectic receptor, platelet glycoprotein IIbIIIa, and disalyl-lacto-N-tetrose) is coated on (table 4) in the elisa plate with optimum concn.Do not detect with non-EGFR is antigenic and combine.ScFv and three kinds of combinations that contain the tumor cell line (people's epidermoid carcinoma A431, human breast carcinoma MDA MB231 and human colon adenocarcinoma HT29) of EGFR have also been tested.The human melanoma cell WM164 that does not express EGFR is as negative control.Clone with the tumour cell bonded by carry out the indirect immunofluorescence analysis test with the on-fixed cell, and carry out quantitatively with facs analysis.Use the on-fixed cell to guarantee the native conformation of membrane receptor.Positive colony shows tangible fluorescence with the A431 cell.A little less than containing the fluorescence of tumor cell line of EGFR with other.Do not detect and combining that negative cells is.The flow cytometry analysis has confirmed these results.Analyzed combining of 17 positive colonies and 3 negative clones and A431, MDA MB231 and HT29 cell with the flow cytometry method.WM164 is as negative cells system.425scFv (P1 clone) is as positive control, and cloning vector (HEN) is as negative control.The result is comprehensively in table 5.Identical with elisa assay, two clones of 4B2 and 5E2 combine with EGFR and are positive, but are negative with the combining of tumor cell line of expressing EGFR.

Table 2: different substratum are to external immunity ^A)Influence

		To the time of A431 screening
						The 3rd day	The 4th day	The 5th day	The 6th day
		Experiment	Antigen	???O.D. c)????d)	?O.D.					?O.D.	OD. ratio
????1	Vesicle PBS					???0.393????2.11 ???0.186	?0.801????3.76 ?0.213	?0.784????3.90 ?0.201	?0.951????10.3 ?0.092
		????2	Vesicle PBS	???0.527????2.50 ???0.210	?0.852????1.76 ?0.482					?0.863????2.75 ?0.313	?1.168????3.94 ?0.296
????3	Vesicle PBS					???0.763????1.48 ???0.513	?1.169????2.01 ?0.581	?1.089????2.07 ?0.525	?1.115????1.91 ?0.581

Experiment 1:M1 adds M ₂, (final concentration of FCS: 2%) experiment 2:M1 adds M to 4%FCS ₂, (final concentration of FCS: 10%) experiment 3:A substratum adds MTC to 20%FCS, and 4%FCS (final concentration of FCS: 2%) a) hatch in 3.5ml M1 at the vesicle or the PBS of the Kong Zhongyu of six orifice plates A431 cell by BALB/c mouse splenocyte (107).Add 3.5ml then and contain 4% or MTC or the M2 of 20%FCS, and hatch.The 3rd, 4,5 or 6 days, from substratum, take out external immune lymphocyte, flushing is removed vesicle and is planted in 96 orifice plates with immobilization A431 cell envelope in HBSS, hatches 48 hours (square method part) again.B) final concentration of FCS in the substratum.C) O.D. is the optical density readings at the 405nm place.It represents the mean value in 16 holes.D) ratio of special replying (vesicle is made antigen)/nonspecific response (PBS makes antigen).

Table 3: the polyspecific of replying after the external immunity ^A)

Screening
	Antigen group A431CELLS EGFR GD3 RNase
Test
	1 EGFR+ 0.512 (b)????0.326???0.140??0.249 ??????EGFR-???0.427???????0.070???0.123??0.304
Test 2 EGFR+, 1.430 0.730 0.233 0.670 EGFR-0.789 0.195 0.118 0.561

A) the lymphocyte vesicle (EGFR that expresses EGFR ⁺) or do not express the vesicle (EGFR of EGFR ^-) external immunity.After hatching 6 days, from culture, remove cell and above-mentioned antigen is screened.B) replying optical density (405nm) expresses.

Table 4: selected scFv fragment is to several antigenic cross reactivities ^(a)

Antigen (b)Package amount [mg/ml] result
	????EGFR????????????2.5???????????????+ ????RNase???????????10????????????????- ????BSA?????????????10????????????????- ????KLH?????????????10????????????????- ????OVA?????????????10????????????????- ????GD 3Sphingolipids,sialo 2-VNR 1-GPIIbIIIa 1-DSLNT 5-

A) carry out elisa assay as mentioned above.

B) Vitronectic receptor (VNR); Platelet glycoprotein IIbIIIa

(GPIIbIIIa); Disialyl-lacto-N-tetrose (DSLNT).

The scFv clone's of the anti-EGFR of table 5 reactivity.Comparative result between the cell counting of use antigenic ELISA method of purification of soluble and clone is analyzed.

The cell counting analysis of clone's tumor cell line (a)The mean value (O.D.) of ELISA experience flat fluorescent
	Positive WM164 A431 MDAAMB231 HT29 EGFR
?7H1?????1.5????112.9????16.4???????2.6????1.2 ?4B2?????1.2????5.3??????4.2????????0.6????2 ?10D2????1.5????145.3????36.3???????4.8????2 ?12D2????1.8????129.5????29.3???????5.7????2 ?5E2?????1.4????2.5??????7.1????????0.5????1.8 ?8E2?????1.5????134.5????47.7???????5.1????1.9 ?5F2?????1.3????146.3????40.6???????5.7????1.9 ?11H2????1.9????152.2????25.3???????2??????1.9 ?1B3?????0.6????105.1????36.4???????5.2????＞2 ?4B3?????0.5????78???????15.8???????2.3????2 ?3D3?????1.2????94.3?????25.1???????4.8????1.9 ?5D3?????0.5????112??????22.2???????5.5????＞2 ?4F3?????0.4????110.3????32.3???????6.2????＞2
	?4G3?????0.4????76.5?????20.4???????2??????＞2 ?1E3?????0.4????118.3????33.8???????5.1????2 ?3H3?????0.6????76.5?????33.7???????4.2????＞2

Table 5 (continuing)

Negative 5F1 2.4 2.3 3.6 1.8 0.2 7G1 1.4 10.2 4 2.8 0.2 1H1 0.5 54 0.75 0.2
	Contrast (b)?HEN????0.4????4.1????3.7????1??????0.2 ?P1?????0.6????85.5???21.3???2.5????1.9

A) clone (A431, MDAAMB231 and HT29) and a kind of clone (WM164) of not expressing EGFR of three kinds of band EGFR are used for described cell counting analysis, detect the binding ability of scFv and tumour cell.B) there is not segmental carrier (HEN) and make feminine gender and positive control respectively from the scFv fragment of 425mAb (P1).

Sequence table (1) general information:

(i) applicant:

(A) title: Merck Patent GmbH

(B) street: Frankfurter str 255

(C) city: Darmstadt

(E) country: Germany

(F) postcode: 64271

(G) phone: 49-6151-727022

(H) fax: 49-6151-727191

(ii) invention exercise question: the single chain variable fragment of anti-epidermal growth factor receptor and antibody is sequence number (iii): 32

(iv) computer-reader form:

(A) medium type: floppy disk

(B) computer: IBM PC compatibility

(C) operating system: PC-DOS/MS-DOS

(D) software: Patent In Release#1.0, the information of Version#1.30 (EPO) (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 327 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: CDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(D) vegetative period: grow up

(F) types of organization; Lymphoglandula

(vii) direct sources:

(B) clone: L2 11C (light chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..327

(xi) sequence description: SEQ ID NO:1GAC ATT GAG CTC ACC CAG TCT CCA GCC TCC CTG GCT GCA TCT GTG GGA 48Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Ala Ser Val Gly 15 10 15GAA ACT GTC ACC ATC ACA TGT CGA GCA AGT GAG AAC ATT TAC TAT AGT 96Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Tyr Ser

20??????????????????25??????????????????30TTA?GCA?TGG?TAT?CAG?CAG?AAG?CAA?GGG?AAA?TCT?CCT?CAG?CTC?CTG?ATC????144Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Gln?Gly?Lys?Ser?Pro?Gln?Leu?Leu?Ile

35??????????????????40??????????????????45TAT?AGT?GCA?AGC?GCC?TTG?GAA?GAT?GGT?GTC?CCA?TCG?AGG?TTC?AGT?GGC????192Tyr?Ser?Ala?Ser?Ala?Leu?Glu?Asp?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50??????????????????55??????????????????60AGT?GGA?TCT?GGG?ACA?CAG?TAT?TCT?TTA?AAG?ATC?AAC?AAC?ATG?CAG?CCT????240Ser?Gly?Ser?Gly?Thr?Gln?Tyr?Ser?Leu?Lys?Ile?Asn?Asn?Met?Gln?Pro?65??????????????????70??????????????????75??????????????????80GAA?GAT?ACC?GCT?ACT?TAC?TTC?TGT?AAA?CAG?ACT?TAT?GAC?GTT?CCG?TGG????288Glu?Asp?Thr?Ala?Thr?Tyr?Phe?Cys?Lys?Gln?Thr?Tyr?Asp?Val?Pro?Trp

85??????????????????90??????????????????95ACG?TTC?GGT?GGA?GGG?ACC?AAG?CTG?GAA?ATA?AAA?CGG?GCG????????????????327Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Ala

The information of 100 105 (2) SEQ ID NO:2

(i) sequence characteristic.

(A) length: 109 amino acid

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:2Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Ala Ser Val Gly 15 10 15Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Tyr Ser

20??????????????????????25??????????????????30Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Gln?GLy?Lys?Ser?Pro?Gln?Leu?Leu?Ile

35??????????????????40??????????????????45Tyr?Ser?Ala?Ser?Ala?Leu?Glu?Asp?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50??????????????????55??????????????????60Ser?Gly?Ser?Gly?Thr?Gln?Tyr?Ser?Leu?Lys?Ile?Asn?Asn?Met?Gln?Pro?65??????????????????70??????????????????75??????????????????80Glu?Asp?Thr?Ala?Thr?Tyr?Phe?Cys?Lys?Gln?Thr?Tyr?Asp?Val?Pro?Trp

85??????????????????90??????????????????95Thr?Phe?G1y?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Ala

The information of 100 105 (2) SEQ ID NO:3

(i) sequence characteristic:

(A) length: 357 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(C) vegetative period: grow up

(F) types of organization: lymphoglandula

(vii) direct sources

(B) clone: L2 11C (heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..357

(xi，SEQUENCE?DESCRIPTION：SEQ?ID?NO：3：CAG?GTG?CAA?CTG?CAG?GAG?TCA?GGG?CCT?GAG?CTG?GTG?AGG?CCT?GGG?GCT????48Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Glu?Leu?Val?Arg?Pro?Gly?Ala110?????????????????115?????????????????120?????????????????125TCA?GTG?AAG?ATG?TCC?TGC?AAG?GCT?TCA?GGC?TAT?ACC?TTC?ACT?ACC?TAC????96Ser?Val?Lys?Met?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Thr?Tyr

130?????????????????135?????????????????140TGG?ATA?CAC?TGG?ATG?AAA?CAG?AGG?CCT?GGA?CAA?GGC?CTT?CAG?TGG?ATT????144Trp?Ile?His?Trp?Met?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Gln?Trp?Ile

145?????????????????150?????????????????155GGC?ATG?ATT?GAT?CCT?TCC?AAT?AGT?GAA?ACT?AGG?TTA?AAT?CAG?AAT?TTC????192Gly?Met?Ile?Asp?Pro?Ser?Asn?Ser?Glu?Thr?Arg?Leu?Asn?Gln?Asn?Phe

160?????????????????165?????????????????170AGG?GAC?AAG?GCC?ACA?TTG?AGT?GTA?GAC?AAA?TCC?TCC?AAT?AAA?GCC?TAC????240Arg?Asp?Lys?Ala?Thr?Leu?Ser?Val?Asp?Lys?Ser?Ser?Ash?Lys?Ala?Tyr???175??????????????????180?????????????????185ATG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?TCT?GCA?ATC?TAT?TAC?TGT????288Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Ile?Tyr?Tyr?Cys190?????????????????195?????????????????200?????????????????205GCA?AGA?TGG?GAC?TAC?GGT?AGT?GGC?CAC?TTT?GAC?TAC?TGG?GGC?CAA?GGG????336Ala?Arg?Trp?Asp?Tyr?Gly?Ser?Gly?His?Phe?Asp?Tyr?Trp?Gly?Gln?Gly

210?????????????????215?????????????????220ACC?ACG?GTC?ACC?GTC?TCC?TCA????????????????????????????????????????????????????????????357Thr?Thr?Val?Thr?Val?Ser?Ser

The information of 225 (2) SEQ ID NO:4

(i) sequence characteristic:

(A) length: 119 bases

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:4:Gln Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Arg Pro Gly Ala 15 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20??????????????????25??????????????????30Trp?Ile?His?Trp?Met?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Gln?Trp?Ile

35??????????????????40??????????????????45Gly?Met?Ile?Asp?Pro?Ser?Asn?Ser?Glu?Thr?Arg?Leu?Asn?Gln?Asn?Phe

50??????????????????55??????????????????60Arg?AspLys?Ala?Thr?Leu?Ser?Val?Asp?Lys?Ser?Ser?Asn?Lys?Ala?Tyr?65?????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Ile?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Arg?Trp?Asp?Tyr?Gly?Ser?Gly?His?Phe?Asp?Tyr?Trp?Gly?Gln?Gly

100????????????????105??????????????????110Thr?Thr?Val?Thr?Val?Ser?Ser

115

The information of SEQ ID NO:5

(i) sequence characteristic:

(A) length: 339 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(C) vegetative period: grow up

(F) types of organization: lymphoglandula

(vii) direct sources

(B) clone: L2 11B (light chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..339

(xi) sequence description: SEQ ID NO:5:GAC ATT GAG CTC ACC CAG TCT CCA GCT TCT TTG GCT GTG TCT CTA GGG 48Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly120 125 130 135CAG AGG GCC ACC ATC TCC TGC AGA GCC AGC GAA AGT GTT GAT AAT TTT 96Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Phe

140?????????????????145?????????????????150GGC?ATT?AGT?TTT?ATG?AAC?TGG?TTC?CAA?CAG?AAA?CCA?GGA?CAG?CCA?CCC????144Gly?Ile?Ser?Phe?Met?Asn?Trp?Phe?Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro

155?????????????????160?????????????????165AAA?CTC?CTC?ATC?TAT?GGT?GCA?TCC?AAC?CAA?GGA?TCC?GGG?GTC?CCT?GCC????192Lys?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Asn?Gln?Gly?Ser?Gly?Val?Pro?Ala

170?????????????????175?????????????????180AGG?TTT?AGT?GGC?AGT?GGG?TCT?GGG?ACA?GAC?TTC?AGC?CTC?AAC?ATC?CAT????240Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ser?Leu?Asn?Ile?His

185?????????????????190?????????????????195CCT?CTG?GAG?GAG?GAT?GAT?ACT?GCA?ATG?TAT?TTC?TGT?CAG?CAA?AGT?AAG????288Pro?Leu?Glu?Glu?Asp?Asp?Thr?Ala?Met?Tyr?Phe?Cys?Gln?Gln?Ser?Lys200?????????????????205?????????????????210?????????????????215GAG?GTT?CCG?CTC?ACG?TTC?GGT?GCT?GGG?ACC?AAG?CTG?GAA?ATA?AAA?CGG????336Glu?Val?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg

220?????????????????225?????????????????230GCG??????????????????????????????????????????????????????????????? 339Ala

(2) information of SEQ ID NO:6

(i) sequence characteristic:

(A) length: 113 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:6:Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 15 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Phe

20??????????????????25??????????????????30Gly?Ile?Ser?Phe?Met?Asn?Trp?Phe?Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro

35??????????????????40??????????????????45Lys?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Asn?Gln?Gly?Ser?Gly?Val?Pro?Ala

50??????????????????55??????????????????60Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ser?Leu?Asn?Ile?His?65??????????????????70??????????????????75??????????????????80Pro?Leu?Glu?Glu?Asp?Asp?Thr?Ala?Met?Tyr?Phe?Cys?Gln?Gln?Ser?Lys

85??????????????????90??????????????????95Glu?Val?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg

100?????????????????105?????????????????110Ala

(2) information of SEQ ID NO:7

(i) sequence characteristic:

(A) length: 357 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(C) vegetative period: grow up

(F) types of organization: lymphoglandula

(vii) direct sources

(B) clone: L2 11B (heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..357

(xi) sequence description: SEQ ID NO:7:CAG GTG CAG CTG CAG GAG TCT GGA CCT GAG CTG GTG AAG CCT GGG GCT 48Gln Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

115?????????????????120?????????????????125TTA?GTG?AAG?ATA?TCC?TGC?AAG?GCT?TCT?GGT?TAC?ACC?TTC?ACC?AGC?TAC????96Leu?Val?Lys?Ile?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser?Tyr130?????????????????135?????????????????140?????????????????145TGG?ATG?CAC?TGG?GTG?AAG?CAG?AGG?CCT?GGA?CAA?GGC?CTT?GAG?TGG?ATC????144Trp?Met?His?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile

150?????????????????155?????????????????160GGA?GAG?ATT?GAT?CCT?TCT?GAT?AGT?TAT?ACT?AAC?TAC?AAT?CAA?AAG?TTC????192Gly?Glu?Ile?Asp?Pro?Ser?Asp?Ser?Tyr?Thr?Asn?Tyr?Asn?Gln?Lys?Phe

165?????????????????170?????????????????175AAG?GGC?AAG?GCC?ACA?TTG?ACT?GTA?GAC?AAA?TCC?TCC?AAC?ACA?GCC?TAC????240Lys?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Asn?Thr?Ala?Tyr

180?????????????????185?????????????????190ATG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?TCT?GCG?GTC?TAT?TAC?TGT????288Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

195?????????????????200?????????????????205GCA?AGA?TCG?GAC?TAC?GGT?AGT?AGC?CAC?TTT?GAC?TAC?TGG?GGC?CAA?GGG????336Ala?Arg?Ser?Asp?Tyr?Gly?Ser?Ser?His?Phe?Asp?Tyr?Trp?Gly?Gln?Gly210?????????????????215?????????????????220?????????????????225ACC?ACG?GTC?ACC?GTC?TCC?TCA????????????????????????????????????????357Thr?Thr?Val?Thr?Val?Ser?Ser

230

(2) information of SEQ ID NO:8

(i) sequence characteristic:

(A) length: 119 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:8:Gln Val Gln Leu Gln Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 15 10 15Leu Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20??????????????????25??????????????????30Trp?Met?His?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35??????????????????40??????????????????45Gly?Glu?Ile?Asp?Pro?Ser?Asp?Ser?Tyr?Thr?Asn?Tyr?Asn?Gln?Lys?Phe

50??????????????????55??????????????????60Lys?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Asn?Thr?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Arg?Ser?Asp?Tyr?Gly?Ser?Ser?His?Phe?Asp?Tyr?Trp?Gly?Gln?Gly

100?????????????????105?????????????????110Thr?Thr?Val?Thr?Val?Ser?Ser

115

The information of SEQ ID NO:9

(i) sequence characteristic:

(A) length: 339 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(bv clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(C) vegetative period: grow up

(F) types of organization: lymphoglandula

(vii) direct sources

(B) clone: L3 11D (light chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..339

(xi) sequence description: SEQ ID NO:9:GAC ATT GAG CTC ACC CAG TCT CCA GCT TCT TTG GCT GTG TCT CTA GGG 48Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly120 125 130 135CAG AGG GCC ACC ATC TCC TGC CGA GCC AGC GAA AGT GTT GAT AAT TTT 96Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Phe

140?????????????????145?????????????????150GGC?ATT?AGT?TTT?ATG?AAC?TGG?TTC?CAA?CAG?AAA?CCA?GGA?CAG?CCA?CCC????144Gly?Ile?Ser?Phe?Met?Asn?Trp?Phe?Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro

155?????????????????160?????????????????165AAA?CTC?CTC?ATC?TAT?GGT?GCA?TCC?AAC?CAA?GGA?TCC?GGG?GTC?CCT?GCC????192Lys?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Asn?Gln?Gly?Ser?Gly?Val?Pro?Ala

170?????????????????175?????????????????180AGG?TTT?AGT?GGC?AGT?GGG?TCT?GGG?ACA?GAC?TTC?AGC?CTC?AAC?ATC?CAT????240Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ser?Leu?Asn?Ile?His

185?????????????????190?????????????????195CCT?TTG?GAG?GAG?GAT?GAT?ACT?GCA?ATG?TAT?TTC?TGT?CAG?CAA?AGT?AAG????288Pro?Leu?Glu?Glu?Asp?Asp?Thr?Ala?Met?Tyr?Phe?Cys?Gln?Gln?Ser?Lys200?????????????????205?????????????????210?????????????????215GAG?GTT?CCG?CTC?ACG?TTC?GGT?GCT?GGG?ACC?AAG?CTG?GAG?CTG?AAA?CGG????336Glu?Val?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu?Leu?Lys?Arg

220?????????????????225?????????????????230GCG????????????????????????????????????????????????????????????????339Ala

(2) information of SEQ ID NO:10

(i) sequence characteristic:

(A) length: 113 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:10:Asp Ile Glu Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 15 10 15Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Phe

20??????????????????25??????????????????30Gly?Ile?Ser?Phe?Met?Asn?Trp?Phe?Gln?Gln?Lys?Pro?Gly?Gln?Pro?Pro

35??????????????????40??????????????????45Lys?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Asn?Gln?Gly?Ser?Gly?Val?Pro?Ala

50??????????????????55??????????????????60Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ser?Leu?Asn?Ile?His?65??????????????????70??????????????????75??????????????????80Pro?Leu?Glu?Glu?Asp?Asp?Thr?Ala?Met?Tyr?Phe?Cys?Gln?Gln?Ser?Lys

85??????????????????90??????????????????95Glu?Val?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu?Leu?Lys?Arg

100?????????????????105?????????????????110Ala

(2) information of SEQ ID NO:1I

(i) sequence characteristic:

(A) length: 357 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(C) vegetative period: grow up

(F) types of organization: lymphoglandula

(vii) direct sources

(B) clone: L3 11D (heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..357

(xi) sequence description: SEQ ID NO:11:GAG GTG CAG CTG CAG CAG TCA GGG GCT GAG CTT GTG AAG CCT GGG GCT 48Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

115?????????????????120?????????????????125TCA?GTG?AAG?CTG?TCC?TGC?AAG?GCT?TCT?GGC?TAC?ACC?TTC?ACC?AGC?TAC????96Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser?Tyr130?????????????????135?????????????????140?????????????????145TGG?ATG?CAC?TGG?GTG?AAG?CAG?AGG?CCT?GGA?CAA?GGC?CTT?GAG?TGG?ATC????144Trp?Met?His?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile

150?????????????????155?????????????????160GGA?GAG?ATT?GAT?CCT?TCT?GAT?AGT?TAT?ACT?AAC?TAC?AAT?CAA?AAG?TTC????192Gly?Glu?Ile?Asp?Pro?Ser?Asp?Ser?Tyr?Thr?Asn?Tyr?Asn?Gln?Lys?Phe

165?????????????????170?????????????????175AAG?GGC?AAG?GCC?ACA?TTG?ACT?GTA?GAC?AAA?TCC?TCC?AGC?ACA?GCC?TAC????240Lys?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr

180?????????????????185?????????????????190ATG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?TCT?GCG?GTC?TAT?TAC?TGT????288Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

195?????????????????200?????????????????205GCA?AGA?TCG?GAC?TAC?GGT?AGT?AGC?CAC?TTT?GAC?TAC?TGG?GGC?CAA?GGG????336Ala?Arg?Ser?Asp?Tyr?Gly?Ser?Ser?His?Phe?Asp?Tyr?Trp?Gly?Gln?Gly210?????????????????215?????????????????220?????????????????225ACC?ACG?GTC?ACC?GTC?TCC?TCA????????????????????????????????????????357Thr?Thr?Val?Thr?Val?Ser?Ser

230

(2) information of SEQ ID NO:12

(ⅰ) sequence characteristic:

(A) length: 119 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: protein

(ⅹ ⅰ) sequence description: SEQ ID NO:12:Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 15 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20??????????????????25??????????????????30Trp?Met?His?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35??????????????????40??????????????????45Gly?Glu?Ile?Asp?Pro?Ser?Asp?Ser?Tyr?Thr?Asn?Tyr?Asn?Gln?Lys?Phe

50??????????????????55??????????????????60Lys?Gly?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Arg?Ser?Asp?Tyr?Gly?Ser?Ser?His?Phe?Asp?Tyr?Trp?Gly?Gln?Gly

100?????????????????105?????????????????110Thr?Thr?Val?Thr?Val?Ser?Ser

115

(2) information of SEQ ID NO:13

(i) sequence characteristic:

(A) length: 327 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(C) vegetative period: grow up

(F) types of organization: lymphoglandula

(vii) direct sources

(B) clone: S4 2D (light chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..327

(xi) sequence description: SEQ ID NO:13:GAC ATT GAG CTC ACC CAG TCT CCA ACC ACC ATG GCT GCA TCT CCC GGG 48Asp Ile Glu Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly120 125 130 135GAG AAG ATC ACT ATC ACC TGC AGT GCC AGC TCA AGT ATA AGT TCC AAT 96Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn

140?????????????????145?????????????????150TAC?TTG?CAT?TGG?TAT?CAG?CAG?AAG?CCA?GGA?TTC?TCC?CCT?AAA?CTC?TTG????144Tyr?Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Phe?Ser?Pro?Lys?Leu?Leu

155?????????????????160?????????????????165ATT?TAT?AGG?ACA?TCC?AAT?CTG?GCT?TCT?GGA?GTC?CCA?GCT?CGC?TTC?AGT????192Ile?Tyr?Arg?Thr?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser

170?????????????????175?????????????????180GGC?AGT?GGG?TCT?GGG?ACC?TCT?TAC?TCT?CTC?ACA?ATT?GGC?ACC?ATG?GAG????240Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Gly?Thr?Met?Glu

185?????????????????190?????????????????195GCT?GAA?GAT?GTT?GCC?ACT?TAC?TAC?TGC?CAG?CAG?GGT?AGT?AGT?ATA?CCA????288Ala?Glu?Asp?Val?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Gly?Ser?Ser?Ile?Pro200?????????????????205?????????????????210?????????????????215CGC?ACG?TTC?GGA?GGG?GGC?ACC?AAG?CTG?GAA?ATC?AAA?CGG????????????????327Arg?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg

220?????????????????225

(2) information of SEQ ID NO:1 4

(ⅰ) sequence characteristic:

(A) length: 109 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ⅱ) molecule type: protein

(ⅹ ⅰ) sequence description: SEQ ID NO:14:Asp Ile Glu Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly 15 10 15Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Asn

20??????????????????25??????????????????30Tyr?Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Phe?Ser?Pro?Lys?Leu?Leu

35??????????????????40??????????????????45Ile?Tyr?Arg?Thr?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser

50??????????????????55??????????????????60Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?Ile?Gly?Thr?Met?Glu?65??????????????????70??????????????????75??????????????????80Ala?Glu?Asp?Val?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Gly?Ser?Ser?Ile?Pro

85??????????????????90??????????????????95Arg?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg

100?????????????????105

(2) information of SEQ ID NO:15

(i) sequence characteristic:

(A) length: 354 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(C) vegetative period: grow up

(F) types of organization: lymphoglandula

() vii direct sources

(B) clone: S4 2D (heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..354

(xi) sequence description: SEQ ID NO:15:GAG GTC AAG CTG CAG CAG TCA GGA CCT GAG CTG GTA AAG CCT GGG GCT 48Glu Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala110 115 120 125TCA GTG AAG ATG TCC TGC AAG GCT TCT GGA TAC GCA TTC ATA AGT TTT 96Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ile Ser Phe

130?????????????????135?????????????????140GTT?ATG?CAC?TGG?GTG?AAG?CAG?AAG?CCT?GGG?CAG?GGC?CTT?GAG?TGG?ATT????144Val?Met?His?Trp?Val?Lys?Gln?Lys?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile

145?????????????????150?????????????????155GGA?TTT?ATT?AAT?CCT?TAC?AAT?GAT?GGT?ACT?AAG?TAC?AAT?GAG?AAG?TTC????192Gly?Phe?Ile?Asn?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn?Glu?Lys?Phe

160?????????????????165?????????????????170AAA?GAC?AAG?GCC?ACA?CTG?ACT?TCA?GAC?AAA?TCC?TCC?AGC?ACA?GCC?TAC????240Lys?Asp?Lys?Ala?Thr?Leu?Thr?Ser?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr

175?????????????????180?????????????????185ATG?GAG?CTC?AGC?AGC?CTG?ACC?TCT?GAG?GAC?TCT?GCG?GTC?TAT?TAC?TGT????288Met?Glu?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys190?????????????????195?????????????????200?????????????????205GCA?AGT?GGG?GAT?TAC?GAC?AGG?GCT?ATG?GAC?TAC?TGG?GGC?CAA?GGG?ACC????336Ala?Ser?Gly?Asp?Tyr?Asp?Arg?Ala?Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr

210?????????????????215?????????????????220ACG?GTC?ACC?GTC?TCC?TCA????????????????????????????????????????????354Thr?Val?Thr?Val?Ser?Ser

225

(2) information of SEQ ID NO:16

(i) sequence characteristic:

(A) length: 118 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:16:Glu Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 15 10 15Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ile Ser Phe

20??????????????????25??????????????????30Val?Met?His?Trp?Val?Lys?Gln?Lys?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35??????????????????40??????????????????45Gly?Phe?Ile?Asn?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn?Glu?Lys?Phe

50??????????????????55??????????????????60Lys?Asp?Lys?Ala?Thr?Leu?Thr?Ser?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Met?Glu?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Ser?Gly?Asp?Tyr?Asp?Arg?Ala?Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr

100?????????????????105?????????????????110Thr?Val?Thr?Val?Ser?Ser

115

(2) information of SEQ ID NO:17

(i) sequence characteristic:

(A) length: 717 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(C) vegetative period: grow up

(F) types of organization: splenocyte

(vii) direct sources

(B) clone: 4B2

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..717

(xi) sequence description: SEQ ID NO:17:GAG GTG AAG CTG CAG GAG TCT GGG GGA GAC TTA GTG AAG CCT GGA GGG 48Glu Val Lys Leu Gln Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

120?????????????????125?????????????????130TCC?CTG?AAA?CTC?TCC?TGT?GCA?GCC?TCT?GGA?TTC?ACT?TTC?AGT?AGC?TAT????96Ser?Leu?Lys?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr135?????????????????140?????????????????145?????????????????150GGC?ATG?TCT?TGG?GTT?CGG?CAG?ACT?CCA?GAC?AAG?AGG?CTG?GAG?TCT?GTC????144Gly?Met?Ser?Trp?Val?Arg?Gln?Thr?Pro?Asp?Lys?Arg?Leu?Glu?Ser?Val

155?????????????????160?????????????????165GCA?ACC?ATT?AGT?AGT?GGT?GGT?GCT?TAC?ATC?TAC?TAT?CCA?GAC?AGT?GTG????192Ala?Thr?Ile?Ser?Ser?Gly?Gly?Ala?Tyr?Ile?Tyr?Tyr?Pro?Asp?Ser?Val

170?????????????????175?????????????????180AAG?GGG?CGA?TTC?ACC?ATC?TCC?AGA?GAC?AAT?GCC?AAG?AAC?ACC?CTG?TAC????240Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Asn?Thr?Leu?Tyr

185?????????????????190?????????????????195CTG?CAA?ATG?AGC?AGT?CTG?AAG?TCT?GAG?GAC?ACA?GCC?ATG?TAT?TAC?TGT????288Leu?Gln?Met?Ser?Ser?Leu?Lys?Ser?Glu?Asp?Thr?Ala?Met?Tyr?Tyr?Cys

200?????????????????205?????????????????210GCA?AGA?CTT?GAA?ACC?GGG?GAC?TAT?GCT?TTG?GAC?TAC?TGG?GGC?CAA?GGG????336Ala?Arg?Leu?Glu?Thr?Gly?Asp?Tyr?Ala?Leu?Asp?Tyr?Trp?Gly?Gln?Gly215?????????????????220?????????????????225?????????????????230ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGC?GGT?GGC?TCG?GGC?GGT?GGT?GGG????384Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly

235?????????????????240?????????????????245TCG?GGT?GGC?GGC?GGA?TCT?GAC?ATT?GAG?CTC?ACC?CAG?TCT?CCA?GCT?TCT????432Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro?Ala?Ser

250?????????????????255?????????????????260TTG?GCT?GTC?TCT?CTA?GGG?CAG?AGG?GCC?ACC?ATA?TTC?TGC?AAG?GAC?AGC????480Leu?Ala?Val?Ser?Leu?Gly?Gln?Arg?Ala?Thr?Ile?Phe?Cys?Lys?Asp?Ser

265?????????????????270?????????????????275CAA?AGT?GTT?GAT?TAT?GAT?GGT?GAT?AGT?TAT?ATG?AAC?TGG?TAC?CAA?CAG????528Gln?Ser?Val?Asp?Tyr?Asp?Gly?Asp?Ser?Tyr?Met?Asn?Trp?Tyr?Gln?Gln

280?????????????????285?????????????????290AAA?CCA?GGA?CAG?CCA?CCC?AAA?CTC?CTC?ATC?TAT?GCT?CGA?TCC?AAT?CTA????576Lys?Pro?Gly?Gln?Pro?Pro?Lys?Leu?Leu?Ile?Tyr?Ala?Arg?Ser?Asn?Leu295?????????????????300?????????????????305?????????????????310GAA?TCT?GGG?GTC?CCT?GCC?AGG?TTT?AGT?GGC?AGT?GGG?TCT?GGG?ACA?GAC????624Glu?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp

315?????????????????320?????????????????325TTC?AGC?CTC?AAC?ATC?CAT?CCT?GTG?GAG?GAG?GAT?GAT?ATT?GCA?ATG?TAT????672Phe?Ser?Leu?Asn?Ile?His?Pro?Val?Glu?Glu?Asp?Asp?Ile?Ala?Met?Tyr

330?????????????????335?????????????????340TTC?TGT?CAG?CAA?AGT?AGG?AAG?GTT?CCG?TGG?TCG?TTC?GGT?GGA?GGG????????717Phe?Cys?Gln?Gln?Ser?Arg?Lys?Val?Pro?Trp?Ser?Phe?Gly?Gly?Gly

345?????????????????350?????????????????355

(2) information of SEQ ID NO:18

(i) sequence characteristic:

(A) length: 239 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:18:Glu Val Lys Leu Gln Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly 15 10 15Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20??????????????????25??????????????????30Gly?Met?Ser?Trp?Val?Arg?Gln?Thr?Pro?Asp?Lys?Arg?Leu?Glu?Ser?Val

35??????????????????40??????????????????45Ala?Thr?Ile?Ser?Ser?Gly?Gly?Ala?Tyr?Ile?Tyr?Tyr?Pro?Asp?Ser?Val

50??????????????????55??????????????????60Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ala?Lys?Asn?Thr?Leu?Tyr?65??????????????????70??????????????????75??????????????????80Leu?Gln?Met?Ser?Ser?Leu?Lys?Ser?Glu?Asp?Thr?Ala?Met?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Arg?Leu?Glu?Thr?Gly?Asp?Tyr?Ala?Leu?Asp?Tyr?Trp?Gly?Gln?Gly

100?????????????????105?????????????????110Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly

115?????????????????120?????????????????125Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro?Ala?Ser

130?????????????????135?????????????????140Leu?Ala?Val?Ser?Leu?Gly?Gln?Arg?Ala?Thr?Ile?Phe?Cys?Lys?Asp?Ser145?????????????????150?????????????????155?????????????????160Gln?Ser?Val?Asp?Tyr?Asp?Gly?Asp?Ser?Tyr?Met?Asn?Trp?Tyr?Gln?Gln

165?????????????????170?????????????????175Lys?Pro?Gly?Gln?Pro?Pro?Lys?Leu?Leu?Ile?Tyr?Ala?Arg?Ser?Asn?Leu

180?????????????????185?????????????????190Glu?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?G1y?Ser?G1y?Thr?Asp

195?????????????????200?????????????????205Phe?Ser?Leu?Asn?Ile?His?Pro?Val?Glu?Glu?Asp?Asp?Ile?Ala?Met?Tyr

210?????????????????215?????????????????220Phe?Cys?Gln?Gln?Ser?Arg?Lys?Val?Pro?Trp?Ser?Phe?Gly?Gly?Gly225?????????????????230?????????????????235

(2) information of SEQ ID NO:19

(i) sequence characteristic:

(A) length: 732 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(F) types of organization: splenocyte

(vii) direct sources

(B) clone: 10D2 (light key adds joint for strand FV, heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..732

(xi) sequence description: SEQ ID NO:19:GAG GTG CAG CTG CAG CAG TCT GGG GCT GAA CTG GTG AAG CCT GGG GCT 48Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala240 245 250 255TCA GTG AAG TTG TCC TGC AAG GCT TCC GGC TAC ACC TTC ACC AGC CAC 96Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

260?????????????????265?????????????????270TGG?ATG?CAC?TGG?GTG?AAG?CAG?AGG?GCT?GGA?CAA?GGC?CTT?GAG?TGG?ATC????144Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile

275?????????????????280?????????????????285GGA?GAG?TTT?AAT?CCC?AGC?AAC?GGC?CGT?ACT?AAC?TAC?AAT?GAG?AAA?TTC????192Gly?Glu?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

290?????????????????295?????????????????300AAG?AGC?AAG?GCC?ACA?CTG?ACT?GTA?GAC?AAA?TCC?TCC?AGC?ACA?GCC?TAC????240Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr

305?????????????????310?????????????????315ATG?CAA?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?TCT?GCG?GTC?TAT?TAC?TGT????288Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys320?????????????????325?????????????????330?????????????????335GCC?AGT?CGG?GAC?TAT?GAT?TAC?GAC?GGA?CGG?TAC?TTT?GAC?TAC?TGG?GGC????336Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

340?????????????????345?????????????????350CAA?GGG?ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGC?GGT?GGC?TCG?GGC?GGT????384Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

355?????????????????360?????????????????365GGT?GGG?TCG?GGT?GGC?GGC?GGA?TCT?GAC?ATT?GAG?CTC?ACC?CAG?TCT?CCA????432Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

370?????????????????375?????????????????380GCA?ATC?ATG?TCT?GCA?TCT?CCA?GGG?GAG?AAG?GTC?ACC?ATG?ACC?TGC?AGT????480Ala?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser

385?????????????????390?????????????????395GCC?AGC?TCA?AGT?GTA?AGT?TAC?ATG?TAC?TGG?TAC?CAG?CAG?AAA?CCA?GGA????528Ala?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys?Pro?Gly400?????????????????405?????????????????410?????????????????415TCC?TCC?CCC?AGA?CTC?CTG?ATT?TAT?GAC?ACA?TCC?AAC?CTG?GCT?TCT?GGA????576Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

420?????????????????425?????????????????430GTC?CCT?GTT?CGC?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACC?TCT?TAC?TCT?CTC????624Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu

435?????????????????440?????????????????445ACA?ATC?AGC?CGA?ATG?GAG?GCT?GAA?GAT?GCT?GCC?ACT?TAT?TAC?TGC?CAG????672Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

450?????????????????455?????????????????460CAG?TGG?AGT?AGT?TAC?CCA?CCC?ATG?TAC?ACG?TTC?GGA?GGG?GGG?ACC?AAG????720Gln?Trp?Ser?Ser?Tyr?Pro?Pro?Met?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys

465?????????????????470?????????????????475CTG?GAA?ATA?AAA????????????????????????????????????????????????????732Leu?Glu?Ile?Lys480

(2) information of SEQ ID NO:20

(i) sequence characteristic:

(A) length: 244 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SIQ ID NO:20:Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 15 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

20??????????????????25??????????????????30Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35??????????????????40??????????????????45Gly?Glu?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

50??????????????????55??????????????????60Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

100?????????????????105?????????????????110Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

115?????????????????120?????????????????125Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

130?????????????????135?????????????????140Ala?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser145?????????????????150?????????????????155?????????????????160Ala?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys?Pro?Gly

165?????????????????170?????????????????175Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

180?????????????????185?????????????????190Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu

195?????????????????200?????????????????205Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

210?????????????????215?????????????????220Gln?Trp?Ser?Ser?Tyr?Pro?Pro?Met?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys225?????????????????230?????????????????235?????????????????240Leu?Glu?Ile?Lys

(2) information of SEQ ID NO:21

(i) sequence characteristic:

(A) length: 732 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(F) types of organization: splenocyte

(vii) direct sources

(B) clone: 3D3 (light key adds joint for strand FV, heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..732

(xi) sequence description: SEQ ID NO:21:GAG GTC CAA CTG CAG CAG TCA GGG GCT GAA CTG GTG AAG CCT GGG GCT 48Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala245 250 255 260TCA GTG AAG TTG TCC TGC AAG GCT TCC GGC TAC ACC TTC ACC AGC CAC 96Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

265?????????????????270?????????????????275TGG?ATG?CAC?TGG?GTG?AAG?CAG?AGG?GCT?GGA?CAA?GGC?CTT?GAG?TGG?ATC????144Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile

280?????????????????285?????????????????290GGA?GAG?TTT?AAT?CCC?AGC?AAC?GGC?CGT?ACT?AAC?TAC?AAT?GAG?AAA?ATC????192Gly?Glu?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Ile

295?????????????????300?????????????????305AAG?AGC?AAG?GCC?ACA?CTG?ACT?GTA?GAC?AAA?TCC?TCC?AGC?ACA?GCC?TAC????240Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr

310?????????????????315?????????????????320ATG?CAA?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?TCT?GCG?GTC?TAT?TAC?TGT????288Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys325?????????????????330?????????????????335?????????????????340GCC?AGT?CGG?GAC?TAT?GAT?TAC?GAC?GGA?CGG?TAC?TTT?GAC?TAC?TGG?GGC????336Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

345?????????????????350?????????????????355CAA?GGG?ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGC?GGT?GGC?TCG?GGC?GGT????384Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

360?????????????????365?????????????????370GGT?GGG?TCG?GGT?GGC?GGC?GGA?TCT?GAC?ATT?GAG?CTC?ACC?CAG?TCT?CCA????432Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

375?????????????????380?????????????????385ACA?ATC?ATG?TCT?GCA?TCT?CCA?GGG?GAG?AAG?GTC?ACC?ATG?ACC?TGC?AGT????480Thr?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser

390?????????????????395?????????????????400GAC?AGC?TCA?AGT?GTA?AGT?TAC?ATG?TAC?TGG?TAC?CAG?CAG?AAG?ACA?GGA????528Asp?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys?Thr?Gly405?????????????????410?????????????????415?????????????????420TCC?TCC?CCC?AGA?CTC?CTG?ATT?TAT?GAC?ACA?TCC?AAC?CTG?GCT?TCT?GGA????576Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

425?????????????????430?????????????????435GTC?CCT?GTT?CGC?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACC?TCT?TAC?TCT?CTC????624Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu

440?????????????????445?????????????????450ACA?ATC?AGC?CGA?ATG?GAG?GCT?GAA?GAT?GCT?GCC?ACT?TAT?TAC?TGC?CAG????672Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

455?????????????????460?????????????????465CAG?TGG?AGT?AGT?TAC?CCA?CCC?ATG?TAC?ACG?TTC?GGA?GGG?GGG?ACC?AAG????720Gln?Trp?Ser?Ser?Tyr?Pro?Pro?Met?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys

470?????????????????475?????????????????480CTG?GAA?ATA?AAA????????????????????????????????????????????????????732Leu?Glu?Ile?Lys485

(2) information of SEQ ID NO:22

(i) sequence characteristic:

(A) length: 244 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:22:Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 15 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

20??????????????????25??????????????????30Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35??????????????????40??????????????????45Gly?Glu?Phe?Ash?Pro?Ser?Ash?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Ile

50??????????????????55??????????????????60Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

100?????????????????105?????????????????110Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

115?????????????????120?????????????????125Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

130?????????????????135?????????????????140Thr?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser145?????????????????150?????????????????155?????????????????160Asp?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys?Thr?Gly

165?????????????????170?????????????????175Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

180?????????????????185?????????????????190Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu

195?????????????????200?????????????????205Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

210?????????????????215?????????????????220Gln?Trp?Ser?Ser?Tyr?Pro?Pro?Met?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys225?????????????????230?????????????????235?????????????????240Leu?Glu?Ile?Lys

(2) information of SEQ ID NO:23

(i) sequence characteristic:

(A) length: 738 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(F) types of organization: splenocyte

(vii) direct sources

(B) clone: lE3 (light key adds joint for strand FV, heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..738

(xi) sequence description: SEQ ID NO:23:GAG GTG CAG CTG CAG CAG TCT GGG GCT GAA CTG GTG AAG CCT GGG GCT 48Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala245 250 255 260TCA GTG AAG TTG TCC TGC AAG GCT TCC GGC TAC ACC TTC ACC AGC CAC 96Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

265?????????????????270?????????????????275TGG?ATG?CAC?TGG?GTG?AAG?CAG?AGG?GCT?GGA?CAA?GGC?CTT?GAG?TGG?ATC????144Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile

280?????????????????285?????????????????290GGA?GAG?TTT?AAT?CCC?AGC?AAC?GGC?CGT?ACT?AAC?TAC?AAT?GAG?AAA?TTC????192Gly?Glu?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

295?????????????????300?????????????????305AAG?AGC?AAG?GCC?ACA?CTG?ACT?GTA?GAC?AAA?TCC?TCC?AGC?ACA?GCT?TAC????240Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr

310?????????????????315?????????????????320ATG?CAA?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?TCT?GCG?GTC?TAT?TAC?TGT????288Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys325?????????????????330?????????????????335?????????????????340GCC?AGT?CGG?GAC?TAT?GAT?TAC?GAC?GGA?CGG?TAC?TTT?GAC?TAC?TGG?GGC????336Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

345?????????????????350?????????????????355CAA?GGG?ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGC?GGT?GGC?TCG?GGC?GGT????384Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

360?????????????????365?????????????????370GGT?GGG?TCG?GGT?GGC?GGC?GGA?TCT?GGA?TCT?GAC?ATT?GAG?CTC?ACC?CAG????432Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln

375?????????????????380?????????????????385TCT?CCA?ACA?ATC?ATG?TCT?GCA?TCT?CCA?GGG?GAG?AAG?GTC?ACC?ATG?ACC????480Ser?Pro?Thr?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr

390?????????????????395?????????????????400TGC?AGT?GAC?AGC?TCA?AGT?GTA?AGT?TAC?ATG?TAC?TGG?TAC?CAG?CAG?AAG????528Cys?Ser?Asp?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys405?????????????????410?????????????????415?????????????????420CCA?GGA?TCC?TCC?CCC?AGA?CTC?CTG?ATT?TAT?GAC?ACA?TCC?AAC?CTG?GCT????576Pro?Gly?Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala

425?????????????????430?????????????????435TCT?GGA?GTC?CCT?GTT?CGC?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACC?TCT?TAC????624Ser?Gly?Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr

440?????????????????445?????????????????450TCT?CTC?ACA?ATC?AGC?CGA?ATG?GAG?GCT?GAA?GAT?GCT?GCC?ACT?TAT?TAC????672Ser?Leu?Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr

455?????????????????460?????????????????465TGC?CAG?CAG?TGG?AGT?AGT?TAC?CCA?CCC?ATG?TAC?ACG?TTC?GGA?GGG?GGG????720Cys?Gln?Gln?Trp?Ser?Ser?Tyr?Pro?Pro?Met?Tyr?Thr?Phe?Gly?Gly?Gly

470?????????????????475?????????????????480ACC?AAG?CTG?GAA?ATA?AAA????????????????????????????????????????????738Thr?Lys?Leu?Glu?Ile?Lys485?????????????????490

(2) information of SEQ ID NO:24

(i) sequence characteristic:

(A) length: 246 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:24:Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 15 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

20?????????????????25??????????????????30Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35??????????????????40??????????????????45Gly?Glu?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

50??????????????????55??????????????????60Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

100?????????????????105?????????????????110Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

115?????????????????120?????????????????125Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln

130?????????????????135?????????????????140Ser?Pro?Thr?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr145?????????????????150?????????????????155?????????????????160Cys?Ser?Asp?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys

165?????????????????170?????????????????175Pro?Gly?Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala

180?????????????????185?????????????????190Ser?Gly?Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr

195?????????????????200?????????????????205Ser?Leu?Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr

210?????????????????215?????????????????220Cys?Gln?Gln?Trp?Ser?Ser?Tyr?Pro?Pro?Met?Tyr?Thr?Phe?Gly?Gly?Gly225?????????????????230?????????????????235?????????????????240Thr?Lys?Leu?Glu?Ile?Lys

245

(2) information of SEQ ID NO:25

(i) sequence characteristic:

(A) length: 726 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(F) types of organization: splenocyte

(vii) direct sources

(B) clone: 5F1 (light key adds joint for strand FV, heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..726

(xi) sequence description: SEQ ID NO:25:CAG GTG AAA CTG CAG GAG TCT GGG GCT GAA CTG GTG AAG CCT GGG GCT 48Gln Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

250?????????????????255?????????????????260TCA?GTG?AAG?TTG?TCC?TGC?AAG?GCT?TCC?GGC?TAC?ACC?TTC?ACC?AGC?CAC????96Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser?His

265?????????????????270?????????????????275TGG?ATG?CAC?TGG?GTG?AAG?CAG?AGG?GCT?GGA?CAA?GGC?CTT?GAG?TGG?ATC????144Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile

280?????????????????285?????????????????290GGA?GAG?ATT?AAT?CCC?AGA?ACG?GCG?CCT?ACT?AAC?TAC?AAT?GAG?AAA?TTC????192Gly?Glu?Ile?Asn?Pro?Arg?Thr?Ala?Pro?Thr?Asn?Tyr?Asn?Glu?Lys?Phe295?????????????????300?????????????????305?????????????????310AAG?AGC?AAG?GCC?ACA?CTG?ACT?GTA?GAC?AAA?TCC?TCC?AGC?ACA?GCC?TAC????240Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr

315?????????????????320?????????????????325ATG?CAA?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?TCT?GCG?GTC?TAT?TAC?TGT????288Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

330?????????????????335?????????????????340GCC?AGT?CGG?GAC?TAT?GAT?TAC?GAC?GGA?CGG?TAC?TTT?GAC?TAC?TGG?GGC????336Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

345?????????????????350?????????????????355CAA?GGG?ACA?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGC?GGT?GGC?TCG?GGC?GGT????384Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

360?????????????????365?????????????????370GGT?GGG?TCG?GGT?GGC?GGC?GGA?TCT?GAC?ATT?GAG?CTC?ACC?CAG?TCT?CCA????432Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro375?????????????????380?????????????????385?????????????????390ACA?ATC?ATG?TCT?GCA?TCT?CCA?GGG?GAG?AAG?GTC?ACC?ATG?ACC?TGC?AGT????480Thr?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser

395?????????????????400?????????????????405GAC?AGC?TCA?AGT?GTA?AGT?TAC?ACG?TAC?TGG?TAC?CAG?CAG?AAG?ACA?GGA????528Asp?Ser?Ser?Ser?Val?Ser?Tyr?Thr?Tyr?Trp?Tyr?Gln?Gln?Lys?Thr?Gly

410?????????????????415?????????????????420TCC?TCC?CCC?AGA?CTC?CTG?ATT?TAT?GAC?ACA?TCC?AAC?CTG?GCT?TCT?GGA????576Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

425?????????????????430?????????????????435GTC?CCT?GTT?CGC?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACC?TCT?TAC?TCT?CTC????624Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu

440?????????????????445?????????????????450ACA?ATC?AGC?CGA?ATG?GAG?GCT?GAA?GAT?GCT?GCC?ACT?TAT?TAC?TGC?CAG????672Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln455?????????????????460?????????????????465?????????????????470CAG?TGG?AGT?AGT?TAC?CCG?CTC?ACG?TTC?GGT?GCT?GGG?ACC?AAG?CTG?GAA????720Gln?Trp?Ser?Ser?Tyr?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu

475?????????????????480?????????????????485ATA?AAA????????????????????????????????????????????????????????????726Ile?Lys

(2) information of SEQ ID NO:26

(i) sequence characteristic:

(A) length: 242 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:26:Gln Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 15 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

20??????????????????25??????????????????30Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35??????????????????40??????????????????45Gly?Glu?Ile?Asn?Pro?Arg?Thr?Ala?Pro?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

50??????????????????55??????????????????60Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

100?????????????????105?????????????????110Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

115?????????????????120?????????????????125Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

130?????????????????135?????????????????140Thr?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser145?????????????????150?????????????????155?????????????????160Asp?Ser?Ser?Ser?Val?Ser?Tyr?Thr?Tyr?Trp?Tyr?Gln?Gln?Lys?Thr?Gly

165?????????????????170?????????????????175Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

180?????????????????185?????????????????190Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu

195?????????????????200?????????????????205Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

210?????????????????215?????????????????220Gln?Trp?Ser?Ser?Tyr?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu225?????????????????230?????????????????235?????????????????240Ile?Lys

(2) information of SEQ ID NO:27

(i) sequence characteristic:

(A) length: 726 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(F) types of organization: splenocyte

(vii) direct sources

(B) library: 7G1 (light key adds joint for strand FV, heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..726

(xi) sequence description: SEQ ID NO:27:GAG GTC AAG CTG CAG CAG TCA GGG GCT GAA CTG GTG AAG CCT GGG GCT 48Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

245?????????????????250?????????????????255TCA?GTG?AAG?TTG?TCC?TGC?AAG?GCT?TCC?GGC?TAC?ACC?TTC?ACC?AGC?CAC????96Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser?His

260?????????????????265?????????????????270TTG?GAT?CAC?TGG?GTG?AAG?CAG?AGG?GGC?TGG?CAA?GGC?CTT?GAG?TGG?ATC????144Leu?Asp?His?Trp?Val?Lys?Gln?Arg?Gly?Trp?Gln?Gly?Leu?Glu?Trp?Ile275?????????????????280?????????????????285?????????????????290GGA?CAG?TTT?AAT?CCC?AGC?AAC?GGC?CGT?ACT?AAC?TAC?AAT?GAG?AAA?TTC????192Gly?Gln?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

295?????????????????300?????????????????305AAG?AGC?AAG?GCC?ACA?CTG?ACT?GTA?GAC?AAA?TCC?TCC?AGC?ACA?GCC?TAC????240Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr

310?????????????????315?????????????????320ATC?GAA?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?TGC?TCG?GTC?TAT?TAC?TGT????288Ile?Glu?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Cys?Ser?Val?Tyr?Tyr?Cys

325?????????????????330?????????????????335GCC?AGT?CGG?GAC?TAT?GAT?TAC?GAC?GGA?CGG?TAC?TTT?GAC?TAC?TGG?GGC????336Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

340?????????????????345?????????????????350CAA?GGG?ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGC?GGT?GGC?TCG?GGC?GGT????384Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly355?????????????????360?????????????????365?????????????????370GGT?GGG?TCG?GGT?GGC?GGC?GGA?TCT?GAC?ATT?GAG?CTC?ACC?CAG?TCT?CCA????432Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

375?????????????????380?????????????????385ACA?ATC?ATG?TCT?GCA?TCT?CCA?GGG?GAG?AAG?GTC?ACC?ATG?ACC?TGC?AGT????480Thr?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser

390?????????????????395?????????????????400GAC?AGC?TCA?AGT?GTA?AGT?TAC?ATG?TAC?TGG?TAC?CAG?CAG?AAG?ACA?GGA????528Asp?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys?Thr?Gly

405?????????????????410?????????????????415TCC?TCC?CCC?AGA?CTT?CTG?ATT?TAT?GAC?ACA?TCC?AAC?CTG?GCT?TCT?GGA????576Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

420?????????????????425?????????????????430GTC?CCT?GTT?CGC?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACC?TCT?TAC?TCT?CTC????624Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu435?????????????????440?????????????????445?????????????????450ACA?ATC?AGC?CGA?ATG?GAG?GCT?GAA?GAT?GCT?GCC?ACT?TAT?TAC?TGC?CAG????672Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

455?????????????????460?????????????????465CAG?TGG?AGT?AGT?TAC?CCG?CTC?ACG?TTC?GGT?GCT?GGG?ACC?AAG?CTG?GAA????720Gln?Trp?Ser?Ser?Tyr?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu

470?????????????????475?????????????????480ATA?AAA????????????????????????????????????????????????????????????726Ile?Lys

(2) information of SEQ ID NO:28

(i) sequence characteristic:

(A) length: 242 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:28:Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 15 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

20??????????????????25??????????????????30Leu?Asp?His?Trp?Val?Lys?Gln?Arg?Gly?Trp?Gln?Gly?Leu?Glu?Trp?Ile

35??????????????????40??????????????????45Gly?Gln?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

50??????????????????55??????????????????60Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Ile?Glu?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Cys?Ser?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

100?????????????????105?????????????????110Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

115?????????????????120?????????????????125Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

130?????????????????135?????????????????140Thr?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser145?????????????????150?????????????????155?????????????????160Asp?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys?Thr?Gly

165?????????????????170?????????????????175Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

180?????????????????185?????????????????190Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu

195?????????????????200?????????????????205Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

210?????????????????215?????????????????220Gln?Trp?Ser?Ser?Tyr?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu225?????????????????230?????????????????235?????????????????240Ile?Lys

(2)

The information of SEQ ID NO:29

(i) sequence characteristic:

(A) length: 726 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(C) vegetative period: grow up

(F) types of organization: splenocyte

(vii) direct sources

(B) clone: 11H1 (light key adds joint for strand FV, heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..726

(xi) sequence description: SEQ ID NO:29:GAG GTC AAG CTG CAG CAG TCA GGG GCT GAA CTG GTG AAG CCT GGG GCT 48Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

245?????????????????250?????????????????255TCA?GTG?AAG?TTG?TCC?TGC?AAG?GCT?TCC?GGC?TAC?ACC?TTC?ACC?AGC?CAC????96Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser?His

260?????????????????265?????????????????270TGG?ATG?CAC?TGG?GTG?AAG?CAG?AGG?GCT?GGA?CAA?GGC?TTG?GAG?TGG?ATC????144Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile275?????????????????280?????????????????285?????????????????290GGA?GAG?TTT?AAT?CCC?AGC?AAC?GGC?CGT?ACT?AAC?TAC?AAT?GAG?AAA?TTC????192Gly?Glu?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

295?????????????????300?????????????????305AAG?AGC?AAG?GCC?ACA?CTG?ACT?GTA?GAC?AAA?TCC?TCC?AGC?ACA?GCC?TAC????240Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr

310?????????????????315?????????????????320ATG?CAA?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?TCT?GCG?GTC?TAT?TAC?TGT????288Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

325?????????????????330?????????????????335GCC?AGT?CGG?GAC?TAT?GAT?TAC?GAC?GGA?CGG?TAC?TTT?GAC?TAC?TGG?GGC????336Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

340?????????????????345?????????????????350CAA?GGG?ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGC?GGT?GGC?TCG?GGC?GGT????384Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly355?????????????????360?????????????????365?????????????????370GGT?GGG?TCG?GGT?GGC?GGC?GGA?TCT?GAC?ATT?GAG?CTC?ACC?CAG?TCT?CCA????432Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

375?????????????????380?????????????????385TCA?ATC?ATG?TCT?GCA?TCT?CCA?GGG?GAG?AAG?GTC?ACC?ATG?ACC?TGC?AGT????480Ser?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser

390?????????????????395?????????????????400GAC?AGC?TCA?AGT?GTA?AGT?TAC?ATG?TAC?TGG?TAC?CAG?CAG?AAG?ACA?GGA????528Asp?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys?Thr?Gly

405?????????????????410?????????????????415TCC?TCC?CCC?AGA?CTC?CTG?ATT?TAT?GAC?ACA?TCC?AAC?CTG?GCT?TCT?GGA????576Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

420?????????????????425?????????????????430GTC?CCT?GTT?CGC?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACC?TCT?TAC?TCT?CTC????624Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu435?????????????????440?????????????????445?????????????????450ACA?ATC?AGC?CGA?ATG?GAG?GCT?GAA?GAT?GCT?GCC?ACT?TAT?TAC?TGC?CAG????672Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

455?????????????????460?????????????????465CAG?TGG?AGT?AGT?TAC?CCA?CAC?ACG?TTC?GGT?GCT?GGG?ACC?AAG?CTG?GAA????720Gln?Trp?Ser?Ser?Tyr?Pro?His?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu

470?????????????????475?????????????????480ATA?AAA????????????????????????????????????????????????????????????726Ile?Lys

(2) information of SEQ ID NO:30

(i) sequence characteristic:

(A) length: 242 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:30:Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 15 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

20??????????????????25??????????????????30Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35??????????????????40??????????????????45Gly?Glu?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

50??????????????????55??????????????????60Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

100?????????????????105?????????????????110Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

115?????????????????120?????????????????125Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

130?????????????????135?????????????????140Ser?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser145?????????????????150?????????????????155?????????????????160Asp?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys?Thr?Gly

165?????????????????170?????????????????175Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

180?????????????????185?????????????????190Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu

195?????????????????200?????????????????205Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

210?????????????????215?????????????????220Gln?Trp?Ser?Ser?Tyr?Pro?His?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu225?????????????????230?????????????????235?????????????????240Ile?Lys

(2) information of SEQ ID NO:31

(i) sequence characteristic:

(A) length: 732 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(ii) molecule type: cDNA

(iii) suppose: do not have

(iv) antisense: do not have

(v) clip types: N-end

(vi) primary source:

(A) biology: mouse

(B) strain: Balb/c

(F) types of organization: splenocyte

(vii) direct sources

(B) clone: 1A1 (light key adds joint for strand FV, heavy chain)

(ix) feature:

(A) title/keyword: CDS

(B) position: 1..732

(xi) sequence description: SEQ ID NO:31:GAG GTG CAG CTG CAG CAG TCT GGG GCT GAA CTG GTG AAG CCT GGG GCT 48Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala

245?????????????????250?????????????????255TCA?GTG?AAG?TTG?TCC?TGC?AAG?GCT?TCC?GGC?TAC?ACC?TTC?ACC?AGC?CAC????96Ser?Val?Lys?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser?His

260?????????????????265?????????????????270TGG?ATG?CAC?TGG?GTG?AAG?CAG?AGG?GCT?GGA?CAA?GGC?CTT?GAG?TGG?ATC????144Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile275?????????????????280?????????????????285?????????????????290GGA?GAG?TTT?AAT?CCC?AGC?AAC?GGC?CGT?ACT?AAC?TAC?AAT?GAG?AAA?TTC????192Gly?Glu?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

295?????????????????300?????????????????305AAG?AGC?AAG?GCC?ACA?CTG?ACT?GTA?GAC?AAA?TCC?TCC?AGC?ACA?GCT?TAC????240Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr

310?????????????????315?????????????????320ATG?CAA?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?TCT?GCG?GTC?TAT?TAC?TGT????288Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

325?????????????????330?????????????????335GCC?AGT?CGG?GAC?TAT?GAT?TAC?GAC?GGA?CGG?TAC?TTT?GAC?TAC?TGG?GGC????336Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?G1y

340?????????????????345?????????????????350CAA?GGG?ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGC?GGT?GGC?TCG?GGC?GGT????384Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly355?????????????????360?????????????????365?????????????????370GGT?GGG?TCG?GGT?GGC?GGC?GGA?TCT?GAC?ATT?GAG?CTC?ACC?CAG?TCT?CCA????432Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

375?????????????????380?????????????????385ACA?ATC?ATG?TCT?GCA?TCT?CCA?GGG?GAG?AAG?GTC?ACC?ATG?ACC?TGC?AGT????480Thr?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser

390?????????????????395?????????????????400GAC?AGC?TCA?AGT?GTA?AGT?TAC?ATG?TAC?TGG?TAC?CAG?CAG?AAG?ACA?GGA????528Asp?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys?Thr?Gly

405?????????????????410?????????????????415TCC?TCC?CCC?AGA?CTC?CTG?ATT?TAT?GAC?ACA?TCC?AAC?CTG?GCT?TCT?GGA????576Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

420?????????????????425?????????????????430GTC?CCT?GTT?CGC?TTC?AGT?GGC?AGT?GGG?TCT?GGG?ACC?TCT?TAC?TCT?CTC????624Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu435?????????????????440?????????????????445?????????????????450ACA?ATC?AGC?CGA?ATG?GAG?GCT?GAA?GAT?GCT?GCC?ACT?TAT?TAC?TGC?CAG????672Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

455?????????????????460?????????????????465CAG?TGG?AGT?AGT?TAC?CCA?CCC?ATG?TAC?ACG?TTC?GGA?GGG?GGG?ACA?AAG????720Gln?Trp?Ser?Ser?Tyr?Pro?Pro?Met?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys

470?????????????????475?????????????????480TTG?GAA?ATA?AAA????????????????????????????????????????????????????732Leu?Glu?Ile?Lys

485

(2) information of SEQ ID NO:32

(i) sequence characteristic:

(A) length: 244 base pairs

(B) type: amino acid

(D) topological framework: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:32:Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 15 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser His

20??????????????????25??????????????????30Trp?Met?His?Trp?Val?Lys?Gln?Arg?Ala?Gly?Gln?Gly?Leu?Glu?Trp?Ile

35??????????????????40??????????????????45Gly?Glu?Phe?Asn?Pro?Ser?Asn?Gly?Arg?Thr?Asn?Tyr?Asn?Glu?Lys?Phe

50??????????????????55??????????????????60Lys?Ser?Lys?Ala?Thr?Leu?Thr?Val?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?65??????????????????70??????????????????75??????????????????80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95Ala?Ser?Arg?Asp?Tyr?Asp?Tyr?Asp?Gly?Arg?Tyr?Phe?Asp?Tyr?Trp?Gly

100?????????????????105?????????????????110Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

115?????????????????120?????????????????125Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln?Ser?Pro

130?????????????????135?????????????????140Thr?Ile?Met?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Ser145?????????????????150?????????????????155?????????????????160Asp?Ser?Ser?Ser?Val?Ser?Tyr?Met?Tyr?Trp?Tyr?Gln?Gln?Lys?Thr?Gly

165?????????????????170?????????????????175Ser?Ser?Pro?Arg?Leu?Leu?Ile?Tyr?Asp?Thr?Ser?Asn?Leu?Ala?Ser?Gly

180?????????????????185?????????????????190Val?Pro?Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu

195?????????????????200?????????????????205Thr?Ile?Ser?Arg?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln

210?????????????????215?????????????????220Gln?Trp?Ser?Ser?Tyr?Pro?Pro?Met?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys225?????????????????230?????????????????235?????????????????240Leu?Glu??Ile?Lys

Claims (10)

1. the single chain variable fragment of anti-epidermal growth factor receptor, it can obtain from the phage antibody library that is made up by immune mammalian cell.

2. the antibody fragment of claim 1, it can obtain from the cell of immune mouse.

3. claim 1 or 2 antibody fragment, it can obtain from following cell:

(i) lymph-node cell,

(ii) splenocyte, or

The (iii) cell of external immunity.

4. arbitrary antibody fragment of claim 1-3, wherein DNA and/or the aminoacid sequence that is selected from one of the heavy chain that provides among the SEQID NO:1-32 and sequence of light chain contained in the variable region of heavy chain and light chain.

5. anti-egfr antibodies, it is by being built into from the dna sequence dna of the antibody fragment of claim 1-4 with from the dna sequence dna of human normal immunoglobulin constant region.

6. the antibody of claim 5, wherein CH comprises the aminoacid sequence of people's γ-1 chain, constant region of light chain comprises the aminoacid sequence of people K chain.

7. the preparation method of the anti-EGFR single-chain Fv of one of claim 1-4, it comprises the following steps:

(i) from the mammalian cell of immunity, isolation of RNA in the preferred mouse cell,

(ii) synthetic first gang of cDNA,

V among the cDNA of immunocyte (iii) increases _HAnd V _KGene,

(iv) described gene is cloned in the phagemid carrier together with suitable restriction site,

(v) transform prokaryotic cell prokaryocyte with connecting mixture,

(vi) from phage library, use the phage antibody of purifying EGFR screening at EGFR, and

(, produce desired strand Fv in the preferred intestinal bacteria vii) at prokaryotic host cell.

8. method for preparing the complete antibody of anti-EGFR, comprise the DNA of coding by the anti-egfr antibodies fragment variable region of claim 7 generation, be cloned at least a carrier for expression of eukaryon, this carrier for expression of eukaryon contains the group group DNA of coding human normal immunoglobulin constant region, with described carrier transformed eukaryotic karyocyte, express and separation antibody.

9. the pharmaceutical composition that contains the anti-EGFR complete antibody of the anti-egfr antibodies fragment of one of claim 1-4 or claim 5 or 6.

10. the anti-egfr antibodies fragment of one of claim 1-4, or the purposes of the anti-EGFR complete antibody of claim 5 or 6, it is used to prepare the medicine at tumour or diagnosis location and assessment tumor growth.

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