CN1812999A

CN1812999A - Pharmaceutical composition comprising a bispecific antibody specific for EpCAM

Info

Publication number: CN1812999A
Application number: CN 200480014479
Authority: CN
Inventors: P·库弗; M·贝里; S·奥夫纳; K·布里施魏因; A·沃尔夫; T·劳姆; B·科勒伊森; U·伦凯里-许茨; P·博伊尔勒
Original assignee: Micromet GmbH
Current assignee: Amgen Research Munich GmbH
Priority date: 2003-05-31
Filing date: 2004-05-26
Publication date: 2006-08-02
Also published as: ZA200508279B

Abstract

The present invention provides a pharmaceutical composition comprising a bispecific single chain antibody construct. Said bispecific single chain antibody construct is characterized to comprise or consist of at least two domains, whereby one of said at least two domains specifically binds to human EpCAM and comprises at least one CDR-H3 region comprising the amino acid sequence NXID antigen and a second domain binds to human CD3 antigen. The invention further provides a process for the production of the pharmaceutical composition of the invention, a method for the prevention, treatment or amelioration of a tumorous disease and the use of the disclosed bispecific single chain antibody construct and corresponding means in the prevention, treatment or amelioration of a tumorous disease.

Description

The pharmaceutical composition that comprises the special construct of EpCAM

The present invention relates to comprise the pharmaceutical composition of bispecific single-chain antibody construct.Described bispecific single-chain antibody construct is characterized as and comprises at least 2 structural domains or be made up of at least 2 structural domains, one of wherein said at least 2 structural domains specific combination is to people EpCAM antigen and comprise the CDR-H3 district that at least one comprises aminoacid sequence NXD, and second structural domain is bonded to people CD3 antigen.The present invention also provides the method for production pharmaceutical composition of the present invention, the method for the method of prevention, treatment or tumor remission disease and the purposes of disclosed bispecific single-chain antibody construct and corresponding prevention, treatment or tumor remission disease.

Whole specification sheets quoted various files.The disclosed content of described file is incorporated herein by reference.

Epithelial cell adhesion molecule (EpCAM, be also referred to as 17-1A antigen, KSA, EGP40, GA733-2, ks1-4 or esa) be that 314 amino acid whose 40-kDa films are integrated glycoprotein, specific expressed (summary in some epithelium and some carcinoid tumors in Balzar, J.Mol.Med.1999,77,699-712).Find EpCAM and by Murine Monoclonal Antibody 17-1A/edrecolomab identification it has been carried out cloning (Goettlinger, Int J Cancer.1986 by it subsequently; 38,47-53 and Simon, Proc.Natl.Acad.Sci.USA.1990; 87,2755-2759).By produced with the human colon cancer cell immune mouse monoclonal antibody 17-1A (Koprowski, SomaticCell Genet.1979,5,957-971).

The EGF-sample iteron of EpCAM is presented at mediation in the preferendum cell adhesion is other and interacts and interaction (Balzar, Mol.Cell.Biol.2001,21,2570-2580) and, because this reason, EpCAM mainly is positioned (Litvinov between the epithelial cell, J Cell Biol.1997,139,1337-1348, Balzar, J Mol Med.1999,77,699-712 and Trebak, J Biol Chem.2001,276,2299-2309).EpCAM with the mode of directed and high-sequential work adhere to chrotoplast (Litvinov, J Cell Biol.1997,139,1337-1348).May in no case can be from the EpCAM in the data presentation healthy tissues of the experiment of transgenic mice that uses skin expressing human EpCAM thereon and rat near the antibody (McLaughlin of systemic administration, Cancer Immunol.Immunother., 1999,48,303-311).In case the epithelial cell vicious transformation, the height cell order that quick growing tumors cell will be discarded epithelium.As a result, the surface arrangement of EpCAM becomes less and is tied and molecule is exposed on the tumour cell more.Because its epithelial source, the tumour cell of most cancer knurls is still at its surface expression EpCAM.

In vivo, the expression of EpCAM and negative relevant with the epithelial proliferation of increase about cytodifferentiation (summary is seen Balzar, 1999, J.Mol.Med.77,699-712).As use anti--EpCAM monoclonal antibody to detect by immunohistochemistry, the expression of EpCAM see basically all main cancer knurls (summarize in Balzar, J Mol Med.1999,77,699-712).Use nonsmall-cell lung cancer (DeBree, Nucl Med Commun.1994,15,613-27) and prostate cancer (Zhang, ClinCancer Res.1998,4,295-302) observe best EpCAM and express, 100% tumour patient sample shows positive EpCAM dyeing there.In these researchs, also reported the tumor tissues of level dyeing, show that this antigen presentation is on most of cell of given tumour.Because its wide expression, EpCAM is called " general cancer knurl " antigen.

Shown that in difference research EpCAM is useful in the diagnosis of multiple cancer knurl and treatment.In addition, in some instances, observe the described cancer knurl higher degree of its parental generation epithelial cell of tumor cell ratio or lower invasion and attack form and express EpCAM.For example, show that being expressed on the tumor tissue He in the gland cancer of EpCAM is significantly higher than normal prostatic epithelial cell (n=76; P＜0.0001), the EpCAM that shows increase express represent the developmental early stage incident of prostate cancer (Poczatek, J Urol., 1999,162,1462-1644).In addition, in most of uterine cervix squamous cell carcinomas and gland cancer strong EpCAM express with the propagation that increases and eventually the end break up the disappearance of mark relevant (Litvinov, Am.J.Pathol.1996,148,865-75).An example is a mammary cancer, wherein EpCAM cross expressing on tumour cell be the survival predictor (Gastl, Lancet.2000,356,1981-1982).And EpCAM has been described as detecting mark (Chaubal, the Anticancer Res 1999 of disperse tumour cell in the patient who suffers from head, neck and lung's squamous cell carcinoma, 19,2237-2242, Piyathilake, HumPathol.2000,31,482-487).As finding in corium, oral cavity, epiglottis, pharynx, larynx and the oesophagus, normal squamous cell significantly do not express EpCAM (Quak, Hybridoma, 1990,9,377-387).

Except above-mentioned cancer knurl, EpCAM has shown to be expressed in and most of former, that shift has gone up (Passlick with NSCLC (non-small cell lung cancer cell) disperse, Int J Cancer, 2000,87,548-552), (Martin on stomach and stomach-oesophagus junction gland cancer, J Clin Pathol 1999,52,701-4) and derive from (Szala in the clone of colorectal carcinoma, carcinoma of the pancreas and mammary cancer, Proc Natl AcadSci USA 1990,87,3542-6, Packeisen, Hybridoma, 1999,18,37-40).

Clinical trial has shown that directly the antibody at 17-1A (EpCAM) is used for the treatment of surgical operation and excises the patient's of colorectal carcinoma knurl purposes fully and cause remarkable profitability (Riethm  ller about total survival rate and far-end transition frequency, Lancet, 1994,343,1177-1183).Find that mouse-anti EpCAM monoclonal antibody can reduce the patient's who suffers from minimal residual disease 5-annual death rate (Riethm  ller, Lancet, 1994,343,1177-1183) or 7-annual death rate (Riethm  ller, Proceedings of the American Society of Clinical Oncology, 1996,15,444).The example of mouse monoclonal antibody of identification EpCAM be Edrecolomab (Panorex) (Koprowski, Somatic Cell Genet.1979,5,957-971 and Herlyn, Cancer Res., 1980,40,717-721).Yet, to use Panorex first during the treatment of colorectal carcinoma adjuvant immunity and cause granulomatous formation of Wei Genashi and deterioration, this shows that mAb 17-1A should be applied to suffer from the patient (Franz of autoimmune disorder modestly, Onkologie, 2000,23,472-474).The limitation of Panorex is to form human anti-mouse antibody (HAMA) fast, this has limited by its mouse IgG2a Fc section and the people's immunological effect interactional ability of mechanism and presented (Frodin, Cancer Res., 1990 of short half life in circulation, 50,4866-4871).In addition, in case duplicate injection in the patient, murine antibody causes immediate allergy and anaphylaxis (Riethm  ller, Lancet.1994,343,1177-1183, Riethm  ller, J Clin Oncol., 1998,16,1788-1794 and Mellstedt, Annals New York Academy of Sciences.2000,910,254-261).

The humanization that is called 3622W94 is anti--and EpCAM antibody causes pancreatitis and increases the amylase serum level, indication as the pancreas epithelial damage, these two kinds of symptoms be this kind high-affinity anti--dose-limiting toxicity (LoBuglio of EpCAM monoclonal antibody, Proceedings of the AmericanSociety of Clinical Oncology (summary) .1997,1562 and Khor, Proceedings of theAmerican Society of Clinical Oncology (summary), 1997,847).

Described and comprised at EpCAM zone with at the bi-specific antibody in CD3 zone.M ller ﹠amp; The hybridoma that the author of Reisfeld 1991 Cancer Immunol.Immunother.33:210-216 has described by producing at the monoclonal antibody of EpCAM constructs two kinds of different bi-specific antibodies with one of two kinds of hybridoma OKT3 and 9.3.In addition, Kroesen, Cancer Research, 1995,55:4409-4415 has described limbs knurl (quadroma) bispecific monoclonal antibody at CD3 (BIS-1) and EpCAM.

Other example at the bi-specific antibody of EpCAM comprises bi-specific antibody, BiUII, (anti--CD3 (rat IgG2b) * anti--EpCAM (mouse IgG2a)), can by its Fc-district in conjunction with and activate the complete Ig molecule (Zeidler of Fc-receptor positive helper (as monocyte/macrophage, NK cell and dendritic cell), J.Immunol., 1999,163:1247-1252) and be arranged as V _L17-1A-V _H17-1A-V _{H resists-CD3}-V _{L resists-CD3}Anti--EpCAM * anti--CD3 bi-specific antibody (Mack, Proc.Natl.Acad.Sci., 1995,92:7021-7025).

In addition, other form antibody construct that comprises EpCAM has been described; For example has structure V _{H resists-CD3}-V _{L resists-EpCAM}-V _{H-resists-EpCAM}-V _{L resists-CD3}Dual specific double antibody (Helfrich, Int.J.Cancer, 1998,76:232-239) with have two kinds of different specific for tumour antigen (in conjunction with two kinds of antigenic two antigen binding domains of difference on the tumour cell) and may have another kind of at the antigenic specific three-specific antibody (DE 195 31 348) that is positioned on the effector cell.

Using display technique of bacteriophage to identify that specificity is bonded in the antigenic antibody of people EpCAM or its segmental prior art, existed multiple description (De Kruif JMB, 1995,248:97-105, WO99/25818).Yet, identify that the antibody at EpCAM is very difficult, described antibody showed cell toxicity activity enough is used for using with the treatment of two special forms.

Therefore the purpose of this invention is to provide the dual specific single chain molecule with EpCAM specific combination structural domain, this molecule has the strong cytotoxicity activity by T cell-targeting specificity activation mediation.

Therefore, the technical problem that implies of the present invention provides to produce and is used for the treatment of and/or the method for the convenience of tumor remission disease and well tolerable medicine.

Solution to described technical problem is by providing the embodiment that characterizes claim to realize.

Correspondingly, the present invention relates to comprise the composition of bispecific single-chain antibody construct, pharmaceutical composition preferably, wherein said construct comprises at least two binding domainss or is made up of at least two binding domainss, one of wherein said structural domain is bonded to people EpCAM antigen and second structural domain is bonded to people CD3 antigen, wherein said EpCAM specific combination structural domain comprises the CDR-H3 district that at least one comprises aminoacid sequence NXD, base acid sequence NXD is preferably SEQ IDNO:80,88 and 96 position 102-104, perhaps be preferably the position 106-108 of SEQ ID NO:84 and 92, wherein X is a die aromatischen Aminosaeuren.

Preferably or alternatively, the present invention relates to comprise the composition of bispecific single-chain antibody construct, pharmaceutical composition preferably, described thus construct comprises at least two binding domainss or is made up of at least two binding domainss, one of wherein said at least two structural domains specificity is bonded to people EpCAM antigen and second structural domain is bonded to people CD3 antigen, and CDR-H3 district and wherein said EpCAM specific combination structural domain that wherein said EpCAM specific combination structural domain comprises at least one at least 9 amino-acid residue have greater than 5 * 10 ^-9The K of M _DValue.

According to the present invention, term " pharmaceutical composition " relates to and is applied to the patient, is preferably the composition of human patients.In preferred embodiments, pharmaceutical composition comprise be used in parenteral, transdermal, the chamber, in the intra-arterial, film or intravenously use or be used to be injected directly into the composition of tumour.Special imagination is applied to the patient with described pharmaceutical composition by infusion or injection.Using of appropriate combination thing can be undertaken by different modes, for example by intravenously, subcutaneous, intraperitoneal, intramuscular, part or intradermal administration.Pharmaceutical composition of the present invention can further comprise pharmaceutically acceptable carrier.Suitably the example of pharmaceutical carrier is well-known in the art and comprises phosphate buffered salt solution, water, emulsion such as oil/aqueous emulsion, various types of wetting agent, sterile solution or the like.Can make the composition that comprises examples of such carriers by prescription by well-known ordinary method.These pharmaceutical compositions can be applied to the experimenter with suitable dosage.Determine dosage regimen by attending doctor and clinical factor.As well-known in the field of medicaments, the dosage that is used for any one patient depends on multiple factor, comprises patient's stature size, body surface area, age, the specific compound with using, sex, time of application and approach, general health and the other medicines of using simultaneously.The preferred dose of using can be in every day per kilogram of body weight 0.24 μ g-48mg unit scope, be preferably 0.24 μ g-24mg, more preferably be 0.24 μ g-2.4mg, even more preferably be 0.24 μ g-1.2mg and be most preferably 0.24 μ g-240 μ g.Narration in the text below the particularly preferred dosage.Can monitor progress by periodical evaluation.Dosage will be change but intravenously to use the preferred dose of DNA be about 10 ⁶-10 ¹²Individual dna molecular copy.The present composition can part or systemic administration.It will be normally parenteral using, and for example intravenously is used; DNA also can directly be applied to target site, for example be delivered to inside or outside target site by biological projectile or by catheter delivery to endarterial site.In preferred embodiments, the pharmaceutical composition subcutaneous administration and in addition the embodiment that is more preferably in be that intravenously is used.Be used for the preparation that parenteral uses and comprise aseptic aqueous solution or non-aqueous solution, suspension and emulsion.The example of non-aqueous solvent is propylene glycol, polyoxyethylene glycol, vegetables oil such as sweet oil and injectable organosilane ester such as ethyl oleate.Aqueous carrier comprises water, ethanol/water solution, emulsion or suspension, comprises salt solution and buffering medium.The parenteral vehicle comprises sodium chloride solution, Ringer ' s glucose, glucose and sodium-chlor, lactic acid Ringer ' s or fixed oil.The intravenously vehicle comprises fluid and nutrition supplement, electrolyte replenisher (as those electrolyte replenishers based on Ringer ' s glucose) or the like.Also there are sanitas and other additive for example biocide, antioxidant, sequestrant and rare gas element or the like.In addition, pharmaceutical composition of the present invention can comprise protein carrier for example serum albumin or immunoglobulin (Ig), and preferably the people originates.It is contemplated that except the nucleic acid molecule or carrier (described in the present invention) of the protein bispecific single-chain antibody construct or the same antibody construct of encoding, pharmaceutical composition of the present invention can also comprise the other biological promoting agent, and this depends on the planned use of pharmaceutical composition.In this area known this type of reagent can be the medicine that acts on gastro-intestinal system, as the medicine of cytostatic agent, prevent hyperuricemic medicine, such as the promoting agent of T-cell co-stimulatory molecules or cytokine, suppress immunoreactive medicine (for example glucocorticosteroid) and/or act on the medicine of the recycle system such as blood pressure.

The possible indication of using the present composition is a tumor disease, particularly is epithelial cancer/cancer knurl such as mammary cancer, colorectal carcinoma, prostate cancer, a neck cancer, skin carcinoma, genitourinary tract cancer for example ovarian cancer, carcinoma of endometrium, cervical cancer and kidney, lung cancer, cancer of the stomach, carcinoma of small intestine, liver cancer, carcinoma of the pancreas, carcinoma of gallbladder, cholangiocarcinoma, esophagus cancer, salivary-gland carcinoma and thyroid carcinoma.The using to specialize of the present composition is applicable to minimal residual disease, is preferably early stage solid tumor, advanced solid tumor or metastatic solid tumors, it is characterized by the part and the non local tumour that cause by the individual cells survival and reproduces.

The present invention further proposes the scheme used jointly with other compound that works by the T cell such as bi-specific antibody construct, targeted toxin or other compound.The clinical protocol of using The compounds of this invention simultaneously is included in when using other compound, before or after use jointly.

Prove that construct validity of the present invention/active possibility method is body inner model such as mouse.Suitable model can be transgenosis and allophenic mice model.The gomphosis mouse model of the mouse model of expressing human CD3 and people EpCAM, expression mouse CD3 and tumor cells expression people EpCAM wherein can be transfected, and the wherein people tumour that comprises nude mice is expressed the gomphosis mouse model of EpCAM and can be transplanted, perhaps the tumour cell of expressing human EpCAM can inject and, extraly, injection human PBMC.Term " bispecific single-chain antibody construct " relates to the construct that comprises 2 kinds of antibody deutero-binding domainss.One of described binding domains is made up of antibody variable region (or its part), antibody fragment or derivatives thereof, and it can specificity be bonded to people EpCAM antigen (target molecule 1)/interact with people EpCAM antigen (target molecule 1).Second binding domains is made up of antibody variable region (or its part), antibody fragment or derivatives thereof, and it can specificity be bonded to people CD3 antigen (target molecule 2)/interact with people CD3 antigen (target molecule 2).As below describing in detail, the part variable region can be at least one CDR (" complementary determining region "), and being most preferably is the CDR3 district at least.Two structural domain/districts are preferably covalently bound as a strand mutually in the described single-chain antibody construct.This kind connection can be directly (structural domain 1[CD3 antigen-specific]-structural domain 2[EpCAM antigen-specific] or structural domain 1[EpCAM antigen-specific]-structural domain 2[CD3 antigen-specific]) or finish by other peptide linker sequence (structural domain 1-joint sequence-structural domain 2).Under the situation of using joint, this joint is preferably its length is enough to guarantee that with sequence first and second structural domains can keep its different binding specificity with being mutually independent.Most preferably and such as in appended embodiment proof, " the bispecific single-chain antibody construct " that is applied to pharmaceutical composition of the present invention is dual specific strand Fv (scFv).The dual specific single chain molecule is well known in the art and is described in WO 99/54440, Mack, J.Immunol. (1997), 158,3965-3970, Mack, PNAS, (1995), 92,7021-7025, Kufer, CancerImmunol.Immunother., (1997), 45,193-197, L ffler, Blood, (2000), 95,6,2098-2103 and Br  hl, J.Immunol., (2001), 166,2420-2426.The particularly preferred molecular form of the present invention provides wherein, and the antibody sources zone comprises a V _HWith a V _LThe polypeptide construct in district.By joint-structural domain V connected to one another _H-structural domain and V _LThe intramolecularly location of-structural domain in the scFv form is not conclusive for described dual specific strand construct.Therefore, have two and may arrange (V _H-structural domain-joint design territory-V _L-structural domain; V _L-structural domain-joint design territory-V _H-structural domain) scFv is the specific embodiments of described dual specific strand construct.Antibody construct can also comprise extra domain, for example is used to separate and/or prepare the structural domain that reorganization produces construct.

The corresponding formal description of bispecific single-chain antibody construct is in appended examples 1.

According to the present invention, employed term " strand " meaning is that first and second structural domains of described dual specific strand construct are covalently bound, preferably with the collinearity aminoacid sequence form of a nucleic acid molecule codified.As employed term in the context of the present invention " be bonded to/with interact " definition at least two " antigen-interaction sites " combining/interacting each other.According to the present invention, term " antigen-interaction sites " definition polypeptide motif, it shows and specific antigens or the interactional ability of antigen special groups specificity.Described combination/interaction also is interpreted as definition " specific recognition ".The term according to the present invention " specific recognition " meaning is that antibody molecule can specificity interact/be bonded to as at least two amino acid of defined everyone target molecule in the literary composition.Described term relates to the specificity of antibody molecule, promptly relates to the ability of its difference as the specific region of defined people's target molecule in the literary composition.The specificity of antigen-interaction-site and its specific antigens interacts can cause the initial of signal, for example owing to the change of inducing antigen conformation, antigenic oligomerization or the like.In addition, the described combination of specificity illustration by " key-lock-principle ".Therefore, since the modification once more of its one-level, secondary or tertiary structure and described structure, the special motif combination each other in antigen-interaction-site and the antigenic aminoacid sequence.The specificity of antigen-interaction-site and its specific antigens interacts and can also cause described site simple knot to be bonded to antigen.

According to term used in the present invention " specificity interaction " meaning be dual specific strand construct not or basically not with (many) peptides cross reaction of analog structure.For example, (see in normal condition by estimation, for example Harlow and Lane, Antibodies:A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1988 and Using Antibodies:A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1999) down described dual specific strand construct group is bonded to purpose (many) peptides and (on the structure and/or on the function) closely-related (many) peptides in a large number more or less, can test the cross reactivity of the dual specific strand construct group of being studied.Only be bonded to purpose (many) peptide/protein but not or basically debond to those antibody of other (many) peptide arbitrarily be considered to special to purpose (many) peptide/protein.Antigen-interaction-site and the interactional example of specific antigens specificity comprise the specificity of part to its acceptor.Described definition particularly comprises the ligand interaction of inducement signal when being bonded to its specific receptors.The example of respective ligand comprises and its specific cell factor acceptor interact/be bonded to cytokine of its specific cell factor acceptor.Particularly described definition comprises that also antigen-interaction-site is bonded to antigen, as selectin family antigen, integrin and growth factor family antigen, as EGF.Described interactional another example, the example that is comprised by described definition is the interaction of the antigen binding site of antigenic determinant (epi-position) and antibody especially.

Term " be bonded to/interact with " can also relate to conformational epitope, by two structure epi-position or discontinuous epi-positions that the zone is formed of people's target molecule or its part.In the context of the present invention, conformational epitope is defined by isolating two or more discontinuous aminoacid sequences in primary sequence, they are gathered in molecular surface (Sela when polypeptide is folded into natural protein, (1969) Science 166,1365 and Laver, (1990) Cell 61,553-6).

Term " discontinuous epi-position " meaning is the non-linear epi-position that is assembled into by the residue from polypeptide chain apart from each other part in the context of the present invention.When polypeptide chain is folded into three-dimensional structure when forming conformation/structure epi-position, these residues accumulate in molecular surface.Construct of the present invention also is envisioned for specificity and is bonded to/interact with conformation/structure epi-position, conformation/structure epi-position by the people CD3 complex body described in the literary composition or as following literary composition in two zone institutes of disclosed its part form and/or comprise this two zones.

Therefore, by method as known in the art and can experimental definite specificity as method disclosed in the literary composition and that describe.These class methods include but not limited to western blotting, ELISA-, RIA-, ECL-, IRMA-, EIA-test and pepscan.

Term " antibody fragment or derivatives thereof " relates to single-chain antibody or its fragment, synthetic antibody, antibody fragment such as Fab, F (ab ₂) ', Fv or scFv fragment or the like, perhaps these segmental chemically modified derivatives arbitrarily.Use routine techniques known in the art, for example be used alone or in combination aminoacid deletion known in the art, insertion, replacement, adding and/or reorganization and/or other modification arbitrarily (for example translation back and chemically modified, for example glycosylation and phosphorylation), can further modify antibody or its corresponding immunoglobulin chain used according to the present invention.Being used for this type of is modified the method that imports in the immunoglobulin (Ig) chain amino acid sequence corresponding DNA sequence is well-known for those skilled in the art; See that for example Sambrook (1989) partly quotes.

Such as in the literary composition use term " (many) peptides " component comprise peptide and polypeptide is described.Peptide is by having 30 amino acid whose molecular compositions of as many as, and polypeptide is by having more than 30 amino acid whose molecular compositions.

Term " antibody fragment or derivatives thereof " is particularly related to (many) peptidic constructs that comprise at least one CDR.The fragment of described antibody molecule or derivative definition (many) peptides, the part that these (many) peptides are above-mentioned antibody molecules and/or by chemical/biological chemistry or molecular biology method modification.Corresponding method is as known in the art and is described in laboratory manual especially and (sees Sambrook etc.; Molecular Cloning:A Laboratory Manual; Cold Spring HarborLaboratory Press, the 2nd edition 1989 and the 3rd editions 2001; Gerhardt etc.; Methods forGeneral and Molecular Bacteriology; ASM Press, 1994; Lefkovits; Immunology Methods Manual:The Comprehensive Sourcebook ofTechniques; Academic Press, 1997; Golemis; Protein-Protein Interactions:A Molecular Cloning Manual; Cold Spring Harbor Laboratory Press, 2002).

In prior art, for example Mack (Proc.Natl.Acad.Sci., 1995, specific recognition EpCAM antigen and the antigenic bi-specific antibody of CD3 have been described in 92:7021-7025).

As mentioned above, connect the described variable domains that is included in the strand of dual specific described in literary composition construct by extra joint sequence.The term according to the present invention " peptide linker " definition aminoacid sequence, the aminoacid sequence by construct that this sequence defines first structural domain and second structural domain is interconnected with one another.The basic technical features of this kind peptide linker is that described peptide linker does not comprise any polymerization activity.Particularly preferred peptide linker is characterized as aminoacid sequence Gly-Gly-Gly-Gly-Ser, i.e. (Gly) 4Ser, or its polymer, i.e. ((Gly) 4Ser) x.The feature that comprises the described peptide linker that lacks the secondary structure promoter action is as known in the art and for example is described in Dall ' Acqua etc. (Biochem. (1998) 37,9266-9273), (Mol Immunol (1992) 29 for Cheadle etc., 21-30) and Raag and Whitlow (FASEB (1995) 9 (1), 73-80).The peptide linker that comprises less amino-acid residue also is particularly preferred.Contemplated have be less than 5 amino acid whose peptide linkers and may comprise 4,3,2 or 1 amino acid.Particularly preferred in " peptide linker " " single " described in literary composition amino acid is Gly.Therefore, described peptide linker can be made up of single amino acids Gly.And the peptide linker that does not promote any secondary structure equally also is preferred.Described in embodiment, can provide being interconnected with one another of described structural domain by for example genetically engineered.That preparation is merged and dual specific strand construct that effectively connect and the method for expressing them in mammalian cell or bacterium are that well-known (for example WO 99/54440 in the art, Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.1989 and 1994 or Sambrook etc., Molecular Cloning:A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, New York, 2001).

Can be humanization or remove the immune antibody construct with following described bispecific single-chain antibody construct above in the literary composition.Be used for humanization and/or remove immunization (many) peptides and, particularly, the method for antibody construct is known for those skilled in the art.

Here be surprisingly found out that structural domain that EpCAM antigen is had specificity and comprises at least one CDR-H3 district that comprises aminoacid sequence NXD (l-asparagine-X-aspartic acid) is useful especially for the particular form of bispecific single-chain antibody construct, aminoacid sequence NXD is preferably SEQ ID NO:80,88 and 96 position 102-104, or the position 106-108 of SEQ ID NO:84 and 92, wherein X is a die aromatischen Aminosaeuren.Because these constructs are favourable for not comprising described amino acid whose construct, these bispecific single-chain antibody constructs are useful especially as pharmaceutical composition.And, find that surprisingly EpCAM antigen is had specificity, comprise the CDR-H3 district of at least one at least 9 amino-acid residue and have greater than 5 * 10 ^-9The K of M _DThe structural domain of value is useful especially for the specificity formation of bispecific single-chain antibody construct.Since these constructs for be less than 9 amino-acid residues and wherein said the special binding domains of EpCAM is had be less than 5 * 10 ^-9The K of M _DThe construct of value is favourable, and these bispecific single-chain antibody constructs are useful especially as pharmaceutical composition.

As shown among the appended embodiment, the prior art construct is characterized as less favourable EC ₅₀Value and/or less effective or complete purifying.Particularly, what surprisingly use according to the present invention is to have highly bioactively to the structural domain of the strand construct of CD3 antigen-specific at N-and C-terminal position, wherein particularly arranges V _{H (anti--CD3)}-V _{L (anti--CD3)}Be preferred.The construct that is applied to pharmaceutical composition of the present invention be characterized as convenient produce and the purifying characteristic with and biological activity highly, i.e. the cellular cytoxicity activity of its expection.Particularly, when the cellular cytoxicity activity of construct of the present invention was compared with the cellular cytoxicity activity of conventional M79 * anti--CD3 and HD70 * anti--CD3 construct, construct of the present invention clearly illustrated higher biological activity (Figure 11 B).As in the cytotoxicity test, determining, low to low-down EC ₅₀Value has been reacted corresponding high biological activity.The EC that molecule is lower ₅₀Value is higher cytotoxicity, and promptly the validity of construct in lysis is higher.On the other hand, the higher EC of molecule in the inducing cell cracking ₅₀Value is effectively low.In the context of the present invention, term " EC ₅₀" corresponding as according to methods known in the art determined and in appended embodiment illustrational EC ₅₀Value: by 4 parameters: the drug level (EC of baseline response (bottom), maximum reaction (top), slope and initiation reaction in the middle of baseline and maximum value ₅₀) definition standard dose response curve.EC ₅₀Be defined as the medicine of initiation reaction in the middle of baseline response (bottom) and maximum reaction (top) or the concentration of molecule.The low K of construct of the present invention _DValue is described higher binding affinity.For example low K _DValue 10 ^-9M shows the high binding affinity of binding constructs.High on the other hand K _DValue for example 10 ^-6M relates to the low binding affinity of construct binding domains.

Especially for example by top disclosed release assay method in the literary composition ⁵¹Cr discharges assay method, LDH release assay method etc. can determine lysis percentage ratio (being cellular cytoxicity activity).Most preferably be to have used fluorochrome release assay method in the context of the present invention as in appended embodiment illustrated.Here, the strong cytotoxicity activity at the EpCAM positive cell of the dual specific strand construct described in the literary composition (seeing the CHO-EpCAM cell among the appended embodiment 3) relates to and comprises EC ₅₀Value preferably≤500pg/ml, more preferably≤400pg/ml even more preferably≤300pg/ml even more preferably≤250pg/ml, most preferably≤200pg/ml ,≤100pg/ml ,≤molecule of 50pg/ml.

Be contained in dual specific construct and prior art M79 * anti--CD3 construct (VL in the pharmaceutical composition of the present invention _17-1A-VH _17-1A-VH _CD3-VL _CD38628pg/ml) compare and show surprising high cell toxicity activity (preferably in the scope of about 10pg/ml-170pg/ml).The technician know the EC50 value with the biological activity determination method difference.The factor that influences the EC50 value comprises effector cell's type, effector cell's activity, target cell type, E: T ratio, incubation time, incubation temperature and other outside atmospheres.The different EC50 values of identical construct in the different experiments can be compared with the EC50 value that contrasts.The construct that has high cell toxicity according to the present invention is the EC50 value of contrast 1/2.5 (toxic at least 2.5 times of control cells) at the most, is preferably at the most 1/3 and more preferably be at the most 1/5 of EC50 value of EC50 value.

And, by surface plasma body resonant vibration (BIAcore ^) measure construct of the present invention with very high avidity in conjunction with EpCAM.In the prior art in conjunction with the K of the construct M79 of EpCAM and CD3 * anti--CD3 _DBe 4 * 10 ^-6M, the K of construct of the present invention _DBe 2.3 * 10 ^-7-2.5 * 10 ^-7M.

Preferably, the X in the described NXD motif is W (tryptophane) or Y (tyrosine).

Further imagination pharmaceutical composition of the present invention comprises the bispecific single-chain antibody construct, and wherein the CDR-H3 in EpCAM specificity structure territory comprises at least 9 amino-acid residues, is preferably at least 14 amino acid.Preferably, CDR-H3 comprises and is less than 18 amino acid, more preferably for being less than 15 amino acid.Therefore, preferably, CDR-H3 comprises 9-17 amino acid, more preferably is 9-15 amino acid and be most preferably 10 or 14 amino acid.

It has surprisingly been found that the bispecific single-chain antibody construct that comprises corresponding EpCAM specific structure territory is better than other EpCAM specificity structure territories known in the art with the form of above-mentioned construct.In appended

embodiment

3,4 and 5, proved this kind effect.Prior art EpCAM binding antibody M79 comprises eight amino acid in its CDR-H3 district and does not comprise sequence NXD (Figure 11 A).

Pharmaceutical composition of the present invention also comprises construct, and wherein said have greater than 5 * 10 the special binding domains of EpCAM ^-9The K of M _DValue.In addition, the pharmaceutical composition binding domains that can be characterized as described CD3 antigen-specific has greater than 10 ^-7The K of M _DThe feature of value.K _DValue is the physical values of definition complex dissociation trend.For binding equilibrium A+B AB, dissociation constant is given as two kinetic rate constant k _OffAnd k _OnRatio: [A] [B] is (kon)/[AB] (koff).More little A of dissociation constant and B combination each other are firm more.In biosystem, good specific combination body dissociation constant is 10 ^-9-10 ^-7In the scope of M.(SPR for example uses BIAcore to use several different methods well known by persons skilled in the art such as surface plasma body resonant vibration ^), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescent spectrometry or can measure K by radioactive mark ligand's binding assay _DValue.The K of construct of the present invention that used surface plasma body resonant vibration (SPR) spectrometry _DValue.Part is expelled to the immobilized antigen chip surface and measure in conjunction with the time chip surface optical density(OD) change.The change that changes the optical density(OD) detected by reflection angle is directly relevant with the amount of the part that is bonded to chip surface-and used biophysics phenomenon is called surface plasma body resonant vibration.

One of interactional mating partner must be fixed in based on surface plasma body resonant vibration (BIAcore for example ^) the sensor chip surface of device.The Real Time Observation part combines and dissociated kinetics with the chip surface immobilized antigen.Binding curve match kinetic rate constant k _OnAnd k _Off, produce apparent equilibrium dissociation constant (KD).

Particularly preferably be that described EpCAM specific combination structural domain has 1 * 10 ^-7-5 * 10 ^-9K between the M in the scope _DValue and CD3 specific combination structural domain had 1 * 10 ^-6-5 * 10 ^-9K between the M in the scope _DValue.

In particularly preferred embodiments, pharmaceutical composition is characterized as the K of the binding domains of described CD3 antigen-specific in addition _DValue＞(more than) 1 * 10 ^-7M.Construct of the present invention has such advantage, promptly because the EpCAM bound fraction has K _DValue is greater than 5 * 10 ^-9The affinity of M is so they can repeatedly use with the kill tumor cell.If the dual specific construct is too high in conjunction with the avidity of the tumour cell of expressing EpCAM, construct is in conjunction with a tumour cell of expressing EpCAM and even be killed when tumour cell and still be retained in its surface and can not continue in conjunction with the killed tumour cell of another desire.Another advantage of construct of the present invention is that the special binding domains of EpCAM is with high affinity combination (corresponding low K _DValue), thus with the tumour cell of circulation T cell guiding dual specific construct mark.Therefore, the K of the EpCAM specific combination structural domain of dual specific construct _DValue is preferably 10 ^-7-5 * 10 ^-9In the M scope and the K of CD3 specific combination structural domain _DValue is preferably 10 ^-6-5 * 10 ^-9In the M scope.In preferred embodiments, the K of EpCAM binding domains _DValue is lower than the K of CD3 binding domains _DValue, corresponding compare with the CD3 binding domains EpCAM binding domains than high affinity.

Further imagination pharmaceutical composition of the present invention comprises the bispecific single-chain antibody construct, and wherein the CDR-H3 in the special structure of EpCAM territory comprises at least 9 amino acid, is preferably at least 14 amino acid.Preferably, CDR-H3 comprises and is less than 18 amino acid, more preferably for being less than 15 amino acid.Therefore, preferably, CDR-H3 comprises 9-17 amino acid, more preferably is 9-15 amino acid and be most preferably 10 or 14 amino acid.

In pharmaceutical composition embodiment preferred of the present invention, the V of people EpCAM antigen-specific structural domain _HChain is selected from:

A) as SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:92 and SEQ ID NO:96 aminoacid sequence as shown in any one;

B) by the aminoacid sequence of nucleic acid sequence encoding as shown in SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:87, SEQ IDNO:91 and SEQ ID NO:95;

C) by with (b) in the complementary strand of the defined nucleotide sequence coded aminoacid sequence of nucleotide sequence of under stringent hybridization condition, hybridizing;

D) by (b) and (c) any one nucleotide sequence because the coded aminoacid sequence of nucleotide sequence that produced of genetic code degeneracy.

Such as in the literary composition use term " hybridization " refer to hybridize the polynucleotide/nucleotide sequence of the polynucleotide of to the literary composition of encoding defined dual specific strand construct or its part.Therefore, described polynucleotide can be used separately as the Northern of RNA or DNA prepared product or the probe in the Southern trace, perhaps can be used as based on the Oligonucleolide primers in its big or small separately pcr analysis.Preferably, described hybridization polynucleotide length comprises at least 10, more preferably is at least 15 Nucleotide, preferably comprises at least 100 and hybridize polynucleotide length as the present invention of probe, be more preferably at least 200, perhaps be most preferably at least 500 Nucleotide.

How using nucleic acid molecule to carry out hybrid experiment is well-known in the art, promptly skilled in the art will recognize that according to the present invention what hybridization conditions she or he must use.At standard textbook such as Molecular Cloning A Laboratory Manual, mention this type of hybridization conditions among Cold Spring HarborLaboratory (2001) N.Y.Preferably under stringent hybridization condition, can hybridize according to the present invention to the polynucleotide of polynucleotide of the present invention or its part.For example, " stringent hybridization condition " refers to containing 50% methane amide, 5 * SSC (750mM NaCl, the 75mM Trisodium Citrate), 42 ℃ of overnight incubation in the solution of shearing salmon sperm DNA of 50mM sodium phosphate (pH7.6), 5 * Denhardt ' s solution and 10% T 500 and 20 μ g/ml sex change, the back with 0.1 * SSC at about 65 ℃ of washing filter membranes.Relate to equally than the nucleic acid molecule of hybridizing under the low stringency condition to polynucleotide of the present invention.Substantially realized the variation of hybridization and signal detection severity by control methane amide concentration (methane amide of low per-cent causes lower severity), salt concn or temperature.For example be included in and contain 6 * SSPE (20 * SSPE=3M NaCl than low stringency condition; 0.2M NaH ₂PO ₄0.02M EDTA pH7.4), 37 ℃ of overnight incubation in the solution of the smart sealing of 0.5%SDS, 30% methane amide, 100 μ g/ml salmons DNA, uses 1 * SSPE, 0.1%SDS in 50 ℃ of washings subsequently.In addition, in order to realize lower severity, can be in higher salt concentrations (for example 5 * SSC) washings of carrying out after strictness is hybridized.Should be understood that by comprising and/or replacing the variation that the another kind of closed reagent that is used to suppress the hybrid experiment background can be realized above-mentioned condition.Typical closed reagent comprises that Denhardt ' s agent, bovine lacto transfer technique optimizer, heparin, sex change salmon sperm DNA and commerce can get the proprietorial preparation of tool.Because the problem of consistency comprises the specificity encapsulant and need change above-mentioned hybridization conditions.Described nucleic acid molecule can be the DNA or the RNA of for example DNA, cDNA, RNA or synthetic generation or comprise the chimeric nucleic acid molecule that these polynucleotide reorganization alone or in combination arbitrarily produces.

Preferably, pharmaceutical composition of the present invention can comprise dual specific strand construct, and wherein the VL chain of people EpCAM antigen-specific structural domain is selected from:

(a) as SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:94 and SEQ ID NO:98 aminoacid sequence as shown in any one;

(b) by the aminoacid sequence of nucleic acid sequence encoding as shown in SEQ ID NO:81, SEQ ID NO:85, SEQ ID NO:89, SEQ IDNO:93 and SEQ ID NO:97;

(c) by with (b) in the complementary strand of the defined nucleotide sequence coded aminoacid sequence of nucleotide sequence of under stringent hybridization condition, hybridizing;

(d) by (b) and (c) any one nucleotide sequence because the coded aminoacid sequence of nucleotide sequence that produced of genetic code degeneracy.

In pharmaceutical composition embodiment preferred of the present invention, the V in described people CD3 specific structure territory _HAnd V _LThe district derives from the CD3 specific antibody that is selected from X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87,12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, WT31 and F101.01.These CD3 specific antibodies are well-known in the art and in addition also are described in Tunnacliffe (1989), Int.Immunol.1,546-550.In the embodiment that is more preferably, the described V in described CD3 specific structure territory _HAnd V _LThe district derives from OKT-3 (as defined above and describe).Even V as described in (and as in appended embodiment illustrated) that be more preferably _HAnd V _LThe district is or derives from Traunecker (1991), EMBO J.10,3655-3659 is described to have specific antibody/antibody derivatives to the CD3 molecule.According to the present invention, described V _HAnd V _LThe district derive from can be under the background condition that other TCR subunits exist the antibody/antibody derivatives etc. of (for example in the genetically modified mouse cell of people CD3-ε chain) specific recognition people CD3-ε chain.These transgenic mices are with natural or approximate native conformation expressing human CD3-ε chain.Therefore, derive from the V of CD3-ε chain specific antibody according to the present invention _HAnd V _LThe district be most preferred and described antibody (parental antibody) should be able to specificity the epi-position of or approximate natural structure natural or the conformational epitope of the people CD3 that under TCR complex body background condition, exists in conjunction with reflection.This kind antibody is classified as " group II " antibody by Tunnacliffe (1989).The further classification of Tunnacliffe (1989) comprises the definition at " the group I " of CD3 and " group III " antibody.For example " group I " antibody recognition of UCHT1 is as the recombinant protein and the CD3-ε chain of expressing as the part of cell surface TCR.Therefore, " group I " antibody has high specific to CD3-ε chain.On the contrary, this paper preferred " group II antibody " is identified in the natural TCR complex body and other TCR subunit bonded CD3-ε chain.Not bound by theory, inferring in the context of the invention needs the TCR background condition for identification CD3-ε chain in " group II " antibody.Also relate to combining of " group II antibody " with the CD3-γ chain and the δ chain of ε chain combination.All these three kinds of subunit expressions can be by carrying out the immunoreceptor tyrosine-based activation motif (ITAM) of tyrosine phosphorylation based on the kinases of protein-tyrosine.Owing to this reason, group II antibody is by CD3-ε chain, γ chain and δ chain inducing T cell signal, causes and compares stronger signal by the group I antibody of CD3-ε chain selective induction T cell signal.Yet, owing to induce strong T cell signal, the V that the dual specific strand construct that comprises at pharmaceutical composition of the present invention is adopted for the treatment application need _{H (anti--CD3)}/ V _{L (anti--CD3)}-district (or its part) preferably derives from the antibody of people CD3 and by Tunnacliffe (1989) (part is quoted) and ranges " group II ".

Relate to pharmaceutical composition in one embodiment of the invention, wherein said bispecific single-chain antibody construct comprises and is selected from following aminoacid sequence:

(a) as SEQ ID NO:2,4,8,10,12,14,16,18,20,30,36,39,42,44,46,48,50,52,54,56,58 and 60 aminoacid sequences shown in any one;

(b) by as SEQ ID NO:1,3,7,9,11,13,15,17,19,29,35,38,41,43,45,47,49,51,53,55, the 57 and 59 coded aminoacid sequences of nucleotide sequence shown in any one;

The present invention also provides and has comprised the pharmaceutical composition that coding as above defines the nucleotide sequence of bispecific single-chain antibody construct.Described nucleic acid molecule can be natural acid molecule and recombinant nucleic acid molecules.Therefore, nucleic acid molecule can be natural origin, synthetic or semisynthetic.Nucleic acid molecule can comprise DNA, RNA and PNA (peptide nucleic acid(PNA)) and can be its heterozygote.Therefore, the present invention relates to comprise the pharmaceutical composition of nucleic acid molecule, described nucleic acid molecule contains and is selected from following nucleotide sequence:

(a) coding comprises the proteinic nucleotide sequence of mature form of the aminoacid sequence of defined bispecific single-chain antibody construct in the literary composition, and described aminoacid sequence is preferably as SEQ ID No:2,4,8,10,12,14,16,18,20,30,36,39,42,44,46,48,50,52, institute is given in 54,56,58 and 60;

(b) comprise or by SEQ ID No:1, in 3,7,9,11,13,15,17,19,29,35,38,41,43,45,47,49,51,53,55,57 and 59 the nucleotide sequence formed of given dna sequence dna;

(c) with (b) in the complementary strand of the defined nucleotide sequence nucleotide sequence of under stringent hybridization condition, hybridizing;

(d) coding by (a) or nucleotide sequence coded protein (b) by by nucleotide sequence (a) or (b) coded aminoacid sequence one or several amino acid replacement, lack and/or add resulting proteinic nucleotide sequence;

(e) coding and nucleotide sequence (a) or (b) coded aminoacid sequence have the proteinic nucleotide sequence of the aminoacid sequence of at least 60% identity;

(f) because (a)-(e) nucleotide sequence that produced of the genetic code degeneracy of the nucleotide sequence of any one;

Term in the context of the invention " mature form protein " is defined as the protein of also randomly modifying subsequently from its corresponding mRNA translation.Defined the term " hybridization " in the context of the invention above this paper.Clearly can be added on the nucleic acid molecule that is contained in the pharmaceutical composition of the present invention for those skilled in the art regulating sequence.For example, can application start, transcriptional enhancer and/or allow the sequence of the abduction delivering of polynucleotide of the present invention.But for example suitable inducible system is that (Proc.Natl.Acad.Sci.USA 89 (1992) as Gossen and Bujard, 5547-5551) and (Trends Biotech.12 (1994) such as Gossen, 58-62) described tsiklomitsin regulate gene expression or J.8 as Crook (1989) EMBO, the described dexamethasone inducible gene expression system of 513-519.And, for further purpose imagination nucleic acid molecule can comprise for example thioester bond and or nucleotide analog.Described modification is useful for the stability of nucleic acid molecule opposing cell inscribe and/or exonuclease.Allow the suitable carrier of the mosaic gene that described nucleic acid molecule transcribes in cell can transcribe described nucleic acid molecule by comprising.In this respect, should also be appreciated that this kind polynucleotide can be used in " gene target " or " gene therapy " method.In another embodiment, described nucleic acid molecule is a mark.The method that is used to detect nucleic acid is well known in the art, for example Southern and Northern trace, PCR or primer extension.This embodiment can be used for the successful importing of screening method with checking above-mentioned nucleic acid molecule during gene therapy method.Described nucleic acid molecule can be the chimeric nucleic acid molecule that reorganization produces, and it comprises any above-mentioned nucleic acid molecule independent or combination.Preferably, nucleic acid molecule is the part of carrier.

Therefore, the invention still further relates to the pharmaceutical composition that comprises the carrier that contains nucleic acid molecule of the present invention.

Many appropriate carriers are known for those technician of biology field, and the selection of carrier is depended on expectation function and carrier comprises plasmid, clay, virus, phage and conventional other carriers that are used for genetic engineering.The well-known method of those skilled in the art can be used in and makes up multiple plasmid and carrier, see (part is quoted) and Ausubel such as for example being described in Sambrook, Current Protocols in Molecular Biology, Green Publishing Associates andWiley Interscience, N.Y. (1989), the technology of (1994).In addition, polynucleotide of the present invention and carrier can be rebuild into liposome to be delivered to target cell.As more detailed discussion below, cloning vector is used to separate different dna sequence dnas.Correlated series can be transferred to the expression vector that needs specific expression of polypeptides.General cloning vector comprises pBluescript SK, pGEM, pUC9, pBR322 and pGBT9.General expression vector comprises pTRE, pCAL-n-EK, pESP-1, pOP13CAT.Preferably, described carrier comprises nucleotide sequence, and wherein nucleotide sequence is the adjusting sequence that effectively is connected to the nucleotide sequence of defined bispecific single-chain antibody construct in the coding literary composition.This type of regulates sequence (controlling elements) is known for the technician and can comprises promotor, montage box, translation initiation codon, be used for importing the translation of insertion sequence and inserting the site at carrier.Preferably, described nucleic acid molecule effectively is connected to the described expression control sequenc that permission is expressed in eucaryon or prokaryotic cell prokaryocyte.

It is contemplated that described carrier is the expression vector that comprises the nucleic acid molecule of bispecific single-chain antibody construct that defines in the coding literary composition.Term " adjusting sequence " is meant the necessary dna sequence dna of expression of the encoding sequence that influence is connected thereto.The characteristic of this type of control sequence depends on host living beings and difference.In prokaryotic organism, control sequence generally includes promotor, ribosome bind site and terminator.In eukaryote, control sequence generally includes promotor, terminator and comprises enhanser, trans-activation or transcription factor in some cases.Term " control sequence " attempts to comprise that its existence is to express necessary whole compositions on bottom line, and can comprise the composition that other are favourable.

Term " effectively connection " is meant that side by side wherein said composition is in its relation that works in the expection mode of permission." effectively connecting " to the control sequence of encoding sequence is to be connected in the mode that realizes the encoding sequence expression under the condition compatible with control sequence.In control sequence is under the situation of promotor, clearly preferably uses double-strandednucleic acid for technicians.Therefore, described carrier expression vector preferably." expression vector " is that the construct that transforms selected host and provide encoding sequence to express in selected host is provided.For example expression vector can be cloning vector, binary vector or integrative vector.Expression comprises that nucleic acid molecule preferably is transcribed into and can translate mRNA.Guarantee that the controlling element of expressing is that those skilled in the art are well-known in protokaryon and/or eukaryotic cell.They generally include the promotor that guarantees transcription initiation and randomly comprise the poly a-signal that guarantees Transcription Termination and transcript stability for eukaryotic cell.The possible controlling element that permission is expressed in prokaryotic host cell comprises the P in the intestinal bacteria (E.coli) for example _L, lac, trp or tac promotor, and the example that allows the controlling element express in eukaryotic host cell is the CMV-in AOX1 or GAL1 promotor or Mammals and other zooblasts in the yeast, SV40-, RSV-promotor (Rous sarcoma virus), CMV-enhanser, SV40-enhanser or globin intron.

Except being responsible for initial element of transcribing, this type of controlling element can also comprise transcription termination signal, for example the SV40 poly A site in polynucleotide downstream or tk poly A site.And, depend on employed expression system, can instruct polypeptide to cellular compartment or be secreted into it on encoding sequence that leader sequence in substratum can be added to described nucleotide sequence and be well-known in the art; Also see for example appended embodiment.Leader sequence is with suitable stage and translation, the assembling of initial sum terminator sequence, and preferably, and leader sequence can instruct the protein of translation or its merocrine secretion in periplasmic space or extracellular substratum.Randomly, the heterologous sequence coding comprises the fused protein of N-terminal evaluation peptide, and N-terminal evaluation peptide is given the stability of its expection characteristic such as expressed recombinant products or simplified purifying; On seeing.In context, suitably expression vector is as known in the art, for example Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pEF-Neo, pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pEF-DHFR and pEF-ADA (Raum etc., Cancer Immunol Immunother (2001) 50 (3), 141-150) or pSPORT1 (GIBCO BRL).

Preferably, expression control sequenc is the eukaryotic promoter system that can transform in the carrier of transfection eukaryotic host cell, but can also use the control sequence that is used for prokaryotic hosts.In case carrier has been integrated into suitable host, the host expresses suitable keeping under the nucleotide sequence high level condition, and as expected, can carry out the collection and the purifying of polypeptide of the present invention subsequently; See for example appended embodiment.

The another kind of expression system that can be used in express cell cycle interacting protein is the insect system.Autographa california nuclear polyhedrosis virus (AcNPV) is as the carrier of expression alien gene in fall army worm cell or Trichoplusia larva in this kind system.The encoding sequence of described nucleic acid molecule can be cloned into for example polyhedron gene of viral nonessential region, and places under the control of polyhedrin promotor.The successful insertion of described encoding sequence causes the polyhedron gene inactivation and produces the recombinant virus that lacks capsid protein matter bag quilt.Then recombinant virus is used to infect fall army worm cell or Trichoplusia larva, in described larva, expresses protein of the present invention (Smith, J.Virol.46 (1983), 584; Engelhard, Proc.Nat.Acad.Sci.USA 91 (1994), 3224-3227).

Extra controlling element can comprise transcribes and translational enhancer.Advantageously, the above-mentioned carrier of the present invention comprises selectivity and/or quantifiable mark.

But to be those skilled in the art well-known and for example comprise and (given the resistance (Reiss of methotrexate as dhfr for the selectable marker gene that is used to screen transformant and for example plant tissue and plant, Plant Physiol. (Life Sci.Adv.) 13 (1994), 143-149)), npt (gives the aminoglycoside Xin Meisu, resistance (the Herrera-Estrella of kantlex and paromycin (paromycin), EMBOJ.2 (1983), 987-995)) and hygro (give the resistance (Marsh of Totomycin, Gene 32 (1984), 481-485)) the basic metabolic antagonist resistance of screening.Other screening-genes have been described, be trpB (allowing cell to utilize indoles to replace tryptophane), hisD (allows cell to utilize histinol to replace Histidine (Hartman, Proc.Natl.Acad.Sci.USA 85 (1988), 8047)), mannose-6-phosphate isomerase (allows cell to utilize seminose (WO 94/20627) and ODC (ornithine decarboxylase, give ornithine decarboxylase inhibitor 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987,: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory volume) resistance) or from the deaminase of terreus (Aspergillusterreus) (gives the resistance (Tamura of blasticidin S, Biosci.Biotechnol.Biochem.59 (1995), 2336-2338)).

Useful quantized mark is also known and be that commerce can get to those skilled in the art.Favourable is that described mark is coding luciferase (Giacomin, Pl.Sci.116 (1996), 59-72; Scikantha, J.Bact.178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett.389 (1996), 44-47) or GRD beta-glucuronidase (Jefferson, EMBO be (1987) J.6, gene 3901-3907).This embodiment is useful especially for cell, tissue and the organism that simple rapid screening comprises described carrier.

As mentioned above, described nucleic acid molecule can use separately or as the part of carrier in cell, to express encoded polypeptide, for example be used for gene therapy.To comprise the nucleic acid molecule or the carrier transfered cell of the dna sequence dna of any one above-mentioned bispecific single-chain antibody construct of encoding, it produces desired polypeptides again.Based on being one of most important application of transgenosis with the gene therapy of therapeutic genes transfered cell by technology in the first external back body or in the body.Suitable carrier, method or the genes delivery system that is used for the treatment of external or vivo gene is described in document and is that those skilled in the art are known; See for example Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ.Res.79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ.Res.77 (1995), 1077-1086; Onodera, Blood 91 (1998), 30-36; Verma, Gene Ther.5 (1998), 692-699; Nabel, Ann.N.Y.Acad.Sci.811 (1997), 289-292; Verzeletti, Hum.Gene Ther.9 (1998), 2243-51; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, and US 5,580, and 859; US 5,589, and 466; Or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640.Described nucleic acid molecule and carrier can be designed for direct importing or pass through liposome or virus vector (for example adenovirus, retrovirus) transfered cell.Preferably, described cell is reproductive tract cell, embryonic cell or ovum or from its deutero-cell, most preferably described cell is a stem cell.The example of embryonic stem cell especially is described in Nagy, and Proc.Natl.Acad.Sci.USA 90 (1993), the stem cell among the 8424-8428.

With top consistent, the present invention relates to drive the method for carrier, described carrier is conventional plasmid, clay, virus and phage of using in the genetic engineering particularly, and they comprise the nucleic acid molecule of peptide sequence of the bispecific single-chain antibody construct of coding this paper definition.Preferably, described carrier is expression vector and/or transgenosis or targeting vector.Derive from virus as the expression vector of retrovirus, vaccinia virus, adeno associated virus, simplexvirus or bovine papilloma virus can be used for as described in polynucleotide or carrier send into the targeted cells group.The well-known method of those skilled in the art can be used in the structure recombinant vectors; See for example to be described in Sambrook etc. (part is quoted) technology in Ausubel (1989, part is quoted) or other standard textbook.Alternatively, described nucleic acid molecule and carrier can be rebuild and enter liposome and be used to send into target cell.The carrier that will comprise nucleic acid molecule of the present invention by well-known method is transferred to host cell, and the method for employing can depend on the type of cell host and change.For example, the calcium chloride transfection is generally used for prokaryotic cell prokaryocyte, and calcium phosphate is handled or electroporation can be used for other cell host, sees Sambrook, and is the same.

Described carrier can be pEF-DHFR, pEF-ADA or pEF-neo.Carrier pEF-DHFR and pEF-ADA have been described in this area, for example at Mack etc., (PNAS (1995) 92,7021-7025) and Raum etc. (Cancer Immunol Immunother (2001) 50 (3) describes in 141-150).

Further imagination pharmaceutical composition of the present invention comprises the host of this paper carrier conversion defined above or transfection.Produce described host by import described at least a above-mentioned carrier or at least a above-mentioned nucleic acid molecule to the host.Mediated the expression of above-mentioned bispecific single-chain antibody construct encoding gene in the existence of at least a carrier described in the host or at least a nucleic acid molecule.

To import host's described nucleic acid molecule or carrier can be integrated into host genome or it can the outer form of karyomit(e) be kept.

Host cell can be any protokaryon or eukaryotic cell.

Term " protokaryon " meaning is to comprise enough protein DNAs of the present invention of whole energy or the bacterium that the RNA molecule transforms or transfection is used to express.Prokaryotic hosts can comprise Gram-negative and gram positive bacterium for example intestinal bacteria, Salmonella typhimurium (S.typhimurium), serratia marcescens (Serratia marcescens) and subtilis (Bacillus subtilis).Term " eucaryon " meaning comprises yeast, higher plant, insect and preferably includes mammalian cell.According to employed host in the recombinant chou production process, can be glycosylated or nonglycosylated by the protein of polynucleotide encoding of the present invention.Particularly preferably be to use the plasmid or virus and hereditary fusion N-terminal FLAG label and/or the C-terminal His label gone up that comprise polypeptid coding sequence of the present invention.Preferably, the length of described FLAG label is about 4-8 amino acid, most preferably 8 amino acid.Any technology of using those skilled in the art to know altogether can be used for above-mentioned polynucleotide transforming or transfecting host.And method that preparation is merged, that effectively connect the method for gene and express these genes in for example mammalian cell and bacterium is well known in the art (Sambrook, part is quoted).Preferably, described host is bacterium, insect, fungi, plant or zooblast.The described host of special imagination can be a mammalian cell, more preferably is people's cell or human cell line.Particularly preferred host cell comprises Chinese hamster ovary celI, myeloma cell line such as SP2/0 or NS/0.

The proteinaceous compound of the activation signals that can provide immune effector cell to be used for cell proliferation or cytositimulation can also be provided pharmaceutical composition of the present invention.Proteinaceous compound is not interpreted as the extra domain of bispecific single-chain antibody construct as defined above, but is a kind of extra composition of pharmaceutical composition of the present invention at least.According to the present invention, further activation signal (for example another kind of costimulatory molecules: B7-family molecule that described " the protein properties compound of immune effector cell activation signals is provided " can be for example T cell, O * 40L, 4.1BBL) or another kind of cytokine: interleukin (for example IL-2) or NKG-2D are in conjunction with (engaging) compound.The preferred form of protein properties compound comprises extra bi-specific antibody and fragment or derivative, for example dual specific scFv.The protein properties compound includes but not limited to TXi Baoshouti or the special scFv fragment of superantigen.Superantigen does not rely on mode directly in conjunction with the TXi Baoshouti variable region of some subtribe with MHC-, thereby mediates elementary t cell activation signal.The protein properties compound can also provide the activation signals of the immune effector cell of non-T cell.The example of the immune effector cell of non-T cell especially comprises the NK cell.

Another technical characterictic of pharmaceutical composition of the present invention is that described pharmaceutical composition is heat-staple in the time of 〉=37 ℃.

Another embodiment of the invention relates to the method that produces pharmaceutical composition of the present invention, and described method is included in cultivates the host who defines in the top literary composition and reclaim the bispecific single-chain antibody construct that is produced from culture under the condition that allows construct to express.

Host transformed can be grown in fermentor tank and cultivates to realize the growth of optimum cell according to technology known in the art.Can separate polypeptide of the present invention from growth medium, cell pyrolysis liquid or cytolemma fraction then.Can separate and the purifying polypeptide of the present invention of microbial expression for example by any ordinary method, described ordinary method is separated for for example preparative chromatography and immunity separates for example to relate to and uses as the mono-clonal of the label of anti-polypeptide of the present invention or those methods or those methods as describing among the appended embodiment of polyclonal antibody.Allow the cultivation host's of expression condition to be known in this area and on this paper, to discuss.These conditions are equally applicable to the purifying/removal process of described construct.Another embodiment of the invention relates to bispecific single-chain antibody construct defined above, nucleotide sequence, carrier, host and/or be used to prepare the purposes of the pharmaceutical composition of prevention, treatment or tumor remission disease by the host that method produced as defined above as defined above as defined above as defined above.Particularly, pharmaceutical composition of the present invention is useful especially in prevention, alleviation and/or treatment cancer.Preferably, described tumor disease is epithelial cancer or minimal residue cancer (minimal residual cancer).

Imagine use standard vector and/or the independent or co-administered bispecific single-chain antibody construct as defined above of genes delivery system, nucleic acid molecule and carrier by the present invention, and randomly use with pharmaceutically acceptable carrier or vehicle.After using, described nucleic acid molecule or carrier can stable integration be gone into experimenter's genome.On the other hand, can use virus vector and its in described cell the lasting existence special to some cell or tissue.Suitable pharmaceutical carrier and vehicle are well known in the art.The disease that the drug prepared composition can be used in prevention or treats or delay to identify above according to the present invention.And, can in gene therapy, use the pharmaceutical composition of the present invention that comprises described nucleic acid molecule or carrier.Suitable genes delivery system comprises liposome, receptor-mediated delivery system, naked DNA and virus vector, as simplexvirus, retrovirus, adenovirus and adeno associated virus or the like.Use biological projectile delivery system for example Williams (Proc.Natl.Acad.Sci.USA 88 (1991), 2726-2729) described system also can realize with delivery of nucleic acids to the health specific site to carry out gene therapy.The additive method of nucleic acid delivery comprises as Verma, Gene Ther.15 (1998), the transgenosis of the described particle mediation of 692-699.

And, the present invention relates to be used to prevent, the method for treatment or tumor remission disease, it comprises to its experimenter of needs uses significant quantity bispecific single-chain antibody construct, nucleotide sequence, carrier, as defined above and/or the host's who produces by method as defined above step as defined above as defined above as defined above.Preferably, described experimenter is the people.

The method that the present invention is used to prevent, treat or alleviate comprises the protein properties compound that can be used in the activation signals of immune effector cell defined above is applied to the experimenter altogether.Using altogether can be to use altogether simultaneously or non-ly use altogether simultaneously.Particularly preferred purposes of the present invention and method are that described tumor disease is an epithelial cancer, preferably gland cancer or minimal residue cancer, preferably early stage solid tumor, advanced solid tumor or shift solid tumor.

At last, the present invention relates to test kit, described test kit comprises as defined above bispecific single-chain antibody construct, nucleotide sequence, carrier and/or host as defined above as defined above as defined above.Also it is contemplated that test kit of the present invention comprise as noted before, be applied to the patient's who needs therapeutic treatment or intervention pharmaceutical composition alone or in combination with other medicines.

Description of drawings

Fig. 1:

Anti-CD3-resists-EpCAM construct A) anti--CD3 VHVL stL * 3-1 VHVL (SEQ IDNO.:11,12), B) anti--CD3 VHVL aL * 4-7 VHVL (SEQ ID NO.:1,2), C) anti--CD3VHVL aL Ser * 4-7VHVL (SEQ ID NO.:7,8), D) anti--CD3 VHVLstL * 4-7 VHVL (SEQ ID NO.:13,14), E) anti--CD3 VHVL stL * 4-7 VLVH (SEQ ID NO.:15,16), F) anti--CD3 VHVL aL * 5-10 VHVL (SEQ IDNO.:3,4), G) anti--CD3 VHVL aL Ser * 5-10VHVL (SEQ ID NO.:9,10), H) anti--CD3VHVL stL * 5-10 VHVL (SEQ ID NO.:17,18), I) anti--CD3 VHVLstL * 5-10 VLVH (SEQ ID NO.:19,20), J) anti--CD3 VHVL aL * 3-1 VHVL (SEQ ID NO.:45,46), K) anti--CD3 VHVL aL Ser * 3-1 VHVL (SEQ IDNO.:47,48), L) anti--CD3 VHVL aL * 3-5 VHVL (SEQ ID NO.:49,50), M) anti--CD3VHVL aL Ser * 3-5 VHVL (SEQ ID NO.:51,52), N) anti--CD3 VHVLstL * 3-5 VHVL (SEQ ID NO.:53,54), O) anti--CD3 VHVL aL * 4-1 VHVL (SEQ ID NO.:55,56), P) anti--CD3 VHVL aL Ser * 4-1 VHVL (SEQ IDNO.:57,58) and Q) DNA and the aminoacid sequence of anti--CD3 VHVL stL * 4-1 VHVL (SEQ ID NO.:59,60).

Fig. 2:

Construct A) anti--CD3 VHVL stL * 5-10 VHVL (SEQ ID NO.:18), B) anti--CD3VHVL stL * 4-7 VHVL (SEQ ID NO.:14), C) anti--CD3 VHVL aL * 5-10VHVL (SEQ ID NO.:4), D) anti--CD3 VHVL aL * 4-7VHVL (SEQ IDNO.:2), E) anti--CD3 VHVL aL Ser * 5-10 VHVL (SEQ ID NO.:10), F) anti--CD3 VHVL aL Ser * 4-7 VHVL (SEQ ID NO.:8), G) anti--CD3 VHVL stL * 3-1 VHVL (SEQ ID NO.:12), H) anti--CD3 VHVL stL * 5-10 VLVH (SEQID NO.:20) and I) facs analysis of anti--CD3 VHVL stL * 4-7 VLVH (SEQ ID NO.:16) in positive Jurkat of CD3 and the positive Kato III of EpCAM-cell.Move to right and show combination.Jurkat and KatoIII cell dotted line represent the migration of negative control (only using secondary antibody), dotted line show anti--EpCAM-anti--combination and the thick line of CD3 control antibodies show purpose dual specific construct.

Fig. 3:

Anti--EpCAM-resists-CD3-construct A) 4-7 VLVH * anti--CD3 VHVL (SEQ IDNO.:41,42), B) 3-5 VLVH * anti--CD3 VHVL (SEQ ID NO.:29,30), C) 3-1VLVH * anti--CD3 VHVL (SEQ ID NO.:35,36), D) 4-1 VLVH * anti--CD3VHVL (SEQ ID NO.:38,39) and E) 5-10 VLVH * anti--CD3 VHVL (SEQ IDNO.:43,44) DNA and aminoacid sequence.

Fig. 4: 4-7 VLVH * anti--CD3 VHVL (SEQ ID NO.:42) construct A), B) 3-5VLVH * anti--CD3 VHVL (SEQ ID NO.:30), C) 3-1 VLVH * anti--CD3 VHVL (SEQ ID NO.:36), D) 4-1 VLVH * anti--CD3 VHVL (SEQ ID NO.:39) and E) 5-10 VLVH * anti--CD3 VHVL (SEQ ID NO.:44) construct at positive Jurkat of CD3 and the intracellular facs analysis of the positive Kato III of EpCAM-.Move to right and show combination.

Fig. 5:

The protein fraction that comprises the EpCAM bi-specific antibody at the 280nm place from the representative elution profile of Zn-ChelatingFractogel  post.The high absorption value at the 280nm place of 50-450ml residence time is because the non-binding protein of the post of flowing through.The EpCAM dual specific construct of representing to comprise the protein fraction that is used to be further purified at the arrow at place, 530.09ml peak.

Fig. 6:

At the 280nm place from the representative protein elution profile of Sephadex  S200 gel-filtration column wash-out.The corresponding approximately molecular weight of 52kD of protein peak at 82.66ml place that comprises the bi-specific antibody of anti-CD3 and EpCAM.Collect fraction from the 40-140ml residence time.

Fig. 7

A) the positive example displacement chromatography figure of 3-1 * anti--CD3 (SEQ ID NO.:36) shows proteinic total charge isotype.On MiniS  (Amersham) post, carry out positive example displacement chromatography.Use after 5.5 washings of 20mMMES pH of buffer, use the gradient of the elution buffer that comprises 1M NaCl: 0-30% elute protein in 60 column volumes.At 23.58ml place wash-out dual specific construct.

Begin with the non-specific protein of 1M NaCl wash-out at the 50ml place.

B) the positive example displacement chromatography figure of 5-10 * anti--CD3 (SEQ ID NO.:44) shows proteinic total charge isotype.Shown in Fig. 7 A, carry out positive example displacement chromatography.At the acromion place of 35.77ml wash-out dual specific construct.Begin with the non-specific protein of 1M NaCl wash-out at the 50ml place.

Fig. 8:

A) the typical SDS-PAGE of EpCAM bispecific single-chain antibody protein fraction analyzes.Swimming lane M: molecular weight standard swimming lane 1: cell culture supernatant liquid; Swimming lane 2:IMAC effluent liquid; Swimming lane 3:IMAC washings; Swimming lane 4:IMAC elutriant; Swimming lane 5: anti-EpCAM that obtains from gel-filtration and the antibody purification of CD3.

B) the typical Western engram analysis of the EpCAM bispecific single-chain antibody protein fraction of purifying.Swimming lane 1: cell culture supernatant liquid; Swimming lane 2:IMAC effluent liquid; Swimming lane 3:IMAC washings; Swimming lane 4:IMAC elutriant; Swimming lane 5: anti-EpCAM that obtains from gel-filtration and the antibody purification of CD3.

Fig. 9:

C-terminal EpCAM binding substances is anti--CD3 * 3-1 (SEQ ID NO.:46), anti--CD3 *-cytotoxic assay of 5-10 (SEQID NO.:4) and resisting-CD3 * 4-7 (SEQ ID NO.:2).CB15T cell clone and CHO-EpCAM cell were with 5: 1 E: T ratio uses.Use PKH26 dyestuff is with the CHO-EpCAM cell dyeing and use the facs analysis counting cells after bispecific single-chain antibody is hatched.

Figure 10:

N-terminal EpCAM binding substances 3-1 * anti--CD3 (SEQ ID NO.:36), and the cytotoxic assay of 5-10 * anti--CD3 (SEQ ID NO.:44).CB15T cell clone and CHO-EpCAM cell were with 5: 1 E: T ratio uses.Use PKH26 dyestuff is with the CHO-EpCAM cell dyeing and use the facs analysis counting cells after bispecific single-chain antibody is hatched.

Figure 11:

A) EpCAM 3-1 (SEQ ID NO.:, 80), EpCAM 4-1 (SEQ ID NO.:88), EpCAM5-10 (SEQ ID NO.:96), EpCAM 3-5 (SEQ ID NO.:84), the CDR3 and the EpCAM M79 of the VH chain of EpCAM 4-7 (SEQID NO.:92), the sequence alignment that the CDR3 of the VH chain of HD70 and 3B10 compares.Runic is represented the NXD motif.

B) 3-1 * anti--CD3 (SEQ ID NO.:36), 5-10 * anti--CD3 (SEQ ID NO.:44), the comparison of the cellular cytoxicity activity of anti--CD3 * 4-7 (SEQ ID NO.:2) and anti--CD3 * 5-10 (SEQ ID NO.:18) and M79 * anti--CD3 and HD70 * anti--CD3 contrast.PBMC cell and KatoIII cell were with 10: 1 E: the T ratio uses.Hatch back facs analysis counting cells with iodate third ingot dyeing KatoIII cell and at bispecific single-chain antibody.

Describe the present invention referring now to following biology embodiment, these embodiment illustrate and are not understood to limitation of the scope of the invention.

The clone and the expression of embodiment 1:EpCAM construct

Produced and comprised with the anti-CD3 of various structures or structural domain arrangement and a large amount of constructs of anti--EpCAM.Anti-EpCAM VH and the VL variable domains of antibody 3-1 are shown in SEQ IDNO.:79,80,81,82, the anti-EpCAM VH of antibody 3-5 and VL variable domains are shown in SEQ ID NO.:83,84,85,86, the anti-EpCAM VH of antibody 4-1 and VL variable domains are shown in SEQ ID NO.:87,88,89,90, the anti-EpCAM VH of antibody 4-7 and VL variable domains be shown in SEQ ID NO.:91,92,93,94 and anti-EpCAM VH and the VL variable domains of antibody 5-10 be shown in SEQ ID NO.:95,96,97,98.Construct is summarized in table 1.

Table 1. is anti--and CD3-is anti--and EpCAM and anti--EpCAM-be anti--the CD3 construct

SEQ ID NO.: construct number.	Construct	Structural domain is arranged	Distinguishing characteristics
SEQ ID NO.: construct number.	Construct	Structural domain is arranged	Distinguishing characteristics	Anti--CD3 * anti--EpCAM construct
SEQ ID NO.：1，2	Anti--CD3 * 4-7	VH-VL×VH-VL		Anti--CD3 * anti--EpCAM construct
SEQ ID NO.：1，2	Anti--CD3 * 4-7	VH-VL×VH-VL		SEQ ID NO.：3，4	Anti--CD3 * 5-10	VH-VL×VH-VL
SEQ ID NO.：45，46	Anti--CD3 * 3-1	VH-VL×VH-VL		SEQ ID NO.：3，4	Anti--CD3 * 5-10	VH-VL×VH-VL
SEQ ID NO.：45，46	Anti--CD3 * 3-1	VH-VL×VH-VL		SEQ ID NO.：49，50	Anti--CD3 * 3-5	VH-VL×VH-VL
SEQ ID NO.：55，56	Anti--CD3 * 4-1	VH-VL×VH-VL		SEQ ID NO.：49，50	Anti--CD3 * 3-5	VH-VL×VH-VL
SEQ ID NO.：55，56	Anti--CD3 * 4-1	VH-VL×VH-VL		SEQ ID NO.：7，8	Anti--CD3 * 4-7Cys-Ser	VH-VL×VH-VL	The Cys-Ser sudden change
SEQ ID NO.：9，10	Anti--CD3 * 5-10Cys-Ser	VH-VL×VH-VL	The Cys-Ser sudden change	SEQ ID NO.：7，8	Anti--CD3 * 4-7Cys-Ser	VH-VL×VH-VL	The Cys-Ser sudden change
SEQ ID NO.：9，10	Anti--CD3 * 5-10Cys-Ser	VH-VL×VH-VL	The Cys-Ser sudden change	SEQ ID NO.：47，48	Anti--CD3 * 3-1	VH-VL×VH-VL	The Cys-Ser sudden change
SEQ ID NO.：51，52	Anti--CD3 * 3-5	VH-VL×VH-VL	The Cys-Ser sudden change	SEQ ID NO.：47，48	Anti--CD3 * 3-1	VH-VL×VH-VL	The Cys-Ser sudden change

SEQ ID NO.：57，58	Anti--CD3 * 4-1	VH-VL×VH-VL	The Cys-Ser sudden change
SEQ ID NO.：57，58	Anti--CD3 * 4-1	VH-VL×VH-VL	The Cys-Ser sudden change	SEQ ID NO.：11，12	Anti--CD3 * 3-1	VH-VL×VH-VL	(G ₄S) ₃-joint
SEQ ID NO.：13，14	Anti--CD3 * 4-7	VH-VL×VH-VL	(G ₄S) ₃-joint	SEQ ID NO.：11，12	Anti--CD3 * 3-1	VH-VL×VH-VL	(G ₄S) ₃-joint
SEQ ID NO.：13，14	Anti--CD3 * 4-7	VH-VL×VH-VL	(G ₄S) ₃-joint	SEQ ID NO.：15，16	Anti--CD3 * 4-7	VH-VL×VL-VH	(G ₄S) ₃-joint
SEQ ID NO.：17，18 1	Anti--CD3 * 5-10	VH-VL×VH-VL	(G ₄S) ₃-joint	SEQ ID NO.：15，16	Anti--CD3 * 4-7	VH-VL×VL-VH	(G ₄S) ₃-joint
SEQ ID NO.：17，18 1	Anti--CD3 * 5-10	VH-VL×VH-VL	(G ₄S) ₃-joint	SEQ ID NO.：19，20	Anti--CD3 * 5-10	VH-VL×VL-VH	(G ₄S) ₃-joint
SEQ ID NO.：53，54	Anti--CD3 * 3-5	VH-VL×VH-VL	(G ₄S) ₃-joint	SEQ ID NO.：19，20	Anti--CD3 * 5-10	VH-VL×VL-VH	(G ₄S) ₃-joint
SEQ ID NO.：53，54	Anti--CD3 * 3-5	VH-VL×VH-VL	(G ₄S) ₃-joint	SEQ ID NO.：59，60	Anti--CD3 * 4-1	VH-VL×VH-VL	(G ₄S) ₃-joint
Anti--EpCAM-resists-the CD3 construct				SEQ ID NO.：59，60	Anti--CD3 * 4-1	VH-VL×VH-VL	(G ₄S) ₃-joint
Anti--EpCAM-resists-the CD3 construct				SEQ ID NO.：29，30	3-5 * anti--CD3	VL-VH×VH-VL
SEQ ID NO.：35，36	3-1 * anti--CD3	VL-VH×VH-VL		SEQ ID NO.：29，30	3-5 * anti--CD3	VL-VH×VH-VL

SEQ ID NO.：38，39	4-1 * anti--CD3	VL-VH×VH-VL
SEQ ID NO.：38，39	4-1 * anti--CD3	VL-VH×VH-VL	SEQ ID NO.：41，42	4-7 * anti--CD3	VL-VH×VH-VL
SEQ ID NO.：43，44	5-10 * anti--CD3	VL-VH×VH-VL	SEQ ID NO.：41，42	4-7 * anti--CD3	VL-VH×VH-VL

1.1C-the clone of terminal EpCAM-combination

1.1.1 the preparation of anti--CD3PCR product

A) have resisting-CD3 construct (SEQ ID NO.:1,2,3 and 4) of initial 18 amino acid joints

Use CD19 * CD3 construct (L ffler A etc., Blood 2000 95:2098-103) N-terminal that has obtained to comprise 18 amino acid joints (SEQ ID NO.:70) by PCR as template and following primer (CD3 VH BsrGI:AGGTGTACACTCCGATATCAAACTGCAGCAG (SEQ ID NO.:5), CD3VL BspEI:AATCCGGATTTCAGCTCCAGCTTGG (SEQ ID NO.:6)) anti--CD3 at first.

B) have initial 18 amino acid joints and have Cys anti--CD3 construct (SEQ ID No.7,8,9 and 10) to Ser sudden change at CDRH3

Use CD19 * anti--CD3 (C → S sudden change) construct to obtain to comprise 18 amino acid joints (Seq ID NO.:70) by PCR and had Cys to resist at first-CD3 to the N-terminal of Ser sudden change as template and primer CD3 VHBsrGI and CD3VL BspEI (Seq ID No.5 and 6).CDRH3 sequence with Cys-Ser sudden change is shown in SEQ ID NO.:78.

C) anti--CD3-that has (G4S) 3 joints resists-EpCAM construct (Seq ID No.11,12,13,14,15,16,17,18,19 and 20)

By using CD19 * CD3 (L ffler A etc., Blood 2000 95:2098-103) to obtain to comprise 15 amino acid standard (G for the PCR of template ₄S) ₃The anti-CD3 of N-terminal of joint (SEQ ID NO.:99).By following primer (CD3 VH:CD3 VH BsrGIAGGTGTACACTCCGATATCAAACTGCAGCAG (SEQ ID NO.:5), 3 ' CD3 VH GS15GGAGCCGCCGCCGCCAGAACCACCACCACCTGAGGAGACTGTGA GAGTGGTGCCTTG (SEQ ID NO.:21); CD3 VL:5 ' CD3 VL GS15GGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATTCAGCTGACCCAGTCTC C (SEQ ID NO.:22), CD3 VL BspEIAATCCGGATTTCAGCTCCAGCTTGG (SEQ ID NO.:6)) anti--CD3 VH district and anti--CD3 VL district have increased respectively.The overlapping complementary sequence that imports the PCR product is used for forming at subsequently fusion PCR and has 15 amino acid (G ₄S) ₃The encoding sequence of (single-letter amino acid code) (SEQ IDNO.:99) joint.Use primer that CD3 VH BsrGI (SEQ ID NO.:5) and CD3 VL BspEI (SEQ ID NO.:6) are carried out amplification step.

1.1.2 with VH _Anti--CD3-VL _Anti--CD3* VH _Anti--EpCAM-VL _Anti--EpCAMDirection clone is anti--CD3 * anti-EpCAM construct (SEQ ID NO.:1,2, SEQ ID NO.:3,4, SEQ ID NO.:7,8, SEQID NO.:9,10, SEQ ID NO.:11,12, SEQ ID NO.:13,14 and SEQ ID NO.:17,18)

Use restriction enzyme BsrG1 and BspE1 enzyme to cut to comprise 18 amino acid joints (SEQID NO.:70) N-terminal anti--CD3 or comprise 15 amino acid standard (G at first ₄S) ₃Joint (SEQID NO.:99) N-terminal is anti--CD3 at first, and (CA), this carrier comprises as the aminoacid sequence of the segmental eucaryon secretion signal of EcoRI/BsrGI (leading peptide) for Stratagene, La Jolla to be cloned into bluescript KS carrier subsequently.Use EcoRI and BspEI enzyme to cut after this construct, that the resulting dna fragmentation that comprises the anti--CD3 scFv that has leading peptide separately is cloned into is that the EcoRI/BspEI enzyme is cut, comprise the terminal EpCAM combination 3-1 (SEQID NO.:79-82) of c among the pEFDHFR, the plasmid of 4-7 (SEQ ID NO.:91-94) or 5-10 (SEQ ID NO.:95-98).PEFDHFR is described in Mack etc., Proc.Natl.Acad.Sci.USA 92 (1995) 7021-7025).

1.1.3. with VH _Anti--CD3-VL _Anti--CD3* VL _Anti--EpCAM-VH _Anti--EpCAMDirection the clone resist-CD3 * anti-EpCAM construct (SEQ ID No.:15,16,19 and 20)

The C-terminal of VLVH direction that has obtained to comprise 15 amino acid modular connections (SEQ ID NO.:99) by PCR is anti--EpCAM antibody 4-7 (SEQ ID NO.:91-94).By following primer (4-7 VL:4-7 VL BspEI FORCTGAAATCCGGAGGTGGTGGATCCGAGCTCGTGATGACCCAGACTCC (SEQ ID NO.:100), 4-7 VL GS15 REVGGAGCCGCCGCCGCCAGAACCACCACCACCTTTGATCTCAAGCTTGGTCCCC (SEQ ID NO.:101); 4-7VH:4-7 VH GS15 FORGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGGTGCAGCTGCTCGAGCAG (SEQ ID NO.:23), 4-7 VH SalI REVTTTTAAGTCGACCTAATGATGATGAT-GATGATGTGAGGAGACGGTGACCGTG G (SEQ ID NO.:24)) 4-7 VH and 4-7 VL district have increased respectively.The overlapping complementary sequence that imports the PCR product is used for forming at subsequently fusion PCR and has 15 amino acid (G ₄S) ₃The encoding sequence of (single-letter amino acid code) joint (SEQID NO.:99).Use primer that 4-7 VL BspEI FOR and 4-7 VH SalIREV (SEQ ID NO.100, SEQ ID NO.:24) are carried out amplification step.

By PCR obtained to comprise the C-terminal of 15 amino acid modular connections (SEQ ID NO.:99) VLVH direction anti--EpCAM antibody 5-10 (SEQ ID NO.:95-98).Use following primer (5-10 VL:5-10 VL BspEI FORCTGAAATCCGGAGGTGGTGGATCCGAGCTCGTGATGACACAGTCTCCAT (SEQ ID NO.:25), 5-10 VL GS15 REVGGAGCCGCCGCCGCCAGAACCACCACCACCTTTGATCTCAAGCTTGGTCCCAG (SEQ ID NO.:26); 5-10 VH:5-10 VH GS15 FORGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGGTGCAGCTGCTCGAGC (SEQ ID NO.:27), 5-10 VH SalI REVTTTTAAGTCGACCTAATGATGATGATGATGATGTGAGGAGACGGTGACCGTGG (SEQ ID NO.:28)) 5-10 VH district and 5-10 VL district have increased respectively.The overlapping complementary sequence that imports the PCR product is used for forming at subsequently fusion PCR and has 15 amino acid (G ₄S) ₃The encoding sequence of joint (SEQ ID NO.:99).Use primer that 5-10 VL BspEIFOR and 5-10 VH SalI REV (SEQ ID NO.:25, SEQ ID NO:28) are carried out amplification step.Cut PCR product (5-10VLVH and 4-7VLVH) and connect into anti--CD3VHVL stL * 5-10VH VL (SEQ IDNO.:17 that the BspEI/SalI enzyme is cut among the pEFDHFR with BspEI and SalI enzyme, 18) or anti--CD3VHVL stL * 4-7 (SEQ ID NO.:13,14) VHVL to replace 5-10 VHVL dna fragmentation.

1.1.4. the expression and the combination of anti--CD3-EpCAM construct

Confirm by order-checking plasmid transfection to be gone into to be used for the DHFR defective type Chinese hamster ovary celI of eukaryotic expression after the sequence of coding dual specific strand.As Kaufmann R.J. (1990) MethodsEnzymol.185, carry out described in 537-566) expressing at the eukaryotic protein of DHFR defective type Chinese hamster ovary celI.Transfectional cell and produce 1 and go up clear liquid then increases.Expression and combination by facs analysis checking dual specific single chain molecule.Use the positive SGC-7901 Kato of EpCAM III for this purpose and (be attained at American type culture collection (ATCC) Manassas, VA20108 USA, ATCC number: HTB-103).In Jurkat cell (ATCC TIB 152), proved the combination of anti--CD3 part.

Hatch about 200000 cells according to supplier's recommendation culturing cell and with 10 μ g/ml constructs among the 50 μ l PBS that contain 2%FCS.Combination with the anti--His antibody of 2 μ g/ml among the 50 μ l PBS that contain 2%FCS (from Quiagen GmbH, Hilden, FRG obtains for Penta-His antibody, no BSA) detection construct.Used the F (ab ') of the affinity purification that the R-phycoerythrin puts together as the second step reagent ₂Fragment, goat anti-mouse IgG, Fc-γ fragment specific antibody to be diluted among the 50 μ l PBS that contain 2%FCS (from Dianova, Hamburg, FRG obtains) at 1: 100.(BD biosciences, Heidelberg FRG) go up working sample at FACSscan.The whole antibody that comprise anti--CD3 and anti--EpCAM show the binding affinity to CD3 and EpCAM (Fig. 2) than anti--EpCAM (M79) in the prior art * anti--the CD3 bi-specific antibody is stronger.

1.2N terminal EpCAM combination

1.2.1 the clone of anti--EpCAM * anti--CD3 construct

The clone of construct 3-5 * anti--CD3 (SEQ ID NOs.29,30):

Obtained C-terminal 3-5 by the PCR that is used to make up 3-5 * anti--CD3 (SEQ ID NO.:29) molecule with the VH-VL direction.By use respectively primer to the pcr amplification of me 81 (SEQ ID NO.:31)/me 90 (SEQ ID NO.:34) and me 83 (SEQ ID NO.:32)/me 84 (SEQ IDNO.:33) fragment I and II.Use the Expand HighFidelity System of Roche Diagnostics to carry out 20 circulations of heat start PCR (94 ℃/30 seconds; 60 ℃/1 minute; 72 ℃/1 minute) be used for amplification, carry out 72 ℃ of circulations of 3 minutes subsequently.

The electrophoresis on 1.5% sepharose with PCR fragment I and II.The template of mixing fragment (every kind of 1ng) and the PCR next time of me 81 (SEQ ID NO.:31) and me 84 (SEQ ID NO::33) being reacted as primer is with amplified fragments III.Carry out PCR as mentioned above.Purifying fragment III is also with BssHII and BspEI (Biolabs) digestion on sepharose, purifying also is cloned into the corresponding site of pEF-dHFR-signal peptide (77/78)-anti--CD3 cloning vector subsequently, this help with anti--front in anti--CD3 district is cloned in the target variable region.Carrier has immediately following in the single BssHII site of signal peptide, follows by BspEI site, joint (G ₄S) and anti--CD3 district.Verified the zone of being cloned by Restriction Enzyme digestion and dna sequencing.

The sequence of the primer that uses:

Me 81：5′-GGA TGC GCG CGA GCT CGT GAT GAC CCA GACTCCA CTC TCC-3′(SEQ ID NO.：31)

Me 83：5′-GGT TCT GGC GGC GGC GGC TCC GGT GGT GGTGGT TCT GAG GTG CAG CTG CTC GA CAG TCT G-3′(SEQ IDNO.：32)

Me 84：5′-GTG CTC CGG AGG AGA CGG TGA CCG TGG TCC CTTGGC CCC AG-3′(SEQ ID NO.：33)

Me 90：5′-CCG GAG CCG CCG CCG CCA GAA CCA CCA CCA CCTTTG ATC TCA AGC TTG GTC CC-3′(SEQ ID NO.：34)

The clone of construct 3-1 * anti--CD3 (SEQ ID NO.:35,36):

The C-terminal 3-1 that has obtained the VH-VL direction by PCR is used to make up 3-1 * anti--CD3 (SEQ ID NO.:35) molecule.By using primer to me 91a (SEQ ID NO.:37)/me 90 (SEQ ID NO.:34) and me 83 (SEQ ID NO.:32)/me 84 (SEQ ID NO.:33) fragment that increased respectively I and II.As above carry out PCR.

The template that the sepharose fragment that will comprise PCR fragment I and II is reacted as the next round PCR that uses primer to me 91a (SEQ ID NO.:37) and me 84 (SEQ ID NO.:33) is with amplified fragments III.Carry out PCR as mentioned above, except at 68 ℃ rather than carry out renaturation at 60 ℃.Also with BsrGI and BspEI (Biolabs) digestion, purifying also is cloned into the corresponding site of pEF-dHFR-M79 * anti--CD3 cloning vector to purifying fragment III subsequently on sepharose.Verified the zone of being cloned by Restriction Enzyme digestion and dna sequencing.

Me 91a：5′-GGA TTG TAC A CTCC GA GCT CGT CAT GAC CCAGTC TCC ATC TTA TCT TGC TGC-3′(SEQ ID NO.：37)

The clone of construct 4-1 * anti--CD3 (SEQ ID NO.:38,39):

The C-terminal 4-1 that has obtained the VH-VL direction by PCR is used to make up 4-1 * anti--CD3 (SEQ ID NO.:38,39) molecule.By using primer to me 92a (SEQ ID NO.:40)/me90 (SEQ ID NO.:34) and me 83 (SEQ ID NO.:32)/me 84 (SEQ ID NO.:33) fragment that increased respectively I and II.As above under 60 ℃ of annealing temperatures, carry out PCR.

The template that the sepharose fragment that will comprise PCR fragment I and II is reacted as the next round PCR that uses primer to me 92a (SEQ ID NO.:40) and me 84 (SEQ ID NO.:33) is with amplified fragments III.Carry out PCR as mentioned above, except at 68 ℃ rather than carry out renaturation at 60 ℃.Also with BsrGI and BspEI (Biolabs) digestion, purifying also is cloned into the corresponding site of pEF-dHFR-M79 * anti--CD3 cloning vector to purifying fragment III subsequently on sepharose.Verified the zone of being cloned by Restriction Enzyme digestion and dna sequencing.

Me 92a：5′-GGA TTG TAC A CTCC GA GCT CGT GAT GAC ACAGTCTCC ATC CTC C-3′(SEQ ID NO.：40)

The clone of construct 4-7 * anti--CD3 (SEQ ID NO.:41,42):

The C-terminal 4-7 that has obtained the VH-VL direction by PCR is used to make up 4-7 * anti--CD3 (SEQ ID NO.:41,42) molecule.By using primer to me 81 (SEQ ID NO.:31)/me90 (SEQ ID NO.:34) and me 83 (SEQ ID NO.:32)/me 84 (SEQ ID NO.:33) fragment that increased respectively I and II.As above under 60 ℃ of annealing temperatures, carry out PCR.

The template that the sepharose fragment that will comprise PCR fragment I and II is reacted as the next round PCR that uses primer to me 81 (SEQ ID NO.:31) and me 84 (SEQ ID NO.:33) is with amplified fragments III.Carry out PCR as mentioned above.Also with BssHII and BspEI (Biolabs) digestion, purifying also is cloned into the corresponding site of pEF-dhft-signal peptide (77/78)-anti--CD3 cloning vector to purifying fragment III subsequently on sepharose.Verified the zone of being cloned by Restriction Enzyme digestion and dna sequencing.

The clone of construct 5-10 * anti--CD3 (SEQ ID NO.:43,44):

The C-terminal 5-10 that has obtained the VH-VL direction by PCR is used to make up 5-10 * anti--CD3 (SEQ ID NO.:43,44) molecule.By using primer to me 92a (SEQ ID NO.:40)/me90 (SEQ ID NO.:34) and me 83 (SEQ ID NO.:32)/me 84 (SEQ ID NO.:33) fragment that increased respectively I and II.As above under 60 ℃ of annealing temperatures, carry out PCR.

1.2.2 the expression of anti--EpCAM * anti--CD3 bispecific molecule

The Chinese hamster ovary celI that lacks the DHFR gene is incubated at added 10% foetal calf serum (LifeTechnologies was 65 ℃ of hot deactivations 30 minutes) and HT (xanthoglobulin and thymidine; LifeTechnologies, catalog number (Cat.No.): 41065-012) α MEM substratum (Life Technologies, catalog number (Cat.No.): 32561).Use Lipofectamine 2000 test kit (Invitrogen; Catalog number (Cat.No.): 11668-019) according to specification sheets that manufacturers provided with pEF-dHFR-3-1 * anti--CD3 (SEQID NO.:35,36), pEF-dHFR-3-5 * anti--CD3 (SEQ ID NO.:29,30), pEF-dHFR-4-1 * anti--CD3 (SEQ ID NO.:38,39), pEF-dHFR-4-7 * anti--CD3 (SEQ ID NO.:41,42) and pEF-dHFR-5-10 * anti--CD3 (SEQ ID NO.:43,44) transfectional cell.After 48 hours, by transfectional cell being transferred to screening culture medium (α MEM substratum (catalog number (Cat.No.): 32561)) the screening cell that comprises heat-inactivated 10% dialysis foetal calf serum (Life Technologies).2-3 is after week, at 2 liters of tissue culture revolving bottles (Falcon (catalog number (Cat.No.): 353068 in screening; Becton Dickinson Labware) grown cell 8-9 days (selecting in the substratum at 500ml) is to produce bispecific molecule in.Tissue culture medium (TCM) was removed cell and cell debris at 4 ℃ in centrifugal 10 minutes with 300g (1300 rev/mins).The supernatant liquor that will comprise secreted bispecific molecule is stored in-20 ℃ up to further analyzing.

1.2.3 the combination of dual specific anti-EpCAM * anti-CD3 variant is measured

For analyze dual specific of the present invention anti--bonding strength of EpCAM * anti--CD3 strand construct, carried out below in conjunction with mensuration.

Hatched 45 minutes with 250000 Jurkat cells (being used to measure CD3 combines) and Kato cell (being used to measure the EpCAM combination) respectively at 4 ℃ with the thick supernatant liquor that comprises the dual specific construct (50 μ l).After this, (Dianova DIA910) was hatched 60 minutes in 4 ℃ with FACS damping fluid (phosphate buffered saline(PBS) that contains 1% foetal calf serum (FCS) and 0.05% sodiumazide) washed cell 2 times and with mouse anti His antibody.As above carry out washing step.

Antibody (IgG) (Sigma, P8547) incubated cell of puting together antibody (BD 550003) or puting together with goat anti-mouse FITC at last with anti-mouse-PE.After the washing step, use FACS Calibur (B ﹠amp; D) analyze 10,000 incidents.All EpCAM constructs all show strong combination (Fig. 4).

The purifying of embodiment 2.EpCAM construct

For purifying comprises the dual specific strand construct of anti--EpCAM and anti--CD3, with cell growth results after 7 days in the revolving bottle that contains HiClone  CHO improvement DMEM substratum (HiQ).Also will comprise expressed proteinic supernatant liquor is stored in-20 ℃ by centrifugal removal cell.

 kta FPLC System  (Pharmacia) and Unicorn Software  are used for chromatographic analysis.All chemical reagent be research grade and available from Sigma (Deisenhofen) or Merck (Darmstadt).

Use and load ZnCl ₂Fractogel  post carry out IMAC according to the scheme of manufacturers.With buffer A 2 (20mM NaPP pH7.5,0.4M NaCl) balance pillar and with cell culture supernatant (500ml) with 3ml/ minute flow applications in pillar (10ml).Remove not in conjunction with sample with buffer A 2 washing pillars.Use the protein of 2 step gradient buffering liquid A2 (20mM NaPP pH7.5,0.4M NaCl, 0.5M imidazoles) elution of bound.In step 1, use 20% buffer B 2 of 10 times of column volumes, in step 2, use 100% buffer B 2 of 10 times of column volumes.Merging is used to be further purified from the protein fraction of wash-out in 100% step.

On with PBS (Gibco) equilibrated Sephade * S200 HiPrep  post (Pharmacia), carry out gel permeation chromatography.The protein example (flow velocity 1ml/ minute) of wash-out is used for SDS-Page and the Western trace detects.

The calibration in advance pillar is used for molecular weight determination (molecular weight standard test kit, Sigma MWGF-200).

(MicroBCA Pierce) and with IgG (Biorad) measures protein concn as standard protein to use the protein determination dyestuff.Proteinic output is shown in table 2.All constructs are producible.

The output of the strand dual specific construct of table 2. comprises anti--EpCAM and anti--CD3

Construct	Output [the protein μ g of purifying in every liter of nutrient solution]
Construct		4-1 * anti--CD3 (SEQ ID NO.:39)	172.5
3-5 * anti--CD3 (SEQ ID NO.:30)	265	4-1 * anti--CD3 (SEQ ID NO.:39)	172.5
3-5 * anti--CD3 (SEQ ID NO.:30)	265	4-7 * anti--CD3 (SEQ ID NO.:42)	37
Anti--CD3 * 4-7 (SEQ ID NO.:2)	112.5	4-7 * anti--CD3 (SEQ ID NO.:42)	37
Anti--CD3 * 4-7 (SEQ ID NO.:2)	112.5	Anti--CD3Cys-Ser * 4-7 (SEQ ID NO.:8)	140
3-1 * anti--CD3 (SEQ ID NO.:36)	265	Anti--CD3Cys-Ser * 4-7 (SEQ ID NO.:8)	140
3-1 * anti--CD3 (SEQ ID NO.:36)	265	5-10 * anti--CD3 (SEQ ID NO.:44)	400
Anti--CD3 * 5-10 (SEQ ID NO.:4)	195	5-10 * anti--CD3 (SEQ ID NO.:44)	400

(Amersham) carries out another time high resolving power sun example displacement chromatography on 20mM MES pH of buffer 5.5 equilibrated MiniS  posts.Before adding pillar, sample is diluted with 1: 3 with same buffer.Use comprises the gradient of the level pad of 1M NaCl: 0-30% elution of bound protein in 60 column volumes.1M NaCl wash-out remaining protein (Fig. 7) with 3 times of column volumes.

Comprising that immobilized metal affinity chromatography (IMAC) (Fig. 5) with in the two-step purifying process of gel-filtration (Fig. 6) separates EpCAM dual specific strand construct protein.As determining that by the gel-filtration among the PBS primary product molecular weight is 52kDa under natural condition.

Under reductive condition, analyze the dual specificity protein matter of purifying with the SDS PAGE of prefabricated 4-12%Bis Tris gel (Invitrogen).Scheme according to manufacturers is carried out specimen preparation and application.Use MultiMark  protein standard (Invitrogen) determining molecular weight.Use colloid Coomassie (Invitrogen method) to dye glue.The purity of institute's isolated protein shows＞95% (Fig. 8 A).Use Optitran BA-S83  film and Invitrogen Blot Module to carry out the Western trace according to the scheme of manufacturers.Used antibody is Penta His (Qiagen) and with alkaline phosphatase (AP) the anti-mouse Ig of labelled goat (Sigma), and chromogenic substrate solution is BCIP/NBT liquid (Sigma).Can specific detection EpCAM dual specificity protein matter (Fig. 8 B) by the Western trace.The main band at 52kD place among the corresponding SDS PAGE of main signal, the bispecific molecule of its corresponding purifying.

Embodiment 3. comprises the cytotoxic assay of the construct of anti--CD3 and anti--EpCAM

In order to test the biological activity that comprises anti--EpCAM and anti--CD3 construct, carried out cytotoxicity test based on FACS.

For the cytotoxicity test, use epithelial cell adhesion molecule (EpCAM) transfection from U.S. typical cells culture collection center (ATCC, Manassas, Chinese hamster ovary celI USA).Come from this cells transfected clone (being called the CHO-EpCAM cell) and be used for experiment.Use PBS washing CHO-EpCAM (1.5 * 10 ⁷) cell 2 times removes serum and hatch with PKH26 dyestuff (Sigma-Aldrich Co.) according to the explanation of manufacturers.Twice of RPMI/10%FCS washed cell in dyeing back.

Counting cells also mixes with CB15 effector cell.CD4 positive T cell clone CB15 is provided by doctor Fickenscher (University of Erlangen/Nuernberg, Germany).Suggestion culturing cell as supplier.The gained cell suspending liquid contains 400,000 target cells and 2 * 10 for every milliliter ⁶Individual effector cell.50 μ l mixtures are used in every hole in 96 hole circle base plates.

Join cell suspending liquid with RPMI/10%FCS dilution antibody to desired concn and with 50 these solution of μ l.Standard reaction is at 37 ℃/5%CO ₂In hatched 16 hours.Adding iodate third ingot to final concentration is 1 μ g/ml.Incubated at room is passed through the facs analysis cell after 10 minutes.PKH26 fluorescence is used for the positive identification of target cell.Cytotoxic assay is the ratio of PI positive cell to whole target cells.As (GraphPad Software Inc., San Diego USA) determine the common R of S shape dose response curve by Prism software ²Value＞0.97 (Fig. 9 and 10).EC by routine analyzer calculating ₅₀Value is used for biological activity relatively.All constructs of the present invention are than the cytotoxicity high at least 50 times (maximum EC50-value 169pg/ml) of prior art construct M79 * anti--CD3 (8628pg/ml).

Embodiment 4. passes through BIAcore ^TM2000 determine to comprise anti--EpCAM and the binding affinity of anti--CD3 construct to EpCAM

In order to show the outstanding binding affinity of construct of the present invention, measured construct and prior art anti--the KD value of EpCAM construct (M79) * anti--CD3.

Use surface plasma body resonant vibration at BIAcore ^TM2000, and Biacore AB (Uppsala, Sweden) enterprising action mechanics is in conjunction with experiment, flow velocity is 5 μ L/ minutes and with HBS-EP (0.01M HEPES, pH7.4,0.15M NaCl, 3mM EDTA, 0.005% tensio-active agent P20) it is 25 ℃ as running buffer and temperature.(residue 17-265) is fixed in the flow cell 2-4 on the CM5 sensor chip with the antigenic extracellular domain of EpCAM.Inject 80 μ L 0.1M N-Hydroxysuccinimide sodium, 0.4M N-ethyl-N ' (3-dimethyl amine propyl group)-carbodiimide (NHS/EDC) activation chip surface.Be dissolved in the 60 μ g/mL EpCAM coupled antigens of 0.01M sodium-acetate pH4.7 by manual injection.

The amount of adjustment manual injection number of times is fixed on the antigen of different densities on the flow cell 2-4.Flow cell 1 is empty and the flow cell the most highdensity EpCAM of 2 usefulness (4100RU) wrap quilt.Flow cell 3 usefulness are fixed on  (974RU) the bag quilt of the antigenic amount on the flow cell 2 and wrap by flow cell 4 with least density Ep-CAM antigen (265RU).Inject the activating surface of 85 μ L 1M thanomins sealings sensor chip and with chip equilibrate overnight in the HBS-EP of 5 μ L/ minute constant currents.

Inject 10 μ L concentration ranges and be the protein soln of 4 μ M-0.07 μ M and monitor and dissociate 100 seconds to measure the binding kinetics of dual specific construct.Protein buffer is in HBS-EP.Use BIAevalution ^TMThe software fitting data uses 1:1 bright wrong you (Langmuir) to determine to dissociate in conjunction with equation (1,2) and the rate constant of binding kinetics.Here A is the concentration and the B[0 of injection of analytes] be Rmax.

dB/dt＝-(ka*[A]*[B]-kd*[AB]) (1)

dAB/dt＝-(ka*[A]*[B]-kd*[AB]) (2)

In 4 kinds of concentration of the every kind of dual specific construct of analyzing, determine the kinetics binding curve.The independent match of raw data produces dissociates and association rate constant, and they are used for calculated equilibrium dissociation constant (KD).The KD value calculated is non-bias for concentration, shows that data analysis is reliable.The mean value and the standard deviation of the independent dissociation constant of determining are summarized in table 3.

The dual specific construct of being analyzed is bonded on the Ep-CAM antigen that is fixed on chip surface in the avidity scope of clearly determining.The standard deviation of the mean dissociation constant that calculates as expected.

Table 3: the dual specific construct is in conjunction with the dissociation constant of EpCAM

	KD(M)
	KD(M)	M79 * anti--CD3 (contrast)	4.0×10 ^-6
4-1 * anti--CD3 (SEQ ID NO.:39)	2.5×10 ^-7	M79 * anti--CD3 (contrast)	4.0×10 ^-6
4-1 * anti--CD3 (SEQ ID NO.:39)	2.5×10 ^-7	3-5 * anti--CD3 (SEQ ID NO.:30)	2.3×10 ^-7

Prior art is anti--and the KD of EpCAM * anti--CD3 construct M79 * CD3 is 4.0 * 10 ^-6M, and be that the KD of construct of the present invention is 2.3 * 10 astoundingly ^-7-2.5 * 10 ^-7In the M scope.Therefore, construct of the present invention has strong binding affinity more than 15 times than prior art construct.

Embodiment 5. compares the cellular cytoxicity activity of construct of the present invention and prior art construct

For the construct that relatively has the NXD motif and the biological activity of conventional M79 * CD3 and HD70 * CD3 construct, carried out following cytotoxic assay.

KatoIII cell (ATCC HTB-103) is as target cell and at 37 ℃, 5%CO ₂Grow in the humidification incubator among the RPMI that adds 10% foetal calf serum.Trypsin treatment branch with 0.25% converges culture, counting and detect cell viability by trypanblue exclusion method on Neubauer chamber slide glass.Only＞95% the culture of cell viability is used for cytotoxic assay.(Sigma-Aldrich GmbH, Germany PKH26-GL) use PKH26 fluorescent screen dyestuff to dye target cell according to manufacturers's handbook.With the RPMI perfect medium cell count is adjusted to 8 * 10 ⁵Cell/ml.

End user's peripheral blood lymphocytes (PBMC) but action effect cell and use phenanthrene density gradient centrifugation from the healthy donors separating periphery blood monocytic cell and subsequently with the centrifugal removal thrombocyte of 100xg.Precipitation is resuspended in the erythrocyte splitting damping fluid of 10 times of volumes and incubated at room 10 minutes.Add PBS and stop scission reaction.PBMC is resuspended in RPMI 1640 to be cultivated fully and concentrates and cell count is adjusted into 8 * 10 ⁶Individual cell/ml.

Equal-volume target cell and effector cell's suspension are mixed and 50 these suspension of μ l are transferred in each hole of 96 hole circle base plates, add 50 μ l EpCAM bi-specific antibody serial dilutions or as the RPMI perfect medium of negative control.At 37 ℃, 5%CO ₂The humidification incubator in hatched plate 16-20 hour.Added 50 μ l iodate, third ingot to final concentration and be 1 μ g/ml and incubated at room 15 minutes.By flow cytometry (FACSCalibur, Becton Dickinson) analytic sample.Counting 2 * 10 ⁴Individual incident.

Also measure the cytotoxicity of this cell colony subsequently by its PKH26 fluorescent marker evaluation target cell.Viable cell is separated with dead cell and the percentage ratio of dead target cell is used to measure cytotoxicity by iodate third ingot dyeing.Mean value is made curve to the logarithm of bi-specific antibody concentration, produces dose response curve (Figure 11 B).Obtain corresponding EC after using GraphPad Prism software with the data nonlinear fitting ₅₀Value.

Cellular cytoxicity activity and conventional construct M79 * anti--CD3 and HD70-* anti--CD3 comparison (Figure 11 B) that will have the construct (SEQ ID NO.:36,44,2 and 18) of NXD motif.Have M79, the 3-1 of HD70 and 3B10,5-10,4-7, the sequence alignment in the VH chain CDR3 district of 3-5 and 4-1 is shown in Figure 11 A.3-1 only, 5-10,4-7,3-5 is different with the length that 4-1 has NXD motif and CDR3 district.Seen in Figure 11 A, 3-1,4-1 and 5-10 have 10 amino acid whose CDR-H3 districts, 3-5 and 4-7 have 14 amino acid whose CDR-H3 districts, and prior art M79 has 8 amino acid whose CDR-H3 districts, and 3B10 has 6 amino acid whose CDR-H3 districts and HD70 has 18 amino acid whose CDR-H3 districts.

SEQ ID NO.:36,44,2 and 18 show and to compare with HD70 with conventional M79 and to have obviously better biological activity (being in a ratio of 2250pg/ml and still less with 71460 and 11327pg/ml of prior art construct respectively), proved the beneficial effect of construct of the present invention.

Sequence table

＜110〉Micromet AG

＜120〉comprise the pharmaceutical composition of the special construct of EpCAM

<130>H1656PCT

<150>EP 03012133.9

<151>2003-05-31

<150>EP 03012134.7

<151>2003-05-31

<160>101

＜170〉PatentIn version 3 .1

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＜213〉artificial sequence

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<223>CD3 VHVL aL×4-7 VHVL

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gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcagtc 360

gaaggtggaa gtggaggttc tggtggaagt ggaggttcag gtggagtcga cgacattcag 420

ctgacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 480

agagccagtt caagtgtaag ttacatgaac tggtaccagc agaagtcagg cacctccccc 540

aaaagatgga tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600

agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc 660

acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg gaccaagctg 720

gagctgaaat ccggaggtgg tggatccgag gtgcagctgc tcgagcagtc tggagctgag 780

ctggcgaggc ctggggcttc agtgaagctg tcctgcaagg cttctggcta caccttcaca 840

aactatggtt taagctgggt gaagcagagg cctggacagg tccttgagtg gattggagag 900

gtttatccta gaattggtaa tgcttactac aatgagaagt tcaagggcaa ggccacactg 960

actgcagaca aatcctccag cacagcgtcc atggagctcc gcagcctgac ctctgaggac 1020

tctgcggtct atttctgtgc aagacgggga tcctacgata ctaactacga ctggtacttc 1080

gatgtctggg gccaagggac cacggtcacc gtctcctcag gtggtggtgg ttctggcggc 1140

ggcggctccg gtggtggtgg ttctgagctc gtgatgaccc agactccact ctccctgcct 1200

gtcagtcttg gagatcaagc ctccatctct tgcagatcta gtcagagcct tgtacacagt 1260

aatggaaaca cctatttaca ttggtacctg cagaagccag gccagtctcc aaagctcctg 1320

atctacaaag tttccaaccg attttctggg gtcccagaca ggttcagtgg cagtggatca 1380

gggacagatt tcacactcaa gatcagcaga gtggaggctg aggatctggg agtttatttc 1440

tgctctcaaa gtacacatgt tccgtacacg ttcggagggg ggaccaagct tgagatcaaa 1500

catcatcacc atcatcatta g 1521

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＜213〉artificial sequence

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Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln

245 250 255

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Leu Ser Cys

260 265 270

Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Leu Ser Trp Val Lys

275 280 285

Gln Arg Pro Gly Gln Val Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg

290 295 300

Ile Gly Asn Ala Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu

305 310 315 320

Thr Ala Asp Lys Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu

325 330 335

Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr

340 345 350

Asp Thr Asn Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr

355 360 365

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

370 375 380

Gly Gly Gly Ser Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro

385 390 395 400

Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

405 410 415

Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys

420 425 430

Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe

435 440 445

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

450 455 460

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe

465 470 475 480

Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys

485 490 495

Leu Glu Ile Lys His His His His His His

500 505

<210>3

<211>1512

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3VHVL aL×5-10 VHVL

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gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcagtc 360

gaaggtggaa gtggaggttc tggtggaagt ggaggttcag gtggagtcga cgacattcag 420

ctgacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 480

agagccagtt caagtgtaag ttacatgaac tggtaccagc agaagtcagg cacctccccc 540

aaaagatgga tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600

agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc 660

acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg gaccaagctg 720

gagctgaaat ccggaggtgg tggatccgag gtgcagctgc tcgagcagtc tggagctgag 780

ctggtaaggc ctgggacttc agtgaagata tcctgcaagg cttctggata cgccttcact 840

aactactggc taggttgggt aaagcagagg cctggacatg gacttgagtg gattggagat 900

attttccctg gaagtggtaa tatccactac aatgagaagt tcaagggcaa agccacactg 960

actgcagaca aatcttcgag cacagcctat atgcagctca gtagcctgac atttgaggac 1020

tctgctgtct atttctgtgc aagactgagg aactgggacg agcctatgga ctactggggc 1080

caagggacca cggtcaccgt ctcctcaggt ggtggtggtt ctggcggcgg cggctccggt 1140

ggtggtggtt ctgagctcgt gatgacacag tctccatcct ccctgactgt gacagcagga 1200

gagaaggtca ctatgagctg caagtccagt cagagtctgt taaacagtgg aaatcaaaag 1260

aactacttga cctggtacca gcagaaacca gggcagcctc ctaaactgtt gatctactgg 1320

gcatccacta gggaatctgg ggtccctgat cgcttcacag gcagtggatc tggaacagat 1380

ttcactctca ccatcagcag tgtgcaggct gaagacctgg cagtttatta ctgtcagaat 1440

gattatagtt atccgctcac gttcggtgct gggaccaagc ttgagatcaa acatcatcac 1500

catcatcatt ag 1512

<210>4

<211>503

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3VHVL aL×5-10 VHVL

<400>4

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln

245 250 255

Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Ile Ser Cys

260 265 270

Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys

275 280 285

Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly

290 295 300

Ser Gly Asn Ile His Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu

305 310 315 320

Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

325 330 335

Thr Phe Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp

340 345 350

Asp Glu Pro Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

355 360 365

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

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

385 390 395 400

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

405 410 415

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

420 425 430

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

435 440 445

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

450 455 460

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

465 470 475 480

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

485 490 495

Lys His His His His His His

500

<210>5

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CD3VH BsrGI primer

<400>5

aggtgtacac tccgatatca aactgcagca g 31

<210>6

<211>25

<212>DNA

＜213〉artificial sequence

<220>

＜223〉CD3 VL BspEI primer

<400>6

aatccggatt tcagctccag cttgg 25

<210>7

<211>1521

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3VHVL aL Ser×4-7 VHVL

<400>7

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actcccttga ctactggggc caaggcacca ctctcacagt ctcctcagtc 360

gaaggtggaa gtggaggttc tggtggaagt ggaggttcag gtggagtcga cgacattcag 420

ctgacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 480

agagccagtt caagtgtaag ttacatgaac tggtaccagc agaagtcagg cacctccccc 540

aaaagatgga tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600

agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc 660

acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg gaccaagctg 720

gagctgaaat ccggaggtgg tggatccgag gtgcagctgc tcgagcagtc tggagctgag 780

ctggcgaggc ctggggcttc agtgaagctg tcctgcaagg cttctggcta caccttcaca 840

aactatggtt taagctgggt gaagcagagg cctggacagg tccttgagtg gattggagag 900

gtttatccta gaattggtaa tgcttactac aatgagaagt tcaagggcaa ggccacactg 960

actgcagaca aatcctccag cacagcgtcc atggagctcc gcagcctgac ctctgaggac 1020

tctgcggtct atttctgtgc aagacgggga tcctacgata ctaactacga ctggtacttc 1080

gatgtctggg gccaagggac cacggtcacc gtctcctcag gtggtggtgg ttctggcggc 1140

ggcggctccg gtggtggtgg ttctgagctc gtgatgaccc agactccact ctccctgcct 1200

gtcagtcttg gagatcaagc ctccatctct tgcagatcta gtcagagcct tgtacacagt 1260

aatggaaaca cctatttaca ttggtacctg cagaagccag gccagtctcc aaagctcctg 1320

atctacaaag tttccaaccg attttctggg gtcccagaca ggttcagtgg cagtggatca 1380

gggacagatt tcacactcaa gatcagcaga gtggaggctg aggatctggg agtttatttc 1440

tgctctcaaa gtacacatgt tccgtacacg ttcggagggg ggaccaagct tgagatcaaa 1500

catcatcacc atcatcatta g 1521

<210>8

<211>506

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3VHVL aL Ser×4-7 VHVL

<400>8

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln

245 250 255

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Leu Ser Cys

260 265 270

Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Leu Ser Trp Val Lys

275 280 285

Gln Arg Pro Gly Gln Val Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg

290 295 300

Ile Gly Asn Ala Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu

305 310 315 320

Thr Ala Asp Lys Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu

325 330 335

Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr

340 345 350

Asp Thr Asn Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr

355 360 365

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

370 375 380

Gly Gly Gly Ser Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro

385 390 395 400

Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

405 410 415

Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys

420 425 430

Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe

435 440 445

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

450 455 460

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe

465 470 475 480

Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys

485 490 495

Leu Glu Ile Lys His His His His His His

500 505

<210>9

<211>1512

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL Ser×5-10 VHVL

<400>9

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actcccttga ctactggggc caaggcacca ctctcacagt ctcctcagtc 360

gaaggtggaa gtggaggttc tggtggaagt ggaggttcag gtggagtcga cgacattcag 420

ctgacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 480

agagccagtt caagtgtaag ttacatgaac tggtaccagc agaagtcagg cacctccccc 540

aaaagatgga tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600

agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc 660

acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg gaccaagctg 720

gagctgaaat ccggaggtgg tggatccgag gtgcagctgc tcgagcagtc tggagctgag 780

ctggtaaggc ctgggacttc agtgaagata tcctgcaagg cttctggata cgccttcact 840

aactactggc taggttgggt aaagcagagg cctggacatg gacttgagtg gattggagat 900

attttccctg gaagtggtaa tatccactac aatgagaagt tcaagggcaa agccacactg 960

actgcagaca aatcttcgag cacagcctat atgcagctca gtagcctgac atttgaggac 1020

tctgctgtct atttctgtgc aagactgagg aactgggacg agcctatgga ctactggggc 1080

caagggacca cggtcaccgt ctcctcaggt ggtggtggtt ctggcggcgg cggctccggt 1140

ggtggtggtt ctgagctcgt gatgacacag tctccatcct ccctgactgt gacagcagga 1200

gagaaggtca ctatgagctg caagtccagt cagagtctgt taaacagtgg aaatcaaaag 1260

aactacttga cctggtacca gcagaaacca gggcagcctc ctaaactgtt gatctactgg 1320

gcatccacta gggaatctgg ggtccctgat cgcttcacag gcagtggatc tggaacagat 1380

ttcactctca ccatcagcag tgtgcaggct gaagacctgg cagtttatta ctgtcagaat 1440

gattatagtt atccgctcac gttcggtgct gggaccaagc ttgagatcaa acatcatcac 1500

catcatcatt ag 1512

<210>10

<211>503

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL Ser×5-10 VHVL

<400>10

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Pha

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln

245 250 255

Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Ile Ser Cys

260 265 270

Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys

275 280 285

Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly

290 295 300

Ser Gly Asn Ile His Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu

305 310 315 320

Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

325 330 335

Thr Phe Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp

340 345 350

Asp Glu Pro Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

355 360 365

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

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

385 390 395 400

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

405 410 415

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

420 425 430

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

435 440 445

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

450 455 460

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

465 470 475 480

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

485 490 495

Lys His His His His His His

500

<210>11

<211>1485

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×3-1 VHVL

<400>11

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcaggt 360

ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca gctgacccag 420

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagagccagt 480

tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc caaaagatgg 540

atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg cagtgggtct 600

gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc cacttattac 660

tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct ggagctgaaa 720

tccggaggtg gtggatccga ggtgcagctg ctcgagcagt ctggagctga gctggtgaaa 780

cctggggcct cagtgaagat atcctgcaag gcttctggat acgccttcac taactactgg 840

ctaggttggg taaagcagag gcctggacat ggacttgagt ggattggaga tcttttccct 900

ggaagtggta atactcacta caatgagagg ttcaggggca aagccacact gactgcagac 960

aaatcctcga gcacagcctt tatgcagctc agtagcctga catctgagga ctctgctgtc 1020

tatttctgtg caagattgag gaactgggac gaggctatgg actactgggg ccaagggacc 1080

acggtcaccg tctcctcagg tggtggtggt tctggcggcg gcggctccgg tggtggtggt 1140

tctgagctcg tcatgaccca gtctccatct tatcttgctg catctcctgg agaaaccatt 1200

actattaatt gcagggcaag taagagcatt agcaaatatt tagcctggta tcaagagaaa 1260

cctgggaaaa ctaataagct tcttatctac tctggatcca ctttgcaatc tggaattcca 1320

tcaaggttca gtggcagtgg atctggtaca gatttcactc tcaccatcag tagcctggag 1380

cctgaagatt ttgcaatgta ttactgtcaa cagcataatg aatatccgta cacgttcgga 1440

ggggggacca agcttgagat caaacatcat caccatcatc attag 1485

<210>12

<211>494

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×3-1 VHVL

<400>12

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile

130 135 140

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser

145 150 155 160

Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser

165 170 175

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro

180 185 190

Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

195 200 205

Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

210 215 220

Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

225 230 235 240

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala

245 250 255

Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser

260 265 270

Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro

275 280 285

Gly His Gly Leu Glu Trp Ile Gly Asp Leu Phe Pro Gly Ser Gly Asn

290 295 300

Thr His Tyr Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp

305 310 315 320

Lys Ser Ser Ser Thr Ala Phe Met Gln Leu Ser Ser Leu Thr Ser Glu

325 330 335

Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Ala

340 345 350

Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly

355 360 365

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val

370 375 380

Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly Glu Thr Ile

385 390 395 400

Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr Leu Ala Trp

405 410 415

Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly

420 425 430

Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser

435 440 445

Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe

450 455 460

Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr Thr Phe Gly

465 470 475 480

Gly Gly Thr Lys Leu Glu Ile Lys His His His His His His

485 490

<210>13

<211>1512

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×4-7 VHVL

<400>13

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcaggt 360

ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca gctgacccag 420

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagagccagt 480

tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc caaaagatgg 540

atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg cagtgggtct 600

gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc cacttattac 660

tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct ggagctgaaa 720

tccggaggtg gtggatccga ggtgcagctg ctcgagcagt ctggagctga gctggcgagg 780

cctggggctt cagtgaagct gtcctgcaag gcttctggct acaccttcac aaactatggt 840

ttaagctggg tgaagcagag gcctggacag gtccttgagt ggattggaga ggtttatcct 900

agaattggta atgcttacta caatgagaag ttcaagggca aggccacact gactgcagac 960

aaatcctcca gcacagcgtc catggagctc cgcagcctga cctctgagga ctctgcggtc 1020

tatttctgtg caagacgggg atcctacgat actaactacg actggtactt cgatgtctgg 1080

ggccaaggga ccacggtcac cgtctcctca ggtggtggtg gttctggcgg cggcggctcc 1140

ggtggtggtg gttctgagct cgtgatgacc cagactccac tctccctgcc tgtcagtctt 1200

ggagatcaag cctccatctc ttgcagatct agtcagagcc ttgtacacag taatggaaac 1260

acctatttac attggtacct gcagaagcca ggccagtctc caaagctcct gatctacaaa 1320

gtttccaacc gattttctgg ggtcccagac aggttcagtg gcagtggatc agggacagat 1380

ttcacactca agatcagcag agtggaggct gaggatctgg gagtttattt ctgctctcaa 1440

agtacacatg ttccgtacac gttcggaggg gggaccaagc ttgagatcaa acatcatcac 1500

catcatcatt ag 1512

<210>14

<211>503

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×4-7 VHVL

<400>14

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile

130 135 140

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser

145 150 155 160

Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser

165 170 175

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro

180 185 190

Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

195 200 205

Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

210 215 220

Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

225 230 235 240

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala

245 250 255

Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser

260 265 270

Gly Tyr Thr Phe Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro

275 280 285

Gly Gln Val Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn

290 295 300

Ala Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp

305 310 315 320

Lys Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu

325 330 335

Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn

340 345 350

Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

355 360 365

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu

385 390 395 400

Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His

405 410 415

Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

420 425 430

Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

435 440 445

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

450 455 460

Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln

465 470 475 480

Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

485 490 495

Lys His His His His His His

500

<210>15

<211>1512

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×4-7 VLVH

<400>15

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcaggt 360

ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca gctgacccag 420

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagagccagt 480

tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc caaaagatgg 540

atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg cagtgggtct 600

gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc cacttattac 660

tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct ggagctgaaa 720

tccggaggtg gtggatccga gctcgtgatg acccagactc cactctccct gcctgtcagt 780

cttggagatc aagcctccat ctcttgcaga tctagtcaga gccttgtaca cagtaatgga 840

aacacctatt tacattggta cctgcagaag ccaggccagt ctccaaagct cctgatctac 900

aaagtttcca accgattttc tggggtccca gacaggttca gtggcagtgg atcagggaca 960

gatttcacac tcaagatcag cagagtggag gctgaggatc tgggagttta tttctgctct 1020

caaagtacac atgttccgta cacgttcgga ggggggacca agcttgagat caaaggtggt 1080

ggtggttctg gcggcggcgg ctccggtggt ggtggttctg aggtgcagct gctcgagcag 1140

tctggagctg agctggcgag gcctggggct tcagtgaagc tgtcctgcaa ggcttctggc 1200

tacaccttca caaactatgg tttaagctgg gtgaagcaga ggcctggaca ggtccttgag 1260

tggattggag aggtttatcc tagaattggt aatgcttact acaatgagaa gttcaagggc 1320

aaggccacac tgactgcaga caaatcctcc agcacagcgt ccatggagct ccgcagcctg 1380

acctctgagg actctgcggt ctatttctgt gcaagacggg gatcctacga tactaactac 1440

gactggtact tcgatgtctg gggccaaggg accacggtca ccgtctcctc acatcatcac 1500

catcatcatt ag 1512

<210>16

<211>503

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×4-7 VLVH

<400>16

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile

130 135 140

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser

145 150 155 160

Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser

165 170 175

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro

180 185 190

Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

195 200 205

Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

210 215 220

Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

225 230 235 240

Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln Thr Pro Leu Ser

245 250 255

Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser

260 265 270

Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu

275 280 285

Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn

290 295 300

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

305 310 315 320

Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val

325 330 335

Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly

340 345 350

Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

355 360 365

Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu

370 375 380

Leu Ala Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly

385 390 395 400

Tyr Thr Phe Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro Gly

405 410 415

Gln Val Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala

420 425 430

Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys

435 440 445

Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp

450 455 460

Ser Ala Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr

465 470 475 480

Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser

485 490 495

Ser His His His His His His

500

<210>17

<211>1503

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×5-10 VHVL

<400>17

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcaggt 360

ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca gctgacccag 420

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagagccagt 480

tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc caaaagatgg 540

atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg cagtgggtct 600

gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc cacttattac 660

tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct ggagctgaaa 720

tccggaggtg gtggatccga ggtgcagctg ctcgagcagt ctggagctga gctggtaagg 780

cctgggactt cagtgaagat atcctgcaag gcttctggat acgccttcac taactactgg 840

ctaggttggg taaagcagag gcctggacat ggacttgagt ggattggaga tattttccct 900

ggaagtggta atatccacta caatgagaag ttcaagggca aagccacact gactgcagac 960

aaatcttcga gcacagccta tatgcagctc agtagcctga catttgagga ctctgctgtc 1020

tatttctgtg caagactgag gaactgggac gagcctatgg actactgggg ccaagggacc 1080

acggtcaccg tctcctcagg tggtggtggt tctggcggcg gcggctccgg tggtggtggt 1140

tctgagctcg tgatgacaca gtctccatcc tccctgactg tgacagcagg agagaaggtc 1200

actatgagct gcaagtccag tcagagtctg ttaaacagtg gaaatcaaaa gaactacttg 1260

acctggtacc agcagaaacc agggcagcct cctaaactgt tgatctactg ggcatccact 1320

agggaatctg gggtccctga tcgcttcaca ggcagtggat ctggaacaga tttcactctc 1380

accatcagca gtgtgcaggc tgaagacctg gcagtttatt actgtcagaa tgattatagt 1440

tatccgctca cgttcggtgc tgggaccaag cttgagatca aacatcatca ccatcatcat 1500

tag 1503

<210>18

<211>500

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×5-10 VHVL

<400>18

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile

130 135 140

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser

145 150 155 160

Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser

165 170 175

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro

180 185 190

Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

195 200 205

Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

210 215 220

Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

225 230 235 240

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala

245 250 255

Glu Leu Val Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser

260 265 270

Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro

275 280 285

Gly His Gly Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn

290 295 300

Ile His Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp

305 310 315 320

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu

325 330 335

Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro

340 345 350

Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly

355 360 365

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val

370 375 380

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

385 390 395 400

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

405 410 415

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

420 425 430

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

435 440 445

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

450 455 460

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

465 470 475 480

Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys His His

485 490 495

His His His His

500

<210>19

<211>1503

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×5-10 VLVH

<400>19

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcaggt 360

ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca gctgacccag 420

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagagccagt 480

tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc caaaagatgg 540

atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg cagtgggtct 600

gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc cacttattac 660

tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct ggagctgaaa 720

tccggaggtg gtggatccga gctcgtgatg acacagtctc catcctccct gactgtgaca 780

gcaggagaga aggtcactat gagctgcaag tccagtcaga gtctgttaaa cagtggaaat 840

caaaagaact acttgacctg gtaccagcag aaaccagggc agcctcctaa actgttgatc 900

tactgggcat ccactaggga atctggggtc cctgatcgct tcacaggcag tggatctgga 960

acagatttca ctctcaccat cagcagtgtg caggctgaag acctggcagt ttattactgt 1020

cagaatgatt atagttatcc gctcacgttc ggtgctggga ccaagcttga gatcaaaggt 1080

ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgaggtgca gctgctcgag 1140

cagtctggag ctgagctggt aaggcctggg acttcagtga agatatcctg caaggcttct 1200

ggatacgcct tcactaacta ctggctaggt tgggtaaagc agaggcctgg acatggactt 1260

gagtggattg gagatatttt ccctggaagt ggtaatatcc actacaatga gaagttcaag 1320

ggcaaagcca cactgactgc agacaaatct tcgagcacag cctatatgca gctcagtagc 1380

ctgacatttg aggactctgc tgtctatttc tgtgcaagac tgaggaactg ggacgagcct 1440

atggactact ggggccaagg gaccacggtc accgtctcct cacatcatca ccatcatcat 1500

tag 1503

<210>20

<211>500

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×5-10 VLVH

<400>20

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile

130 135 140

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser

145 150 155 160

Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser

165 170 175

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro

180 185 190

Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

195 200 205

Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

210 215 220

Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

225 230 235 240

Ser Gly Gly Gly Gly Ser Glu Leu Val Met Thr Gln Ser Pro Ser Ser

245 250 255

Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser

260 265 270

Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr

275 280 285

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

290 295 300

Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly

305 310 315 320

Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala

325 330 335

Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala

340 345 350

Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

355 360 365

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala

370 375 380

Glu Leu Val Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser

385 390 395 400

Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro

405 410 415

Gly His Gly Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn

420 425 430

Ile His Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp

435 440 445

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu

450 455 460

Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro

465 470 475 480

Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser His His

485 490 495

His His His His

500

<210>21

<211>57

<212>DNA

＜213〉artificial sequence

<220>

＜223〉3 ' CD3 VH GS15 primer

<400>21

ggagccgccg ccgccagaac caccaccacc tgaggagact gtgagagtgg tgccttg 57

<210>22

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉5 ' CD3 VL GS15 primer

<400>22

ggcggcggcg gctccggtgg tggtggttct gacattcagc tgacccagtc tcc 53

<210>23

<211>51

<212>DNA

＜213〉artificial sequence

<220>

<223>4-7 VH GS15 FOR

<400>23

ggcggcggcg gctccggtgg tggtggttct gaggtgcagc tgctcgagca g 51

<210>24

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉4-7 VH SalI REV primer

<400>24

ttttaagtcg acctaatgat gatgatgatg atgtgaggag acggtgaccg tgg 53

<210>25

<211>49

<212DNA

＜213〉artificial sequence

<220>

＜223〉5-10 VLBspEI38 primer

<400>25

ctgaaatccg gaggtggtgg atccgagctc gtgatgacac agtctccat 49

<210>26

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉5-10 VLGS15REV primer

<400>26

ggagccgccg ccgccagaac caccaccacc tttgatctca agcttggtcc cag 53

<210>27

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉5-10VH GS15 FOR primer

<400>27

ggcggcggcg gctccggtgg tggtggttct gaggtgcagc tgctcgagc 49

<210>28

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉5-10 VHSalIREV primer

<400>28

ttttaagtcg acctaatgat gatgatgatg atgtgaggag acggtgaccg tgg 53

<210>29

<211>1581

<212>DNA

＜213〉artificial sequence

<220>

＜223〉3-5 (VL-VH) * anti--CD3

<400>29

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgcg 60

cgcgagctcg tgatgaccca gactccactc tccctgcctg tcagtcttgg agatcaagcc 120

tccatctctt gcagatctag tcagagcctt gtacacagta atggaaacac ctatttacat 180

tggtacctgc agaagccagg ccagtctcca aagctcctga tctacaaagt ttccaaccga 240

ttttctgggg tcccagacag gttcagtggc agtggatcag ggacagattt cacactcaag 300

atcagcagag tggaggctga ggatctggga gtttatttct gctctcaaag tacacatgtt 360

ccgtacacgt tcggaggggg gaccaagctt gagatcaaag gtggtggtgg ttctggcggc 420

ggcggctccg gtggtggtgg ttctgaggtg cagctgctcg agcagtctgg agctgagctg 480

gtaaggcctg ggacttcagt gaagctgtcc tgcaaggctt ctggctacac cttcacaagc 540

tatggtttaa gctgggtgaa gcagagaact ggacagggcc ttgagtggat tggagaggtt 600

tatcctagaa ttggtaatgc ttactacaat gagaagttca agggcaaggc cacactgact 660

gcagacaaat cctccagcac agcgtccatg gagctccgca gcctgacatc tgaggactct 720

gcggtctatt tctgtgcaag acggggatcc tacggtagta actacgactg gtacttcgat 780

gtctggggcc aagggaccac ggtcaccgtc tcctccggag gtggtggatc cgatatcaaa 840

ctgcagcagt caggggctga actggcaaga cctggggcct cagtgaagat gtcctgcaag 900

acttctggct acacctttac taggtacacg atgcactggg taaaacagag gcctggacag 960

ggtctggaat ggattggata cattaatcct agccgtggtt atactaatta caatcagaag 1020

ttcaaggaca aggccacatt gactacagac aaatcctcca gcacagccta catgcaactg 1080

agcagcctga catctgagga ctctgcagtc tattactgtg caagatatta tgatgatcat 1140

tactgccttg actactgggg ccaaggcacc actctcacag tctcctcagt cgaaggtgga 1200

agtggaggtt ctggtggaag tggaggttca ggtggagtcg acgacattca gctgacccag 1260

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagagccagt 1320

tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc caaaagatgg 1380

atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg cagtgggtct 1440

gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc cacttattac 1500

tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct ggagctgaaa 1560

catcatcacc atcatcatta g 1581

<210>30

<211>526

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3-5 (VL-VH) * anti--CD3

<400>30

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Ala Arg Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu

20 25 30

Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln

35 40 45

Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg

65 70 75 80

Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr

100 105 110

Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu

145 150 155 160

Val Arg Pro Gly Thr Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Ser Tyr Gly Leu Ser Trp Val Lys Gln Arg Thr Gly Gln

180 185 190

Gly Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr

195 200 205

Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

210 215 220

Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser

225 230 235 240

Ala Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Gly Ser Asn Tyr Asp

245 250 255

Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

260 265 270

Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu

275 280 285

Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr

290 295 300

Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln

305 310 315 320

Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn

325 330 335

Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser

340 345 350

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

355 360 365

Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp

370 375 380

Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly

385 390 395 400

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile

405 410 415

Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys

420 425 430

Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp

435 440 445

Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr

450 455 460

Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser

465 470 475 480

Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala

485 490 495

Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly

500 505 510

Ala Gly Thr Lys Leu Glu Leu Lys Hi s His His His His His

515 520 525

<210>31

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Me81 primer

<400>31

ggatgcgcgc gagctcgtga tgacccagac tccactctcc 40

<210>32

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Me83 primer

<400>32

ggttctggcg gcggcggctc cggtggtggt ggttctgagg tgcagctgct cgacagtctg 60

<210>33

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Me84 primer

<400>33

gtgctccgga ggagacggtg accgtggtcc cttggcccca g 41

<210>34

<211>53

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Me90 primer

<400>34

ccggagccgc cgccgccaga accaccacca cctttgatct caagcttggt ccc 53

<210>35

<211>1548

<212>DNA

＜213〉artificial sequence

<220>

＜223〉3-1 (VLVH) * anti--CD3

<400>35

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60

ctcgtcatga cccagtctcc atcttatctt gctgcatctc ctggagaaac cattactatt 120

aattgcaggg caagtaagag cattagcaaa tatttagcct ggtatcaaga gaaacctggg 180

aaaactaata agcttcttat ctactctgga tccactttgc aatctggaat tccatcaagg 240

ttcagtggca gtggatctgg tacagatttc actctcacca tcagtagcct ggagcctgaa 300

gattttgcaa tgtattactg tcaacagcat aatgaatatc cgtacacgtt cggagggggg 360

accaagcttg agatcaaagg tggtggtggt tctggcggcg gcggctccgg tggtggtggt 420

tctgaggtgc agctgctcga gcagtctgga gctgagctgg tgaaacctgg ggcctcagtg 480

aagatatcct gcaaggcttc tggatacgcc ttcactaact actggctagg ttgggtaaag 540

cagaggcctg gacatggact tgagtggatt ggagatcttt tccctggaag tggtaatact 600

cactacaatg agaggttcag gggcaaagcc acactgactg cagacaaatc ctcgagcaca 660

gcctttatgc agctcagtag cctgacatct gaggactctg ctgtctattt ctgtgcaaga 720

ttgaggaact gggacgaggc tatggactac tggggccaag ggaccacggt caccgtctcc 780

tccggaggtg gtggatccga tatcaaactg cagcagtcag gggctgaact ggcaagacct 840

ggggcctcag tgaagatgtc ctgcaagact tctggctaca cctttactag gtacacgatg 900

cactgggtaa aacagaggcc tggacagggt ctggaatgga ttggatacat taatcctagc 960

cgtggttata ctaattacaa tcagaagttc aaggacaagg ccacattgac tacagacaaa 1020

tcctccagca cagcctacat gcaactgagc agcctgacat ctgaggactc tgcagtctat 1080

tactgtgcaa gatattatga tgatcattac tgccttgact actggggcca aggcaccact 1140

ctcacagtct cctcagtcga aggtggaagt ggaggttctg gtggaagtgg aggttcaggt 1200

ggagtcgacg acattcagct gacccagtct ccagcaatca tgtctgcatc tccaggggag 1260

aaggtcacca tgacctgcag agccagttca agtgtaagtt acatgaactg gtaccagcag 1320

aagtcaggca cctcccccaa aagatggatt tatgacacat ccaaagtggc ttctggagtc 1380

ccttatcgct tcagtggcag tgggtctggg acctcatact ctctcacaat cagcagcatg 1440

gaggctgaag atgctgccac ttattactgc caacagtgga gtagtaaccc gctcacgttc 1500

ggtgctggga ccaagctgga gctgaaacat catcaccatc atcattag 1548

<210>36

<211>515

<212>PRT

＜213〉artificial sequence

<220>

＜223〉3-1 (VLVH) * anti--CD3

<400>36

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Leu Val Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala

20 25 30

Ser Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile

35 40 45

Ser Lys Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys

50 55 60

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

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

85 90 95

Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu

100 105 110

Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln

130 135 140

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

145 150 155 160

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu

165 170 175

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

180 185 190

Leu Phe Pro Gly Ser Gly Asn Thr His Tyr Asn Glu Arg Phe Arg Gly

195 200 205

Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe Met Gln

210 215 220

Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

225 230 235 240

Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr

245 250 255

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln

260 265 270

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys

275 280 285

Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys

290 295 300

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pr0Ser

305 310 315 320

Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu

325 330 335

Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

340 345 350

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp

355 360 365

His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

370 375 380

Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

385 390 395 400

Gly Val Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala

405 410 415

Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val

420 425 430

Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg

435 440 445

Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe

450 455 460

Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met

465 470 475 480

Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn

485 490 495

Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His

500 505 510

His His His

515

<210>37

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Me91a primer

<400>37

ggattgtaca ctccgagctc gtcatgaccc agtctccatc ttatcttgct gc 52

<210>38

<211>1566

<212>DNA

＜213〉artificial sequence

<220>

＜223〉4-1 (VLVH) * anti--CD3

<400>38

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60

ctcgtgatga cacagtctcc atcctccctg agtgtgtcag caggagagaa ggtcactatg 120

agctgcaagt ccagtcagag tctgttaaac agtggaaatc aaaagaacta cttggcctgg 180

taccagcaga aaccagggca gcctcctaaa ctgttgatct acggggcatc cactagggaa 240

tctggggtcc ctgatcgctt cacaggcagt ggatctggaa cagatttcac tctcaccatc 300

agcagtgtgc aggctgaaga cctggcagtt tattactgtc agaatgatta tagttatccg 360

tacacgttcg gaggggggac caagcttgag atcaaaggtg gtggtggttc tggcggcggc 420

ggctccggtg gtggtggttc tgaggtgcag ctgctcgagc agtctggagc tgagctggta 480

aggcctggga cttcagtgaa gatatcctgc aaggcttctg gatacgcctt cactaactac 540

tggctaggtt gggttaagca gaggcctgga catggacttg aatgggttgg agatattttc 600

cctggaagtg gtaatgctca ctacaatgag aagttcaagg gcaaagccac actgactgca 660

gacaagtcct cgtacacagc ctatatgcag ctcagtagcc tgacatctga ggactctgct 720

gtctatttct gtgcaagatt gcggaactgg gacgaggcta tggactactg gggccaaggg 780

accacggtca ccgtctcctc cggaggtggt ggatccgata tcaaactgca gcagtcaggg 840

gctgaactgg caagacctgg ggcctcagtg aagatgtcct gcaagacttc tggctacacc 900

tttactaggt acacgatgca ctgggtaaaa cagaggcctg gacagggtct ggaatggatt 960

ggatacatta atcctagccg tggttatact aattacaatc agaagttcaa ggacaaggcc 1020

acattgacta cagacaaatc ctccagcaca gcctacatgc aactgagcag cctgacatct 1080

gaggactctg cagtctatta ctgtgcaaga tattatgatg atcattactg ccttgactac 1140

tggggccaag gcaccactct cacagtctcc tcagtcgaag gtggaagtgg aggttctggt 1200

ggaagtggag gttcaggtgg agtcgacgac attcagctga cccagtctcc agcaatcatg 1260

tctgcatctc caggggagaa ggtcaccatg acctgcagag ccagttcaag tgtaagttac 1320

atgaactggt accagcagaa gtcaggcacc tcccccaaaa gatggattta tgacacatcc 1380

aaagtggctt ctggagtccc ttatcgcttc agtggcagtg ggtctgggac ctcatactct 1440

ctcacaatca gcagcatgga ggctgaagat gctgccactt attactgcca acagtggagt 1500

agtaacccgc tcacgttcgg tgctgggacc aagctggagc tgaaacatca tcaccatcat 1560

cattag 1566

<210>39

<211>521

<212>PRT

＜213〉artificial sequence

<220>

＜223〉4-1 (VLVH) * anti--CD3

<400>39

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Val

20 25 30

Ser Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val

145 150 155 160

Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala

165 170 175

Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly

180 185 190

Leu Glu Trp Val Gly Asp Ile Phe Pro Gly Ser Gly Asn Ala His Tyr

195 200 205

Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser

210 215 220

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

225 230 235 240

Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

275 280 285

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

290 295 300

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

305 310 315 320

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

325 330 335

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

340 345 350

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

355 360 365

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

370 375 380

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

385 390 395 400

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

405 410 415

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

420 425 430

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

435 440 445

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

450 455 460

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

465 470 475 480

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

485 490 495

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

500 505 510

Glu Leu Lys His His His His His His

515 520

<210>40

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Me92a primer

<400>40

ggattgtaca ctccgagctc gtgatgacac agtctccatc ctcc 44

<210>41

<211>1581

<212>DNA

＜213〉artificial sequence

<220>

＜223〉4-7 (VL-VH) * anti-CD3

<400>41

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgcg 60

cgcgagctcg tgatgaccca gactccactc tccctgcctg tcagtcttgg agatcaagcc 120

tccatctctt gcagatctag tcagagcctt gtacacagta atggaaacac ctatttacat 180

tggtacctgc agaagccagg ccagtctcca aagctcctga tctacaaagt ttccaaccga 240

ttttctgggg tcccagacag gttcagtggc agtggatcag ggacagattt cacactcaag 300

atcagcagag tggaggctga ggatctggga gtttatttct gctctcaaag tacacatgtt 360

ccgtacacgt tcggaggggg gaccaagctt gagatcaaag gtggtggtgg ttctggcggc 420

ggcggctccg gtggtggtgg ttctgaggtg cagctgctcg agcagtctgg agctgagctg 480

gcgaggcctg gggcttcagt gaagctgtcc tgcaaggctt ctggctacac cttcacaaac 540

tatggtttaa gctgggtgaa gcagaggcct ggacaggtcc ttgagtggat tggagaggtt 600

tatcctagaa ttggtaatgc ttactacaat gagaagttca agggcaaggc cacactgact 660

gcagacaaat cctccagcac agcgtccatg gagctccgca gcctgacctc tgaggactct 720

gcggtctatt tctgtgcaag acggggatcc tacgatacta actacgactg gtacttcgat 780

gtctggggcc aagggaccac ggtcaccgtc tcctccggag gtggtggatc cgatatcaaa 840

ctgcagcagt caggggctga actggcaaga cctggggcct cagtgaagat gtcctgcaag 900

acttctggct acacctttac taggtacacg atgcactggg taaaacagag gcctggacag 960

ggtctggaat ggattggata cattaatcct agccgtggtt atactaatta caatcagaag 1020

ttcaaggaca aggccacatt gactacagac aaatcctcca gcacagccta catgcaactg 1080

agcagcctga catctgagga ctctgcagtc tattactgtg caagatatta tgatgatcat 1140

tactgccttg actactgggg ccaaggcacc actctcacag tctcctcagt cgaaggtgga 1200

agtggaggtt ctggtggaag tggaggttca ggtggagtcg acgacattca gctgacccag 1260

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagagccagt 1320

tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc caaaagatgg 1380

atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg cagtgggtct 1440

gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc cacttattac 1500

tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct ggagctgaaa 1560

catcatcacc atcatcatta g 1581

<210>42

<211>526

<212>PRT

＜213〉artificial sequence

<220>

＜223〉4-7 (VL-VH) * anti-CD3

<400>42

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Ala Arg Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu

20 25 30

Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln

35 40 45

Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln

50 55 60

Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg

65 70 75 80

Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr

100 105 110

Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu

145 150 155 160

Ala Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Asn Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro Gly Gln

180 185 190

Val Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr

195 200 205

Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser

210 215 220

Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser

225 230 235 240

Ala Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp

245 250 255

Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

260 265 270

Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu

275 280 285

Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr

290 295 300

Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln

305 310 315 320

Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn

325 330 335

Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser

340 345 350

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

355 360 365

Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp

370 375 380

Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly

385 390 395 400

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile

405 410 415

Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys

420 425 430

Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp

435 440 445

Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr

450 455 460

Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser

465 470 475 480

Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala

485 490 495

Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly

500 505 510

Ala Gly Thr Lys Leu Glu Leu Lys His His His His His His

515 520 525

<210>43

<211>1566

<212>DNA

＜213〉artificial sequence

<220>

＜223〉5-10 (VLVH) * anti--CD3

<400>43

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggtgt acactccgag 60

ctcgtgatga cacagtctcc atcctccctg actgtgacag caggagagaa ggtcactatg 120

agctgcaagt ccagtcagag tctgttaaac agtggaaatc aaaagaacta cttgacctgg 180

taccagcaga aaccagggca gcctcctaaa ctgttgatct actgggcatc cactagggaa 240

tctggggtcc ctgatcgctt cacaggcagt ggatctggaa cagatttcac tctcaccatc 300

agcagtgtgc aggctgaaga cctggcagtt tattactgtc agaatgatta tagttatccg 360

ctcacgttcg gtgctgggac caagcttgag atcaaaggtg gtggtggttc tggcggcggc 420

ggctccggtg gtggtggttc tgaggtgcag ctgctcgagc agtctggagc tgagctggta 480

aggcctggga cttcagtgaa gatatcctgc aaggcttctg gatacgcctt cactaactac 540

tggctaggtt gggtaaagca gaggcctgga catggacttg agtggattgg agatattttc 600

cctggaagtg gtaatatcca ctacaatgag aagttcaagg gcaaagccac actgactgca 660

gacaaatctt cgagcacagc ctatatgcag ctcagtagcc tgacatttga ggactctgct 720

gtctatttct gtgcaagact gaggaactgg gacgagccta tggactactg gggccaaggg 780

accacggtca ccgtctcctc cggaggtggt ggatccgata tcaaactgca gcagtcaggg 840

gctgaactgg caagacctgg ggcctcagtg aagatgtcct gcaagacttc tggctacacc 900

tttactaggt acacgatgca ctgggtaaaa cagaggcctg gacagggtct ggaatggatt 960

ggatacatta atcctagccg tggttatact aattacaatc agaagttcaa ggacaaggcc 1020

acattgacta cagacaaatc ctccagcaca gcctacatgc aactgagcag cctgacatct 1080

gaggactctg cagtctatta ctgtgcaaga tattatgatg atcattactg ccttgactac 1140

tggggccaag gcaccactct cacagtctcc tcagtcgaag gtggaagtgg aggttctggt 1200

ggaagtggag gttcaggtgg agtcgacgac attcagctga cccagtctcc agcaatcatg 1260

tctgcatctc caggggagaa ggtcaccatg acctgcagag ccagttcaag tgtaagttac 1320

atgaactggt accagcagaa gtcaggcacc tcccccaaaa gatggattta tgacacatcc 1380

aaagtggctt ctggagtccc ttatcgcttc agtggcagtg ggtctgggac ctcatactct 1440

ctcacaatca gcagcatgga ggctgaagat gctgccactt attactgcca acagtggagt 1500

agtaacccgc tcacgttcgg tgctgggacc aagctggagc tgaaacatca tcaccatcat 1560

cattag 1566

<210>44

<211>521

<212>PRT

＜213〉artificial sequence

<220>

＜223〉5-10 (VLVH) * anti--CD3

<400>44

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val

145 150 155 160

Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala

165 170 175

Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly

180 185 190

Leu Glu Trp Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr

195 200 205

Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser

210 215 220

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala

225 230 235 240

Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

275 280 285

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

290 295 300

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

305 310 315 320

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

325 330 335

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

340 345 350

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

355 360 365

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

370 375 380

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

385 390 395 400

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

405 410 415

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

420 425 430

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

435 440 445

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

450 455 460

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

465 470 475 480

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

485 490 495

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

500 505 510

Glu Leu Lys His His His His His His

515 520

<210>45

<211>1494

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL×3-1 VHVL

<400>45

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcagtc 360

gaaggtggaa gtggaggttc tggtggaagt ggaggttcag gtggagtcga cgacattcag 420

ctgacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 480

agagccagtt caagtgtaag ttacatgaac tggtaccagc agaagtcagg cacctccccc 540

aaaagatgga tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600

agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc 660

acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg gaccaagctg 720

gagctgaaat ccggaggtgg tggatccgag gtgcagctgc tcgagcagtc tggagctgag 780

ctggtgaaac ctggggcctc agtgaagata tcctgcaagg cttctggata cgccttcact 840

aactactggc taggttgggt aaagcagagg cctggacatg gacttgagtg gattggagat 900

cttttccctg gaagtggtaa tactcactac aatgagaggt tcaggggcaa agccacactg 960

actgcagaca aatcctcgag cacagccttt atgcagctca gtagcctgac atctgaggac 1020

tctgctgtct atttctgtgc aagattgagg aactgggacg aggctatgga ctactggggc 1080

caagggacca cggtcaccgt ctcctcaggt ggtggtggtt ctggcggcgg cggctccggt 1140

ggtggtggtt ctgagctcgt catgacccag tctccatctt atcttgctgc atctcctgga 1200

gaaaccatta ctattaattg cagggcaagt aagagcatta gcaaatattt agcctggtat 1260

caagagaaac ctgggaaaac taataagctt cttatctact ctggatccac tttgcaatct 1320

ggaattccat caaggttcag tggcagtgga tctggtacag atttcactct caccatcagt 1380

agcctggagc ctgaagattt tgcaatgtat tactgtcaac agcataatga atatccgtac 1440

acgttcggag gggggaccaa gcttgagatc aaacatcatc accatcatca ttag 1494

<210>46

<211>497

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL×3-1 VHVL

<400>46

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln

245 250 255

Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys

260 265 270

Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys

275 280 285

Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp Leu Phe Pro Gly

290 295 300

Ser Gly Asn Thr His Tyr Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu

305 310 315 320

Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe Met Gln Leu Ser Ser Leu

325 330 335

Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp

340 345 350

Asp Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

355 360 365

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Glu Leu Val Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly

385 390 395 400

Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr

405 410 415

Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile

420 425 430

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly

435 440 445

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

450 455 460

Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr

465 470 475 480

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys His His His His His

485 490 495

His

<210>47

<211>1494

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL Ser×3-1 VHVL

<400>47

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actcccttga ctactggggc caaggcacca ctctcacagt ctcctcagtc 360

gaaggtggaa gtggaggttc tggtggaagt ggaggttcag gtggagtcga cgacattcag 420

ctgacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 480

agagccagtt caagtgtaag ttacatgaac tggtaccagc agaagtcagg cacctccccc 540

aaaagatgga tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600

agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc 660

acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg gaccaagctg 720

gagctgaaat ccggaggtgg tggatccgag gtgcagctgc tcgagcagtc tggagctgag 780

ctggtgaaac ctggggcctc agtgaagata tcctgcaagg cttctggata cgccttcact 840

aactactggc taggttgggt aaagcagagg cctggacatg gacttgagtg gattggagat 900

cttttccctg gaagtggtaa tactcactac aatgagaggt tcaggggcaa agccacactg 960

actgcagaca aatcctcgag cacagccttt atgcagctca gtagcctgac atctgaggac 1020

tctgctgtct atttctgtgc aagattgagg aactgggacg aggctatgga ctactggggc 1080

caagggacca cggtcaccgt ctcctcaggt ggtggtggtt ctggcggcgg cggctccggt 1140

ggtggtggtt ctgagctcgt catgacccag tctccatctt atcttgctgc atctcctgga 1200

gaaaccatta ctattaattg cagggcaagt aagagcatta gcaaatattt agcctggtat 1260

caagagaaac ctgggaaaac taataagctt cttatctact ctggatccac tttgcaatct 1320

ggaattccat caaggttcag tggcagtgga tctggtacag atttcactct caccatcagt 1380

agcctggagc ctgaagattt tgcaatgtat tactgtcaac agcataatga atatccgtac 1440

acgttcggag gggggaccaa gcttgagatc aaacatcatc accatcatca ttag 1494

<210>48

<211>497

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL Ser×3-1 VHVL

<400>48

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln

245 250 255

Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys

260 265 270

Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys

275 280 285

Gln Arg Pro Gly His Gly Leu Glu Trp Ile Gly Asp Leu Phe Pro Gly

290 295 300

Ser Gly Asn Thr His Tyr Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu

305 310 315 320

Thr Ala Asp Lys Ser Ser Ser Thr Ala Phe Met Gln Leu Ser Ser Leu

325 330 335

Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp

340 345 350

Asp Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

355 360 365

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Glu Leu Val Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly

385 390 395 400

Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr

405 410 415

Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile

420 425 430

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly

435 440 445

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

450 455 460

Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr

465 470 475 480

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys His His His His His

485 490 495

His

<210>49

<211>1521

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL×3-5 VHVL

<400>49

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcagtc 360

gaaggtggaa gtggaggttc tggtggaagt ggaggttcag gtggagtcga cgacattcag 420

ctgacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 480

agagccagtt caagtgtaag ttacatgaac tggtaccagc agaagtcagg cacctccccc 540

aaaagatgga tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600

agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc 660

acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg gaccaagctg 720

gagctgaaat ccggaggtgg tggatccgag gtgcagctgc tcgagcagtc tggagctgag 780

ctggtaaggc ctgggacttc agtgaagctg tcctgcaagg cttctggcta caccttcaca 840

agctatggtt taagctgggt gaagcagaga actggacagg gccttgagtg gattggagag 900

gtttatccta gaattggtaa tgcttactac aatgagaagt tcaagggcaa ggccacactg 960

actgcagaca aatcctccag cacagcgtcc atggagctcc gcagcctgac atctgaggac 1020

tctgcggtct atttctgtgc aagacgggga tcctacggta gtaactacga ctggtacttc 1080

gatgtctggg gccaagggac cacggtcacc gtctcctcag gtggtggtgg ttctggcggc 1140

ggcggctccg gtggtggtgg ttctgagctc gtgatgaccc agactccact ctccctgcct 1200

gtcagtcttg gagatcaagc ctccatctct tgcagatcta gtcagagcct tgtacacagt 1260

aatggaaaca cctatttaca ttggtacctg cagaagccag gccagtctcc aaagctcctg 1320

atctacaaag tttccaaccg attttctggg gtcccagaca ggttcagtgg cagtggatca 1380

gggacagatt tcacactcaa gatcagcaga gtggaggctg aggatctggg agtttatttc 1440

tgctctcaaa gtacacatgt tccgtacacg ttcggagggg ggaccaagct tgagatcaaa 1500

catcatcacc atcatcatta g 1521

<210>50

<211>506

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL×3-5 VHVL

<400>50

Asn Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu ThrPhe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln

245 250 255

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

260 265 270

Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Gly Leu Ser Trp Val Lys

275 280 285

Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg

290 295 300

Ile Gly Asn Ala Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu

305 310 315 320

Thr Ala Asp Lys Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu

325 330 335

Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr

340 345 350

Gly Ser Asn Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr

355 360 365

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

370 375 380

Gly Gly Gly Ser Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro

385 390 395 400

Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

405 410 415

Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys

420 425 430

Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe

435 440 445

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

450 455 460

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe

465 470 475 480

Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys

485 490 495

Leu Glu Ile Lys His His His His His His

500 505

<210>51

<211>1521

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL Ser×3-5 VHVL

<400>51

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actcccttga ctactggggc caaggcacca ctctcacagt ctcctcagtc 360

gaaggtggaa gtggaggttc tggtggaagt ggaggttcag gtggagtcga cgacattcag 420

ctgacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 480

agagccagtt caagtgtaag ttacatgaac tggtaccagc agaagtcagg cacctccccc 540

aaaagatgga tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600

agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc 660

acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg gaccaagctg 720

gagctgaaat ccggaggtgg tggatccgag gtgcagctgc tcgagcagtc tggagctgag 780

ctggtaaggc ctgggacttc agtgaagctg tcctgcaagg cttctggcta caccttcaca 840

agctatggtt taagctgggt gaagcagaga actggacagg gccttgagtg gattggagag 900

gtttatccta gaattggtaa tgcttactac aatgagaagt tcaagggcaa ggccacactg 960

actgcagaca aatcctccag cacagcgtcc atggagctcc gcagcctgac atctgaggac 1020

tctgcggtct atttctgtgc aagacgggga tcctacggta gtaactacga ctggtacttc 1080

gatgtctggg gccaagggac cacggtcacc gtctcctcag gtggtggtgg ttctggcggc 1140

ggcggctccg gtggtggtgg ttctgagctc gtgatgaccc agactccact ctccctgcct 1200

gtcagtcttg gagatcaagc ctccatctct tgcagatcta gtcagagcct tgtacacagt 1260

aatggaaaca cctatttaca ttggtacctg cagaagccag gccagtctcc aaagctcctg 1320

atctacaaag tttccaaccg attttctggg gtcccagaca ggttcagtgg cagtggatca 1380

gggacagatt tcacactcaa gatcagcaga gtggaggctg aggatctggg agtttatttc 1440

tgctctcaaa gtacacatgt tccgtacacg ttcggagggg ggaccaagct tgagatcaaa 1500

catcatcacc atcatcatta g 1521

<210>52

<211>506

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL Ser×3-5 VHVL

<400>52

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln

245 250 255

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

260 265 270

Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Gly Leu Ser Trp Val Lys

275 280 285

Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg

290 295 300

Ile Gly Asn Ala Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu

305 310 315 320

Thr Ala Asp Lys Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu

325 330 335

Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr

340 345 350

Gly Ser Asn Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr

355 360 365

Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

370 375 380

Gly Gly Gly Ser Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro

385 390 395 400

Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

405 410 415

Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys

420 425 430

Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe

435 440 445

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

450 455 460

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe

465 470 475 480

Cys Ser Gln Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys

485 490 495

Leu Glu Ile Lys His His His His His His

500 505

<210>53

<211>1512

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×3-5 VHVL

<400>53

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcaggt 360

ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca gctgacccag 420

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagagccagt 480

tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc caaaagatgg 540

atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg cagtgggtct 600

gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc cacttattac 660

tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct ggagctgaaa 720

tccggaggtg gtggatccga ggtgcagctg ctcgagcagt ctggagctga gctggtaagg 780

cctgggactt cagtgaagct gtcctgcaag gcttctggct acaccttcac aagctatggt 840

ttaagctggg tgaagcagag aactggacag ggccttgagt ggattggaga ggtttatcct 900

agaattggta atgcttacta caatgagaag ttcaagggca aggccacact gactgcagac 960

aaatcctcca gcacagcgtc catggagctc cgcagcctga catctgagga ctctgcggtc 1020

tatttctgtg caagacgggg atcctacggt agtaactacg actggtactt cgatgtctgg 1080

ggccaaggga ccacggtcac cgtctcctca ggtggtggtg gttctggcgg cggcggctcc 1140

ggtggtggtg gttctgagct cgtgatgacc cagactccac tctccctgcc tgtcagtctt 1200

ggagatcaag cctccatctc ttgcagatct agtcagagcc ttgtacacag taatggaaac 1260

acctatttac attggtacct gcagaagcca ggccagtctc caaagctcct gatctacaaa 1320

gtttccaacc gattttctgg ggtcccagac aggttcagtg gcagtggatc agggacagat 1380

ttcacactca agatcagcag agtggaggct gaggatctgg gagtttattt ctgctctcaa 1440

agtacacatg ttccgtacac gttcggaggg gggaccaagc ttgagatcaa acatcatcac 1500

catcatcatt ag 1512

<210>54

<211>503

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×3-5 VHVL

<400>54

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile

130 135 140

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser

145 150 155 160

Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser

165 170 175

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro

180 185 190

Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

195 200 205

Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

210 215 220

Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

225 230 235 240

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala

245 250 255

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

260 265 270

Gly Tyr Thr Phe Thr Ser Tyr Gly Leu Ser Trp Val Lys Gln Arg Thr

275 280 285

Gly Gln Gly Leu Glu Trp Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn

290 295 300

Ala Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp

305 310 315 320

Lys Ser Ser Ser Thr Ala Ser Met Glu Leu Arg Ser Leu Thr Ser Glu

325 330 335

Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Gly Ser Tyr Gly Ser Asn

340 345 350

Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val

355 360 365

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

370 375 380

Ser Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu

385 390 395 400

Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His

405 410 415

Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

420 425 430

Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

435 440 445

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

450 455 460

Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln

465 470 475 480

Ser Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

485 490 495

Lys His His His His His His

500

<210>55

<211>1512

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL×4-1 VHVL

<400>55

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcagtc 360

gaaggtggaa gtggaggttc tggtggaagt ggaggttcag gtggagtcga cgacattcag 420

ctgacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 480

agagccagtt caagtgtaag ttacatgaac tggtaccagc agaagtcagg cacctccccc 540

aaaagatgga tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600

agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc 660

acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg gaccaagctg 720

gagctgaaat ccggaggtgg tggatccgag gtgcagctgc tcgagcagtc tggagctgag 780

ctggtaaggc ctgggacttc agtgaagata tcctgcaagg cttctggata cgccttcact 840

aactactggc taggttgggt taagcagagg cctggacatg gacttgaatg ggttggagat 900

attttccctg gaagtggtaa tgctcactac aatgagaagt tcaagggcaa agccacactg 960

actgcagaca agtcctcgta cacagcctat atgcagctca gtagcctgac atctgaggac 1020

tctgctgtct atttctgtgc aagattgcgg aactgggacg aggctatgga ctactggggc 1080

caagggacca cggtcaccgt ctcctcaggt ggtggtggtt ctggcggcgg cggctccggt 1140

ggtggtggtt ctgagctcgt gatgacacag tctccatcct ccctgagtgt gtcagcagga 1200

gagaaggtca ctatgagctg caagtccagt cagagtctgt taaacagtgg aaatcaaaag 1260

aactacttgg cctggtacca gcagaaacca gggcagcctc ctaaactgtt gatctacggg 1320

gcatccacta gggaatctgg ggtccctgat cgcttcacag gcagtggatc tggaacagat 1380

ttcactctca ccatcagcag tgtgcaggct gaagacctgg cagtttatta ctgtcagaat 1440

gattatagtt atccgtacac gttcggaggg gggaccaagc ttgagatcaa acatcatcac 1500

catcatcatt ag 1512

<210>56

<211>503

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL×4-1 VHVL

<400>56

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln

245 250 255

Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Ile Ser Cys

260 265 270

Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys

275 280 285

Gln Arg Pro Gly His Gly Leu Glu Trp Val Gly Asp Ile Phe Pro Gly

290 295 300

Ser Gly Asn Ala His Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu

305 310 315 320

Thr Ala Asp Lys Ser Ser Tyr Thr Ala Tyr Met Gln Leu Ser Ser Leu

325 330 335

Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp

340 345 350

Asp Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

355 360 365

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

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

385 390 395 400

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

405 410 415

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

420 425 430

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val

435 440 445

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

450 455 460

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

465 470 475 480

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

485 490 495

Lys His His His His His His

500

<210>57

<211>1512

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL Ser×4-1 VHVL

<400>57

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actcccttga ctactggggc caaggcacca ctctcacagt ctcctcagtc 360

gaaggtggaa gtggaggttc tggtggaagt ggaggttcag gtggagtcga cgacattcag 420

ctgacccagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 480

agagccagtt caagtgtaag ttacatgaac tggtaccagc agaagtcagg cacctccccc 540

aaaagatgga tttatgacac atccaaagtg gcttctggag tcccttatcg cttcagtggc 600

agtgggtctg ggacctcata ctctctcaca atcagcagca tggaggctga agatgctgcc 660

acttattact gccaacagtg gagtagtaac ccgctcacgt tcggtgctgg gaccaagctg 720

gagctgaaat ccggaggtgg tggatccgag gtgcagctgc tcgagcagtc tggagctgag 780

ctggtaaggc ctgggacttc agtgaagata tcctgcaagg cttctggata cgccttcact 840

aactactggc taggttgggt taagcagagg cctggacatg gacttgaatg ggttggagat 900

attttccctg gaagtggtaa tgctcactac aatgagaagt tcaagggcaa agccacactg 960

actgcagaca agtcctcgta cacagcctat atgcagctca gtagcctgac atctgaggac 1020

tctgctgtct atttctgtgc aagattgcgg aactgggacg aggctatgga ctactggggc 1080

caagggacca cggtcaccgt ctcctcaggt ggtggtggtt ctggcggcgg cggctccggt 1140

ggtggtggtt ctgagctcgt gatgacacag tctccatcct ccctgagtgt gtcagcagga 1200

gagaaggtca ctatgagctg caagtccagt cagagtctgt taaacagtgg aaatcaaaag 1260

aactacttgg cctggtacca gcagaaacca gggcagcctc ctaaactgtt gatctacggg 1320

gcatccacta gggaatctgg ggtccctgat cgcttcacag gcagtggatc tggaacagat 1380

ttcactctca ccatcagcag tgtgcaggct gaagacctgg cagtttatta ctgtcagaat 1440

gattatagtt atccgtacac gttcggaggg gggaccaagc ttgagatcaa acatcatcac 1500

catcatcatt ag 1512

<210>58

<211>503

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL aL Ser×4-1 VHVL

<400>58

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln

245 250 255

Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Ile Ser Cys

260 265 270

Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys

275 280 285

Gln Arg Pro Gly His Gly Leu Glu Trp Val Gly Asp Ile Phe Pro Gly

290 295 300

Ser Gly Asn Ala His Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu

305 310 315 320

Thr Ala Asp Lys Ser Ser Tyr Thr Ala Tyr Met Gln Leu Ser Ser Leu

325 330 335

Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp

340 345 350

Asp Glu Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

355 360 365

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

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

385 390 395 400

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

405 410 415

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

420 425 430

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val

435 440 445

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

450 455 460

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

465 470 475 480

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

485 490 495

Lys His His His His His His

500

<210>59

<211>1503

<212>DNA

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×4-1 VHVL

<400>59

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctcaggt 360

ggtggtggtt ctggcggcgg cggctccggt ggtggtggtt ctgacattca gctgacccag 420

tctccagcaa tcatgtctgc atctccaggg gagaaggtca ccatgacctg cagagccagt 480

tcaagtgtaa gttacatgaa ctggtaccag cagaagtcag gcacctcccc caaaagatgg 540

atttatgaca catccaaagt ggcttctgga gtcccttatc gcttcagtgg cagtgggtct 600

gggacctcat actctctcac aatcagcagc atggaggctg aagatgctgc cacttattac 660

tgccaacagt ggagtagtaa cccgctcacg ttcggtgctg ggaccaagct ggagctgaaa 720

tccggaggtg gtggatccga ggtgcagctg ctcgagcagt ctggagctga gctggtaagg 780

cctgggactt cagtgaagat atcctgcaag gcttctggat acgccttcac taactactgg 840

ctaggttggg ttaagcagag gcctggacat ggacttgaat gggttggaga tattttccct 900

ggaagtggta atgctcacta caatgagaag ttcaagggca aagccacact gactgcagac 960

aagtcctcgt acacagccta tatgcagctc agtagcctga catctgagga ctctgctgtc 1020

tatttctgtg caagattgcg gaactgggac gaggctatgg actactgggg ccaagggacc 1080

acggtcaccg tctcctcagg tggtggtggt tctggcggcg gcggctccgg tggtggtggt 1140

tctgagctcg tgatgacaca gtctccatcc tccctgagtg tgtcagcagg agagaaggtc 1200

actatgagct gcaagtccag tcagagtctg ttaaacagtg gaaatcaaaa gaactacttg 1260

gcctggtacc agcagaaacc agggcagcct cctaaactgt tgatctacgg ggcatccact 1320

agggaatctg gggtccctga tcgcttcaca ggcagtggat ctggaacaga tttcactctc 1380

accatcagca gtgtgcaggc tgaagacctg gcagtttatt actgtcagaa tgattatagt 1440

tatccgtaca cgttcggagg ggggaccaag cttgagatca aacatcatca ccatcatcat 1500

tag 1503

<210>60

<211>500

<212>PRT

＜213〉artificial sequence

<220>

<223>CD3 VHVL stL×4-1 VHVL

<400>60

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile

130 135 140

Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser

145 150 155 160

Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser

165 170 175

Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro

180 185 190

Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

195 200 205

Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp

210 215 220

Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

225 230 235 240

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Gln Ser Gly Ala

245 250 255

Glu Leu Val Arg Pro Gly Thr Ser Val Lys Ile Ser Cys Lys Ala Ser

260 265 270

Gly Tyr Ala Phe Thr Asn Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro

275 280 285

Gly His Gly Leu Glu Trp Val Gly Asp Ile Phe Pro Gly Ser Gly Asn

290 295 300

Ala His Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp

305 310 315 320

Lys Ser Ser Tyr Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

325 330 335

Asp Ser Ala Val Tyr Phe Cys Ala Arg Leu Arg Asn Trp Asp Glu Ala

340 345 350

Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly

355 360 365

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Leu Val

370 375 380

Met Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ala Gly Glu Lys Val

385 390 395 400

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

405 410 415

Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

420 425 430

Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

435 440 445

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

450 455 460

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

465 470 475 480

Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys His His

485 490 495

His His His His

500

<210>61

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CDRH3 M1 mutant

<400>61

His Tyr Asp Asp His Tyr Cys Leu Asp Tyr

1 5 10

<210>62

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CDRH3 M4 mutant

<400>62

Tyr Ser Asp Asp His Tyr Cys Leu Asp Tyr

1 5 10

<210>63

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CDRH3 M7 mutant

<400>63

Tyr Tyr Asp Ala His Tyr Cys Leu Asp Tyr

1 5 10

<210>64

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CDRH3 M9 mutant

<400>64

Tyr Tyr Asp Asp Gln Tyr Cys Leu Asp Tyr

1 5 10

<210>65

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CDRH3 M10 mutant

<400>65

Tyr Tyr Asp Asp Pro Tyr Cys Leu Asp Tyr

1 5 10

<210>66

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CDRH3 M11 mutant

<400>66

Tyr Phe Asn Asp His Tyr Cys Leu Asp Tyr

1 5 10

<210>67

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CDRH3 M13 mutant

<400>67

Tyr Tyr Asn Asp Gln Tyr Cys Leu Asp Tyr

1 5 10

<210>68

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CDRH3 M20 mutant

<400>68

Tyr His Asp Asp Pro Tyr Cys Leu Asp Tyr

1 5 10

<210>69

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉CDRH3 M76 mutant

<400>69

Tyr Tyr Asp Asp Asn Tyr Cys Leu Asp Tyr

1 5 10

<210>70

<211>18

<212>PRT

＜213〉artificial sequence

<220>

＜223〉initial joint

<400>70

Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

1 5 10 15

Val Asp

<210>71

<211>357

<212>DNA

＜213〉artificial sequence

<220>

＜223〉anti--CD3VH

<400>71

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt ctcctca 357

<210>72

<211>119

<212>PRT

＜213〉artificial sequence

<220>

＜223〉anti--CD3VH

<400>72

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210>73

<211>318

<212>DNA

＜213〉artificial sequence

<220>

＜223〉anti--CD3 VL

<400>73

gacattcagc tgacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60

atgacctgca gagccagttc aagtgtaagt tacatgaact ggtaccagca gaagtcaggc 120

acctccccca aaagatggat ttatgacaca tccaaagtgg cttctggagt cccttatcgc 180

ttcagtggca gtgggtctgg gacctcatac tctctcacaa tcagcagcat ggaggctgaa 240

gatgctgcca cttattactg ccaacagtgg agtagtaacc cgctcacgtt cggtgctggg 300

accaagctgg agctgaaa 318

<210>74

<211>106

<212>PRT

＜213〉artificial sequence

<220>

＜223〉anti--CD3 VL

<400>74

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210>75

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉vH CDR1 resists-CD3

<400>75

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

1 5 10

<210>76

<211>357

<212>DNA

＜213〉artificial sequence

<220>

＜223〉vH resists-CD3 cys-〉ser

<400>76

gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60

tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120

cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180

aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240

atgcaactga gcagcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300

gatgatcatt actcccttga ctactggggc caaggcacca ctctcacagt ctcctca 357

<210>77

<211>119

<212>PRT

＜213〉artificial sequence

<220>

＜223〉vH resists-CD3 cys-〉ser

<400>77

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210>78

<211>10

<212>PRT

＜213〉artificial sequence

<220>

＜223〉VH CDR3 resists-CD3cys-〉ser

<400>78

Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr

1 5 10

<210>79

<211>360

<212>DNA

＜213〉artificial sequence

<220>

<223>EpCAM 3-1VH

<400>79

gaggtgcagc tgctcgagca gtctggagct gagctggtga aacctggggc ctcagtgaag 60

atatcctgca aggcttctgg atacgccttc actaactact ggctaggttg ggtaaagcag 120

aggcctggac atggacttga gtggattgga gatcttttcc ctggaagtgg taatactcac 180

tacaatgaga ggttcagggg caaagccaca ctgactgcag acaaatcctc gagcacagcc 240

tttatgcagc tcagtagcct gacatctgag gactctgctg tctatttctg tgcaagattg 300

aggaactggg acgaggctat ggactactgg ggccaaggga ccacggtcac cgtctcctca 360

<210>80

<211>120

<212>PRT

＜213〉artificial sequence

<220>

<223>EpCAM 3-1VH

<400>80

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

1 5 10 15

Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn

20 25 30

Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp

35 40 45

Ile Gly Asp Leu Phe Pro Gly Ser Gly Asn Thr His Tyr Asn Glu Arg

50 55 60

Phe Arg Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala

65 70 75 80

Phe Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe

85 90 95

Cys Ala Arg Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>81

<211>321

<212>DNA

＜213〉artificial sequence

<220>

<223>EpCAM 3-1 VL

<400>81

gagctcgtca tgacccagtc tccatcttat cttgctgcat ctcctggaga aaccattact 60

attaattgca gggcaagtaa gagcattagc aaatatttag cctggtatca agagaaacct 120

gggaaaacta ataagcttct tatctactct ggatccactt tgcaatctgg aattccatca 180

aggttcagtg gcagtggatc tggtacagat ttcactctca ccatcagtag cctggagcct 240

gaagattttg caatgtatta ctgtcaacag cataatgaat atccgtacac gttcggaggg 300

gggaccaagc ttgagatcaa a 321

<210>82

<211>107

<212>PRT

＜213〉artificial sequence

<220>

<223>EpCAM 3-1 VL

<400>82

Glu Leu Val Met Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly

1 5 10 15

Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Tyr

20 25 30

Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile

35 40 45

Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Glu Tyr Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>83

<211>372

<212>DNA

＜213〉artificial sequence

<220>

<223>EpCAM 3-5VH

<400>83

gaggtgcagc tgctcgagca gtctggagct gagctggtaa ggcctgggac ttcagtgaag 60

ctgtcctgca aggcttctgg ctacaccttc acaagctatg gtttaagctg ggtgaagcag 120

agaactggac agggccttga gtggattgga gaggtttatc ctagaattgg taatgcttac 180

tacaatgaga agttcaaggg caaggccaca ctgactgcag acaaatcctc cagcacagcg 240

tccatggagc tccgcagcct gacatctgag gactctgcgg tctatttctg tgcaagacgg 300

ggatcctacg gtagtaacta cgactggtac ttcgatgtct ggggccaagg gaccacggtc 360

accgtctcct ca 372

<210>84

<211>124

<212>PRT

＜213〉artificial sequence

<220>

<223>EpCAM 3-5 VH

<400>84

Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly

1 5 10 15

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

20 25 30

Tyr Gly Leu Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp

35 40 45

Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala

65 70 75 80

Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe

85 90 95

Cys Ala Arg Arg Gly Ser Tyr Gly Ser Asn Tyr Asp Trp Tyr Phe Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>85

<211>336

<212>DNA

＜213〉artificial sequence

<220>

<223>EpCAM 3-5 VL

<400>85

gagctcgtga tgacccagac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300

tacacgttcg gaggggggac caagcttgag atcaaa 336

<210>86

<211>112

<212>PRT

＜213〉artificial sequence

<220>

<223>EpCAM 3-5 VL

<400>86

Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>87

<211>360

<212>DNA

＜213〉artificial sequence

<220>

<223>EpCAM 4-1 VH

<400>87

gaggtgcagc tgctcgagca gtctggagct gagctggtaa ggcctgggac ttcagtgaag 60

atatcctgca aggcttctgg atacgccttc actaactact ggctaggttg ggttaagcag 120

aggcctggac atggacttga atgggttgga gatattttcc ctggaagtgg taatgctcac 180

tacaatgaga agttcaaggg caaagccaca ctgactgcag acaagtcctc gtacacagcc 240

tatatgcagc tcagtagcct gacatctgag gactctgctg tctatttctg tgcaagattg 300

cggaactggg acgaggctat ggactactgg ggccaaggga ccacggtcac cgtctcctca 360

<210>88

<211>120

<212>PRT

＜213〉artificial sequence

<220>

<223>EpCAM 4-1 VH

<400>88

Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly

1 5 10 15

Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn

20 25 30

Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp

35 40 45

Val Gly Asp Ile Phe Pro Gly Ser Gly Asn Ala His Tyr Asn Glu Lys

50 55 60

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

65 70 75 80

Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe

85 90 95

Cys Ala Arg Leu Arg Asn Trp Asp Glu Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>89

<211>339

<212>DNA

＜213〉artificial sequence

<220>

<223>EpCAM 4-1 VL

<400>89

gagctcgtga tgacacagtc tccatcctcc ctgagtgtgt cagcaggaga gaaggtcact 60

atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttggcc 120

tggtaccagc agaaaccagg gcagcctcct aaactgttga tctacggggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300

ccgtacacgt tcggaggggg gaccaagctt gagatcaaa 339

<210>90

<211>113

<212>PRT

＜213〉artificial sequence

<220>

<223>EpCAM 4-1 VL

<400>90

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

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210>91

<211>372

<212>DNA

＜213〉artificial sequence

<220>

<223>EpCAM 4-7 VH

<400>91

gaggtgcagc tgctcgagca gtctggagct gagctggcga ggcctggggc ttcagtgaag 60

ctgtcctgca aggcttctgg ctacaccttc acaaactatg gtttaagctg ggtgaagcag 120

aggcctggac aggtccttga gtggattgga gaggtttatc ctagaattgg taatgcttac 180

tacaatgaga agttcaaggg caaggccaca ctgactgcag acaaatcctc cagcacagcg 240

tccatggagc tccgcagcct gacctctgag gactctgcgg tctatttctg tgcaagacgg 300

ggatcctacg atactaacta cgactggtac ttcgatgtct ggggccaagg gaccacggtc 360

accgtctcct ca 372

<210>92

<211>124

<212>PRT

＜213〉artificial sequence

<220>

<223>EpCAM 4-7 VH

<400>92

Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly

1 5 10 15

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

20 25 30

Tyr Gly Leu Ser Trp Val Lys Gln Arg Pro Gly Gln Val Leu Glu Trp

35 40 45

Ile Gly Glu Val Tyr Pro Arg Ile Gly Asn Ala Tyr Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala

65 70 75 80

Ser Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe

85 90 95

Cys Ala Arg Arg Gly Ser Tyr Asp Thr Asn Tyr Asp Trp Tyr Phe Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>93

<211>336

<212>DNA

＜213〉artificial sequence

<220>

<223>EpCAM 4-7 VL

<400>93

gagctcgtga tgacccagac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120

tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg 300

tacacgttcg gaggggggac caagcttgag atcaaa 336

<210>94

<211>112

<212>PRT

＜213〉artificial sequence

<220>

<223>EpCAM 4-7 VL

<400>94

Glu Leu Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser

85 90 95

Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>95

<211>360

<212>DNA

＜213〉artificial sequence

<220>

<223>EpCAM 5-10 VH

<400>95

gaggtgcagc tgctcgagca gtctggagct gagctggtaa ggcctgggac ttcagtgaag 60

atatcctgca aggcttctgg atacgccttc actaactact ggctaggttg ggtaaagcag 120

aggcctggac atggacttga gtggattgga gatattttcc ctggaagtgg taatatccac 180

tacaatgaga agttcaaggg caaagccaca ctgactgcag acaaatcttc gagcacagcc 240

tatatgcagc tcagtagcct gacatttgag gactctgctg tctatttctg tgcaagactg 300

aggaactggg acgagcctat ggactactgg ggccaaggga ccacggtcac cgtctcctca 360

<210>96

<211>120

<212>PRT

＜213〉artificial sequence

<220>

<223>EpCAM 5-10 VH

<400>96

Glu Val Gln Leu Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly

1 5 10 15

Thr Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn

20 25 30

Tyr Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp

35 40 45

Ile Gly Asp Ile Phe Pro Gly Ser Gly Asn Ile His Tyr Asn Glu Lys

50 55 60

Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala

65 70 75 80

Tyr Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Phe

85 90 95

Cys Ala Arg Leu Arg Asn Trp Asp Glu Pro Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>97

<211>339

<212>DNA

＜213〉artificial sequence

<220>

<223>EpCAM 5-10 VL

<400>97

gagctcgtga tgacacagtc tccatcctcc ctgactgtga cagcaggaga gaaggtcact 60

atgagctgca agtccagtca gagtctgtta aacagtggaa atcaaaagaa ctacttgacc 120

tggtaccagc agaaaccagg gcagcctcct aaactgttga tctactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg gaacagattt cactctcacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga ttatagttat 300

ccgctcacgt tcggtgctgg gaccaagctt gagatcaaa 339

<210>98

<211>113

<212>PRT

＜213〉artificial sequence

<220>

<223>EpCAM 5-10 VL

<400>98

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

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

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

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

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

100 105 110

Lys

<210>99

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉modular connection

<400>99

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>100

<211>47

<212>DNA

＜213〉artificial sequence

<220>

<223>4-7VL BspEI FOR

<400>100

ctgaaatccg gaggtggtgg atccgagctc gtgatgaccc agactcc 47

<210>101

<211>52

<212>DNA

＜213〉artificial sequence

<220>

<223>4-7 VL GS15 REVs148

<400>101

ggagccgccg ccgccagaac caccaccacc tttgatctca agcttggtcc cc 52

Claims

1. the pharmaceutical composition that comprises the bispecific single-chain antibody construct, wherein said construct comprises or is made up of at least two structural domains, one of wherein said structural domain in conjunction with people EpCAM antigen and second structural domain in conjunction with people CD3 antigen, wherein the special described binding domains of EpCAM is comprised at least one CDR-H3 district, the CDR-H3 district is preferably at SEQ ID NO:80,88 and 96 102-104 position or preferably comprise aminoacid sequence NXD in the 106-108 position of SEQ ID NO:84 and 92, wherein X is a die aromatischen Aminosaeuren.

2. the pharmaceutical composition of claim 1, wherein X is W or Y.

3. claim 1 or 2 pharmaceutical composition, wherein CDR-H3 comprises at least 9 amino-acid residues.

4. any one pharmaceutical composition of claim 1-3, the K of the binding domains that wherein said EpCAM is special _DValue is greater than 5 * 10 ^-9M.

5. any one pharmaceutical composition of claim 1-4, the K of the binding domains that wherein said EpCAM is special _DValue is 1 * 10 ^-7To 5 * 10 ^-9In the M scope and the K of described CD3 specific combination structural domain _DValue is 1 * 10 ^-6To 5 * 10 ^-9In the M scope.

6. the pharmaceutical composition that comprises the bispecific single-chain antibody construct, wherein said construct comprises or is made up of at least two structural domains, one of wherein said at least two structural domains specific combination people EpCAM antigen and second structural domain be in conjunction with people CD3 antigen, wherein the special described binding domains of EpCAM comprised the CDR-H3 district of at least one at least 9 amino-acid residue and wherein to the K of the special described binding domains of EpCAM _DValue is greater than 5 * 10 ^-9M.

7. any one pharmaceutical composition of claim 1-6, the K of wherein said CD3 specific combination structural domain _DValue is greater than 10 ^-7M.

8. any one pharmaceutical composition of claim 1-7, wherein the CDR-H3 district comprises at least 14 amino acid.

9. any one pharmaceutical composition of claim 1-8, wherein the VH chain structure territory of people EpCAM antigen-specific is selected from:

(a) as SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:92 and the SEQ ID NO:96 aminoacid sequence as shown in any one;

(b) by the coded aminoacid sequence of nucleotide sequence as shown in SEQ ID NO:79, SEQ ID NO:83, SEQ ID NO:87, SEQ IDNO:91 and SEQ ID NO:95;

(d) by (b) and (c) any one nucleotide sequence because the aminoacid sequence of the nucleic acid sequence encoding that produced of genetic code degeneracy.

10. any one pharmaceutical composition of claim 1-9, wherein people EpCAM antigen-specific V _LThe chain structure territory is selected from:

(a) as SEQ ID NO:82, SEQ ID NO:86, SEQ ID NO:90, SEQ ID NO:94 and the SEQ ID NO:98 aminoacid sequence as shown in any one;

(b) by the aminoacid sequence of the nucleic acid sequence encoding as shown in SEQ ID NO:81, SEQ ID NO:85, SEQ ID NO:89, SEQ IDNO:93 and SEQ ID NO:97;

11. the pharmaceutical composition that claim 1-10 is any, wherein CD3 antigen-specific binding domains derives from and is selected from following antibody: X35-3, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87,12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, WT31 and F101.01.

12. comprising, the pharmaceutical composition that claim 1-11 is any, wherein said bispecific single-chain antibody construct be selected from following aminoacid sequence:

(a) as the aminoacid sequence of SEQ ID NO:2,4,8,10,12,14,16,18,20,30,36,39,42,44,46,48,50,52,54,56,58 and 60 as shown in any one;

(b) by the coded aminoacid sequence of nucleotide sequence as shown in SEQ ID NO:1,3,7,9,11,13,15,17,19,29,35,38,41,43,45,47,49,51,53,55,57 and 59;

13. pharmaceutical composition, wherein said pharmaceutical composition comprise the nucleotide sequence of coding as any defined bispecific single-chain antibody construct among the claim 1-12.

14. pharmaceutical composition, wherein said pharmaceutical composition comprises the carrier that contains nucleotide sequence as defined in claim 13.

15. the pharmaceutical composition of claim 14, wherein said carrier also comprise the adjusting sequence that effectively is connected to defined described nucleotide sequence in the claim 13.

16. the pharmaceutical composition of claim 14 or 15, wherein said carrier is an expression vector.

17. pharmaceutical composition, wherein said pharmaceutical composition comprise the host with any defined carrier conversion or transfection among the claim 14-16.

18. the pharmaceutical composition any according to claim 1-17, it also comprises can provide the protein properties of immune effector cell activation signals compound.

19. the pharmaceutical composition any according to claim 1-18, wherein pharmaceutical composition is heat-staple in the time of 〉=37 ℃.

20. produce the method according to any one pharmaceutical composition of claim 1-19, wherein said method is included in the bispecific single-chain antibody construct that allows under the condition that any one institute of the claim 1-12 bispecific single-chain antibody construct that define expresses defined host in the cultivation claim 17 and produced from the culture recovery.

21. as any defined bispecific single-chain antibody construct of claim 1-12, as defined in claim 13 nucleotide sequence, as any defined carrier of claim 14-16, as defined in claim 17 and/or according to the host's that method produced of claim 20 purposes, be used to prepare the pharmaceutical composition of prevention, treatment or tumor remission disease.

22. the method for prevention, treatment or tumor remission disease, described method comprise that the experimenter to the prevention of this kind of needs, treatment or alleviation uses the step of any one pharmaceutical composition of claim 1-19.

23. the method for claim 22, wherein said experimenter is the people.

24. the method for the purposes of claim 21 or claim 22 or 23, wherein said tumor disease are epithelial cancer or minimal residue cancer.

25. test kit, wherein said test kit comprise as any defined bispecific single-chain antibody construct of claim 1-12, as defined in claim 13 nucleotide sequence, such as claim 14-16 any one the definition carrier, as defined in claim 17 and/or according to the host that method produced of claim 20.