CN1615315A

CN1615315A - Tumor specific monoclonal antibodies

Info

Publication number: CN1615315A
Application number: CNA028270959A
Authority: CN
Inventors: J·D·瓦特金斯
Original assignee: Applied Molecular Evolution Inc
Current assignee: Applied Molecular Evolution Inc
Priority date: 2001-11-19
Filing date: 2002-11-19
Publication date: 2005-05-11
Also published as: WO2003044036A1; JP2005517388A; CA2467020A1; BR0214288A; EP1446416A1; EP1446416A4; AU2002365978A1; MXPA04004658A

Abstract

The invention provides tumor-specific human monoclonal antibodies and functional fragments. Also provided are nucleic acids encoding tumor-specific human monoclonal antibodies and functional fragments. A method for reducing neoplastic cell proliferation is also provided. The method consists of administering an effective amount of a tumor-specific human monoclonal antibody or functional fragment. Also provided is a method of detecting a neoplastic cell in a sample. The method consists of contacting a cell with a tumor-specific monoclonal antibody or functional fragment and detecting the specific binding of the human monoclonal antibody or functional fragment to the sample.

Description

The tumour-specific monoclonal antibody

Technical field

The present invention relates generally to cancer, and more specifically relates to the human monoclonal antibodies that is used for the treatment of with diagnosing cancer.

Background technology

At american cancer is to cause one of main causes of death.Annual American above 500,000 dies from cancer, and has the million people of surpassing to be diagnosed as this disease recently.In cancer, neoplastic cell is fled from out from its normal growth regulation mechanism and with uncontrolled mode hyperplasia, causes developing into malignant tumour.If not thoroughly before the essence development of this disease or also do not begin, tumour cell can be transferred on second site so to the treatment of primary tumo(u)r.Thereby early diagnosis and effective treatment malignant tumour thereby very necessary to survival.

The existing method of treatment cancer comprises surgical operation, radiotherapy and chemotherapy.The subject matter of each in these therapeuticss is that they lack specificity to cancer cells.For example, to remove tumour often not thorough for surgical operation.Even several residual neoplastic cells also can be fatal, because their hyperplasia and transfer to other positions fast.Radiotherapy and chemotherapy also have a serious limitation.All grown cells in these therapy aiming healths comprise normal cell and neoplastic cell.Because these therapies are to the toxicity of healthy tissues, the exit dose that can use safely or chemotherapy dosage often are not enough to kill all neoplastic cells.In addition, they are showed by the undesirable side effect that comprises nauseating and alopecia the toxicity of healthy tissues, and these side effects have seriously reduced the cancer patients's who accepts these treatments quality of life.Obviously need have more the mode of the treatment cancer of selectivity and validity.

Monoclonal antibody is the preparation of the same race of specific recognition and zone that is attached to its corresponding antigens or epi-position immunoglobulin (Ig).Neoplastic cell is optionally expressed the antigen of not being presented in the normal cell.Therefore, can produce monoclonal antibody at tumour specific antigen.These tumour specific antigens can be connected to and kill or stop on the treatment part of growth of neoplastic cell.In addition, described tumour specific antigen can be connected on the diagnosis part that allows the neoplastic cell imaging.Therefore, at can be favourable by tumour cell (comparing) selective expression's antigenic monoclonal antibody with normal cell be used for early detection and effective treatment cancer.

To the most popular immunotherapy strategy of cancer owing to its dependence to mouse monoclonal antibody has limited effectiveness.Utilize hybridoma technology to be easy to and quantitatively reality make mouse monoclonal antibody without restriction.Yet when being applied to human body, they can be identified as heterogeneous by the human immune system and be neutralized before illing tissue is brought into play its therapeutic action.And the Muridae immunity system is often preferentially discerned the antigenic immunodominance epi-position of normal human (immunodominant epitope) that is present in the tumour cell.Therefore, the human tumor specific antigens often can not produce treatment and go up useful Muridae antibody.

Human monoclonal antibodies can overcome all these two kinds of limitation.The most important thing is that human monoclonal antibodies causes immunity like that not as Muridae antibody.Therefore, the tumour-specific human monoclonal antibodies can more effective aiming and elimination neoplastic cell.And it is littler that the human immune system produces antigenic possibility at being present in Normocellular epi-position, increased the probability that produces and successfully differentiate tumour specific antigen.In addition, human immune system's integral part is different from the integral part of mouse, contains novel antibodies specific potentially.

At present in order to the general lymphocyte of the program that produces the tumour-specific human monoclonal antibodies to obtain on one's body from lotus knurl patient.These programs depend on and in vivo stimulate and be amplified in tumour-reactive lymphocyte.These programs are subjected to the serious restriction of narrow range of antigen-specific of cancer patients's activated B-cell.Owing to obviously can not make individual immunity with tumour cell in vivo, and to mouse can, therefore also can not produce tumour-specific human monoclonal antibodies easily to any given antigen or tumor cell type.The program that is used for producing any specific tumor specific antibody of wanting may extremely help effective immunotherapy and immunodiagnosis.

Therefore, exist being used for the needs of the treatment for cancer and the improved tumour-specific human monoclonal antibodies of diagnosis.The present invention satisfies these needs and relevant advantage is provided.

Summary of the invention

The invention provides isolating human monoclonal antibodies or its functional fragment, comprise a complementary determining region with aminoacid sequence of the CDR that is selected from the group that forms by following SEQ ID NO substantially: 10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42 and 44, wherein human monoclonal antibodies or its functional fragment are attached on neoplastic cell or its antigen specifically.

The present invention also provides a kind of isolating nucleic acid of encode human monoclonal antibodies or its functional fragment, comprise that an aminoacid sequence to following CDR carries out nucleotide sequence coding, this CDR is encoded by the nucleotide sequence that is selected from the group that is made up of following SEQ ID NO substantially: 9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43 and 45.

The present invention also provides a kind of outgrowth method of neoplastic cell that is used to reduce.Described method is by the human monoclonal antibodies of the present invention of throwing significant quantity or functional fragment and form.The method of neoplastic cell in a kind of test sample also is provided.Described method by cell and monoclonal antibody of the present invention or functional fragment are contacted and detect human monoclonal antibodies or functional fragment to the specificity of sample in conjunction with and form.Compare with normal cell, the existence of monoclonal antibody or functional fragment is arranged or the content interpret sample that increases in have neoplastic cell.

Description of drawings

Fig. 1 shows the combination to the H3464 cell surface of living of LH11238 and LH13 antibody.Figure 1A shows that (fluorescent activated cellsorting FACS) analyzes the cell divide method utilize the fluorescence-activation that LH11238 antibody done.Figure 1B shows the facs analysis that utilizes LH13 antibody to be done.

Fig. 2 illustrates that LH13 antigen is by H3396 emiocytosis.Fig. 2 A demonstration is competed the combination of LH13 antibody to the fixed cell individual layer from the conditioned medium of H3396 (solid torus) cell.Fig. 2 B demonstration LH13 antigen is come out by H3396 emiocytosis and is attached on the culture dish.

Fig. 3 shows by anion-exchange chromatography purification LH13 antigen on Q sepharose post.

Fig. 4 illustrates that LH13 antigen is vulnerable to the influence of trypsinase and endo-glycosidase-F/ peptide-N-Glycosylase (lycosidase) F treatment.

Fig. 5 shows the sign with recombinant chou LH13, LH11238 and the variant Fab of ELISA form.Picture A shows LH13 antibody (cavity ring), HCDR3 variant S97N (ring type filling), LCDR3 variant R91Y (hollow square), the LH13 antigen titration that reaches mutant 4H7 (filling square) partial concentration of respectively hanging oneself that makes up and the result who detects with goat Anti-Human K alkaline phosphatase conjugation.Picture B shows LH11238 antibody (cavity ring) and LCDR3 mutant Q89W (hollow square), N93C (hollow triangle), N94C (filling square) and P95aR (ring type filling) respectively hang oneself the fixedly individual layer titration of H3396 tumor cell line and the result who detects with goat Anti-Human K alkaline phosphatase.

Embodiment

The present invention is directed to tumour-specific human monoclonal antibodies and functional fragment thereof.The present invention's human monoclonal antibodies and functional fragment specificity thereof are attached on the neoplastic cell of comparing with normal cell, and can be used for optionally aiming at tumour.These antibody sources are in human body, and can not produce immune response when offeing medicine human body target.Therefore, can make them conjugate to cytotoxic agent or suppress the cell agent and be used for optionally aiming at cancer cells for eliminating tumour.The tumour-specific human monoclonal antibodies also can be used in and differentiates neoplastic cell in the diagnostor.Detect cancer early and improved the individual chance that survives this disease greatly.

In one embodiment, the invention provides with the method that generates the hybridoma that produces the tumour-specific human monoclonal antibodies.In mixed lymphocyte reacion, make normal human's cell immunity of spleen in vitro with tumour cell or tumor cell membrane.Then make the unlimited hyperplasiaization of described immune spleen cell produce the hybridoma that the tumour-specific of unlimited supply human monoclonal antibodies is provided.Use normal cell as the hybridoma source greatly amplified to the treatment cancer the integral part of obtainable different tumor specific antibodies.In addition, can optionally change so that effectively produce the antibody that is used for different human body treatment and diagnostic use as antigenic cell or cytolemma type in the method for the invention.

In another embodiment, the present invention is directed to the nucleic acid of coding human tumor monoclonal antibody specific.These nucleic acid can be used to express encoded human body antibody or its segment.In addition, coding nucleic acid can be produced by recombined engineering tumour cell is shown higher affinity or higher optionally through the human body antibody or the functional fragment of upgrading, or increases other functional characters of encoded antibody.Described antibody through upgrading can be by additional configurations to contain upgrading favourable on the other treatment, for example gets a promotion with the cognation of cytotoxic agent or immune stimulation is enhanced.

In a further embodiment, the present invention is directed to the antigen of discerning by the tumour-specific human monoclonal antibodies.This antigen can be used in the cancer diagnosis program and grow the specific cell toxin agent that is used for cancer therapy.Effective immune response that tumour specific antigen also can be used as vaccine and offers medicine to and have on the cancered individuality at neoplastic cell to develop.The nucleic acid of codes for tumor specific antigens can be used as the probe in the diagnostor, or by recombination method in addition upgrading development antigenic special inhibitor.

The basic structure of immunoglobulin (Ig) or antibody molecule is made up of with two identical heavy chains two identical light chains, and they are associated and can connect by disulfide linkage with non-covalent.Each bar heavy chain and light chain contain have an appointment 110 amino acid whose amidos-terminal variable region and the constant series in the remainder of chain.The variable region comprises several hypervariable regions or forms the antigen-binding site of antibody molecule and determine its specific complementation-determining area (CDR).At the CDR of heavy chain and light chain either side is the skeleton district, i.e. the relative conservative aminoacid sequence of grappling and directed CDR.Constant region is made up of (κ or λ) in five sequence of heavy chain (μ, γ, δ, α or ε) and two light chains.The CH sequence determines the homotype of this antibody and the effector function of this molecule.

Term used herein " human monoclonal antibodies " is used to refer to monoclonal antibody, and the heavy chain of being found among its heavy chain that comprises and light chain cdr amino acid sequence and the specific human immunoglobulin is identical substantially with light chain cdr amino acid sequence.Compare the antibody institute specificity bonded antigen that the sequence identity that shows a great deal of or degree and favourable promotion had reference sequences with reference sequences with the roughly the same aminoacid sequence of heavy chain or light chain CDR and carry out the specificity combination.Described identity is known or discernible for determining when describing the aminoacid sequence of specific human monoclonal antibodies.As long as kept the ability in conjunction with specific antigen, identical substantially heavy chain and light chain cdr amino acid sequence can be carried out (for example) amino acid whose small upgrading or conservative the replacement.Term " human monoclonal antibodies " is used for comprising the monoclonal antibody with people's cdr amino acid sequence substantially that produces by recombination method with lymphocyte or hybridoma (for example).

Term used herein " identical substantially " when being used in reference to aminoacid sequence, be used to refer to one have with the competent structure identity of reference amino acid sequence or similarity to be thought aminoacid sequence by the those skilled in the art with reference amino acid sequence encoded polypeptide or its pulsating specific function.This term can comprise the identity or the similarity of primary structure, and it also is called sequence identity or sequence similarity in affiliated field.The aminoacid sequence identical substantially with the reference sequences of antibody or its functional fragment can have the primary structure identical with reference sequences at least 70%, and it comprises (for example) and reference sequences at least 80%, at least 83%, at least 85%, at least 90%, at least 95%, at least 97% or at least 98% identical sequence.Described term can comprise tertiary structure identity or similarity, wherein tertiary structure is interpreted as to refer to functionally active antibodies or its pulsating three-dimensional structure.The specific function that comprises in the described term can be specific to come encoded polypeptides or its pulsating any biological activity by reference amino acid sequence, and it comprises that (for example) is to neoplastic cell or its antigenic specificity combination.

The nucleotide sequence identical substantially with reference sequences comprises the nucleotide sequence that identical polypeptid acid sequence is encoded.In affiliated field, generally different the but nucleotide sequence identical aminoacid sequence of encoding is each other called owing to genetic code is degenerated and had inactive otherness (silent difference).Described term can comprise complete sequence or its any part (as specific cryptosystem).Can use following method to differentiate identical substantially amino acid or nucleotide sequence.

Term used herein " CDR " is used to refer to the non-antigen combination site of finding of adjoining in the variable region of heavy chain and light chain polypeptide.This specific region has been described in people such as Kabat, U.S.Dept.ofHealth and Human Services, and people such as " Sequences of Proteins of Immunological Interest " (1983) and Chothia, J.Mol.Biol.People such as 196:901-917 (1987) and MacCallum, J. Mol.Biol.252:732-745 (1996), it is incorporated herein by reference, and definition wherein is when the overlapping or subclass of carrying out comprising when comparing mutually amino-acid residue.However, any in using two kinds define the CDR that refers to antibody or its functional fragment all be used in defined herein and the term category that uses in.The suitable amino-acid residue that has held the CDR that defines by above each piece document of quoting is listed in the table 1 as a comparison.Holding the structure that the definite residue quantity of specific CDR will depend on CDR changes.Which residue the those skilled in the art can be routinely decides comprise specific CDR according to the variable region amino acid sequence of antibody.The those skilled in the art can define the zone or the independent amino acid position of sequence separately by the identical CDR that be described in further detail enough and hereinafter, contrasts two or more antibody sequences by this.

Table 1:CDR definition

	??Kabat ¹	??Chothia ²	??MacCallum ³
	??Kabat ¹	??Chothia ²	??MacCallum ³	????VH ?CDR1	????31-35	????26-32	????30-35
????VH ?CDR2	????50-65	????52-56	????47-58	????VH ?CDR1	????31-35	????26-32	????30-35
????VH ?CDR2	????50-65	????52-56	????47-58	????VH ?CDR3	????95-102	????95-102	????93-101
????VL ?CDR1	????24-34	????24-34	????30-36	????VH ?CDR3	????95-102	????95-102	????93-101
????VL ?CDR1	????24-34	????24-34	????30-36	????VL ?CDR2	????50-56	????50-56	????46-55
????VL ?CDR3	????89-97	????89-97	????89-96	????VL ?CDR2	????50-56	????50-56	????46-55

¹The residue numbering is followed people such as Kabat, the nomenclature of supra

²The residue numbering is followed people such as Chothia, the nomenclature of supra

³The residue numbering is followed people such as MacCallum, the nomenclature of supra

When term used herein " functional fragment " when being used to refer to human monoclonal antibodies, its be used to refer to can the specificity conjugated antigen a part of monoclonal antibody, described antigen-specific is incorporated into reference antibody.Functionally active also can be the effector function that (for example) provided by antibody constant region.The human monoclonal antibodies functional fragment comprises (for example): independent heavy chain or light chain and segment thereof, as VL, VH and Fd; For example Fv, Fab, and the monovalent fragments of Fab '; F (ab ') for example ₂The divalence segment; Strand Fv (scFv); Fc segment and CDR territory.Described term description is in (for example) Harlow and Lane, Antibodies:A Laboratory Manual.Cold Spring Harbor Laboratory, New York (1989); Molec.Biology and Biotechnology:A Comprehensive Desk Reference(Myers, R.A. (ed.), New York:VCH Publisher, Inc.); People such as Huston, Cell Biophysics.22:189-224 (1993); Pl ü ckthun and Skerra, Meth.Enzymol..178:497-515 (1989) and Day, E.D., Advanced Immunochemistry, second edition, Wiley-Liss, Inc., New York, NY (1990), they are incorporated herein by reference.The term functional fragment is in order to comprise that (for example) is by protease digestion or the segment that reduces human monoclonal antibodies and produced by the known recombinant DNA method of those skilled in the art.

Term used herein " VL segment " is meant the light chain segment of the human monoclonal antibodies of the variable region of light chain (it comprises CDR) that comprises all or part.The VL segment can further comprise the constant region of light chain sequence.

Term used herein " Fd segment " is meant the heavy chain segment of the human monoclonal antibodies of the variable region of heavy chain (it comprises CDR) that comprises all or part.The Fd segment may further include the CH sequence.

Term used herein " Fv segment " is meant the monovalent antigen-binding fragment of human monoclonal antibodies, and it comprises all or part variable region of heavy chain and light chain, and lacks the constant region of heavy chain and light chain.The variable region of heavy chain and light chain comprises (for example) CDR.For example, the Fv segment comprises the terminal variable region of about 110 amino acid whose all or part amidos of heavy chain and light chain.

Term used herein " Fab segment " is meant the monovalent antigen-associativity segment greater than the pulsating human monoclonal antibodies of Fv.For example, the Fab segment comprises the variable region of heavy chain and light chain and first constant domain of all or part.Therefore, the Fab segment additionally comprises about 110 to about 220 amino-acid residue of (for example) heavy chain and light chain.

Term used herein " Fab ' segment " is meant the monovalent antigen-binding fragment greater than the pulsating human monoclonal antibodies of Fv.For example, Fab ' segment comprises first and second constant domains of all or part of all variable regions of all light chains, heavy chain and heavy chain.For example, Fab ' segment can additionally comprise 220 to 330 amino-acid residues of some or all of heavy chains.

Term used herein " F (ab ') ₂Segment " be meant the divalence antigen-binding fragment of human monoclonal antibodies.F (ab ') ₂Segment comprises all or part variable region of (for example) two heavy chains and two light chains, and may further include first constant domain of all or part of two heavy chains and two light chains.The those skilled in the art knows that the pulsating definite border of human monoclonal antibodies has change, as long as described segment keeps functionally active.The recombination method that utilization is known, the those skilled in the art can designing nucleic acid expresses and has the functional fragment of wanting end points that is used for specific end use.

Term used herein " marker " is in order to refer to be attached to the part of human monoclonal antibodies of the present invention or other molecules.Part can be used for (for example) treatment or diagnostor.

The treatment marker comprises that (for example) can be attached to molecule of the present invention and be used for reducing the part of uncontrolled hyperplasia of neoplastic cell or survival ability.The marker that can reduce hyperplasia or survival can be (for example) cytotoxic agent or cytostatics, growth factor, necrocytosis acceptor shrinking agent or immune modulator.

Diagnostic marker comprises that (for example) can be by the part of analytical procedure detection.Analytical procedure comprises (for example) qualitative and quant program.Method for qualitative analysis comprises (for example) immunohistochemistry and indirect immunofluorescence.Quantitative analysis method comprises (for example) immune affinity program, as radioimmunoassay, ELISA or facs analysis.Analytical procedure also comprises image forming program in vitro and in vivo.The specific examples of the diagnostic marker that can detect by analytical procedure comprises enzyme, radio isotope, chromophoric group, fluorescence dye, chemiluminescent labeling, and vitamin H.

Marker can directly be attached on the molecule of the present invention, or is attached on the secondary wedding agent of specificity in conjunction with molecule of the present invention.Described secondary wedding agent can be (for example) secondary antibody.Secondary antibody can be polyclone or monoclonal, and derives from the mankind, rodent or mosaic.

Term used herein " cytotoxic agent or cytostatics " is in order to refer to reduce the survival ability of cell or the medicament of hyperplasia potentiality.Described medicament can be attached on (for example) human monoclonal antibodies of the present invention or other molecules and be used for aiming at cell or tissue.Cell that is aimed at and tissue can comprise (for example) neoplastic cell and tumour.The cell that is aimed at and the example of tissue comprise cell and the tissue derived from breast, lung and ovary tissue.Survival ability or hyperplasia ability that cytotoxic agent or cytostatics can play a role in many ways and reduce cell.Described function comprises (for example): suppress DNA and synthesize, suppress cell fission, cell death inducing or induce non-apoptosis cell killing.The specific examples of cytotoxic agent and cytostatics comprises Pokeweed antiviral protein, abrin, ricin and their A chain, adriamycin (doxorubicin), cis-platinum, iodine-131, Yttrium-90, rhenium-188,. bismuth-212, taxol, 5 FU 5 fluorouracil VP-16, bleomycin, methotrexate, desacetyl vinblastine amide, Zorubicin (adriamycin), vincristine(VCR), vinealeucoblastine(VLB), BCNU, mitomycin and endoxan and some cytokine of TNF-α and TNF-β for example.Cytotoxic agent or cytostatics can comprise (for example) radioactive nuleus, chemotherapeutics, protein and lectin.

Term used herein " specificity combination " is in order to refer to human monoclonal antibodies or its functional fragment and antigenic selectivity interaction.Because described interaction has selectivity, human monoclonal antibodies can be not substantially in conjunction with or be made into can not be attached to substantially on the mark except that specific antigen.Specificity is in conjunction with comprising (for example) about 10 ⁵M ^-1Or higher dependent constant (K _a).Therefore, specificity is in conjunction with comprising at least about 1 * 10 ⁶M ^-1, 1 * 10 ⁷M ^-1, or 1 * 10 ⁸M ^-1, 1 * 10 ⁹M ^-1, 1 * 10 ¹⁰M ^-1, 1 * 10 ¹¹M ^-1Or 1 * 10 ¹²M ^-1Dependent constant.Specificity is in conjunction with also comprising (for example) high reactivity interaction.

Term used herein " cancer " is meant a kind of pathological condition, it is characterized in that existing neoplastic cell.Neoplastic cell is the cell that shows improper morphology or hyperplasia phenotype.Described cell is characterised in that (for example) do not rely on the cell growth of anchorage, hyperplasia and forfeiture contact inhibition in the blood serum medium that reduces.Described cell is also with the improper update of (for example) tissue growth (as tumour, impel the vascular system of angiogenesis) and to intrude into surrounding tissue be feature.Neoplastic cell also can be transferred to other positions the human body from primary tumo(u)r.For example, form, can transfer on other organs though the tumour of breast, lung or gonad cell still can be identified as by breast, lung or gonad cell.Term when therefore, relating to breast, lung or ovary " tumour " or " cancer " are in order to comprise the transfer of these tumours to other organ of health.

Term used herein " significant quantity " is used to refer to the amount that can reduce the outgrowth molecule of the present invention of neoplastic cell.Can be considered to depend on (for example) some factors, as the bioavailability or the selectivity of affinity, reactivity, stability, molecule, be attached to part, pharmaceutical carriers and dosing way on the molecule for the actual amount of a concrete significant quantity of using.Utilize the known method of those skilled in the art can determine or infer significant quantity.Described method comprises that (for example) use through cultured cells or organize vivisection and the in vitro analysis done of animal model reliably.

Term used herein " separated " is when being used in reference to antibody, be meant from one or more compounds and separate, described compound is found to have antibody in essence or be used for producing antibody in building-up reactions, comprises (for example) reactant, presoma or other reaction product.Separated reagent also can comprise purified substantially reagent.This term can comprise spontaneous molecule (as the product of biosynthesizing reaction) or synthetic molecules.

The invention provides human monoclonal antibodies or functional fragment, its at least one CDR that has is substantially the aminoacid sequence of the CDR of SEQ ID NO:2 or SEQ ID NO:4.The present invention also provides human monoclonal antibodies or functional fragment, and its at least one CDR that has is substantially the aminoacid sequence of the CDR of SEQ ID NO:6 or SEQ IDNO:8.The present invention further provides the human monoclonal antibodies or the functional fragment that produce by hybridoma cell line H1140, H2420 and H935.Hybridoma cell line H1140, H2420 and H935 also are provided.

The human monoclonal antibodies that is produced by hybridoma cell line LH11238, LH13, H1140, H2420 and H935 all shows (comparing with normal cell) specificity combination to neoplastic cell, and therefore becomes the tumour-specific human monoclonal antibodies.Specifically, human monoclonal antibodies of the present invention is all optionally in conjunction with breast cancer cell and demonstrate comparatively speaking hardly in conjunction with normal fiber.For example, compare with normal fiber archeocyte, peripheral blood lymphocyte, melanoma cells or lung's cancer cells, the LH11238 antibodies specific is in conjunction with the antigen of chamber between surface that appears at breast and ovarian cancer cell and lysosome.Compare with melanoma cells with the normal fiber archeocyte, the LH13 antibodies specific is in conjunction with the product that is produced by breast, lung and ovarian cancer cell.

By hybridoma is that the human monoclonal antibodies that H1140, H2420, H935 and LH13 produce is IgM homotype and lambda light chain class, is that the human monoclonal antibodies that LH11238 produces is IgM homotype and K light chain class by hybridoma still.The further characteristic of tumour-specific human monoclonal antibodies has been described in following example.

It is determined and be appointed as SEQ ID NO:1 that the variable region of heavy chain (VH) of the human monoclonal antibodies that produced by LH11238 clone is carried out nucleotide sequence coding.The VH aminoacid sequence of the human monoclonal antibodies that will be produced by LH11238 clone is appointed as SEQ ID NO:2.It is also determined and be appointed as SEQ ID NO:3 that the variable region of light chain (VL) of the human monoclonal antibodies that produced by LH11238 clone is carried out nucleotide sequence coding.The VL aminoacid sequence of the human monoclonal antibodies that will be produced by LH11238 clone is appointed as SEQ ID NO:4.

The nucleotide sequence of the variable region of heavy chain (VL) of the human monoclonal antibodies that coding is produced by LH13 clone is also determined and be appointed as SEQ ID NO:5.The VL aminoacid sequence of the human monoclonal antibodies that is produced by LH13 clone is designated as SEQ ID NO:6.The nucleotide sequence of the variable region of light chain (VL) of the human monoclonal antibodies that coding is produced by LH13 clone is also determined and be appointed as SEQ ID NO:7.The VL aminoacid sequence of the human monoclonal antibodies that is produced by LH13 clone is designated as SEQ ID NO:8.

The present invention further provides separated human monoclonal antibodies or its functional fragment, its at least one CDR that has has the cdr amino acid sequence of the sequence that is selected from the group that is made up of following SEQ ID NO substantially: 10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42 and 44, and wherein human monoclonal antibodies or its functional fragment specificity are in conjunction with neoplastic cell or its antigen.

The variant of the human monoclonal antibodies that is produced by hybridoma cell line of the present invention (as LH13 and LH11238 clone) can show (comparing with normal cell) specificity combination to neoplastic cell, and therefore becomes the tumour-specific human monoclonal antibodies.The functional variant and their method of production of the human monoclonal antibodies that is produced by hybridoma cell line LH13 and LH11238 have been described in example VIII and table 5.

List in the table 5 and comprise antibody fragment that (for example) produced by clone body S97G, S97T or S97N by the functional variant that LH13 clone produces, specificity is incorporated into the antigenic human monoclonal antibodies of neoplastic cell, its each have without the VH of upgrading in addition on 101 sites of the VL of upgrading and the sequence that in SEQ ID NO:6, sets, according to the described upgrading of people's such as Kabat numbering system on 97 sites of HCDR3.Have a glycine on 97 sites of the Kabat HCDR3 that the antibody fragment that is produced by clone body S97G sets in the VH aminoacid sequence as SEQ ID NO:10, the VH aminoacid sequence of SEQ ID NO:10 is encoded by the nucleotide sequence that sets in SEQ ID NO:9.Has a Threonine on 97 sites of the KabatHCDR3 that the antibody fragment that is produced by clone S97T sets and by encoding in the VH aminoacid sequence as SEQ ID NO:12 as the nucleotide sequence that in SEQ ID NO:11, sets.Has a l-asparagine on 97 sites of the Kabat HCDR3 that the antibody fragment that is produced by clone body S97N sets and by encoding in the VH aminoacid sequence as SEQ ID NO:14 as the nucleotide sequence that in SEQ ID NO:13, sets.

Other functional variant of the human monoclonal antibodies that is produced by LH13 clone also comprises the antibody fragment that is produced by clone body R91Y or R91F, its each have without the VL of upgrading in addition on 90 sites of the VH of upgrading and the sequence that in SEQ IDNO:8, sets, according to the described upgrading of people's such as Kabat numbering system on 91 sites of LCDR3.Have a tyrosine on 91 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone body R91Y sets in the VL aminoacid sequence as SEQID NO:16, the VL aminoacid sequence of SEQ ID NO:16 is encoded by the nucleotide sequence that sets in SEQ ID NO:15.Have a phenylalanine on 91 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone body R91F sets in the VL aminoacid sequence as SEQ ID NO:18, the VL aminoacid sequence of SEQ IDNO:18 is encoded by the nucleotide sequence that sets in SEQ ID NO:17.

As illustrated in the antibody fragment that produces by clone body V97Y and V97F, upgrading in addition on 98 sites of the VL sequence that other functional variant of the human monoclonal antibodies that is produced by LH13 clone can set in SEQ ID NO:8, keep a VH simultaneously without upgrading, according to the described upgrading of people's such as Kabat numbering system on 97 sites of LCDR3.Have a tyrosine on 97 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone body V97Y sets in the VL aminoacid sequence as SEQ IDNO:20, the VL aminoacid sequence of SEQ ID NO:20 is encoded by the nucleotide sequence that sets in SEQ ID NO:19.Have a tyrosine on 97 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone body V97Y sets in the VL aminoacid sequence as SEQ ID NO:22, the VL aminoacid sequence of SEQ ID NO:22 is encoded by the nucleotide sequence that sets in SEQ ID NO:21.

The combinatory variants that is produced by LH13 clone, specificity is attached to the human monoclonal antibodies on the antigen of neoplastic cell also is listed in the table 5, and comprises antibody fragment that (for example) produced by clone body 4D5,4E2,4H7,4G11 or 3G4.Compare with the human monoclonal antibodies that produces by LH13 clone, by the antibody fragment of clone body 4D5 generation in addition upgrading to have a tyrosine having on 97 sites of Kabat LCDR3 on a Threonine and 91 sites at Kabat LCDR3.The aminoacid sequence of 4D5 VH is set among the SEQ ID NO:12 and by the nucleotide sequence that is set in SEQ ID NO:11 and encodes, and the VL aminoacid sequence is set among the SEQ ID NO:16 and by the nucleotide sequence that is set in SEQ IDNO:15 and encodes.

Compare with the human monoclonal antibodies that produces by LH13 clone, by the combinatory variants of clone body 4E2 generation in addition upgrading to have a phenylalanine having on 97 sites of Kabat HCDR3 to have on tyrosine and 97 sites on a Threonine, 91 sites at Kabat LCDR3 at KabatLCDR3.The aminoacid sequence of 4E2 VH is set among the SEQ ID NO:12 and by being set in that nucleotide sequence among the SEQ ID NO:11 is encoded and the VL aminoacid sequence is set among the SEQ IDNO:24 and by the nucleotide sequence that is set in SEQ ID NO:23 and encodes.

Compare with the human monoclonal antibodies that produces by LH13 clone, by the combinatory variants of clone 4H7 generation in addition upgrading to have a phenylalanine having on 97 sites of Kabat HCDR3 to have on a phenylalanine and 97 sites on a Threonine, 91 sites at Kabat LCDR3 at Kabat LCDR3.The aminoacid sequence of 4H7 VH is set among the SEQ ID NO:12 and by being set in that nucleotide sequence among the SEQID NO:11 is encoded and the VL aminoacid sequence is set among the SEQ ID NO:26 and by the nucleotide sequence that is set in SEQ ID NO:25 and encodes.

Compare with the human monoclonal antibodies that produces by LH13 clone, by the combinatory variants of clone 4G11 generation in addition upgrading to have a phenylalanine having on 91 sites at Kabat LCDR3 on a phenylalanine and 97 sites at Kabat LCDR3.The aminoacid sequence of 4G11 VH is set among the SEQ ID NO:6 and by being set in that nucleotide sequence among the SEQ ID NO:5 is encoded and the VL aminoacid sequence is set among the SEQ ID NO:26 and by the nucleotide sequence that is set in SEQ ID NO:25 and encodes.

Compare with the human monoclonal antibodies that produces by LH13 clone, by the combinatory variants of clone 3G4 generation in addition upgrading to have a phenylalanine having on 91 sites at Kabat LCDR3 on tyrosine and 97 sites at KabatLCDR3.The aminoacid sequence of 3G4 VH is set among the SEQID NO:6 and by being set in that nucleotide sequence among the SEQ ID NO:5 is encoded and the VL aminoacid sequence is set among the SEQ ID NO:24 and by the nucleotide sequence that is set in SEQ ID NO:23 and encodes.

The functional variant that produces, is incorporated into the human monoclonal antibodies on the neoplastic cell by LH13 clone is listed in the table 5 and is comprised antibody fragment that (for example) produced by clone body Q89L, Q89G, Q89V, Q89F or Q89W, its each have one without the VL of upgrading in addition on 97 sites of the VH of upgrading and the sequence in being set in SEQ ID NO:4, according to the described upgrading of people's such as Kabat numbering system on 89 sites of LCDR3.Have a leucine (1uecine) on 89 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone body Q89L sets in the VL aminoacid sequence as SEQ ID NO:28, the VL aminoacid sequence of SEQ ID NO:28 is encoded by the nucleotide sequence that is set among the SEQ ID NO:27.Have a glycine on 89 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone Q89G sets in the VL aminoacid sequence as SEQ ID NO:30, the VL aminoacid sequence of SEQ IDNO:30 is encoded by the nucleotide sequence that is set among the SEQ ID NO:29.Have a Xie Ansuan on 89 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone Q89V sets in the VL aminoacid sequence as SEQ ID NO:32, the VL aminoacid sequence of SEQ ID NO:32 is encoded by the nucleotide sequence that is set among the SEQ ID NO:31.Have a phenylalanine on 89 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone Q89F sets in the VL aminoacid sequence as SEQ ID NO:34, the VL aminoacid sequence of SEQ ID NO:34 is encoded by the nucleotide sequence that is set among the SEQ IDNO:33.Have a tryptophane on 89 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone Q89W sets in the VL aminoacid sequence as SEQ IDNO:36, the VL aminoacid sequence of SEQ ID NO:36 is encoded by the nucleotide sequence that is set among the SEQ ID NO:35.

Other functional varianies of the human monoclonal antibodies that is produced by LH13 clone also comprise the antibody fragment that is produced by clone P95aF or P95aR, its each have one without the VL of upgrading in addition on 103 sites of the VH of upgrading and the sequence in being set in SEQID NO:4, according to the described upgrading of people's such as Kabat numbering system on 95 sites of LCDR3.Have a phenylalanine on 95 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone P95aF sets in the VL aminoacid sequence as SEQ IDNO:42, the VL aminoacid sequence of SEQ ID NO:42 is encoded by the nucleotide sequence that is set among the SEQ ID NO:41.Have a phenylalanine on 95 sites of the Kabat LCDR3 that the antibody fragment that is produced by clone P95aR sets in the VL aminoacid sequence as SEQ ID NO:44, the VL aminoacid sequence of SEQ IDNO:44 is encoded by the nucleotide sequence that is set among SEQ ID NO:43 or the SEQ ID NO:45.

As being demonstrated by clone's antibody fragment that N93C produced, on 100 sites of the VL sequence that can in SEQ ID NO:4, set the functional variant of the human monoclonal antibodies that produces by LH13 clone in LCDR3 upgrading in addition, keep VH simultaneously without upgrading.Have a halfcystine on 93 sites of the Kabat LCDR3 that in VL aminoacid sequence, sets by clone's antibody fragment that N93C produced and encode by the nucleotide sequence that is set among the SEQ ID NO:37 as SEQ ID NO:38.As by shown in clone's antibody fragment that N94C produced, the functional variant of the human monoclonal antibodies that is produced by LH13 clone can keep one without the VH of upgrading and can be through upgrading have a halfcystine on 101 sites (94 sites of Kabat LCDR3) of the VL aminoacid sequence among the SEQ IDNO:40 and encoded by the nucleotide sequence that is set among the SEQ ID NO:39 so that be set in.

Immunity generates the hybridoma that produces tumour-specific human monoclonal antibodies of the present invention by carrying out the human spleen cell culture in vitro with breast cancer cell.Concise and to the point, by being cultivated altogether, the single-cell suspension liquid of separating sets up mixed lymphocyte reacion (MLR) from the allogenic human spleen.Subsequently by with individual layer or cultivate the MLR culture with the enrichment membrane plasmapheresis of breast cancer cell and come enrichment tumour-reactive lymphocyte.For the lasting source of human monoclonal antibodies of the present invention is provided, by merging with K6H6/B5 hybridization myeloma cell line or merging the unlimited hyperplasiaization of lymphocyte that makes through immunity with the K6H6/B5 cell subsequently with the EBV conversion.Antigenic particular source has more fully been described and in order to produce the unlimited hyperplasia program of each hybridoma cell line of the present invention in following example.

Tumour-specific human monoclonal antibodies of the present invention also can generate by the known method of those skilled in the art.These methods comprise (for example): tumour-reactive lymphocyte in vivo and in vitro enrichment.For example, the individuality that has breast, lung or ovarian tumor can have the lymphocyte of expression specificity in conjunction with the antibody of tumour specific antigen, and described antibody comprises (for example) LH13, LH11238, H1140, H2420 or H935 antigen.Described lymphocyte can be separated from (for example) peripheral blood or from patient's spleen, and the unlimited hyperplasiaization of quilt as mentioned below.

Also knowing in the affiliated field utilizes tumour specific antigen to come the human lymphocytic method of in vitro enrichment tumour-reactivity.The antigen source can be (for example) purified substantially antigen, tumour cell or tumour cell cut.Purified substantially antigen be can prepare by any method that the those skilled in the art knows, (for example) chromatogram, electrophoretic separation and immunity isolation comprised.The antigen that can be used for preparing human monoclonal antibodies of the present invention can be (for example) neoplastic cell.Neoplastic cell can be cell, the primary tumo(u)r cell through cultivating or the clone through forming that (for example) directly obtains from tumour.Described cell can be risen in any organ, tissue or the liquid of human body, comprises (for example) breast, lung or ovary.Cancer cell can be undressed, fixed or grown catching.Can use and comprise being fixed of (for example) chemical fixation by the known many methods of those skilled in the art.Can be used for the fixed chemical and comprise (for example) many tools formaldehyde, glutaraldehyde, methyl alcohol or acetone.Another is chosen as and can uses the cytostatics cell of growing-catch.The specific examples of cytostatics is an ametycin.

The antigen that can be used for preparation human monoclonal antibodies of the present invention also can be the cut of tumour cell.The tumour cell cut can be (for example) cytolemma, tenuigenin content or karyon.Being used for the isolating method of cell branch is known by the people in affiliated field.The antigen that is used to prepare monoclonal antibody of the present invention also can be the antigen by tumor cell secretion.Can use the conditioned medium or the cell matrix of known field separating tumor cell in the affiliated field to prepare described antigen by (for example).

The antigen for preparing as mentioned above can be used for stimulating human lymphocyte to generate tumour-specific human monoclonal antibodies of the present invention.Can (for example) from living individual peripheral blood or obtain human lymphocyte from dead spleen individual or that carry out operating individuality.Can directly cultivate lymphocyte with antigen.Another is chosen as, and can cultivate lymphocyte with antigen in mixed lymphocyte reacion in the presence of the allos lymphocyte.Can determine easily specific antigen and lymphocytic suitable culture condition by the those skilled in the art.

If necessary, come primed (antigen-primed) lymphocyte of enrichment to carry out unlimited hyperplasiaization by in vivo or in vitro stimulating as mentioned above by the known many programs of those skilled in the art.Unlimited hyperplasiaization provides the lasting source of tumour-specific human monoclonal antibodies.Can merge with unlimited outgrowth clone by (for example) and finish lymphocytic unlimited hyperplasiaization.The described unlimited outgrowth clone that can be used for cytogamy can be the female B clone of (for example) human myeloma cell or human lymph.Fusion partners also can be rodent myeloma cell or people: rodent hybridization myeloma cell line.The hybridization myeloma cell line can be (for example) people: the different hybridoma cell line K6H6/B5 of mouse.Another is chosen as, and can use (for example) virus to come the unlimited hyperplasiaization of primed lymphocyte via transformation.The virus that can be used for carrying out via viral transformation unlimited hyperplasiaization is EBV.Primed lymphocyte also can carry out merging after the unlimited hyperplasiaization via viral transformation.With merging after the viral transformation can be that (for example) merges with K6H6/B5 clone after with the EBV transformation.The those skilled in the art can be identified for the culture condition of lymphocyte fusion or viral transformation easily.Use the known immunoassay of those skilled in the art can screening to be attached to the human monoclonal antibodies of human body tumour cell to be used for the production specificity through the lymphocyte of unlimited hyperplasiaization.Described immunoassay comprise (for example) quantitative and qualitative immunoassay.Qualitative immunoassay comprise (for example): precipitin method, lectin reaction, immunohistochemistry, immunofluorescence technique, the immunity point method of the use of ink and water and immunoprecipitation.Quantitatively immunoassay comprise (for example) immune affinity method, for example radioimmunoassay, facs analysis, and elisa assay.Elisa assay can be for (sandwich) of direct, interlayer or competition.(for example) used in immunoassay can be direct through the human monoclonal antibodies of mark.Another is chosen as described method use (for example) can be indirect through Anti-Human's secondary antibody of mark.Marker can be (for example) fluorescent marker, enzyme, radio isotope or vitamin H.Decide according to immunoassay, can detect by spectrophotometry, X-ray radiophotography x or chemiluminescence method.Described method also can be used for screening through improved antibody or its functional fragment, and they have the affinity that has strengthened to neoplastic cell or its antigen.

Also can be according to differentiating through improved antibody or its functional fragment in screening with neoplastic cell or its antigenic contacted antigen.Specifically, can improve correlation ratio differentiating through improved antibody or its functional fragment to compare with the female antibody that is derived from for it.Use described screening, the antibody or its functional fragment that have improved therapeutic efficacy owing to the association rate raising can be from having increase K because association rate reduces for identical antigen _aCome in conjunction with polypeptide difference, described association rate reduces and does not connect each other with therapeutic efficacy.In this, the those skilled in the art will be appreciated that according to K _A=k _On/ k _OffRelation, k _OnIncrease or k _OffReduce or both carry out increasing K simultaneously _A

Can any non--measure association rate in the equilibrium mixture, described mixture comprises that (for example) contacts or contain by quick change the formed mixture of solution temperature of binding partner fast by making antibody or its functional fragment with antigen.Non--equilibrium mixture can be pre--equilibrium mixture.Can pass through soluble antibody or its functional fragment to be contacted with soluble antigen form the pre-equilibration mixture under the amount constant condition of (for example) total antigen in detection case and total antibody or its functional fragment.Measurement in advance-association rate in the equilibrium mixture can carry out in form, described form provides with antibody or its functional fragment and antigen short mix and at millisecond or rapid detection antibody or its functional fragment or antigenic characteristic changing on the hour range faster.Provide the facilitated method of measurement nonequilibrium kinetics such as hereinafter described with quick quencher mobile instrument with stopping to flow.Also can measure the association rate in non--equilibrium mixture, this non-equilibrium mixture comprises that (for example) contain the solution of the antibody of solubility kind or its functional fragment or contain total antigen of variable concentrations or the solution of total antibody or its functional fragment.The association rate of measurement in non-equilibrium mixture can be carried out in form, described form provides antigen is attached on the solution surface and Continuous Flow that contains antibody or its functional fragment from the teeth outwards, or combined with the characteristic changing on rapid detection antibody or its functional fragment, antigen or surface conversely, so that measure at millisecond or carry out on the hour range faster.

Be used for comprising (for example): stop flowing dynamics instrument and the mobile instrument of quick quencher at the form of pre-equilibration mixture measurement association rate.The instrument that stops to flow can be used to the solution and the antigen that contain antibody or its functional fragment were shifted onto in the blending bin from the storage tank that separates before by detection cell.Follow change that described instrument can detect one or more above-mentioned characteristics and monitor the progress of binding events.The mobile instrument of quencher can be used to the solution that contains antibody or its functional fragment and contains antigenic solution short mix quencher association reaction after the limited amount time of process subsequently fast.Then can be to detect the change of one or more above-mentioned characteristics by the quencher mixture that is produced in the different time quencher with the mixing back.Can be by (for example) freezing or add chemical quenching reagent and carry out quencher, as long as described quencher step can not suppress to measure for combination rate the detection of the characteristic that is relied on.Therefore, fast the quencher instrument can be used on (for example) and is inconvenient to carry out under the situation of spectral detection.Can from for example KinTek Corp. (State College, PA) and Hi-Tech Scientific (Salisbury, UK) supplier buys multiple instrument in the place.

Be used for comprising (for example): surface plasma resonance and decaying wave instrument at the form of non-equilibrium mixture measurement association rate.Surface plasma resonance and decaying wave technology are utilized antigen or antibody or its functional fragment attached to a biosensor surface.Make the solution that contains binding partner separately pass biosensor surface and also can with the mode beasurement base that depends on the time in conjunction with and occur in the solution change of refractive of chip surface.For example, surface plasma resonance is based on the phenomenon that is taken place when at metal/liquid surface excitating surface plasma wave.The got along well contacted surface orientation of sample or of light from its reflection, and SPR causes that the light intensity that is reflected at the particular combinations place of angle and wavelength reduces.The biomolecules binding events causes the upper layer change of refractive, and it is as the variation of spr signal and detected.Binding events can be acceptor-ligand between bonded in conjunction with or dissociate.Therefore basically can measure change of refractive immediately and allow to measure the individual composition of affinity constant.More particularly, described method makes it possible to accurately measure association rate constant (k _On) and the rate constant (k that dissociates _Off).Available surface plasma resonance instrument device comprises that (for example) BIAcore instrument, IBIS system, SPR-CELLIA system, Spreeta and plasma SPR and resonance wave technology can be used in the Iasys system in the affiliated field, it is described in (for example) Rich and Myszka Curr.Qpin.Biotech.11:54-61 (2000).

In the binding events process, use (for example) method mentioned above to locate at one or more discrete times intervals (discreet time interval) by measuring ligand or measuring correlation ratio in conjunction with the characteristic of polypeptide.Measurement in the discrete time measuring space in the binding events process can be used to determine the quantitative measurment of association rate or the relative measurement of association rate.The quantitative measurment of association rate can comprise (for example) association rate value or k _OnValue.Can measure association rate or k from the mathematical analysis or the pattern analysis of time dependent measurement _OnQuantitative values.Described analysis is known for the people in affiliated field and is comprised and be used for fitting data to the algorithm of the summation of index or linear term or be used for computer simulation and arrive combination model with fitting data, and it is described in (for example) Johnson, Cur.Opin.Biotech.9:87-89 (1998).

If in reaction, count the influence of mass transport, use mathematical analysis for example mentioned above or pattern analysis or all to measure association rate so from whole ligands of containing solubility kind or variable concentrations in conjunction with the mixture of polypeptide.The technician in described field can come the calculated mass transmission by the association rate under having the restrictive condition similar to mass transport relatively or by the association rate that obtainable model adjustment in according to affiliated field calculates, described model comprises that (for example) is described in people such as Myszka Biophys.J.75:583-594 the model in (1998).

Not needing with being used to make the antigen of human lymphocyte immune identical to be used at the tumour specific antigen that tumor response carries out the screening monoclonal antibody.Be used in antigen in the screening and can be (for example) substantially the antigen through purifying, live or through fixed tumour cell individual layer, section that live or that detect through the cut or the tumour living tissue of fixed tumor cell suspension, tumour cell, it depends on used routine analyzer.Tumour cell can be (for example) human breast, ovary or lung's cancer cells.

Tumour-specific human monoclonal antibodies of the present invention and functional fragment debond or MIN combination or be made into only MIN being attached on the normal cell antigen.Normal cell or Normocellular cut can be used as the contrast to the screening human monoclonal antibodies.Described normal cell can be (for example) live or through the fixed healthy tissues or through cultured cells be.The normal cell system through cultivating that can be used as contrast comprises (for example) people fibroblast and peripheral blood lymphocyte.Though any normal cell all can be used as contrast, the selection of concrete contrast part is based on by the specificity of the concrete tumour-specific monoclonal antibody of screening.For example, if produce and screening human tumor-specific monoclonal antibody at cancer cells, a class normal cell that then can be used as comparison so is normal epithelium cell culture or clone.Proximate, if produce and screening human tumor-specific monoclonal antibody at sarcoma cell, a class normal cell that can be used as comparison so is normal fiber archeocyte or clone.Also can provide normal control from identical types of organization or from the normal cell culture or the clone of individuals with same.The those skilled in the art can know what is the normal cell of adequate types, and it can be used as measures the contrast that specificity is attached to the tumour cell of particular type.

Can purify and quantification tumour-specific human monoclonal antibodies or its functional fragment to be used for immunodiagnosis and immunotherapy program.The source and concrete application of human monoclonal antibodies known and depended on to described method of purification for the those skilled in the art.Method of purification can comprise (for example) precipitation, electrophoresis, chromatogram, reach the immune affinity purification.Under can using in the field known spectrophotometry or immunoassay antibody quantification and known standard control through purifying are compared.

In order further to characterize the tomour specific human monoclonal antibodies, also can measure their kind and subclass by the existence that immunoassay is measured independent heavy chain and light chain polypeptide.Described immunoassay comprises that ELISA measures and is that the those skilled in the art is known.The present invention also comprises the functional fragment of tumour-specific human monoclonal antibodies of the present invention.The functional fragment of human monoclonal antibodies keeps the biological activity of for example specificity combination or effector function.Therefore functional fragment can advantageously be used for detecting and the treatment cancer.

Functional fragment comprises having the heavy chain identical substantially with tumour-specific human monoclonal antibodies of the present invention and the segment of variable region of light chain.For example, functional fragment comprises the functional fragment that at least one CDR sequence wherein is made up of the aminoacid sequence identical substantially with the CDR sequence of tumour-specific human monoclonal antibodies of the present invention.Described functional fragment comprises (for example): VL, Fd, Fv, Fab, Fab ', F (ab ') ₂, Fc and CDR segment.For example, functional fragment may have the combination of one or more or VL and VH CDR in three the CDR sequences of one or more, the VH in three CDR sequences of VL of human monoclonal antibodies of the present invention.Can by the those skilled in the art rely on functional fragment the affinity of wanting and specificity and desirable purposes decide the suitable quantity and the combination of VL and VH CDR sequence.

The method of using the those skilled in the art to know can produce and separate the functional fragment of human monoclonal antibodies of the present invention at an easy rate.Described method comprises (for example): proteolysis method, recombination method and chemosynthesis.Be used for the pulsating proteolysis method of separation function end user monoclonal antibody as parent material.The enzyme that is suitable for the proteolysis human monoclonal antibodies comprises (for example): papoid, stomach en-and elastin.Needing monovalent fragments according to (for example) still is divalence segment and deciding, and can select suitable enzyme easily by the those skilled in the art.For example, the papoid division obtains two conjugated antigens and the pulsating unit price Fab of Fc ¹Segment.Stomach en-(for example) causes divalence F (ab ') ₂Segment.Can use further reduction F of the present invention (ab ') of (for example) DTT or beta-mercaptoethanol ₂Segment also produces two unit price Fab ¹Segment.

Can purify by the functional fragment of proteolysis generation by affinity and column chromatography program.For example, undigested antibody and Fc segment often remove by being attached to a-protein.In addition, use (for example) ion-exchange and gel filtration chromatography to utilize the electric charge of functional fragment and size that they are purified.The those skilled in the art knows described method.

The recombination method that is used to produce the functional fragment of human monoclonal antibodies starts from the separated nucleic acid in the zone of wanting of heavy chain immunoglobulin and light chain.Described zone can comprise the heavy chain of (for example) all or part and the variable region of light chain.Described zone can specifically comprise the CDR of heavy chain and light chain.

The invention provides the separated nucleic acid of coding human monoclonal antibodies or its functional fragment, its aminoacid sequence that has substantially at least one following CDR carries out nucleotide sequence coding, and described CDR encodes by the nucleotide sequence that is selected from the group that is made up of following SEQ ID NO: 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43 and 45.The present invention also comprises the separated nucleic acid to the Variable Area coding, its aminoacid sequence that has following CDR carries out nucleotide sequence coding, and this CDR encodes by the nucleotide sequence that is selected from the group that is made up of following SEQ ID NO: 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43 and 45.The separated nucleic acid of coding CDR also is provided, and it has the cdr amino acid sequence by following SEQ ID NO coding substantially: 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43 and 45.

Can use the encode nucleic acid of human monoclonal antibodies of the present invention or its functional fragment of the known method of those skilled in the art.The useful program that is used for separating described DNA start from can from the RNA of the hybridoma that produces the special monoclonal antibody of tumour-specific oppositely-cDNA that transcribes.Being used for cDNA synthetic method is known by the people in affiliated field.Use (for example) polymerase chain reaction (PCR) can amplify the cDNA that the functional fragment of heavy chain or light chain is encoded.Round pcr is a United States Patent (USP) the 4th, 683, the 195, the 4th, 800, and No. 159, the 4th, 754, No. 065, and the 4th, 683, No. 202 theme, described all patents are incorporated herein by reference.It is known and can use the lateral conserved sequence of concrete function segment that is positioned at heavy chain or light chain to decide by the those skilled in the art to be used for the suitable introduction of PCR.Suitable substance P CR condition is known and also can be decided by the those skilled in the art similarly.

Also can come the nucleic acid of the functional fragment of direct composite coding human monoclonal antibodies of the present invention by known oligonucleotide synthetic method in the affiliated field.Another is chosen as, and known recombination method can synthesize and connect littler segment to form bigger functional fragment in the affiliated field of use.

The nucleic acid of functional fragment of human monoclonal antibodies of the present invention of encoding can be cloned in the suitable expression and in suitable host and be expressed.Appropriate carriers and host cell systems can allow the coexpression and the assembling of the functional fragment of (for example) heavy chain and light chain.The those skilled in the art can determine to be used for the pulsating suitable system of expressing antibodies, and it comprises (for example) M13 antibiotic immunity expression vector.The known method recombination function segment of the present invention of purifying substantially in the field under can using, described method depends on used concrete carrier and host expression system.

The separated nucleic acid that also can adjust codes for tumor human monoclonal antibody specific or functional fragment has antibody as the optimal performance of affinity, selectivity, reactivity, stability or bioavailability with generation.Described upgrading can comprise (for example): the replacement of the interpolation of amino-acid residue ground, deletion or replacement or D-amino acid or amino acid, as long as described segment keeps the functional response of (for example) antigen-binding specificity.

In addition, the invention provides the difference storehouse (distinct libraries) of LH13 and LH11238 variant.The variant that has at least one amino acid change in Kabat light chain CDR3 (LCDR3) on the 88-98 site corresponding to SEQ ID NO:8 can be contained in the LH13 storehouse, has the variant of at least one amino acid change on the 99-107 site corresponding to SEQ ID NO:6 in Kabat heavy chain CDR3 (HCDR3).The variant that has at least one amino acid change in Kabat light chain CDR3 on the 97-106 site corresponding to SEQ ID NO:4 can be contained in the LH11238 storehouse.Though above the storehouse of being demonstrated is based on people's such as Kabat coding scheme, the those skilled in the art can use the coding scheme that had before set to produce similar storehouse according to any CDR definition that comprises people such as people such as (for example) Chothia or MacCallum.According to the coding scheme that had before set, the variant that has at least one amino acid change in the zone corresponding to the CDR of one or more LH13 or LH11238 can be contained in storehouse of the present invention, and described CDR comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3.Can use the storehouse of LH13 or LH11238 variant to have the antigen of increase or the antibody or the functional fragment of neoplastic cell binding reactive with screening, it is as further set forth in detail hereinafter.

Can by in the several different methods known in this technology prepare and have sufficient diversity and have antibody variants or its pulsating storehouse of enhanced in conjunction with affinity to contain.The those skilled in the art can know that great storehouse size and diversity are essential or competent in order to reach its intended purposes.For example, antibody or antibody fragment variant storehouse can be prepared to contain in female antibody one or each position of many CDR in undiscovered 19 kinds of basic amino acid each, and screening goes out to have in the gained colony enhanced in conjunction with active variant.

Another is chosen as, utilize the structure relevant, biochemistry with antibody described herein, and model information prepare storehouse through concentrating.Should be appreciated that to be relevant to and measure or prediction all can be used for designing storehouse through concentrating for any information in conjunction with activity or structural integrity or stable crucial residue or zone of antibody of the present invention.Therefore, after measured or through prediction to its antigenic antibody of living in conjunction with neoplastic cell in conjunction with activity or structural integrity or stability on crucial one or more amino acid position particular locations that are arranged in a zone, the antibody variants of forming described storehouse can contain the amino acid change.The bias combinatorial libraries (focusedlibrary) of antibody variants provides to be reduced to differentiates to have enhanced needs the variant quantity of screening in conjunction with the variant of activity or structural stability favourable condition.

Combination approach synthetic and screening bias combinatorial libraries can further provide the favourable condition of discriminating through improved antibody variants or its pulsating time and efficient.Shown in example VIII, the multiple variation in the LH13 variant is an additivity.The additivity that amino acid changes in other incoherent monoclonal antibody has formerly had report, and it is as people such as (for example) Yelton, J.Immunol.155:1994-2004 (1995) are described in the people such as Wu, supra (1998), and people such as Wu, supra (1999).Differentiate that by the synthetic combinatorial libraries that changes based on the monamino acid of finding from HCDR3 storehouse and LCDR3 storehouse additional amino acid combination permitted the antibody affinity to strengthen fast in very effective mode.

Shown in example VIII, synthetic in the first step what is made up of 171 HCDR3 mutant and 209 LCDR3 mutant only is 416 differential protein variants synthetic 36 combinatory variants in second step subsequently, finishes two of LH13 thus and goes on foot the affinity maturations.On the contrary, confirm needs to be contained 19 with the total randomization that influences active LCDR3 S97, HCDR3R91 and three CDR residues of HCDR3 V97 ³Or the expression in the storehouse of 6,859 variants.Therefore, the combination approach described in the example VIII provides the progressively improvement to the additivity of catching independent mutation, and it is for simplifying the effective ways of affinity ripening process.The those skilled in the art will recognize from the result of example VIII by synthetic can further improve antibody of the present invention (as through enhanced classification conversion variant (class-switched variant)) affinity corresponding to the additional LH13 storehouse of HCDR1 and HCDR2 and LCDR1 and LCDR2.And, with the mode of combination can use the favourable sudden change from these storehouses, differentiated with differentiate to neoplastic cell or its antigen have raising affinity through improved antibody or antibody fragment variant.

Can by known several different methods in the affiliated field measure or predict for antibodies of the present invention to neoplastic cell or crucial independent residue of its antigen or zone, and it can be used for concentrating the synthetic of antibody or antibody fragment variant storehouse.Can carry out independent residue or zone that the structure contrast of sequence (primary structure) or three-dimensional structure (tertiary structure) or the two combination is used to suddenly change with discriminating between in conjunction with same antigen or approximate antigenic antibody.For example, the structural models based on antibody the 3rd structure can disclose topology, electrostatics, hydrophobicity or the hydrophilic environments that relates to antigen bonded binding site or concrete CDR.The total characteristic of the binding site that combines antigenic two or more antibody similar on common antigen or the structure is compared can be in order to position or the zone of differentiating that sudden change forms.Can use the sequence control methods to come collating sequence and differentiate and have the homology residue or having position in two or more antibody of the residue of sharing similar spaces, static, hydrophobic or water-wet behavior.Can use the correlated combination of primary structure and tertiary structure to differentiate the position of being changed.For example, can in tertiary structure is analyzed, differentiate in primary structure away from each other in conjunction with crucial position, and described position is focused on to inquire about the database of the elementary sequence of antibody or compare with the elementary sequence of one or more other antibody being used in elementary sequence.

Under know the method for the elementary sequential structure of contrast that can be used to determine that two sequences is identical substantially in the field.For example, a kind of usefulness decides two sequences, and whether identical substantially method is BLAST, (BasicLocal Alignment Search Tool, local comparison basic search instrument), and it can be according to being described in people such as Tatiana, FEMS Microbial Lett.Among the 174:247-250 (1999) or national center about the webpage of biotechnology information on the default parameter of ncbi.nlm.gov/BLAST/ use described instrument.BLAST is a cover similarity searching program, and it is designed to detect all obtainable sequence libraries and can moves with the similarity in search amino acid or the accounting sequence.Blast search provides the mark of the search with well-defined descriptive statistics.And BLAST uses heuristic algorithm, its seek local comparison and also therefore can detect relation between the sequence of only sharing the similarity separated region that comprises (for example) protein territory (people such as Altschul, J.Mol.Biol.215:403-410 (1990)).Except original described BLAST people such as (, supra, 1990) Altschul, to algorithm did to revise (people such as Altschul, Nucleic Acids Res.25:3389-3402 (1997)).A kind of modification is Gapped BLAST, and it allows space, insertion or deletes in two kinds any and introduce and compare.In comparison, allow the space to help more closely to reflect biological relation.For example, can use gapped BLAST in the similar regions of two or more polypeptide, to differentiate sequence identity.Second kind of modification is PSI-BLAST, and it is the sensitive method of search sequence homologue.PSI-BLAST carries out initial Gapped blast search and uses information from any important comparison with special minute matrix number of construction location, and its replacement is used for the search sequence of next round database search.The PSI-BLAST search often sequence similarity to faint but biologically relevant is more sensitive.

Can be used for determining whether identical substantially second source is the PROSITE that can obtain to two sequences on the Global Internet of ExPASy.PROSITE be measure method from genomic or the translation of cDNA sequence without the polypeptide function of characterization (people such as Bairoch, Nucleic Acids Res.25:217-221 (1997)).PROSITE is made up of the database of significant site and pattern biologically, and described site and pattern can be used to differentiate which (if existence) known peptide family is new sequence belong to.Use this or similar algorithm, can be by the specific clusters (comprising, for example those bunches of in similar territory, finding) of the generation (this can be described as pattern, decorative pattern, symbol or finger mark) of specific clusters amino-acid residue in peptide sequence and amino-acid residue in reference polypeptide identically substantially differentiate identical with another polypeptide substantially polypeptide.The PROSITE algorithm that uses a computer differentiates to be family member's figure with search with polypeptide.PROSITE also keeps the editor of the decorative pattern through differentiating before, and it can be in order to determine whether a nearest polypeptide through differentiating is the member of a known family.

Sequence contrast can comprise that gene, cDNA or its are through the full sequence of expressed products or can comprise its one or more specific regions.Can differentiate the specific region to determine the high relatively homology or the zone of similarity by range estimation sequence alignment discriminating specific region.As mentioned above, can be with which zone of structured data cross reference to find the dependency between ad hoc structure territory and the homology zone.Also can in algorithm, use reference antibody or its pulsating structural models, this algorithm will include (for example) SCOP, CATH or FSSP (at Hadley and Jones, Structure7:1099-1112 has commentary in (1999)) and have particular fold or structure and compare as polypeptide structure and second polypeptide in the zone of sequence contrast district, with the similar substantially zone of discriminating.

Except that the structural models of antibody, can use physicochemical data to measure or predict in conjunction with neoplastic cell or the crucial antibody location of its antigen or zone and these position or zones of in the bias combinatorial libraries of preparation antibody variants, being aimed at.In this, about combination rate constant (k _On), dissociation rate constant (k _Off) or balance in conjunction with affinity constant (K _dOr K _a) the female antibody or the sign of its variant can be used to differentiate to being attached to neoplastic cell or the crucial zone of its antigen.In order to generate the antibody variants storehouse, can be used alone or in combination different kinds of information to measure or to predict to being attached to the zone of neoplastic cell or the crucial aminoacid sequence of its antibody.For example, can the information (as relatively for the affinity that has the variant of variation on the structurally conservative position) based on structural models and biochemical analysis be made up to measure the amino acid sequence region in conjunction with active crucial antibody.Predicted in conjunction with crucial zone because can make up the information that obtains by several different methods, it will be understood by one of ordinary skill in the art that the approximate but not strict boundary line of representative, described zone self.The result is can be determined or be predicted as directly change antibody variants storehouse, position beyond the zone that is harmonious with antigen.For example, the skeleton district may antagonist or its pulsating structural stability is very important maybe may influence in conjunction with affinity by long-range or via the steric interaction that influences the binding site characteristic.Similarly, can be in conjunction with neoplastic cell or its antigenic antibody or its pulsating variant or identify herein directly with and change in conjunction with having amino acid outside other relevant zone at CDR.

Should be further understood that based on used Forecasting Methodology and correlated structure, predictedly go out to change quantity or location in conjunction with active crucial amino acid position.For example, as indicated above, can use different CDR to define and contrast antibody sequence.The technician in described field should understand and can use identical CDR to define to differentiate zone or the position that will compare between two or more antibody structures.For example, can define with the CDR3 of the people such as Kabat described in the example VII and contrast two or more antibody and be shown in the Table V.Can decide the suitable CDR definition that is used to estimate antibody based on the primary structure or the tertiary structure of antibody.Similarly, can decide the CDR definition that is used to contrast two or more antibody structures based on the original observed of each antibody structure with to the discriminating of the definition of the most suitable correlated all structures.In selecting suitable CDR definition, also may take into account other factors, comprise the functional performance of (for example) female antibody or its variant.The those skilled in the art can use different CDR to define to carry out the separately contrast of same antibody and therefore according to finding the zone or the position is similar or the homologous frequency is differentiated zone or the position that is changed in contrast separately.

The storehouse that is used for making all kinds molecule (as peptide, peptoid and peptidomimetics) that contains diversified colony well known in affiliated field (for example referring to Ecker and Crooke, Biotechnology13:351-360 (1995) reaches people such as Blondelle, Trends Anal.Chem.14:83-92 (1995), and the document of wherein being quoted; Also referring to Goodman and Ro, Peptidomimetics for Drug Design, in " Burger ' s Medicinal Chemistry and DrugDiscovery ", the 1st volume (ed.M.E.Wolff; John Wiley ﹠amp; Sons 1995), the 803-861 page or leaf reaches people such as Gordon, J.Med.Chem.37:1385-1401 (1994)).When molecule was peptide, protein or its segment, described molecule can in vitro directly produce or can be from the expression of nucleic acid that can in vitro produce.The method of synthetic chemistry of peptides is well known in affiliated field.

Can (for example) produce the antibody variants storehouse by the expression of nucleic acid storehouse of constructing the encoding antibody variant.The method that is used for producing described storehouse well known in affiliated field (for example referring to people such as Sambrook, Molecular Cloning:A Laboratory Manual, second edition, Cold Spring Harbor Press, Plainview, New York (1989); People such as Sambrook, Molecular Cloning:A Laboratory Manual.The third edition, Cold Spring Harbor Press, Plainview, New York (2001); People such as Ausubel Current Protocols in Molecular Biology(Supplement 47), JohnWiley ﹠amp; Sons, New York (1999)).The nucleic acid library of encoding antibody variant can be made up of DNA, RNA or its analogue.Can (for example) construct the storehouse of containing the RNA molecule by chemosynthesis RNA molecule.Can generate the nucleic acid library of encoding antibody or antibody fragment variant by any way that the user wants.The those skilled in the art can know that available which kind of method generates the nucleic acid library of encoding antibody or antibody fragment variant.For example, use the known method of those skilled in the art (people such as Sambrook, supra (1989); People such as Sambrook, supra (2001); People such as Ausubel, supra (1999)) can carry out mutagenesis by nucleic acid and generate the antibody variants storehouse female antibody of coding such as LH13 or LH11238.The nucleic acid library of antibody of the present invention or antibody fragment variant but randomization is encoded is so that it has sufficient diversity to contain the abiogenous amino acid whose nucleic acid of each possibility of coding on each amino acid position of one or more CDR.Another is chosen as, can make nucleic acid library make its contain only encode each at locational each the amino acid place that is positioned at the CDR zone may abiogenous amino acid whose nucleic acid, described CDR zone as example VIII described in through predicting or be determined as for being attached to neoplastic cell or its antigen is crucial.

Fixed point (site-directed) mutagenesis of use (for example) site (referring to: Wu (Ed.), Meth.In Enzymol.Vol.217, San Diego:Academic Press (1993); Higuchi, " Recombinant PCR " inInnis et al. (Ed.), PCR Protocols.San Diego:Academic Press, Inc. (1990), its each piece of writing is incorporated herein by reference) can introduce one or more sudden changes in nucleic acid molecule of encoding antibodies or antibody fragment variants to produce nucleic acid molecule through upgrading.Can use described mutagenesis to introduce amino acid change specific, that wanted.Therefore, can be produced on one or more after measured in conjunction with the difference storehouse of containing amino acid change in the active crucial zone and the single storehouse of in several or All Ranges, containing sudden change.

Can be as people such as Wu, Proc.Natl.Acad.Sci.USA, 95:6037-6042 (1998); People such as Wu, J. Mol.Biol..294:151-162 (1999); And Kunkel, Proc.Natl.Acad.Sci.USA.82:488-492 (1985) were before described, used the mutagenesis of oligonucleotide-orientation to finish effectively synthesizing and expression of antibody or antibody fragment variant.The program of sudden change (not relying on their phenotype) is known and effectively be used for introducing systemicly in the oligonucleotide-directed mutagenesis institute that behaves, and so be ideally suited orthogenesis approach in protein engineering.In order to carry out oligonucleotide-directed mutagenesis, the strand that the nucleic acid library of the sudden change that making encodes wants hybridizes to wild-type sequence contains on the template of uridylic.Described method is very flexible, allows not use restriction enzyme and introduces accurate sudden change, and if use based on the mutagenesis synthesis of oligonucleotides Nucleotide of codon then described method is relatively cheap.Known method in the field under using can be introduced aminoacid replacement by the nucleic acid codon mutation that makes the coding specific amino acids.As long as segment reservation function activity can be changed single or multiple codons.Being used for making fast method with a plurality of simultaneous variations of screening knows for the people in affiliated field and can be used to produce to containing that the nucleic acid all possible or variation that all are wanted is encoded and then expression and the human monoclonal antibodies of screening reservation function or the storehouse in segment storehouse.Described method comprises (for example): based on the mutagenesis of codon, the oligonucleotide synthetic and that part is degenerated of oligonucleotide is synthetic at random.

Mutagenesis based on codon is United States Patent (USP) the 5th, 264, No. 563 and the 5th, 523, No. 388 theme and help said procedure is because its allows to produce any basically and all frequencies of wanting that how reach the locational encoded amino-acid residue of all specific cryptosystems in office in oligonucleotide.The described frequency of wanting comprises that the merging of predetermined deflection of the merging of (for example) all 20 amino acid or its specified subclass true random and one or more specific amino acids is to merge and other compares the higher or lower frequency of described residue through deflection through the amino-acid residue that merges.

Can be similar to oligonucleotide at random synthetic be used to produce with the screening function on amino acid of equal value change.Yet because the degeneration of genetic code, described method can merge redundant (for example referring to United States Patent (USP) the 5th, 723, No. 323) at the amino acid position of wanting.Oligonucleotide synthetic is included in the codon all four Nucleotide that are coupled in each nucleotide position place at random.Also have other synthetic random device, it can cause the oligonucleotide (for example referring to United States Patent (USP) the 5th, 723, No. 323) of oligonucleotide of degenerating or part is degenerated or the aminoacid sequence of encoding completely random.

The oligonucleotide that part is degenerated is synthetic to be the equality part and equality part at two Nucleotide of the 3rd position that is coupling in all four Nucleotide of two initial nucleotide sites of (for example).Can find that these synthetic methods afterwards all are described in people such as (for example) Cwirla, Proc.Natl.Acad.Sci.USA87:6378-6382, people such as (1990) and Devlin, Science249:404-406, (1990).

Can produce the synthetic correct based on codon of various some variant sequences to mentioned above being used to, it can be used for producing antibody as herein described or antibody fragment variant storehouse.This modification is based on and makes synthetic two above-mentioned vessel process and the permission user who is partial to auxiliary sequence variant is divided into the colony of containing set amount codon position (it has random cipher and changes).

Concise and to the point, carry out this and synthesize by reaction vessel is separated into two new containers behind each codon position synthetic.Separately,, mix from each reaction product of right reaction vessel continuously since second container.Mix the mixture of the codon position reaction that has random variation with equal amts is taken to together.By then with first and the product of last container separate and be separated into again in two new containers with new mixed product and proceed to synthesize from each continuous right reaction vessel.In one of them of new container synthetic female codon and in second container synthetic random cipher.For example, make a reaction vessel bear the synthetic of female codon and second reaction vessel born the synthetic of random cipher first codon position synthetic.For for second codon position synthetic, each all is separated into two initial reaction vessels and produces two pairs of containers in two containers.For each to, therein in container synthetic female codon and also in second container synthetic random cipher.When by linear array, the mixing of the reaction product in the second and the 3rd container is taken to together with those products that will have the random cipher subsequence in the sub-position of single password.This mixing also makes the product group reduce to three, and they are to be used in the initial group of next round in synthetic.Similarly, for the 3rd, the 4th and each remaining position, separately and synthetic female codon and random cipher each the reaction product group who is used for aforementioned location.

With based on after the above-mentioned modification of the synthetic of codon, can be separated in easily on one, two, three and four positions and other position and contain the group that random cipher changes, and described group can be used based on the needs of individuality.And this synthesis flow allows also that enrichment is used to be subjected to randomized sequence group on auxiliary sequence, is separated from random cipher is synthetic similarly because only contain auxiliary sequence synthetic container.Can use this method to come the nucleic acid library of composite coding antibody variants, described antibody variants has the amino acid change one or more being predicted to be for being attached among neoplastic cell or the crucial CDR of its antigen.

Another is chosen as, and also can use the gene mixing to generate the nucleic acid library of encoding antibody or antibody fragment variant.Gene mixing or DNA mixing be used for by reorganization generate the multifarious directed method that develops (for example referring to Stemmer, Proc.Natl.Acad.Sci.USA91:10747-10751 (1994); Stemmer, Nature370:389-391 (1994); People such as Crameri, Nature391:288-291 (1998); People such as Stemmer, United States Patent (USP) the 5th, 830, No. 721, on November 3rd, 1998 issued).Gene mixing or DNA mixing are to use the in vitro method in the mutator gene pond of homologous recombination through selecting.For example, can use the point mutation pond of specific gene.For example, use Dnase smashes gene randomness, and is ressembled by PCR.If need, can use homologous gene from different organisms to carry out the DNA mixing to generate diversity people such as (, supra, 1998) Crameri.Need carry out many wheel fragmentations and ressemble if having.The genomic constitution of gained through ressembling can be used in antibody or the antibody fragment variant storehouse in the present composition and the method.

For some treatment and diagnostic use, may better use antibody or its segment that has identical antigen-specific but have different homotypes or special-shaped determinative.Described antibody may have immunogenicity, the stability of increase or the effector function of more the bests that (for example) reduces.Therefore, functional fragment of the present invention may comprise that those pass through the functional fragment that the CDR sequence clone of human monoclonal antibodies of the present invention is obtained to different skeleton districts.Can obtain described different skeleton district from different sorts, different people individuality or from the heavy chain or the light chain class of identical or different individuality.Described CDR transfer methods is known by the those skilled in the art.In example VIII, described from the IgM immunoglobulin (Ig) and transplanted the example of CDR sequence to IgG skeleton district.Shown in example VIII, in vitro the affinity maturation can allow the improvement of any in fact antibody, comprises having the low affinity IgMs of embryonal system sequence basically.

Can estimate the functionally active of antibody of the present invention or antibody fragment variant by the above-mentioned immunocompetent method that is used to measure human monoclonal antibodies.The useful especially method that is used to measure the segment functionally active comprises that competition radioimmunoassay and competitive ELISA measure.Described method can be used for screening to neoplastic cell or its antigen affinity through improved variant.Decide according to the variant quantity of institute's screening, strict degree that can be different uses described method.Also can adjust strict degree based on the cycle index or the number of occurrence that screening is carried out.For example, can use low strict degree and in screening step after a while, improve strict degree in the screening step in early days.Can in the screening antibody library, use the affinity standard of low strict degree need not create the clone of bigger storehouse or the bigger quantity of screening to amplify the diversity of being surveyed and can cause from the Ig integral part of previous institute screening, finding novel antibody.

The invention provides the medical composition that contains human monoclonal antibodies of the present invention or functional fragment and medical supporting agent.Available described composition gives human monoclonal antibodies or segment to reduce hyperplasia or the survival ability of neoplastic cell with throwing.Also can use described composition to detect neoplastic cell.The suitable medical supporting agent that is used for the inventive method behave know and comprise (for example) aqueous solution such as the physiology buffer saline, and other solvents or substratum are as ethylene glycol, glycerine, oils or injectable organic ester.The medicine supporting agent can contain acceptable compound on the physiology, and it is as solubility stable or the increase medical composition.Acceptable compound can be (for example) on the described physiology: carbohydrate, as glucose, sucrose or dextran; Antioxidant is as xitix or glutathione; Sequestrant; Low molecular weight protein; Or another kind of stablizer or vehicle.The medical supporting agent that comprises stablizer and sanitas is described in (for example) Martin, Remington ' s Pharm.Sci.. the 15th edition (Mack Publ.Co., Easton, 1975), it incorporates this paper by reference into.Medical supporting agent that all are above-mentioned and substratum may be for being defined as pharmaceutical grade in affiliated field, it means that they have competent purity and quality to be used for human body and to be different from comparable reagent in the research grade formulation.

The those skilled in the art can know select medical substratum and suitably the described composition of preparation will depend on the purposes wanted and the pattern of dispensing.

The invention provides multiple heavy chain territory or its functional fragment with CDR, described CDR has the substantive CDR sequence of the CDR of the sequence that is selected from the group that is made up of following SEQ ID NO: 2,6,10,12 and 14.The present invention also provides variable light chain territory or its functional fragment with CDR, and described CDR has the cardinal principle CDR sequence of the CDR of the sequence that is selected from the group that is made up of following SEQ ID NO: 4,8,16,18,20,22,24,26,28,30,32,34,36,38,40,42 and 44.The present invention also provides CDR or its functional fragment with the aminoacid sequence that is essentially the CDR that is selected from the group that is made up of following SEQ ID NO: 2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42 and 44.

Can produce CDR of the present invention or its functional fragment by method known and as indicated above in the affiliated field.For example, can produce CDR of the present invention by recombination method or chemosynthesis.Use that can be favourable CDR of the present invention (for example) is to generate optionally the antiidiotypic antibody in conjunction with tumour-specific human monoclonal antibodies of the present invention.Produce and use the method that is used for diagnosing with the antiidiotypic antibody of therapeutic purpose to be known by the people in affiliated field.

Antibody fragment of the present invention can be variable heavy chain territory or variable light chain territory.Use method as herein described or various under in the field known additive method can in recombination system, express described segment.For example, proteantigen is had under being expressed in of variable heavy chain territory (it is similar to the affinity of those desired monoclonal antibodies for proteantigen) of affinity in the field known as people such as (for example) Ward, NaturePeople such as 341:544-546 (1989) and Williams, Proc Natl.Acad.Sci.USA86:5537-5541 (1989).In description.Can (for example) can come upgrading segment of the present invention to produce multivalent binding fragment at two described cysteine residues that between the segment of upgrading, form intermolecular cross-linking by merging, it is described in people such as williams at the variable heavy chain territory, supra (1989).Also can (for example) its restricted generation on conformation be had in conjunction with active CDR segment by expressing the CDR territory.People such as (for example) Ditzel, The J.Immunol.Described the expression to restricted CDR on the conformation among the 157:739-749 (1996), described CDR forms by adding to form disulfide linkage and the antigen of female antibody is had specific two halfcystines.Can be or in replacing by described various interpolations herein, deletion to amino acid or other parts (as marker) anyly come upgrading antibody fragment of the present invention.

The present invention also provides by throwing the human monoclonal antibodies or the functional fragment (it is produced by the clone that is selected from the group that is made up of H1140, H2420 and H935) that give significant quantity to cell and reduces the outgrowth method of neoplastic cell.Also provide by throwing the human monoclonal antibodies or the functional fragment that give significant quantity and reduce the outgrowth method of neoplastic cell to cell, described human monoclonal antibodies or functional fragment have at least one CDR, and it has the aminoacid sequence of the CDR that is selected from the sequence in the group that is made up of following SEQ ID NO substantially: 2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42 and 44.Be used to reduce outgrowth human monoclonal antibodies of neoplastic cell or functional fragment and may further include marker as cytotoxic agent or cytostatics, and can be combined with medical supporting agent.

The significant quantity that is used to reduce the outgrowth antibody of neoplastic cell or its functional fragment be known or can by the those skilled in the art use in vitro method or reliably animal model determine easily.In vitro method can comprise that (for example) decision is used to reduce the significant quantity of neoplastic cell growth or neoplastic cell transfer compositions.The used neoplastic cell of in vitro method in the minimizing that is used for detecting growth or shifts can be external (ex vivo) culture of (for example) tumor cell line or tumour.Clone or tumour can be clone or the tumours that (for example) originates from breast, lung or ovary tissue.Be used to suppress the neoplastic cell growth significant quantity can for (for example) suppress DNA synthetic, suppress cell fission, inducing apoptosis, bring out non-apoptosis deactivation or suppress the significant quantity of vasculogenesis.Being used to suppress significant quantity that neoplastic cell shifts can be (for example) and is used to suppress cell mobility, cell migration, cell attachment, cell invasion or hyperplasia.

Also can from rodent, decide the significant quantity that is used to reduce the outgrowth antibody of neoplastic cell or its functional fragment by the heterograft of people's tumour.Described rodent can be (for example) rat or mouse.Mouse can be (for example) normal or immune disorder.The mouse of immune disorder can be (for example) nude mice or severe severe combined immunodeficiency mouse (scid mouse).Described kind is known by the people and can be obtained from commercial source in affiliated field kind.Can by comprise through subcutaneous, through vein, and human cancer cell is introduced in the animal through a large amount of routes in abdominal cavity.

After the tumour establishment, described animal can be treated with of the present invention a kind of molecule of various dose, and can be determined tumor quality or volume.Effect can be used as the partially or completely decline of tumour and measures.The effective dose that is used for the treatment of cancer causes more part of tumour and decline fully.

Can decide the significant quantity of molecule of the present invention by the those skilled in the art, and described significant quantity will depend on age, body weight, sex and medical conditions as individuality and the factor of throwing the particular course of giving therapeutical agent.Throwing give composition of the present invention with the useful route of treatment cancer comprise in (for example) muscle, tumour, route in the blood vessel, in the intraperitoneal, subcutaneous or nose.Can decide effect by the those skilled in the art to cancer patients's concrete treatment.For example, can use in vivo or in vitro diagnostic method as mentioned below measure tumour restored or receive treatment after be eliminated.In addition, can use normal emissary sign, as for cancer patients's the survival rate and the quality of life of increase.

The invention provides by sample is contacted with human monoclonal antibodies or functional fragment (it is produced by the clone that is selected from the group that is made up of H1140, H2420 and H935) and detect neoplastic cell, and detect human monoclonal antibodies or functional fragment specificity bonded method to this sample, wherein compare with normal cell, the human monoclonal antibodies or the functional fragment of the content of existence or increase represent to exist cancer or cancer-prone physique.The present invention also provides by making sample and having and is selected from the NO2 by SEQ ID, 4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40, the human monoclonal antibodies of the cdr amino acid sequence of the sequence in 42 and 44 groups that formed or functional fragment contact and detect neoplastic cell, and detecting human monoclonal antibodies or functional fragment specificity bonded method to this sample, the human monoclonal antibodies of the content that exists or increase of wherein comparing with normal cell or the expression of functional fragment exist cancer or cancer-prone physique.

Term used herein " sample " is used to refer to any biological fluid, cell, tissue, organ or its part, and it comprises or comprise potentially neoplastic cell.Biological fluid can be (for example) blood or lymph liquid.Tissue can be (for example) breast, ovary or lung.Sample can be a sample in vivo or in vitro.In vitro sample can be (for example) tissue slice, the sample that detect to obtain by living tissue or the cell that is substituted into or is adapted to tissue culture.Can prepare sample by the method for known in the affiliated field, as to be suitable for detection method concrete form.

Can make sample with human monoclonal antibodies of the present invention or functional fragment contacts and can detect the specificity combination of human monoclonal antibodies to sample.As the those skilled in the art according to used detection method form determined, described connection can be in vivo or sv.Can measure the specificity combination of human monoclonal antibodies by above-mentioned immunoassay to sample.Described immunoassay comprise that (for example) in vivo and in vitro immunoassay.In vivo immunoassay comprise (for example) radiant image (radioimaging).Described method comprises intraindividual sample is contacted with monoclonal antibody of the present invention, and comes the detection specificity combination by (for example) roentgenogramX imaging.In vitro immunoassay and comprise that qualitative test also comprises quantitative assay are such as (for example) immunohistochemistry mentioned above, immunofluorescence technique, radioimmunoassay, facs analysis, immunity the point method of the use of ink and water, immunoprecipitation and elisa assay.

Can determine to exist neoplastic cell by determining the content of comparing existence with normal cell or being in increase of the present invention through specificity bonded human monoclonal antibodies or functional fragment.As mentioned above, the those skilled in the art may determine that suitable normal cell is used for comparing with the neoplastic cell of particular type.

The present invention further provides purified substantially human tumor-specific antigen.Term " tumour specific antigen " is used to refer to the antigen of being expressed according to qualifications by human tumor cells.Term " is expressed according to qualifications by human tumor cells " and is used to refer to by the human tumor cells expression and by the tumour specific antigen of normal human cell with lower substantially horizontal expression.The tumour cell of described expression tumour specific antigen of the present invention can be the tumour cell in (for example) breast, lung or ovary tissue source.

Human tumor-specific antigen of the present invention and the specific reaction of human monoclonal antibodies of the present invention.Described antigen can carry out specific reaction by human monoclonal antibodies or the functional fragment that LH11238, LH13, H1140, H2420 or H935 hybridoma cell line are produced with (for example).Described antigen also can carry out specific reaction with human monoclonal antibodies or the functional fragment of the CDR with at least one cdr amino acid sequence with the sequence that is selected from the group that is made up of following SEQ IDNO: 2,4,6,8,10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42 and 44, perhaps with have at least one cdr amino acid sequence that has SEQ ID NO:6 or SEQ ID NO:8 substantially and carry out specific reaction.With the antigen of LH11238 human monoclonal antibodies or its pulsating variant reaction is to compare with normal people fibroblast, peripheral blood lymphocyte, melanoma cells or lung's cancer cells to be present in cell surface and the protein in the chamber between the lysosome of breast and ovarian cancer cell.With the antigen of LH13 human monoclonal antibodies reaction is the secretor type glycoprotein that is produced by the breast of comparing with normal fiber archeocyte or melanoma cells, lung and ovarian cancer cell.LH11238 and the further characteristic of LH13 antigen have been described in example.

Human tumor-specific antigen of the present invention can be advantageously used in treatment for cancer and the diagnosis.For example, can use described antigen with generation be used in the treatment and diagnostor in specificity in conjunction with the antigenic additional knot mixture of human tumor-specific.Described wedding agent energy (for example) suppresses or stimulates the function of tumour specific antigen or regulate immunity system, so that neoplastic cell is grown-catches or deactivation.Also can make described wedding agent conjugation to the marker such as (for example) cytotoxic agent or cytostatics, it causes the dead of tumour cell or catches.The useful reagent that is attached to tumour specific antigen of the present invention comprises (for example) ligand, receptor antagonist and antibody.Also can be in cancer therapy by carrying out preventive vaccination with the antigen of significant quantity and use purified substantially tumour specific antigen to suffering from cancer or having the patient of developing cancer risk.After preventive vaccination, individual immunity system can prevent, reduce the hyperplasia of expressing described antigenic neoplastic cell or eliminate this neoplastic cell.

Also can in the bonded method that detects tumour-specific wedding agent (as human monoclonal antibodies of the present invention or functional fragment), advantageously use the purified substantially tumour specific antigen of the present invention.For example, can in as the immunoassay of competitive ELISA, use as described in antigen.

The immune affinity program of knowing in the field under can using identifies by (for example) screening one plate (panel) human tumor cells and to be used to separate the purified substantially antigenic suitable parent material of the present invention.For example, as described in example II, can use elisa assay to be identified for the antigenic useful cell source of the effect of purifying subsequently.Antigenic useful cell source can be (for example) breast, ovary or pulmonary malignant tumour.The useful especially enlightenment material of tumour specific antigen of being used to purify is the H3396 breast cancer cell line.

Be used for separating purified substantially antigenic proper method and depend on described antigenic cellular localization.For example, can in secretor type substratum, cell surface membrane, vesicular membrane, tenuigenin or the nucleus of neoplastic cell, mainly express antigen of the present invention.Can determine antigenic cellular localization by the immunoassay in the field under (for example).For example, as described in example IV and VI, can use indirect immunofluorescence and elisa assay to set up antigenic main location.

Can monitor the purification of antagonist by known immune affinity program in the affiliated field.The method that is particularly useful that monitoring is purified comprises (for example) elisa assay and the immunity point method of the use of ink and water.

Can be by the biochemical program known in the affiliated field from the particular source tumour specific antigen of purifying.For example, purification can comprise centrifuging, red, orange, green, blue, yellow (ROGBY), electrophoretic method and immune affinity method, and it can be selected according to the feature of specific antigen by the those skilled in the art.Can use centrifuging to concentrate or enrichment contains the subcellular fraction of the antibody of substantial amount.Described subcellular fraction program is known by the people in affiliated field.Red, orange, green, blue, yellow (ROGBY) is also known by the people in affiliated field, and comprise based on antigenic size or to the different affinitys of specific resin with its isolating method from pollutent.Described resin can comprise (for example): size exclusion resin, ion exchange resin, and lectin post.As described in example VI, the antigenic resin that is particularly useful of the present invention that is used to purify is a Q SEPHAROSE FAST FLOW  agarose resin.Electrophoretic method is also known by the people in affiliated field, and comprises the peacekeeping two dimensional electrophoresis via (for example) acrylamide or sepharose.The immune affinity method is also known for the people in affiliated field and is comprised through the compound of conjugation to human monoclonal antibodies of the present invention.For immune affinity purification tumour specific antigen conjugation comprises chromatography resin and a-protein to the useful compound on the antibody.

Therefore, follow the biochemical program of knowing, the those skilled in the art can separate easily purified substantially tumour specific antigen of the present invention be used for the treatment of with diagnostor in.

Also the recombination method that can know by the those skilled in the art is from the nucleic acids for preparation of the tumour specific antigen of the present invention of encoding purified substantially tumour specific antigen of the present invention.

The invention provides the antigenic separated nucleic acid of coding human tumor-specific.Can separate the antigenic nucleic acid of coding human tumor-specific by the method that the those skilled in the art knows.Described method comprises that (for example) use monoclonal antibody of the present invention to come the screening expression library.

Other method comprises that the oligonucleotide that (for example) use to degenerate comes screening cDNA or genomic library as hybridization probe.Can be by separated tumour specific antigen of the present invention or the little serializing of its segment being determined the oligonucleotide sequence of described degeneration.Known other method of those skilled in the art that is used to produce the nucleic acid of tumour specific antigen comprises (for example): use the polymerase chain reaction (PCR) that carries out from the oligonucleotide introduction of the degeneration that aminoacid sequence obtained of tumour specific antigen of the present invention.Can rely on PCR to begin to amplify the sequence of wanting by index law from the copy of individual gene only.Aforesaid method is for known to the those skilled in the art and be described in people such as (for example) Sambrook, Molecular Cloning:A Laboratory ManualCold Spring Harbor Laboratory, NewYork (1992) and people such as document of wherein being quoted and Ansubel, Current Protocols in Molecular Biology.John Wiley and Sons, Baltimore, MD (1989); And people such as Harlow, Antibodies:A Laboratory Manual.Cold Spring Harbor Laboratory, New York (1989).These documents and the publication of wherein being quoted are incorporated herein by reference.

Should understand in the range of definition of the present invention that this paper provides and also comprise the active upgrading that does not influence various embodiments of the present invention substantially.Therefore, following example is used for illustrating and unrestricted the present invention.

Example I

Produce the tumour-specific human monoclonal antibodies

This example has shown the production of hybridoma of the human monoclonal antibodies of secretion and the specific reaction of tumour cell.Described a kind of unlimited hyperplasia turn into before make the in vitro program of immunization of normal lymphocyte with tumour cell or cytolemma.This program allows the lymphocyte that produces novel tumour-active human monoclonal antibodies is carried out enrichment.These human monoclonal antibodies will help cancer immunotherapy and immunodiagnosis program.

Prepare lymphocyte as following step.With the spleen tissue from unexpected victim separate, smash, and by force by the no.50 mesh wire sifter (Bellco, Vineland, NJ).By on 250xg, coming collecting cell and removed RBC in centrifugal 10 minutes by chloride leach.Remaining cell through the washing, in refrigerant (40%RPMI, 50%FCS and 10%DMSO) with 100 to 300 * 10 ⁶The concentration resuspending of individual cell/ml, with the 1.5-ml aliquot through freezing, and in liquid nitrogen, preserve.Adherent cell separates by this program with the lymphocyte both.

Follow as following foundation through blended lymphocyte reaction (MLR).Will from two kinds of different donors through refrigerated unit cell splenocyte preparation (as mentioned above) by thawing 37 ℃ of following slight wobble, collecting with the RPMI washed twice and by centrifugal 10min on 250xg.After washing for the second time, will be from 3 * 10 of each source ⁶Individual splenocyte be combined to the 1.5mM HEPES (FisherScientific), the 10%FBS (HyClone) that contain pH 7.4,2mM L-glutaminate, non-basic amino acid, 1mM Sodium.alpha.-ketopropionate, and the 2ml RPMI of 100 μ g/ml garamycin sulfates in, and be placed in the tissue culture ware of 24-hole.This MLR stimulates (as described below) to produce desired lymphokine internally together with antigenicity.

Then use the H3396 tumour cell handled through mitomycin, stimulate the MLR culture from the plasma membrane preparation of H3396 cell or through Paraformaldehyde 96 fixed H3922 tumour cell.H3922 and H3396 be from the metastasis of human breast carcinoma set up through cultured cells system, it can be transplanted in culture and preserve.Each clone is all derived from different transplanting.

By hereinafter preparing the H3396 cell of handling through mitomycin.The H3396 cell is placed in the tissue culture ware of 24-hole, grows to fusion and handles 12-15h with 0.1 μ g/ml ametycin.The cell fission of approximately catching over these days in the tumor cell line of this concentration.After 12-15h cultivates, remove the substratum that contains ametycin and also with cell with 2ml phosphoric acid normal saline solution (PBS) washing.

After removing substratum, by preparing tumour cell in the PBS of 2% Paraformaldehyde 96 solution, cultivating cell 15min under 25 ℃ through Paraformaldehyde 96 fixed H2922.Then wash described cell four times with PBS before use.

As hereinafter separating plasma membrane from the H3396 cell.With the 150-mm of ten H3396 cells that converge dish each with freezing (Tris damping fluid) twice of the TBS rinsing of 10ml and contain 1 μ g/ml leupeptin, 1 μ g/ml Gastric inhibitory polypeptide, and the 2ml/150-mm dish of 2mM AEBSF (Tris physiological saline) in gather in the crops.Using A type beater to knock with 40 times in 15ml Du Ensi homogenizer (Dounce homogenizer) breaks cell.Under 4 ℃ with 800xg with the centrifugal 5min of lysate to remove cell and the nucleus of not broken.Keep supernatant liquor and with spherolite resuspending, homogenizing and centrifugal in the TS of 0.5 volume snubber at 800xg.Supernatant liquor and the first supernatant liquor group are incorporated under 4 ℃ with 10 the centrifugal 2h of 000xg.Abandon supernatant liquor and with the spherolite resuspending in 2ml water.Use 2ml Du Ensi homogenizer and Type B beater with resuspending film spherolite.In 6.4% polymer system, mix by polyoxyethylene glycol, the 0.20ml 0.2 M potassiumphosphate of pH 7.2,0.8ml1M sucrose, 2.16ml water and carry out being separated of film 2.56ml20% dextran, 1.28ml 40% on ice.In this mixture, add the 1ml film and test tube is turned upside-down 20 times.By under 4 ℃, separating each phase with the centrifugal 5min of 800xg.Discharge the upper strata mutually and with its with from the low layer of blank (water) sample recovery mutually.Similarly, the material that is included in the interface is mixed with the upper strata of recovering from blank sample mutually at interior low layer.Two-phase is mixed, is turned upside-down 20 times and separated with the centrifugal 5min of 800xg under 4 ℃.The combination of materials that will recover mutually from the upper strata of two samples is adjusted to 21ml to volume with TBS, by under 4 ℃ with 10,000xg is centrifugal, and 2h comes collection membrane.Abandon upper strata liquid and contain resuspending spherolite in the TS damping fluid of proteinase inhibitor at 1-2ml with 2ml Du Ensi homogenizer and B beater.Preserve described film 12h down at 4 ℃, afterwards by under 4 ℃, coming from upper strata liquid separatin non-soluble material with the centrifugal 10min of 800xg.1-2ml contain suspend for the second time in the TS damping fluid of proteinase inhibitor described spherolite and with its as suspension 4 ℃ of storages down.Described upper strata liquid is called part 1 and the particulate matter through resuspending is called part 2.By measure 5 '-phosphonuclease or phosphodiesterase activity determine the plasma membrane in the part 1 is enriched to greater than 10 times.Described preparation has the minimum cytochrome C-reductase that depends on succinate (plastosome) activity or depends on cytochrome C-reductase (endoplasmic reticulum) activity of NADPH.

By make the in vitro immunity of MLR culture over three days with a kind of cultivate in the following material: the H339S cell monolayer of handling through mitomycin C, through Paraformaldehyde 96 fixed H3922 cell monolayer, 5mg from the plasma membrane part 1 of H3396 cell or 10mg plasma membrane part 2 from the H3396 cell.

By any makes through the in vitro immune unlimited hyperplasiaization of lymphocyte in two kinds of alternative methods.In first method, merge lymphocyte with the K6H6/B5 hybridoma.Stir culture is supported the K6H6/B5 cell in 1: 1 mixture of the Iscoves of RPMI and DMEM upgrading thing, and described mixture is with 10%FCS, 1% non-basic amino acid, 2mM glutamine, and 1mM Sodium.alpha.-ketopropionate.With about 4 * 10 ⁶Individual lymphocyte and 2 * 10 ⁶Logarithm phase K6H6/B5 groups of cells merges uses the RPMI washed twice.Add the mixture 1ml of 35% polyoxyethylene glycol (approximately m.w.1450) and 7.5%DMSO through 1min, then with RPMI be diluted to gradually 4ml and then the usefulness RPMI that replenished 10%FCS be diluted to 16ml.Then lymphocyte concentration is adjusted to 5 * 10 ⁴Individual cell/ml, and containing HAT (13.6 μ g/ml xanthoglobulin, 3.8 μ g/ml Thymine deoxyriboside, 1 μ g/ml azaserine) and the hybridoma substratum of 1.0 μ M ouabains (ouabain) (with the 15mM HEPES of 10%FCS, non-basic amino acid, 2 mM glutamine, 1mM Sodium.alpha.-ketopropionate, pH 7.4, and the RPMI that replenishes of 0.1mg/ml gentamicin) in the 0.2ml cell inoculation in the culture dish of 96-hole picking out reactivity without the cell screening as mentioned below of merging, and amplify the clone who is concerned about.Use the unlimited hyperplasia method in a this step, per 10 ⁶Lymphocyte through merging produces 10-50 hybridoma; Yet, be stable through after the three-wheel cell cultures 5% clone who is concerned about only being arranged.Hybridoma cell clone H1140, H2420 and H935 have been obtained by this unlimited hyperplasia program.

In the process of being devoted to improve clone's stability, to have estimated and be used to make through second kind of two-stage process of the unlimited hyperplasiaization of lymphocyte of immunity in vitro, it relates at first with EBV transformant and the clone that then is concerned about with the fusion of K6H6/B5 hybridoma.With isopyknic lymphocyte (8 * 10 ⁴Individual cell/ml) and the 1A2/C7 cell (8 * 10 that transforms through EBV ⁶The combination of individual cell/ml) and cumulative volume is doubled by adding the hybridoma culture.Add xanthoglobulin, Thymine deoxyriboside and azaserine so that their ultimate density is respectively 13.6 μ g/ml, 3.8 μ g/ml, reaches 1 μ g/ml.The described cell mixture of deposition two hectolambdas is to produce final 4000 lymphocyte and 1 * 10 altogether in each hole in each hole of 96-porocyte culture dish ⁵Individual 1A2/C7 cell.Raise described cell with hybridoma substratum-HAT in per three days and when 2 Zhou Houneng see cell colony, measured antibody product (being described in hereinafter).

Transforming lymphocyte with EBV generates than the antibody-secreting clone (per 10 of merging the higher per-cent of secretory antibody clone that lymphocyte obtained with the hybridization myeloma cell line ⁶Individual lymphocyte 50-100 secretory antibody clone).Yet, the general secretion of the female clone of class lymph lower level antibody (less than 10 μ g/ml), and as viewed with hybridoma, it demonstrates very poor long-range stability (it is stable amplifying the initial clone about 5% who is concerned about through three-wheel).For handling these limitation, before many wheels amplify, merge the female clone of tumor response class lymph with the K6H6/B5 cell as mentioned above.

Forming hybridoma from lymphoblastoid has improved stable so that keep stablizing through the female clone of the class lymph of three-wheel amplification 40% after fusion.The combination of following the EBV transformation after the fusion that carries out with the hybridization myeloma cell line causes and utilizes any one approach to obtain the corresponding lymphocytic higher unlimited hyperplasia frequency of unlimited hyperplasia frequency separately.In addition, these clones generally secrete the antibody greater than 20 μ g/ml.Produce hybridoma clone LH11238 and LH13 by this unlimited hyperplasia method of two steps.

The immunization and the unlimited hyperplasia condition that are used for producing hybridoma cell line H1140, H2420, H935 LH11238 and LH13 are summarized in table 2.

Table 2 is used to generate and produces the tumour-specific human monoclonal antibodies

The immunization of hybridoma cell line and unlimited hyperplasia condition

Hybridoma	The unlimited hyperplasia of immunization effect turns usefulness into
Hybridoma			??H1140	5mg H3396 plasma membrane part 1	Use the K6H6/B5 cytogamy
??H2420	5mg H3396 plasma membrane part 1	Use the K6H6/B5 cytogamy	??H1140	5mg H3396 plasma membrane part 1	Use the K6H6/B5 cytogamy
??H2420	5mg H3396 plasma membrane part 1	Use the K6H6/B5 cytogamy	??H935	The confluent monolayer of the H339S cell of handling through mitomycin	Use the K6H6/B5 cytogamy
??LH11238	10mg H339S plasma membrane part 2	EBV transformation and merge with the K6HS/B5 cell	??H935		Use the K6H6/B5 cytogamy
??LH11238	10mg H339S plasma membrane part 2	EBV transformation and merge with the K6HS/B5 cell	??LH13	4×10 ⁴Individual through Paraformaldehyde 96 fixed H3922 cell monolayer	EBV transformation and merge with the K6HS/B5 cell

The initial ELISA of use measure screening the lymphocytic culture supernatants of the unlimited hyperplasiaization of hanging oneself to the reactivity of the primary tumour cell individual layer of living.This has guaranteed that reactive antibody identifies surface antigen, and has avoided the artifact that is associated with the screening fixed cell.Deposit tumour cell in 96-porocyte culture dish, its cell concn is enough to produce the confluent monolayer of 90-95% behind 12-24h.Just remove substratum before use and cultivate 2h down adding in the hole and at 4 ℃ from hybridoma or through 50 μ l supernatant liquors of EBV cell transformed.Cultivate control wells (background) with the hybridoma substratum that 50 μ l are fresh.

With after cultivating, sucking-off supernatant liquor and with 200 μ l PBS rinsing cells three times.Then cultivate cell 1-2h with 50 μ l goat Anti-Human 1g (H+L) the alkaline phosphate ester conjugated bodies that in 1%BSA-PBS, dilute 1000 times.Sucking-off detects antibody and as above-mentioned soft rinsing cell four times.Under 25 ℃, cultivate these dishes 1h by the 0.2 M 2-amido-2-methyl isophthalic acid-propanol solution, the 0.5MTris of pH 10.2, the 0.1% sodiumazide furnishing that add 50 μ l 10mM phenolphthalein monophosphates.Stop this reaction and in 560nm place mensuration absorbancy by 50 μ l 30mMTris, the 15mM EDTA that adds pH 10.2.The background value of this mensuration is generally A560＜0.060.Selection has 4 times and is used for further characterizing to background or bigger clone body.

Also analyzed to show and be used for combining fibroblast through fixed HF235 normal people with reactive antibody of H3396 cell.After removing substratum, in the PBS of 2% Paraformaldehyde 96 solution, descend fixedly HF235 cells at 25 ℃.Then wash described cell four times with PBS before use.Be generally A560＜0.080 for using through the ELISA of fixed fibroblast background value.Minimal reaction with fibroblast is defined as on background associativity less than 2 times.

For the homotype antibody of determining to produce, with goat Anti-Human IgM or the goat Anti-Human IgG coating microtiter plates of 0.5 μ g/ml by hybridoma.Detect antibody-binding with goat Anti-Human Ig alkaline phosphate conjugated body.When producing a positive signal with one of capture antibody, supernatant liquor stops the carrying out measured.Similarly, by also using goat Anti-Human γ or the special alkaline phosphatase conjugation of κ chain with suitable heavy chain reagent capture antibody.

Carry out similarly immunoglobulin (Ig) quantitatively, the supernatant liquor of Ig that contains unknown quantity except serial dilution is up to detecting reactivity.Diluent value in the linearity range that drops on typical curve calculates Ig concentration, defines this typical curve by the standard model of the polyclone people IgM through purifying that uses in 0.01 to 2.0 μ g/ml scope.

By at the centrifugal 10min of 800xg with by filtering the classification of carrying out, from LH11238 hybridoma supernatant liquor, be settled out immunoglobulin (Ig) through 0.45 μ m cellulose acetate filter.Supernatant liquor is placed in the dialysis tubing (Mr separation point 12-14kDa) and by with diquat (Aquacide) (Calbiochem) apply this pipe and 4 ℃ down placement 6-8h with its concentrated two to four times.Remove unnecessary diquat, rinsing dialysis tubing, and the multiple variation of freezing relatively distilled water and the sample 12-24h that dialyses.By 10, the centrifugal 30min of 000xg collects through sedimentary antibody.Remove supernatant liquor and concentrate resuspending spherolite among 10 times the warm PBS at the warp of minimum volume.Behind the most of spherolites of dissolving, buffer concentration is adjusted to PBS and by 10,000xg is centrifugal, and 30min removes insoluble material.Precipitating antibody H1140, H2420 and H935 in low ionic strength buffer liquid similarly.Therefore, in order to obtain, utilize the YM-30 film to reduce through clarifying supernatant liquor volume with the Amicon instrument through spissated antibody.By this approach antibody is concentrated greater than 50 times.

5 percent the hybridoma through screening (372/7216) combines the TSA greater than four times of backgrounds.55 (15%) in these 372 clone body have produced the antibody of obvious debond fibroblast.Though never with tumour cell or membrane portions cultivate contrast MLR in isolate the hybridoma of a large amount of secretory antibodies, none demonstrates preferential tumor response in these antibody.About 20% (11/55) the hybridoma that produces the antibody that shows tumour-specific is stable through the multiple amplification in culture.Five antibody of secreting greater than 20 μ g/ml in these clone body.Summed up the characteristic of these clone body in the table 2.All these five antibody are that IgM is isostructural and show as being determined at the H3396 cell through the determined immunoreactivity of fixed individual layer.From employed each culture condition and unlimited hyperplasia program, generate at least one antibody.Further (hereinafter) characterized and shown maximum immunoreactive two antibody LH13 and LH11238.

The characteristic of the tumour-specific human monoclonal antibodies that is produced by hybridoma cell line H1140, H2420, H935 LH11238 and LH13 is summarized in the table 3.

Table 3: the feature of tumour-specific human monoclonal antibodies

Hybridoma	Homotype/light chain class	Secretion level (μ g/ml) ^a	Immunoreactivity ^b	Precipitating action ^c
Hybridoma	Homotype/light chain class	Secretion level (μ g/ml) ^a	Immunoreactivity ^b	Precipitating action ^c	??H1140	????IgM/λ	????40	?????1.0	????+
??H2420	????IgM/λ	????S3	?????1.3	????+	??H1140	????IgM/λ	????40	?????1.0	????+
??H2420	????IgM/λ	????S3	?????1.3	????+	??H935	????IgM/λ	????36	?????1.4	????+
??LH11238	????IgM/K	????29	?????5.7	????+	??H935	????IgM/λ	????36	?????1.4	????+
??LH11238	????IgM/K	????29	?????5.7	????+	??LH13	????IgM/λ	????26	?????341	????-

^aSecretion level is the standard value that supernatant liquor obtained from final cultures

^bImmunoreactivity (AA560/[(μ g Ig) is (min)] be normalized to the immunoreactivity of H1140 in the mensuration of converging on Paraformaldehyde 96 fixed H3396 individual layer and with it

^cPrecipitating action is represented can (+) or can not (-) precipitating antibody in the low ionic strength buffer device

In a word, this case representation can be used for generating the human monoclonal antibodies that reacts with novel tumour antigen without the people's immunity integral part (from non-lotus knurl patient's splenocyte) that excites.With whole tumour cells or derived from any of the membrane portions of tumour cell via stimulating the MLR culture to reach this purpose.By generating tumor specific antibody and by lacking the specificity that shows antigenic stimulation from the tumor specific antibody of the culture of handling without tumour cell or membrane portions.

Example II The immunoreactivity of LH13 and LH11238 antibody

This example has shown the immunoreactivity of LH13 and a LH11238 antibody and a plate human tumor cells.

For LH13 and LH11238 antibody being used for the purpose of immunodiagnosis and immunotherapy, need to differentiate the scope of the tumor type of expressing corresponding antibodies.Tumour cell is the representative of corresponding tumor type, and normal fibroblast is the representative of non--neoplastic cell tissue.Derive tumor cell line from melanoma, lung cancer, ovarian cancer and mastocarcinoma.Tested the immunoreactivity of human monoclonal antibodies of the present invention and human carcinoma cell line and normal fiber archeocyte.

H3396, H3464, H3477 and H3922 be from the metastasis of human breast carcinoma set up through cultured cells system, they are transplanted and be stored in the culture.H2981 and H2987 be from people's lung cancer set up through cultured cells system, they are transplanted and be stored in the culture.H3639 and H3723 be from human ovarian cancer set up through cultured cells system, they are transplanted and be stored in the culture.Each clone is derived from different explants.By using the antibody concentration of cultivating wide region through fixed tumour cell individual layer, measure immunoreactivity by above-mentioned ELISA program.Detection antibody with saturation capacity is measured antibody-binding.In the setting-out line scope with AA560/[(μ g Ig) (min)] measure antibody-binding.The tumour-specific human monoclonal antibodies is shown in Table 4 at a plate tumour and Normocellular immunoreactivity.

Table 4: at the immunocompetence of tumour and Normocellular human monoclonal antibodies LH13 and LH11228

Immunocompetence
Immunocompetence	(ΔA560/[(μg?Ig)(min)])
Clone	(ΔA560/[(μg?Ig)(min)])	Describe	????LH13???????LH1123?8

??HF285	The normal fiber archeocyte	????0	????0
??HF285	The normal fiber archeocyte	????0	????0	??H2669	Melanoma	????0	????0
??H3774	Melanoma	????0	????0	??H2669	Melanoma	????0	????0
??H3774	Melanoma	????0	????0	??H3396	Breast cancer	????1.136	????0.019
??H34S4	Breast cancer	????0.507	????0.022	??H3396	Breast cancer	????1.136	????0.019
??H34S4	Breast cancer	????0.507	????0.022	??H3477	Breast cancer	????0.046	????0.008
??H3922	Breast cancer	????0 ^a	????0	??H3477	Breast cancer	????0.046	????0.008
??H3922	Breast cancer	????0 ^a	????0	??H2981	Lung cancer	????0.584	????0
??H2987	Lung cancer	????0	????0	??H2981	Lung cancer	????0.584	????0
??H2987	Lung cancer	????0	????0	??H3S39	Ovarian cancer	????0 ^a	????0.011
??H3723	Ovarian cancer	????2.602	????0.026	??H3S39	Ovarian cancer	????0 ^a	????0.011

^aShown in substantially immunoreactivity according to the Premeabilisation of cells effect of carrying out with 0.1% digitonin

As shown in table 4, LH13 on the tumour cell plate, demonstrate have height (H3723 and H3396), in (H2981 and H3464), and low (H3477) immunoreactive broad intersection-reactivity.LH13 antigen is gone up at undamaged normal fiber archeocyte (HF285), melanoma (H2669 and H3774) and is lacked or exist with the level that can't detect, and has tested several undamaged cancers (H3922, H2987, and H3639).Lack with the reactivity of H3922 cell especially amazing because LH13 separates from the lymphocyte of having crossed with the H3922 cytositimulation.In order further to detect this result, permeate each clone with 0.1% digitonin and also detect their reactivity again.When measuring intact cell, be entirely under the condition of negative two clone H3922 and H3639 chamber between antibody can arrive in the cell in conjunction with LH13 antibody.And, when measuring under non--infiltration condition, have the immunoreactive H2981 cell of medium LH13 and under infiltration condition (immunoreactivity is greater than 7.0), have hyperergy.Under the existence and shortage of 0.1% digitonin, because variant from the Tissue Culture Dish loss cell in cultivating process, obtain can not directly be compared about immunoreactive data.

LH11238 demonstrates similar reactivity and distributes, though its immunoreactivity degree always is significantly smaller than the immunoreactivity degree of LH13 antibody.With undamaged H3396, H3464, H3477, H3639, and H3723 observing response.The cell of other tested person is negative, comprises fibroblast and cancerous cell line.Based on LH13 and LH11238 the immunoreactivity of each clone made being measured, these antibody seem to identify identical epi-position.For example, LH13 demonstrates with the medium immunoreactivity of H2981 lung cancerous cell line and does not detect LH11238 combining this clone.Similarly, LH11238 combines undamaged H3639 ovarian cancer cell, though can not detect the associativity of LH13 under same breeding condition.

Example III

The combination of LH13 and LH11238 human monoclonal antibodies is active

This example has shown that human monoclonal antibodies LH13 and LH11238 are active with combining of normal cell and tumour cell.

In order to measure human monoclonal antibodies and tumour cell and Normocellular immunoreactivity, used fluidic cell surveying (facs analysis).Selected the H34S4 breast cancer cell to be because they had not only been expressed LH13 but also had expressed LH11238 antigen and be easy to be separated as single-cell suspension liquid by elisa assay.Also detect normal peripheral blood lymphocyte and represent healthy tissues.Facs analyzes the cell mass heterogeneity that allows extraly about antigen presentation.

From culture dish, remove the H3464 cell with trypsinase or sodium ethylene diamine tetracetate (EDTA), with its with PBS washing, in the tumour substratum with 2 * 10 ⁶Individual cell/ml resuspending, and under 37 ℃, recover 2h.With 1 * 10 ⁶Individual tumour cell is cultivated antibody 30min at 5.0 μ g/ml with the cumulative volume of 50 μ l on ice cube.With cell once, on ice cube, cultivate 30min through 2 μ g/ml FITC labelled goat Anti-Human IgM with what be diluted in 1%BSA-PBS with the freezing PBS of 1.0ml washing, and again with the freezing PBS washing of 1.0ml once.Analyze the antibody that is attached on the cell with Becton Dickinson FACSort.Consistent with ELISA result, show the dyeing pattern (Figure 1A) that moves with respect to incoherent contrast people IgM with LH11238 with through the H3464 cell that the anti-people IgM of FITC mark cultivates.The cell greater than 90% of tested person combines LH11238, although viewed wide region fluorescence intensity is consistent in the heterogenous expression level of these tumour cells with antigen.With H935, H2420, and H1140 also observe similar FACS dye distribution.

Surprisingly, measure (table 4) as ELISA and on the H3464 cell, show displacement (Figure 1B) hardly than the reactive big 23 times LH13 antibody of LH11238.In order to determine whether LH13 antigen is especially responsive with the proteolyzing condition of the tumour cell of (trypsinase) to being used for compartment analysis, allow cell to be arranged longer recovery period or utilize non--proteoclastic cell isolation method, discharge as sodium ethylene diamine tetracetate (EDTA) with after separating.These two kinds of methods all do not cause significantly bigger dyeing of tumour cell.

The Facs analysis (as peripheral blood lymphocyte) of normal cell is negative to the antibody of each tested person, and it is consistent with the ELISA result who is obtained with the normal fiber archeocyte.

Example IV

The antigenic sign of LH11238

This case description the antigenic ubcellular of LH11238 characterize.ELISA screening and facs analysis to viable cell mentioned above all shows the expression of LH11238 antigen on the tumour cell plasma membrane.Yet these approach do not detect the distribution of antigen on the cell inner structure of cancer derived cell.Used the immunofluorescence technique analysis to locate to detect in the antigenic cell of LH11238.

On 12-mm dome slide, inoculated the H3464 cell monolayer in one day before use and its PBS solution with 2% Paraformaldehyde 96 has been descended fixedly 15min at 25 ℃.Cell is also used 50 μ l/ml antibody cultivation cell 2h under 4 ℃ of the 1%BSA-PBS (through what permeate) that is diluted in 1%BSA-PBS (without what permeate) or contains 0.1% digitonin for twice with the PBS rinsing.With cell with twice of PBS rinsing and then in the dark in the damping fluid identical, cultivate this cell through FITC labelled goat Anti-Human IgM with dilution in 1: 500 with former antibody.Be installed in Fluoromount-G (Southern Biotechnology) with PBS rinsing cell 4 times and with cover glass.Olympus microscope with optics that is equipped with surface fluorescence (epifluorescent) and Olympus Splan 40X (NA 0.70) object lens makes cell imaging.With the LH11238 antibody staining without the infiltration, demonstrate and navigate to the corresponding to padding of plasma membrane through Paraformaldehyde 96 fixed H3464 cell.Consistent with facs analysis, greater than 70% combine LH11238 through fixed H3464 cell.When the H3464 cell arrives the cell inner structure through infiltration with permission, observe dyeing point-like, the nucleus outside.The LH11238 that is attached to the H3464 cell is specific, because the contrast of carrying out with people's antibody of uncorrelated homotype and concentration coupling is cultivated and do not dyeed unbroken or through the cell of 0.1%. digitonin infiltration.Hinted the location of antigen in lysosome in dyeing observed point-like, the nucleus outside on the cell of infiltration.For verifying this conclusion, with the antibody staining of cell to a kind of known lysosome gC D63.Be similar to those cell inner structures with the LH11238 mark with the antibody of CD63 being cultivated the painted cell inner structure of H3464 cell institute, its lysosome with the interior LH11238 of cell is located consistent.

Reach a conclusion based on these results: LH11238 antigen not only is present on the plasma membrane but also be present in the lysosome of H3464 cell.

Example V

The antigenic internalization of LH11238

This example show LH11238 antigen from the plasma membrane internalization to chamber lysosome.

Immunofluorescence technique experiment mentioned above shows that LH11238 antigen not only is present in cell surface but also be present in the lysosome of tumour cell.May be by at the LH11238 antigen of lysosome and cell surface coexpression or by having been produced dual location to chamber endosome/lysosome by internalization.In order to determine that LH11238 antigen whether by internalization, has carried out the modification of immunofluorescence locating procedure.

The H3464 cell monolayer quick-frozen of living on ice 30-60min to suppress endocytosis fully.Follow cell washing, cultivate, wash once more and use through FITC labelled goat Anti-Human IgM antibody and cultivate with LH11238 antibody.In all steps of carrying out, keep the temperature of cell under 4 ℃, to suppress endocytosis fully with assurance always.Then with the cell culture medium of pre-warm with under the cell transfer to 37 ℃.At the different timed interval places cell transfer being returned 4 ℃ is fixed down and with 2% Paraformaldehyde 96.Original observed is to the padding of diffusion, its with use fixed cell viewed the same without permeating.Behind 37 ℃ of following 10min, observe bunch collection of LH11238 antibody in a plurality of sites of cell surface.With the longer cultivation time, LH11238 at first navigates on several sites of cell surface, and then begins to carry out internalization.If cultivate cell, only cultivate cell, do not observe these dyeing patterns under 4 ℃ so if perhaps in experimentation, cell maintained with secondary antibody with incoherent former antibody.These results are consistent by the LH11238 of internalization in conjunction with also passing through the H3464 cell with LH11238.

Generally speaking, at first based on the facs analysis of the elisa assay of damaged cell not, viable cell, and the immunolocalization that uses the cell without infiltration to carry out characterize LH11238 antibody.Yet, use the part of this antibody of further immunolocalization research explanation that the cell through infiltration carries out also to be positioned chamber between the nucleus outside of point-like.Arrive similar dyeing with the cell observation of cultivating through anti--CD63 antibody (a kind of lysosomal protein).This result is consistent at plasma membrane and the dual location in lysosome with LH11238 antigen, description taken in conjunction to the antibody on viable cell surface by internalization.Before observed tumour antigen to plasma membrane and lysosomal dual location.For example, the BR96 antibody membrane protein lamp-1 that combination is associated with lysosome on not damaged surface of tumor cells is although lamp-1 normally mainly is positioned at the integral membrane proteins matter of chamber between lysosome.In addition, also verified go out the soluble protein of elevated levels, as kethepsin (cathespin) B, D, and L from tumor cell secretion.The part of oozy cathepsin D is by combination of seminose 6-phosphate acceptor and internalization.The membrane protein whether present not clear LH11238 is solvable or complete.Yet, in the soluble fractions that the TX-114 of cell extract is separated, having found this antigen, this and LH11238 are associated consistent and opposite with complete membrane protein with film.

Example VI

The antigenic sign of LH13

This example has shown the antigenic location of LH13.

When as indicated above, through fixed by cancer deutero-clone individual layer on when the elisa assay LH13 antibody, it shows effective immunoreactivity.Yet facs analysis hints out that LH13 only is attached on the cell surface rarely.Further elisa assay explanation LH13 antigen mainly be present in when with 0.1% digitonin processing cell, only can arrive antibody between indoor.For further detecting LH13 antigen, immunofluorescence analysis and direct elisa assay have been adopted indoor between ubcellular or secreting the expression that in the substratum.

In order to detect the antigenic Subcellular Localization of LH13, on Paraformaldehyde 96 fixed H3464 cell, carry out immunofluorescence technique in as indicated above.Use the slight stain that causes cell surface without the H3464 cell culture LH13 antibody (use LH11238 antibody as indicated above) of infiltration, it is with consistent by the viewed small displacement of facs analysis.Detect less than dyeing with cultivating LH13 antibody result by immunofluorescence technique through the H3464 cell of infiltration.May be almost do not have LH13 to be associated with the cell inner structure of H3464 cell or the cell of LH13 in form do not arrived by the antibody recognition in this clone.

These results can not illustrate main secretion LH13 antigen.In order to observe oozy LH13 antigen, the LH13 antibody (0.01ug/ml) of constant is diluted in the substratum of the increasing amount that from the confluent monolayer of H3396 cell, has removed out (conditioned medium).Then the condition for surveys substratum is to the associativity through fixed H3396 individual layer.The concentration that increases conditioned medium is observed LH13 antibody the associativity of this individual layer is suitably reduced (about 15%) (Fig. 2 A), and it is consistent with the LH13 antigen in being present in described substratum.

Used direct ELISA form to observe the LH13 antigen in the conditioned medium of H3396 cell.Conditioned medium is cultivated 10-12h, removed out under 4 ℃ in 96-porocyte culture dish, and for the hole of LH13 antibody-binding check through washing.The antigen that is present in from the conditioned medium that the H3396 cell is obtained is attached to culture dish and is easy to detect (Fig. 2 B).By cultivating the specificity that LH13 antibody shows antibodies with the cell cultures hole, following preprocessing substance mistake has been used in described cell cultures hole: (1) is from the conditioned medium of the tumour system (H3922) of expressing LH13 that can not detection limit, (2) fresh culture (substratum), or (3) no material (blank).What these pre-breeding conditions did not all cause being attached to this hole can detected LH13 (Fig. 2 B).

In a word, the antigenic sign of LH13 illustrates that this antibody great majority are found in the conditioned medium.Based on this observation, sum up LH13 and come out from tumor cell secretion.

Example VII

Purify and the further LH13 of sign antigen

This case description the antigenic purification of LH13 and further characterizing.

As mentioned above, LH13 antigen is secreted out and is demonstrated the surprising high affinity of pair cell culture dish from breast tumor cell.These observe explanation purification LH13 antigen from the H3396 conditioned medium at an easy rate, utilize its mode in conjunction with its purification of conduct monitoring to culture dish.In case LH13 antigen after purifying, can be easy to measure its to the susceptibility of all ingredients with its characteristic of further sign.

As the antigenic the first step of enrichment LH13, H3396 clone is adapted to middle growth of substratum [use TCH (Celox, the blood serum substituting product of regulation), 2mM L-glutaminate, non-basic amino acid, reach the Iscove substratum that ketone acid sodium replenishes in the 1mM] with the additional serum-free of the protein of minimum.Cultured cells secretes and the suitable antigen of level of cultured cells in the presence of serum under these conditions.Conditioned medium is collected, planned as a whole, and water is diluted to 1 liter to 250ml to reduce ionic strength.Diluted substratum is applied on 11cm * 1.5cm post of using the PBS isostatic Q SEPHAROSE FAST FLOW agarose resin through concentrating 0.25-times (0.25X) with 3ml/min.Lacked shown in the reactivity by the part that flows through, most of antigens are attached on the post.After washing this post, use from the linear gradient of the 0.25X PBS solution of 100%0.25X PBS to 70%0.5M NaCl and wash dike protein with the flow velocity of 6ml/min with 200ml 0.25X PBS.

With using BSA to come the protein concn of measuring column cut as the BCA protein determination of standard.The LH13 antigen of following measuring column cut.Each cut is diluted 10 times and 50 μ l/ holes are transferred in the aseptic 96-porocyte culture dish at 4 ℃ cultivate 12h down in water.Wash this dish twice and cultivate 2h down at 37 ℃ with PBS with the 10 μ g/ml LH13 antibody that are diluted among the 1%BSA-PBS.Then will coil with PBS washing four times, cultivate 1h to the goat Anti-Human Ig alkaline phosphatase conjugation among the 1%BSA-PBS down at 25 ℃ with 1000 times of dilutions, and as indicated above the development.

Most protein is washed dike come out (Fig. 3, closed hoop) in 150mM and 250mM NaCl, and most of LH13 antigen is washed dike come out (Fig. 3, cavity ring) more than 250mM NaCl.The specific activity of summit antigen cut (ELISA signal/[protein]) is big 200 times compared with the beginning material.In contrast, dilute LH13 antigen with NaCl concentration range (50mM is to 400mM) and whether influenced the combination of pair cell culture dish to measure ionic concn.For from post in conjunction with and wash the NaCl concentration range that dike LH13 applied and do not influence the antigen that is attached to Tissue Culture Dish.Therefore, directly ELISA has accurately reflected the distribution of antigen in multiple post cut.

Further decompose the post cut by on 4-20%SDS-polyacrylamide gradient gel, carrying out electrophoresis.Coomassie brilliant blue staining (Coomassie blue staining) discloses the single main protein in great majority reaction cut, but the west ink dot of each post cut (Western blot) can not be differentiated this to be the LH13-reactive antigen.

In order further to characterize LH13 antigen, use direct ELISA method to detect its susceptibility to various processing.As mentioned above LH13 antigen is coated on the Tissue Culture Dish and with it and cultivates under various conditions.Cultivate LH13 antigen 30min with 5mg/ml trypsinase down at 37 ℃.Another is chosen as; by using 2U/ml sialidase (Oxford GlycoSystems), 400U/ml endo-glycosidase-F/ peptide-N-Glycosylase F (Endo F; PNGase F) in (Oxford GlycoSystems), the 60mU/ml-α-N-ethanoyl galactosamindase (O-glycanase) (Oxford GlycoSystems) or 2U/ml sialidase add in the 60mU/ml-α-N-ethanoyl galactosamindase (sialidase; the O-glycanase) before 37 ℃ are handled 24h down, handles 30min with the 10%SDS+2% beta-mercaptoethanol down at 37 ℃ and make the sex change of LH13 antigen.Culture dish is washed three times and estimation LH13 antibody-binding as indicated above with PBS.With the influence handled each time with under the same conditions separately with the control sample of damping fluid processing compare (Fig. 4).

Handle the almost not influence of final associativity of LH13 antigen antagonist with 10%SDS+2% beta-mercaptoethanol, sialidase or interior-α-N-ethanoyl galactosamindase (O-glycanase).Before processing, make the antigen sex change or handle with two kinds of Glycosylases simultaneously and can not influence the resistibility of antigen the processing done with sialidase and O-glycanase.Directly passing through with trypsin 79% among the ELISA, Fig. 4) or with endo-glycosidase-F/ peptide-N-Glycosylase F (36%) handling the associativity that has reduced antibody without showing.Antibody is further reduced the combination with the LH13 of 10%SDS+2% beta-mercaptoethanol sex change before handling with endo-glycosidase-F/ peptide-N-Glycosylase F, and the associativity of comparing with undressed sample reduces 75% (Fig. 4).

Because the range of specificity of endo-glycosidase-F/ peptide-N-Glycosylase F is wide, can not obtain concrete conclusion according to the structure of carbohydrate.Yet the associativity of LH13 antibody is not benefited from the influence of the processing (it has removed N-acyl group or O-acyl group, non-reduced terminal sialic acid) that sialidase does fully.Do not influence antibodies with processing (it removes the Gal β 1-3GalNAc that is associated with Serine or Threonine) that interior-α-N-ethanoyl galactosamindase is done or the processing of doing with the combination of sialidase and interior-α-N-ethanoyl galactosamindase yet.The trypsinase susceptibility that is attached to antigenic antibody illustrates that this epi-position is associated with protein component.

In a word, use ion exchange chromatography never to contain the LH13 antigen of purifying in the conditioned medium of H3396 serum greater than 200 times.Preliminary study shows that the LH13 antigen through purifying is excluded outside Superdex 75 gel-filtration columns, and it is consistent with the antigen greater than 70kDa.Handling the LH13 antigen be attached to Tissue Culture Dish with all ingredients provides proof explanation carbohydrate and protein component all to exist, and with epi-position to the combination of LH13 antibody or that LH13 antigen is attached to the tissue culture ware is relevant.Can not between these two kinds of possibilities, make differentiation at present.

Example VIII

Antibody maturation in vitro

This examples show the clone of antagonist variable region, contain the variable region of being cloned variant the Fab storehouse synthetic, and have the screening of the variant of improved affinity in order to discriminating.

The DNA ordering of LH13 and LH11238 shows H chain and L chain and people's embryonal system sequence height homology of two kinds of antibody.LH11238 VH is identical with embryonal system sequence D P-63 (VH4.21) and LH13 VH and DP-10/hv1051 (Vh1-69) are identical.And LH11238 VL and LH13 VL identical with embryonal system sequence D PK21/humkv328h5 and DPL16/VL3.1 height homology contain mononucleotide and change in the V-J junction.Height homology between antibody and the embryonal system sequence shows that antibody may not carry out tangible affinity maturation in cell culture.In order to improve the physical attribute of LH13 and LH11238 antibody fast by protein engineering, as described below with antibody cloning in bacterial expression system.

As hereinafter coming clonal antibody VH and VL Variable Area.From 5 * 10 of each clone body ⁷Individual cellular segregation goes out total RNA and prepares first gang of cDNA.By PCR use group and people's signal sequence homologous 5 ' introduction down and corresponding to 3 ' introduction of people CH1 (5 '-AGACGAGGGGGAAAAGGGTT-3 ', SEQ IDNO:47) amplifies H chain variable region: ATGGAGTTTGGGCTGAGCTGG (SEQ ID NO:48), ATGGACTGGACCTGGAG (G/C) (A/T/GJTC (SEQ ID NO:49), ATGAA (A/G) CA (C/T) CTGTGGTTCTT (SEQ ID NO:50), ATGGGGTCAACCGCCATCCTC (SEQ ID NO:51), ATGGGATGGAGCTGTATCATC (SEQ ID NO:52), ATGTCTGTCTCCTTCCTCATC (SEQ ID NO:53), and ATG (A/G) AC (C/A) TACTTTGTT (G/C) C (SEQ ID NO:54).(CT (C/T) CT (G/C) is (G/T) (G/C) CTCCTGCT (A/G) CTCTGG (G/T) to use one group of 5 ' introduction to signal sequence encoding, SEQ ID NO:55 and CT (C/T) CT (G/C) is (G/T) (G/C) (C/G/A/T) T (A/G) CTCTGG of CT (C/G) CT (G/A) (G/T), SEQ ID NO:56) and corresponding to 3 ' introduction (CATCAGATGGCCGGGAAGAT, SEQ ID NO:57) of constant region amino acid/11 17-122 amplify the K variable region of light chain.Similarly, (ATG (A/G) CCTG (C/G) is (A/T) (C/T) C of C (C/T) CCTCTC (C/T) T (C/T) CT (C/G) (A/T) to use 5 ' introduction of a group coding signal sequence, SEQ ID NO:58) and corresponding to 3 ' introduction (CTCCTCAGAGGAGGGCGGGAACAGAGTGAC, SEQ ID NO:59) of constant region amino acid/11 15-124 amplify the lambda light chain variable district.After amplifying with PCR, purification dna segment and it is cloned in the pCR2.1 carrier (Invitrogen, Carlsbad CA) on sepharose.Use the preposition introduction of M13 and be inverted that (CA) the polyclone body with each sample all sorts introduction for Perkin-Elmer, Foster City on two strands by fluorescence di-deoxynucleoside acid cessation method.Reduction on the specific position in a sequence between the Nucleotide is in order to show that these Nucleotide are at this locational molar mixture that waits.

For IgM being converted to more useful therapeutic mAb, make described homotype be transformed into IgG1 from IgM by the H chain variable region being grafted directly to γ 1 constant region.Carried out in vitro classification conversion with the expression of the better characterization of the control that improves the antagonist effector function and being used for the treatment of property of exploitation mAb, production, and method of purification.By hybridization mutagenesis with LH11238 antibody through clone VH and VL regional cloning (be described in people such as Wu to the phage vector M13IX104CS that is configured in human IgG CH1 and the KCL sequence J.Mol.Biol.People such as 294:151-162 (1999) and Kristensson, Vaccines 95.ColdSprings Harbor Laboratory Press, Cold Springs Harbor, NY 39-43 page or leaf (1995)) go up (be described in people such as Rosok, J.Biol.Chem.271:22611-22618 (1996)).To identical carrier, K CL is replaced by λ CL sequence in this carrier with the LH13 antibody cloning.

In order to improve the affinity of antibody in vitro fast, the potentiality that will change epitope specificity simultaneously minimizes, and has synthesized with screening the HCDR3 and the LCDR3 variant storehouse that are closely related with auxiliary sequence.Have a plurality of mechanism in vivo, make the diversity degree of introducing HCDR3 and LCDR3 greater than the diversity among other CDR of antibody by them, it serves as the regional consistent of keying action with these in antigen recognition.And without before in vitro in the affinity ripening process of relevant mAb, the favourable sudden change of maximum quantity is arranged in HCDR3 and LCDR3 usually.Therefore, initial storehouse concentrates on H chain and L chain and is changed and from the 3rd CDR of the variant of auxiliary sequence change by single amino acid.

Use the numbering decorum of people supra such as Kabat, the residue of selecting for mutagenesis LH13 CDR is: middle Asn89 to Va197 of L chain CDR3 (LCDR3) and the middle Glu95 to Tyr102 of H chain CDR3 (HCDR3) and the residue of selecting for mutagenesis LH11238 CDR are: Glu95-Tyr102 among Gln89-Thr97 and the HCDR3 among the LCDR3.As people such as Wu, Proc.Natl.Acad.Sci.USA95:6037-6042 (1998) is described to be suddenlyd change to synthesize initial storehouse and makes up the LH13 storehouse and be shown in Table 5 based on favourable LCDR3 and HCDR3.

Table 5

Sequence

Antibody library clone body heavy chain CDR3 light chain CDR3

LH13 HCDR3 wild-type EDS S G W Y H Y

S97G??????G

S97T??????T

S97N??????N

LCDR3 wild-type N S R D S S G N P V V

R91Y???????????????????????????????????????????????Y

R91F???????????????????????????????????????????????F

V97Y??????????????????????????????????????????????????????????Y

V97F??????????????????????????????????????????????????????????F

Combination 4DS T Y

4E2???????T????????????????????????Y??????????F

4H7???????T????????????????????????F??????????F

4G11???????????????????????????????F??????????F

3G4????????????????????????????????Y??????????F

LH1238 LCDR3 wild-type Q Q Y N N W P P Y T

Q89L?????????????????L

QS9G?????????????????G

Q89V?????????????????V

QS9F?????????????????F

Q89W?????????????????H

N93C?????????????????C

N94C?????????????????C

P95aF????????????????F

P95aR????????????????R

Each contains single sudden change antibody variants in these bias combinatorial libraries, and introduces all 20 amino acid on each residue of two CDR.Therefore, 228 (HCDR3) and 190 (LCDR3) unique non-wild-type variant is contained and the variant of 171 (HCDR3) and 209 (LCDR3) individual uniquenesses is contained in the LH13 storehouse in the LH11238 storehouse.

As people such as Wu, in intestinal bacteria (E.coli) XL1-blue cells (Stratagene), express Fab described in the supra (1999) and as people such as Watkins, Anal.Biochem,253:37-45 isolates Fab and it is quantitative from peripheral space described in (1997).Screening solubility Fab in the ELISA form.Detected the LH13 antigenic combination of solubility LH13 Fab with goat Anti-Human λ chain alkaline phosphatase conjugation to purifying through part.The LH13 antigen of described in example VII, partly purifying.As people such as Watkins, the described use of supra (1997) is measured LH11238 Fab associativity through fixed H3396 tumour cell.

Shown in (cavity ring) among Fig. 5, in ELISA, demonstrate relative low reactivity through the LH13 and the LH11238 Fab of bacterial expression, its explanation antibody does not carry out the antibody maturing of significance in vivo.These results are further consistent with the observation that the combination of IgM molecule is ordered about by multivalence to a great extent.Shown in the comparison of cavity ring among Fig. 5 A and the 5B and hollow square, the screening of bias combinatorial libraries identifies a plurality of variants of each antibody that contains the monamino acid variation and demonstrates the associativity more similar greatly 100 times than wild-type antibody.In HCDR3 and LCDR3, identified the favourable sudden change of LH13 antibody.Shown in Fig. 5 A, clone body S97N (ring type filling) compares the associativity with raising with clone body R91Y with the LH13 parent.Shown in Fig. 5 B, in the LCDR3 of the LH11238 that comprises clone body N94C (filling square), clone body N93C (hollow triangle) and clone body P95aR (ring type filling), also identify a plurality of favourable sudden changes.

The variant ordering that demonstrates the associativity of raising is caused the sudden change of higher affinity with discriminating, and it is listed in the table 5 also.The LH13 variant (Fig. 5 A, hollow square) that demonstrates high combination activity contains the R91Y sudden change and has the K of about 1.8nM in LCDR3 _dThe LH11238 mutant (Fig. 5 B, hollow square) that demonstrates maximum increasing amount on affinity is a Q89W results of mutation and have the K of about 11nM among the LCDR3 _aThe limited screening in described storehouse identifies and shows raising seven LH13 variants and nine LH11238 variants greater than 2 times associativity, and they are listed in the table 5 all.In S97 and the R91 of LCDR3 and the favourable sudden change that the V97 place identifies LH13 CDR of HCDR3, and improved LH11238 variant Q89, the N93 of LCDR3, N94, and the P95a place contain favourable variation.These presentation of results single amino acid in the LCDR3 of LH13 change improved greater than 50 times affinity and among the LCDR3 of LH11238 single amino acid change the affinity that has improved greater than 35 times.The combinatorial libraries that synthesizes the favourable sudden change that all identifies in the HCDR3 of LH13 and LCDR3 is with the combination idiovariation and further improve the antibody affinity.This storehouse contain each possible combination of idiovariation or three CDR positions the wild-type amino acid of the everywhere of (S97 of HCDR3 and the R91 of LCDR3 and V97), its generation contains the storehouse of 36 unique variant.Shown in the comparison of combination mutant 4H7 among Fig. 5 A (filling square) and LCDR3 variant R91Y (hollow square) or HCDR3 variant S97N (ring type filling), the variant that is easy to identify than containing the monamino acid variation demonstrates the more combination clone body of high affinity.Combination mutant 4H7 has the K of about 1.1nM _dThe DNA ordering of combination clone body shows that all clone body contain at least two mutant (as shown in table 5), and two clone body (clone body 4E2 and 4H7) contain three sudden changes.Five all combination clone bodies demonstrate the bigger combination activity of variant that contains single sudden change than any.

This examples show by the synthetic affinity maturation that the LH13 that 416 particular protein variants finish is only arranged in two steps.State that as mentioned the first step is formed then 36 combinatory variants of screening in second step by 171 HCDR3 mutant of screening and 209 LCDR3 mutant.In contrast, the total randomization that demonstrates active three the CDR residues of influence (LCDR3 S97, HCDR3 R91, and HCDR3 V97) in this research may need to contain 19 ³, or the storehouse of 6,859 variants express.Therefore, progressively improving the additivity of having captured independent mutation and further providing affinity sophisticated effective ways of affinity is provided result described herein.

Run through the application's case with reference to many pieces of open cases.The disclosure of these open cases is incorporated herein so that describe the position of the present invention in affiliated field more fully by reference in full in the application's case.

Though described the present invention with reference to the embodiment that disclosed, the those skilled in the art should recognize easily that the auspicious concrete experiment of stating of institute only is an illustration of the present invention.Should understand and to make multiple modification and do not deviate from spirit of the present invention.Therefore, the present invention only is subjected to the restriction of claim mentioned above.

Sequence table

＜110〉Applied Molecular Evolution In

＜120〉tumour-specific monoclonal antibody

<130>66797-162(IX?5518)

<140>PCT/US02/37134

<141>2002-11-19

<150>US?09/989,901

<151>2001-11-19

<160>59

<170>FastSEQ?for?Windows?Version?4.0

<210>1

<211>417

<212>DNA

＜213〉class people

<220>

<221>CDS

<222>(1)...(417)

<400>1

atg?aaa?cac?ctg?tgg?ttc?ttc?ctc?ctc?ctg?gtg?gca?gct?ccc?aga?tgg????48

Met?Lys?His?Leu?Trp?Phe?Phe?Leu?Leu?Leu?Val?Ala?Ala?Pro?Arg?Trp

1???????????????5???????????????????10??????????????????15

gtc?ctg?tcc?cag?gtg?cag?cta?cag?cag?tgg?ggc?gca?gga?ctg?ttg?aag????96

Val?Leu?Ser?Gln?Val?Gln?Leu?Gln?Gln?Trp?Gly?Ala?Gly?Leu?Leu?Lys

20??????????????????25??????????????????30

cct?tcg?gag?acc?ctg?tcc?ctc?acc?tgc?gct?gtc?tat?ggt?ggg?tcc?ttc????144

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

35??????????????????40??????????????????45

agt?ggt?tac?tac?tgg?agc?tgg?atc?cgc?cag?ccc?cca?ggg?aag?ggg?ctg????192

Ser?Gly?Tyr?Tyr?Trp?Ser?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu

50??????????????????55??????????????????60

gag?tgg?att?ggg?gaa?atc?aat?cat?agt?gga?agc?acc?aac?tac?aac?ccg????240

Glu?Trp?Ile?Gly?Glu?Ile?Asn?His?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro

65??????????????????70??????????????????75??????????????????80

tcc?ctc?aag?agt?cga?gtc?acc?ata?tca?gta?gac?acg?tcc?aag?aac?cag????288

Ser?Leu?Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln

85??????????????????90??????????????????95

ttc?tcc?ctg?aag?ctg?agc?tct?gtg?acc?gcc?gcg?gac?acg?gct?gtg?tat????336

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

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

tac?tgt?gcg?aga?gaa?ata?gca?gct?cgt?cct?cac?cga?tac?ttt?gac?tac????384

Tyr?Cys?Ala?Arg?Glu?Ile?Ala?Ala?Arg?Pro?His?Arg?Tyr?Phe?Asp?Tyr

115?????????????????120?????????????????125

tgg?ggc?cag?gga?acc?ctg?gtc?acc?gtc?tcc?tca????????????????????????417

Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

130?????????????????135

<210>2

<211>139

<212>PRT

＜213〉class people

<400>2

Met?Lys?His?Leu?Trp?Phe?Phe?Leu?Leu?Leu?Val?Ala?Ala?Pro?Arg?Trp

1???????????????5??????????????????10???????????????????15

Val?Leu?Ser?Gln?Val?Gln?Leu?Gln?Gln?Trp?Gly?Ala?Gly?Leu?Leu?Lys

20??????????????????25??????????????????30

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

35??????????????????40??????????????????45

Ser?Gly?Tyr?Tyr?Trp?Ser?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu

50??????????????????55??????????????????60

Glu?Trp?Ile?Gly?Glu?Ile?Asn?His?Ser?Gly?Ser?Thr?Asn?Tyr?Asn?Pro

65??????????????????70??????????????????75??????????????????80

Ser?Leu?Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln

85??????????????????90??????????????????95

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

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

Tyr?Cys?Ala?Arg?Glu?Ile?Ala?Ala?Arg?Pro?His?Arg?Tyr?Phe?Asp?Tyr

115?????????????????120???????????????125

Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser

130?????????????????135

<210>3

<211>351

<212>DNA

＜213〉class people

<220>

<221>CDS

<222>(1)...(351)

<400>3

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Set?Gln?Set?Val?Ser?Set?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?agc?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

cag?cag?tat?aat?aac?tgg?cct?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Gln?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

ctg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>4

<211>117

<212>PRT

＜213〉class people

<400>4

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Gln?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>5

<211>354

<212>DNA

＜213〉class people

<220>

<221>CDS

<222>(1)...(354)

<400>5

cag?gtg?cag?ctg?gtg?cag?tct?ggg?gct?gag?gtg?aag?aag?cct?ggg?tcc????48

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

l???????????????5???????????????????10??????????????????15

tcg?gtg?aag?gtc?tcc?tgc?aag?gct?tct?gga?ggc?acc?ttc?agc?agc?tat????96

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

20??????????????????25??????????????????30

gct?atc?agc?tgg?gtg?cga?cag?gcc?cct?gga?caa?ggg?ctt?gag?tgg?atg????144

Ala?Ile?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met

35??????????????????40??????????????????45

gga?ggg?atc?atc?cct?atc?ttt?ggt?aca?gca?aac?tac?gca?cag?aag?ttc????192

Gly?Gly?Ile?Ile?Pro?Ile?Phe?Gly?Thr?Ala?Asn?Tyr?Ala?Gln?Lys?Phe

50??????????????????55??????????????????60

cag?ggc?aga?gtc?acg?att?acc?gcg?gac?gaa?tcc?acg?agc?aca?gcc?tac????240

Gln?Gly?Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Ser?Thr?Ala?Tyr

65??????????????????70??????????????????75??????????????????80

atg?gag?ctg?agc?agc?ctg?aga?tct?gag?gac?acg?gcc?gtg?tat?tac?tgt????288

Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

gcg?aga?gaa?gat?agc?agt?ggc?tgg?tat?cac?tac?tgg?ggc?cag?gga?acc????336

Ala?Arg?Glu?Asp?Ser?Ser?Gly?Trp?Tyr?His?Tyr?Trp?Gly?Gln?Gly?Thr

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

ctg?gtc?acc?gtc?tcc?tca????????????????????????????????????????????354

Leu?Val?Thr?Val?Ser?Ser

115

<210>6

<211>118

<212>PRT

＜213〉class people

<400>6

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

1???????????????5??????????????????10??????????????????15

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

20??????????????????25??????????????????30

Ala?Ile?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met

35??????????????????40??????????????????45

Gly?Gly?Ile?Ile?Pro?Ile?Phe?Gly?Thr?Ala?Asn?Tyr?Ala?Gln?Lys?Phe

50??????????????????55??????????????????60

Gln?Gly?Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Ser?Thr?Ala?Tyr

65??????????????????70??????????????????75??????????????????80

Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Ala?Arg?Glu?Asp?Ser?Ser?Gly?Trp?Tyr?His?Tyr?Trp?Gly?Gln?Gly?Thr

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

Leu?Val?Thr?Val?Ser?Ser

115

<210>7

<211>333

<212>DNA

＜213〉class people

<220>

<221>CDS

<222>(1)...(333)

<400>7

tct?tct?gag?ctg?act?cag?gac?cct?gct?gtg?tet?gtg?gcc?ttg?gga?cag????48

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

aca?gtc?agg?atc?aca?tgc?caa?gga?gac?agc?ctc?aga?agc?tat?tat?gca????96

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

agc?tgg?tac?cag?cag?aag?cca?gga?cag?gcc?cct?gta?ctt?gtc?atc?tat????144

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

ggt?aaa?aac?aac?cgg?ccc?tca?ggg?atc?cca?gac?cga?ttc?tct?ggc?tcc????192

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

agc?tca?gga?aac?aca?gct?tcc?ttg?acc?atc?act?ggg?gct?cag?gcg?gaa????240

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

gat?gag?gct?gac?tat?tac?tgt?aac?tcc?cgg?gac?agc?agt?ggt?aac?ccc????288

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Arg?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

gtg?gta?ttc?ggc?gga?ggg?acc?aag?ctg?acc?gtc?cta?ggt?cag?ccc????????333

Val?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>8

<211>111

<212>PRT

＜213〉class people

<400>8

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

Ser?Ser?6ly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Arg?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

Val?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>9

<211>354

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(354)

<400>9

cag?gtg?cag?ctg?gtg?cag?tct?ggg?gct?gag?gtg?aag?aag?cct?ggg?tcc????48

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

1???????????????5???????????????????10??????????????????15

tcg?gtg?aag?gtc?tcc?tgc?aag?gct?tct?gga?ggc?acc?ttc?agc?agc?tat????96

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

20??????????????????25??????????????????30

gct?atc?agc?tgg?gtg?cga?cag?gcc?cct?gga?caa?ggg?ctt?gag?tgg?atg????144

Ala?Ile?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met

35??????????????????40??????????????????45

gga?ggg?atc?atc?cct?atc?ttt?ggt?aca?gca?aac?tac?gca?cag?aag?ttc????192

Gly?Gly?Ile?Ile?Pro?Ile?Phe?Gly?Thr?Ala?Asn?Tyr?Ala?Gln?Lys?Phe

50??????????????????55??????????????????60

cag?ggc?aga?gtc?acg?att?acc?gcg?gac?gaa?tcc?acg?agc?aca?gcc?tac????240

Gln?Gly?Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Ser?Thr?Ala?Tyr

65??????????????????70??????????????????75??????????????????80

atg?gag?ctg?agc?agc?ctg?aga?tct?gag?gac?acg?gcc?gtg?tat?tac?tgt????288

Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

gcg?aga?gaa?gat?ggt?agt?ggc?tgg?tat?cac?tac?tgg?ggc?cag?gga?acc????336

Ala?Arg?Glu?Asp?Gly?Ser?Gly?Trp?Tyr?His?Tyr?Trp?Gly?Gln?Gly?Thr

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

ctg?gtc?acc?gtc?tcc?tca????????????????????????????????????????????354

Leu?Val?Thr?Val?Ser?Ser

115

<210>10

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>10

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

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

Ala?Ile?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met

35??????????????????40??????????????????45

Gly?Gly?Ile?Ile?Pro?Ile?Phe?Gly?Thr?Ala?Asn?Tyr?Ala?Gln?Lys?Phe

50??????????????????55??????????????????60

Gln?Gly?Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Ser?Thr?Ala?Tyr

65??????????????????70??????????????????75??????????????????80

Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Ala?Arg?Glu?Asp?Gly?Ser?Gly?Trp?Tyr?His?Tyr?Trp?Gly?Gln?Gly?Thr

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

Leu?Val?Thr?Val?Ser?Ser

115

<210>1l

<211>354

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(354)

<400>11

cag?gtg?cag?ctg?gtg?cag?tct?ggg?gct?gag?gtg?aag?aag?cct?ggg?tcc????48

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

1???????????????5???????????????????10??????????????????15

tcg?gtg?aag?gtc?tcc?tgc?aag?gct?tct?gga?ggc?acc?ttc?agc?agc?tat????96

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

20??????????????????25??????????????????30

gct?atc?agc?tgg?gtg?cga?cag?gcc?cct?gga?caa?ggg?ctt?gag?tgg?atg????144

Ala?Ile?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met

35??????????????????40??????????????????45

gga?ggg?atc?atc?cct?atc?ttt?ggt?aca?gca?aac?tac?gca?cag?aag?ttc????192

Gly?Gly?Ile?Ile?Pro?Ile?Phe?Gly?Thr?Ala?Ash?Tyr?Ala?Gln?Lys?Phe

50??????????????????55??????????????????60

cag?ggc?aga?gtc?acg?att?acc?gcg?gac?gaa?tcc?acg?agc?aca?gcc?tac????240

Gln?Gly?Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Ser?Thr?Ala?Tyr

65??????????????????70??????????????????75??????????????????80

atg?gag?ctg?agc?agc?ctg?aga?tct?gag?gac?acg?gcc?gtg?tat?tac?tgt????288

Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

gcg?aga?gaa?gat?act?agt?ggc?tgg?tat?cac?tac?tgg?ggc?cag?gga?acc????336

Ala?Arg?Glu?Asp?Thr?Ser?Gly?Trp?Tyr?His?Tyr?Trp?Gly?Gln?Gly?Thr

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

ctg?gtc?acc?gtc?tcc?tca????????????????????????????????????????????354

Leu?Val?Thr?Val?Ser?Ser

115

<210>12

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>12

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

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

Ala?Ile?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met

35??????????????????40??????????????????45

Gly?Gly?Ile?Ile?Pro?Ile?Phe?Gly?Thr?Ala?Asn?Tyr?Ala?Gln?Lys?Phe

50??????????????????55??????????????????60

Gln?Gly?Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Ser?Thr?Ala?Tyr

65??????????????????70??????????????????75??????????????????80

Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Ala?Arg?Glu?Asp?Thr?Ser?Gly?Trp?Tyr?His?Tyr?Trp?Gly?Gln?Gly?Thr

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

Leu?Val?Thr?Val?Ser?Ser

115

<210>13

<211>354

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(354)

<400>13

cag?gtg?cag?ctg?gtg?cag?tct?ggg?gct?gag?gtg?aag?aag?cct?ggg?tcc????48

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

1???????????????5???????????????????10??????????????????15

tcg?gtg?aag?gtc?tcc?tgc?aag?gct?tct?gga?ggc?acc?ttc?agc?agc?tat????96

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

20??????????????????25??????????????????30

gct?atc?agc?tgg?gtg?cga?cag?gcc?cct?gga?caa?ggg?ctt?gag?tgg?atg????144

Ala?Ile?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met

35??????????????????40??????????????????45

gga?ggg?atc?atc?cct?atc?ttt?ggt?aca?gca?aac?tac?gca?cag?aag?ttc????192

Gly?Gly?Ile?Ile?Pro?Ile?Phe?Gly?Thr?Ala?Asn?Tyr?Ala?Gln?Lys?Phe

50??????????????????55??????????????????60

cag?ggc?aga?gtc?acg?att?acc?gcg?gac?gaa?tcc?acg?agc?aca?gcc?tac????240

Gln?Gly?Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Ser?Thr?Ala?Tyr

65??????????????????70??????????????????75??????????????????80

atg?gag?ctg?agc?agc?ctg?aga?tct?gag?gac?acg?gcc?gtg?tat?tac?tgt????288

Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

gcg?aga?gaa?gat?aat?agt?ggc?tgg?tat?cac?tac?tgg?ggc?cag?gga?acc????336

Ala?Arg?Glu?Asp?Asn?Ser?Gly?Trp?Tyr?His?Tyr?Trp?Gly?Gln?Gly?Thr

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

ctg?gtc?acc?gtc?tcc?tca????????????????????????????????????????????354

Leu?Val?Thr?Val?Ser?Ser

115

<210>14

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>14

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

1???????????????5???????????????????10??????????????????15

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

20??????????????????25??????????????????30

Ala?Ile?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met

35??????????????????40??????????????????45

Gly?Gly?Ile?Ile?Pro?Ile?Phe?Gly?Thr?Ala?Asn?Tyr?Ala?Gln?Lys?Phe

50??????????????????55??????????????????60

Gln?Gly?Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Ser?Thr?Ala?Tyr

65??????????????????70??????????????????75??????????????????80

Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Ala?Arg?Glu?Asp?Asn?Ser?Gly?Trp?Tyr?His?Tyr?Trp?Gly?Gln?Gly?Thr

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

Leu?Val?Thr?Val?Ser?Ser

115

<210>15

<211>333

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(333)

<400>15

tct?tct?gag?ctg?act?cag?gac?cct?gct?gtg?tct?gtg?gcc?ttg?gga?cag????48

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

aca?gtc?agg?atc?aca?tgc?caa?gga?gac?agc?ctc?aga?agc?tat?tat?gca????96

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

agc?tgg?tac?cag?cag?aag?cca?gga?cag?gcc?cct?gta?ctt?gtc?atc?tat????144

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

ggt?aaa?aac?aac?cgg?ccc?tca?ggg?atc?cca?gac?cga?ttc?tct?ggc?tcc????192

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

agc?tca?gga?aac?aca?gct?tcc?ttg?acc?atc?act?ggg?gct?cag?gcg?gaa????240

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65?????????????????70??????????????????75??????????????????80

gat?gag?gct?gac?tat?tac?tgt?aac?tcc?tat?gac?agc?agt?ggt?aac?ccc????288

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Tyr?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

gtg?gta?ttc?ggc?gga?ggg?acc?aag?ctg?acc?gtc?cta?ggt?cag?ccc?????????333

Val?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>16

<211>111

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>16

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5??????????????????10??????????????????15

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Tyr?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

Val?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>17

<211>333

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(333)

<400>17

tct?tct?gag?ctg?act?cag?gac?cct?gct?gtg?tct?gtg?gcc?ttg?gga?cag????48

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

aca?gtc?agg?atc?aca?tgc?caa?gga?gac?agc?ctc?aga?agc?tat?tat?gca????96

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25?????????????????30

agc?tgg?tac?cag?cag?aag?cca?gga?cag?gcc?cct?gta?ctt?gtc?atc?tat????144

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

ggt?aaa?aac?aac?cgg?ccc?tca?ggg?atc?cca?gac?cga?ttc?tct?ggc?tcc????192

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

agc?tca?gga?aac?aca?gct?tcc?ttg?acc?atc?act?ggg?gct?cag?gcg?gaa????240

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

gat?gag?gct?gac?tat?tac?tgt?aac?tcc?ttt?gac?agc?agt?ggt?aac?ccc????288

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Phe?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

gtg?gta?ttc?ggc?gga?ggg?acc?aag?ctg?acc?gtc?cta?ggt?cag?ccc????????333

Val?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>18

<211>111

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>18

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Phe?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

Val?Val?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>19

<211>333

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(333)

<400>19

tct?tct?gag?ctg?act?cag?gac?cct?gct?gtg?tct?gtg?gcc?ttg?gga?cag????48

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

aca?gtc?agg?atc?aca?tgc?caa?gga?gac?agc?ctc?aga?agc?tat?tat?gca????96

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

agc?tgg?tac?cag?cag?aag?cca?gga?cag?gcc?cct?gta?ctt?gtc?atc?tat????144

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

ggt?aaa?aac?aac?cgg?ccc?tca?ggg?atc?cca?gac?cga?ttc?tct?ggc?tcc????192

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

agc?tca?gga?aac?aca?gct?tcc?ttg?acc?atc?act?ggg?gct?cag?gcg?gaa????240

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

gat?gag?gct?gac?tat?tac?tgt?aac?tcc?cgg?gac?agc?agt?ggt?aac?ccc????288

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Arg?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

gtg?tat?ttc?ggc?gga?ggg?acc?aag?ctg?acc?gtc?cta?ggt?cag?ccc????????333

Val?Tyr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>20

<211>111

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>20

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Arg?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

Val?Tyr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>21

<211>333

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(333)

<400>21

tct?tct?gag?ctg?act?cag?gac?cct?gct?gtg?tct?gtg?gcc?ttg?gga?cag????48

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5??????????????????10???????????????????15

aca?gtc?agg?atc?aca?tgc?caa?gga?gac?agc?ctc?aga?agc?tat?tat?gca????96

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

agc?tgg?tac?cag?cag?aag?cca?gga?cag?gcc?cct?gta?ctt?gtc?atc?tat????144

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

ggt?aaa?aac?aac?cgg?ccc?tca?ggg?atc?cca?gac?cga?ttc?tct?ggc?tcc????192

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

agc?tca?gga?aac?aca?gct?tcc?ttg?acc?atc?act?ggg?gct?cag?gcg?gaa????240

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

gat?gag?gct?gac?tat?tac?tgt?aac?tcc?cgg?gac?agc?agt?ggt?aac?ccc????288

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Arg?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

gtg?ttt?ttc?ggc?gga?ggg?acc?aag?ctg?acc?gtc?cta?ggt?cag?ccc????????333

Val?Phe?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>22

<211>111

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>22

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Arg?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

Val?Phe?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>23

<211>333

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(333)

<400>23

tct?tct?gag?ctg?act?cag?gac?cct?gct?gtg?tct?gtg?gcc?ttg?gga?cag????48

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

aca?gtc?agg?atc?aca?tgc?caa?gga?gac?agc?ctc?aga?agc?tat?tat?gca????96

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

agc?tgg?tac?cag?cag?aag?cca?gga?cag?gcc?cct?gta?ctt?gtc?atc?tat????144

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

ggt?aaa?aac?aac?cgg?ccc?tca?ggg?atc?cca?gac?cga?ttc?tct?ggc?tcc????192

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

agc?tca?gga?aac?aca?gct?tcc?ttg?acc?atc?act?ggg?gct?cag?gcg?gaa????240

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

gat?gag?gct?gac?tat?tac?tgt?aac?tcc?tat?gac?agc?agt?ggt?aac?ccc????288

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Tyr?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

gtg?ttt?ttc?ggc?gga?ggg?acc?aag?ctg?acc?gtc?cta?ggt?cag?ccc????????333

Val?Phe?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>24

<211>111

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>24

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Tyr?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

Val?Phe?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>25

<211>333

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(333)

<400>25

tct?tct?gag?ctg?act?cag?gac?cct?gct?gtg?tct?gtg?gcc?ttg?gga?cag????48

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5???????????????????10??????????????????15

aca?gtc?agg?atc?aca?tgc?caa?gga?gac?agc?ctc?aga?agc?tat?tat?gca????96

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25??????????????????30

agc?tgg?tac?cag?cag?aag?cca?gga?cag?gcc?cct?gta?ctt?gtc?atc?tat????144

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

ggt?aaa?aac?aac?cgg?ccc?tca?ggg?atc?cca?gac?cga?ttc?tct?ggc?tcc????192

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

agc?tca?gga?aac?aca?gct?tcc?ttg?acc?atc?act?ggg?gct?cag?gcg?gaa????240

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65?????????????????70??????????????????75??????????????????80

gat?gag?gct?gac?tat?tac?tgt?aac?tcc?ttt?gac?agc?agt?ggt?aac?ccc????288

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Phe?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

gtg?ttt?ttc?ggc?gga?ggg?acc?aag?ctg?acc?gtc?cta?ggt?cag?ccc????????333

Val?Phe?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>26

<211>111

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>26

Ser?Ser?Glu?Leu?Thr?Gln?Asp?Pro?Ala?Val?Ser?Val?Ala?Leu?Gly?Gln

1???????????????5??????????????????10??????????????????15

Thr?Val?Arg?Ile?Thr?Cys?Gln?Gly?Asp?Ser?Leu?Arg?Ser?Tyr?Tyr?Ala

20??????????????????25?????????????????30

Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Val?Leu?Val?Ile?Tyr

35??????????????????40??????????????????45

Gly?Lys?Asn?Asn?Arg?Pro?Ser?Gly?Ile?Pro?Asp?Arg?Phe?Ser?Gly?Ser

50??????????????????55??????????????????60

Ser?Ser?Gly?Asn?Thr?Ala?Ser?Leu?Thr?Ile?Thr?Gly?Ala?Gln?Ala?Glu

65??????????????????70??????????????????75??????????????????80

Asp?Glu?Ala?Asp?Tyr?Tyr?Cys?Asn?Ser?Phe?Asp?Ser?Ser?Gly?Asn?Pro

85??????????????????90??????????????????95

Val?Phe?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Thr?Val?Leu?Gly?Gln?Pro

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

<210>27

<21l>351

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(351)

<400>27

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55?????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?agc?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

ttg?cag?tat?aat?aac?tgg?cct?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Leu?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

ctg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>28

<211>117

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>28

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Leu?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>29

<211>351

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(351)

<400>29

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?agc?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

ggt?cag?tat?aat?aac?tgg?cct?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Gly?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

ctg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>30

<211>117

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>30

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40?????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Gly?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>31

<211>351

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(351)

<400>31

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?agc?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

gtg?cag?tat?aat?aac?tgg?cct?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Val?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

ctg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>32

<211>117

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>32

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Val?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>33

<211>351

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(351)

<400>33

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25?????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?agc?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

ttt?cag?tat?aat?aac?tgg?cct?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Phe?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

ctg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>34

<211>117

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>34

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5??????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20?????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Phe?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>35

<211>351

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(351)

<400>35

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?age?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

tgg?cag?tat?aat?aac?tgg?cct?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Trp?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

ctg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>36

<211>117

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>36

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5??????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Trp?Gln?Tyr?Asn?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>37

<211>351

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(351)

<400>37

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?agc?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

cag?cag?tat?tgt?aac?tgg?cct?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Gln?Gln?Tyr?Cys?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

ctg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>38

<211>117

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>38

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Gln?Gln?Tyr?Cys?Asn?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>39

<211>351

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(351)

<400>39

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?agc?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

cag?cag?tat?aat?tgt?tgg?cct?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Gln?Gln?Tyr?Asn?Cys?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

etg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>40

<211>117

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>40

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Gln?Gln?Tyr?Asn?Cys?Trp?Pro?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>41

<211>351

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(351)

<400>41

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?agc?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

cag?cag?tat?aat?aac?tgg?ttt?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Gln?Gln?Tyr?Asn?Asn?Trp?Phe?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

ctg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>42

<211>117

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>42

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1????????????????5?????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Gln?Gln?Tyr?Asn?Asn?Trp?Phe?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>43

<211>351

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(351)

<400>43

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?agc?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

cag?cag?tat?aat?aac?tgg?cgg?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Gln?Gln?Tyr?Asn?Asn?Trp?Arg?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

ctg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>44

<211>117

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>44

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Gln?Gln?Tyr?Asn?Asn?Trp?Arg?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>45

<211>351

<212>DNA

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<221>CDS

<222>(1)...(351)

<400>45

ctc?tgg?ctc?cca?gat?acc?act?gga?gaa?ata?gtg?atg?acg?cag?tct?cca????48

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5???????????????????10??????????????????15

gcc?acc?ctg?tct?gtg?tct?cca?ggg?gaa?aga?gcc?acc?ctc?tcc?tgc?agg????96

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

gcc?agt?cag?agt?gtt?agc?agc?aac?tta?gcc?tgg?tac?cag?cag?aaa?cct????144

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

ggc?cag?gct?ccc?agg?ctc?ctc?atc?tat?ggt?gca?tcc?acc?agg?gcc?act????192

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

ggt?atc?cca?gcc?agg?ttc?agt?ggc?agt?ggg?tct?ggg?aca?gag?ttc?act????240

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

ctc?acc?atc?agc?agc?ctg?cag?tct?gaa?gat?ttt?gca?gtt?tat?tac?tgt????288

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

cag?cag?tat?aat?aac?tgg?cgt?ccg?tac?act?ttt?ggc?cag?ggg?acc?aag????336

Gln?Gln?Tyr?Asn?Asn?Trp?Arg?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

ctg?gag?atc?aaa?cga????????????????????????????????????????????????351

Leu?Glu?Ile?Lys?Arg

115

<210>46

<211>117

<212>PRT

＜213〉artificial sequence

<220>

＜223〉reorganization variant

<400>46

Leu?Trp?Leu?Pro?Asp?Thr?Thr?Gly?Glu?Ile?Val?Met?Thr?Gln?Ser?Pro

1???????????????5??????????????????10??????????????????15

Ala?Thr?Leu?Ser?Val?Ser?Pro?Gly?Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg

20??????????????????25??????????????????30

Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro

35??????????????????40??????????????????45

Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala?Ser?Thr?Arg?Ala?Thr

50??????????????????55??????????????????60

Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr

65??????????????????70??????????????????75??????????????????80

Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser?Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys

85??????????????????90??????????????????95

Gln?Gln?Tyr?Asn?Asn?Trp?Arg?Pro?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys

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

Leu?Glu?Ile?Lys?Arg

115

<210>47

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>47

agacgagggg?gaaaagggtt????????????????????????????????????????????????20

<210>48

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>48

atggagtttg?ggctgagctg?g??????????????????????????????????????????????21

<210>49

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<221>misc_feature

<222>18

＜223〉molar mixture of n=g and c.

<221>misc_feature

<222>19

＜223〉n=a, the molar mixture of t and g.

<400>49

atggactgga?cctggagnnt?c?????????????????????????????????????????????21

<210>50

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<221>misc_feature

<222>6

＜223〉molar mixture of n=a and g.

<221>misc_feature

<222>9

＜223〉molar mixture of n=c and t.

<400>50

atgaancanc?tgtggttctt????????????????????????????????????????????????20

<210>51

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>51

atggggtcaa?ccgccatcct?c??????????????????????????????????????????????21

<210>52

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>52

atgggatgga?gctgtatcat?c??????????????????????????????????????????????21

<210>53

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>53

atgtctgtct?ccttcctcat?c??????????????????????????????????????????????21

<210>54

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<221>misc_feature

<222>4

＜223〉molar mixture of n=a and g.

<221>misc_feature

<222>7

＜223〉molar mixture of n=c and a.

<221>misc_feature

<222>17

＜223〉molar mixture of n=g and c.

<400>54

atgnacntac?tttgttnc??????????????????????????????????????????????????18

<210>55

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<221>misc_feature

<222>3

＜223〉molar mixture of n=c and t.

<221>misc_feature

<222>6，9

＜223〉molar mixture of n=g and c.

<221>misc_feature

<222>7，8

＜223〉molar mixture of n=g and t.

<221>misc_feature

<222>18

＜223〉molar mixture of n=a and g.

<400>55

ctnctnnnnc?tcctgctnct?ctgg???????????????????????????????????????????24

<210>56

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<221>misc_feature

<222>3

＜223〉molar mixture of n=e c and t.

<221>misc_feature

<222>6，9，12

＜223〉molar mixture of n=g and c.

<221>misc_feature

<222>7，8

＜223〉molar mixture of n=g and t.

<221>misc_feature

<222>15，18

＜223〉molar mixture of n=g and a.

<221>misc_feature

<222>16

＜223〉n=c, g, the molar mixture of a and t.

<400>56

ctnctnnnnc?tnctnntnct?ctgg???????????????????????????????????????????24

<210>57

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>57

catcagatgg?ccgggaagat????????????????????????????????????????????????20

<210>58

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<221>misc_feature

<222>4

＜223〉molar mixture of n=a and g.

<221>misc_feature

<222>9，24

＜223〉molar mixture of n=c and g.

<221>misc_feature

<222>10，25

＜223〉molar mixture of n=a and t.

<221>misc_feature

<222>12，19，21，26

＜223〉molar mixture of n=c and t.

<400>58

atgncctgnn?cncctctcnt?nctnnnc????????????????????????????????????????27

<210>59

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>59

ctcctcagag?gagggcggga?acagagtgac?????????????????????????????????????30

Claims

1. a separated human monoclonal antibodies or its functional fragment, it comprises the complementary determining region of the aminoacid sequence with the CDR that is selected from the group that is made up of following SEQ ID NO substantially: 10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42 and 44, and wherein said human monoclonal antibodies or its functional fragment are specifically in conjunction with neoplastic cell or its antigen.

2. separated human monoclonal antibodies according to claim 1, wherein said functional fragment are selected from by Fv, Fab, Fab ' or F (ab ') ₂The group that forms.

3. separated human monoclonal antibodies according to claim 1 or functional fragment further comprise marker.

4. separated human monoclonal antibodies according to claim 3 or functional fragment, wherein said marker contains cytotoxic agent or cytostatics.

5. medical composition, it comprises separated human monoclonal antibodies according to claim 1 or functional fragment and medical supporting agent.

6. separated human monoclonal antibodies according to claim 1 or functional fragment, it comprises three complementary determining regions, they have substantially to be stated in the sequence that is selected from the group that is formed by following SEQ ID NO: 10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42 and 44.

7. separated human monoclonal antibodies according to claim 1 or functional fragment further comprise acceptable compound on the physiology.

8. a separated CDR or its functional fragment, the aminoacid sequence that comprises the CDR that is selected from the group that is formed by following SEQ ID NO substantially: 10,12,14,16,18,20,22,24,26,28,30,32,34,36,38,40,42 and 44, wherein said CDR or its functional fragment are specifically in conjunction with neoplastic cell or its antigen.

9. the separated nucleic acid of encode human monoclonal antibodies or its functional fragment, it comprises the nucleotide sequence of the aminoacid sequence of the following CDR that encodes substantially, and this CDR is coded through the Nucleotide that is selected from the group that is made up of following SEQ ID NO: 9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43 and 45.

10. separated nucleic acid according to claim 9, wherein said functional fragment are to be selected from by Fv, Fab, Fab ' or F (ab ') ₂The group that is formed.

11. separated nucleic acid according to claim 9, it comprises substantially the aminoacid sequence of three CDR in the sequence that is selected from the group that is made up of following SEQ ID NO of being stated is carried out nucleotide sequence coding: 9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43 and 45.

12. the separated nucleic acid of encode CDR or its functional fragment, its comprise substantially to through be selected from by

The aminoacid sequence of the nucleotide sequence coded CDR of the group that following SEQ ID NO is formed carries out nucleotide sequence coding: 9,11,13,15,17,19,21,23,25,27,29,31,33,35,37,39,41,43 and 45.

13. one kind is reduced the outgrowth method of neoplastic cell, it comprises to described cell throws human monoclonal antibodies according to claim 1 or the functional fragment that gives significant quantity.

14. method according to claim 13, wherein said neoplastic cell are to be selected from the group that is made up of breast cancer, lung cancer and ovarian cancer cell.

15. the method for the neoplastic cell in the test sample, it comprises:

(a) sample is contacted with human monoclonal antibodies according to claim 1 or functional fragment; And

(b) detect the specificity combination of described human monoclonal antibodies or functional fragment, wherein compare to exist or have the described human monoclonal antibodies or the functional fragment that increase content and in described sample, have neoplastic cell with normal cell to described sample.

16. method according to claim 15, wherein said neoplastic cell are to be selected from the group that is made up of breast cancer, lung cancer and ovarian cancer cell.