CN1721445A - Degraded TPO agonist antibody - Google Patents

Abstract

The invention relates to a modified antibody which contains two or more H chain V regions and two or more L chain V regions of monoclonal antibody and can transduce a signal into cells by crosslinking TPO receptor to thereby exert TPO agonist action. The modified antibody can be used as a TPO signal transduction agonist and, therefore, useful as a preventive and/or remedy for various diseases such as platelet-reduction-related blood diseases, thrombopenia following chemotherapy for cancer or leukemia, etc.

Description

Degraded TPO agonist antibody

The application is that the application number of October 22 calendar year 2001 application is dividing an application of 01817544.9 Chinese invention patent " degraded TPO agonist antibody ".

Technical field

The antibody that the present invention relates to change, it comprises two or more H chain V districts and two or more L chains V district of antibody, and this antibody shows agonist activity by crosslinked TPO acceptor.The antibody of this change has that transduction signal enters the TPO agonist activity of cell by crosslinked TPO acceptor, and as multi-purpose medicine.

Background technology

Thrombopoietin (TPO) is that a kind of thrombocyte of finding in 1994 generates regulatory factor, and the known glycoprotein that is 70-80k by the main molecular weight that generates in liver is formed.Thrombopoietin is a kind of cytokine, promotes platelet precursors cell survival, propagation, differentiation and maturation in marrow, promptly promotes megalokaryocyte differentiation and propagation.Thrombopoietin (TPO) acceptor is confirmed as a kind of acceptor of regulating thrombopoietic atopen early than TPO, c-Mpl (people such as M.Souyri, Cell 63:1137 (1990)).It was reported that c-Mpl mainly is distributed in the platelet precursors cell, megalokaryocyte and platelet cell suppress c-Mpl and express selectivity and suppress megalokaryocyte and generate people such as (, Blood 82:1395 (1993)) M.Methia.It was reported that at the proliferation assay of the cell of c-Mpl aglucon and the result who utilizes this aglucon of c-Mpl purifying, the aglucon of c-Mpl is TPO (people such as F.de Sauvage, Nature369:533 (1994) according to specificity; People such as TD.Bartley, Cell 77:1117 (1994)).Mpl is called as the TPO acceptor at present.Therefore, expectation TPO and TPO receptor stimulant be as the medicine of thrombocytopenia, for example, and as the medicine that alleviates thrombocytopenia due to cancer patient bone marrow depression or the marrow excision treatment.

On the other hand, improve the permeability of tissue and tumour and utilize recombinant methods to develop the antibody of change by reducing molecular size, particularly the antibody of molecular size decline, for example strand Fv.Recently, the dimer of strand Fv, particularly the dual specific dimer has been used for crosslinked cell.This dimeric exemplary is heterodimer people such as (, Int.J.Cancer, 77,9763-9772,1998) Kipriyanov of identification cancer cell antigen and the antigenic strand Fv of host cell (as NK cell and neutrophil).Their utilize with strand Fv be configured to change antibody technology and prepare, by crosslinked between inducing cell and more effectively treat cancer.Thought by antibody and fragment (for example Fab fragment) thereof, crosslinked between the antibody that dual specific changes and even the strand Fv dimer inducing cell of monospecific.

For passing through crosslinked cell surface molecule and the antibody of transduction signal, known have the antibody (JP-A 2000-95800) of cytodifferentiation and the EPO acceptor that relates to of propagation, the antibody of a MuSK acceptor (people such as Xie, Nature Biotech.15,768-771,1997) and other.Known agonist antibody, its fragment and the strand Fv (WO99/17364) that also has the TPO acceptor.Yet, still do not have strand Fv dimer and the antibody of change, for example report of strand bivalent antibody with agonist activity.

Notice that the strand Fv monomer that derives from monoclonal antibody (inventor preparation MABL-1 antibody and MABL-2 antibody) induces the apoptosis that contains IAP, cell death inducing not, and dimer is apoptosis-induced, the inventor finds the IAP acceptor of dimer crosslinked (Dimerized) cell surface, thereby transduction signal enters cell, causes apoptosis-induced.This prompting, the crosslinked cell surface molecule of monospecific strand Fv dimer (for example acceptor), and as the aglucon transduction signal, thereby as agonist.

The crosslinked discovery of research iuntercellular, above-mentioned strand Fv dimer does not cause hemagglutination reaction, said monoclonal antibody then can.Strand bivalent antibody (single chain polypeptide that comprises two H chain V districts and two L chain V districts) is also observed identical result.This prompting, it is crosslinked that monoclonal antibody may form iuntercellular, and the antibody that changes, as strand Fv dimer and strand bivalent antibody, crosslinked cell surface molecule but not form iuntercellular crosslinked.

Based on these observationss, the inventor finds recently, the antibody that changes, for example strand Fv dimer and strand bivalent antibody, except be used for known iuntercellular crosslinked, molecule in also crosslinked isocellular cell surface molecule or the cell is suitable as the aglucon aglucon of the natural aglucon effect of simulation (particularly as) of this molecule.

The inventor further finds, antibody molecule (IgG fully) can change and become the strand of crosslinked cell surface molecule Fv dimer, strand bivalent antibody etc., thereby reduce the crosslinked side effect that causes of iuntercellular, and the new drug of an inducing cell purpose effect is provided, finished the present invention.Compare the complete antibody (IgG) that has identical V district with the antibody of this change, the antibody that the present invention changes has significantly high activity.Because its molecular size descends than antibody molecule, and lacks constant region, it has the tissue permeability of raising.

Summary of the invention

One object of the present invention is to provide the antibody of the agonist change of hanging down molecular size, and it comprises two or more H chain V districts and two or more L chains V district of monoclonal antibody, and has the effect of TPO agonist by crosslinked TPO acceptor.

Therefore, the antibody that the present invention relates to change, it comprises two or more H chain V districts and two or more L chains V district, preferred every kind of 2-6, preferred especially every kind of 2-4, most preferably two every kind, and show the TPO agonist activity by crosslinked TPO acceptor.

" antibody of change " is meant any material that comprises two or more H chain V districts and two or more L chains V district in this specification sheets, and wherein said V district directly connects or connects with covalent linkage or non covalent bond by joint.For example, connect polypeptide and the compound that each V district of antibody prepares by peptide linker or chemical cross-linking agent etc.Two or more H chain V districts of utilization of the present invention and two or more L chains V district can derive from same antibody or different antibodies.

The antibody of change of the present invention can be specific recognition and crosslinked TPO acceptor thereby any material that can transduction signal enters cell.They comprise the partial amino-acid series in antibody V district of further this change of modification and the antibody of the change for preparing.

The preferred embodiment of the antibody that the present invention changes is the polymer that comprises the strand Fv in H chain V district and L chain V district, and for example dimer, tripolymer or the tetramer perhaps comprise the single chain polypeptide in two or more H chain V districts and two or more L chains V district.If the antibody of change of the present invention is the polymer that comprises the strand Fv in H chain V district and L chain V district, for example dimer, tripolymer or the tetramer etc. are preferably placed at H chain V district and L chain V district debond formation antigen binding site on the same chain.

Preferred example be comprise H chain V district and L chain V district strand Fv dimer or comprise two H chain V districts and the single chain polypeptide in two L chain V districts.The H chain V district and the L chain V district of the antibody that changes preferably link by joint.

Above-mentioned strand Fv polymer comprises non covalent bond polymer, the covalent linkage polymer by crosslinking group and the polymer (antibody that antibody, antibody fragment or two price-reform become) by linking agent.The conventional crosslinking group that is used for crosslinked peptide can be as forming polymeric crosslinking group.For example by the crosslinked disulphide of cysteine residues, other crosslinking group is as C ₄-C ₁₀Alkylene (for example tetramethylene, pentamethylene, hexa-methylene, heptamethylene and eight methylene radical etc.) or C ₄-C ₁₀Alkenylene (suitable/anti--3-crotonylidene, suitable/anti--2-inferior pentenyl, suitable/anti--3-inferior pentenyl, suitable/inferior hexenyl of anti--3-etc.).

In addition, can have, for example can arbitrarily introduce the aminoacid sequence of Fv, the antibody of for example anti-FLAG sequence etc. or its fragment or derive from the antibody of the change of antibody, for example strand Fv in conjunction with the linking agent of strand Fv.

This specification sheets " effect of TPO agonist " is meant the biological action of transduceing and taking place in the cell of signal by crosslinked TPO acceptor, for example breeds, differentiation or Megakaryocytic growth-stimulating or thrombocyte generate.

Utilize the currently known methods of measuring the agonist effect, determine the ED50 of TPO agonist of the present invention effect.The example of measuring has, utilize the clone (for example BaF/mpl or UT7/TPO) of TPO sensitivity to carry out analysis of cell proliferation, measure the proteinic phosphorylation of MPL, by medullary cell differentiation carrying out megakaryocyte colony analysis, platelet recovery synthesis analysis in the mouse body, utilize human leukemia megakaryoblast system (CMK) to measure the induced expression of platelet antigen GPIIbIIIa (anti-GPIIbIIIa), perhaps measure the polyploidy of megakaryoblast system (DAMI) and induce.ED50 is for reaching 50% maximum reaction (being made as 100% in the dose-response curve) required dosage.

The antibody that the present invention preferably changes, its TPO agonist effect (ED50) is equal to or better than the antibody with antigen binding domain identical with the antibody of this change, promptly have with the H chain V district of the antigen binding domain of the antibody that forms this change and L chain V district to the right complete antibody (following " parental antibody ") in identical H chain V district and L chain V district, as IgG.More preferably its TPO agonist effect (ED50) is higher than parental antibody more than two times, further preferred more than 5 times, the antibody of the change more than 10 times most preferably.The present invention comprises the antibody of the change with the effect of TPO agonist, and it comprises H chain V district and the L chain V district that forms the antigen binding domain identical with parental antibody (can in conjunction with the TPO acceptor, but this molecule is not had the agonist effect).

The compound that comprises two or more H chain V districts and two or more L chains V district of the present invention can be any compound that the agonist effect (ED50) that comprises two or more H chain V districts of antibody and two or more L chains V district, show is equal to or better than thrombopoietin (TPO).Be preferably its TPO agonist effect (ED50) be higher than TPO more than 2 times, more preferably more than 5 times, the compound more than 10 times most preferably.

" compound " described herein not only comprises the antibody of change of the present invention, also comprise comprise two or more, preferred 2-6, more preferably 2-4,2 antigen binding domains (for example complete antibody or F (ab ') most preferably ₂) any compound.

The intercellular adhesion effect (ED50) that antibody or compound had that the present invention comprises the preferred change in two or more H chain V districts of antibody and two or more L chains V district is no more than 1/10 of parental antibody, does not most preferably have the intercellular adhesion effect basically.

Utilize the currently known methods of measuring the intercellular adhesion effect, for example measure the cohesion of the cell of expressing the TPO acceptor, determine the ED50 of above-mentioned intercellular adhesion effect.

The DNA of antibody of this change the present invention relates to encode.

The present invention relates to produce the zooblast or the microorganism of the antibody of this change.

The present invention relates to the purposes of the antibody of this change as the TPO agonist.

The present invention relates to utilize the antibody of this change, transduction signal enters cell by crosslinked TPO acceptor, thereby induces the method for TPO agonist effect (phosphorylation of for example megakaryocyte proliferation, induction or growth-stimulating, thrombocyte generation, TPO receptor protein etc.).

The antibody that the present invention relates to comprise this change is the medicine of treatment thrombocytopenia of activeconstituents etc.

The present invention relates to the purposes of the antibody of this change as medicine.

The present invention relates to the screening or the measuring method of the antibody of the change that comprises two or more H chain V districts and two or more L chain V districts of antibody and show the effect of TPO agonist by crosslinked TPO acceptor, it comprises 1) preparation comprises two or more H chain V districts of antibody and two or more L chain V districts and specificity antibody in conjunction with the change of TPO acceptor, the cell of TPO acceptor is expressed in the antibody that 2) will change contact, and 3) measure the TPO agonist effect that in cell, takes place by crosslinked TPO acceptor.This measuring method is used for preparing the quality control of the present invention as the antibody of the change of medicine and other purpose.

The antibody of this change can be the antibody of the change of the antibody of change of monospecific or polyspecific, for example antibody of the change of dual specific.The antibody of the change of preferred monospecific.

The invention still further relates to H chain V district and/or L chain V district and be the antibody of change that derives from the H chain V district of people's antibody and/or derive from the L chain V district of people's antibody.As described in WO99/10494, can obtain to derive from the H chain V district and/or the L chain V district of people's antibody by screening human monoclonal antibodies library.Also comprise the H chain V district and the L chain V district that derive from by the human monoclonal antibodies of preparations such as transgenic mice.

The invention further relates to H chain V district and/or L chain V district is the antibody of the change in humanized H chain V district and/or humanized L chain V district.Particularly, the antibody of this humanization change is made up of humanized L chain V district (complementary determining region (following " CDR ") that it comprises the framework region (FR) that derives from human monoclonal antibodies L chain V district and derives from non-human mammal (for example mouse, rat, ox, sheep, ape) monoclonal antibody L chain V district) and/or humanized H chain V district (CDR that it comprises the FR that derives from human monoclonal antibodies H chain V district and derives from non-human mammal (for example mouse, rat, ox, sheep, ape) monoclonal antibody H chain V district).In this case, can partly change the aminoacid sequence of CDR and FR, for example lack, replace or add.

The H chain V district and/or the L chain V district of the antibody that the present invention changes can be H chain V district and/or the L chain V districts that derives from non-human animal's (for example mouse, rat, ox, sheep, ape, chicken etc.) monoclonal antibody.In this case, can partly change the aminoacid sequence of CDR and FR, for example lack, replace or add.

The DNA of antibody of above-mentioned various changes and the genetic engineering technique that is used to prepare the recombinant vectors that comprises this DNA the present invention also relates to encode.

The invention still further relates to this recombinant vectors transformed host cells.The example of host cell is a zooblast, for example people's cell, mouse cell etc., and microorganism, for example intestinal bacteria ( E. Coli), subtilis ( Bacillus Subtilis), yeast etc.

The present invention relates to the method for mutagenic antibody, comprise and cultivate above-mentioned host, and from its culture, extract the antibody of this change.

The present invention relates to the dimeric method of strand Fv that produces in addition, and it is included in and cultivates the host animal cell that can produce this strand Fv in the serum free medium, with secretion strand Fv in substratum, and the strand Fv dimer that forms in the isolation medium.

The invention still further relates to the purposes of the antibody of this change as the TPO agonist.Promptly relate to the signal transduction agonist, its antibody of change that comprises above-mentioned acquisition is as activeconstituents.

Therefore, comprising the antibody that TPO agonist of the present invention changes is the pharmaceutical preparation of activeconstituents, can be used as the medicine that prevents and/or treats of thrombocytopenia due to thrombopenia relevant hematologic disease, cancer or the leukemia chemotherapy etc.

The antibody of change of the present invention comprises 2 or a plurality of H chains V district and 2 or a plurality of L chain V district that comes from antibody.The structure of the antibody of this change may be the dimer that comprises the strand Fv in 1 H chain V district and 1 L chain V district, or comprises the polypeptide in 2 H chain V districts and 2 L chain V districts.In the antibody of change of the present invention, the V district of H chain and L chain preferably links to each other by the peptide linker of being made up of one or more amino acid.The antibody of the change that produces comprises the variable region of antibody, with the specificity conjugated antigen identical with original monoclonal antibody.

H chain V district

In the present invention, derive from the H chain V district identification TPO acceptor and the oligomerize of antibody, for example by crosslinked described molecule and Dimerized, thereby transduction signal enters cell.H chain V of the present invention district comprises the H chain V district that derives from Mammals (for example people, mouse, rat, ox, sheep, ape etc.), and the H chain V district that partly changes of the aminoacid sequence in H chain V district.More preferably humanized H chain V district, it comprises the FR in human monoclonal antibodies H chain V district, and the CDR in mouse monoclonal antibody H chain V district.The H chain V district that also preferably has the aminoacid sequence that derives from the people, it can utilize the recombinant technology preparation.H chain V of the present invention district may be the fragment that keeps the above-mentioned H chain V district of antigen binding capacity.

L chain V district

In the present invention, this L chain V district identification TPO acceptor and oligomerize, for example by crosslinked described molecule and Dimerized, thereby transduction signal enters cell.L chain V of the present invention district comprises the L chain V district that derives from Mammals (for example people, mouse, rat, ox, sheep, ape etc.), and the L chain V district that partly changes of the aminoacid sequence in L chain V district.More preferably humanized L chain V district, it comprises the FR in human monoclonal antibodies L chain V district, and the CDR in mouse monoclonal antibody L chain V district.The L chain V district that also preferably has the aminoacid sequence that derives from the people, it can utilize the recombinant technology preparation.L chain V of the present invention district may be the fragment that keeps the L chain V district of antigen binding capacity.

Complementary determining region (CDR)

Each V district of L chain and H chain forms antigen binding site.(Kabat is formed by 4 the quite conservative common framework regions that are connected 3 hypervariable regions or complementary determining region (CDR) in the variable region of L and H chain, E.A. wait the people, " Sequences of Protein of ImmunologicalInterest ", US Dept.Health and Human Services, 1983).

Major portion in 4 framework regions (FR) forms β-laminated structure, thereby 3 CDR form ring.Under some situation, CDR can form the part of β-laminated structure.3 CDR are near each other by FR on the locus, form antigen in conjunction with node together with 3 CDR.

According to Kabat, the empirical rule among the people such as E.A. " Sequences of Protein ofImmunological Interest " compares the antibody V region amino acid sequence of acquisition and the known amino acid sequence in known antibodies V district, identifies these CDR.

Strand Fv

Strand Fv is a polypeptide monomer, comprises interconnected H chain V district and the L chain V district that derives from monoclonal antibody.The strand Fv that produces comprises the variable region of original antibody, and keeps its complementary determining region, thereby this strand Fv is with the specificity conjugated antigen (JP-Appl.11-63557) identical with original antibody.Part variable region and/or the CDR of strand Fv of the present invention, or its partial amino-acid series may be that part changes, and for example lacks, replaces or add.Before addressed H chain V district and the L chain V district that constitutes strand Fv of the present invention, they can directly connect or be continuous by joint, the preferred peptide joint.The structure of strand Fv may be [H chain V district]-[L chain V district], perhaps [L chain V district]-[H chain V district].Among the present invention, might make strand Fv form dimer, tripolymer or the tetramer, therefrom form change antibody of the present invention.

The antibody that strand changes

The present invention comprises the antibody that two or more H chains V district and two or more L chain V district, preferred every kind of 2-4, preferred especially 2 every kind strand change, and comprises above-mentioned two or more H chains V district and L chain V district.Should arrange each district of peptide, make the single-chain antibody of change form specific space structure, accurately simulate the space structure that forms by strand Fv dimer.For example, arrange the V district in the following order: [H chain V district]-[L chain V district]-[H chain V district]-[L chain V district]; Perhaps [L chain V district]-[H chain V district]-[L chain V district]-[H chain V district], wherein these districts link to each other by peptide linker respectively.

Joint

Among the present invention, connecting joint between H chain V district and the L chain V district and may be can be by any peptide linker of gene engineering method introducing, or any joint of chemosynthesis.For example, the present invention may use disclosed joint in the document, as Protein Egineering, and 9 (3), 299-305,1996.These joints can be identical or different in a part.Peptide linker if desired, below joint example for quoting:

Ser

Gly-Ser

Gly-Gly-Ser

Ser-Gly-Gly

Gly-Gly-Gly-Ser

Ser-Gly-Gly-Gly

Gly-Gly-Gly-Gly-Ser

Ser-Gly-Gly-Gly-Gly

Gly-Gly-Gly-Gly-Gly-Ser

Ser-Gly-Gly-Gly-Gly-Gly

Gly-Gly-Gly-Gly-Gly-Gly-Ser

Ser-Gly-Gly-Gly-Gly-Gly-Gly

(Gly-Gly-Gly-Gly-Ser) _nAnd

(Ser-Gly-Gly-Gly-Gly) _n

Wherein n is not less than 1 integer.Preferred peptide linker length is according to changing as antigenic acceptor, for strand Fv, in preferred usually 1-20 the amino acid scope.Antibody for the strand change that comprises two or more H chains V district and two or more L chain V district, the peptide linker length that is connected to form the same antigen binding site that comprises [H chain V district]-[L chain V district] (or [L chain V district]-[H chain V district]) with it is 1-30 amino acid, preferred 1-20 amino acid, more preferably 3-18 amino acid.Be connected with it and do not form the peptide linker of the same antigen binding site that comprises [H chain V district]-[L chain V district] (or [L chain V district]-[H chain V district]), its length is 1-40 amino acid, preferred 3-30 amino acid, more preferably 5-20 amino acid.In the explanation of the DNA construct of the antibody that code book invention changes, the method for introducing these joints will be described.

According to the present invention, the joint of chemosynthesis, promptly chemical cross-linking agent can be any joint that routine is used for connection peptides.The example of joint may comprise N-hydroxy-succinamide (NHS), disuccinimidyl suberate (DSS), two (sulfosuccinimide the ester) (BS of suberic acid ³), dithio two (propionic acid amber imines ester) (DSP), dithio two (propionic acid sulfosuccinimide ester) (DTSSP), ethylene glycol bis (succsinic acid succinimide ester) (EGS), ethylene glycol bis (succsinic acid sulfosuccinimide ester) (sulfo-EGS), tartrate two succinimide esters (DST), tartrate disulfo succinimide ester (sulfo-DST), two [2-(succinimido oxygen ketonic oxygen base) ethyl] sulfone (BSOCOES), two [2-(sulfosuccinimide base oxygen ketonic oxygen base) ethyl] sulfone (sulfo-BSOCOES) etc.These all are commercial.Preferably identical chemosynthesis joint with peptide linker length.

Be to form the dimer of strand Fv, preferred joint is suitable for making strand Fv that host cell produces (for example in the substratum) is Dimerized in solution and surpasses 20%, preferably surpasses 50%, more preferably surpasses 80%, most preferably surpasses 90%.Especially, preferred joint is made up of 2-12 amino acid, preferred 3-10 amino acid, or other joint of its correspondence.

The preparation of the antibody that changes

Derive from H chain V district and the L chain V district of specificity by connecting, can prepare the antibody of change in conjunction with the known or novel monoclonal antibody of TPO acceptor with above-mentioned joint.The example of the strand Fv that quotes has the antibody 12B5 that describes among the WO99/10494 and H chain V district and the L chain V district of antibody 12E10.15 amino acid), sc12E10 dimer (joint: 15 amino acid), db12B5 dimer (joint: 5 amino acid), db12E10 dimer (joint: 5 amino acid), sc12B5sc (FV) example of antibody that the present invention has a change in two or more H chain V districts and two or more L chains V district is the sc12B5 dimer that comprises the H chain V district that derives from a said monoclonal antibody and L chain V district (joint: ₂And sc12E10sc (FV) ₂

For preparing the antibody of this change, be the secretion peptide if wish this polypeptide, then can connect signal peptide at its N end.For effective this polypeptide of purifying, can connect the well-known aminoacid sequence that is used for peptide purification, for example the FLAG sequence.In the case, utilize anti-FLAG antibody can form dimer.

For preparing the antibody of change of the present invention, must obtain DNA, the DNA of the DNA of the strand Fv that promptly encodes or the single chain polypeptide of coding and rebuilding.The DNA of these DNA, particularly sc12B5, db12B5, sc12E10 and/or db12E10 can obtain from the H chain V district of the described Fv that encodes and the DNA in L chain V district.Utilize these DNA to be template, and the primer of corresponding its two end is right, increasing by polymerase chain reaction (PCR) method wherein comprises the DNA part of coding purpose aminoacid sequence, also can obtain them.

When the aminoacid sequence of wishing each V district partly changes, can utilize PCR to pass through methods known in the art, obtain the V district of one of them or some amino acid changes (promptly lack, replace or add).For preparing the antibody that specific antigen is had abundant active change, preferably utilize PCR method known in the art to change the partial amino-acid series in V district.

For determining the pcr amplification primer,, determine the type of H chain and L chain if monoclonal antibody is then utilized classifying method known in the art as raw material.

For the L chain V district of pcr amplification antibody 12B5 and antibody 12E10, determine the Oligonucleolide primers of 5 ' end and 3 ' end as stated above.In kind determine used 5 ' end and 3 ' the end Oligonucleolide primers in H chain V district of amplification antibody 12B5 and antibody 12E10.

In embodiment of the present invention, 5 ' end primer of use comprises " GANTC " sequence, provides Restriction Enzyme Hinf I recognition site near its 5 ' end, and 3 ' end primer comprises nucleotide sequence " CCCGGG ", provides XmaI recognition site near its 5 ' end.As long as can be used for the target DNA fragment subclone in cloning vector, can use these sites other Restriction Enzyme recognition site in addition.

Use specially designed PCR primer, 5 ' end and 3 ' end at the V district of antibody 12B5 and 12E10 code cDNA provide suitable nucleotide sequence, thereby make this cDNA insert expression vector easily and in expression vector, have appropriate functional (for example, the present invention is designed to improve translation efficiency by inserting the Kozak sequence).Utilize these primers to carry out pcr amplification, obtain the V district of antibody 12B5 and 12E10, be inserted into the HEF expression vector (seeing WO92/19759) that comprises purpose people C district.Can utilize any ordinary method order-checking clone's DNA, for example utilize automated DNA sequenator (Applied Biosystems).

Can be by following method, with a joint, for example peptide linker is introduced the antibody of change of the present invention.Design has part complementary sequence and the N end of coding joint or the primer of C end of above-mentioned H chain V district and L chain V district primer.Then, utilize these primers to carry out the PCR program, the preparation coding has the DNA of the peptide linker of purpose aminoacid sequence and length.The DNA that utilization obtains connects the DNA in coding H chain V district and L chain V district, and the preparation code book is invented the DNA of the antibody of the change with purpose peptide linker.In case prepared the DNA of the antibody of the change of encoding, by the various primers of designed joint, utilize this primer and above-mentioned DNA to carry out PCR for template, can be easy to prepare the DNA of antibody that coding is with or without the change of purpose peptide linker.

Utilize routine techniques (for example, people such as Sato K., Cancer Res., 53,1-6 (1993)), each V district of the antibody that can change the present invention carries out humanization.In case prepared the DNA of each humanization Fv that encodes, can be easy to according to ordinary method the fragment of fragment, Humanized monoclonal antibodies and the Humanized monoclonal antibodies of preparation Humanized single chain Fv, Humanized single chain Fv.Preferably, if desired, can carry out part to its V region amino acid sequence and change.

In addition, utilize above-mentioned ordinary method, encoding according to preparation derives from H chain V district and the used the same manner of L chain V district DNA of mouse, can prepare to derive from other mammiferous DNA, the DNA in each V district of the people's antibody of for example encoding.The DNA that obtains can be used for preparing monoclonal antibody and the fragment thereof that other Mammals particularly derives from H chain V district and L chain V district, the strand Fv that derives from the people and the fragment and the people source of people's antibody.

If the antibody of change of the present invention is the antibody of the change of dual specific, then can prepare (for example described method of WO9413804) by currently known methods.

As mentioned above, in case prepared the target DNA in the V district of the V district of the antibody that coding changes and the antibody that humanization changes, just can comprise their expression vector and utilize this carrier host transformed according to the ordinary method acquisition.In addition, can cultivate the host, the strand Fv that preparation is rebuild, Humanized single chain Fv, Humanized monoclonal antibodies and the fragment thereof of reconstruction according to ordinary method.It can separate from cell or substratum, and purifying is a uniform substance.For this purpose, if necessary, can unite the conventional any method that is used for protein separation and purifying of using, for example chromatography, ultrafiltration, saltout and dialyse, without limits it.

When in serum free medium, cultivating zooblast (for example COS7 cell or Chinese hamster ovary celI, preferred Chinese hamster ovary celI), when preparing reconstruction strand Fv of the present invention, can stablize reclaim and high yield purifying substratum in the dimer of the described strand Fv that forms.The dimer of purifying can steady in a long-termly be preserved like this.The serum free medium that the present invention uses can be any substratum that routine is used to prepare recombinant protein, to it without limits.

For preparing the antibody of change of the present invention, can utilize any expression system, for example eukaryotic cell such as zooblast (for example mammal cell line of Jian Liing), filamentous fungus and yeast, and prokaryotic cell prokaryocyte such as bacterial cell are (for example E. Coli).The antibody of change of the present invention is preferably expressed in mammalian cell, for example COS7 cell or Chinese hamster ovary celI.

In these cases, can use the conventional promotor that is used for mammalian cell expression.The preferred immediate early promoter that uses Human cytomegalic inclusion disease virus (HCMV).The expression vector that comprises the HCMV promotor comprises (WO92/19759) such as HCMV-VH-HC γ 1, HCMV-VL-HCK that come from pSV2neo.

In addition, other promotor of the mammalian cell genetic expression that the present invention may use comprises the viral promotors that derives from retrovirus, polyoma virus, adenovirus and simian virus 40 (SV40), and derive from mammiferous promotor, for example the human polypeptides chain extension factor 1 α (HEF-1 α).(Nature 277 can to use the SV40 promotor easily according to people's such as Mulligan R.C. method, 108-114 (1979)), also can use HEF-1 α promotor (Nucleic Acids Research, 18,5322 (1990)) according to people's methods such as Mizushima S..

Can be used for replication orgin of the present invention (ori) and comprise the starting point that derives from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) etc.Expression vector may comprise phosphotransferase APH (3 ') II or I (neo) gene, thymidine kinase (TK) gene, E. ColiXanthine-guanine phosphoribosyl transferase (Ecogpt) gene or Tetrahydrofolate dehydrogenase (DHFR) gene are as selection marker.

Utilize ordinary method, for example radioimmunoassay (RIA), Enzyme Linked Immunoadsorbent Assay (ELISA) or surperficial cytogene resonance technique (surface plasmonresonance) can be estimated the antigen-binding activity of antibody of the change of above-mentioned preparation.Also can utilize the combination-inhibition ability of original antibody to estimate for index (for example whether having the concentration dependent restraining effect with antigenic the combination) according to described monoclonal antibody.

In more detail, cultivate zooblast, for example COS7 cell or Chinese hamster ovary celI with the expression vector conversion of the antibody dna that comprises code book invention change.Cultured cells and/or culture supernatant or the antibody of the change of purifying therefrom are used for measuring and combine with antigenic.The substratum supernatant of cultivating the cell that only transforms expression vector is with comparing.As for antigen, for example antibody 12B5 and antibody 12E10, the specimen or the contrast supernatant of the antibody that the present invention is changed add in the Ba/F3 cell of expressing human MPL, for example carry out flow cytometry then and measure, to estimate antigen-binding activity.

Carry out the in-vitro evaluation of signal transduction effect (phosphorylation of for example megakaryocyte proliferation, induction or growth-stimulating, thrombocyte generation or TPO receptor protein) in accordance with the following methods.The specimen of the antibody of above-mentioned change is added the cell of expressing antibodies or introduced in the cell of antibody gene, and utilize the variation that ordinary method causes by signal transduction (for example people MPL antigen-specific propagation, measure protein phosphorylation or express platelet-specific antigen) to estimate.

Give the antibody or the PBS contrast of the monoclonal antibody of mouse identification MPL, change of the present invention, estimate activity intensity by the variation of platelet counts in the mice serum and carry out interior evaluating.As mentioned above, comprise two or more H chain V districts and two or more L chain V districts and specificity antibody by preparation in conjunction with the change of TPO acceptor, and, can obtain the antibody of change of the present invention by in the above-mentioned body or the antibody that changes of in-vitro evaluation method screening.

The present invention comprises two or more H chain V districts and two or more L chains V district, preferred every kind of 2-4, more preferably 2 every kind the antibody of change, may be the dimer that comprises the strand Fv in a H chain V district and a L chain V district, or the single chain polypeptide that wherein has two or more H chains V district to link to each other with two or more L chains V district.Can think, because such structure makes the three-dimensional structure of peptide simulation TPO, thereby has good antigen binding characteristic and TPO agonist activity.

The antibody of change of the present invention is compared parental antibody molecule (for example IgG), has significantly low molecular size, thereby has the strong permeability to tissue or tumour, has the activity than parent's monoclonal anti height.Therefore, the antibody of the change of the present invention TPO signal of can effectively transduceing enters cell.The pharmaceutical preparation that comprises them is used for the treatment of thrombocytopenia due to the relevant hemopathy of thrombopenia and cancer or the leukemia chemotherapy.Also expect to can be used as contrast medium after antibody of the present invention is through the RI mark.Can strengthen this effect by connecting RI compound or toxin.

Optimum mode of operation of the present invention

The present invention will specifically illustrate according to following examples, but it does not limit the scope of the invention.

For illustrating the preparation method of the antibody that the present invention changes, the embodiment of preparation strand Fv is as follows.Use mouse anti human IAP antibody among the embodiment of the antibody that preparation changes, MABL-1 and MABL-2.On September 11st, 1997, the hybridoma MABL-1 and the MABL-2 that produce them have respectively been carried out international preservation, deposit in microorganism with FERM BP-6100 and FERM BP-6101 respectively and authorize preservation mechanism-state-run life science and human body technical institute, the industrial science Technical Board, Ministry of International Trade and Industry (National Institute of BiosCience andhuman Technology, Agency of Industrial Science andTechnology, Minister of International Trade and Industry) (1-3Higasi 1-Chome, Tsukuba-shi, Ibaraki-Ken, Japan).

Embodiment

Embodiment 1 (the V district coding DNA of clone's mouse anti human IAP monoclonal antibody)

The coding DNA of the variable region of following clone's mouse anti human IAP monoclonal antibody MABL-1 and MABL-2.

1.1 Preparation messenger RNA(mRNA) (mRNA)

Utilize mRNA Purification test kit (Pharmacia Biotech) to obtain the mRNA of hybridoma MABL-1 and MABL-2.

1.2 Synthetic double chain cDNA

Utilize Marathon cDNA Amplification test kit (CLONTECH) from about 1 μ g mRNA synthetic double chain cDNA, and connect a joint.

1.3 The encoding gene of pcr amplification antibody variable region

Utilize Thermal Cycler (PERKIN ELMER) to carry out PCR.

(1) Amplification MABL-1 L chain V district encoding gene

The primer that is used for PCR method is Adapter Primer-1 (CLONTECH) shown in the SEQ ID No.1, the partial sequence hybridization of it and joint, and MKC shown in the SEQ ID No.2 (mouse Kappa is constant) primer (Bio/Technology, 9,88-89,1991), it and mouse Kappa type L chain V district hybridization.

50 μ l PCR solution comprise 5 μ l, 10 * PCR damping fluid II, 2mM MgCl ₂, 0.16mMdNTPs (dATP, dGTP, dCTP and dTTP), the 2.5 archaeal dna polymerase AmpliTaqGold of unit (PERKIN ELMER), 0.2 μ M SEQ ID No.1 joint primer, 0.2 μ M SEQID No.2MKC primer and 0.1 μ g derive from the double-stranded cDNA of MABL-1.Solution is in 94 ℃ of preheatings of starting temperature 9 minutes, then 1 minute, 60 ℃ 1 minute, 72 ℃ of 94 ℃ of heating 1 minute and 20 seconds successively.This temperature cycle repeats 35 times, and 72 ℃ of reheat reaction mixtures are 10 minutes then.

(2) The code cDNA in amplification MABL-1H chain V district

MHC-γ 1 (murine heavy chain is constant) primer (Bio/Technology, 9,88-89,1991) shown in AdapterPrimer-1 shown in the SEQ ID No.1 and the SEQ ID No.3 is as the PGR primer.

Amplification method according to the described L chain of embodiment 1.3-(1) V district gene carries out the cDNA amplification, only is to use 0.2 μ M MHC-γ, 1 primer, but not 0.2 μ M MKC primer.

(3) The code cDNA in amplification MABL-2L chain V district

The MKC primer of the AdapterPrimer-1 of SEQ ID No.1 and SEQ ID No.2 is as the PCR primer.

Carry out the cDNA amplification according to the amplification method of the described MABL-1 L of embodiment 1.3-(1) chain V district gene, only be to use 0.1 μ g to derive from the double-stranded cDNA of MABL-2, but not the double-stranded cDNA of 0.1 μ gMABL-1.

(4) The code cDNA in amplification MABL-2 H chain V district

MHC-γ 2a primer (Bio/Technology, 9,88-89,1991) shown in the AdapterPrimer-1 of SEQ ID No.1 and the SEQ ID No.4 is as the PCR primer.

Amplification method according to the described L chain of embodiment 1.3-(3) V district gene carries out the cDNA amplification, only is to use 0.2 μ M MHC-γ 2a primer, but not 0.2 μ M MKC primer.

1.4 Purified pcr product

Utilize the dna fragmentation of the above-mentioned pcr amplification of QIAquick PCR Purification test kit (QIAGEN) purifying, and be dissolved in the 10mM Tris-HCl (pH8.0) that contains 1mM EDTA.

1.5 Connect and conversion

Above-mentioned preparation comprise the about 140ng of dna fragmentation that comes from MABL-1 mouse Kappa type L chain V district encoding gene, be connected 3 hours with 50ng pGEM-T Easy carrier (Promega) in 15 ℃, reaction buffer comprises 30mM Tris-HCl (pH7.8), 10mM MgCl ₂, 10mM dithiothreitol (DTT), 1mM ATP and 3 T4DNA of unit ligase enzymes (Promega).

Then 1 μ l reaction mixture is added 50 μ l E. ColiIn the DH5 α competent cell (Toyobo Inc.), cell is in preserving 30 minutes on ice, and 42 ℃ of incubations 1 minute were preserved 2 minutes on ice again.Add 100 μ l SOC substratum (GIBCO BRL).Will E. ColiCell inoculation is (MolecularCloning:A laboratory Manual in the LB nutrient agar that contains 100 μ g/ml penbritins (SIGMA), people such as Sambrook, Cold SpringHarbor laboratory Press, 1989), 37 ℃ of overnight incubation obtain E. ColiTransformant.

Transformant contains in the LB substratum of 50 μ g/ml penbritins in 3ml, and 37 ℃ of overnight incubation use QIAprep Spin Minprep test kit (QIAGEN) to prepare plasmid DNA from culture.

Gained comprises the plasmid of the encoding gene in the mouse Kappa type L chain V district that comes from hybridoma MABL-1, called after pGEM-M1L.

According to above-mentioned same procedure, prepare plasmid from the dna fragmentation of purifying, it comprises the mouse H chain V district encoding gene that comes from hybridoma MABL-1, called after pGEM-M1H.

Prepare plasmid from the dna fragmentation of purifying, it comprises the mouse kappa type L chain V district encoding gene that comes from hybridoma MABL-2, called after pGEM-M2L.

Prepare plasmid from the dna fragmentation of purifying, it comprises the mouse H chain V district encoding gene that comes from hybridoma MABL-2, called after pGEM-M2H.

Embodiment 2 (dna sequencing)

Utilize Auto DNA Sequencer (Applied Biosystem) and ABI PRISMDye Terminator Cycle Sequencing Ready Reaction test kit (Applied Biosystem), according to manufacturers's rules, measure the nucleotide sequence of cDNA coding region in the above-mentioned plasmid.

The nucleotide sequence of the L chain V district encoding gene of the mouse antibodies MABL-1 that comprises among the plasmid pGEM-M1L is shown in SEQ ID No.5.

The nucleotide sequence of the H chain V district encoding gene of the mouse antibodies MABL-1 that comprises among the plasmid pGEM-M1H is shown in SEQ ID No.6.

The nucleotide sequence of the L chain V district encoding gene of the mouse antibodies MABL-2 that comprises among the plasmid pGEM-M2L is shown in SEQ ID No.7.

The nucleotide sequence of the H chain V district encoding gene of the mouse antibodies MABL-2 that comprises among the plasmid pGEM-M2H is shown in SEQ ID No.8.

Embodiment 3 (determining CDR)

The common structural similitude in V district of L chain and H chain, its per four framework regions are by three hypervariable regions, and promptly complementary determining region (CDR) links to each other.The aminoacid sequence of framework is relatively very conservative, and the aminoacid sequence of CDR alterable height (people such as Kabat E.A. extremely, " Sequencesof Proteins of Immunological Interest ", US Dept.Health andHuman Services, 1983).

Based on these facts,, be applied to study its homology in the antibody amino acid sequence database that people such as Kabat sets up with the aminoacid sequence of mouse anti human IAP variable region of mab.Table 1 is depicted as the CDR district that determines according to homology.

Table 1

Plasmid SEQ ID No. CDR (1) CDR (2) CDR (3)

pGEM-M1L 5 43-58 74-80 113-121

pGEM-M1H 6 50-54 69-85 118-125

pGEM-M2L 7 43-58 74-80 113-121

pGEM-M2H 8 50-54 69-85 118-125

Embodiment 4 (identifying expression, the chimeric MABL-1 antibody of preparation and the chimeric MABL-2 antibody of clone cDNA)

4.1 The carrier of chimeric MABL-1 antibody is expressed in preparation

The cDNA clone pGEM-M1L and the pGEM-M1H in the L chain of encoding murine antibody MABL-1 and H chain V district after the PCR method change, introduce HEF expression vector (WO92/19759) respectively, and the carrier of chimeric MABL-1 antibody is expressed in preparation.

The forward primer MLS (SEQ ID No.9) in design L chain V district and the forward primer MHS (SEQ ID No.10) in H chain V district, make it and to hybridize with the coding DNA that each V district leader sequence begins to locate, and comprise Kozak consensus sequence (J.Mol.Biol., 196,947-950,1987) and HindIII Restriction Enzyme site.Design L chain V district reverse primer MLAS (SEQID No.11) and H chain V district reverse primer MHAS (SEQ ID No.12) make it and can hybridize with the terminal coding DNA in J district, and comprise donor splicing site sequence and BamHI Restriction Enzyme site.

To comprise 10 μ l, 10 * PCR damping fluid II, 2mM MgCl ₂, 0.16mM dNTPs (dATP, dGTP, dCTP and dTTP), 100 μ l PCR solution of the 5 archaeal dna polymerase AmpliTaq Gold of unit, every kind of primer of 0.4uM and 8ng template DNA (pGEM-M1L or pGEM-M1H), 94 ℃ of preheatings of starting temperature 9 minutes, then successively 94 ℃ the heating 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute 20 seconds.This temperature cycle repeats 35 times, and 72 ℃ of reheat reaction mixtures are 10 minutes then.

With QIAquick PCR Purification test kit (QIAGEN) purified pcr product, then with HindIII and BamHI digestion.The product cloning in L chain V district is to HEF expression vector HEF-κ, and the product cloning in H chain V district is to HEF expression vector HEF-γ.Behind the dna sequencing, contain the plasmid of the dna fragmentation of correct dna sequence dna, respectively called after HEF-M1L and HEF-M1H.

4.2 The carrier of chimeric MABL-2 antibody is expressed in preparation

According to identical method as described in embodiment 4.1, cDNA is changed and clones, only being to use pGEM-M2L and pGEM-M2H is template DNA, but not pGEM-M1L and pGEM-M1H.Behind the dna sequencing, contain the plasmid of the dna fragmentation of correct dna sequence dna, respectively called after HEF-M2L and HEF-M2H.

4.3 Rotaring redyeing COS 7 cell

In the COS7 cell, test above-mentioned expression vector, observe the transient expression of chimeric MABL-1 and MABL-2 antibody.

(1) The gene of the chimeric MABL-1 antibody of transfection

Use Gene Pulser device (BioRad), by electroporation with HEF-M1L and HEF-M1H carrier cotransformation COS7 cell.Adding every kind of DNA (10 μ g) and 0.8ml contain 1 * 10 in the cuvette ⁷The PBS of cell/ml.Mixture is handled with 1.5KV, 25 μ F capacitor pulses.

Room temperature was recovered after 10 minutes, electroporation of cells was forwarded in the DMEM substratum of the foetal calf serum that comprises 10% no gamma globulin (GIBCO BRL).Cultivate after 72 hours, collect supernatant, centrifugal removal cell debris also reclaims.

(2) The encode gene of chimeric MABL-2 antibody of transfection

Utilize as same procedure as described in the embodiment 4.3-(1), the encoding gene cotransfection COS7 cell with chimeric MABL-2 antibody only is to use HEF-M2L and HEF-M2H carrier, but not HEF-M1L and HEF-M1H carrier.In kind reclaim supernatant.

4.4 Flow cytometry

Utilize above-mentioned COS7 cells and supernatant to carry out flow cytometry, measure its antigen combination.To express the COS7 cell of chimeric MABL-1 antibody or express the culture supernatant of cell of the COS7 of chimeric MABL-2 antibody, perhaps human IgG antibody in contrast (SIGMA) adds 4 * 10 ⁵Among the L-1210 L1210 of expressing human IAP, ice bath.After the washing, to the anti-human IgG antibody (Cappel) who wherein adds the FITC mark.After incubation and the washing, (BECTON DICKINSON) measures its fluorescence intensity with the FACScan device.

Because the L1210 cell of chimeric MABL-1 and MABL-2 antibodies specific associative list intelligent IAP can determine that these chimeric antibodies have the correct V plot structure (Fig. 1-3) of mouse monoclonal antibody MABL-1 and MABL-2 respectively.

Embodiment 5 (the reconstruction strand Fv (scFv) of preparation antibody MABL-1 and antibody MABL-2)

5.1 The reconstruction strand Fv of preparation antibody MABL-1

Be prepared as follows the reconstruction strand Fv of antibody MABL-1.Utilize PCR method increase respectively H chain V district and the L chain V district of antibody MABL-1, and joint, and link together, with the reconstruction strand Fv of preparation antibody MABL-1.The preparation method as shown in Figure 4.Utilize the strand Fv of six kinds of primers (A-F) preparation antibody MABL-1.Primer A, C and E have just sequence, and primer B, D and F have antisense sequences.

The forward primer VHS in H chain V district (primer A, SEQ ID No.13) is designed to and can hybridizes with H chain V district N end coding DNA, and comprises NcoI Restriction Enzyme recognition site.The reverse primer VHAS in H chain V district (primer B, SEQ ID No.14) is designed to and can hybridizes with H chain V district C end coding DNA, and overlapping with joint.

The forward primer LS of joint (primer C, SEQ ID No.15) is designed to and can hybridizes with joint N end coding DNA, and overlapping with H chain V district C end coding DNA.The reverse primer LAS of joint (primer D, SEQ ID No.16) is designed to and can hybridizes with joint C end coding DNA, and overlapping with L chain V district N end coding DNA.

The forward primer VLS in L chain V district (primer E, SEQ ID No.17) is designed to and can hybridizes with joint C end coding DNA, and overlapping with L chain V district N end coding DNA.Reverse primer VLAS-FLAG (the primers F in L chain V district, SEQ ID No.18) is designed to and hybridizes with L chain V district C end coding DNA, and has a FLAG peptide-coding sequence (people such as Hopp.T.P., Bio/Technology, 6,1204-1210,1988), two terminator codons and EcoRI Restriction Enzyme recognition site.

In first PCR step, carry out A-B, C-D, three reactions of E-F, its PCR product of purifying.Three kinds of PCR products that the first step PCR obtains are assembled according to its complementarity.Add primer A and F then, the total length coding DNA of the reconstruction strand Fv of amplification antibody MABL-1 (the second step PCR).Among the first step PCR respectively with the plasmid pGEM-M1H (referring to embodiment 2) in encoding antibody MABL-1H chain V district, (this connector area comprises: (people such as Huston J.S. Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly GlyGly Ser (SEQ ID No.19)) to comprise the plasmid pSC-DP1 of connector area DNA sequences encoding, Proc.Natl.Acad.Sci.USA, 85,5879-5883,1988) and the plasmid pGEM-M1L (referring to embodiment 2) in encoding antibody MABL-1L chain V district make template.

50 μ l solution of the first step PCR reaction comprise 5 μ l, 10 * PCR damping fluid II, 2mMMgCl ₂, 0.16mM dNTPs, the 2.5 archaeal dna polymerase AmpliTaq Gold of unit (PERKINELMER), every kind of primer of 0.4 μ M and every kind of template DNA of 5ng.PCR solution is 94 ℃ of preheatings of starting temperature 9 minutes, then 1 minute, 65 ℃ 1 minute, 72 ℃ of 94 ℃ of heating 1 minute and 20 seconds successively.This temperature cycle repeats 35 times, and 72 ℃ of reheat reaction mixtures are 7 minutes then.

Utilize QIApuick PCR Purification test kit (QIAGEN) purified pcr product A-B (371bp), C-D (63bp) and E-F (384bp), and in the second step PCR, assemble.Among the second step PCR, will comprise 120ng the first step PCR product A-B, 20ng PCR product C-D and 120ng PCR product E-F, 10 μ l, 10 * PCR damping fluid II, 2mMMgCl ₂, 0.16mM dNTPs, the 5 archaeal dna polymerase AmpliTaq Gold of unit (PERKINELMER) 9 μ l PCR solution, 94 ℃ of preheatings of starting temperature 8 minutes, then successively 94 ℃ the heating 2 minutes, 65 ℃ 2 minutes, 72 ℃ 2 minutes.This temperature cycle repeats 2 times, adds every kind of primer A and F 0.4 μ M respectively in the reaction then.Mixture is 94 ℃ of preheatings of starting temperature 1 minute, then 1 minute, 65 ℃ 1 minute, 72 ℃ of 94 ℃ of heating 1 minute and 20 seconds successively.This temperature cycle repeats 35 times, and 72 ℃ of reheat reaction mixtures are 7 minutes then.

The 843bp dna fragmentation of second step of purifying PCR preparation is with NcoI and EcoRI digestion.The dna fragmentation that obtains is cloned into the pSCFVT7 carrier.Expression vector pSCFVT7 comprises and is suitable for E. ColiThe pelB signal sequence of periplasmic expression system (people such as Lei S.P., J.Bacteriology, 169,4379-4383,1987).Behind the dna sequencing, will comprise the segmental plasmid of coding DNA of the correct aminoacid sequence of reconstruction strand Fv of antibody MABL-1, called after " pscM1 " (referring to Fig. 5).The nucleotide sequence of the antibody MABL-1 reconstruction strand Fv that plasmid pscM1 comprises and aminoacid sequence are shown in SEQ ID No.20.

Utilize PCR method to change the pscM1 carrier, rebuild the carrier of strand Fv with preparation expressing antibodies MABL-1 in mammalian cell.The dna fragmentation that obtains is introduced the pCHO1 expression vector.Expression vector pCHO1 is through EcoRI and SmaI digestion DHFR-Δ E-rvH-PM1-f (WO92/19759), remove antibody gene, and connects EcoRI-NotI-BamHI adapter (Takara Shuzo) and make up.

Sal-VHS primer shown in the design SEQ ID No.21, as the PCR forward primer, it can be hybridized with the coding DNA of H chain V district N end, and comprises Sal I Restriction Enzyme recognition site.Design is the FRHlanti primer shown in SEQ ID No.22, and as the PCR reverse primer, it can be hybridized with the coding DNA of the first frame sequence end.

To comprise 10 μ l, 10 * PCR damping fluid II, 2mM MgCl ₂, 0.16mM dNTPs, the 5 archaeal dna polymerase AmpliTaq Gold of unit, 0.4 every kind of primer of μ l M and 8ng template DNA (pscM1) 100 μ l PCR solution, 95 ℃ of preheatings of starting temperature 9 minutes, then successively 95 ℃ the heating 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute 20 seconds.This temperature cycle repeats 35 times, and 72 ℃ of reheat reaction mixtures are 7 minutes then.

Utilize QIAquick PCR Purification test kit (QIAGEN) purified pcr product,, obtain the N end coding DNA fragment of the reconstruction strand Fv of antibody MABL-1 with SalI and MboII digestion.With MboII and EcoRI digestion pscM1 carrier, obtain the C end coding DNA fragment of the reconstruction strand Fv of antibody MABL-1.This SalI-MboII dna fragmentation and MboII-EcoRI dna fragmentation are cloned into the pCHO1-IgS carrier.Behind the dna sequencing, will comprise the plasmid called after " pCHOM1 " (referring to Fig. 6) of target DNA sequence.Expression vector pCHO1-Igs comprises the mouse IgG1 signal sequence (Nature, 322,323-327,1988) that is suitable for mammalian cell secreting, expressing system.The nucleotide sequence of the antibody MABL-1 reconstruction strand Fv that plasmid pCHOM1 comprises and aminoacid sequence are shown in SEQ ID No.23.

5.2 The reconstruction strand Fv of preparation antibody MABL-2

According to the foregoing description 5.1 methods, the reconstruction strand Fv of preparation antibody MABL-2.Utilize the plasmid pGEM-M2H (referring to embodiment 2) in the H chain V district of coding MABL-2 among the first step PCR, but not pGEM-M1H, and the plasmid pGEM-M2L (referring to embodiment 2) in the L chain V district of coding MABL-2, but not pGEM-M1L, thereby obtain plasmid pscM2, wherein comprise the coding DNA fragment of the strand Fv purpose aminoacid sequence of antibody MABL-2.The nucleotide sequence of the reconstruction strand Fv of the antibody MABL-2 that comprises among the plasmid pscM2 and aminoacid sequence are shown in SEQ ID No.24.

Utilize PCR method to change the pscM2 carrier, preparation carrier pCHOM2 is used for expressing at mammalian cell, wherein comprises the coding DNA fragment of the correct aminoacid sequence of reconstruction strand Fv of antibody MABL-2.The nucleotide sequence of the reconstruction strand Fv of the antibody MABL-2 that comprises among the plasmid pCHOM2 and aminoacid sequence are shown in SEQ ID No.25.

5.3 Rotaring redyeing COS 7 cell

Test pCHOM2 carrier in the COS7 cell, the transient expression of the reconstruction strand Fv of observation antibody MABL-2.

Utilize Gene Pulser device (BioRad), pCHOM2 is transformed the COS7 cell by electroporation.Adding every kind of DNA (10 μ g) and 0.8ml contain 1 * 10 in the cuvette ⁷The PBS of cell/ml.Mixture is handled with 1.5KV, 25 μ F capacitor pulses.

Room temperature is recovered electroporation of cells to be forwarded to the IMDM substratum (GIBCO BRL) that contains 10% foetal calf serum after 10 minutes.Cultivate after 72 hours, collect supernatant, centrifugal removal cell debris also reclaims.

5.4 In the COS7 cells and supernatant, detect the reconstruction strand Fv of antibody MABL-2

By Western trace method, determine to exist in the culture supernatant of COS7 cell of pCHOM2 carrier transfection the strand Fv of antibody MABL-2.

With the culture supernatant of the COS7 cell of pCHOM2 carrier transfection, and the COS7 cells and supernatant of the pCHO1 transfection that compares, carry out the SDS electrophoresis, and forward (Schleicher ﹠amp on the REINFORCED NC film to; Schuell).With 5% skimmed milk (Morinaga Nyu-gyo) closing membrane,, mix with anti-FLAG antibody (SIGMA) with 0.05% Tween20-PBS washing.Film is incubation, washing and mixed-alkali Phosphoric acid esterase link coupled mouse IgG antibody (Zymed) at room temperature.After incubation and the washing, add substrate solution (Kirkegaard Perry Laboratories) colour developing (Fig. 7) under the room temperature.

Only in the culture supernatant of the COS7 cell of introducing the pCHOM2 carrier, detect FLAG peptide specific protein, thereby the reconstruction strand Fv of proof antibody MABL-2 is secreted in this culture supernatant.

5.5 Flow cytometry

Utilize above-mentioned COS7 cells and supernatant, measure the antigen combination by flow cytometry.With the COS7 cells and supernatant of the reconstruction strand Fv of expressing antibodies MABL-2, the COS7 cells and supernatant that transforms through the pCHO1 carrier that perhaps compares adds 2 * 10 ⁵Among the L-1210 L1210 of expressing human integrin associated protein matter (IAP), perhaps add in the L1210 clone of the pCOS1 conversion that compares.After ice bath and the washing, add mouse anti FLAG antibody (SIGMA).Incubation cell and washing then.The anti-mouse IgG antibody (BECTON DICKINSON) that adds the FITC mark then, incubation and washed cell once more.Measure fluorescence intensity with FACScan device (BECTON DICKINSON) subsequently.

Because the strand Fv of antibody MABL-2 combines with the L1210 cell-specific of expressing human IAP, can determine that the reconstruction strand Fv of antibody MABL-2 has avidity (referring to Fig. 8-11) to people's integrin associated protein matter (IAP).

5.6 Competitive ELISA

According to inhibition activity, measure the combination activity of the reconstruction strand Fv of antibody MABL-2 to the mouse monoclonal antibody conjugated antigen.

To resist FLAG antibody to be adjusted into 1 μ g/ml, add in every hole of 96 orifice plates, 37 ℃ of incubations 2 hours.After the washing, seal with 1%BSA-PBS.After incubation and the washing, will introduce the COS7 cells and supernatant of secretor type people IAP antigen gene (SEQ ID No.26) under the room temperature, be diluted to two volumes, add in every hole with PBS.Under the room temperature after incubation and the washing, 50 μ l are adjusted into the mixture of COS7 cells and supernatant of reconstruction strand Fv of the expressing antibodies MABL-2 of the biotinylation MABL-2 antibody of 100ng/ml and 50 μ l serial dilutions, add in every hole.After incubation and the washing, add alkaline phosphatase link coupled streptavidin (Zymed) in every hole under the room temperature.After incubation and the washing, add substrate solution (SIGMA) under the room temperature, measure in every hole reaction mixture in the absorbancy at 405nm place.

The result shows, compares with the COS7 cells and supernatant of introducing pCHO1, and by contrast, reconstruction strand Fv (MABL2-scFv) the concentration dependent ground of antibody MABL-2 significantly suppresses mouse antibodies MABL-2 and combines (Figure 12) with people IAP is antigenic.Therefore prompting among the reconstruction strand Fv of antibody MABL-2, has each V district that derives from mouse monoclonal antibody MABL-2 of correct structure.

5.7 External apoptosis induction effect

The L1210 cell of end user IAP gene transfection, L1210 cell and the CCRF-CEM cell that compare through the transfection of pCOS1 carrier, by Annexin-V dyeing (Boehninger Mannheim), detect the apoptosis-induced effect of the reconstruction strand Fv of antibody MABL-2.

1 * 10 ⁵In above-mentioned every kind of cell, add the COS7 cells and supernatant of reconstruction strand Fv of expressing antibodies MABL-2 or the COS7 cells and supernatant through the transfection of pCHO1 carrier that compares by 50% final concentration, mixture was cultivated 24 hours.Carry out Annexin-V dyeing then, measure fluorescence intensity with FACScan device (BECTON DICKINSON).

The coloration result of Annexin-V is respectively shown in Figure 13-18.The point in lower-left district is represented viable cell, and the point in bottom right district is represented the early stage cell of apoptosis, and the point in upper right district is represented apoptosis cell in late period.The result shows that significantly inducing specific is at the antigenic L1210 necrocytosis of people IAP (Figure 13-16) for the reconstruction strand Fv (MABL2-scFv) of antibody MABL-2, and compared with the control, the strand Fv of reconstruction also significantly induces CCRF-CEM necrocytosis (Figure 17-18).

5.8 The strand Fv that comes from MABL-2 that purifying CHO cell produces

With pCHOM2 carrier transfection CHO cell, set up the Chinese hamster ovary celI system of the strand Fv (polypeptide) that constant expression comes from antibody MABL-2.

Use Gene Dulser device (BioRad), the pCHOM2 carrier is transformed Chinese hamster ovary celI by electroporation method.Add DNA (10 μ g) and 0.7ml in the cuvette and contain Chinese hamster ovary celI (1 * 10 ⁷The mixture of the PBS of cell/ml).Mixture is handled with 1.5KV, 25 μ F capacitor pulses.Room temperature was recovered after 10 minutes, electroporation of cells is forwarded in the free nucleic acid α-MEM substratum (GIBCO BRL) that contains 10% foetal calf serum cultivate.The clone who obtains selects the high clone of expression level, as the production clone of the strand Fv that comes from antibody MABL-2 with the expression that SDS-PAGE confirms target protein matter.In containing the serum free medium CHO-S-SFM II (GIBCO BRL) of 10nM methotrexate (SIGMA), cultivate this clone.Then, collect culture supernatant, centrifugal removal cell debris also reclaims.

5.9 The strand Fv that comes from MABL-2 that purifying CHO cell produces

The culture supernatant of the Chinese hamster ovary celI system of the expression strand Fv that embodiment 5.8 is obtained utilizes hemodialysis's post (PAN130SF, ASAHI MEDICALS) to concentrate nearly 20 times.Concentrated solution-20 ℃ preservation, and when purifying, melt.

Utilize three kinds of chromatography methods, i.e. Blue-sepharose, hydroxyapatite and gel-filtration, purification of single stranded Fv from the Chinese hamster ovary celI culture supernatant.

(1) The Blue-sepharose column chromatography

With 20mM acetate buffer (pH6.0) spissated supernatant is diluted 10 times, centrifugal removal insolubles (10,000 * rev/min, 30 minutes).With same buffer balance Blue-sepharose post (20ml), with sample on the supernatant.After washing post with same buffer, utilize in the same buffer NaCl progressively gradient 0.1,0.2,0.3,0.5 to up to 1.0M, the protein that adsorbs in the wash-out post.Cross component and each elution fraction with the SDS-PAGE analysis stream.With confirming that the component (0.1-0.3M NaCl elution fraction) that contains strand Fv mixes, utilize Centri Prep-10 (AMICON) to concentrate up to about 20 times.

(2) Hydroxyapatite

(1) concentrated solution that obtains in dilutes 10 times with 10mM phosphate buffered saline buffer (pH7.0), and last sample hydroxyapatite column (20ml, BIORAD).Wash post with 60ml 10mM phosphate buffered saline buffer (pH7.0).Then, use linear gradient, the protein that adsorbs in the wash-out post (referring to Figure 19) up to the sodium phosphate buffer of 200mM.Analyze each component with SDS-PAGE, confirm in component A and B component, to exist strand Fv.

(3) Gel-filtration

Concentrate component A and B in (2) respectively with CentriPrep-10, and go up sample to using 20mM acetate buffer (pH6.0) the equilibrated TSKgelG3000SWG post (21.5 * 600mm) that contains 0.15M NaCl.Tomographic map as shown in figure 20.Analyze each component through SDS-PAGE, confirmed that two main peaks (AI and BI) are purpose strand Fv.During gel-filtration was analyzed, component A was at 36KDa apparent molecular weight place wash-out, and B component is at 76kDa place wash-out.Analyze the strand Fv (AI, BI) of purifying with the 15%SDS polyacrylamide gel.Having or not having and handle sample in the presence of the reductive agent, carry out electrophoresis according to Laemmli ' s method.Use coomassie brilliant blue staining protein then.As shown in figure 21, no matter whether reductive agent exists, and AI and BI present the strand at 35kDa apparent molecular weight place.Can infer that from the above AI is the monomer of strand Fv, BI is the non-covalent bonded dimer of strand Fv.(7.5 * 60mm) by gel-filtration analysis component AI and BI, shows only to detect the monomer peak in component AI, only detects dimer peak (Figure 22) in B component I to use TSKgel G3000SW post.Dimer component (B component I) accounts for 4 percent of total strand Fv.Dimer above 90% in the dimer component can 4 ℃ of stable preservations above 1 month.

5.10 Be structured in the E.coli cell and express carrying of the strand Fv that comes from antibody MABL-2 Body

Utilize PCR method to change the pscM2 carrier, exist with preparation E. ColiThe carrier of the strand Fv of effective expression antibody MABL-2 in the cell.The dna fragmentation that obtains is introduced the pSCFVT7 expression vector.

Design is the Nde-VHSm02 primer shown in SEQ ID No.27, and as the PCR forward primer, it can be hybridized with H chain V district N end coding DNA, and comprises initiator codon and NdeI Restriction Enzyme recognition site.Design is the VLAS primer shown in SEQ ID No.28, and as the PCR reverse primer, it can be hybridized with L chain V district C end coding DNA, and comprises two terminator codons and EcoRI Restriction Enzyme recognition site.For E. ColiMiddle effective expression comprises 5 point mutation in forward primer Nde-VHSm02 with in the interfertile part of H chain V district N end coding DNA.

To comprise 10 μ l, 10 * PCR damping fluid #1,1mM MgCl ₂, 0.2mM dNTPs, 5 unit K OD archaeal dna polymerases (all from TOYOBO), every kind of primer of 1 μ M and 100ng template DNA (pscM2) 100 μ l PCR solution, 15 seconds, 65 ℃ of 98 ℃ of heating 2 seconds and 74 ℃ are 30 seconds successively.This temperature cycle repeats 25 times.

Utilize QIAquick PCR Purification test kit (QIAGEN) purified pcr product, with NdeI and EcoRI digestion, the dna fragmentation that obtains is cloned into the pSCFVT7 carrier then, has removed pel B signal sequence with NdeI and EcoRI digestion in this carrier.Behind dna sequencing, with the plasmid called after " pscM2DEm02 " (referring to Figure 23) that comprises the dna fragmentation that has the target DNA sequence that produces.The nucleotide sequence of the strand Fv that comes from antibody MABL-2 that comprises among the plasmid pscM2DEm02 and aminoacid sequence are shown in SEQ ID No.29.

5.11 In the E.coli cell, express the strand Fv that comes from antibody MABL-2

Transform with the pscM2DEm02 carrier E. ColiBL21 (DE3) pLysS (STRATAGENE) obtains expressing the strand Fv's that comes from antibody MABL-2 E. ColiBacterial strain.Check the expression of target protein matter among the clone who obtains with SDS-PAGE, select the high clone of expression level, as the production bacterial strain of the strand Fv that comes from antibody MABL-2.

5.12 The strand Fv that comes from antibody MABL-2 that produces among the purifying E.coli

Conversion is obtained E. ColiMono-clonal was cultivated 7 hours for 28 ℃, then 28 ℃ of overnight incubation in 70ml LB substratum in 3ml LB substratum.

Pre-culture is transferred in the 7L LB substratum, 300rpm (rev/min) stir, with 28 ℃ of cultivations of Jar fermentor tank.When the substratum absorbancy reaches O.D.=1.5, induce bacterium, cultivated then 3 hours with 1mM IPTG.

Substratum centrifugal (10,000 * g, 10 minutes) reclaims the bacterial precipitation thing.In bacterium, add the 50mM Tris-HCl damping fluid (pH8.0) that contains 5mM EDTA, 0.1M NaCl and 1% Triton X-100, the ultrasonic disruption bacterium (work output: 4, working cycle: 70%, 1 minute * 10 times).The suspension of centrifugal broken bacterium (12,000xg, 10 minutes) is with the precipitation inclusion body.Isolating inclusion body is mixed with the 50mM Tris-HCl damping fluid (pH8.0) that contains 5mM EDTA, 0.1M NaCl and 4%Triton X-100, supersound process (work output: 4 once more, working cycle: 50%, 30 seconds * 2 times), centrifugal (12,000xg, 10 minutes) separate target protein matter precipitation, remove the protein that comprises in the supernatant.

Cracking contains the inclusion body of target protein matter in containing the 50mM Tris-HCl damping fluid (pH8.0) of 6M urea, 5mM EDTA and 0.1M NaCl, last sample is to Sephacryl S-300 gel-filtration column (5 * 90cm, Amersharm Pharmacia), this post 50mM Tris-HCl damping fluid (pH8.0) balance that contains 4M urea, 5mM EDTA, 0.1M NaCl and 10mM mercaptoethanol, flow velocity is 5ml/ minute, to remove bonded high molecular strand Fv.Analyze the component that obtains through SDS-PAGE, highly purified protein component is diluted to O.D with the used damping fluid of gel-filtration ₂₈₀=0.25.With the 50mM Tris-HCl damping fluid (pH8.0) that contains 5mM EDTA, 0.1M NaCl, 0.5MArg, 2mM reduced glutathione and 0.2mM oxidized form Triptide, this component of dialysing 3 times is so that protein refolding then.Further with dialyse this component three times of the 20mM acetate buffer (pH6.0) that contains 0.15M NaCl, with exchange buffering liquid.

To dialyse sample on the product to Superdex 200pg gel-filtration column (2.6 * 60cm, Amersharm Pharmacia), this post is with 20mM acetate buffer (pH6.0) balance that contains 0.15M NaCl, and is a small amount of by the crosslinked high molecular weight protein of intermolecular S-S key to remove.As shown in figure 24, go out two peaks, i.e. main peak and submaximum at broad peak (be contemplated to high molecular and assemble thing) back wash-out.The wash-out position at these two peaks is analyzed in SDS-PAGE analysis (referring to Figure 21) and gel-filtration, and the prompting main peak is a strand Fv monomer, the covalently bound strand Fv of submaximum right and wrong dimer.Non-covalent bonded dimer accounts for 4% of total strand Fv.

5.13 Come from the external apoptosis induction activity of the strand Fv of antibody MABL-2

Utilize the L1210 cell (hIAP/L1210) introduced people IAP gene, according to two kinds of Annexin-V dyeing (Boehringer Mannheim) methods, detect Chinese hamster ovary celI and E. ColiThe apoptosis-induced effect of the strand Fv (MABL2-scFv) that comes from antibody MABL-2 that produces.

In the first method, to 5 * 10 ⁴Add sample antibody in the individual hIAP/L1210 cell, final concentration is 3 μ g/ml, cultivates 24 hours.Analytic sample antibody, i.e. monomer and the dimer of the MABL-2 strand Fv that obtains from Chinese hamster ovary celI among the embodiment 5.9, among the embodiment 5.12 from E. ColiMonomer and the dimer of the MABL-2 strand Fv that obtains, and the mouse IgG antibody that compares.Carry out Annexin-V dyeing after the cultivation, (BECTONDICKINSON) measures its fluorescence intensity with the FACScan device.

In the second method, to 5 * 10 ⁴Add sample antibody in the individual hIAP/L1210 cell, final concentration is 3 μ g/ml, cultivates 2 hours, and mixes with anti-FLAG antibody (SIGMA), and final concentration is 15 μ g/ml, cultivates 22 hours again.Analyzing samples antibody, they are monomers of the MABL-2 strand Fv that obtains from Chinese hamster ovary celI among the embodiment 5.9, and the mouse IgG antibody that compares.Carry out Annexin-V dyeing after the cultivation, use its fluorescence intensity of FACScan measurement device.

The result of Annexin-V staining analysis is shown in Figure 25-31.The result shows, Chinese hamster ovary celI and E. ColiIn the dimer of the MABL-2 strand Fv polypeptide that produces, (Figure 25) compared with the control, remarkable inducing cell death (Figure 26,27), and Chinese hamster ovary celI with E. ColiThe monomer of the middle MABL-2 strand Fv polypeptide that produces is not then observed apoptosis-induced effect (Figure 28,29).When using anti-FLAG antibody together, (Figure 30) compared with the control, the remarkable inducing cell death of monomer (Figure 31) of the strand Fv polypeptide that comes from MABL-2 antibody that produces in the Chinese hamster ovary celI.

5.14 The monomer of scFv/CHO polypeptide and dimer are anti-swollen to the human myeloma model mice The knurl effect

(1) Human IgG in the quantitative measurment mice serum

The human IgG (M protein) that utilizes following ELISA method to measure to contain in the mice serum, produce by human myeloma cell.(BIOSOURCE Lot#7902) is diluted to 1 μ g/ml, and every hole adds 100 μ l in 96 orifice plates (Nunc), and 4 ℃ are incubated overnight, make antibody immobilization with the anti-human IgG antibody of goat with 0.1% bicarbonate buffer (pH9.6).After the sealing, add mice serum that 100 μ l progressively dilute or (CAPPEL, Lot#00915), incubation is 2 hours under the room temperature as the human IgG of standard to every hole.After the washing, (BIOSOURCE, Lot#6202), incubation is 1 hour under the room temperature to add the anti-human IgG antibody of the alkali phosphatase enzyme mark that 100 μ l5000 doubly dilute.The washing back adds substrate solution.Behind the incubation, use MICROPLATE READER Model 3550 (BioRad) to measure the absorbancy at 405nm place.Based on the working curve that the absorbancy by the human IgG standard substance obtains, calculate the concentration of human IgG in the mice serum.

(2) Preparation administrable antibody

On administration same day, the monomer and the dimer of scFv/CHO polypeptide is diluted to 0.4mg/ml or 0.25mg/ml respectively, preparation administrable sample with the PBS (-) of sterile filtration.

(3) The mouse model of preparation human myeloma

Be prepared as follows the mouse model of human myeloma.To be suspended in the RPMI1640 substratum that contains 10% foetal calf serum (GIBCO-BRL) (GIBCO-BRL) at the KPMM2 cell (JP-Appl.7-236475) of SCID mouse (Japan Clare) interior generation, be adjusted into 3 * 10 ⁷Cell/ml.By the tail vein with 200 μ l KPMM2 cell suspensions (6 * 10 ⁶Cell/mouse) transplant to SCID mouse (male, 6 ages in week), this SCID mouse takes off sialic acid GM1 antibody (WAKO JUNYAKU, 1 bottle is dissolved in 5ml) in transplanting subcutaneous injection the day before yesterday.

(4) Antibody administration

With the antibody sample of preparation in (2), monomer (250 μ l) and dimer (400 μ l) are administered intravenously in the human myeloma cell model mice of preparation in (3) by tail.Transplant the KPMM2 cell and begin administration after 3 days, every day 2 times, carried out 3 days.In contrast, by the PBS (-) of tail intravenously administrable 200 μ l sterile filtrations, one day 2 times, carried out 3 days equally.Every group comprises 7 mouse.

(5) Estimate the monomer of scFv/CHO polypeptide and dimeric with the human myeloma model mice Anti-tumour effect

According to (M protein) variation of concentration of human IgG in the mice serum and the survival time of mouse, estimate scFv/CHO polypeptide monomer and dimeric anti-tumour effect with the human myeloma model mice.Collect mice serum in 24 days behind the transplanting KPMM2 cell, utilize ELISA method described in above (1) to measure the variation of human IgG concentration.The amount of human IgG in the serum of PBS (-) administration group (contrast) (M protein) is brought up to about 8500 μ g/ml, and the amount of the human IgG of scFv/CHO dimer administration group is obviously lower, is equal to or less than 1/10th of control group.So this result shows scFv/CHO dimer strongly inhibited KPMM2 cell growth (Figure 32).As shown in figure 33, compare, obviously prolong in the observed survival time of scFv/CHO dimer administration group with PBS (-) administration group.

Can confirm that as mentioned above the scFv/CHO dimer has anti-tumour effect for the human myeloma model mice.Can think that scFv/CHO dimer, the i.e. antibody of change of the present invention, its anti-tumour effect are because the antibody of this change has apoptosis-induced effect.

5.15 The hemagglutination reaction experiment

Publish according to Tokyo Kagaku Dojin, the Zoku-Seikagaku Jikken Koza " Immuno-Biochemical Investigation " that Japanese biochemical society edits carries out the hemagglutination reaction test and determines hemagglutination reaction.

Get healthy donor blood with the syringe of handling through heparin,, use the red cell suspension of PBS (-) preparation final concentration 2% then with PBS (-) washing three times.Test sample book comprises, MABL-2 antibody, and the monomer and the dimer of the strand Fv polypeptide that Chinese hamster ovary celI produces, E. ColiThe monomer and the dimer of the strand Fv polypeptide that produces are contrast (ZYMED) with mouse IgG.Utilization is available from the round bottom 96 orifice plates research hemagglutination effect of Falcon.Add the above-mentioned antibody sample of 50 μ l in every hole, and 50 μ l, 2% red cell suspension, Yu Kongzhong mixes.37 ℃ of incubations after 2 hours spend the night 4 ℃ of preservations of reaction mixture, measure its hemagglutination reaction.Every hole 50 μ l PBS (-) are used in contrast, by carry out the hemagglutination reaction test with quadrat method.Mouse IgG that uses and the antibody final concentration of antibody MABL-2 are 0.01,0.1,1.0,10.0 or 100.0 μ g/ml.The strand Fv final concentration that uses is 0.004,0.04,0.4,4.0,40.0 or 80.0 μ/ml, only exists E. ColiUnder the polypeptide dimer situation that produces, use 160.0 μ g/ml in addition.The result is as shown in table 2.For antibody MABL-2, under greater than 0.1 μ/ml concentration, observe hemagglutination reaction, and the monomer of strand Fv and dimer are not all observed hemagglutination reaction.

Table 2 hemagglutination reaction test

Contrast 0.01 0.1 1 10 100 μ g/mL mIgG------MABL-2--+++++++++ (complete)
	-----monomer scFv/CHO------dimer that contrasts 0.004 0.04 0.4 4 40 80 μ g/mL scFv/CHO
Contrast 0.004 0.04 0.4 4 40 80 160 μ g/mL scFv/E.coli------monomer scFv/E.coli--------dimer

Embodiment 6 comprises the antibody sc (Fv) of the change in 2 H chain V districts and 2 L chain V districts ₂, and the antibody MABL-2scFv with different lengths joint

6.1 Make up table expressing antibodies MABL-2sc (Fv) ₂ Plasmid

Utilize following PCR method to change above-mentioned pcHOM2 (coding DNA that wherein comprises the scFv that comes from above-mentioned MABL-2), the dna fragmentation that produces is inserted pcHOM2, with the antibody [sc (Fv) of preparation change ₂] expression plasmid of (wherein comprising two H chain V districts and two L chain V districts of coming from antibody MABL-2).

The PCR the primer comprises, EF1 primer (SEQ ID NO:30) is a forward primer, be designed to and hybridize with EF1 α coding DNA, and reverse primer (SEQ ID NO:19), be designed to and hybridize with L chain V district C end coding DNA, and comprise the DNA sequences encoding of connector area and the VLLAS primer (SEQ ID NO:31) that comprises SalI Restriction Enzyme recognition site.

100 μ l PCR solution comprise 10 μ l, 10 * PCR damping fluid #1,1mM MgCl ₂, 0.2mM dNTPs (dATP, dGTP, dCTP and dTTP), 5 unit K OD archaeal dna polymerases (Toyobo, Inc.), every kind of primer of 1 μ M and 100ng template DNA (pCHOM2).With this PCR solution 94 ℃ of heating 30 seconds, 50 ℃ 30 seconds and 74 ℃ successively 1 minute.This temperature cycle repeats 30 times.

Utilize QIAquick PCR Purification test kit (QIAGEN) purified pcr product, and digest with SalI.The dna fragmentation that produces is cloned into pBluescript KS ⁺Carrier (Toyobo, Inc.).Behind the dna sequencing, digest the plasmid that comprises the target DNA sequence, the dna fragmentation that obtains is connected with the pCHOM2 that digests through SalI with rapid DNA Ligation test kit (BOEHRINGER MANNHEIM) with SalI.Behind the dna sequencing, will comprise the plasmid called after " pCHOM2 (Fv) of target DNA sequence ₂" (referring to Figure 34).Plasmid pCHOM2 (Fv) ₂In the antibody MABL-2sc (Fv) that comprises ₂The nucleotide sequence in district and aminoacid sequence are shown in SEQ ID No.32.

6.2 Preparation has the expression plasmid of the antibody MABL-2scFv of different lengths joint

H chain and L chain encoding cDNA that utilization comes from MABL-2 make template, and preparation scFv as described below wherein comprises different lengths joint and V district, by [H chain]-[L chain] (hereinafter claiming " HL ") or [L chain]-[H chain] (hereinafter claiming " LH ") order design.

Utilize pCHOM2 (Fv) ₂For template is carried out the PCR program, make up HL type scFv.In the PCR step, use primer, or primer is to CFHL-F2 (SEQ ID NO:35) and CFHL-R1 (SEQ IDNO:36) to CFHL-F1 (SEQ ID NO:33) and CFHL-R2 (SEQ IDNO:34), and the KOD polysaccharase.Carry out the PCR program, temperature cycle is 94 ℃ 30 seconds, 60 ℃ 30 seconds and 72 ℃ 1 minute, repeats 30 times, and producing the H chain cDNA that its 5 ' end contains leader sequence, or its 3 ' end contains the L chain cDNA of FLAG sequence.Mix H chain and the L chain cDNA produced, utilize mixture to carry out PCR for template and KOD polysaccharase, temperature cycle for successively 94 ℃ 30 seconds, 60 ℃ 30 seconds and 721 minutes, repeat 5 times.In reaction mixture, add CFHL-F1 and CFHL-R1 primer, repeat said temperature circulation 30 times then, carry out the PCR reaction, do not have the HL-0 type cDNA of joint with generation.

For making up LH type scFv, utilize pGEM-M2L and pGEM-M2H to be template, wherein comprise the code cDNA in L chain V district and the H chain V district of antibody MABL-2 respectively, carry out PCR reaction (referring to JP-Appl.11-63557).Use primer to T7 (SEQ ID NO:37) and CFLH-R2 (SEQ ID NO:38), or primer is to CFLH-F2 (SEQ ID NO:39) and CFLH-R1 (SEQ ID NO:40), and KOD polysaccharase (Toyobo Inc.).Carry out PCR reaction, temperature cycle is 94 ℃ 30 seconds, 60 ℃ 30 seconds and 72 ℃ in turn 1 minute, repeats 30 times, holds the L chain cDNA that comprises leader sequence to produce 5 ', or it 3 ' holds the H chain cDNA that comprises the FLAG sequence.Mixing L chain and the H chain cDNA produced, is template with this mixture, uses the KOD polysaccharase to carry out PCR, and temperature cycle comprises successively 94 ℃ 30 seconds, 60 ℃ 30 seconds and 72 ℃ 1 minute, repeats 5 times.Add T7 and CFLH-R1 primer in the reaction mixture, the said temperature circulation repeats 30 times and reacts.With the reaction product is template, utilizes primer that CFLH-F4 (SEQ ID NO:41) and CFLH-R1 are carried out PCR, and temperature cycle comprises successively 94 ℃ 30 seconds, 60 ℃ 30 seconds and 72 ℃ 1 minute, repeats 30 times, does not have the LH-0 type cDNA of joint with generation.

LH-0 that produces and HL-0 type cDNA introduce the cDNA that digests respectively among the Mammals expression plasmid INPEP4 with Ligation High (ToyoboInc.) with EcoRI and BamHI Restriction Enzyme (Takara Shuzo) digestion.With each plasmid transformed competence colibacillus E. ColiJM109 (Nippon Gene) utilizes QIAGEN Plasmid Maxi test kit (QUIAGEN) from transforming E. ColiThe middle purpose plasmid that separates.Thereby preparation plasmid pCF2LH-0 and pCF2HL-0.

The HL type expression plasmid that comprises different banjo connectors for structure, with pCF2HL-0 is template, the sense primer that uses is CFHL-X3 (SEQ ID NO:42), CFHL-X4 (SEQID NO:43), CFHL-X5 (SEQ ID NO:44), CFHL-X6 (SEQ ID NO:45) or CFHL-X7 (SEQ ID NO:46), antisense primer is BGH-1 (SEQ ID NO:47), itself and the complementation of carrier sequence.Use the KOD polysaccharase to carry out the PCR reaction, temperature cycle comprises successively 94 ℃ 30 seconds, 60 ℃ 30 seconds and 72 ℃ 1 minute, repeats usefulness Restriction Enzyme XhoI and BamHI (Takara Shuzo) digestion reaction product 30 times.The fragment of digestion uses Ligation High (Toyobo Inc.) to introduce between the XhoI and BamHI site of pCF2HL-0 respectively.With each plasmid transformed competence colibacillus E. ColiJM109 utilizes Qiagen Plasmid Maxi test kit from transforming E. ColiThe middle purpose plasmid that separates.Thereby preparation expression plasmid pCF2LH-3, pCF2LH-4, pCF2LH-5, pCF2LH-6 and pCF2LH-7.

For being structured in the expression plasmid of transient expression in the COS7 cell, with Restriction Enzyme EcoRI and BamHI (Takara Shuzo) digested plasmid pCF2HL-0, pCF2HL-3, pCF2HL-4, pCF2HL-5, pCF2HL-6 and pCF2HL-7, with about 800bp fragment of agarose gel electrophoresis purifying generation.Utilize Ligation High (Toyobo Inc.) respectively the fragment that obtains to be introduced between the EcoRI and BamHI site of expression plasmid pCOS1, be used for expressing at mammalian cell.Each plasmid transformed competence colibacillus E. ColiDH5 α (Toyobo Inc.) uses Qiagen Plasmid Maxi test kit from transforming E. ColiThe middle purpose plasmid that separates.Thereby preparation expression plasmid CF2HL-0/pCOS1, CF2HL-3/pCOS1, CF2HL-4/pCOS1, CF2HL-5/pCOS1, CF2HL-6/pCOS1 and CF2HL-7/pCOS1.

As the representative instance of these plasmids, the structure of plasmid CF2HL-0/pCOS1 as shown in figure 35, the MABL2-scFv＜HL-0 that comprises in this plasmid〉nucleotide sequence and aminoacid sequence shown in SEQ ID No.48.The nucleotide sequence in these plasmid center tap districts and aminoacid sequence are also as shown in figure 36.

The LH type expression plasmid that comprises different banjo connectors for structure, use pCF2LH-0 to be template, CFLH-X3 (SEQ ID NO:49), CFLH-X4 (SEQ ID NO:50), CFLH-X5 (SEQ ID NO:51), CFHL-X6 (SEQ ID NO:52) or CFLH-X7 (SEQID NO:53) are sense primer, BGH-1 is an antisense primer, itself and the complementation of carrier sequence.Use the KOD polysaccharase to carry out the PCR reaction, temperature cycle comprises successively 94 ℃ 30 seconds, 60 ℃ 30 seconds and 72 ℃ 1 minute, repeats usefulness Restriction Enzyme XhoI and BamHI digestion reaction product 30 times.Utilize Ligation High respectively the fragment of digestion to be introduced between the XhoI and BamHI site of pCF2LH-0.Each plasmid transformed competence colibacillus E. ColiDH5 α (Toyobo Inc.) uses Qiagen Plasmid Maxi test kit from transforming E. ColiThe middle purpose plasmid that separates.Thereby preparation expression plasmid pCF2LH-3, pCF2LH-4, pCF2LH-5, pCF2LH-6 and pCF2LH-7.

For being structured in the expression plasmid of transient expression in the COS7 cell, with Restriction Enzyme EcoRI and BamHI (Takara Shuzo) digested plasmid pCF2LH-0, pCF2LH-3, pCF2LH-4, pCF2LH-5, pCF2LH-6 and pCF2LH-7, with about 800bp fragment of agarose gel electrophoresis purifying generation.Utilize Ligation High respectively the fragment that obtains to be introduced between the XhoI and BamHI site of mammalian cell expression plasmid pCOS1.With each plasmid transformed competence colibacillus E. ColiDH-5 α (Toyobo Inc.) utilizes Qiagen Plasmid Maxi test kit from transforming E. ColiThe middle purpose plasmid that separates.Prepare expression plasmid CF2LH-0/pCOS1, CF2LH-3/pCOS1, CF2LH-4/pCOS1, CF2LH-5/pCOS1, CF2LH-6/pCOS1 and CF2LH-7/pCOS1 subsequently.

As the representative instance of these plasmids, the structure of plasmid CF2LH-0/pCOS1 as shown in figure 37, the MABL2-scFv＜LH-0 that comprises in this plasmid〉nucleotide sequence and aminoacid sequence shown in SEQ ID No.54.The nucleotide sequence in these plasmid center tap districts and aminoacid sequence are also as shown in figure 38.

6.3 In the COS7 cell, express scFv and sc (Fv) ₂

(1) prepares culture supernatant with containing blood serum medium

The scFv and the sc (Fv) of (JCRB9127, Japan Health SciencesFoundation) transient expression HL type and LH type in the COS7 cell ₂At CO ₂In the gas incubator, with the DMEM substratum (GIBCOBRL) that contains 10% foetal calf serum (HyClone), 37 ℃ of cultivation COS7 cells that go down to posterity.Use Gene Pulser device (BioRad), by CF2HL-0,3～7/pCOS1 or CF2LH-0, the 3～7/pCOS1 of electroporation with embodiment 6.2 preparations, or pCHOM2 (Fv) ₂The carrier rotaring redyeing COS 7 cell.Add DNA (10 μ g) and 0.25ml in the cuvette and contain 2 * 10 ⁷The DMEM substratum of cell/ml, 10%FBS and 5mMBES (SIGMA).After keeping 10 minutes, with 0.17kv, 950 μ F capacitor pulse treating mixture.Room temperature is recovered to change electroporation of cells over to 75cm after 10 minutes ³DMEM substratum (10%FBS) in the culturing bottle.Cultivate after 72 hours, collect culture supernatant, centrifugal removal cell debris.Culture supernatant is filtered with 0.22 μ m strainer (FALCON), to obtain culture supernatant (hereinafter claiming " CM ").

(2) prepare culture supernatant with serum free medium

Will be by changing 75cm over to (1) same procedure cells transfected ³DMEM substratum (10%FBS) in the culturing bottle, overnight incubation.Cultivate the back supernatant discarded, use the PBS washed cell, add CHO-S-SFM II substratum (GIBCO BRL) then.Cultivate after 72 hours, collect culture supernatant, centrifugal removal cell debris filters to obtain CM with 0.22 μ m strainer (FALCON).

6.4 Detect scFv and sc (Fv) among the COS7 CM ₂

With various MABL2-scFv and the sc (Fv) among the COS7 CM of preparation in Western trace method detection the foregoing description 6.3 (2) ₂

The CM of each COS7 of SDS-PAGE electrophoretic analysis transfers to REINFORCED NC film (Schleicher ﹠amp; Schuell).With 5% skimmed milk (Morinaga Nyu-gyo) closing membrane, the TBS washing.Then to wherein adding anti-FLAG antibody (SIGMA).Film is incubation and washing at room temperature.The mouse IgG antibody (JachsonImmuno Research) that adds peroxidase labelling.After incubation and the washing, add substrate solution (Kirkegaard Perry Laboratories) colour developing (Figure 39) under the room temperature.

6.5 Flow cytometry

Use the COS7 cells and supernatant of embodiment 6.3 (1) preparations, carry out flow cytometry, to measure MABL2-scFv and sc (Fv) ₂With antigenic combination of people's integrin associated protein matter (IAP).With culture supernatant to be measured or the COS7 cells and supernatant that compares, add 2 * 10 ⁵The L-1210 L1210 of individual expressing human IAP.After ice bath and the washing, add 10 μ g/ml mouse anti FALG antibody (SIGMA), incubation and washed cells then.The anti-mouse IgG antibody (BECTON DICKINSON) that wherein adds the FITC mark then, incubation and washed cell once more.Utilize FACScan device (BECTON DICKINSON) to measure fluorescence intensity.The flow cytometry result shows in the COS7 culture supernatant, to have the MABL2-scFv and the sc (Fv) of different lengths joint ₂People IAP had high affinity (referring to Figure 40 a and 40b).

6.6 External apoptosis induction effect

By Annexin-V dyeing (Boehringer Mannheim), utilize the L1210 cell (hIAP/L1210) of transfection people IAP gene, detect the apoptosis-induced effect of the COS7 culture supernatant of embodiment 6.3 (1) preparations.

To 5 * 10 ⁴Add COS7 cells and supernatant in the individual hIAP/L1210 cell with each carrier transfection, or the COS7 cells and supernatant that compares, final concentration is 10%, mixture was cultivated 24 hours.Carry out Annexin-V/PI dyeing then, utilize FACScan device (BEKTON DICHINSON) to measure fluorescence intensity.The result shows, the scFv＜HL3 among the COS7CM, 4,6,7, LH3,4,6,7〉and sc (Fv) ₂Significantly induce the death of hIAP/L1210 cell.These results as shown in figure 41.

6.7 Structure is used for Chinese hamster ovary celI and expresses scFv and sc (Fv) ₂ Carrier

Be separation, purifying MABL2-scFv and sc (Fv) from culture supernatant ₂, following structure is used for the carrier that Chinese hamster ovary celI is expressed.

Use Ligation High,, introduce between the EcoRI and BamHI site of expressing cho cell carrier pCHO1 the pCF2HL-0 of embodiment 6.2 preparations, 3～7 and pCF2LH-0,3～7 EcoRI-BamHI fragment.Use its transformed competence colibacillus E. ColiDH5 α.Use QIAGEN Plasmind Midi test kit (QIAGEN) from transforming E. ColiIn separation quality grain, preparation expression plasmid pCHOM2HL-0,3～7 and pCHOM2LH-0,3～7.

6.8 MABL2-scFv＜HL-0,3～7 is expressed in preparation 〉, MABL2-scFv＜LH-0, 3～7〉and sc (Fv) ₂ Chinese hamster ovary celI, and prepare its culture supernatant

Each expression plasmid pCHOM2HL-0 that makes up with embodiment 6.7,3～7 and pCHOM2LH-0,3～7, and pCHOM2 (Fv) ₂Carrier transforms Chinese hamster ovary celI, with the Chinese hamster ovary celI of the antibody for preparing every kind of change of constant expression.As its representative instance, constant expression MABL2-scFv＜HL-5〉or sc (Fv) ₂Being prepared as follows of Chinese hamster ovary celI shown in.

By Restriction Enzyme PvuI digestion, linearizing expression plasmid pCHOM2HL-5 and pCHOM2 (Fv) ₂, use Gene Pulser device (BioRad) by the electroporation transfection Chinese hamster ovary celI.In cuvette, add DNA (10 μ g) and 0.75ml 1 * 10 ⁷The PBS of cell/ml handles with 1.5KV, 25 μ F capacitor pulses.Room temperature was recovered after 10 minutes, electroporation of cells was forwarded to (GIBCOBRL) cultivation in the α that the comprises nucleic acid-MEM substratum that contains 10% foetal calf serum.After the overnight incubation, supernatant discarded.Use the PBS washed cell, add the free nucleic acid α-MEM substratum (GIBCO BRL) that contains 10% foetal calf serum.After cultivating for two weeks, cell being cultivated in the substratum that contains 10nM (final concentration) methotrexate (SIGMA), is the methotrexate of 50nM and 100nM then.The cell that obtains is at CHO-S-SFM II serum free medium (GIBCO BRL) transfer flask culture.Collect culture supernatant, centrifugal removal cell debris with 0.22 μ m aperture membrane filtration, obtains CM respectively.

As mentioned above, obtained constant expression MABL2-scFv＜HL-0 ,-3 ,-4 ,-6 ,-7〉and＜LH-0 ,-3 ,-4 ,-5 ,-6 ,-7 Chinese hamster ovary celI and CM thereof.

6.9 Purifying MABL2-scFv＜HL-5〉dimer and sc (Fv) ₂

Utilize following two kinds of purification process, purifying MABL2-scFv＜HL-5 from the CM of embodiment 6.8 preparations〉and sc (Fv) ₂

＜purification process 1 〉

Utilization is positioned at the FLAG sequence of peptide C end, by anti-FLAG antibody affinity column chromatography and gel-filtration purifying HL-5 and sc (Fv) ₂With anti-FLAG M2 affinity gel (SIGMA) preparation pillar (7.9ml), with the 50mM Tris-HCl damping fluid (TBS, pH7.5) balance, the sample on 1 liter of CM that will in 6.8, obtain that contain 150mM NaCl.After washing post with TBS, by 0.1M glycine-HCl damping fluid (pH3.5) wash-out scFv.Utilize SDS-PAGE to analyze resulting component, the scFv that confirmed wash-out.The scFv component can be mixed up to 0.01% Tween 20 with final concentration, and concentrate with Centricon-10 (MILIPORE).(7.5 * 600mm) with after containing 20mM acetate buffer (pH6.0) balance of 150mM NaCl and 0.01%Tween 20, with sample on the concentrated solution for TSKgel G3000SWG post.Flow velocity is the 0.4ml/ branch, by the absorbance detection scFv of 280nm.Main ingredient at dimer place wash-out is HL-5, the wash-out sc of monomer place (Fv) ₂

＜purification process 2 〉

Use ion exchange chromatography, hydroxyapatite and gel-filtration, three step purifying HL-5 and sc (Fv) ₂In ion exchange chromatography, purifying HL-5 uses Q Sepharose FastFlow post (Pharmacia), purifying sc (Fv) ₂Use SP-sepharose Fast Flow post.After among second step, reaching, handle HL-5 and sc (Fv) with same program ₂

The first step of HL-5

The CM of HL-5 transfers pH to 9.0 with 1M Tris then with 2 times of 20mM Tris-HCl damping fluid (pH9.0) dilutions that contains 0.02% Tween 20.Behind 20mM Tris-HCl damping fluid (pH8.5) the balance Q-sepharosefast flow post that contains 0.02%Tween 20, with sample on the solution.By containing the same buffer of 0.1～0.55M NaCl linear gradient, bonded polypeptide on the wash-out post.Through SDS-PAGE monitoring elution fraction, collected the component that contains HL-5, on the hydroxyapatite in last sample to the second step.

Sc (Fv) ₂ The first step

Sc (Fv) ₂CM with 2 times of 20mM acetate buffer (pH5.5) dilutions that contains 0.02% Tween 20, transfer pH to 5.5 with 1M acetate.Behind 20mM acetate buffer (pH5.5) the balance SP-sepharose fast flow post that contains 0.02% Tween 20, with sample on the solution.By bonded polypeptide on the buffer solution elution post that contains 0～0.5M NaCl linear gradient.Through SDS-PAGE monitoring elution fraction, collection comprises sc (Fv) ₂Component, on the hydroxyapatite in last sample to the second step.

Second step: HL-5 and sc (Fv) ₂Hydroxyapatite chromatography

(the I type BIORAD) with after containing 10mM phosphate buffered saline buffer (pH7.0) balance of 0.02% Tween 20, goes up HL-5 and sc (Fv) that the sample the first step obtains respectively to hydroxyapatite column ₂Component.After washing post with same buffer, utilize phosphate buffered saline buffer, bonded polypeptide on the wash-out post up to the 0.5M linear gradient.Through the component of SDS-PAGE monitoring wash-out, collect the component that comprises desired polypeptides.

The 3rd step: HL-5 and sc (Fv) ₂Gel-filtration

Concentrate each component that second step obtained respectively with CentriPrep-10 (MILIPORE), and go up sample to usefulness contain 0.02% Tween 20 and 0.15M NaCl 20mM acetate buffer (pH6.0) equilibrated Superdex 200 posts (2.6 * 60cm, Pharmacia).The place is eluted to HL-5 at dimer, sc (Fv) HL-5 and sc (Fv) ₂Respectively at the monomer place as main peaks by wash-out.

Two kinds of purification process all are difficult to detect the HL-5 monomer, when confirming that joint as strand Fv comprises about 5 amino acid, and high yield ground formation strand Fv dimer.In addition, the dimer of HL-5 and sc (Fv) ₂Can be behind purifying 4 ℃ of stable preservations one month.

6.10 Estimating the scFv＜HL-5 of purifying〉dimer and sc (Fv) 2 combine work with antigenic The property

Utilize the MABL2-scFv＜HL-5 of purifying〉sc (Fv) of dimer and purifying ₂Carry out flow cytometry, to estimate itself and antigenic combination of people's integrin associated protein matter (IAP).MABL2-scFv＜HL-5 with 10 μ g/ml purifying〉sc (Fv) of dimer, purifying ₂, positive control antibody MABL-2 or negative control mouse IgG (Zymed), add 2 * 10 ⁵The mouse leukemia L1210 clone (hIAP/L1210) of individual expressing human IAP, or the L1210 clone (pCOS1/L1210) that transforms through pCOS1 compares.After ice bath and the washing, add 10 μ g/ml mouse anti FLAG antibody (SIGMA), incubation and washed cells then.To the anti-mouse IgG antibody that wherein adds the FITC mark (BECTION DICKINSON), incubation and washed cell once more.Utilize FACScan device (BECTON DICKINSON) to measure fluorescence intensity then.

Because the MABL2-scFv＜HL-5 of purifying〉sc (Fv) of dimer and purifying ₂Specificity can confirm scFv＜HL-5 in conjunction with the hIAP/L1210 cell〉dimer and sc (Fv) ₂To people IAP high-affinity (referring to Figure 42).

6.11 ScFv＜the HL-5 of purifying〉dimer and sc (Fv) ₂ Live at external apoptosis induction The property

Use L1210 cell (hIAP/L1210) and the human leukemia cell line CCRF-CEM cell of introducing people IAP gene, by Annexin-V dyeing (BoehringerMannheim), detect the MABL2-scFv＜HL-5 of purifying〉sc (Fv) of dimer and purifying ₂Apoptosis-induced effect.

MABL2-scF＜HL-5 with the purifying of different concns〉MABL2-sc (Fv) of dimer, purifying ₂, positive control antibody MABL-2 or negative control mouse IgG, add 5 * 10 ⁴Individual hIAP/L1210 cell or 1 * 10 ⁵In the individual CCRF-CEM cell.Cultivate and carry out Annexin-V dyeing after 24 hours, use FACScan device (BECTON DICKLNSON) to measure its fluorescence intensity.As a result, MABL2-scFv＜HL-5〉dimer and MABL2-sc (Fv) ₂Rely on mode with concentration and significantly induce hIAP/L1210 and CCRF-CEM necrocytosis (referring to Figure 43).The result shows, MABL2-scFv＜HL-5〉dimer and MABL2-sc (Fv) ₂Apoptosis induction render a service and to be higher than original antibody MABL-2.

6.12 ScFv＜the HL-5 of purifying〉dimer and sc (Fv) ₂ Hemagglutination reaction test

According to embodiment 5.15 methods, use the scFv＜HL-5 of the purifying of different concns〉sc (Fv) of dimer and purifying ₂, carry out the hemagglutination reaction test.

As positive control, observe hemagglutination reaction with antibody MABL-2, and to single-chain antibody MABL2-sc (Fv) ₂And MABL2-scFv＜HL-5〉all do not observe hemagglutination reaction.In addition, antibody MABL-2 uses two kinds of damping fluids to carry out hemagglutination reaction, does not have essential difference.These results are as shown in table 3.

Table 3

The hemagglutination reaction test

Thinner: PBS (μ g/ml)

MABL2- sc(Fv) 2	Contrast-	28.9 -	14.45 -	7.225 -	3.6125 -	1.8063 -	0.9031 -	0.4516 -	0.2258 -	0.1129 -	0.0564 -	0.0282 -	0.0141 -	0.0071 -	0.0035 -	0.0018 -
																	MABL2- sc(Fv) <HL5>	Contrast-	28.0 -	14.0 -	7.0 -	3.5 -	1.75 -	0.875 -	0.4375 -	0.2188 -	0.1094 -	0.0547 -	0.0273 -	0.0137 -	0.0068 -	0.0034 -	0.0017 -
MABL2 (complete) mlgG	Contrast--	80 + -	40 + -	20 + -	10 + -	5 + -	2.5 + -	1.25 + -	0.625 + -	0.3125 + -	0.1563 ± -	0.0781 - -	0.0391 - -	0.0195 - -	0.0098 - -	0.0049 - -

Thinner: acetate buffer

(μg/ml)

MABL2 (complete)

Contrast-

80 +

40 +

20 +

10 +

5 +

2.5 +

1.25 +

0.625 +

0.3125 +

0.1563 +

0.0781 +

0.0391 -

0.0195 -

0.0098 -

0.0049 -

6.13 ScFv＜the HL-5 of purifying〉dimer and sc (Fv) ₂ To the human myeloma model mice Anti-tumour effect

ScFv＜the HL-5 of test implementation example 6.8 and 6.9 preparations and purifying〉dimer and sc (Fv) ₂Anti-tumour effect.The human myeloma mouse model that utilizes embodiment 5.1 to produce, ELISA measures the proteinic amount of M that human myeloma cell produces in the mice serum, and detects the survival time of mouse, tests.According to the survival time of proteinic number change of M in the mice serum and mouse, estimate scFv＜HL-5 then〉dimer and sc (Fv) ₂Anti-tumour effect.

Use 0.01,0.1 or HL-5 and the sc (Fv) of 1mg/ml in the test ₂, they are dissolved in the carrier that comprises 150mM NaCl, 0.02% Tween and 20mM acetate buffer (pH6.0), by 0.1,1 or 10mg/kg dosage to the mouse administration.Control group mice only gives carrier.

Transplant human myeloma cell after 26 days, collect mice serum, press embodiment 5.14 methods, utilize ELISA to measure the proteinic quantity of M in the serum.As a result, give the serum M protein quantity of two groups of mouse of HL-5 dimer and sc (Fv) 2, all with dose-dependently mode descend (referring to Figure 44).In addition, compare, observe and give HL-5 (Figure 45) and sc (Fv) with the control group that gives carrier ₂(Figure 46) two groups, equal significant prolongation of its survival time.These results show, HL-5 of the present invention and sc (Fv) ₂Has fabulous anti-tumor in vivo effect.

Embodiment 7

Comprise the H chain V district of people's antibody 12B5 of anti-people MPL and the strand Fv in L chain V district

The V district coding DNA of the human monoclonal antibodies 12B5 of the anti-people MPL of following structure:

7.1 Make up the gene in coding 12B5H chain V district

Nucleotide sequence (SEQ ID NO:55) by its gene connects leader sequence (SEQ ID the NO:56) (Eur.J.Immunol.1996 that derives from human immunoglobulin gene at 5 ' end; 26:63-69), design is in conjunction with the encoding gene in the H chain V district of people's antibody 12B5 of people MPL.The nucleotide sequence of design is divided into four oligonucleotide (12B5VH-1,12B5VH-2,12B5VH-3,12B5VH-4) that respectively have the 15bp overlap.Respectively, 12B5VH-1 (SEQ ID NO:57) and 12B5VH-3 (SEQ ID NO:59) are that just direction is synthetic, and 12B5VH-2 (SEQ ID NO:58) and 12B5VH-4 (SEQ ID NO:60) are synthetic for antisense orientation.After assembling each synthetic oligonucleotide according to complementarity separately, add outside primer (12B5VH-S and 12B5VH-A), the amplification full-length gene.Respectively, 12B5VH-S (SEQ ID NO:61) is designed to 5 ' end hybridization by forward primer and leader sequence, and have Hind III Restriction Enzyme recognition site and a Kozak sequence, and 12B5VH-A (SEQ ID NO:62) is designed to by the nucleotide sequence hybridization of reverse primer with coding H chain V district C end, and has donor splicing site sequence and BamHI Restriction Enzyme recognition site.

100 μ l PCR solution comprise 10 μ l, 10 * PCR Gold Buffer II, 1.5mMMgCl ₂, 0.08mM dNTPs (dATP, dGTP, dCTP and dTTP), the 5 archaeal dna polymerase AmpliTaq Gold of unit (all from PERKIN ELMER), every kind of synthetic oligonucleotide of 2.5pmol (12B5VH-1 is to-4), 94 ℃ of starting temperatures heating 9 minutes was 94 ℃ 2 minutes, 55 ℃ 2 minutes and 72 ℃ 2 minutes.After this circulation repeats 2 times, add outside primer 12B5VH-S of each 100pmol and 12B5VH-A.This mixture comprises 94 ℃ 30 seconds, 55 ℃ 30 seconds and 72 ℃ of circulations of 1 minute 35 times, and 72 ℃ of reheat 5 minutes.

Utilize 1.5% low melting point temperature sepharose (Sigma) purified pcr product, utilize Restriction Enzyme BamHI and Hind III digestion, and be cloned into people H chain expression vector HEF-g γ 1.Behind the dna sequencing, will comprise the plasmid called after HEF-12B5H-g γ 1 of correct dna sequence dna.

HEF-12B5H-g γ 1 usefulness Restriction Enzyme EcoRI and BamHI digestion, the gene of generation coding 12B5VH is cloned into human Fab H chain expression vector pCOS-Fd then, produces pFd-12B5H.Utilize pcr amplification to comprise to include the DNA (SEQ ID NO:63) of the encoding gene of subarea and people H chain portion constant region, this intron district is positioned between the encoding gene of people's heavy chain of antibody V district and constant region, this PCR product is inserted animal cell expression carrier pCOS1, make up human Fab H chain expression vector.With HEF-g γ 1 is template, forward primer G1CH1-S (SEQ ID NO:64) (is designed to 5 ' the terminal sequence hybridization with introne 1, and have Restriction Enzyme recognition site EcoRI and a BamHI), reverse primer G1CH1-A (SEQ ID NO:65) (is designed to 3 ' end DNA hybridization with people H chain constant region CH1 structural domain, and have the sequence of a part of hinge area of coding, 2 terminator codons and a Restriction Enzyme recognition site BglII), in above-mentioned the same terms amplification people H chain constant region gene down.

The nucleotide sequence of the 12B5H chain variable region of the reconstruction that comprises among plasmid HEF-12B5H-g γ 1 and the pFd-12B5H and aminoacid sequence are shown in SEQ ID NO:66.

7.2 Make up the gene in coding 12B5L chain V district

Human immunoglobulin gene 3D6 (Nuc.Acid Res.1990:18 is held and derived to nucleotide sequence (SEQ ID NO:67) by gene 5 '; 4927) leader sequence (SEQID NO:68) connects, and design is in conjunction with the encoding gene in the L chain V district of people's antibody 12B5 of people MPL.By above-mentioned same procedure, the nucleotide sequence of design is divided into four oligonucleotide (12B5VL-1,12B5VL-2,12B5VL-3,12B5VL-4) that respectively have the 15bp overlap, synthesizes respectively.Respectively, 12B5VL-1 (SEQ ID NO:69) and 12B5VL-3 (SEQ ID NO:71) have just sequence, and 12B5VL-2 (SEQ ID NO:70) and 12B5VL-4 (SEQ ID NO:72) have antisense sequences.After assembling each synthetic oligonucleotide according to complementarity separately, (12B5VL-S and 12B5VL-A) mixes with outside primer, the amplification full-length gene.12B5VL-S (SEQ ID NO:73) is designed to 5 ' end hybridization by forward primer and leader sequence, and has Hind III Restriction Enzyme recognition site and Kozak sequence.12B5VL-A (SEQ ID NO:74) is designed to by the nucleotide sequence hybridization of reverse primer with coding L chain V district C end, and has donor splicing site sequence and BamHI Restriction Enzyme recognition site.

Carry out PCR as mentioned above, utilize 1.5% low melting point temperature sepharose (Sigma) purified pcr product, utilize Restriction Enzyme BamHI and Hind III digestion, and be cloned into people L chain expression vector HEF-g κ.Behind the dna sequencing, will comprise the plasmid called after HEF-12B5L-g κ of correct dna sequence dna.The nucleotide sequence in the 12B5 L chain V district of the reconstruction that comprises among the plasmid HEF-12B5L-g κ and aminoacid sequence are shown in SEQ ID NO:75.

7.3 The 12B5 strand Fv (scFv) that preparation is rebuild

The 12B5 antibody strand Fv that rebuilds is designed to 12B5VH-joint-12B5VL order, and the C end has FLAG sequence (SEQ ID NO:76), so that detect and purifying.Utilization is by (Gly ₄Ser) ₃The joint sequence that 15 amino acid of representative are formed makes up the 12B5 strand Fv (sc12B5) of this reconstruction.

(1) The 12B5 strand that the joint sequence preparation that utilization is made up of 15 amino acid is rebuild Fv

Utilize PCR increase respectively 12B5 H chain V district, connector area and 12B5L chain V district and be connected, preparation comprises the encoding gene of 12B5 antibody strand Fv of the reconstruction of the joint sequence of being made up of 15 amino acid.This method diagram is shown in Figure 47.The 12B5 strand Fv that utilizes six kinds of primers (A-F) preparation to rebuild.Primer A, C and E have just sequence, and primer B, D and F have antisense sequences.

The forward primer 12B5-S in H chain V district (primer A, SEQ ID No.77) is designed to and can hybridizes with 5 ' end of H chain leader sequence, and has EcoRI Restriction Enzyme recognition site.The coding DNA that the reverse primer HuVHJ3 in H chain V district (primer B, SEQ ID No.78) is designed to hold with the C in H chain V district is hybridized.

The forward primer RHuJH3 of joint (primer C, SEQ ID No.79) is designed to the coding DNA hybridization with joint N end, and overlapping with the coding DNA of H chain V district C end.The reverse primer RHuVK1 of joint (primer D, SEQ ID No.80) is designed to and can hybridizes with the coding DNA of joint C end, and overlapping with the coding DNA of L chain V district N end.

The forward primer HuVK1.2 in L chain V district (primer E, SEQ ID No.81) is designed to the coding DNA hybridization with L chain V district N end.Reverse primer 12B5F-A (the primers F in L chain V district, SEQID No.82) is designed to and hybridizes with the coding DNA of L chain V district C end, and has a FLAG peptide-coding sequence (people such as Hopp T.P., Bio/Technology, 6,1204-1210,1988), two Transcription Termination codons and NotI Restriction Enzyme recognition site.

In first PCR step, carry out A-B, C-D, three reactions of E-F, three kinds of PCR products that the first step PCR is obtained are assembled according to complementarity separately.Add after primer A and the F, amplification has the total length coding DNA (the second step PCR) of 12B5 strand Fv joint, that rebuild that is made up of 15 amino acid.Among the first step PCR respectively with the plasmid HEF-12B5H-g γ 1 (referring to embodiment 7.1) in the 12B5H chain V district of coding and rebuilding, plasmid pSCFVT7-hM21 (humanized ONS-M21 the antibody) (people such as Ohtomo who comprises connector area coding DNA (SEQ ID NO:83), Anticancer Res.18 (1998), 4311-4316) (this connector area is made up of Gly Gly Gly Gly Ser Gly Gly Gly Gly SerGly Gly Gly Gly Ser) (people such as Huston, Proc.Natl.Acad.Sci.USA, 85,5879-5883,1988), and the plasmid HEF-12B5L-g κ (referring to embodiment 7.2) in the 12B5L chain V district of coding and rebuilding makes template.

50 μ l solution of the first step PCR reaction comprise 5 μ l, 10 * PCR Gold BufferII, 1.5mM MgCl ₂, 0.08mM dNTPs, the 5 archaeal dna polymerase AmpliTaq Gold of unit (all from PERKIN ELMER), every kind of primer of 100pmol and every kind of template DNA of 100ng.PCR solution was 94 ℃ of starting temperatures heating 9 minutes, 94 ℃ 30 seconds, 55 ℃ 30 seconds and 72 ℃ 1 minute.This temperature cycle repeats after 35 times, 72 ℃ of reheat reaction mixtures 5 minutes.

Utilize second step PCR assembling PCR product A-B, C-D and E-F.The second step PCR mixture solution 98 μ l, comprising 1 μ l the first step PCR product A-B, 0.5 μ l PCR product C-D and 1 μ l PCR product E-F is template, 10 μ l, 10 * PCR Gold Buffer II, 1.5mMMgCl ₂, 0.08mM dNTPs, the 5 archaeal dna polymerase AmpliTaq Gold of unit (all from PERKIN ELMER), 94 ℃ of starting temperatures heating 9 minutes, 94 ℃ 2 minutes, 65 ℃ 2 minutes and 72 ℃ 2 minutes.This circulation repeats to add every kind of primer A and each 100pmol of F after 2 times.Repetition by 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ circulations of forming in 1 minute 35 times after, reaction mixture 72 ℃ the heating 5 minutes.

Utilize the dna fragmentation of 1.5% low melting point temperature sepharose purifying, second step PCR preparation,, be cloned into pCHO1 carrier and pCOS1 carrier (Japanese patent application No. 8-255196) with EcoRI and NotI digestion.Expression vector pCHO1 utilizes EcoRI and SmaI digestion DHFR-Δ E-rvH-PM1-f (referring to WO92/19759), remove antibody gene, and connects EcoRI-NotI-BamHI joint (TAKARA SHUZO) and make up.Behind the dna sequencing, will comprise the coding DNA segmental plasmid called after pCHO-sc12B5 and the pCOS-sc12B5 of the correct aminoacid sequence of 12B5 strand Fv of reconstruction.The nucleotide sequence of the 12B5 strand Fv of the reconstruction that comprises among plasmid pCHO-sc12B5 and the pCOS-sc12B5 and aminoacid sequence are shown in SEQ TD NO:84.

7.4 Utilize animal cell expression antibody 12B5 (IgG, Fab) and strand Fv polypeptide

Utilize COS-7 cell or expressing cho cell antibody 12B5 (IgG, Fab) and derive from the strand Fv of antibody 12B5.

The following COS7 of utilization cell carries out transient expression.Use Gene Pulser device (BioRad), carry out transfection by electroporation method.In being suspended in the COS-7 cell of 0.8ml PBS (1 * 10 ⁷Cell/ml), for expressing antibodies 12B5 (IgG) adds above-mentioned expression vector HEF-12B5H-g γ 1 and each 10 μ g of HEF-12B5L-g κ, add pFd-12B5H and each 10 μ g of HEF-12B5L-g κ for expressing 12B5 Fab fragment, add 10 μ g pCOS-sc12B5 for expressing strand Fv.Utilize 1.5KV, 25 μ FD capacitor pulses to handle the mixture in the cuvette.Room temperature was recovered after 10 minutes, electroporation of cells was added to (GIBCO BRL) cultivation in the DMEM substratum that contains 10% foetal calf serum.After the overnight incubation, with the PBS washed cell once, be added among the serum free medium CHO-S-SFM II and cultivated 2 days.The centrifugal removal cell debris of substratum, with 0.22 μ m membrane filtration, the preparation culture supernatant.

For setting up the Chinese hamster ovary celI system that stably express derives from the strand Fv (polypeptide) of antibody 12B5, following with expression vector pCHO-sc12B5 introducing Chinese hamster ovary celI.

Use Gene Pulser device (BioRad), this expression vector is introduced Chinese hamster ovary celI by electroporation method.Utilize Restriction Enzyme PvuI digestion, obtain linearizing DNA (100 μ g), with the Chinese hamster ovary celI (1 * 10 that is suspended in 0.8ml PBS ⁷Cell/ml) mix in cuvette left standstill 10 minutes on ice, utilized 1.5KV, 25 μ FD capacitor pulses to handle.Room temperature was recovered after 10 minutes, electroporation of cells is added among the CHO-S-SFMII (GIBCO BRL) that contains 10% foetal calf serum cultivates.Cultivate after 2 days, in the CHO-S-SFM II that contains 5nM methotrexate (SIGMA) and 10% foetal calf serum (GIBCO BRL), continue to cultivate.From the clone of such acquisition, select high expression level rate clone, as the production clone of 12B5 strand Fv.After cultivating in containing the serum free medium CHO-S-SFM II (GIBCO BRL) of 5nM methotrexate (SIGMA), the centrifugal separating cell fragment obtains culture supernatant.

7.5 The strand Fv that derives from 12B5 that purifying CHO cell produces

With the culture supernatant of the Chinese hamster ovary celI system of the expression 12B5 strand Fv that obtains in 7.4, utilize anti-FLAG antibody column and gel-filtration column purification.

(1) Anti-FLAG antibody column

Culture supernatant is added the anti-FLAG M2 of PBS equilibrated affinity gel (Sigma).Utilize same buffer to wash after the post, by the protein that adsorbs on 0.1M glycine-hydrochloride buffer (pH3.5) wash-out post.Elution fraction adds 1M Tris-HCl damping fluid (pH8.0) immediately and neutralizes.Analyze elution fraction with SDS-PAGE, utilize Centricon-10 (MILLIPORE) to concentrate through confirming to contain the component of strand Fv.

(2) Gel-filtration

(1) concentrated solution that obtains in adds by the PBS equilibrated Superdex200 post (10 * 300mm, AMERSHAM PHARMACIA) that contains 0.01% Tween20.

Product sc12B5 is wash-out (A, B) (referring to Figure 48) in 2 peaks.Utilize the 14%-SDS-polyacrylamide gel to analyze component A and B.According to the Laemmli method, having or do not having in the presence of the reductive agent sample is carried out electrophoresis, use coomassie brilliant blue staining behind the electrophoresis.As shown in figure 49, no matter whether reductive agent exists, and component A and B produce the single band of the about 31kD of performance molecular weight.Utilize Superdex200 PC 3.2/3.0 (3.2 * 300mm, AMERSHAM PHARMACIA) to analyze component A and B by gel-filtration, component A produces the eluted product of the about 44kD of apparent molecular weight, and B component produces the product (referring to Figure 50 a and b) of 22kD.This result shows that component A is the non covalent bond dimer of sc12B5 strand Fv, and B component is a monomer.

7.6 Measure the TPO sample agonist activity of various strand Fv

By measuring proliferation activity, estimate the TPO sample activity of anti-MPL single-chain antibody to the Ba/F3 cell (BaF/mpl) of expressing human TPO acceptor (MPL).After utilization contains twice in RPMI1640 substratum (GIBCO) the washing BaF/Mpl cell of 10% foetal calf serum (GIBCO), with cell by 5 * 10 ⁵The cell density of cell/ml is suspended in the substratum.Dilute anti-MPL single-chain antibody and people TPO (R﹠amp with substratum respectively; D Systems).The antibody of 50 μ l cell suspensions and 50 μ l dilution or people TPO are added in the 96 hole microwell plates (flat) (Falcon), CO ₂(CO in the insulation can ₂Concentration: 5%) cultivated 24 hours.After the incubation, add 10 μ l WST-8 reagent and (be used to measure the number of initiating cell SF: Nacalai Tesque), utilize fluorescent absorption photometer SPECTRA Fluor (TECAN) to measure absorbancy immediately, measure wavelength 450nm, reference wavelength 620nm.At CO ₂(CO in the insulation can ₂Concentration: 5%) incubation utilized SPECTRA Fluor to measure absorbancy once more after 2 hours, measured wavelength 450nm, reference wavelength 620nm.Because WST-8 reagent produces color reaction according to the viable cell number at wavelength 450nm place, by the ED50 of following calculating, based on the variation of absorbancy after 2 hours, the proliferation activity of evaluation BaF/Mpl.In the proliferative response curve, be that ordinate zou, antibody concentration are that X-coordinate is drawn with the absorbancy, the absorbancy of plateau is made as 100% reactivity.Based near the drawn numerical value of 50% reactivity, utilize line fitting method (straight line approximation) to obtain fitting formula, calculate the antibody concentration of 50% reactivity, be adopted as ED50.

Utilize the COS-7 cells and supernatant of expressing various 12B5 antibody molecules, mensuration is to the agonist activity of MPL, and the result shows shown in Figure 51, has the 12B5IgG of two valency antigen binding sites, its absorbancy improves in the concentration dependent mode, and has TPO sample agonist activity (ED50; 29nM), the very weak (ED50 of agonist activity that has the 12B5Fab of monovalent antigen binding site; 34,724nM).On the contrary, have the strand Fv (sc12B5) of monovalent antigen binding site as Fab, show strong agonist activity, the ED50 level is 75nM.Yet the variable region of known strand Fv H chain and L chain is by the non covalent bond combination, and therefore separate each variable region in solution, can combine with the variable region of other molecule, forms polymer, as dimer.When measuring the molecular weight of sc12B5 of gel-filtration purifying, alleged occurrence is identified as monomer and dimeric molecule (referring to Figure 48).Separate sc12B5 monomer and sc12B5 dimer (referring to Figure 50) then, measure its agonist activity MPL.Shown in Figure 51 and 52, the monomeric ED50 of sc12B5 is 4438.7nM, confirms to compare with the result who utilizes the COS-7 cells and supernatant, and its agonist activity descends.On the contrary, have the strand Fv (sc12B5 dimer) of two valency antigen binding sites, the agonist activity of demonstration exceeds about 400 times of (ED50 than unit price sc12B5; 10.1nM).In addition, the agonist activity of two valency strand Fv demonstrations is equal to or higher than the agonist activity of people TPO and 12B5IgG.

Embodiment 8

Make up the variable region encoding gene of people's antibody 12E10 of anti-people MPL

The variable region coding DNA of the human monoclonal antibodies 12E10 of the anti-people MPL of following structure:

8.1 Make up the encoding gene in 12E10 H chain V district

The nucleotide sequence of SEQ ID NO:86 is to be the encoding gene in conjunction with the H chain V district of people's antibody 12E10 of people MPL of basic design with the described aminoacid sequence of WO99/10494 (SEQ ID NO:85).Connect the leader sequence (SEQ ID NO:87) that derives from human immunoglobulin gene (GeneBank accession number AF062252) by its 5 ' end, design full length nucleotide sequence.The nucleotide sequence of design is divided into four oligonucleotide (12E10VH1,12E10VH2,12E10VH3,12E10VH4) that respectively have the 15bp overlap.Respectively, 12E10VH1 (SEQ ID NO:88) and 12E10VH3 (SEQ ID NO:90) are that just direction is synthetic, and 12E10VH2 (SEQ ID NO:89) and 12E10VH4 (SEQ ID NO:91) are synthetic for antisense orientation.After assembling each synthetic oligonucleotide according to complementarity separately, add outside primer (12E10VHS and 12E10VHA), the amplification full-length gene.Respectively, 12E10VHS (SEQ ID NO:92) is designed to 5 ' end hybridization by forward primer and leader sequence, and have Hind III Restriction Enzyme recognition site and a Kozak sequence, and 12E10VHA (SEQ ID NO:93) is designed to by the nucleotide sequence hybridization of reverse primer with coding H chain V district C end, and has donor splicing site sequence and BamHI Restriction Enzyme recognition site.

100 μ l PCR solution comprise 10 μ l, 10 * PCR Gold Buffer II, 1.5mMMgCl ₂, 0.08mM dNTPs (dATP, dGTP, dCTP and dTTP), the 5 archaeal dna polymerase AmpliTaq Gold of unit (all from PERKIN ELMER), every kind of synthetic oligonucleotide of 2.5pmol (12E10VH-1 is to-4), 94 ℃ of starting temperatures heating 9 minutes was 94 ℃ 2 minutes, 55 ℃ 2 minutes and 72 ℃ 2 minutes.After this circulation repeats 2 times, add outside primer 12E10VHS of each 100pmol and 12E10VHA.This mixture carries out by 94 ℃ 30 seconds, 55 ℃ 30 seconds and 72 ℃ of circulations of forming in 1 minute 35 times, and 72 ℃ of reheat 5 minutes.

Utilize 1.5% low melting point temperature sepharose (Sigma) purified pcr product, utilize Restriction Enzyme BamHI and Hind III digestion, and be cloned into people H chain expression vector HEF-g γ 1.Behind the dna sequencing, will comprise the plasmid called after HEF-12E10H-g γ 1 of correct dna sequence dna.

HEF-12E10H-g γ 1 usefulness Restriction Enzyme EcoRI and BamHI digestion, the encoding gene of generation 12E10VH is cloned into human Fab H chain expression vector pCOS-Fd then, produces pFd-12E10H.Utilize pcr amplification to comprise to include the DNA (SEQ ID NO:63) of the encoding gene of subarea and people H chain part constant region, this intron district is positioned between the encoding gene of people's heavy chain of antibody V district and constant region, this PCR product inserts animal cell expression carrier pCOS1, makes up human Fab H chain expression vector.With HEF-g γ 1 is template, forward primer G1CH1-S (SEQ IDNO:64) (is designed to 5 ' the terminal sequence hybridization with introne 1, and have Restriction Enzyme recognition site EcoRI and a BamHI), reverse primer G1CH1-A (SEQ ID NO:65) (is designed to 3 ' end DNA hybridization with people H chain constant region CH1 structural domain, and have the sequence of a part of hinge area of coding, 2 terminator codons and a Restriction Enzyme recognition site Bgl II), in above-mentioned the same terms amplification people H chain constant region gene down.

The nucleotide sequence of the 12E10H chain variable region of the reconstruction that comprises among plasmid HEF-12E10H-g γ 1 and the pFd-12E10H and aminoacid sequence are shown in SEQ ID NO:94.

8.2 Make up the gene in coding 12E10 L chain V district

The nucleotide sequence of SEQ ID NO:96 is to be the encoding gene in conjunction with the L chain V district of people's antibody 12E10 of people MPL of basic design with the described aminoacid sequence of WO99/10494 (SEQ ID NO:95).Derive from human immunoglobulin gene (Mol.Immunol.1992 by its 5 ' end connection; Leader sequence 29:1515-1518) (SEQ ID NO:97) further designs.By above-mentioned same procedure, the nucleotide sequence of design is divided into four oligonucleotide (12E10VL1,12E10VL2,12E10VL3,12E10VL4) that respectively have the 15bp overlap, synthesizes respectively.Respectively, 12E10VL1 (SEQ ID NO:90) and 12E10VL3 (SEQ ID NO:100) have just sequence, and 12E10VL2 (SEQ ID NO:99) and 12E10VL4 (SEQ ID NO:101) have antisense sequences.After assembling each synthetic oligonucleotide according to complementarity separately, (12E10VLS and 12E10VLA) mixes with outside primer, the amplification full-length gene.12E10VLS (SEQ ID NO:102) is designed to 5 ' end hybridization by forward primer and leader sequence, and has EcoR I Restriction Enzyme recognition site and Kozak sequence.12E10VLA (SEQ ID NO:103) is designed to by the nucleotide sequence hybridization of reverse primer with coding L chain V district C end, and has BlnI Restriction Enzyme recognition site.

Carry out PCR as mentioned above, utilize 1.5% low melting point temperature sepharose (Sigma) purified pcr product, utilize Restriction Enzyme EcoR I and BlnI digestion, and be cloned among the pUC19 that comprises people λ chain constant region gene.Behind the dna sequencing, the plasmid that will comprise correct dna sequence dna digests with EcoR I, produces the gene of coding 12E10 L chain V district and people λ chain constant region, inserts expression vector pCOS1 then.Plasmid called after DCOS-12E10L with 12E10 L chain gene (SEQ ID NO:104).

8.3 The 12E10 strand Fv that preparation is rebuild

The 12E10 antibody strand Fv that rebuilds is designed to 12E10VH-joint-12E10VL order, and the C end has FLAG sequence (SEQ ID NO:105) so that detect and purifying.Utilization is by (Gly ₄Ser) ₃15 amino acid of representative or (Gly ₄Ser) ₁The joint sequence that 5 amino acid of representative are formed makes up the 12E10 chain Fv (sc12E10 and db12E10) of this reconstruction.

(1) utilize the joint sequence of forming by 5 amino acid to prepare the 12E10 strand Fv that rebuilds

With (Gly ₄Ser) ₁The nucleotide sequence of joint is introduced 3 ' end of 12E10H chain V district encoding gene and 5 ' end of 12E10 L chain V district encoding gene, each gene that utilizes pcr amplification to obtain like this, and connect the gene that is increased, make up the encoding gene of the 12E10 strand Fv of the reconstruction that comprises the joint sequence of forming by 5 amino acid.The 12E10 strand Fv that utilizes four kinds of PCR primers (A-D) preparation to rebuild.Primer A and C have just sequence, and primer B and D have antisense sequences.

The forward primer in H chain V district is 12E10S (primer A, SEQ ID NO:106).The reverse primer DB2 in H chain V district (primer B, SEQ ID NO:107) is designed to the coding DNA hybridization with H chain V district C end, and has (Gly ₄Ser) ₁The coding nucleotide sequence of the coding nucleotide sequence of joint and L chain V district N end.

The forward primer DB1 in L chain V district (primer C, SEQ ID NO:108) is designed to the coding DNA hybridization with L chain V district N end, and has (Gly ₄Ser) ₃The coding nucleotide sequence of the coding nucleotide sequence of joint and H chain V district C end.The reverse primer 12E10FA in L chain V district (primer D, SEQ ID NO:109) is designed to and can hybridizes with the coding DNA of L chain V district C end, and has FLAG encoding sequence and NotI Restriction Enzyme recognition site.

In first PCR step, carry out two reactions of A-B, C-D, two kinds of PCR products that the first step PCR is obtained are assembled according to complementarity separately.Add after primer A and the D, amplification has the total length coding DNA (the second step PCR) of 12E10 strand Fv joint, that rebuild that is made up of 5 amino acid.Use the plasmid HEF-12E10H-g γ 1 (referring to embodiment 8.1) in the 12E10H chain V district of coding and rebuilding among the first step PCR respectively, and the plasmid pCOS-12E10L (referring to embodiment 8.2) in the 12E10 L chain V district of coding and rebuilding makes template.

50 μ l solution of the first step PCR reaction comprise 5 μ l, 10 * PCR Gold BufferII, 1.5mM MgCl ₂, 0.08mM dNTPs, the 5 archaeal dna polymerase AmpliTaq Gold of unit (from PERKIN ELMER), every kind of primer of 100pmol and every kind of template DNA of 100ng.PCR solution was 94 ℃ of starting temperatures heating 9 minutes, 94 ℃ 30 seconds, 55 ℃ 30 seconds and 72 ℃ 1 minute.This temperature cycle repeats after 35 times, 72 ℃ of reheat reaction mixtures 5 minutes.

Utilize second step PCR assembling PCR product A-B (429bp) and the C-D (395bp).It is template, every kind of each 100pmol of primer, 10 μ l, 10 * PCR Gold BufferII, 1.5mM MgCl that the second step PCR mixture solution (98 μ l) comprises the first step PCR product A-B and every kind 1 μ l of C-D ₂, 0.08mM dNTPs, the 5 archaeal dna polymerase AmpliTaq Gold of unit (from PERKIN ELMER), under above-mentioned the same terms, react.

The 795bpDNA fragment of utilizing 1.5% low melting point temperature sepharose purifying, second step PCR to produce with EcoRI and NotI digestion, is cloned into pCHO1 carrier or pCOS1 carrier.Expression vector pCHO1 utilizes EcoRI and SmaI digestion DHFR-Δ E-RVH-PM1-f (referring to WO92/19759), remove antibody gene, and connects EcoRI-NotI-BamHI joint (TAKARA SHUZO) and make up.Behind the dna sequencing, will comprise the segmental plasmid of coding DNA of the correct aminoacid sequence of 12E10 strand Fv of reconstruction, called after pCHO-db12E10 and pCOS-db12E10.The nucleotide sequence of the 12E10 strand Fv of the reconstruction that comprises among plasmid pCHO-db12E10 and the pCOS-db12E10 and aminoacid sequence are shown in SEQ ID NO:110.

(2) utilize the joint sequence of forming by 15 amino acid to prepare the 12E10 strand Fv that rebuilds

With (Gly ₄Ser) ₃The nucleotide sequence of joint is introduced 3 ' end of 12E10 H chain V district encoding gene and 5 ' end of 12E10 L chain V district encoding gene, each gene that utilizes PCR to increase respectively and obtain like this, and connect the gene that is increased, make up the encoding gene of the reconstruction 12E10 antibody strand Fv that comprises the joint sequence of forming by 15 amino acid.The 12E10 strand Fv that utilizes four kinds of PCR primers (A-D) preparation to rebuild.Primer A and C have just sequence, and primer B and D have antisense sequences.

The forward primer in H chain V district is 12E10S (primer A, SEQ ID NO:106).The reverse primer sc4.3 in H chain V district (primer B, SEQ ID NO:111) is designed to the coding DNA hybridization with H chain V district C end, and has (Gly ₄Ser) ₃The coding nucleotide sequence of the coding nucleotide sequence of joint and L chain V district N end.

The forward primer sc1.3 in L chain V district (primer C, SEQ ID NO:112) is designed to the coding DNA hybridization with L chain V district N end, and has (Gly ₄Ser) ₃The coding nucleotide sequence of the coding nucleotide sequence of joint and H chain V district C end.The reverse primer 12E10FA in L chain V district (primer D, SEQ ID NO:109) is designed to and can hybridizes with the coding DNA of L chain V district C end, and has FLAG coding nucleotide sequence and NotI Restriction Enzyme recognition site.

In first PCR step, carry out two reactions of A-B, C-D, two kinds of PCR products that the first step PCR is obtained are assembled according to complementarity separately.Add after primer A and the D, amplification has the total length coding DNA (the second step PCR) of 12E10 strand Fv joint, that rebuild that is made up of 15 amino acid.Among the first step PCR, the coding plasmid pCOS-db12E10 of the 12E10 strand Fv of reconstruction (referring to embodiment 8.3 (1)) is as template.

50 μ l solution of the first step PCR reaction comprise 5 μ l, 10 * ExTaq Buffer, 0.4mM dNTPs, the 2.5 archaeal dna polymerase TaKaRa ExTaq of unit (from TAKARA), every kind of each 100pmol of primer and every kind of template DNA of 10ng.PCR solution is 94 ℃ of starting temperatures heating 30 seconds, 94 ℃ 15 seconds, 72 ℃ 2 minutes, this circulation repeats 5 times.Repeat after 28 times 72 ℃ of reheat reaction mixtures 5 minutes 94 ℃ of 15 seconds, 72 ℃ circulations of 2 minutes.

Utilize second step PCR assembling PCR product A-B (477bp) and the C-D (447bp).It is template, every kind of primer A and each 100pmol of D, 5 μ l, 10 * ExTaq Buffer, 0.4mM dNTPs, the 2.5 archaeal dna polymerase TaKaRa ExTaq of unit (from TAKARA) that the second step PCR mixture solution (98 μ l) comprises each 1 μ l the first step PCR product A-B and C-D, reacts under above-mentioned the same terms.

The 825bpDNA fragment of utilizing 1.0% low melting point temperature sepharose purifying, second step PCR to produce is with EcoRI and NotI digestion.The dna fragmentation of Huo Deing is cloned into pCHO1 carrier or pCOS1 carrier like this.Behind the dna sequencing, will comprise the segmental plasmid of coding DNA of the correct aminoacid sequence of 12E10 strand Fv of reconstruction, called after pCHO-sc12E10 and pCOS-sc12E10.The nucleotide sequence of the 12E10 strand Fv of the reconstruction that comprises among plasmid pCHO-sc12E10 and the pCOS-sc12E10 and aminoacid sequence are shown in SEQ ID NO:113.

8.4 Utilize animal cell expression antibody 12E10 (IgG, Fab) and strand Fv polypeptide

Utilize COS-7 cell or expressing cho cell antibody 12E10 (IgG, Fab) and derive from the strand Fv (5 amino acid of joint sequence, 15 amino acid) of antibody 12E10.

Utilize the following transient expression that carries out of COS7 cell.Use Gene Pulser II device (BioRad), carry out transfection by electroporation method.In being suspended in the COS-7 cell of 0.8ml PBS (1 * 10 ⁷Cell/ml), for expressing antibodies 12E10 (IgG) adds above-mentioned expression vector HEF-12E10H-g γ 1 and each 10 μ g of pCOS-12E10L, add pFd-12E10H and each 10 μ g of pCOS-12E10L for expressing the 12E10Fab fragment, add pCOS-sc12E10 (10 μ g) or pCOS-db12E10 (10 μ g) for expressing strand Fv.Utilize 1.5KV, 25 μ FD capacitor pulses to handle the mixture in the cuvette.Room temperature was recovered after 10 minutes, electroporation of cells is added in the DMEM substratum (GIBCO BRL) that contains 10% foetal calf serum cultivates.After the overnight incubation, with the PBS washed cell once, be added among the serum free medium CHO-S-SFMII (GIBCO BRL) and cultivated 3 days.The centrifugal removal cell debris of culture supernatant is with 0.22 μ m membrane filtration.

For setting up the Chinese hamster ovary celI system that stably express derives from the strand Fv (polypeptide) of antibody 12E10, expression vector pCHO-sc12E10 or pCOS-ds12E10 are introduced Chinese hamster ovary celI respectively.

Use Gene Pulser II device (BioRad), every kind of expression vector is introduced Chinese hamster ovary celI by electroporation method.Utilize Restriction Enzyme PvuI digestion, obtain linearizing DNA (100 μ g), with the Chinese hamster ovary celI (1 * 10 that is suspended in 0.8ml PBS ⁷Cell/ml) mix in cuvette left standstill 10 minutes on ice, utilized 1.5KV, 25 μ FD capacitor pulses to handle.Room temperature was recovered after 10 minutes, electroporation of cells is added in the CHO-S-SFM II substratum (GIBCO BRL) that contains 10% foetal calf serum of dialysing and nucleic acid cultivates.Cultivate after 2 days, in containing the free nucleic acid CHO-S-SFM II substratum (GIBCOBRL) of 10% foetal calf serum of dialysing, continue to cultivate.From the clone of such acquisition, select high expression level rate clone, as the production clone of 12E10 strand Fv.After cultivating in serum-free CHO-S-SFM II substratum (GIBCO BRL), centrifugal culture supernatant is removed cell debris, with 0.22 μ m membrane filtration.

8.5 The strand Fv that derives from 12E10 that purifying CHO cell produces

With the Chinese hamster ovary celI of the expression 12E10 strand Fv (sc12E10, db12E10) of acquisition among the embodiment 8.4 is the culture supernatant that produces, and utilizes anti-FLAG antibody column and gel-filtration column purification respectively, the strand Fv of preparation purifying.

(1) utilizes anti-FLAG antibody column purifying

Every kind of culture supernatant (sc12E10, db12E10) is added with 50mM Tris-HCl damping fluid (pH7.4) the equilibrated anti-FLAG M2 affinity gel post (Sigma) that contains 150mM NaCl.Utilize same buffer to wash after the post, by the protein that adsorbs on 100mM glycine buffer (pH3.5) the wash-out post.Elution fraction adds 1M Tris-HCl damping fluid (pH8.0) immediately and neutralizes, and analyzes with SDS-PAGE.With confirming to contain the component mixing of strand Fv, utilize Centricon-10 (AMICON) to concentrate about 20 times.

(2) gel-filtration

(1) concentrated solution that obtains in adds with the PBS equilibrated Superdex200 post HR (10 * 300mm, AMERSHAM PHARMACIA) that contains 0.01% Tween20.Tomographic map is shown in Figure 53 and 54.Product sc12E10 wash-out is 2 peaks (A, B) (referring to Figure 53).Product db12E10 wash-out is 2 peaks (C, D) (referring to Figure 54).Collect each peak component,,, use coomassie brilliant blue staining behind the electrophoresis according to Laemmli method electrophoresis having or not having in the presence of the reductive agent and handle.Shown in Figure 55, no matter whether reductive agent exists, and all components A, B, C and D produce the single band of the about 31kD of apparent molecular weight.When utilizing Superdex200HR to analyze these components by gel-filtration, component A produces the product of the about 42kD of apparent molecular weight place wash-out, B component produces the product (referring to Figure 56) of 20kD, and component C produces the product of 69kD, and component D produces the product (referring to Figure 57) of 41kD.This results suggest, the component A in sc12E10 source is the non covalent bond dimer of strand Fv, and B component is the monomer of strand Fv, and the component C in db12E10 source is the non covalent bond tripolymer of strand Fv, and component D is the non covalent bond dimer of strand Fv.

8.6 Measure the TPO sample agonist activity of various strand Fv

By measuring proliferation activity, estimate the TPO sample activity of anti-mpl single-chain antibody to the Ba/F3 cell (BaF/mpl) of expressing human TPO acceptor (MPL).

After utilization contained twice in RPMI1640 substratum (GIBCO) the washing BaF/mpl cell of 1% foetal calf serum (GIBCO), cell was by 5 * 10 ⁵The cell density of cell/ml is suspended in the substratum.Dilute anti-MPL single-chain antibody or people TPO (R﹠amp respectively with substratum; D Systems).The antibody of 50 μ l cell suspensions and 50 μ l dilution or people TPO are added in the 96 hole microwell plates (flat) (Falcon), CO ₂(CO in the insulation can ₂Concentration: 5%) cultivated 24 hours.Add 10 μ l WST-8 reagent behind the incubation and (measure the reagent of initiating cell SF number: Nacalai Tesque), utilize absorption spectrophotometry Benchmark Plus (BioRad) to measure absorbancy immediately, measure wavelength 450nm, reference wavelength 655nm.At CO ₂(CO in the insulation can ₂Concentration: 5%) incubation utilized Benchmark Plus to measure absorbancy once more after 2 hours, measured wavelength 450nm, reference wavelength 655nm.Because WST-8 reagent produces color reaction according to the viable cell number at wavelength 450nm place, based on the variation of absorbancy after 2 hours, the proliferation activity of evaluation BaF/mpl.

Utilize the COS-7 cells and supernatant of expressing various 12E10 antibody molecules, measure agonist activity, shown in Figure 58 MPL.Have the strand Fv of 5 amino acid joints (ds12E10) and 15 amino acid joints (sc12E10), its absorbancy improves in the concentration dependent mode, shows TPO sample agonist activity (ED50; Difference 9pM and 51pM), and 12E10IgG and 12E10Fab do not have activity.

Known strand Fv H chain and the not only intramolecularly combination of L chain, and intermolecular in conjunction with forming polymer, for example dimer.The culture supernatant of the Chinese hamster ovary celI of expression 12E10 strand Fv is measured the agonist activity to MPL through gel-filtration.The result is shown in Figure 59.The a small amount of dimer that comprises among the sc12E10, TPO sample agonist activity (sc12E10 dimer, the ED50 of demonstration; 1.9pM) than monomer (sc12E10 monomer, ED50;＞10nM) high about 5000 times.Activity is higher than TPO (ED50; 27pM).The dimer of db12E10 (db12E10 dimer, ED50; 2.0pM) the demonstration activity suitable with the sc12E10 dimer.The dimer of db12E10 (db12E10 dimer, ED50; 2.0pM) the demonstration high reactivity suitable with the sc12E10 dimer.The tripolymer of db12E10 (molecular weight that obtains from gel-filtration is speculated as tripolymer) (ED50; 7.4pM) show and to be lower than the dimeric high reactivity of db12E10.These results suggest, two valency antigen binding sites (dimer) are important for the activity of agonist antibody 12E10.In view of 12E10IgG does not have activity, infer that the other factors outside two valencys is important, for example the location of antigen binding site, distance or angle.

Description of drawings

Fig. 1 represents the flow cytometry result, shows the L1210 cell (hIAP/L1210) of human IgG antibody's debond expressing human IAP.

Fig. 2 represents the flow cytometry result, shows the L1210 cell (hIAP/L1210) of chimeric MABL-1 antibodies specific associative list intelligent IAP.

Fig. 3 represents the flow cytometry result, shows the L1210 cell (hIAP/L1210) of chimeric MABL-2 antibodies specific associative list intelligent IAP.

The process of Fig. 4 diagram strand Fv produced according to the present invention.

Fig. 5 illustrates the structure of expression plasmid, and it is used in E. ColiMiddle coding DNA of expressing strand Fv of the present invention.

Fig. 6 illustrates the structure of expression plasmid, and it is used in the coding DNA of expressing strand Fv of the present invention in the mammalian cell.

Fig. 7 shows the figure as a result of Western trace among the embodiment 5.4.Begin to, molecular weight sign (from top demonstration 97.4,66,45,31,21.5 and 14.5kDa) from the left side, introduce the COS7 cells and supernatant of pCHO1 and the COS7 cells and supernatant of introducing pCHOM2.Show the reconstruction strand Fv (arrow) that in the cells and supernatant of introducing pCHOM2, comprises antibody MABL-2.

Fig. 8 represents the flow cytometry result, shows the antibody in the pCHO1/COS7 cells and supernatant that compares, debond pCOS1/L1210 control cells.

Fig. 9 represents the flow cytometry result, shows the antibody in the MABL2-scFv/COS7 cells and supernatant, debond pCOS1/L1210 control cells.

Figure 10 represents the flow cytometry result, shows the antibody debond hIAP/L1210 cell in the pCOS1/COS7 cells and supernatant that compares.

Figure 11 represents the flow cytometry result, shows that antibodies specific in the MABL2-scFv/COS7 cells and supernatant is in conjunction with the hIAP/L1210 cell.

Figure 12 represents competitive ELISA result among the embodiment 5.6, be indication wherein with restraining effect to mouse monoclonal antibody MABL-2 conjugated antigen, compare with the pCHO1/COS7 cells and supernatant of contrast, illustrate the antigen-binding activity of strand Fv of the present invention (MABL2-scFv).

Figure 13 represents the result of apoptosis induction effect among the embodiment 5.7, shows the antibody in the pCHO1/COS7 cells and supernatant that compares, and can not induce pCOS1/L1210 control cells apoptosis.

Figure 14 represents the result of apoptosis induction effect among the embodiment 5.7, shows the antibody in the MABL2-scFv/COS7 cells and supernatant, does not induce pCOS1/L1210 control cells apoptosis.

Figure 15 represents the result of apoptosis induction effect among the embodiment 5.7, shows the antibody in the pCHO1/COS7 cells and supernatant that compares, and does not induce the hIAP/L1210 apoptosis.

Figure 16 represents the result of apoptosis induction effect among the embodiment 5.7, shows the antibody in the MABL2-scFv/COS7 cells and supernatant, and specificity is induced the hIAP/L1210 apoptosis.

Figure 17 represents the result of apoptosis induction effect among the embodiment 5.7, shows the antibody in the pCHO1/COS7 cells and supernatant that compares, and does not induce CCRF-CEM apoptosis (final concentration 50%).

Figure 18 represents the result of apoptosis induction effect among the embodiment 5.7, shows the antibody in the MABL2-scFv/COS7 cells and supernatant, and specificity is induced CCRF-CEM apoptosis (final concentration 50%).

Figure 19 represents the resulting tomographic results of strand Fv that comes from antibody MABL-2 that embodiment 5.9 purifying CHO cells produce, when showing with the hydroxyapatite column purification from the component of Blue-sepharose post, and the main peaks of acquisition, component A and B component.

Figure 20 represents the component A that embodiment 5.9-(2) obtains and the gel-filtration purification result of B component, show component A at apparent molecular weight about 36kD place and B component in the main peaks (being respectively AI and BI) of about 76kD place wash-out.

Figure 21 shows the single band of observing the about 35kD of molecular weight in two components for the SDS-PAGE of the strand component that Fv obtains that comes from antibody MABL-2 that embodiment 5.9 purifying CHO cells produce analyzes.

Figure 22 represents that the strand Fv that comes from antibody MABL-2 of gel-filtration purifying CHO cell generation obtains the analytical results of component AI and BI, and wherein component AI comprises monomer, and B component I comprises dimer.

Figure 23 illustrates the structure of expression plasmid, and it is used in E. ColiMiddle coding DNA of expressing strand Fv of the present invention.

Figure 24 represents embodiment 5.12 gel-filtration column purification E. ColiThe result of the strand Fv polypeptide crude product that comes from antibody MABL-2 that produces, wherein each peak is represented respectively E. ColiMonomer or the dimer of the strand Fv that produces.

Figure 25 represents the result of apoptosis induction effect among the embodiment 5.13, shows the mouse IgG antibody that compares, and does not induce hIAP/L1210 apoptosis (final concentration 3 μ g/ml).

Figure 26 represents the result of apoptosis induction effect among the embodiment 5.13, shows the MABL2-scFv dimer that CHO produces, and significantly induces hIAP/L1210 apoptosis (final concentration 3 μ g/ml).

Figure 27 represents the result of apoptosis induction effect among the embodiment 5.13, shows E. ColiThe MABL2-scFv dimer that produces is significantly induced hIAP/L1210 apoptosis (final concentration 3 μ g/ml).

Figure 28 represents the result of apoptosis induction effect among the embodiment 5.13, shows that MABL2-scFv monomer that Chinese hamster ovary celI produces is in par (final concentration 3 μ g/ml) to the apoptosis-induced effect and the contrast of hIAP/L1210 cell.

Figure 29 represents the result of apoptosis induction effect among the embodiment 5.13, shows E. ColiThe MABL2-scFv monomer that produces is in par (final concentration 3 μ g/ml) to the apoptosis-induced effect and the contrast of hIAP/L1210 cell.

Figure 30 represents the result of apoptosis induction effect among the embodiment 5.13, shows the mouse IgG antibody that compares, even add anti-FLAG antibody, does not also induce hIAP/L1210 apoptosis (final concentration 3 μ g/ml).

Figure 31 represents the result of apoptosis induction effect among the embodiment 5.13, shows the MABL2-scFv monomer that Chinese hamster ovary celI produces, and when adding anti-FLAG antibody, significantly induces hIAP/L1210 apoptosis (final concentration 3 μ g/ml).

Figure 32 is illustrated in the quantitative measurment result of human IgG in the serum that human myeloma cell line KPMM2 transplants mouse, shows the human IgG quantity that is produced by human myeloma cell in the mouse.Show that the scFv/CHO dimer significantly suppresses the growth of KPMM2 cell.

Figure 33 represents the survival time of mouse after transplantation tumor, shows scFv/CHO dimer administration group survival time significant prolongation.

Figure 34 illustrates the structure of expression plasmid, and it can express the antibody [sc (Fv) of the change that comprises two H chain V districts coming from antibody MABL-2 and two L chain V districts ₂].

Figure 35 illustrates the structure of expressing scFv (HL type) plasmid, and wherein the V district connects by [H chain]-[L chain] mode that does not have peptide linker.

Figure 36 illustrates the structure of HL type polypeptide and the aminoacid sequence of peptide linker.

Figure 37 illustrates the structure of expressing scFv (LH type) plasmid, and wherein the V district connects by [L chain]-[H chain] mode that does not have peptide linker.

Figure 38 illustrates the structure of LH type polypeptide and the aminoacid sequence of peptide linker.

Figure 39 represents the result of embodiment 6.4 Western traces, shows the antibody sc (Fv) that has expressed the change that comprises two H chain V districts and two L chain V districts ₂, and MABL2-scFv with different lengths peptide linker.

Figure 40 a and 40b represent the flow cytometry result of the COS7 cells and supernatant of embodiment 6.3 (1) preparations, show MABL2-scFv and the sc (Fv) with different lengths peptide linker ₂People IAP had high-affinity.

Figure 41 a and 41b represent the result of embodiment 6.6 apoptosis induction effects, show scFv＜HL3,4,6,7, LH3,4,6 and 7〉and sc (Fv) ₂Significantly induce the hIAP/L1210 necrocytosis.

Figure 42 represents the evaluation result of embodiment 6.10 antigen binding capacities, shows scFv＜HL5〉dimer and sc (Fv) ₂People IAP had high-affinity.

Figure 43 represents embodiment 6.11 ExternalThe result of apoptosis induction effect shows scFv＜HL5〉dimer and sc (Fv) ₂Induce hIAP/L1210 cell and CCRF-CEM apoptosis in concentration dependence mode.

Figure 44 is illustrated in the mice serum of transplanting human myeloma cell, by the proteinic quantitative measurment result of M of human myeloma cell line KPMM2 generation.Show scFv＜HL5〉dimer and sc (Fv) ₂Significantly suppress the growth of KPMM2 cell.

Figure 45 represent mouse after the tumour transplatation survival time (my god), show scFv＜HL5 the survival time significant prolongation of administration group.

Figure 46 represent mouse after the tumour transplatation survival time (my god), show sc (Fv) ₂The survival time significant prolongation of administration group.

Figure 47 illustrates the segmental construction process of coding DNA and the structure thereof of rebuilding 12B5 strand Fv (comprising the joint sequence of being made up of 15 amino acid).

Figure 48 represents the gel-filtration purification result of every kind of 12B5 strand Fv of acquisition among the embodiment 7.5 (1), shows that sc12B5 is divided into two peaks (component A and B).

Figure 49 represents each the component A that carries out among the embodiment 7.5 (2) and the SDS-PAGE analytical results of B.

Figure 50 represents each the component A that carries out among the embodiment 7.5 (2) and the Superdex200 post analytical results of B, the main peak that shows component A is at the wash-out of about 44kD apparent molecular weight place shown in (a), and the main peak of B component is at the wash-out of about 22kD apparent molecular weight place shown in (b).

Figure 51 represents the measuring result of the TPO sample agonist activity of sc12B5 and antibody 12B5 (IgG, Fab), shows that 12B5IgG and unit price strand Fv (sc12B5) show TPO sample agonist activity in the concentration dependent mode.

Figure 52 represents the measuring result of sc12B5 monomer and dimeric TPO sample agonist activity, the agonist activity that shows the strand Fv (sc12B5 dimer) with two valency antigen binding sites is higher than about 400 times of unit price sc12B5, and effectiveness is equal to or higher than people TPO.

Figure 53 represents to utilize the purification result of Superdex200HR post by the sc12E10 single-chain antibody of gel permeation chromatography acquisition, shows that sc12E10 is divided into two peaks (component A and B).

Figure 54 represents to utilize the purification result of Superdex200HR post by the db12E10 single-chain antibody of gel permeation chromatography acquisition, shows that db12E10 is divided into two peaks (component C and D).

Figure 55 represents component A and B (sc12E10) and component C and the SDS-PAGE analysis of D (db12E10) under reduction or non-reduced condition.

Figure 56 represents to utilize the Superdex200HR post by the component A of gel permeation chromatography and the analytical results of B, and the main peak that shows (1) component A is at about 42kD apparent molecular weight place wash-out, and the main peak of (2) B component is at about 20kD apparent molecular weight place wash-out.

Figure 57 represents to utilize the Superdex200HR post by the component C of gel permeation chromatography and the analytical results of D, and the main peak that shows (1) component C is at about 69kD apparent molecular weight place wash-out, and the main peak of (2) component D is at about 41kD apparent molecular weight place wash-out.

Figure 58 illustrates the agonist activity of various 12E10 antibody molecules to MPL, show that strand Fv (sc12E10, db12E10) shows TPO sample agonist activity, and 12E10 IgG and 12E10 Fab does not show.

Figure 59 illustrates sc12E10 monomer and dimer and db12E10 dimer and the tripolymer agonist activity to MPL, shows that the TPO sample agonist activity that sc12E10 dimer and db12E10 dimer and tripolymer show is higher than TPO.

Commercial Application

Change antibody of the present invention has can transduction signal enters the activator effect of cell by crosslinked cell surface molecule, and advantage is that the molecular size of comparing antibody molecule (fully IgG) because of it descends to the permeability height of tissue and tumour. The invention provides the antibody that its agonist activity is significantly higher than the change of TPO or parental antibody (fully IgG). Particularly even without the parental antibody of agonist activity, also can change into the antibody of the agonist activity change higher than TPO. Therefore, the antibody of this change can be used as the signal transduction activator. The change of antibody molecule causes the crosslinked reduced side effects that causes of iuntercellular, and the newtype drug of only inducing required effect by crosslinked cell surface molecule is provided. The antibody that comprises change of the present invention is the pharmaceutical preparation of active component, can be used as the medicine that prevents and/or treats of thrombopenia due to decrease of platelet relevant hematologic disease, cancer or the leukemia chemotherapy etc.

Sequence table

<110>CHUGAI SEIYAKU KABUSHIKI KAISHA

＜120〉degraded TPO agonist antibody

<130>FP1033

<141>2001-10-22

<150>JP2000-321821

<151>2000-10-20

<150>PCT/JP01/03288

<151>2001-04-17

<150>JP2001-277314

<151>2001-09-12

<160>113

<210>1

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>1

ccatcctaat acgactcact atagggc 27

<210>2

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>2

ggatcccggg tggatggtgg gaagatg 27

<210>3

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>3

ggatcccggg ccagtggata gacagatg 28

<210>4

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>4

ggatcccggg agtggataga ccgatg 26

<210>5

<211>394

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(393)

＜223〉pGEM-M1L.1-57; Signal peptide, 58-394; Mature peptide

<400>5

atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct gcg 48

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 15

tcc agc agt gat gtt gtg atg acc caa act cca ctc tcc ctg cct gtc 96

Ser Ser Ser Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val

20 25 30

agt ctt gga gat caa gcc tcc atc tct tgc aga tct agt cag agc ctt 144

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

35 40 45

cta cac agt aaa gga aac acc tat tta caa tgg tac cta cag aag cca 192

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

50 55 60

ggc cag tct cca aag ctc ctg atc tac aaa gtt tcc aac cga ttt tct 240

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

65 70 75 80

ggg gtc cca gac agg ttc agt ggc agt gga tca ggg aca gat ttc aca 288

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

85 90 95

ctc aag atc agc aga gtg gag gct gag gat ctg gga gtt tat ttc tgc 336

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

100 105 110

tct caa agt aca cat gtt ccg tac acg tcc gga ggg ggg acc aag ctg 384

Ser Gln Ser Thr His Val Pro Tyr Thr Ser Gly Gly Gly Thr Lys Leu

115 120 125

gaa ata aaa c 394

Glu Ile Lys

130

<210>6

<211>409

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(408)

＜223〉pGEM-M1H.1-57; Signal peptide, 58-409; Mature peptide

<400>6

atg gaa tgg agc tgg ata ttt ctc ttc ctc ctg tca gga act gca ggt 48

Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly

1 5 10 15

gtc cac tcc cag gtc cag ctg cag cag tct gga cct gac ctg gta aag 96

Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys

20 25 30

cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc 144

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

35 40 45

gtt aac cat gtt atg cac tgg gtg aag cag aag cca ggg cag ggc ctt 192

Val Asn His Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu

50 55 60

gag tgg att gga tat att tat cct tac aat gat ggt act aag tac aat 240

Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

gag aag ttc aag ggc aag gcc aca ctg act tca gag aaa tcc tcc agc 288

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

85 90 95

gca gcc tac atg gag ctc agc agc ctg gcc tct gag gac tct gcg gtc 336

Ala Ala Tyr Met Glu Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val

100 105 110

tac tac tgt gca aga ggg ggt tac tat agt tac gac gac tgg ggc caa 384

Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Ser Tyr Asp Asp Trp Gly Gln

115 120 125

ggc acc act ctc aca gtc tcc tca g 409

Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210>7

<211>394

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(393)

＜223〉pGEM-M2L.1-57; Signal peptide, 58-394; Mature peptide

<400>7

atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct ggt 48

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Gly

1 5 10 15

tcc agc agt gat gtt gtg atg acc caa agt cca ctc tcc ctg cct gtc 96

Ser Ser Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val

20 25 30

agt ctt gga gat caa gcc tcc atc tct tgo aga tca agt cag agc ctt 144

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

35 40 45

gtg cac agt aat gga aag acc tat tta cat tgg tac ctg cag aag cca 192

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

50 55 60

ggc cag tct cca aaa ctc ctg atc tac aaa gtt tcc aac cga ttt tct 240

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

65 70 75 80

ggg gtc cca gac agg ttc agt ggc agt gga tca gtg aca gat ttc aca 288

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

85 90 95

ctc atg atc agc aga gtg gag gct gag gat ctg gga gtt tat ttc tgc 336

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

100 105 110

tct caa agt aca cat gtt ccg tac acg ttc gga ggg ggg acc aag ctg 384

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

115 120 125

gaa ata aaa c 394

Glu Ile Lys

130

<210>8

<211>409

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(408)

＜223〉pGEM-M2H.1-57; Signal peptide, 58-409; Mature peptide

<400>8

atg gaa tgg agc tgg ata ttt ctc ttc ctc ctg tca gga act gca ggt 48

Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly

1 5 10 15

gtc cac tcc cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag 96

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

20 25 30

cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc 144

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

35 40 45

gct aac cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt 192

Ala Asn His Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu

50 55 60

gag tgg att gga tat att tat cct tac aat gat ggt act aag tat aat 240

Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

gag aag ttc aag gac aag gcc act ctg act tca gac aaa tcc tcc acc 288

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

85 90 95

aca gcc tac atg gac ctc agc agc ctg gcc tct gag gac tct gcg gtc 336

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

100 105 110

tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa 384

Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln

115 120 125

ggc acc act ctc aca gtc tcc tca g 409

Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210>9

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>9

cccaagcttc caccatgaag ttgcctgtta gg 32

<210>10

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>10

cccaagcttc caccatggaa tggagctgga ta 32

<210>11

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>11

cgcggatcca ctcacgtttt atttccagct tggt 34

<210>12

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>12

cgcggatcca ctcacctgag gagactgtga gagt 34

<210>13

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>13

catgccatgg cgcaggtcca gctgcagcag 30

<210>14

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>14

accaccacct gaggagactg tgagagt 27

<210>15

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>15

gtctcctcag gtggtggtgg ttcgggt 27

<210>16

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>16

cacaacatcc gatccgccac cacccga 27

<21O>17

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>17

ggcggatcgg atgttgtgat gacccaa 27

<210>18

<211>57

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>18

ccggaattct cattatttat cgtcatcgtc tttgtagtct tttatttcca gcttggt 57

<210>19

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉joint aminoacid sequence and nucleotide sequence

<400>19

ggt ggt ggt ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg 45

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

5 10 15

<210>20

<211>828

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(822)

<223>pscM1.MABL1-scFv

<400>20

atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc gct 48

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

gcc caa cca gcc atg gcg cag gtc cag ctg cag cag tct gga cct gac 96

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

20 25 30

ctg gta aag cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga 144

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

35 40 45

tac acc ttc gtt aac cat gtt atg cac tgg gtg aag cag aag cca ggg 192

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

50 55 60

cag ggc ctt gag tgg att gga tat att tat cct tac aat gat ggt act 240

Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr

65 70 75 80

aag tac aat gag aag ttc aag ggc aag gcc aca ctg act tca gag aaa 288

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

85 90 95

tcc tcc agc gca gcc tac atg gag ctc agc agc ctg gcc tct gag gac 336

Ser Ser Ser Ala Ala Tyr Met Glu Leu Ser Ser Leu Ala Ser Glu Asp

100 105 110

tct gcg gtc tac tac tgt gca aga ggg ggt tac tat agt tac gac gac 384

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

115 120 125

tgg ggc caa ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg 432

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

130 135 140

ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa 480

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln

145 150 155 160

act cca ctc tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct 528

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

165 170 175

tgc aga tct agt cag agc ctt cta cac agt aaa gga aac acc tat tta 576

Cys Arg Ser Ser Gln Ser Leu Leu His Ser Lys Gly Asn Thr Tyr Leu

180 185 190

caa tgg tac cta cag aag cca ggc cag tct cca aag ctc ctg atc tac 624

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

195 200 205

aaa gtt tcc aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt 672

Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

210 215 220

gga tca ggg aca gat ttc aca ctc aag atc agc aga gtg gag gct gag 720

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

225 230 235 240

gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg 768

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

245 250 255

tcc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac 816

Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp Asp

260 265 270

gat aaa taatga 828

Asp Lys

<210>21

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>21

acgcgtcgac tcccaggtcc agctgcagca g 31

<210>22

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>22

gaaggtgtat ccagaagc 18

<210>23

<211>819

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(813)

<223>pCHOM1.MABL1-scFv

<400>23

atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48

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

1 5 10 15

gtc gac tcc cag gtc cag ctg cag cag tct gga cct gac ctg gta aag 96

Val Asp Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Asp Leu Val Lys

20 25 30

cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc 144

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

35 40 45

gtt aac cat gtt atg cac tgg gtg aag cag aag cca ggg cag ggc ctt 192

Val Asn His Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu

50 55 60

gag tgg att gga tat att tat cct tac aat gat ggt act aag tac aat 240

Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

gag aag ttc aag ggc aag gcc aca ctg act tca gag aaa tcc tcc agc 288

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

85 90 95

gca gcc tac atg gag ctc agc agc ctg gcc tct gag gac tct gcg gtc 336

Ala Ala Tyr Met Glu Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val

100 105 110

tac tac tgt gca aga ggg ggt tac tat agt tac gac gac tgg ggc caa 384

Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Ser Tyr Asp Asp Trp Gly Gln

115 120 125

ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt 432

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

130 135 140

ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa act cca ctc 480

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

145 150 155 160

tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct tgc aga tct 528

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

165 170 175

agt cag agc ctt cta cac agt aaa gga aac acc tat tta caa tgg tac 576

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

180 185 190

cta cag aag cca ggc cag tct cca aag ctc ctg atc tac aaa gtt tcc 624

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

195 200 205

aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tca ggg 672

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

aca gat ttc aca ctc aag atc agc aga gtg gag gct gag gat ctg gga 720

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

225 230 235 240

gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg tcc gga ggg 768

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

245 250 255

ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac gat aaa taa 816

Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp Asp Asp Lys

260 265 270

tga 819

<210>24

<211>828

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(822)

<223>pscM2.MABL2-scFv

<400>24

atg aaa tac cta ttg cct acg gca gcc gct gga ttg tta tta ctc gct 48

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

gcc caa cca gcc atg gcg cag gtc cag ctg cag cag tct gga cct gaa 96

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

20 25 30

ctg gta aag cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga 144

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

35 40 45

tac acc ttc gct aac cat gtt att cac tgg gtg aag cag aag cca ggg 192

Tyr Thr Phe Ala Asn His Val Ile His Trp Val Lys Gln Lys Pro Gly

50 55 60

cag ggc ctt gag tgg att gga tat att tat cct tac aat gat ggt act 240

Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr

65 70 75 80

aag tat aat gag aag ttc aag gac aag gcc act ctg act tca gac aaa 288

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

85 90 95

tcc tcc acc aca gcc tac atg gac ctc agc agc ctg gcc tct gag gac 336

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

100 105 110

tct gcg gtc tat tac tgt gca aga ggg ggt tac tat act tac gac gac 384

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

115 120 125

tgg ggc caa ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg 432

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

130 135 140

ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa 480

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln

145 150 155 160

agt cca ctc tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct 528

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

165 170 175

tgc aga tca agt cag agc ctt gtg cac agt aat gga aag acc tat tta 576

Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Lys Thr Tyr

180 185 190

cat tgg tac ctg cag aag cca ggc cag tct cca aaa ctc ctg atc tac 624

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

195 200 205

aaa gtt tcc aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt 672

Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser

210 215 220

gga tca gtg aca gat ttc aca ctc atg atc agc aga gtg gag gct gag 720

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

225 230 235 240

gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg 768

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

245 250 255

ttc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac 816

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

260 265 270

gat aaa taa tga 828

Asp Lys

<210>25

<211>819

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(813)

<223>pCHOM2.MABL2-scFv

<400>25

atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48

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

1 5 10 15

gtc gac tcc cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag 96

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

20 25 30

cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc 144

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

35 40 45

gct aac cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt 192

Ala Asn His Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu

50 55 60

gag tgg att gga tat att tat cct tac aat gat ggt act aag tat aat 240

Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

gag aag ttc aag gac aag gcc act ctg act tca gac aaa tcc tcc acc 288

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

85 90 95

aca gcc tac atg gac ctc agc agc ctg gcc tct gag gac tct gcg gtc 336

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

100 105 110

tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa 384

Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln

115 120 125

ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt 432

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

130 135 140

ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa agt cca ctc 480

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

145 150 155 160

tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct tgc aga tca 528

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

165 170 175

agt cag agc ctt gtg cac agt aat gga aag acc tat tta cat tgg tac 576

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

180 185 190

ctg cag aag cca ggc cag tct cca aaa ctc ctg atc tac aaa gtt tcc 624

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

195 200 205

aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tca gtg 672

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Val

210 215 220

aca gat ttc aca ctc atg atc agc aga gtg gag gct gag gat ctg gga 720

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

225 230 235 240

gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg ttc gga ggg 768

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

245 250 255

ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac gat aaa taa 816

Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp Asp Asp Lys

260 265 270

tga 819

<210>26

<211>456

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(450)

＜223〉pCHO-shIAP. soluble human IAP

<400>26

atg tgg ccc ctg gta gcg gcg ctg ttg ctg ggc tcg gcg tgc tgc gga 48

Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly

1 5 10 15

tca gct cag cta cta ttt aat aaa aca aaa tct gta gaa ttc acg ttt 96

Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe

20 25 30

tgt aat gac act gtc gtc att cca tgc ttt gtt act aat atg gag gca 144

Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala

35 40 45

caa aac act act gaa gta tac gta aag tgg aaa ttt aaa gga aga gat 192

Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp

50 55 60

att tac acc ttt gat gga gct cta aac aag tcc act gtc ccc act gac 240

Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp

65 70 75 80

ttt agt agt gca aaa att gaa gtc tca caa tta cta aaa gga gat gcc 288

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

85 90 95

tct ttg aag atg gat aag agt gat gct gtc tca cac aca gga aac tac 336

Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr

100 105 110

act tgt gaa gta aca gaa tta acc aga gaa ggt gaa acg atc atc gag 384

Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu

115 120 125

cta aaa tat cgt gtt gtt tca tgg ttt tct cca aat gaa aat gac tac 432

Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Asp Tyr

130 135 140

aag gac gac gat gac aag tgatag 456

Lys Asp Asp Asp Asp Lys

145 150

<210>27

<211>46

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>27

ggaattccat atgcaagtgc aacttcaaca gtctggacct gaactg 46

<210>28

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>28

ggaattctca ttattttatt tccagcttgg t 31

<210>29

<211>741

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(735)

<223>pscM2DEm02.MABL2-scFv

<400>29

atg caa gtg caa ctt caa cag tct gga cct gaa ctg gta aag cct ggg 48

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

1 5 10 15

gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc gct aac 96

Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asn

20 25 30

cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt gag tgg 144

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

35 40 45

att gga tat att tat cct tac aat gat ggt act aag tat aat gag aag 192

Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys

50 55 60

ttc aag gac aag gcc act ctg act tca gac aaa tcc tcc acc aca gcc 240

Phe Lys Asp Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Thr Thr Ala

65 70 75 80

tac atg gac ctc agc agc ctg gcc tct gag gac tct gcg gtc tat tac 288

Tyr Met Asp Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr

85 90 95

tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa ggc acc 336

Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln Gly Thr

100 105 110

act ctc aca gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt ggt tcg 384

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

115 120 125

ggt ggt ggc gga tcg gat gtt gtg atg acc caa agt cca ctc tcc ctg 432

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

130 135 140

cct gtc agt ctt gga gat caa gcc tcc atc tct tgc aga tca agt cag 480

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

145 150 155 160

agc ctt gtg cac agt aat gga aag acc tat tta cat tgg tac ctg cag 528

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

165 170 175

aag cca ggc cag tct cca aaa ctc ctg atc tac aaa gtt tcc aac cga 576

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

180 185 190

ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tca gtg aca gat 624

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

195 200 205

ttc aca ctc atg atc agc aga gtg gag gct gag gat ctg gga gtt tat 672

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

210 215 220

ttc tgc tct caa agt aca cat gtt ccg tac acg ttc gga ggg ggg acc 720

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

225 230 235 240

aag ctg gaa ata aaa taatga 741

Lys Leu Glu Ile Lys

245

<210>30

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>30

cagacagtgg ttcaaagt 18

<210>31

<211>72

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>31

cgcgtcgacc gatccgccac cacccgaacc accaccaccc gaaccaccac caccttttat 60

ttccagcttg gt 72

<210>32

<211>1605

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(1599)

<223>pCHOM2(Fv)2.MABL2-sc(Fv)2

<400>32

atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt 48

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

1 5 10 15

gtc gac tcc cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag 96

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

20 25 30

cct ggg gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc 144

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

35 40 45

gct aac cat gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt 192

Ala Asn His Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu

50 55 60

gag tgg att gga tat att tat cct tac aat gat ggt act aag tat aat 240

Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

gag aag ttc aag gac aag gcc act ctg act tca gac aaa tcc tcc acc 288

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

85 90 95

aca gcc tac atg gac ctc agc agc ctg gcc tct gag gac tct gcg gtc 336

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

100 105 110

tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg ggc caa 384

Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln

115 120 125

ggc acc act ctc aca gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt 432

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

130 135 140

ggt tcg ggt ggt ggc gga tcg gat gtt gtg atg acc caa agt cca ctc 480

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

145 150 155 160

tcc ctg cct gtc agt ctt gga gat caa gcc tcc atc tct tgc aga tca 528

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

165 170 175

agt cag agc ctt gtg cac agt aat gga aag acc tat tta cat tgg tac 576

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

180 185 190

ctg cag aag cca ggc cag tct cca aaa ctc ctg atc tac aaa gtt tcc 624

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

195 200 205

aac cga ttt tct ggg gtc cca gac agg ttc agt ggc agt gga tca gtg 672

Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Val

210 215 220

aca gat ttc aca ctc atg atc agc aga gtg gag gct gag gat ctg gga 720

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

225 230 235 240

gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg ttc gga ggg 768

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

245 250 255

ggg acc aag ctg gaa ata aaa ggt ggt ggt ggt tcg ggt ggt ggt ggt 816

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

260 265 270

tcg ggt ggt ggc gga tcg gtc gac tcc cag gtc cag ctg cag cag tct 864

Ser Gly Gly Gly Gly Ser Val Asp Ser Gln Val Gln Leu Gln Gln Ser

275 280 285

gga cct gaa ctg gta aag cct ggg gct tca gtg aag atg tcc tgc aag 912

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

290 295 300

gct tct gga tac acc ttc gct aac cat gtt att cac tgg gtg aag cag 960

Ala Ser Gly Tyr Thr Phe Ala Asn His Val Ile His Trp Val Lys Gln

305 310 315 320

aag cca ggg cag ggc ctt gag tgg att gga tat att tat cct tac aat 1008

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

325 330 335

gat ggt act aag tat aat gag aag ttc aag gac aag gcc act ctg act 1056

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

340 345 350

tca gac aaa tcc tcc acc aca gcc tac atg gac ctc agc agc ctg gcc 1104

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

355 360 365

tct gag gac tct gcg gtc tat tac tgt gca aga ggg ggt tac tat act 1152

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

370 375 380

tac gac gac tgg ggc caa ggc acc act ctc aca gtc tcc tca ggt ggt 1200

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

385 390 395 400

ggt ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gat gtt gtg 1248

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

405 410 415

atg acc caa agt cca ctc tcc ctg cct gtc agt ctt gga gat caa gcc 1296

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

420 425 430

tcc atc tct tgc aga rca agt cag agc crt gtg cac agt aat gga aag 1344

Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Lys

435 440 445

acc tat tta cat tgg tac ctg cag aag cca ggc cag tct cca aaa ctc 1392

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

450 455 460

ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca gac agg ttc 1440

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

465 470 475 480

agt ggc agt gga tca gtg aca gat ttc aca ctc atg atc agc aga gtg 1488

Ser Gly Ser Gly Ser Val Thr Asp Phe Thr Leu Met Ile Ser Arg Val

485 490 495

gag gct gag gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt 1536

Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val

500 505 510

ccg tac acg ttc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa 1584

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

515 520 525

gac gat gac gat aaa taatga 1605

Asp Asp Asp Asp Lys

530

<210>33

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>33

tgaggaattc ccaccatggg atg 33

<210>34

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>34

cacgacgtca ctcgagactg tgagagtggt gccttggccc 40

<210>35

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>35

agtctcgagt gacgtcgtga tgacccaaag tccactctcc 40

<210>36

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>36

gactggatcc tcattattta tcgtcatcgt c 31

<210>37

<211>22

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>37

cgcgtaatac gactcactat ag 22

<210>38

<211>46

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>38

gcaattggac ctgttttatc tcgagcttgg tcccccctcc gaacgt 46

<210>39

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>39

gctcgagata aaacaggtcc aattgcagca gtctggacct gaact 45

<210>40

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>40

gactggatcc tcattattta tcgtcatcgt ctttgtagtc tgaggagact gtgagagtgg 60

<210>41

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>41

gactgaattc ccaccatgaa gttgcctgtt ag 32

<210>42

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>42

cagtctcgag tggtggttcc gacgtcgtga tgacccaaag 40

<210>43

<211>43

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>43

cagtctcgag tggtggtggt tccgacgtcg tgatgaccca aag 43

<210>44

<211>46

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>44

cagtctcgag tggtggtggt ggttccgacg tcgtgatgac ccaaag 46

<210>45

<211>49

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>45

cagtctcgag tggtggtggt ggtggttccg acgtcgtgat gacccaaag 49

<210>46

<211>52

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>46

cagtctcgag tggtggtggt ggtggtggtt ccgacgtcgt gatgacccaa ag 52

<210>47

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>47

ggccgcatgt tgtcacgaat 20

<210>48

<211>780

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(768)

<223>CF2HL-0/pCOS1.MABL2-scFv<HL-0>

<400>48

atg gga tgg agc tgt atc atc ctc ttc ttg gta gca aca gct aca ggt gtc 51

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

5 10 15

gac tcc cag gtc cag ctg cag cag tct gga cct gaa ctg gta aag cct ggg 102

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

20 25 30

gct tca gtg aag atg tcc tgc aag gct tct gga tac acc ttc gct aac cat 153

Ala Ser Val Lys MET Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asn His

35 40 45 50

gtt att cac tgg gtg aag cag aag cca ggg cag ggc ctt gag tgg att gga 204

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

55 60 65

tat att tat cct tac aat gat ggt act aag tat aat gag aag ttc aag gac 255

Tyr Ile Tyr Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Asp

70 75 80 85

aag gcc act ctg act tca gac aaa tcc tcc acc aca gcc tac atg gac ctc 306

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

90 95 100

agc agc ctg gcc tct gag gac tct gcg gtc tat tac tgt gca aga ggg ggt 357

Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly

105 110 115

tac tat act tac gac gac tgg ggc caa ggc acc act ctc aca gtc tcg agt 408

Tyr Tyr Thr Tyr Asp Asp Trp Gly Gln Gly Thr Thr Leu Thr yal Ser Ser

120 125 130 135

gac gtc gtg atg acc caa agt cca ctc tcc ctg cct gtc agt ctt gga gat 459

Asp Val Val MET Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly Asp

140 145 150

caa gcc tcc atc tct tgc aga tca agt cag agc ctt gtg cac agt aat gga 510

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

155 160 165 170

aag acc tat tta cat tgg tac ctg cag aag cca ggc cag tct cca aaa ctc 561

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

175 180 185

ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca gac agg ttc agt 612

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

190 195 200

ggc agt gga tca gtg aca gat ttc aca ctc atg atc agc aga gtg gag gct 663

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

205 210 215 220

gag gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg tac acg 714

Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Val Pro Tyr Thr

225 230 235

ttc gga ggg ggg acc aag ctg gaa ata aaa gac tac aaa gac gat gac gat 765

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

240 245 250 255

aaa taa tga gga tcc 780

Lys

<210>49

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>49

caagctcgag ataaaatccg gaggccaggt ccaattgcag cagtc 45

<210>50

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>50

caagctcgag ataaaatccg gaggtggcca ggtccaattg cagcagtc 48

<210>51

<211>51

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>51

caagctcgag ataaaatccg gaggtggtgg ccaggtccaa ttgcagcagt c 51

<210>52

<211>54

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>52

caagctcgag ataaaatccg gaggtggtgg tggccaggtc caattgcagc agtc 54

<210>53

<211>57

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>53

caagctcgag ataaaatccg gaggtggtgg tggtggccag gtccaattgc agcagtc 57

<210>54

<211>780

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(768)

<223>CF2LH-0/pCOS1.MABL2-scFv<LH-0>

<400>54

atg aag ttg cct gtt agg ctg ttg gtg ctg atg ttc tgg att cct ggt tcc 51

MET Lys Leu Pro Val Arg Leu Leu Val Leu MET Phe Trp Ile Pro Gly Ser

5 10 15

agc agt gat gtt gtg atg acc caa agt cca ctc tcc ctg cct gtc agt ctt 102

Ser Ser Asp Val Val MET Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu

20 25 30

gga gat caa gcc tcc atc tct tgc aga tca agt cag agc ctt gtg cac agt 153

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

35 40 45 50

aat gga aag acc tat tta cat tgg tac ctg cag aag cca ggc cag tct cca 204

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

55 60 65

aaa ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca gac agg 255

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

70 75 80 85

ttc agt ggc agt gga tca gtg aca gat ttc aca ctc atg atc agc aga gtg 306

Phe Ser Gly Ser Gly Ser Val Thr Asp Phe Thr Leu MET Ile Ser Arg Val

90 95 100

gag gct gag gat ctg gga gtt tat ttc tgc tct caa agt aca cat gtt ccg 357

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

105 110 115

tac acg ttc gga ggg ggg acc aag ctc gag ata aaa cag gtc caa ttg cag 408

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gln Val Gln Leu Gln

120 125 130 135

cag tct gga cct gaa ctg gta aag cct ggg gct tca gtg aag atg tcc tgc 459

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

140 145 150

aag gct tct gga tac acc ttc gct aac cat gtt att cac tgg gtg aag cag 510

Lys Ala Ser Gly Tyr Thr Phe Ala Asn His Val Ile His Trp Val Lys Gln

155 160 165 170

aag cca ggg cag ggc ctt gag tgg att gga tat att tat cct tac aat gat 561

Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Tyr Asn Asp

175 180 185

ggt act aag tat aat gag aag ttc aag gac aag gcc act ctg act tca gac 612

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

190 195 200

aaa tcc tcc acc aca gcc tac atg gac ctc agc agc ctg gcc tct gag gac 663

Lys Ser Ser Thr Thr Ala Tyr MET Asp Leu Ser Ser Leu Ala Ser Glu Asp

205 210 215 220

tct gcg gtc tat tac tgt gca aga ggg ggt tac tat act tac gac gac tgg 714

Ser Ala Val Tyr Tyr Cys Ala Arg Gly Gly Tyr Tyr Thr Tyr Asp Asp Trp

225 230 235

ggc caa ggc acc act ctc aca gtc tcc tca gac tac aaa gac gat gac gat 765

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

240 245 250 255

aaa taa tga gga tcc 780

Lys

<210>55

<211>351

<212>DNA

＜213〉people

<220>

<221>CDS

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

＜223〉12B5HV.1-351 peptide

<400>55

cag gtg cag ctg gtg cag tct ggg gga ggc ttg gtc cgg ccc ggg ggg 48

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

tcc ctg agt ctc tcc tgt gca gtc tct gga atc acc ctc agg acc tac 96

Ser Leu Ser Leu Ser Cys Ala Val Ser Gly Ile Thr Leu Arg Thr Tyr

20 25 30

ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

gca ggt ata tcc ttt gac gga aga agt gaa tac tat gca gac tcc gtg 192

Ala Gly Ile Ser Phe Asp Gly Arg Ser Glu Tyr Tyr Ala Asp Ser Val

50 55 60

cag ggc cga ttc acc atc tcc aga gac agt tcc aag aac acc ctg tat 240

Gln Gly Arg Phe Thr Ile Ser Arg Asp Ser Ser Lys Asn Thr Leu Tyr

65 70 75 80

ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt 288

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

85 90 95

gcg aga gga gca cat tat ggt ttc gat atc tgg ggc caa ggg aca atg 336

Ala Arg Gly Ala His Tyr Gly Phe Asp Ile Trp Gly Gln Gly Thr Met

100 105 110

gtc acc gtc tcg agt 351

Val Thr Val Ser Ser

115

<210>56

<211>57

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)...(57)

＜223〉leader sequence

<400>56

atg gag ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt tta aga ggt 48

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

5 10 15

gtc cag tgt 57

Val Gln Cys

<210>57

<211>115

<212>DNA

＜213〉artificial sequence

<220>

<223>12B5VH-1

<400>57

atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60

gtgcagctgg tgcagtctgg gggaggcttg gtccggcccg gggggtccct gagtc 115

<210>58

<211>115

<212>DNA

＜213〉artificial sequence

<220>

<223>12B5VH-2

<400>58

aaggatatac ctgccaccca ctccagcccc ttgcctggag cctggcggac ccagtgcatg 60

ccgtaggtcc tgagggtgat tccagagact gcacaggaga gactcaggga ccccc 115

<210>59

<211>115

<212>DNA

＜213〉artificial sequence

<220>

<223>12B5VH-3

<400>59

ggcaggtata tcctttgacg gaagaagtga atactatgca gactccgtgc agggccgatt 60

caccatctcc agagacagtt ccaagaacac cctgtatctg caaatgaaca gcctg 115

<210>60

<211>108

<212>DNA

＜213〉artificial sequence

<220>

<223>12B5VH-4

<400>60

actcgagacg gtgaccattg tcccttggcc ccagatatcg aaaccataat gtgctcctct 60

cgcacagtaa tacacagccg tgtcctcggc tctcaggctg ttcatttg 108

<210>61

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉1285VH-S, the PCR primer

<400>61

ttcaagcttc caccatggag tttgggctga gc 32

<210>62

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉1285VH-A, the PCR primer

<400>62

ttgggatcca ctcaccactc gagacggtga ccat 34

<210>63

<211>588

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(236)...(558)

＜223〉1-235: intron, 236-558: human IgG constant region (part)

<400>63

gaattcgtga gtggatccca agctagcttt ctggggcagg ccaggcctga ccttggcttt 60

ggggcaggga gggggctaag gtgaggcagg tggcgccagc caggtgcaca cccaatgccc 120

atgagcccag acactggacg ctgaacctcg cggacagtta agaacccagg ggcctctgcg 180

ccctgggccc agctctgtcc cacaccgcgg tcacatggca caacctctct tgca gcc 237

Ala

1

tcc acc aag ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc 285

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu A1a Pro Ser Ser Lys Ser

5 10 15

acc tct ggg ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc 333

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

20 25 30

ccc gaa ccg gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc 381

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

35 40 45

gtg cac acc ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc 429

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

50 55 60 65

agc agc gtg gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac 477

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

70 75 80

atc tgc aac gtg aat cac aag ccc agc aac acc aag gtg gac aag aaa 525

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

85 90 95

gtt gag ccc aaa tct tgt gac aaa act cac aca 558

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

100 105

<210>64

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉G1CH1-S, the PCR primer

<400>64

tgagaattcg tgagtggatc ccaagct 27

<210>65

<211>60

<212>DNA

＜213〉artificial sequence

<220>

＜223〉G1CH1-A, the PCR primer

<400>65

aaaagatctt tatcatgtgt gagttttgtc acaagatttg ggctcaactt tcttgtccac 60

<210>66

<211>432

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(12)...(419)

＜223〉HEF-12B5H-g gamma.12-419 peptide

<400>66

aagcttccac c atg gag ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt 50

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu

1 5 10

tta aga ggt gtc cag tgt cag gtg cag ctg gtg cag tct ggg gga ggc 98

Leu Arg Gly Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Gly Gly

15 20 25

ttg gtc cgg ccc ggg ggg tcc ctg agt ctc tcc tgt gca gtc tct gga 146

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

30 35 40 45

atc acc ctc agg acc tac ggc atg cac tgg gtc cgc cag gct cca ggc 194

Ile Thr Leu Arg Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly

50 55 60

aag ggg ctg gag tgg gtg gca ggt ata tcc ttt gac gga aga agt gaa 242

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

65 70 75

tac tat gca gac tcc gtg cag ggc cga ttc acc atc tcc aga gac agt 290

Tyr Tyr Ala Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Ser

80 85 90

tcc aag aac acc ctg tat ctg caa atg aac agc ctg aga gcc gag gac 338

Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

95 100 105

acg gct gtg tat tac tgt gcg aga gga gca cat tat ggt ttc gat atc 386

Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala His Tyr Gly Phe Asp Ile

110 115 120 125

tgg ggc caa ggg aca atg gtc acc gtc tcg agt ggtgagtgga tcc 432

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

130 135

<210>67

<211>321

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)...(321)

＜223〉12B5LV.1-321 peptide

<400>67

gac atc cag atg acc cag tct cct tcc acc ctg tct gea tct att gga 48

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

1 5 10 15

gac aga gtc acc atc acc tgc cgg gcc agc gag ggt att tat cac tgg 96

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Gly Ile Tyr His Trp

20 25 30

ttg gcc tgg tat cag cag aag cca ggg aaa gcc cct aaa ctc ctg atc 144

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

35 40 45

tat aag gcc tct agt tta gcc agt ggg gcc cca tca agg ttc agc ggc 192

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

50 55 60

agt gga tct ggg aca gat ttc act ctc acc atc agc age ctg cag cct 240

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

65 70 75 80

gat gat ttt gca act tat tac tgc caa caa tat agt aat tat ccg ctc 288

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

85 90 95

act ttc ggc gga ggg acc aag ctg gag atc aaa 321

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>68

<211>66

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)...(66)

＜223〉leader sequence

<400>68

atg gac atg agg gtc ccc gct cag ctc ctg ggg ctc ctg ctg ctc tgg 48

MET Asp MET Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

5 10 15

ctc cca ggt gcc aaa tgt 66

Leu Pro Gly Ala Lys Cys

20

<210>69

<211>110

<212>DNA

＜213〉artificial sequence

<220>

<223>12B5VL-1

<400>69

atggacatga gggtccccgc tcagctcctg gggctcctgc tgctctggct cccaggtgcc 60

aaatgtgaca tccagatgac ccagtctcct tccaccctgt ctgcatctat 110

<210>70

<211>110

<212>DNA

＜213〉artificial sequence

<220>

<223>12B5VL-2

<400>70

ggagtttagg ggctttccct ggcttctgct gataccaggc caaccagtga taaataccct 60

cgctggcccg gcaggtgatg gtgactctgt ctccaataga tgcagacagg 110

<210>71

<211>110

<212>DNA

＜213〉artificial sequence

<220>

<223>12B5VL-3

<400>71

aagcccctaa actcctgatc tataaggcct ctagtttagc cagtggggcc ccatcaaggt 60

tcagcggcag tggatctggg acagatttca ctctcaccat cagcagcctg 110

<210>72

<211>103

<212>DNA

＜213〉artificial sequence

<220>

<223>12B5VL-4

<400>72

tttgatctcc agcttggtcc ctccgccgaa agtgagcgga taattactat attgttggca 60

gtaataagtt gcaaaatcat caggctgcag gctgctgatg gtg 103

<210>73

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12B5VL-S, the PCR primer

<400>73

ttcaagcttc caccatggac atgagggtcc cc 32

<210>74

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12B5VL-A, the PCR primer

<400>74

tctaggatcc actcacgttt gatctccagc ttggt 35

<210>75

<211>415

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(12)...(398)

＜223〉HEF-12B5H-g kappa.12-398 peptide

<400>75

aagcttccac c atg gac atg agg gtc ccc gct cag ctc ctg ggg ctc ctg 50

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu

1 5 10

ctg ctc tgg ctc cca ggt gcc aaa tgt gac atc cag atg acc cag tct 98

Leu Leu Trp Leu Pro Gly Ala Lys Cys Asp Ile Gln Met Thr Gln Ser

15 20 25

cct tcc acc ctg tct gca tct att gga gac aga gtc acc atc acc tgc 146

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

30 35 40 45

cgg gcc agc gag ggt att tat cac tgg ttg gcc tgg tat cag cag aag 194

Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln Lys

50 55 60

cea ggg aaa gcc cct aaa ctc ctg atc tat aag gcc tct agt tta gcc 242

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

65 70 75

agt ggg gcc cca tca agg ttc agc ggc agt gga tct ggg aca gat ttc 290

Ser Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

80 85 90

act ctc acc atc agc agc ctg cag cct gat gat ttt gca act tat tac 338

Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr

95 100 105

tgc caa caa tat agt aat tat ccg ctc act ttc ggc gga ggg acc aag 386

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

110 115 120 125

ctg gag atc aaa cgtgagtgga tcctaga 415

Leu Glu Ile Lys

<210>76

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉FLAG sequence label

<400>76

gac tac aag gat gac gac gat aag 24

Asp Tyr Lys Asp Asp Asp Asp Lys

5

<210>77

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12B5-S, the PCR primer

<400>77

atagaattcc accatggagt ttgggctgag c 31

<210>78

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉HuVHJ3, the PCR primer

<400>78

tgaagagacg gtgaccattg tccc 24

<210>79

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RhuJH3, the PCR primer

<400>79

ggacaatggt caccgtctct tcaggtgg 28

<210>80

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉RhuVK1, the PCR primer

<400>80

ggagactggg tcatctggat gtccgatccg cc 32

<210>81

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉HuVK1.2, the PCR primer

<400>81

gacatccaga tgacccagtc tcc 23

<210>82

<211>59

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12B5F-A, the PCR primer

<400>82

attgcggccg cttatcactt atcgtcgtca tccttgtagt ctttgatctc cagcttggt 59

<210>83

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉joint aminoacid sequence and nucleotide sequence

<400>83

ggt ggt ggt ggt tcg ggt ggt ggt ggt tcg ggt ggt ggc gga tcg 45

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

5 10 15

<210>84

<211>823

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(12)...(809)

＜223〉sc12B5, strand Fv

<400>84

aagcttccac c atg gag ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt 50

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu

1 5 10

tta aga ggt gtc cag tgt cag gtg cag ctg gtg cag tct ggg gga ggc 98

Leu Arg Gly Val Gln Cys Gln Val Gln Leu Val Gln Ser Gly Gly Gly

15 20 25

ttg gtc cgg ccc ggg ggg tcc ctg agt ctc tcc tgt gca gtc tct gga 146

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

30 35 40 45

atc acc ctc agg acc tac ggc atg cac tgg gtc cgc cag gct cca ggc 194

Ile Thr Leu Arg Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly

50 55 60

aag ggg ctg gag tgg gtg gca ggt ata tcc ttt gac gga aga agt gaa 242

Lys Gly Leu Glu Trp yal Ala Gly Ile Ser Phe Asp Gly Arg Ser Glu

65 70 75

tac tat gca gac tcc gtg cag ggc cga ttc acc atc tcc aga gac agt 290

Tyr Tyr Ala Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Ser

80 85 90

tcc aag aac acc ctg tat ctg caa atg aac agc ctg aga gcc gag gac 338

Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

95 100 105

acg gct gtg tat tac tgt gcg aga gga gca cat tat ggt ttc gat atc 386

Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala His Tyr Gly Phe Asp Ile

110 115 120 125

tgg ggc caa ggg aca atg gtc acc gtc tcg agt ggt ggt ggt ggt tcg 434

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

130 135 140

ggt ggt ggt ggt tcg ggt ggt ggc gga tcg gac atc cag atg acc cag 482

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln

145 150 155

tct cct tcc acc ctg tct gca tct att gga gac aga gtc acc atc acc 530

Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile Thr

160 165 170

tgc cgg gcc agc gag ggt att tat cac tgg ttg gcc tgg tat cag cag 578

Cys Arg Ala Ser Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln

175 180 185

aag cca ggg aaa gcc cct aaa ctc ctg atc tat aag gcc tct agt tta 626

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

190 195 200 205

gcc agt ggg gcc cca tca agg ttc agc ggc agt gga tct ggg aca gat 674

Ala Ser Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

210 215 220

ttc act ctc acc atc agc agc ctg cag cct gat gat ttt gca act tat 722

Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr

225 230 235

TAC TGC CAA CAA TAT AGT AAT TAT CCG CTC ACT TTC GGC GGA GGG ACC 770

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

240 245 250

aag ctg gag atc aaa gac tac aag gat gac gac gat aag tgataagcgg c 820

Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp Asp Asp Lys

255 260 265

cgc 823

<210>85

<211>114

<212>PRT

＜213〉people

<400>85

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Ser Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg Gly Thr Met Val Thr Val

100 105 110

Ser Ser

<210>86

<211>342

<212>DNA

＜213〉people

<400>86

caggtgcagc tgcagcagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60

acctgcactg tctctggtga ctccatcagt agttactact ggagctggat tcggcagccc 120

ccagggaagg gactggagtg gattgggtat atctattaca gtgggagcac caactacaac 180

ccctccctca agagtcgagt caccatatca gtagacacgt ccaagagcca gttctccctg 240

aagctgagct ctgtgaccgc cgcagacacg gccgtgtatt actgtgcgag agggcggtac 300

ttcgatgtct ggggccgtgg caccatggtc actgtctcct ca 342

<210>87

<211>57

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)...(57)

＜223〉leader sequence

<308>GenBank No.AF062252

<400>87

atg aaa cat ctg tgg ttc ttc ctt ctc ctg gtg gca gct ccc aga tgg 48

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

1 5 10 15

gtc ctg tcc 57

Val Leu Ser

<210>88

<211>110

<212>DNA

＜213〉artificial sequence

<220>

<223>12E10VH1

<400>88

atgaaacatc tgtggttctt ccttctcctg gtggcagctc ccagatgggt cctgtcccag 60

gtgcagctgc agcagtcggg cccaggactg gtgaagcctt cggagaccct 110

<210>89

<211>110

<212>DNA

＜213〉artificial sequence

<220>

<223>12E10VH2

<400>89

acccaatcca ctccagtccc ttccctgggg gctgccgaat ccagctccag tagtaactac 60

tgatggagtc accagagaca gtgcaggtga gggacagggt ctccgaaggc 110

<210>90

<211>110

<212>DNA

＜213〉artificial sequence

<220>

<223>12E10VH3

<400>90

tggagtggat tgggtatatc tattacagtg ggagcaccaa ctacaacccc tccctcaaga 60

gtcgagtcac catatcagta gacacgtcca agagccagtt ctccctgaag 110

<210>91

<211>114

<212>DNA

＜213〉artificial sequence

<220>

<223>12E10VH4

<400>91

tgaggagaca gtgaccatgg tgccacggcc ccagacatcg aagtaccgcc ctctcgcaca 60

gtaatacacg gccgtgtctg cggcggtcac agagctcagc ttcagggaga actg 114

<210>92

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12E10VHS, the PCR primer

<400>92

ttcaagcttc caccatgaaa catctgtggt tc 32

<210>93

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12E10VHA, the PCR primer

<400>93

ttgggatcca ctcacctgag gagacagtga ccat 34

<210>94

<211>426

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(12)...(417)

＜223〉12E10H, H chain V district

<400>94

aagcttccac c atg aaa cat ctg tgg ttc ttc ctt ctc ctg gtg gca gct 50

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala

1 5 10

ccc aga tgg gtc ctg tcc cag gtg cag ctg cag cag tcg ggc cca gga 98

Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly

15 20 25

ctg gtg aag cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggt 146

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

30 35 40 45

gac tcc atc agt agt tac tac tgg agc tgg att cgg cag ccc cca ggg 194

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

50 55 60

aag gga ctg gag tgg att ggg tat atc tat tac agt ggg agc acc aac 242

Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn

65 70 75

tac aac ccc tcc ctc aag agt cga gtc acc ata tca gta gac acg tcc 290

Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser

80 85 90

aag agc cag ttc tcc ctg aag ctg agc tct gtg acc gcc gca gac acg 338

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

95 100 105

gcc gtg tat tac tgt gcg aga ggg cgg tac ttc gat gtc tgg ggc cgt 386

Ala Val Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg

110 115 120 125

ggc acc atg gtc act gtc tcc tca ggtgagtgga tcccaa 426

Gly Thr Met Val Thr Val Ser Ser

130

<210>95

<211>110

<212>PRT

<213>Mus

<400>95

Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

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

85 90 95

Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210>96

<211>330

<212>DNA

<213>Mus

<400>96

tcctatgtgc tgactcagcc accctcggtg tcagggtctc ctggacagtc gatcaccatc 60

tcctgcactg gaaccagcag tgacgttggt ggttataact atgtctcctg gtaccaacag 120

cacccaggca aagcccccaa actcatgatt tatgagggca gtaaacggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240

caggctgagg acgaggctga ttattactgc agctcatata caaccagaag cactcgggtg 300

ttcggcggag ggaccaagct gaccgtccta 330

<210>97

<211>57

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)...(57)

＜223〉leader sequence

<310>

<400>97

atg gcc tgg acc gtt ctc ctc ctc ggc ctc ctc tct cac tgc aca ggc 48

Met Ala Trp Thr Val Leu Leu Leu Gly Leu Leu Ser His Cys Thr Gly

1 5 10 15

tct gtg acc 57

Ser Val Thr

<210>98

<211>110

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12E10VL1, the PCR primer

<400>98

atggcctgga ccgttctcct cctcggcctc ctctctcact gcacaggctc tgtgacctcc 60

tatgtgctga ctcagccacc ctcggtgtca gggtctcctg gacagtcgat 110

<210>99

<211>62

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12E10VL2, the PCR primer

<400>99

tcatgagttt gggggctttg cctgggtgct gttggtacca ggagacatag ttataaccac 60

caacgtcact gctggttcca gtgcaggaga tggtgatcga ctgtccagga 110

<210>100

<211>110

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12E10VL3, the PCR primer

<400>100

cccccaaact catgatttat gagggcagta aacggccctc aggggtttct aatcgcttct 60

ctggctccaa gtctggcaac acggcctccc tgaccatctc tgggctccag 110

<210>101

<211>102

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12E10VL4, the PCR primer

<400>101

taggacggtc agcttggtcc ctccgccgaa cacccgagtg cttctggttg tatatgagct 60

gcagtaataa tcagcctcgt cctcagcctg gagcccagag at 102

<210>102

<211>31

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12E10VLS, the PCR primer

<400>102

atcaagcttc caccatggcc tggaccgttc t 31

<210>103

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12E10VLA, the PCR primer

<400>103

ctaggatccg ggctgaccta ggacggtcag cttggt 36

<210>104

<211>387

<212>DNA

<213>Mus

<220>

<221>CDS

<222>(1)...(387)

＜223〉12E10L, L chain V district

<310>

<400>104

atg gcc tgg acc gtt ctc ctc ctc ggc ctc ctc tct cac tgc aca ggc 48

Met Ala Trp Thr Val Leu Leu Leu Gly Leu Leu Ser His Cys Thr Gly

1 5 10 15

tct gtg acc tcc tat gtg ctg act cag cca ccc tcg gtg tca ggg tct 96

Ser Val Thr Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ser

20 25 30

cct gga cag tcg atc acc atc tcc tgc act gga acc agc agt gac gtt 144

Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val

35 40 45

ggt ggt tat aac tat gtc tcc tgg tac caa cag cac cca ggc aaa gcc 192

Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala

50 55 60

ccc aaa ctc atg att tat gag ggc agt aaa cgg ccc tca ggg gtt tct 240

Pro Lys Leu Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Ser

65 70 75 80

aat cgc ttc tct ggc tcc aag tct ggc aac acg gcc tcc ctg acc atc 288

Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile

85 90 95

tct ggg ctc cag gct gag gac gag gct gat tat tac tgc agc tca tat 336

Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr

100 105 110

Aca acc aga agc act cgg gtg ttc ggc gga ggg acc aag ctg acc gtc 384

Thr Thr Arg Ser Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val

115 120 125

cta 387

Leu

<210>105

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(24)

＜223〉FLAG, leader sequence

<400>105

gac tac aag gat gac gac gat aag 24

Asp Tyr Lys Asp Asp Asp Asp Lys

<210>106

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12E10S, the PCR primer

<400>106

tatgaattcc accatgaaac atctgtggtt 30

<210>107

<211>38

<212>DNA

＜213〉artificial sequence

<220>

＜223〉DB2, the PCR primer

<400>107

taggagctac cgcctccacc tgaggagaca gtgaccat 38

<210>108

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉DB1, the PCR primer

<400>108

gtctcctcag gtggaggcgg tagctcctat gtgctgactc agcc 44

<210>109

<211>59

<212>DNA

＜213〉artificial sequence

<220>

＜223〉12E10FA, the PCR primer

<400>109

attgcggccg cttatcactt atcgtcgtca tccttgtagt ctaggacggt cagcttggt 59

<210>110

<211>792

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(11)...(778)

＜223〉12E10, strand Fv

<400>110

gaattccacc atg aaa cat ctg tgg ttc ttc ctt ctc ctg gtg gca gct 49

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala

1 5 10

ccc aga tgg gtc ctg tcc cag gtg cag ctg cag cag tcg ggc cca gga 97

Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly

15 20 25

ctg gtg aag cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggt 145

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

30 35 40 45

gac tcc atc agt agt tac tac tgg agc tgg att cgg cag ccc cca ggg 193

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

50 55 60

aag gga ctg gag tgg att ggg tat atc tat tac agt ggg agc acc aac 241

Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn

65 70 75

tac aac ccc tcc ctc aag agt cga gtc acc ata tca gta gac acg tcc 289

Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser

80 85 90

aag agc cag ttc tcc ctg aag ctg agc tct gtg acc gcc gca gac acg 337

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

95 100 105

gcc gtg tat tac tgt gcg aga ggg cgg tac ttc gat gtc tgg ggc cgt 385

Ala Val Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg

110 115 120 125

ggc acc atg gtc act gtc tcc tca ggt gga ggc ggt agc tcc tat gtg 433

Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Ser Tyr Val

130 135 140

ctg act cag cca ccc tcg gtg tca ggg tct cct gga cag tcg atc acc 481

Leu Thr Gln Pro Pro Ser Val Ser Gly Ser Pro Gly Gln Ser Ile Thr

145 150 155

atc tcc tgc act gga acc agc agt gac gtt ggt ggt tat aac tat gtc 529

Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val

160 165 170

tcc tgg tac caa cag cac cca ggc aaa gcc ccc aaa ctc atg att tat 577

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

175 180 185

gag ggc agt aaa cgg ccc tca ggg gtt tct aat cgc ttc tct ggc tcc 625

Glu Gly Ser Lys Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser

190 195 200 205

aag tct ggc aac acg gcc tcc ctg acc atc tct ggg ctc cag gct gag 673

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

210 215 220

gac gag gct gat tat tac tgc agc tca tat aca acc aga agc act cgg 721

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

225 230 235

gtg ttc ggc gga ggg acc aag ctg acc gtc cta gac tac aag gat gac 769

Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Asp Tyr Lys Asp Asp

240 245 250

gac gat aag tgataagcgg ccgc 792

Asp Asp Lys

255

<210>111

<211>62

<212>DNA

＜213〉artificial sequence

<220>

＜223〉sc4.3, the PCR primer

<400>111

ggtggctgag tcagcacata ggacgatccg ccaccacccg aaccaccacc acccgaacca 60

cc 62

<210>112

<211>61

<212>DNA

＜213〉artificial sequence

<220>

＜223〉sc1.3, the PCR primer

<400>112

gcaccatggt cactgtctcc tcaggtggtg gtggttcggg tggtggtggt tcgggtggtg 60

g 61

<210>113

<211>822

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(11)...(807)

＜223〉sc12E10, strand Fv

<400>113

gaattccacc atg aaa cat ctg tgg ttc ttc ctt ctc ctg gtg gca gct 49

Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala

1 5 10

ccc aga tgg gtc ctg tcc cag gtg cag ctg cag cag tcg ggc cca gga 97

Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly

15 20 25

ctg gtg aag cct tcg gag acc ctg tcc ctc acc tgc aet gtc tct ggt 145

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

30 35 40 45

gac tcc atc agt agt tac tac tgg agc tgg att cgg cag ccc cca ggg 193

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

50 55 60

aag gga ctg gag tgg att ggg tat atc tat tac agt ggg agc acc aac 241

Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn

65 70 75

tac aac ccc tcc ctc aag agt cga gtc acc ata tca gta gac acg tcc 289

Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser

80 85 90

aag agc cag ttc tcc ctg aag ctg agc tct gtg acc gcc gca gac acg 337

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

95 100 105

gcc gtg tat tac tgt gcg aga ggg cgg tac ttc gat gtc tgg ggc cgt 385

Ala Val Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Val Trp Gly Arg

110 115 120 125

ggc acc atg gtc act gtc tcc tca ggt ggt ggt ggt tcg ggt ggt ggt 433

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

130 135 140

ggt tcg ggt ggt ggc gga tcg tcc tat gtg ctg act cag cca ccc tcg 481

Gly Ser Gly Gly Gly Gly Ser Ser Tyr Val Leu Thr Gln Pro Pro Ser

145 150 155

gtg tca ggg tct cct gga cag tcg atc acc atc tcc tgc act gga acc 529

Val Ser Gly Ser Pro Gly Gln Ser Ile Thr Ile Ser Cys Thr Gly Thr

160 165 170

agc agt gac gtt ggt ggt tat aac tat gtc tcc tgg tac caa cag cac 577

Ser Ser Asp Val Gly Gly Tyr Asn Tyr Val Ser Trp Tyr Gln Gln His

175 180 185

cca ggc aaa gcc ccc aaa ctc atg att tat gag ggc agt aaa cgg ccc 625

Pro Gly Lys Ala Pro Lys Leu Met Ile Tyr Glu Gly Ser Lys Arg Pro

190 195 200 205

tca ggg gtt tct aat cgc ttc tct ggc tcc aag tct ggc aac acg gcc 673

Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala

210 215 220

tcc ctg acc atc tct ggg ctc cag gct gag gac gag gct gat tat tac 721

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

225 230 235

tgc agc tca tat aca acc aga agc act cgg gtg ttc ggc gga ggg acc 769

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

240 245 250

aag ctg acc gtc cta gac tac aag gat gac gacgat aag tgataagcgg 818

Lys Leu Thr Val Leu Asp Tyr Lys Asp Asp Asp Asp Lys

255 260 265

ccgc 822

Claims (22)

1. increase the method for TPO agonist activity, it is undertaken by the antibody of antibody being changed into change, and the antibody of this change comprises two or more H chain V districts and two or more L chains V district.

2. signal transduction is gone into the method for cell, it is undertaken by using the antibody linked TPO acceptor that changes, and wherein the antibody of this change comprises two or more H chains V district and two or more L chain V district.

3. claim 1 or 2 method, wherein H chain V district links to each other by joint with L chain V district.

4. each method of claim 1-3, wherein this joint is to comprise at least one amino acid whose peptide linker.

5. each method among the claim 1-4, wherein the antibody of this change is the polymer that comprises the strand Fv in a H chain V district and a L chain V district.

6. the method for claim 5 wherein is somebody's turn to do the antibody that changes and is made up of the tetramer, tripolymer or the dimer of strand Fv.

7. the method for claim 6 wherein is somebody's turn to do the antibody that changes and is made up of the dimer of strand Fv.

8. each method among the claim 5-7 wherein is in H chain V district and L chain V district debond formation antigen binding site on the same chain.

9. each method among the claim 1-4, wherein the antibody of this change is the single chain polypeptide that comprises two or more H chains V district and two or more L chains V district.

10. the method for claim 9, wherein the antibody of this change is the single chain polypeptide that comprises two H chain V districts and two L chain V districts.

11. each method among the claim 1-10, wherein the antibody of this change comprises the aminoacid sequence that is used for peptide purification in addition.

12. each method among the claim 1-12, wherein the antibody that should change is purified.

13. each method among the claim 1-12, wherein H chain V district and/or L chain V district are H chain V district and/or the L chain V districts that derives from people's antibody.

14. each method among the claim 1-12, wherein H chain V district and/or L chain V district are humanized H chain V district and/or L chain V district.

15. each method among the claim 1-14, wherein the antibody of this change is the antibody of the change of monospecific.

16. each method among the claim 1-14, wherein the antibody of this change is the antibody of the change of polyspecific.

17. the method for claim 16, wherein the antibody of this change is the antibody of the change of dual specific.

18. the method for claim 15, wherein this L chain V district and H chain V district are from same monoclonal antibody.

19. each method among the claim 1-18, it shows identical with parent's monoclonal antibody or better agonist effect (ED50).

20. the method for claim 19, it compares the agonist effect (ED50) that parent's monoclonal antibody shows at least 2 times.

21. the method for claim 20, it compares the agonist effect (ED50) that parent's monoclonal antibody shows at least 10 times.

22. each method among the claim 1-18, it derives from the parental antibody that does not have the agonist effect substantially.

Images