CN101218257A - GITR binding molecules and uses therefor - Google Patents

Abstract

The present invention provides binding molecules that specifically bind to GITR, e.g., human GITR (hGITR), on T cells and dendritic cells. Binding molecules of the invention are characterized by binding to hGITR with high affinity, in the presence of a stimulating agent, e.g., CD3, are agonistic, and abrogate the suppression of Teff cells by Treg cells. Various aspects of the invention relate to binding molecules, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such binding molecules. Methods of using a binding molecule of the invention to detect human GITR or to modulate human GITR activity, either in vitro or in vivo, are also encompassed by the invention.

Description

GITR binding molecule and uses thereof

Related application

The application enjoys the right of priority of the U.S. Provisional Patent Application 60/687265 of U.S. Provisional Patent Application of submitting on March 25th, 2,005 60/665322 that is called " GITR binding molecule and uses thereof " and " GITR binding molecule and uses thereof " by name of submitting on June 3rd, 2005, and the full content of these two parts of application cases is incorporated this paper by reference into.

Background technology

The member of tumour necrosis factor and TNF acceptor (TNFR) superfamily can regulate multiple biological function, comprises cell proliferation, differentiation and survival.Utilization variance shows to be identified and is synthesized the inductive T of glucocorticosteroid dexamethasone institute cell mRNA, and Nocentini et al. ((1997) Proc.Natl.Acad.Sci.USA 94:6216-6221997) identifies a coding TNFR newcomer's of family mouse cDNA.Its corresponding gene is named as GITR, represents glucocorticosteroid-inductive TNFR family-genes involved (being called TNFRSF18 again).The same with other TNFR, the proteinic ectodomain of the CITR of this expectation contains the homocysteine repeated fragment.In addition, born of the same parents' intracellular domain homology of the born of the same parents' intracellular domain of GITR and mouse and people TNFR, 4-1BB and CD27 is very high.Nocentini et al. ((1997) Proc.Natl.Acad.Sci.USA 94:6216-6221997) confirmation GITR gene is induced by dexamethasone and other cell-stimulating sexual stimulus in the T cell.GITR expresses and can protect the T cell to avoid handling the apoptosis that is brought out by anti-cd 3 antibodies, but can not protect the apoptosis that is caused by other apoptosis agent.

Shimizu et al. ((2002) Nat Immunol 3:135-42) finds that GITR expresses on the CD4+CD25+ regulatory T cells.But GITR also has expression on conventional CD4+ and CD8+T cell, and it is expressed in the enhancing rapidly of activation back.In vitro study shows that GITR plays a significant role in the periphery immunological tolerance cell-mediated by these, can eliminate inhibit feature (Shimizu et al. (2002) the Nat Immunol 3:135-42 of CD4+CD25+ regulatory T cells; McHughet al. (2002) Immunity16:311-23).

The reagent that exploitation can be used to regulate the signal conduction of carrying out via GITR will be very useful.

Summary of the invention

The invention provides such binding molecule, the GITR of these molecules on can specific combination cell (such as T cell and dendritic cell), for example people GITR (hGITR).Binding molecule of the present invention is characterised in that can be with high-affinity in conjunction with hGITR; Under the situation that has stimulant (for example CD3) to exist, antagonistic action is arranged, can eliminate regulatory T (Treg) cell pairing effect T (Teff) cell inhibiting.

One aspect of the present invention provides the binding molecule of the aminoacid sequence that comprises SEQ ID NO:1, the optional homing sequence that also comprises.

Another aspect the invention provides the binding molecule of the aminoacid sequence that comprises SEQ ID NO:66, the optional homing sequence that also comprises.

Another aspect the invention provides the binding molecule of the aminoacid sequence that comprises SEQ ID NO:2, the optional homing sequence that also comprises.

Another aspect the invention provides the binding molecule of the aminoacid sequence that comprises SEQ ID NO:58, the optional homing sequence that also comprises.

One aspect of the present invention provides the binding molecule of the aminoacid sequence that comprises SEQ ID NO:59, the optional homing sequence that also comprises.

Another aspect the invention provides the binding molecule of the aminoacid sequence that comprises SEQ ID NO:60, the optional homing sequence that also comprises.

One aspect of the present invention provides the binding molecule of the aminoacid sequence that comprises SEQ ID NO:61, the optional homing sequence that also comprises.

On the other hand, invention provides the binding molecule of the aminoacid sequence that comprises SEQ ID NO:62, the optional homing sequence that also comprises.

One aspect of the present invention provides the binding molecule of the aminoacid sequence that comprises SEQ ID NO:63, the optional homing sequence that also comprises.

One side more of the present invention provides such binding molecule, described molecule to comprise complementary determining region (CDR) aminoacid sequence that at least one is selected from SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO:19 and SEQ ID NO.5.In one embodiment, described binding molecule comprises at least two complementary determining region (CDR) aminoacid sequences that are selected from SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO:19 and SEQ ID NO.5.In another embodiment, described binding molecule comprises at least three complementary determining region (CDR) aminoacid sequences that are selected from SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO:19 and SEQ ID NO.5.

The present invention provides such binding molecule on the other hand, and described molecule comprises at least one complementary determining region (CDR) aminoacid sequence that is selected from SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.In one embodiment, described binding molecule comprises at least two complementary determining region (CDR) aminoacid sequences that are selected from SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.In another embodiment, described binding molecule comprises at least three complementary determining region (CDR) aminoacid sequences that are selected from SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

The present invention provides the binding molecule that comprises CDR shown in the SEQ ID NO:3,4,5,6,7 and 8 on the other hand.Invention the binding molecule that comprises CDR shown in the SEQ ID NO:3,19,5,6,7 and 8 is provided on the other hand.

One aspect of the present invention provides such binding molecule, and this molecule comprises the variable region of heavy chain of the aminoacid sequence that contains SEQ IDNO:1, also comprises the variable region of light chain of the aminoacid sequence that contains SEQ ID NO:2.Invention such binding molecule is provided on the other hand, this molecule comprises the variable region of heavy chain of the aminoacid sequence that contains SEQ ID NO:66, and also comprises the variable region of light chain of the aminoacid sequence that contains SEQ ID NO:2.In more than one embodiment, described binding molecule comprises the people or is people's heavy chain and light chain framework region basically.In another embodiment, one or more people's framework region amino-acid residue is mutated into corresponding mouse amino-acid residue.In another embodiment, constant region comprises the IgG2b CH.In another embodiment, constant region comprises the people, for example human IgG1's CH.In another embodiment, described binding molecule has been changed so that reduce effector function and/or glycosylation.In one embodiment, binding molecule is in conjunction with people GITR.In one embodiment, binding molecule can not bring out apoptosis.In another embodiment, binding molecule can not be blocked elementary mixed lymphocyte reacion.In an embodiment again, binding molecule can be offset regulatory T cells pairing effect T cell inhibiting.In one embodiment, binding molecule can be regulated the propagation of effector T cell.In one embodiment, binding molecule is from mouse.In another embodiment, binding molecule comprises mouse IgG2b heavy chain.In one embodiment, binding molecule is a humanized antibody.In the embodiment, binding molecule is a chimeric antibody again.Also have in the embodiment, binding molecule can be adjusted the activity of people GITR.In another embodiment, binding molecule weakens the degraded of I-κ B in the T cell.

It can be 1 * 10 in conjunction with GITR on human T-cell and the human dendritic cell and binding constant (Kd) that the present invention provides on the other hand ^-9Or lower binding molecule.In one embodiment, this binding molecule can be offset regulatory T cells pairing effect T cell inhibiting.In another embodiment, described binding molecule is a humanized antibody.

Further aspect of the present invention provides the composition that comprises binding molecule of the present invention and pharmaceutically acceptable carrier.In one embodiment, described composition also comprises at least a other therapeutical agent that is used to dispose experimenter's cancer.In one embodiment, composition also comprise at least a be used to dispose the experimenter other therapeutical agent of infecting of ill poison.In another embodiment, composition also comprises at least a tumour antigen that is used to dispose experimenter's cancer.In the embodiment, described composition also comprises at least a antigen from pathogenic agent again.

One aspect of the present invention provides a kind of method of eliminating regulatory T cells pairing effect T cell inhibiting, and this method comprises people's immunocyte is contacted with binding molecule of the present invention, thereby makes regulatory T cells pairing effect T cell inhibiting be cancelled.

The present invention provides a kind of method of regulating TXi Baoshouti institute inductive signal conduction in the effector T cell on the other hand, this method comprises described cell is contacted with binding molecule of the present invention, thereby makes the receptor signal conduction of induced t cell in the effector T cell adjusted.In one embodiment, this method has been regulated the degraded of I-κ B.In one embodiment, described T cell is the Th1 cell.In another embodiment, described T cell is the CD4+ cell.In the embodiment, described T cell is the CD8+ cell again.

Further aspect of the present invention provides the method for a kind of experimenter's of raising immunne response, and this method comprises cell is contacted with binding molecule of the present invention, thereby makes experimenter's immunne response be enhanced.

The present invention provides a kind of method of the experimenter's of disposal cancer on the other hand, and this method comprises cell is contacted with binding molecule of the present invention, thereby makes experimenter's cancer obtain medical treatment.In one embodiment, the type of cancer is selected from: carcinoma of the pancreas (pancreatic cancer), melanoma (melanomas), mammary cancer (breast cancer), lung cancer (lung cancer), bronchogenic carcinoma (bronchialcancer), colorectal cancer (colorectal cancer), prostate cancer (prostate cancer), cancer of the stomach (stomachcancer), ovarian cancer (ovarian cancer), Urinary Bladder cancer (urinary bladder cancer), brain or central nervous system cancer (brain or central nervous system cancer), peripheral nervous system cancer (peripheral nervous system cancer), the esophageal carcinoma (esophageal cancer), cervical cancer (cervicalcancer), uterus or carcinoma of endometrium (uterine or endometrial cancer), oral carcinoma or pharynx cancer (cancer of the oral cavity or pharynx), liver cancer (liver cancer), kidney (kidney cancer), carcinoma of testis (testicular cancer), cholangiocarcinoma (biliary tract cancer), small intestine or adnexal carcinoma (smallbowel or appendix cancer), salivary-gland carcinoma (salivary gland cancer), thyroid carcinoma (thyroidgland cancer), adrenal carcinoma (adrenal gland cancer), osteosarcoma (osteosarcoma), the cancer of chondrosarcoma (chondrosarcoma) and hemopoietic tissue (cancer of hematological tissues).

The present invention provides the method for the infection that a kind of treatment causes by pathogenic agent on the other hand in subject, this method comprises cell is contacted with the binding molecule of claim 1, thereby the infection that is caused by pathogenic agent in the subject is obtained medical treatment.In one embodiment, described pathogenic agent is a virus, for example be selected from: hepatitis A virus (HAV) (hepatitis type A), hepatitis B virus (hepatitis type B), hepatitis C virus (hepatitis type C), influenza virus (influenza), varicella virus (varicella), adenovirus (adenovirus), I herpes simplex virus type (herpes simplex type I, HSV I), II herpes simplex virus type (HSV II), rinderpest virus (rinderpest), rhinovirus (rhinovirus), Chinese mugwort can virus (echovirus), rotavirus (rotavirus), respiratory syncytial virus (respiratory syncytial virus), papilloma virus (papilloma virus), papovavirus (papova virus), cytomegalovirus (cytomegalovirus), echinovirus, arboviruses (arbovirus), Hantaan virus (hantavirus), Coxsackie virus (coxsackie virus), mumps virus (mumps virus), Measles virus (measlesvirus), rubella virus (rubella virus), poliovirus (polio virus), I type human immunodeficiency virus (human immunodeficiency virus type I, HIV I) and II type human immunodeficiency virus (HIV II), any picornavirus (picornaviridae), enterovirus (enteroviruses), Calicivirus (caliciviridae), any of norwalk virus group (Norwalk group of viruses), togavirus (togaviruses) (such as Alphavirus (alphaviruses)), flavivirus (flaviviruses), coronavirus (coronaviruses), rabies virus (rabies virus), Marburg virus (Marburg viruses), Ebola virus (ebola viruses), parainfluenza virus (parainfluenza virus), orthomyxovirus (orthomyxoviruses), bunyavirus (bunya viruses), sand grains virus (arenaviruses), reovirus (reoviruses), rotavirus, Orbivirus (orbiviruses), I type human T-cell leukemia virus (human T cell leukemia virus), II type human T-cell leukemia virus, simian immunodeficiency virus (simian immunodeficiency virus), slow virus (lentiviruses), polyomavirus (polyomaviruses), parvovirus (parvoviruses), Epstein-Barr virus (Epstein Barr virus), human herpes virus type 6 (human herpesvirus 6), cercopithecid herpesvirus 1 type (cercopithecine hernesvirus 1, B virus) and poxvirus (poxviruses).In one embodiment, described method is used to dispose chronic viral infection.

In another embodiment, pathogenic agent is a bacterium, for example is selected from: Neisseria gonorrhoeae species (Neisseria spp), suis species (Streptococcus spp), Streptococcus mutans S.mutans), hemophilic bacterium species (Haemophilus spp.), catarrhalis species (Moraxella spp), Bordetella species (Bordetella spp), mycobacterium species (Mycobacterium spp), legionella species (Legionella spp), Escherichia species (Escherichia spp), vibrios species (Vibrio spp), Yersinia species (Yersinia spp), crooked fungus kind (Campylobacter spp), Salmonellas species (Salmonella spp), listeria spp species (Listeria spp.), Helicobacter pylori species (Helicobacterspp), pseudomonas species (Pseudomonas spp), staphylococcus species (Staphylococcus spp.), faecalis species (Enterococcus spp), clostridium species (Clostridium spp.), bacillus species (Bacillus spp), rod bacillus species (Corynebacterium spp.), burgdorferi species (Borreliaspp.), ehrlichiosis body species (Ehrlichia spp), Rickettsiae species (Rickettsia spp), chlamydozoan species (Chlamydia spp.), Leptospira species (Leptospira spp.), treponema species (Treponema spp.).

The present invention provides a kind of method of the GITR of adjusting function on the other hand, when being included in immunoactivator, people GITR is contacted with binding molecule of the present invention, thereby adjusts the GITR function.

One aspect of the present invention is embodied in such binding molecule, and it comprises at least one cdr amino acid sequence that is selected from SEQID NO.3, SEQ ID NO.4, SEQ ID NO:19, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.In one embodiment, described composition also comprises the therapeutical agent of at least a disposal experimenter cancer.In another embodiment, described binding molecule comprises at least one CDR derived from the 6C8 binding molecule.In another embodiment, binding molecule comprises at least two CDR derived from the 6C8 binding molecule.In another embodiment, binding molecule comprises at least three CDR derived from the 6C8 binding molecule.In another embodiment, binding molecule comprises at least four CDR derived from the 6C8 binding molecule.In another embodiment, binding molecule comprises at least five CDR derived from the 6C8 binding molecule.In another embodiment, binding molecule comprises at least six CDR derived from the 6C8 binding molecule.

The present invention is embodied in the binding molecule that comprises six CDR shown in the SEQ ID NOs.3,4 or 19,5,6,7 and 8 on the other hand.

One side more of the present invention is embodied in such binding molecule, and it comprises the variable region of heavy chain of the aminoacid sequence that contains SEQ ID NO:1, and also comprises the variable region of light chain of the aminoacid sequence that contains SEQ ID NO:2.In one embodiment, this binding molecule comprises the people or is people's heavy chain and light chain framework region substantially.In another embodiment, people's framework region that binding molecule of the present invention comprises, wherein one or more people's framework region amino-acid residue is become corresponding mouse amino-acid residue by reverse mutation, perhaps is mutated into other amino-acid residue.In another embodiment, binding molecule of the present invention comprises the constant region of immunoglobulin molecules, for example the IgG2b CH.Also have in the embodiment, binding molecule can be in conjunction with people GITR (hGITR).In one embodiment, binding molecule can not bring out apoptosis.In another embodiment, binding molecule can not blocked elementary mixed lymphocyte reacion.Also have in the embodiment, binding molecule has been offset regulatory T cells pairing effect T cell inhibiting.In one embodiment, binding molecule has strengthened the propagation of effector T cell.In another embodiment, the binding molecule activity of people GITR that neutralized.Also have in the embodiment, binding molecule has weakened the degraded of I-κ B in the T cell.

An aspect, the present invention is embodied in such binding molecule, and it is in conjunction with the GITR on human T-cell and the human dendritic cell, and binding constant (Kd) is 1 * 10 ^-9Or it is lower.In one embodiment, described binding molecule has been offset the restraining effect of regulatory T cells.In another embodiment, binding molecule derives from mouse or comprises mouse CDR.In the embodiment, binding molecule comprises the IgG2b heavy chain again.In one embodiment, binding molecule is a humanized antibody.In further embodiment, binding molecule is a chimeric antibody.

The present invention provides the composition that comprises binding molecule of the present invention and pharmaceutically acceptable carrier on the other hand.In one embodiment, described composition also comprises at least a other the therapeutical agent of treatment experimenter cancer.

One aspect of the present invention provides a kind of inhibiting method of eliminating regulatory T cells to T effector cell, and this method comprises people's immunocyte is contacted with binding molecule of the present invention, thereby eliminates the restraining effect of regulatory T cells.

The present invention provides a kind of method that TXi Baoshouti inductive signal conducts of regulating on the other hand in effector T cell, this method comprises cell contacted with binding molecule of the present invention, thereby makes in the effector T cell conduction of TXi Baoshouti inductive signal adjusted.In one embodiment, described method has been regulated the degraded of I-κ B.In one embodiment, the T cell is the Th1 cell.

Further aspect of the present invention provides a kind of experimenter's of enhancing immunoreactive method, and this method comprises cell is contacted with binding molecule of the present invention, thus the immune response that improves study subject.

The present invention provides a kind of method of the experimenter's of disposal cancer on the other hand, and this method comprises cell is contacted with binding molecule of the present invention, thereby cancer is obtained medical treatment.In one embodiment, the cancer kind is selected from: the cancer of carcinoma of the pancreas, melanoma, mammary cancer, lung cancer, bronchogenic carcinoma, colorectal carcinoma, prostate cancer, cancer of the stomach, ovarian cancer, Urinary Bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, the esophageal carcinoma, cervical cancer, uterus or carcinoma of endometrium, oral carcinoma or pharynx cancer, liver cancer, kidney, carcinoma of testis, cholangiocarcinoma, small intestine or adnexal carcinoma, salivary-gland carcinoma, thyroid carcinoma, adrenal carcinoma, osteosarcoma, chondrosarcoma and hemopoietic tissue.

The present invention provides a kind of method of the GITR of inhibition function on the other hand, and this method comprises makes people GITR and binding molecule of the present invention contact having under the situation of stimulant, thereby makes the GITR function be suppressed.

One aspect of the invention provides isolated nucleic acid molecule, and this nucleic acid molecule comprises the nucleotide sequence that contains nucleotide sequence SEQ ID NO:9 of encoding heavy chain variable region, the optional homing sequence that also comprises.The present invention provides isolated nucleic acid molecule on the other hand, and this nucleic acid molecule comprises the nucleotide sequence that contains nucleotide sequence SEQ ID NO:67 of encoding heavy chain variable region, the optional homing sequence that also comprises.

The present invention provides isolated nucleic acid molecule on the other hand, and this nucleic acid molecule comprises the nucleotide sequence that contains nucleotide sequence SEQ ID NO:10 of encoded light chain variable region, the optional homing sequence that also comprises.

Further aspect of the present invention provides isolated nucleic acid molecule, and this nucleic acid molecule comprises at least one nucleotide sequence that is selected from the coding CDR of SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO:65 and SEQ ID NO.13.In one embodiment, nucleotide sequence coded at least two CDR of comprising of described isolated nucleic acid molecule derived from the 6C8 binding molecule.In another embodiment, nucleotide sequence coded at least three CDR of comprising of described isolated nucleic acid molecule derived from the 6C8 binding molecule.

The present invention provides isolated nucleic acid molecule on the other hand, and this nucleic acid molecule comprises at least one nucleotide sequence that is selected from the coding CDR of SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16.In one embodiment, nucleotide sequence coded at least two CDR of comprising of described isolated nucleic acid molecule derived from the 6C8 binding molecule.In another embodiment, nucleotide sequence coded at least three CDR that described isolated nucleic acid molecule comprised derived from the 6C8 binding molecule.

One aspect of the present invention provides isolated nucleic acid molecule, and it comprises the nucleotide sequence shown in SEQ ID NO:11-16 and the SEQ ID NO:65.

One aspect of the invention provides and has comprised the recombinant expression vector of inventing described nucleic acid molecule.In one embodiment, the recombinant expression vector that is provided comprises the nucleic acid molecule of the nucleotide sequence with code book invention binding molecule.In another embodiment, the invention provides and imported the host cell of inventing described recombinant expression vector.The present invention provides the preparation can be in conjunction with the method for the binding molecule of people GITR on the other hand, method be included in cultivate in the substratum that the described host cell of invention produces until this cell can be in conjunction with the binding molecule of people GITR.

The accompanying drawing summary

Fig. 1 shows the mouse of purifying and the SDS-PAGE trace of people GITR binding molecule.12 micrograms of protein have been added in each hole.

Fig. 2 shows size exclusion chromatography (SE-HPLC) result of the people GITR binding molecule of purifying.50 micrograms of protein are expelled in the SE-HPLC pillar with the flow velocity of 0.6ml/min.Binding molecule through the SE-HPLC purifying produces binding molecule colony, and wherein 99.8% is monomeric form, and 0.2% is aggregate.

Fig. 3 show transfection the L-M cell (l cell) of GITR gene carry out painted facs analysis result with the supernatant liquor that 50 μ l express the hybridoma of GITR.The GITR binding molecule makes by the GITR-cells transfected and dyes, and the not dyeing of the L-M cell of untransfected.

Fig. 4 shows facs analysis, and it shows that GITR mainly expresses on activated lymphocytes.The 6C8 binding molecule makes CD4+, CD8+, the dyeing of CD25+ lymphocyte, the weak dyeing of CD103+ cell.

Fig. 5 show the 6C8 binding molecule in conjunction with saturation curve, with biotin labeled 6C8 titration and assessing out on the CD3 activated lymphocytes.

Fig. 6 shows that the 6C8 binding molecule has collaborative stimulating activity to the T lymphocyte, and described T lymphocyte is to optimize (sub-optimal) OKT3 (anti-CD3 through the Asia; 0.01 the cell that stimulates and be incubated with anti-CD28 or anti-GITR μ g/ml).Also used isotype contrast (IgG2b).

Fig. 7 A and 7B show that the 6C8 binding molecule does not bring out apoptosis.Lymphocyte is with PHA activation 3 days, add again 10 μ g/ml YTH655 (a kind of known on activated lymphocyte the anti-CD2 antibody of cell death inducing; Friend, P.et al. (1987) Transplant.Proc.19:4317), 6C8 or isotype contrast (IgG2b).Apoptosis is measured by cell survival rate (A), annexin V dyeing (B), flow cytometry.

Fig. 8 shows that the 6C8 binding molecule do not block elementary mixed lymphocyte reacion (MLR).Under the situation that different concns TRX1 (anti-people CD4), 6C8 or MOPC (the isotype contrast of TRX1) arranged, will mix from the lymphocyte of allogeneic donor.Cell insulation 3 days is used ³H-thymidine pulse 18 hours is gathered in the crops then and is counted.

Fig. 9 shows the restraining effect to T effector cell that 6C8 binding molecule blocking-up regulatory T cells (Treg) brings out.The CD4+/CD25+ cell is added in the CD4+/CD25-cell with various ratios.Cell stimulates with the anti-CD3 and the anti-CD28 that are combined on the microwell plate.Ratio is 1: 1 o'clock, and the propagation of CD4+/CD25-cell is suppressed.Add two kinds of dilution 6C8 of difference and can block the restraining effect that the CD4+/CD25+ regulatory T cells brings out CD4+T effector cell.

Figure 10 shows that the 6C8 binding molecule has collaborative stimulating activity, even also be like this when stimulating the T cell with anti-CD3 under the situation of anti-CD28 not.The CD4+/CD25+ cell together is incubated with different cells ratios with the CD4+/CD25-cell.Cell only stimulates with the anti-CD3 that is combined on the microwell plate.6C8 is added in the cell, under described situation, show collaborative stimulating activity.

Figure 11 shows the influence of anti-GITR to the degraded of I-κ B in CD3 activated T cell.

Figure 12 shows the influence of anti-GITR to the phosphorylation of I-κ B in CD3 activated T cell.

Figure 13 shows the influence of anti-GITR to the degraded of I-κ B in CD3+CD28 activated T cell.

Figure 14 shows the influence of anti-GITR to the phosphorylation of I-κ B in CD3+CD28 activated T cell.

Figure 15 shows, 6C8 and R﹠amp; D Systems (Minneapolis, the epi-position of antibody recognition uniqueness MN).On through OKT3 and concanavalin A (Con A) activated lymphocytes, carry out competitive assay.The competitive R﹠amp of 1 μ g/ml 6C8 and different amounts; D Systems antibody (GITT/TNFRSF18 monoclonal antibody) uses together.Observe some competitions at the maximum concentration of antibody, but this may be caused very by sterically hindered.

Figure 16 shows the relative R﹠amp of the anti-GITR antibody of 6C8; The dynamic analysis of D Systems GITR antibody.

Figure 17 shows, the mouse of having injected B1 6 cells that ametycin handled is with the survival rate after anti-GITR antibody (the 2F8 rat anti-mouse GITR binding molecule) disposal.

Figure 18 A-18D shows the variable heavy chain (VHD) (being respectively A and B) of 6C8 binding molecule and the nucleotide sequence and the aminoacid sequence of variable light chain (VKA) (being respectively C and D).Homing sequence shows with runic; The framework sequence is added with underscore; The CDR sequence is an italic.

Figure 19 A and 19B demonstration, 2F8 and 2F8 F (ab ') ₂Fragment has strengthened the humoral response to HA.

Figure 20 A and 20B demonstration, 2F8 and 2F8 F (ab ') ₂Fragment has strengthened the humoral response to Ova.

Detailed Description Of The Invention

The invention provides can specific bond T cell and BMDC on GITR, people for example The binding molecule of GITR (hGITR). Binding molecule characteristics of the present invention are can be with the high-affinity combination HGITR, and in the situation that stimulant (for example CD3) arranged, they have antagonism, can Offset the Treg cell to the inhibitory action of T effect (Teff) cell. Various aspects of the present invention relate to combination Molecule, its pharmaceutical composition, and the nucleic acid of the described binding molecule of encoding, for the preparation of this in conjunction with branch Recombinant expression carrier and the host cell of son. Utilize the external or interior people of detection of body of binding molecule of the present invention The method of GITR or mediator GITR activity also contains within the scope of the present invention.

The understanding of the present invention at first defines some terms for convenience.

I. definition

Term " the TNF acceptor of glucocorticoid inducible " (being abbreviated as " GITR " in the literary composition) is called again TNF Receptor superfamily 18 (TNFRSF18) is used for this paper, refers to TNF/nerve growth factor The member of receptor family. It is one 241 amino acid whose I type transmembrane proteins, and characteristics are outside the born of the same parents The false iterons of three cysteines are arranged in the domain, and special Cell protection avoids that φt cell receptor induces Apoptosis, although it can not avoid other Apoptosis signal by Cell protection, comprise Fas bring out, Sai Misong disposes or UV radiation (Nocentini, G, et al. (1 997) Proc.Natl.Acad.Sci.USA 94:6216-622). The nucleotide sequence of people GITR (hGITR) shown in SEQ ID NO:17, its ammonia The base acid sequence is shown in SEQ ID NO.18.

Term " binding molecule " is used for this paper and comprises and contain the antigen that at least one can specific bond GITR The molecule of binding site. The described binding molecule of " specific bond " expression only shows non-GITR molecule is basic Go out the background combination. But the binding molecule of the separation of specific bond GITR is to from other species The GITR molecule may have cross reactivity.

Binding molecule of the present invention can comprise immunoglobulin molecules any isotype (for example, IgG, IgE, IgM, IgD, IgA and IgY), type (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) Or the heavy chain immunoglobulin of hypotype. Binding molecule can also have light chain by existing heavy chain. Be used for this paper, The term binding molecule also comprises antibody (comprising full length antibody); Monoclonal antibody (comprises the total length monoclonal Antibody); Polyclonal antibody; Multi-specificity antibody (for example, bispecific antibody); People, humanization or Chimeric antibody antibody; And antibody fragment, for example Fab fragment, F (ab ') fragment, Fab expression library The fragment, above all epi-position binding fragments and the engineered forms of antibody, for example scFv that produce Molecule, as long as they show required activity, for example can be in conjunction with GITR.

" antigen " is the entity (for example, albumen entity or peptide) of binding molecule institute specific bond.

Term " epi-position " or " antigenic determinant " refer to the binding molecule specific bond to antigen on the position The point. Epi-position can be made of continuous amino acid, also can be that discontinuous amino acid is by protein Level Four folds and is arranged in together. The epi-position that is made of continuous amino acid is logical when the contact metamorphism solvent Often can keep, and can lose when usually processing with the sex change solvent by the folding epi-position that consists of of level Four. Epi-position generally includes has 3,4,5,6,7,8,9,10,11,12,13,14 or 15 at least Amino acid whose unique three-dimensional conformation. The method of determining the three-dimensional conformation of epi-position comprises that for example the X-ray is tied Brilliant learning and 2-dimension nuclear magnetic resonance. Referring to for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol.66, G.E.Morris, Ed. (1996).

Can by simple immune detection, show that a kind of antibody can be blocked another kind of antibody and target is anti-The binding molecule that combined techniques comes the identical epi-position of Identification is namely competed in former combination. The competition combination is logical Cross such detection and determine, wherein binding molecule to be measured can stop with reference to binding molecule with total anti-The specific bond of former (such as GITR). Known competition has many types in conjunction with detection, and for example solid phase is straight Connect or indirect radioimmunoassay (RIA); The direct or indirect enzyme immunoassays of solid phase method (EIA) is sandwich Competition checking method (seeing Stahli et al.Methods in Enzymology 9:242 (1983)); Solid phase is direct Biotin-avidin EIA (referring to Kirkland et al.J. Immunol.137:3614 (1986)); The direct mark checking method of solid phase, the sandwich checking method of the direct mark of solid phase (referring to Harlow and Lane, Antibodies:A Laboratory Manual, Cold Spring Harbor Press (1988)); Use I-125 The direct mark RIA of the solid phase that mark carries out is (referring to Morel et al.Mol.Immunol.25 (1): 7 (1988)); The direct biotin of solid phase-avidin EIA (Cheung et al.Virology 176:546 (1990)); And direct mark RIA (Moldenhauer et al.Scand.J.Immunol.32:77 (1990)). Usually, this class detection can relate to and use the purifying that is combined on the surface of solids or the cell anti-Former, with unmarked binding molecule to be measured and mark with reference to binding molecule. By determine have to be measured In the situation of binding molecule, the amount that is attached to the mark on the surface of solids and the cell is weighed the competition inhibition. In general, binding molecule to be measured is excessive. Usually, be excessive when competing binding molecule, it Can suppress with reference to the specific bond of binding molecule and total antigen at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more than.

Epi-position can also be by immunocyte, and for example B cell and/or T cell are identified. The cell of epi-position Identification can determine by the external checking method of measuring antigen dependence propagation, resemble by³The H-thymidine is mixed Enter, cytokine secretion, antibody-secreting or antigen dependence kill and wound (cytotoxic T lymphocyte calibrating Method) determines.

Term " monoclonal binding molecule " is used for this paper and refers to from the binding molecule colony of homogeneous basically The binding molecule that obtains. The monoclonal binding molecule is high special, and is directed for single antigen position The point. In addition, from the polyclone combination that usually comprises for the different binding molecules of different determinants (epi-position) Molecule prepared product difference, every kind of monoclonal binding molecule are for the single determinant on the antigen. Qualifier " monoclonal " shows that this class binding molecule is characterised in that the binding molecule group who derives from basic homogeneous Body needs any concrete grammar to prepare this binding molecule and should not be construed as. For example, with the present invention Consistent monoclonal binding molecule can be by by (Nature 256:495 (1975)) such as Kohler first The hybridoma method preparation of describing, perhaps can pass through recombinant DNA method (referring to, U.S. for example Patent 4,816,567). " monoclonal binding molecule " can also utilize for example Clackson, et al.Nature The skill of describing among 352:624-628 (1991) and the Marks et al.J.Mol Biol.222:581-597 (1991) Art is separated from phage library.

The combination that term " chimeric binding molecule " refers to comprise the amino acid sequence that derives from different plant species divides Son. Chimeric binding molecule can for example pass through genetic engineering by the binding molecule genetic fragment of different plant species Make up.

Monoclonal binding molecule in the literary composition is particularly including " chimeric " binding molecule, its heavy chain and/or light chain A part with derive from concrete species, perhaps belong to the phase in the binding molecule of antibody specific type or hypotype Answering sequence is identical or homology, and the other parts of chain with derive from another species or belong to that another is anti-The binding molecule of build or hypotype, and the corresponding sequence in the fragment of such binding molecule is identical Or homology, as long as they show required biologically active (United States Patent (USP) 4,816,567; With Morrison, et al.Proc.Natl.Acad. Sci.USA 81:6851-6855 (1984)), such as with the people GITR (hGITR) combination.

Light chain and heavy chain all are divided into zones of different according to the 26S Proteasome Structure and Function homology. Term " constant " and " variable " is for representation function. With regard to this respect, be understood that the varistructure of light chain part The variable domains (VH) of territory (VL) and heavy chain part has determined antigen recognizing and specificity. On the contrary, light The constant domain of the constant domain of chain (CL) and heavy chain (CH1, CH2 or CH3) give important life The thing characteristic, such as secretion property, pass placenta, Fc receptors bind, complement in conjunction with etc. Traditionally, The constant region domain along with from antigen binding site or antibody amino terminal more away from its numbering more big. The N end End is the variable region, and the C end is constant region; CH3 and CL domain comprise in fact respectively heavy chain and light The c-terminus of chain.

" variable region " refers to the amino terminal part of binding molecule, this one when being used for describing binding molecule Divide and impel antigen to be attached on the described binding molecule, and be not constant region. This term comprise keep whole The functional fragment of some or all binding functions of individual variable region.

Term " hypervariable region " is used for this paper and refers to the zone that the binding molecule variable domains is such, and it is in order List super variable and/or form the annular that is confined on the structure together. Hypervariable region comprises from " complementation The amino acid residue of determining area " or " CDR ".

Be used for this paper, term " CDR " or " complementary determining region " mean at heavy chain and light chain polypeptide can Become the discontinuous antigen binding site that to find in the district. Kabat, et al.J.Biol.Chem.252, 6609-6616 (1977) and Kabat, et al.Sequences of protein of immunological Interest. (1991), and Chothia, et al.J.Mol.Biol.196:901-917 (1987) and MacCallum, et al.J.Mol.Biol.262:732-745 (1996) described these specifically zones, wherein Definition be included in the overlapping or subgroup of amino acid residue when mutually comparing. In a word, fixed with these In the justice any one refer to binding molecule or transplant binding molecule or the CDR of its variant all in the text In the scope of the term that defines and use.

Be used for this paper, term " framework region " or " FR " mean in the framework by CDR separated each Domain. Therefore, the variable region framework is about 100-120 amino acid, but only refers to outside the CDR that A little amino acid.

" humanization " form of inhuman (for example mouse) binding molecule is to contain minimum dividing from inhuman combination The chimeric antibody of subsequence. Most cases, humanization binding molecule be such human binding molecules (acceptor/ Accept binding molecule), wherein from the residue of hypervariable region by from inhuman species (donor binding molecule), Possess required specificity, compatibility and ability such as mouse, rat, rabbit or non-human primates hypervariable region Residue replace. In some instances, Fv framework region (FR) residue of human binding molecules is changed, For example replace, replacement or back mutation become corresponding inhuman residue. In addition, the humanization binding molecule can Can comprise the residue that does not all have in receptors bind molecule or the donor binding molecule. This class modify generally be for The performance of the molecule of further optimizing integration. Usually, the humanization binding molecule can comprise basically whole At least one, generally be two variable regions, wherein all or basically all hypermutation ring correspondences non-The hypermutation ring of human binding molecules, and whole or basic all FR districts are the FR of human binding molecules sequence. The binding molecule constant region (Fc) that the humanization binding molecule is optional also to comprise human binding molecules normally extremely A few part. Detailed content more, referring to Jones, et al.Nature 321:522-525 (1986); Riechmann, et al.Nature 332:323-329 (1988); Presta, Curr.Op. Struct.Biol. 2:593-596 (1992).

Preferably, humanization binding molecule of the present invention comprises at least one and is selected from SEQ ID NO.3 (GFSLSTSGMGVG (HC CDR1)), SEQ ID NO.4 (HIWWDDDKYYNPSLKS (HC CDR2N)), SEQ ID NO.5 (TRRYFPFAY (HC CDR3)), SEQ ID NO.6 (KASQNVGTNVA (LC CDR1)), SEQ ID NO.7 (SASYRYS (LC CDR2)), SEQ ID NO.8 (QQYNTDPLT (LC CDR3)) and SEQ ID NO:19 The CDR of (HIWWDDDKYYQPSLKS (HC CDR2Q)).

Term " through engineering approaches " or " restructuring " binding molecule are used for this paper and comprise by recombinant means system Standby, as to express, create or separate binding molecule is transfected into the recombinant expressed of host cell such as utilization The binding molecule of vector expression, the binding molecule that from restructuring, combination binding molecule library, separates, from The binding molecule that separates in human immunoglobulin gene's the transgenic animals (for example mouse) (referring to for example, Taylor, L.D.et al. (1992) Nucl.Acids Res.20:6287-6295), perhaps relate to by other The montage of human binding molecules gene order is prepared, expresses, creates to the means on other dna sequence dna Or the binding molecule that separates. But in some embodiments, this class recombinant human binding molecule has stood External sudden change (perhaps, when the transgenic animals of end user Ig sequence, being somatic mutation in the body), The amino acid sequence in the VH of binding molecule and VL district derives from and with ethnic group is although therefore recombinate VH and VL Serial relation may not be to plant in the system natural being present in the human binding molecules body.

" binding molecule that separates " is used for this paper and refers to such binding molecule, and this binding molecule basically Do not contain (for example, the separation of specific bond GITR of other binding molecule with different antigentic specificities Binding molecule is substantially devoid of the binding molecule of the antigen outside the specific bond GITR). In addition, divide From binding molecule may be substantially devoid of other cellular material and/or chemical substance. " separate " knot Close molecule and be identify in the composition from its natural surroundings and separate and/or reclaim in conjunction with dividing Son. The impurity component of natural surroundings comprises, for example can disturb the diagnosis of binding molecule or therapeutical uses Material can comprise enzyme, hormone, and other albumen or non-albumen solute. In preferred enforcement side In the case, binding molecule can reach following degree through purifying: (1) is pressed the Lowry method and is measured, and reaches this More than 95% of compound weight, more than 99% of weight most preferably, (2) are enough to through the rotary-cup type sequenator (spinning cup sequenator) measures-terminal amino acid sequence or internal amino acid sequence at least 15 residues, perhaps (3) through the reduction or non--reducing condition under SDS-PAGE and Coomassie blue or Preferred silver dyeing is measured and is homogeneous. The binding molecule that separates comprises the original position binding molecule in the recombinant cell, Because lack at least one composition of natural surroundings of this binding molecule. But usually, separation in conjunction with dividing Son is by at least one purification step preparation.

Be used for this paper, term " binding constant " " (kd) " (being called again " affinity costant ") is used for weighing two kinds The degree of Reversible binding between the molecule comprises actual binding affinity and apparent binding affinity. Actual Binding affinity is by calculating M^-1S ^-1Binding constant and S^-1The ratio of dissociation constant determine that unit is " M^-1". Therefore, give or the compatibility of optimizing integration comprises one in these two compositions Change, perhaps both make the binding affinity that change reaches desired level. Apparent compatibility Can comprise, for example the affinity of reaction. For example, two valency heteromerism variable region binding fragments may because Its valence state shows binding affinity variation or optimised. Binding affinity can for example pass through Utilize BIAcore systematic survey surface plasma resonance to determine.

Term " nucleic acid molecules " is used for this paper, comprises dna molecular and RNA molecule. Nucleic acid molecules can Being strand or two strands, but double-stranded DNA preferably.

Term " nucleic acid molecules that separates " is used in this article description encoding and can divides in conjunction with the combination of GITR During the nucleic acid of son, refer in this nucleic acid molecules that the nucleotide sequence of the described binding molecule of coding does not contain Other nucleotide sequence of natural this nucleic acid of side joint among the human gene group DNA. This class sequence is optional comprise right Regulate or 5 ' or 3 ' important nucleotide sequence of protein stability.

Term " carrier " is used for the nucleic acid branch that this paper refers to shift another nucleic acid that is connected in above it Son. One class of carrier is " plasmid ", refers to the circle that can connect therein other dna fragmentation The double-stranded DNA ring. Another kind of carrier is viral vectors, and wherein other dna fragmentation can be connected to In the viral genome. Some carriers can in the host cell that imports, automatically copy (for example, with The bacteria carrier of bacterium origin of replication and sequestered mammal carrier). Other carrier is (for example, non-Sequestered mammal carrier) after importing host cell, can be incorporated in the genome of host cell, Thereby copy with host genome. In addition, also have some carriers can instruct with them and can handle The expression of the gene of property connection. This class carrier is referred to herein as " recombinant expression carrier " (or referred to as " table Reach carrier "). In general, the expression vector that uses in recombinant DNA technology is plasmid normally. In this specification, " plasmid " and " carrier " can Alternates, because the most frequently used carrier format is exactly Plasmid. But, the present invention includes the expression vector of other form, (for example, copy such as viral vectors Defective retrovirus, adenovirus and adeno-associated virus), they can play identical function.

Term " recombinant host cell " (perhaps being called for short " host cell ") is used for this paper, refers to import heavy The cell of group expression vector. Should be appreciated that this class term not only refers to concrete described cell, also The offspring who comprises this cell. Because in the follow-up generation, may repair because of sudden change or ambient influnence Decorations, these offsprings may be actually and be different from parental cell, but still comprise in the text used art In the scope of language " host cell ".

Be used for this paper, term " T cell " (being the T lymphocyte) comprises from mammal (for example people) T clone in all cells, comprise thymocyte, prematurity T cell, mature T cells etc. Deng. Preferably, described T cell is to express CD4 or CD8, but is not both to express, and T The mature T cells of cell receptor. The various T cell colonys of describing in the literary composition can be according to their cell Factor characteristics and function define, and are known to those skilled in the art.

Be used for this paper, term " BMDC " refers to can activate originally (naive) T cell, stimulates B The full-time antigen presenting cell (APCs) of Growth of Cells and differentiation.

Be used for this paper, term " originally (naive) T cell " comprises also not contact pass associated antigen, therefore It is the T cell that is not activated or memory is arranged. Originally the T cell does not circulate, and people originally T cell is CD45RA+. If T cell recognition antigen and accepted other signal originally, these signals can Be based on but be not limited to antigen amount, give approach and give opportunity, originally the T cell may be bred also Be divided into various T cell subsets, for example effector T cell.

Be used for this paper, term " effector T cell " or " Teff cell " comprise that function (for example, is Cell factor by producing the activation can regulate other cell or pass through cytotoxic activity) remove antigen The T cell. Term " effector T cell " comprises helper T lymphocyte (for example, Th1 and Th2 cell) And cytotoxic T cell. The cell-mediated delayed hypersensitivity of Th1 and giant cell activation, and Th2 Cell can be assisted the B cell, very crucial in allergic reaction (Mosmann and Coffman, 1989, Annu.Rev.Immunol.7,145-173; Paul and Seder, 1994, Cell 76,241-251; Arthur And Mason, 1986, J.Exp.Med.163,774-786; Paliard, et al.1988, J.Immunol. 141,849-855; Finkelman, et al.1988, J.Immunol.141,2335-2341).

Be used for this paper, term " reaction of 1 type helper T lymphocyte " (Th1 reaction) refer to so instead Should, it is characterized in that producing one or more and be selected from the thin of IFN-γ, IL-2, TNF and lymphotoxin (LT) Intracellular cytokine, and other is preferential or special by the Th1 cell but not the cell factor that the Th2 cell produces. Be used for this paper, " reaction of 2 type helper T lymphocytes " (Th2 reaction) refers to the reaction of CD4+T cell, It is characterized in that producing the cell factor that one or more is selected from IL-4, IL-5, IL-6 and IL-10, and Relevant (for example, the generation of IgG1 and/or IgE of B cell " assistance " that provides with effective Th2 cell Increase).

Be used for this paper, term " regulatory T cells " or " Treg cell " comprise produce low-level IL-2, The T cell of IL-4, IL-5 and IL-12. Regulatory T cells produce TNF α, TGF β, IFN-γ and IL-10, but be lower than the level that effector T cell produces. Although being regulatory T cells, TGF β produces Major cytokine, the level of the cell factor that it produces will be lower than or be equal to Th1 or Th2 is thin Born of the same parents produce, for example than the low order of magnitude in Th1 or the Th2 cell. Regulatory T cells as seen In the CD4+CD25+ cell mass (referring to, Waldmann and Cobbold.2001. for example Immunity.14:399). Regulatory T cells can actively suppress Th1, Th2 or T originally on one's own initiative The propagation of cell and produce cell factor, wherein these originally the T cell in cultivation with activating letter Number (for example, antigen and antigen presenting cell, perhaps usefulness is simulated antigen (for example, the anti-CD3 among the MHC Antibody adds anti-CD28 antibody)) stimulated.

Be used for this paper, term " tolerance " comprises the refractoriness to the stimulation of activated receptor mediation. This Refractoriness is antigentic specificity normally, with still exist after contacting of toleranceization antigen ended. Example As, the characteristics of tolerance show as to lack and produce cell factor (for example IL-2), perhaps can utilize mixing Lymphocyte is cultivated to estimate. Tolerance can take place autoantigen or exotic antigen.

" mixed lymphocytes cultivation " (" MLC ") is that a kind of lymphopoiesis detects, will cultivate together from the lymphocyte of two individualities, by³It is (" mixed that breeder reaction is measured in the picked-up of the thymidine of H-mark Close lymphocyte reaction ").

Be used for this paper, term " Apoptosis " is called again programmed cell death (PCD), is such Cell death, its feature includes, but are not limited to examine heterochromatic cohesion, cellular contraction, cytoplasm Cohesion, and in the Apoptosis later stage, the cell DNA of endonuclease mediation is cut into concrete sheet Section. When the cell DNA of apoptosis carries out electrophoretic analysis, may form obvious spy Levying property dna fragmentation " gradient ".

" disposal " both comprised the therapeutic disposal, also comprised prevention or preventive measure. Need certain disposal Comprise and suffer from certain disorder, comprise also that also performance is disorderly.

" disorder " is anyly may benefit from the situation of disposing with binding molecule of the present invention. It comprises slowly Property and acute disorder or disease or the pathological state relevant with too high or too low immune response.

Following part has described in further detail various aspects of the present invention.

The II.GITR binding molecule

The invention provides isolating GITR binding molecule.Exemplary binding molecule of the present invention comprises 6C8 antibody and 2F8 antibody.6C8 antibody is a kind of anti-GITR antibody, can with T cell and dendritic cell, such as the GITR high-affinity combination on human T-cell and the dendritic cell.Preferably, such binding molecule is offset the Treg cell to the Teff cell inhibiting, and external under the situation that has stimulant (for example CD3) to exist is antagonism to part activated T cell.

In one embodiment, the VH structural domain of binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:1 (6C8 VH structural domain " N " comprises homing sequence).Comprise homing sequence though should be appreciated that some sequences of binding molecule described herein, binding molecule of the present invention also can not comprise homing sequence, and this point is chosen wantonly.For example, in one embodiment, binding molecule of the present invention comprises the aminoacid sequence of mature protein shown in the SEQ ID NO:1, for example the amino acid 20-138 among the SEQ ID NO:1.

In one embodiment, the VH structural domain of binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:66 (6C8 VH structural domain " Q " comprises homing sequence).

In one embodiment, the VL structural domain of binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:2 (6C8 VL structural domain comprises homing sequence).

In one embodiment, the VH structural domain of binding molecule of the present invention comprises the amino-acid residue 20-138 (6C8 VH structural domain " N " does not contain homing sequence) among the SEQ ID NO:1.

In one embodiment, the VH structural domain of binding molecule of the present invention comprises the amino-acid residue 20-138 (6C8 VH structural domain " Q " does not contain homing sequence) among the SEQ ID NO:66.

In one embodiment, the VL structural domain of binding molecule of the present invention comprises the amino-acid residue 21-127 (6C8 VL structural domain does not contain homing sequence) among the SEQ ID NO:2.

In an embodiment of the present invention, the VL chain comprises guiding and/or signal sequence, i.e. amino-acid residue 1-20 among the SEQ IDNO:2 (SEQ ID NO:59).In one embodiment, the VH chain comprises guiding and/or signal sequence, i.e. amino-acid residue 1-19 among the SEQ ID NO:1 (SEQ IDNO:64).

In one embodiment, the VH structural domain that comprises of binding molecule of the present invention contains the CDR shown in the SEQ ID NO:3 (6C8 VH CDR1).

In one embodiment, the VH structural domain that comprises of binding molecule of the present invention contains the CDR shown in the SEQ ID NO:4 (6C8 VH CDR2-" N ").

In one embodiment, the VH structural domain that comprises of binding molecule of the present invention contains the CDR shown in the SEQ ID NO:5 (6C8 VH CDR3).

In one embodiment, the VH structural domain that comprises of binding molecule of the present invention contains the CDR shown in the SEQ ID NO:19 (6C8 VH CDR2-alternate " Q ").

In one embodiment, the VL structural domain that comprises of binding molecule of the present invention contains the CDR shown in the SEQ ID NO:6 (6C8 VL CDR1).

In one embodiment, the VL structural domain that comprises of binding molecule of the present invention contains the CDR shown in the SEQ ID NO:7 (6C8 VL CDR2).

In one embodiment, the VL structural domain that comprises of binding molecule of the present invention contains the CDR shown in the SEQ ID NO:8 (6C8 VL CDR3).

The invention still further relates to the nucleic acid molecule of the above-mentioned aminoacid sequence of coding.

In one embodiment, the VH structural domain of binding molecule of the present invention comprises the nucleotide sequence shown in the SEQ ID NO:9 (6C8 VH structural domain, " N " comprises homing sequence).

In one embodiment, the VH structural domain of binding molecule of the present invention comprises the nucleotide sequence shown in the SEQ ID NO:65 (6C8 VH structural domain, " Q " comprises homing sequence).

In one embodiment, the VH structural domain of binding molecule of the present invention comprises the Nucleotide 58-414 (6C8 VH structural domain, " N " do not contain homing sequence) among the SEQ ID NO:9.

In one embodiment, the VH structural domain of binding molecule of the present invention comprises the Nucleotide 58-414 (6C8 VH structural domain, " Q " do not contain homing sequence) among the SEQ ID NO:65.

In one embodiment, the VL structural domain of binding molecule of the present invention comprises the nucleotide sequence shown in the SEQ ID NO:10 (6C8 VL structural domain comprises homing sequence).

In one embodiment, the VL structural domain of binding molecule of the present invention comprises the Nucleotide 61-381 (6C8 VL structural domain does not contain homing sequence) among the SEQ ID NO:10.

In one embodiment, the VH structural domain that comprises of binding molecule of the present invention contains the CDR (6C8 VH CDR1) of its nucleotide sequence shown in SEQ ID NO:11.

In one embodiment, the VH structural domain that comprises of binding molecule of the present invention contains the CDR (6C8 VH CDR2-" AAT ") of its nucleotide sequence shown in SEQ ID NO:12.

In one embodiment, the VH structural domain that comprises of binding molecule of the present invention contains the CDR (6C8 VH CDR3) of its nucleotide sequence shown in SEQ ID NO:13.

In one embodiment, the VH structural domain that comprises of binding molecule of the present invention contains the CDR (6C8 VH CDR2-alternate " CAA ") of its nucleotide sequence shown in SEQ ID NO:65.

In one embodiment, the VL structural domain that comprises of binding molecule of the present invention contains the CDR (6C8 VL CDR1) of its nucleotide sequence shown in SEQ ID NO:14.

In one embodiment, the VL structural domain that comprises of binding molecule of the present invention contains the CDR (6C8 VL CDR2) of its nucleotide sequence shown in SEQ ID NO:15.

In one embodiment, the VL structural domain that comprises of binding molecule of the present invention contains the CDR (6C8 VL CDR3) of its nucleotide sequence shown in SEQ ID NO:16.

In one embodiment, the CL structural domain of binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:20 (mouse IgG2a constant region of light chain).

In one embodiment, the CH structural domain of binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:21 (mouse IgG2a CH).

In one embodiment, binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:22 (chimeric-6C8VL/ people CL IgG1).

In one embodiment, binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:23 (chimeric Gly-6C8 VH/ people CH IgG1).

In one embodiment, binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:24 (chimeric Agly-6C8 VH/ people CH IgG1).

In one embodiment, binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:44 (humanization 6C8 VL).

In one embodiment, binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:53 (humanization 6C8 VH " N ").

In one embodiment, binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:54 (humanization 6C8 VH " Q ").

In one embodiment, the CL structural domain of binding molecule of the present invention comprises the aminoacid sequence (human IgG1 Gly CH) shown in SEQ ID NO:55.

In one embodiment, the CH structural domain of binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:56 (human IgG1 Agly CH).

In one embodiment, the CL structural domain of binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:57 (human IgG1's constant region of light chain).

In one embodiment, binding molecule of the present invention comprises the aminoacid sequence shown in the SEQ ID NO:58 (full-length human 6C8 light chain).

In one embodiment, binding molecule of the present invention comprises (the full-length human 6C8 heavy chain-HuN6C8-Gly) of the aminoacid sequence shown in the SEQ ID NO:60.

In one embodiment, binding molecule of the present invention comprises (the full-length human 6C8 heavy chain-HuN6C8-Agly) of the aminoacid sequence shown in the SEQ ID NO:61.

In one embodiment, binding molecule of the present invention comprises (the full-length human 6C8 heavy chain-HuQ6C8-Gly) of the aminoacid sequence shown in the SEQ ID NO:62.

In one embodiment, binding molecule of the present invention comprises (the full-length human 6C8 heavy chain-HuQ6C8-Agly) of the aminoacid sequence shown in the SEQ ID NO:63.

In one embodiment, binding molecule of the present invention has VL and the VH sequence shown in Figure 18 A-18D; SEQ ID NO:1 has also shown the aminoacid sequence in 6C8 VH district; The aminoacid sequence in 6C8 VL district is shown in SEQ ID NO:2.In another embodiment, binding molecule of the present invention has LC shown in SEQ ID NO:20 and 21 and HC sequence; ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWT DQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNE (SEQ ID NO:20); AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO：21)。

In an embodiment of the present invention, the VL chain comprises guiding and/or signal sequence, for example the amino-acid residue 1-20 of SEQ IDNO:2.In one embodiment, the VH chain comprises guiding and/or signal sequence, for example the amino-acid residue 1-19 among the SEQ ID NO:1.In another embodiment, binding molecule of the present invention does not contain guiding and/or signal sequence.

In one aspect, the present invention relates to the 6C8 binding molecule binding molecule suitable with the 6C8 characteristic with other, described characteristic in conjunction with GITR, and is offset the Treg cell to the effect of Teff cell inhibiting such as high-affinity.In addition, binding molecule of the present invention can not bring out apoptosis, can not suppress mixed lymphocyte reacion yet.Accordingly, the binding molecule that is equal to of the present invention is the GITR antagonist, and promptly they can bring out the signal transduction that carries out via GITR.GITR is the member of TNFR superfamily.Because the member of TNFR family participates in cell survival and apoptosis by the signal transduction that carries out via NF-κ B, in one embodiment, binding molecule of the present invention can weaken the degraded of I-κ B.

In one embodiment, the invention provides isolating hGITR binding molecule, its variable region of light chain (VL) comprises the aminoacid sequence of SEQ ID NO:2, and optional homing sequence; Variable region of heavy chain (VH) comprises the aminoacid sequence of SEQ ID NO:1, and optional homing sequence.In some embodiments, described binding molecule comprises CH, such as IgG1, and IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region.In addition, described binding molecule can comprise constant region of light chain, i.e. κ constant region of light chain or lambda light chain constant region.Described binding molecule preferably comprises the κ constant region of light chain.In one embodiment, binding molecule of the present invention comprises the constant region of light chain shown in the SEQ ID NO:20.In one embodiment, binding molecule of the present invention comprises the CH shown in the SEQ ID NO:21.In one embodiment, binding molecule of the present invention comprises the CH shown in the SEQ ID NO:55.In one embodiment, binding molecule of the present invention comprises the CH shown in the SEQ ID NO:56.In one embodiment, binding molecule of the present invention comprises the CH shown in the SEQ ID NO:57.

In another embodiment, the invention provides binding molecule with the relevant VL CDR structural domain of 6C8, the binding molecule that for example has such variable region of light chain (VL), this variable region of light chain has at least one CDR structural domain, and the aminoacid sequence that the latter comprises is selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8.In another embodiment, variable region of light chain (VL) has at least two CDR structural domains, and the aminoacid sequence of structural domain is selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8.Also have in the embodiment, the aminoacid sequence that the CDR structural domain of variable region of light chain (VL) comprises is selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8.

Also have in some embodiments, the invention provides the binding molecule with the relevant VH CDR structural domain of 6C8-, for example the CDR structural domain of the variable region of light chain of binding molecule (VH) comprises the aminoacid sequence that is selected from SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:19.In another embodiment, variable region of heavy chain (VH) has at least two CDR structural domains, and its aminoacid sequence that comprises is selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ IDNO:19.Also have in the embodiment, the CDR structural domain that variable region of heavy chain (VH) has comprises the aminoacid sequence that is selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:19.

In another embodiment, binding molecule of the present invention comprises at least one and derives from mouse anti human GITR binding molecule, for example CDR of 6C8 binding molecule.Be used for this paper, term " derives from " source that a specified protein is represented polypeptide.In one embodiment, polypeptide or the aminoacid sequence that derives from concrete initial polypeptide is CDR sequence or its correlated series.In another embodiment, polypeptide or the aminoacid sequence that derives from concrete initial polypeptide is FR sequence or its correlated series.In one embodiment, deriving from concrete initial amino acid sequence of polypeptide is not successive.

For example, in one embodiment, there is 1,2,3,4,5 or 6 CDR to derive from mouse 6C8 antibody.In one embodiment, binding molecule of the present invention comprises at least one heavy chain or the light chain CDR of mouse 6C8 antibody.In another embodiment, binding molecule of the present invention comprises at least two CDR from mouse 6C8 antibody.In another embodiment, binding molecule of the present invention comprises at least three CDR from mouse 6C8 antibody.In another embodiment, binding molecule of the present invention comprises at least four CDR from mouse 6C8 antibody.In another embodiment, binding molecule of the present invention comprises at least five CDR from mouse 6C8 antibody.Binding molecule of the present invention comprises at least six CDR from mouse 6C8 antibody.

It is different on aminoacid sequence to it will be understood by those skilled in the art that binding molecule of the present invention can be modified to the 6C8 molecule of originating with them.For example, can cause the Nucleotide or the aminoacid replacement of conservative property replacement, or non-essential amino acid residue (for example, the residue in CDR and/or framework) is changed, and keep its ability in conjunction with GITR (for example people GITR).

In one embodiment, at least one CDR (perhaps at least one among above 6C8 CDR in the binding molecule) is modified to different with the sequence of natural 6C8 binding molecule, but has kept the ability in conjunction with 6C8.For example, in one embodiment, one or more CDR in the 6C8 antibody is modified so that remove the potential glycosylation site.For example, because it is the consensus sequence of glycosylation site that aminoacid sequence Asn-X-(Ser/Thr) estimates, may have influence on the generation of binding molecule, and the CDR2 of 6C8 heavy chain has sequence A sn-Pro-Ser, is replaced to glutamine (Gln) so the l-asparagine (Asn) that second version of this heavy chain is prepared to amino-acid residue 62 among the SEQ IDNO:53 guards.

In one embodiment, binding molecule of the present invention comprises such polypeptide or aminoacid sequence, the part of itself and 6C8 antibody or this antibody is basic identical, described part by 3-5 amino acid at least, at least 5-10 amino acid, at least 10-20 amino acid, at least 20-30 amino acid or at least 30-50 amino acid form, perhaps those skilled in the art it derives from homing sequence as can be seen.

In another embodiment, derive from the polypeptide of concrete initial polypeptide or aminoacid sequence or aminoacid sequence and have about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% aminoacid sequence homogeny, perhaps those skilled in the art it derives from described homing sequence as can be seen.

The isolated nucleic acid molecule of the non-natural variant of coded polypeptide can replace, add or disappearance by introduce one or more Nucleotide in the nucleotide sequence of binding molecule, produces thereby introduce one or more aminoacid replacement, interpolation or disappearance in coded protein.Can pass through routine techniques, introduce sudden change such as the sudden change of rite-directed mutagenesis and PCR mediation.In one embodiment, done the conservative amino acid replacement at one or more non-key amino-acid residue place." conservative amino acid replacement " is that amino-acid residue is replaced with another amino-acid residue that similar side chain is arranged.The amino-acid residue family that similar side chain arranged is existing clearly defining in this area, comprise that basic side chain (for example, Methionin, arginine, Histidine), acid side-chain (for example, aspartic acid, L-glutamic acid), uncharged polar side chain (for example, glycine, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), non-polar sidechain (for example, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), β-branched building block (for example, Threonine, Xie Ansuan, Isoleucine) and aromatic side chain (for example, tyrosine, phenylalanine, tryptophane, Histidine).Therefore, a non-key amino-acid residue in the binding molecule polypeptide can replace to another amino-acid residue from identical side chain family.In another embodiment, a row amino acid can replace to similar, but order and/or the different amino acid string of side chain man's group composition.

Alternate in another embodiment, can be introduced sudden change along binding molecule encoding sequence all or part of.

Preferred binding molecule of the present invention comprises framework and the constant region aminoacid sequence that derives from the human amino acid sequence.But described binding molecule can comprise framework and/or the constant region sequence that derives from another mammalian species.For example, can comprise framework region (for example, non-human primates), the heavy chain part of primates in the described binding molecule, and/or hinge fraction.In one embodiment, can contain one or more mouse amino acid in conjunction with the framework region of polypeptide, for example people or non-human primates framework amino acid sequence can comprise one or more aminoacid replacement and/or reverse mutation, and corresponding mouse amino-acid residue is wherein arranged.The immunogenicity of preferred binding molecule of the present invention is low compared with the 6C8 murine antibody that begins.

The present invention also provides specific chimeric and/or the humanization binding molecule (that is chimeric and/or Humanized immunoglobulin) of GITR.Chimeric and/or humanization binding molecule has identical or similar binding specificity and affinity with mouse or other non-human binding molecules as the starting raw material that makes up this chimeric or humanization binding molecule are provided.

Chimeric binding molecule generally is by genetically engineered, is built into its light chain and heavy chain gene by the immunoglobulin gene fragment that belongs to different plant species.For example, can with variable (V) fragment of mouse monoclonal binding molecule gene and constant (C) fragment of people (such as, IgG1 or IgG4, preferred people's isotype IgG1) connect together.Therefore, exemplary chimeric binding molecule is by from the V of mouse binding molecule or antigen binding domains and the hybridization protein that constitutes from the C or the effector structural domain of human binding molecules.

In one embodiment, the polypeptide that the present invention relates to the variable region of humanization 6C8 binding molecule and comprise such humanization variable region.In one embodiment, binding molecule of the present invention comprises at least one humanization 6C8 binding molecule variable region, for example light chain or variable region of heavy chain.

Term " humanization binding molecule " is meant such binding molecule, it comprises at least one chain that contains the variable region framework residue that derives from human binding molecules chain (being called receptor immunoglobulin or binding molecule) and at least one derives from the mouse binding molecule complementary determining region of (being called donor immunity sphaeroprotein or binding molecule).The humanization binding molecule can adopt the recombinant DNA technology of discussing below to prepare.Referring to for example, Hwang, W.Y.K.et al. (2005) Methods 36:35; Queen et al.Proc.Natl.Acad.Sci.USA, (1989), 86:10029-10033; Jones et al.Nature, (1986), 321:522-25; Riechmann et al.Nature, (1988), 332:323-27; Verhoeyen et al.Science, (1988), 239:1534-36; Orlandi et al.Proc.Natl.Acad.Sci.USA, (1989), 86:3833-37; United States Patent (USP) 5,225,539; 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370, Selick et al.WO 90/07861 and Winter, US 5,225,539 (mode with reference is all incorporated this paper into).Constant region if any, preferably also derives from human normal immunoglobulin.

When having selected preferred inhuman donor binding molecule and carried out humanization, can be from the human immunoglobulin gene sequence library of for example expressing, to plant be that the consensus sequence of Ig sequence or a plurality of human binding molecules obtain suitable people's receptors bind molecule.

In one embodiment, be used to carry out humanization (referring to, Hwang for example, W.Y.K.et al. (2005) Methods 36:35, this article content is all incorporated this paper by reference into) based on the method for CDR homology.This method is usually directed to according to the mouse of similar structures and people CDR, rather than the mouse of similar structures and people's framework substitute onto mouse CDR in people's variable domains framework.The similarity of determining mouse and people CDR generally is the people's gene of the chain (light chain or heavy chain) that is tested and appraised same type, and described chain has identical standard CDR textural association with the mouse binding molecule, thereby keeps the three-dimensional conformation of CDR peptide backbone.The second, each is had candidate's variable gene of the norm structure of coupling, between mouse and candidate CDR, estimate the homology of residue and residue.At last, in order to produce the humanization binding molecule, CDR residues different with mouse CDR among the people candidate CDR that chooses is changed over the sequence of mouse.In one embodiment, do not introduce the sudden change of people's framework in the humanization binding molecule.

In one embodiment, having estimated ethnic group is the CDR homology of sequence and GITR binding molecule CDR.For example, to mouse 6C8 antibody, be that light chain κ chain V gene and 6C8 antibody sequence compare with all kinds that have the 2-1-1 norm structure in the IMGT database.To heavy chain is that heavy chain V gene and 6C8 aminoacid sequence compare equally with all 3-1 kinds.Accordingly, in one embodiment, binding molecule of the present invention comprises the people κ chain V district framework with 2-1-1 norm structure.In another embodiment, binding molecule of the present invention comprises the people's heavy chain V district framework with 3-1 norm structure.

We identify following potential people light chain kind is sequence, can be incorporated in the binding molecule of the present invention:

The IMGT accession number of IGKV3-15 gene is M23090.Aminoacid sequence is:

EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP(SEQ?ID?NO：25)。

The IMGT accession number of IGKV3D-11 gene is X17264.Aminoacid sequence is:

EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH(SEQID?NO：26)。

The IGKV3-11 gene has two allelotrope.The allelotrope of IGKV3-11 gene ^*01 IMGT accession number is X01668.Aminoacid sequence is:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP(SEQID?NO：27)。

Allelotrope ^*02 IMGT accession number is X02768.Aminoacid sequence is:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGRDFTLTISSLEPEDFAVYYCQQRSNWP(SEQID?NO：28)。

The IMGT accession number of IGKV1D-43 gene is X72817.Aminoacid sequence is:

AIRMTQSPFSLSASVGDRVTITCWASQGISSYLAWYQQKPAKAPKLFIYYASSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTP(SEQID?NO：29)。

The IGKV1-39 gene has two allelotrope.The allelotrope of IGKV1-39 gene ^*01 IMGT accession number is X59315.Aminoacid sequence is:

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP(SEQID?NO：30)。

The allelotrope of IGKV1-39 gene ^*02 IMGT accession number is X59318.Aminoacid sequence is:

DIQMTQSPSFLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQCGYSTP(SEQID?NO：31)。

The IMGT accession number of IGKV1-33 gene is M64856.Aminoacid sequence is:

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLP(SEQID?NO：32)。

The IMGT accession number of IGKV1-27 gene is X63398.Aminoacid sequence is:

DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAP(SEQ?ID?NO：33)。

The IGKV1-17 gene has two allelotrope.The allelotrope of IGKV1-17 gene ^*01 IMGT accession number is X72808.Aminoacid sequence is:

DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYP(SEQID?NO：34)。

The allelotrope of IGKV1-17 gene ^*02 IMGT accession number is D88255.Aminoacid sequence is:

DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCLQHNSYP(SEQID?NO：35)。

The IGKV1D-16 gene has two allelotrope.The allelotrope of IGKV1D-16 gene ^*01 IMGT accession number is K01323.Aminoacid sequence is:

DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP(SEQID?NO：36).

The allelotrope of IGKV1D-16 gene ^*02 IMGT accession number is J00244.Aminoacid sequence is:

DIQMTQSPSSLSASVGDRVTITCRARQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP(SEQID?NO：37)。

The IMGT accession number of IGKV1-16 gene is J00248.Aminoacid sequence is:

DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP(SEQID?NO：38)。

The IGKV1-12 gene has two allelotrope.The allelotrope of IGKV1-12 gene ^*01 IMGT accession number is V01577.Aminoacid sequence is:

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP(SEQID?NO：39)。

The allelotrope of IGKV1-12 gene ^*02 IMGT accession number is V01576.Aminoacid sequence is:

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP(SEQID?NO：40)。

The IMGT accession number of IGKV1-9 gene is Z00013.Aminoacid sequence is:

DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYP(SEQID?NO：41)。

The IMGT accession number of IGKV1-6 gene is M64858.Aminoacid sequence is:

AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYP(SEQID?NO：42)。

The IGKV1-5 gene has three allelotrope.The allelotrope of IGKV1-5 gene ^*01 IMGT accession number is Z00001.Aminoacid sequence is:

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYS(SEQID?NO：43)。

We identify following potential people heavy chain kind is sequence, can be incorporated in the binding molecule of the present invention:

The IGHV2-5 gene has ten allelotrope.The allelotrope of IGHV2-5 gene ^*01 IMGT accession number is X62111.Aminoacid sequence is:

QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIYWNDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYY(SEQ?ID?NO：45)。

The IMGT accession number of IGHV2-26 gene is M99648.Aminoacid sequence is: QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAHIFSN DEKSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCARI (SEQ ID NO:46).

The IGHV2-70 gene has 13 allelotrope.The allelotrope of IGHV2-70 gene ^*01 IMGT accession number is L21969.Aminoacid sequence is:

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCVSWIRQPPGKALEWLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARI(SEQ?ID?NO：47)。

The IGHV4-30-2 gene has 4 allelotrope.The allelotrope of IGHV4-30-2 gene ^*01 IMGT accession number is L10089.Aminoacid sequence is:

QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGGYSWSWIRQPPGKGLEWIGYIYHSGSTYYNPSLKSRVTISVDRSKNQFSLKLSSVTAADTAVYYCAR(SEQ?ID?NO：48)。

The IGHV4-30-4 gene has six allelotrope.The allelotrope of IGHV4-30-4 gene ^*01 IMGT accession number is Z14238.Aminoacid sequence is:

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ?ID?NO：49)。

The IGHV4-31 gene has ten allelotrope.The allelotrope of IGHV4-31 gene ^*01 IMGT accession number is L10098.Aminoacid sequence is:

QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLKSLVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ?ID?NO：50)。

The IGHV4-39 gene has six allelotrope.The allelotrope of IGHV4-39 gene ^*01 IMGT accession number is L10094.Aminoacid sequence is:

QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ?ID?NO：51)。

The IGHV4-61 gene has eight allelotrope.The allelotrope of IGHV4-61 gene ^*01 IMGT accession number is M29811.Aminoacid sequence is:

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ?ID?NO：52)。

These kinds be sequence each may be used to provide framework region and one or more 6C8 CDR to use together.

Be used for this paper, " norm structure " is the conservative hypermutation ring conformation that is formed by different CDR, and binding molecule produces with antigen by these conformations and contacts.Utilize the software of public sphere new binding molecule can be divided the norm structure type.

In another embodiment, it is to carry out on the basis of the correct stereochemical orientation that can keep mouse variable domains framework that mouse CDR is substituted onto in people's variable domains framework, this is to be tested and appraised such people's variable domains framework, and these people's variable domains frameworks can maintain the identical conformation of mouse variable domains framework of being originated with CDR.In one embodiment, the realization of this point is by obtaining people's variable domains that those its framework sequences and CDR are originated in the human binding molecules mouse variset territory shows height sequence homogeny.Referring to Kettleborough et al.ProteinEngineering 4:773 (1991); Kolbinger et al.Protein Engineering 6:971 (1993) and Carter et al.WO 92/22653.

Preferred people's receptors bind molecule keeps the standard and the interface residue of donor binding molecule.In addition, described people's receptors bind molecule is preferably having suitable similarity aspect the CDR ring length.Referring to, Kettleborough et al.Protein Engineering 4:773 (1991); Kolbinger et al.ProteinEngineering 6:971 (1993) and Carter et al.WO 92/22653.

In another embodiment, can with the basis of the homology of the framework region of 6C8 binding molecule on select suitable people's receptor sequence.For example, the aminoacid sequence of 6C8 binding molecule and the aminoacid sequence of other known binding molecule can be compared, for example by the FR district of 6C8 aminoacid sequence or public's database of variable region and known binding molecule are compared, select those variable regions or the highest sequence of FR district amino acid same percentage, promptly reach 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical.In one embodiment, framework series (QVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWD DDKYNPSLKSRLTISKDTSSNQVFLKITSVDTRDTATYYCARTRRYFPFAYWGEGT SVTVTS (the SEQ ID NO:67 that can use SEQ ID NO:67 to list; The framework residue is represented with runic)).In another embodiment, framework sequence (QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWD DDKYNP SLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSS (the SEQ ID NO:68 that can use SEQ ID NO:68 to list; The framework residue is represented with runic)).

After identifying the complementary determining region and suitable people's receptor immunoglobulin of mouse donor immunity sphaeroprotein, next step is to determine which residue in these compositions if desired should be substituted so that the characteristic of gained humanization binding molecule is optimized.Usually, the amino-acid residue that replaces the people with mouse should be reduced as far as possible, binding molecule causes human anti-mouse antibody (HAMA) reaction in human body danger can be improved because import the mouse residue.The definite immunoreactive method that can use this area approval monitors that the HAMA in concrete patient or the clinical trial process reacts.Can in the beginning of carrying out this therapy and process, carry out the immunogenicity evaluation to the patient who is given the humanization binding molecule.For example utilize method known to those skilled in the art, comprise surface plasma resonance technology (BIACORE) and/or solid phase elisa assay method, weigh the HAMA reaction at the antibody of this humanization therapeutical agent by detecting in patient's serum sample.

If necessary, one or more residue in people's framework region can be changed or is replaced to the residue in corresponding site in the murine antibody, thereby keep humanized antibody antigenic binding affinity.This change is called as " reverse mutation " sometimes.To may the influencing of CDR conformation and/or conjugated antigen, some amino acid of selecting in the framework residue of people variable region are done reverse mutation based on them.Choose variable framework region replaces mouse CDR district may cause conformation to limit, if do not correct by replacing some amino-acid residues, can cause losing of binding affinity.

In one embodiment, the amino-acid residue that carries out reverse mutation can partly utilize the technology of this area approval to make up by computer model to select.Usually, be to be that starting point produces molecular model with the structure that immunoglobulin chain or its structural domain have solved.Relatively treat chain and the chain of known three-dimensional structure or the aminoacid sequence similarity of structural domain of modeling, select and show the homophylic chain of highest serial or structural domain starting point as the molecule modeling.Selection has at least 50% sequence homogeny, preferably have at least 60%, 70%, 80%, 90% or the chain or the structural domain of higher sequence homogeny carry out modeling.Known initial structure is changed, allow to wait to simulate real amino acid in immunoglobulin chain or the structural domain and the difference between the amino acid in the initial structure.The structural group that to change is dressed up the combination immunoglobulin (Ig) then.At last, model is carried out accurate adjustment, comprise by energy minimization, and confirm that all atoms are in the suitable distance, bond distance and bond angle are in chemically acceptable scope.

The amino-acid residue that selection replaces also can be partly by the amino acid whose characteristics on the check particular location, the perhaps influence that may cause by the concrete aminoacid replacement of empiric observation or sudden change.For example, when the amino acid of mouse variable region framework residue and selected people variable region framework residue not simultaneously, if can reasonably expect directly conjugated antigen of the non-covalent ground of this amino acid (1), (2) approaching with the CDR district, (3) or with the CDR district there is interaction (for example, to determine that by microcomputer modelling itself and CDR district are at about 3-6

In), perhaps (4) participate in the VL-VH interface, people's framework amino acid can be replaced with the framework amino acid that is equal in the mouse binding molecule.

The residue of " non-covalent ground directly conjugated antigen " comprises the amino acid on the framework region site, they have very big may with amino acid on the antigen according to chemical force (for example hydrogen bond, Van der Waals force, the hydrophobic interaction etc.) direct interaction of having set up.

The residue in " near the CDR district " comprises the amino-acid residue on the site that is close to one or more CDR in the Humanized immunoglobulin chain primary sequence, for example be in and be close to the defined CDR of Kabat, perhaps on the site of the CDR of Chothia definition (referring to, Chothia and Lesk JMB 196:901 (1987) for example).These amino acid may interact with the amino acid among the CDR especially, if select from acceptor, may make donor CDR distortion, reduce affinity.In addition, adjacent amino acid may with antigen direct interaction (Amit et al.Science, 233:747 (1986), this article is incorporated this paper in the reference mode), just may wish to select these amino acid in the donor, so that keep providing in original binding molecule all antigens contacts of affinity.

The residue that " perhaps interacts with the CDR district " comprises that those are defined as being in the residue of the stereochemical orientation that enough influences the CDR district by secondary structure analysis.In one embodiment, the residue that " perhaps interacts with the CDR district " is to identify by the three-dimensional model (for example model of computer generation) of analyzing the donor immunity sphaeroprotein.Three-dimensional model, the three-dimensional model of normally original donor binding molecule shows some outer amino acid of CDR near CDR, and the very big amino acid interaction that may pass through among hydrogen bond, Van der Waals force, hydrophobic interaction etc. and the CDR is arranged.On these amino acid sites, can select donor immunity sphaeroprotein amino acid rather than receptor immunoglobulin amino acid.Meeting this standard amino acid has some atoms among side chain atom and the CDR to be in about 3 usually

In, and must contain atom can with the CDR atom according to the chemical force set up, interact such as above-named those chemical forces.

For the atom that may form hydrogen bond, 3

Be the observed value of its internuclear distance, but for the atom of Cheng Jian not, 3

It is the observed value of the distance between its Van der Waals surface.Therefore, in the later case, nucleus must be about 6

With interior (3 Add van der Waals radius and), atom just is believed to interact.In many situations, nucleus is at a distance of 4 or 5 to 6

When whether definite amino acid have interaction with CDR, preferably last 8 amino acid of heavy chain CDR are not thought the part of CDR, because on structure, these 8 amino acid are more as if the part of framework region.

Can also identify by another kind of method with the interactional amino acid of the amino acid among the CDR.The solvent that calculates each framework amino acid in two ways can be near surface-area: (1) in complete binding molecule and (2) removed in the illusion molecule that the binding molecule of CDR constitutes.10 square angstroms of having an appointment between these two numerals or bigger difference show that framework amino acid blocked by CDR with contacting at least in part of solvent, so this amino acid contacts with CDR.Amino acid whose solvent can be on the basis of the three-dimensional model of binding molecule near surface-area, utilize algorithm known in the art (for example to calculate, Connolly, J.Appl.Cryst.16:548 (1983) and Lee and Richards, J.Mol.Biol.55:379 (1971), these two pieces of articles are all incorporated this paper in the mode of reference).Framework amino acid has the conformation of the framework amino acid that contacts also may interact indirectly with CDR once in a while by influencing another with CDR.

Known (the Chothia and Lesk that in many binding molecules, can interact of amino acid in the framework on several sites with CDR, ditto, the same and Tramontano et al.J.Mol.Biol.215:175 (1990) of Chothia et al., these articles are all incorporated this paper in the mode of reference).Specifically, the amino acid in the light chain on the site 2,48,64 and 71, heavy chain site 26-30,71 and 94 amino acid (numbering is according to Kabat's) be known can be interactional in many binding molecules with CDR.The amino acid of light chain site 35 and heavy chain site 93 and 103 also may interact with CDR.In all these numbering sites, preferably in Humanized immunoglobulin, select donor amino acid rather than acceptor amino acid (when they not simultaneously).On the other hand, some residues can interact with the CDR district, such as 5 amino acid of beginning of light chain, can from receptor immunoglobulin, select sometimes, and the affinity that can not lose the humanization binding molecule.

The residue of " participate in VL-VH interface " or " accumulation residue " comprise that those are positioned at the VL that for example Novotny and Haber (Proc.Natl.Acad.Sci.USA, 82:4592-66 (1985)) or Chothia etc. (ditto) define and the residue at the interface between the VH.In general, should in the humanization binding molecule, keep uncommon accumulation residue, if they with people's framework in different.

Usually, one or more satisfies above-mentioned standard amino acid and is substituted.In some embodiments, all or major part satisfy above standard amino acid and be substituted.Once in a while, be difficult to judge whether a concrete amino acid satisfies above-mentioned standard, can produce alternate variant binding molecule, and one of them has this concrete replacement, and another does not have.The activity of the alternate variant binding molecule that test produces like this in the arbitrary analytical method that can describe in the text, thus preferred that binding molecule selected.

In general, the CDR district of humanization binding molecule is substantially the same with the corresponding CDR district in the donor binding molecule, and perhaps more frequent is exactly identical.Though do not need usually, can do one or more conserved amino acid replacement to the CDR residue sometimes, and not influence the binding affinity of gained humanization binding molecule substantially.That conservative replacement is represented is Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; And Phe, these combinations of Tyr.

Other candidate replaces be those for this human normal immunoglobulin site is seldom to see or the acceptor people framework amino acid of " rare (rare) ".These amino acid can be used the medium amino acid with the site of mouse donor binding molecule, and the amino acid that perhaps more common human normal immunoglobulin is equal on the site replaces.For example, the amino acid of people's framework region is rare for this site in receptor immunoglobulin, and the corresponding amino acid in the donor immunity sphaeroprotein is when being common to this site of human normal immunoglobulin sequence; Perhaps the amino acid in the receptor immunoglobulin is rare to this site, the corresponding amino acid in the donor immunity sphaeroprotein relatively other day sequence this site when also being rare, just may wish to replace.These standards have guaranteed that the atypia amino acid in people's framework can not destroy the structure of binding molecule.And being used in the donor binding molecule is that typical amino acid replaces uncommon people's acceptor amino acid for human binding molecules by chance, and the humanization binding molecule possibility immunogenicity that obtains is lower.

Term " rare " is used for this paper and shows, amino acid is being less than about 20% but be less than usually in about 10% the representative series sample and occur in this site, term " common " is used for this paper, and expression amino acid is surpassing about 25% but surpass in about 50% the representative sample usually and occur.For example, everyone light chain and weight chain variabl area sequence all are divided into respectively homology each other especially, and in the identical sequence of some critical sites upper amino acids " subgroup " (Kabat etc., the same).When the amino acid in decider's receptor sequence is " rare " or " common " for the human sequence, often preferred the human sequence in the identical subgroup is thought receptor sequence.

What other candidate replaced is the acceptor people framework amino acid that can be identified as the part in CDR district according to the definition that (ditto) such as Chothia proposes.What other candidate replaced is the acceptor people framework amino acid that can be identified as the part in CDR district according to AbM and/or contact definition.Particularly the CDR1 in the variable heavy chain is defined as and comprises residue 26-32.

Other is available, and what replace is the acceptor framework residue corresponding with rare or uncommon donor framework residue.Rare or uncommon donor framework residue is the residue of for this site of mouse binding molecule rare or uncommon (as defining in the literary composition).For the mouse binding molecule, can divide subgroup according to Kabat and by the identification residue site different with total residue.The distinctive difference of these donors may show the somatic mutation that those feasible activity are improved in the mouse sequence.Expectation can influence the uncommon residue of bonded and be retained, and estimates being substituted in conjunction with unessential residue.

Other is available, and what replace is that the non-kind that appears in the acceptor framework region is a residue.For example, receptors bind molecular chain (the human binding molecules chain that obvious sequence homogeny is promptly arranged with the donor chain binding molecule) and kind bound close molecular chain (same obvious sequence identity is arranged with the donor chain) when carrying out sequence alignment, unmatched residue can be that residue in the sequence replaces with corresponding kind between receptor chain framework and the kind tethers framework.

Except special aminoacid replacement discussed above, humanization binding molecule framework region usually basically, the framework region that perhaps is exactly the human binding molecules of originating with it is identical.Certainly, the many amino acid in the framework region are to the specificity of binding molecule or affinity has seldom or not directly contribution.Therefore, can tolerate that the single conservative property of many framework residues replaces, and the specificity or the affinity of not obvious change gained humanization binding molecule.So in one embodiment, the consensus sequence of the variable framework region of humanization binding molecule and the variable framework region sequence of people or these sequences has at least 85% sequence homogeny.In another embodiment, the consensus sequence of the variable framework region of humanization binding molecule and the variable framework region sequence of people or these sequences has at least 90%, and is preferred 95%, more preferably 96%, 97%, 98% or 99% sequence homogeny.But, in general, be not wish to carry out this class to replace.

In one embodiment, binding molecule of the present invention also comprises at least one reverse mutation from the human amino acid residue to corresponding mouse amino-acid residue, and described residue is that residue is piled up at the interface." residue (interface packing residue) is piled up at the interface " comprises the residue at the interface between VL and VH, and for example Novotny and Haber defines among the Proc.Natl.Acad.Sci.USA, 82:4592-66 (1985).

In one embodiment, binding molecule of the present invention also comprises the reverse mutation of at least one human amino acid residue to corresponding mouse amino-acid residue, and wherein said amino-acid residue is a standard residue." standard residue (canonical residue) " is the framework residue of guarding, they belong to known to very important standard of CDR conformation or structure type (Tramontano et al.J.Mol.Biol.215:175 (1990), this article is incorporated this paper in the reference mode in full).The standard residue comprises residue 2,25, the 27B, 28,29,30,33,48,51,52,64,71,90,94 and 95 in the light chain, and the residue 24,26,27,29,34,54,55,71 and 94 of heavy chain.Other residue (for example, CDR structures shape residue) can be identified according to the method for Martin and Thorton (1996) J.Mol.Biol.263:800.

In one embodiment, binding molecule of the present invention also comprises at least one human amino acid residue to the reverse mutation of corresponding mouse amino-acid residue, wherein said amino-acid residue be positioned at can with the interactional site of CDR.Especially, light chain site 2,48,64 and 71 and heavy chain site 26-30,71 and 94 amino acid (following the numbering of Kabat) be known can be interactional in many antibody with CDR.The amino acid of light chain site 35 and heavy chain site 93 and 1 03 also may interact with CDR.

Analyze based on CLUSTAL W, a plurality of amino-acid residues that identify in people's framework may replace with the corresponding amino-acid residue in the 6C8 light chain for example.These comprise the residue on site 1,8,9,10,11,13,15,17,19,20,21,22,43,45,46,58,60,63,70,76,77,78,79,83,85,87,100 and 104.

In one embodiment, the variable light chain framework of binding molecule of the present invention also comprises at least one human amino acid residue to the replacement of corresponding mouse amino-acid residue, and described residue is selected from: (that is, the E that comprises site 1 in the CDR grafted antibody in mouse CDR and people FR district is mutated into D to E1D, the latter is the corresponding amino-acid residue in the 6C8 antibody), P8Q, A9K, T10F, L11M, V13T, P15V, E17D, A19V, T20S, L21V, S22T, A43S, R45K, L46A, I58V, A60D, S63T, E70D, S76N, S77N, L78V, Q79H, F83L, V85E, Y87F, G100A and V104L.

Analyze based on CLUSTAL W, we have identified the several amino acid residue that may replace with the corresponding amino-acid residue of for example 6C8 heavy chain in people's framework.They comprise site 5,10,11,12,15,19,23,43,46,68,77,81,83,84,86,87,89,90 and 92.

In one embodiment, the variable heavy chain framework of binding molecule of the present invention also comprises at least one human amino acid residue to the replacement of corresponding mouse amino-acid residue, and described residue is selected from: (that is, the R that comprises site 5 in the CDR grafted antibody in mouse CDR and people FR district is mutated into K to R5K, the latter is the corresponding amino-acid residue in the 6C8 antibody), A10G, L11I, V12L, T15S, T19S, T23S, P43S, A46G, R68Q, K77R, V81F, T83K, M84I, N86S, M87V, P89T, V90A and T92A.

Preferred described humanization binding molecule is at least 10 to antigenic specific combination affinity ⁷, 10 ⁸, 10 ⁹Or 10 ¹⁰M ^-1Usually, the humanization binding molecule to the upper limit of antigenic binding affinity in 3,4 or 5 times of the donor immunity sphaeroprotein binding affinity upper limit.The lower limit of binding affinity is also in 3,4 or 5 times of donor immunity sphaeroprotein binding affinity lower limit.Alternate, binding affinity can be compared with the binding affinity of the humanization binding molecule that does not have to replace (for example, have donor CDR and acceptor FR, but do not have the binding molecule of FR replacement).In this case, the combination of the binding molecule that process is optimized is preferably high at least 2 to 3 times than not replacing binding molecule, or 3 to 4 times.In order to compare, the activity of various binding molecules can be determined in conjunction with method of inspection by for example BIACORE (promptly carrying out surface plasma resonance with unmarked reagent) or competition.

After having selected the CDR and frame components of humanization binding molecule theoretically, there are many methods can supply the such binding molecule of preparation.Because the degeneracy of password, each binding molecule aminoacid sequence has multiple nucleic acid sequence encoding.Required nucleotide sequence is can be by solid phase DNA again synthetic or the variant of the required polynucleotide of previous preparation is carried out PCR mutagenesis produce.

The sudden change of oligonucleotide-mediation is the preferred method of replacement, disappearance and the insertion variant of preparation target polypeptid DNA.Referring to Adelman et al. (DNA 2:183 (1983)).In brief, oligonucleotide and the single stranded DNA template of target polypeptid DNA by the required sudden change of will encoding hybridized and is changed.After the hybridization, with total length second complementary strand of the synthetic template of archaeal dna polymerase, described template has been introduced Oligonucleolide primers, the selected change of coding target polypeptid DNA.

The variable fragment of Zhi Bei binding molecule (for example, the heavy chain and variable region of light chain of chimeric, humanization or human binding molecules) can be connected at least a portion constant region for immunoglobulin (Fc) usually as mentioned above, generally is the constant region of human normal immunoglobulin.The human constant region dna sequence dna can separate from the various human cell according to well-known method, but preferred from the B cell of immortalization, the separation (the same and Liu et al.WO87/02671 referring to Kabat et al.) (two Wen Jun all incorporate this paper into way of reference).In general, binding molecule had both contained light chain and had also contained CH.CH generally comprises CH1, hinge, CH2, CH3 and CH4 district.Binding molecule described herein includes the antibody of all types constant region, comprises IgM, IgG, IgD, IgA and IgE; All isotypes comprise IgG1, IgG2, IgG3 and IgG4.Depend on whether need dependent complement of binding molecule and/or cell-mediated toxicity on the selection part of constant region.For example, isotype IgG1 and IgG3 have complement activity, and isotype IgG2 and IgG4 do not have.When showing cytotoxic activity, constant domain is the complement fixation(CF) constant domain normally when wishing binding molecule (for example, the humanization binding molecule), and type generally is IgG1.When not needing this cytotoxic activity, constant domain can be an IgG2 type for example.The selection of isotype also may influence antibody and enter brain.Preferred people's isotype IgG1.Constant region of light chain can be λ or κ.The humanization binding molecule can comprise the sequence from an above type or isotype.Binding molecule can be expressed as the tetramer that contains two light chains and two heavy chains, the heavy chain that separates, light chain, Fab, Fab ', F (ab ') 2 and Fv, perhaps be expressed as the strand binding molecule that its heavy chain and light chain variable structural domain connect together by spacerarm.

III. prepare binding molecule

The invention provides GITR, for example people GITR has specific binding molecule.This class binding molecule can be used for the described various medical compositions of preparation invention, and the complement determining area (hereinafter having a detailed description) of preparation humanization or chimeric binding molecule perhaps preferably is provided.Preparing inhuman (for example mouse, cavy, primates, rabbit or rat) mono-clonal binding molecule can be by for example realizing to animal inoculation pvaccination with the nucleic acid molecule of GITR or coding GITR.For example, can insert expression vector by the gene of the people GITR that will encode, and prepare the 6C8 binding molecule to animal inoculation pvaccination.Also can use the longer polypeptide of the antiidiotype binding molecule of the immunogenic fragments that comprises GITR or GITR or GITR.(referring to, Harlow﹠amp for example; Lane, the same, incorporate this paper into way of reference).This para-immunity is former can to derive from natural origin, synthetic or recombinant expressed through peptide.Choose wantonly, immunogen as mentioned below can or otherwise be combined with each other with the carrier proteins fusion and give.Choose wantonly, described immunogen can give with adjuvant.Term " adjuvant " is meant a kind of like this compound, and when with antigen combined giving, it can strengthen this antigenic immune response, but causes this antigenic immune response giving Shi Buhui separately.Adjuvant can comprise and convene lymphocyte, stimulation B and/or T cell, and stimulate scavenger cell by several machine-processed enhancing immunity reactions.As will be explained hereinafter, there is the adjuvant of several types to use.The complete Freund's adjuvant full freund's adjuvant that toos many or too much for use subsequently is the preferred method of immunization experiment animal.

Usually prepare the polyclone binding molecule with rabbit or cavy.Exemplary preparation for example is used for the polyclone binding molecule of passive protection and can followingly operates.Animal with 100 μ g GITR, is added the adjuvant inoculation, sentence euthanasia in the time of 4-5 month.Collect blood, from other blood ingredient, separate IgG.Immunogenic specific combination molecule can be done partial purification by affinity chromatography.Every animal on average can obtain the immunogen specific binding molecules of about 0.5-1.0mg, can reach 60-120mg altogether.

Usually use mouse to prepare the mono-clonal binding molecule.Can be expelled in the mouse by fragment or the more microscler formula with GITR at certain segmental monoclonal preparation, the preparation hybridoma also screens the binding molecule that hybridoma is sought specific combination GITR.Choose wantonly, the binding molecule that is screened can be in conjunction with concrete zone or the target fragment of GITR, and other non-overlapped fragment of debond GITR.The screening in back can be by determining the situation that combines of binding molecule and a series of deletion mutants of GITR peptide, and definite which mutant can combine with binding molecule and realizes.Estimate in conjunction with being undertaken by for example Western trace or ELISA.Demonstrate the epi-position that the minimal segment of the specific combination of binding molecule has been defined binding molecule.Alternate can be by the competition checking method, and is promptly to be measured and determine epitope specificity with reference to binding molecule competition in conjunction with GITR.If to be measured and compete with reference to binding molecule, then they are in conjunction with identical epi-position (or the bonded epi-position is enough near), have therefore disturbed and another combine with combining of a binding molecule.The preferred isotype of this class binding molecule is the reciprocity isotype in mouse isotype IgG2a or other species.Mouse isotype IgG2a is the equivalent of people's isotype IgG1.

In another embodiment, the DNA of coding binding molecule can adopt ordinary method (for example, by use can with the gene specific bonded oligonucleotide probe of coding mouse binding molecule heavy chain and light chain) easily separate and check order.Separating also, the hybridoma of subclone is the source of preferred this class DNA.Once separation, described DNA can be put into expression vector, then the expression vector transfection can not produced in the protokaryon or eukaryotic host cell of immunoglobulin (Ig) originally to those, such as Bacillus coli cells, monkey COS cell, Chinese hamster ovary (CHO) cell or myeloma cell.More particularly, DNA isolation (can resemble described herein is synthetic DNA) can resemble the United States Patent (USP) 5 that Newman etc. submits to January 25 nineteen ninety-five, 658, describe in 570 (this patent is incorporated this paper in the mode of reference), be used to clone constant and variable region sequences so that the preparation binding molecule.Basically, this patent has been set forth from selected cell and has been extracted RNA, changes into cDNA, utilizes the Ig Auele Specific Primer to increase through PCR.The primer that is applicable to this purpose is at United States Patent (USP) 5,658, description also arranged in 570.Can produce the transformant that to express required antibody relatively in large quantities so that guarantee the clinical and commercial offers of binding molecule.

Those skilled in the art can understand, the DNA of coding binding molecule or its fragment (being antigen binding site) for example can also utilize pd phage or Fd phasmid technology to derive from the antibody phage library.At for example EP 368 684 B1; United States Patent (USP) 5,969,108, Hoogenboom, H.R.and Chames.2000.Immunol.Today 21:371; Nagy et al.2002.Nat.Med.8:801; Huie etal.2001.Proc.Natl.Acad.Sci.USA 98:2682; Lui et al.2002.J.Mol.Biol.315:1063 has enumerated exemplary method in (these articles are all incorporated this paper in the mode of reference).Many parts of publications (for example, Marks et al.Bio/Technology10:779-783 (1992)) have been described by reorganization in chain reorganization, combination infection and the body and have been produced the high-affinity human binding molecules as the strategy that makes up big phage library.In another embodiment, can with ribosomal display replace phage as display platform (referring to, Hanes et al.2000.Nat.Biotechnol.18:1287 for example; Wilson etal.2001.Proc.Natl.Acad.Sci.USA 98:3750; Perhaps Irving et al.2001 J.Immunol.Methods 248:31).Also have in the embodiment, can in the cell surface library, screen binding molecule (Boder et al.2000.Proc.Natl.Acad.Sci.USA97:10701; Daugherty et al.2000 J.Immunol.Methods 243:211).These programs provide the alternative method of separating and cloning traditional hybridoma technology of mono-clonal binding molecule.

Other embodiment of the present invention also comprised those can not produce produce the people in the transgenic animal (for example, mouse) of endogenous immunoglobulin (Ig) or be basically the people binding molecule (referring to, for example United States Patent (USP) 6,075,181,5,939,598,5,591,669 and 5,589,369, every part of patent is all incorporated this paper in the mode of reference).For example, existing people described in chimeric and germ line mutation mouse homotype disappearance heavy chain of antibody joining region and causes endogenous antibody to produce being suppressed fully.Human immunoglobulin gene's array transferred to cause when attacking producing human binding molecules in such germ line mutation mouse with antigen.Another preferably utilizes means that the SCID mouse produces human binding molecules at United States Patent (USP) 5,811, is disclosed in 524, and this patent is incorporated this paper in the mode of reference.Should be appreciated that the genetic material that is associated with these human binding molecules also can resemble separates described herein and operates.

Another high efficiency method that produces the reorganization binding molecule is at Newman, and Biotechnology has open among the 10:1455-1460 (1992).Specifically, this technology has caused producing the long source of the spirit binding molecule that contains monkey variable domains and people's constant series.This piece reference is incorporated this paper by reference in full into.In addition, this technology is at United States Patent (USP) 5,658, description also arranged in 570,5,693,780 and 5,756,096, and these patents are all incorporated this paper by reference into.

In another embodiment, can select lymphocyte, and separate variable gene by micrurgy.For example, can be from the Mammals that inoculated separating periphery blood monocytic cell, about 7 days of vitro culture.The screening and culturing thing is to be met the specific IgG of screening criteria.Can separate the cell in the positive aperture.The B cell of single generation Ig can separate by FACS, perhaps by identifying in the haemolysis patch test of complement-mediated.Produce the B cell of Ig can micrurgy in test tube, for example utilize RT-PCR increase VH and VL gene.Can be in antibody expression vector with VH and VL gene clone, transfectional cell (for example, eucaryon or prokaryotic cell prokaryocyte) is expressed.

In addition, the gene order that is used to produce polypeptide of the present invention can derive from multiple different sources.For example, resemble that extensive discussions above crosses, many human immunoglobulin genes can the retrievable preservation form of the public obtain.The sequence of the gene of lot of antibodies and encoding antibody is disclosed, can utilize the technology of this area approval to come the suitable antibody gene of chemosynthesis by these sequences.Oligonucleotide synthetic technology compatible with this one side of the present invention is well-known to those skilled in the art, can utilize any commodity automatization synthesizer to carry out.In addition, the encode dna sequence dna of a few class heavy chain listed in the text and light chain can be obtained by the synthetic service of the DNA of commerce.Then, thus the genetic material that utilizes any aforesaid method to obtain can be changed or synthetic provide polypeptide of the present invention.

Alternate can adopt technology well known to those skilled in the art to select and cultivate antibody produced cell system.This class technology all has description in many laboratory manuals and one-level publication.In this respect, be applicable to that technology of the present invention is at Current Protocols in Immunology, Coligan et al.Eds.GreenPublishing Associates and Wiley-Interscience, John Wiley and Sons, New York has description in (1991), and this article is incorporated this paper into the mode full text (comprising supplementary material) of reference.

Well-known, can from initial hybridoma or in other transformant, come isolation of RNA by routine techniques, extract and precipitation centrifugal subsequently or chromatography such as guanidinium isothiocyanate.If desired, can pass through standard technique, come separating mRNA from total RNA such as on few dT Mierocrystalline cellulose, carrying out chromatography.Suitable technique is that those skilled in the art are familiar with.

In one embodiment, the method that can know according to people utilizes reversed transcriptive enzyme and archaeal dna polymerase to prepare the cDNA of coding binding molecule light chain and heavy chain at the same time or separately.Can on the basis of disclosed heavy chain and light chain DNA and aminoacid sequence, utilize total constant region primer or more special primer to carry out PCR.Discussed as mentioned, can also utilize PCR to separate to encode the light chain of binding molecule and the dna clone of heavy chain.In this situation, can come the library is screened such as mouse constant region probe by total primer or big homology probe.

DNA, normally plasmid DNA can be separated from cell with technology known in the art, limits restriction enzyme mapping and order-checking according to the standard of describing in detail in the aforementioned document relevant with recombinant DNA technology, well-known technology.Certainly, according to the present invention separate or follow-up analytic process in whenever DNA can be a synthetic.

In one embodiment, binding molecule of the present invention comprises antigen-binding fragments of antibodies, perhaps is made up of antigen-binding fragments of antibodies.Term " Fab " is meant in immunoglobulin (Ig) or the antibody can conjugated antigen, perhaps with the polypeptide fragment of complete antibody (that is the complete antibody of originating with them) competition conjugated antigen (that is, specificity in conjunction with).Be used for this paper, " fragment " of term antibody molecule comprises antigen-binding fragments of antibodies, for example, light chain of antibody (VL), heavy chain of antibody (VH), single-chain antibody (scFv), F (ab ') 2 fragments, Fab fragment, Fd fragment, Fv fragment, and single domain antibody fragment (DAb).These fragments can perhaps obtain by recombinant means by for example complete or whole antibody or antibody chain being carried out chemistry or enzymatic treatment.

In one embodiment, binding molecule of the present invention is antibody through engineering approaches or adorned.The antibody of engineered forms comprises, for example miniantibody (minibodies), bifunctional antibody, the bifunctional antibody that has merged the CH3 molecule, tetravalent antibody, the interior bifunctional antibody (intradiabodies) of born of the same parents are (for example, Jendreyko et al.2003.J.Biol.Chem.278:47813), bi-specific antibody, fusion rotein (for example, antibody cell factor fusion protein) or bi-specific antibody.Other immunoglobulin (Ig) (Ig) and their some variants are at for example United States Patent (USP) 4,745,055; EP 256,654; Faulkner et al.Nature 298:286 (1982); EP 120,694; EP 125,023; Morrison, J.Immun.123:793 (1979); Kohler et al.Proc.Natl.Acad.Sci.USA 77:2197 (1980); Raso et al.Cancer Res.41:2073 (1981); Morrison et al.Ann.Rev.Immunol.2:239 (1984); Morrison, Science 229:1202 (1985); Morrison et al.Proc.Natl.Acad.Sci.USA81:6851 (1984); EP 25 5, and 694; EP 266,663; With among the WO 88/03559 description is arranged.The immunoglobulin chain that reconfigures also is known.Referring to, for example United States Patent (USP) 4,444, and 878; WO 88/03565; The reference of quoting with EP 68,763 and they.

In one embodiment, modified antibodies of the present invention is a miniantibody.Miniantibody is the dimer molecule that is made of two polypeptide chains, an each self-contained ScFv molecule (single polypeptide that comprises one or more antigen binding site of these two polypeptide chains, for example the VL structural domain is connecting the VH structural domain by flexible joint, and the latter is merged the CH3 structural domain through connection peptides.

The ScFv molecule can be built into VH-joint-VL orientation or VL-joint-VH orientation.

Preferably comprise about 10 to 50 amino-acid residues with constituting the VL of antigen binding site and the flexible hinge that the VH structural domain links together.The connection peptides example that is used for this purposes is (Gly4Ser) 3 (SEQ IDNO:17) (Huston et al.1988.Proc.Natl.Acad.Sci.USA 85:5879).Other connection peptides is known in the art.

The method for preparing single-chain antibody is well-known in the art, for example Ho et al.1989.Gene77:51; Bird et al.1988 Science 242:423; Pantoliano et al.1991.Biochemistry30:10117; Milenic et al.1991.Cancer Research 51:6363; Takkinen et al.1991.Protein Engineering 4:837.

Miniantibody (minibody) can utilize means known in the art (referring to, for example United States Patent (USP) 5,837,821 or WO 94/09817A1), prepare by making up ScFv component and connection peptides-CH3 component.These components can be used as restricted fragment separates from the plasmid that separates, and connects then and lays equal stress on new clone in suitable carriers.Whether assembling correctly can be confirmed by restriction enzyme digestion and dna sequence analysis.

Bifunctional antibody and scFv molecule are similar, but have (being less than 10, preferred 1-5) amino-acid residue joint of a weak point that two V structural domains are connected together usually, and therefore the VL and the VH structural domain of same polypeptide chain can not interact.Opposite, the VH of the VL of a polypeptide chain and VH structural domain and second polypeptide chain and VL structural domain (difference) interaction (WO 02/02781).In one embodiment, binding molecule of the present invention is the bifunctional antibody that has merged at least one heavy chain part.In preferred embodiments, binding molecule of the present invention is the bifunctional antibody that has merged the CH3 structural domain.

The modified antibodies of other form is also included within (for example, WO 02/02781 A1 in the scope of the invention; 5,959,083; 6,476,198 B1; US 2002/0103345 A1; WO 00/06605; Byrn et al.1990.Nature.344:667-70; Chamow and Ashkenazi.1996.TrendsBiotechnol.14:52).

In one embodiment, binding molecule of the present invention comprises constant region for immunoglobulin.Constant region known in the art is mediating multiple effector function.For example, combine with binding molecule can the activating complement system for the C1 component of complement.The activation of complement is very important for the opsonization and the cracking of cytopathy substance.The activation of complement also stimulates inflammatory reaction, also may participate in the autoimmunization allergy.In addition, binding molecule combines with cell by the Fc district, and the Fc acceptor site in binding molecule Fc district is in conjunction with the Fc acceptor (FcR) of cell.Having many is special Fc acceptors to dissimilar binding molecules, comprises IgG (γ acceptor), IgE (epsilon receptor), IgA (α acceptor) and IgM (μ acceptor).Binding molecule is attached on the Fc acceptor of cell surface can cause multiple important different biological respinse, the target cell of removing, the combined molecule of bag that comprises the particulate endocytosis of combined molecule and destruction, immunocomplex by killer cell cracking (cytotoxicity that is called the antibody dependent cellular mediation, perhaps ADCC), discharge the generation of inflammatory mediator, placental transport and control immunoglobulin (Ig).

In one embodiment, utilize the constant region of IgG4 binding molecule, this constant region is considered to eliminate target cell, perhaps by (for example utilizing technology known in the art, United States Patent (USP) 5,585,097) Key residues of effector function in the Fc district is suddenlyd change prepare the Fc variant, can remove or reduce effector function.For example, constant region structural domain disappearance or deactivation (realizing by point mutation or other means) can weaken the Fc acceptor and modify combining of binding molecule with round-robin, thereby improve the location to tumour.In other situation, can be that the constant region consistent with the present invention modified and can be regulated the complement combination, therefore reduce the serum half life and with link coupled cytotoxic non-specific related (association).Can also utilize the modification of other constant region to modify disulfide linkage or oligosaccharides part, thereby make the location strengthen because of flexible raising of antigen-specific or binding molecule.In short, those skilled in the art can understand that the binding molecule of describing modification according to this paper can bring into play multiple delicate effect, and these effects may be easy to or be not easy recognize.But these modify last physiological change spectrum, bioavailability and other the biochemical effect that produces, and can utilize known immunological technique such as tumor-localizing ability, bio distribution and serum half life, do not need too much experiment just easily to measure and quantitatively.

In one embodiment, upward another functional molecular (for example, another peptide or protein) can be derived or connect to binding molecule of the present invention.Accordingly, binding molecule of the present invention comprises deriving of the anti-GITR binding molecule described in the literary composition and other modified forms, comprises immunoadhesin molecule.For example, binding molecule of the present invention can functionally connect (by chemical coupling, gene fusion, non-covalent association or alternate manner) and go up one or more other molecular entity, such as another binding molecule (for example, bi-specific antibody or bifunctional antibody) but detection reagent, cytotoxic agent, medicament, and/or can mediate the albumen of getting in touch or the peptide (such as Streptavidin core area or polyhistidine mark) of this binding molecule and another molecule.

One class derivatize binding molecule is by the crosslinked generation of two or more binding molecules (same type or dissimilar is for example when producing bi-specific antibody).Suitable cross-linking agent comprises that xenogenesis is bifunctional, two different reactive groups that separated by suitable spacerarm (for example ,-maleimide phenalgin methyl-N-hydroxyl succinimide ester (m-maleimidobenzoyl-N-hydroxysuccinimide ester)) are promptly arranged; Or bifunctional (for example, suberic acid two succinimide esters (disuccinimidyl suberate)) of the same race.These coupling agents are at Pierce Chemical Company, Rockford, and IL is on sale.

But binding molecule of the present invention can be derived and be had useful detection reagent, comprises fluorescent chemicals.But exemplary fluorescence detection reagent comprises fluorescein, lsothiocyanates fluorescein, rhodamine (rhodamine), dansyl chloride (5-dimethylamine-1-napthalenesulfonyl chloride), phycoerythrin (phycoerythrin) etc.Binding molecule can also be derived detectable enzyme, such as alkaline phospholipase, horseradish peroxidase, glucose oxidase etc.Binding molecule is derived to be had in the time of can detecting enzyme, but can detect by adding the reagent that other this enzyme produces the detection reaction product with it.For example, but when having the detection reagent horseradish peroxidase, add hydrogen peroxide and diaminobenzidine and can produce colored reaction product, just can detect.Binding molecule can also be derived vitamin H, detects by indirect measurement avidin or Streptavidin combination.

IV. express binding molecule

Binding molecule of the present invention can prepare by recombinant expressed light chain immunoglobulin and heavy chain in host cell.For recombinant expressed binding molecule, have the recombinant expression vector transfection host cell of the dna fragmentation of the light chain immunoglobulin of the binding molecule of encoding and heavy chain with one or more, make light chain and heavy chain in host cell, be expressed, and preferably be secreted in the substratum of growth host cell, from this substratum, just can reclaim binding molecule.Utilize the standard recombinant dna technology, such as Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), Ausubel, F.M.et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, the United States Patent (USP) 4 of (1989) and Boss etc., 816, the technology of describing in 397 is obtained heavy chain of antibody and light chain gene, and these genes are imported recombinant expression vector, and the latter introduces host cell.

In order to express binding molecule of the present invention, the DNA of encoding part or full-length light chains and heavy chain can be inserted expression vector, make gene and transcribe operably to link together with the translational control sequence.In the linguistic context here, the described binding molecule gene of term " operably connect " expression is connected in the carrier, thus transcribing and translating control sequence and can bring into play the function that they regulate binding molecule gene transcription and translation in the works in the carrier.In one embodiment, the expression vector of selection and expression regulation sequence are compatible with used expression host cell.Binding molecule light chain gene and binding molecule heavy chain gene can insert in the carrier separately, and be more common, is that two genes are inserted in the same expression vector.Can the binding molecule gene be inserted expression vector by standard method (for example, the complementary restriction site on binding molecule gene fragment and the carrier is coupled together,, put down terminal the connection) if perhaps without limits during restriction enzyme site.Before inserting binding molecule light chain or sequence of heavy chain, expression vector may carry the constant region sequence of binding molecule.For example, a strategy that VH and VL sequence is converted to total length binding molecule gene is their to be inserted can distinguish in the expression vector of encoding heavy chain and constant region of light chain, make that the VH fragment operably is connected with the CH fragment in the carrier, the VL fragment operably is connected with the CL fragment.In addition or alternate, the recombinant expression vector signal peptide that can encode and to assist chain binding molecule from host cell, to secrete to come out.Can with the chain binding molecule gene clone in carrier, make the N-terminal of signal peptide and binding molecule chain gene connect together with meeting frame.Described signal peptide can be immunoglobulin (Ig) signal peptide or allos the signal peptide signal peptide of NIg (that is, from).

Except the binding molecule chain gene, recombinant expression vector of the present invention also has the regulating and controlling sequence that can control the expression of binding molecule chain gene in host cell.Term " regulation and control series " comprises the expression regulation element (for example, poly-adenosine signal) of promotor, enhanser and other control chain binding molecule gene transcription or translation.This class regulating and controlling sequence is at for example Goeddel; Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, CA has description in (1990).It will be understood by those skilled in the art that the design expression vector, comprise and select regulating and controlling sequence to depend on some factors so possibly, such as alternative transformed host cells that is used for, the expression level of target protein etc.Preferably being used for regulating and controlling sequence that mammalian host cell expresses comprises and instructs the viral element of protein at the mammalian cell high level expression, such as the promotor that derives from cytomegalovirus (CMV) and/or enhanser (such as CMV promotor/enhanser), simian virus 40 (SV40) (such as SV40 promotor/enhanser), adenovirus (for example, adenovirus major late promoter (AdMLP) and polyomavirus.About further describing of viral controlling element and sequence thereof, referring to the United States Patent (USP) 4,968,615 of the United States Patent (USP) 4,510,245 of the United States Patent (USP) 5,168,062 of for example Stinski, Bell etc. and Schaffner etc.

Except binding molecule chain gene and regulation and control series, recombinant expression vector of the present invention can have other sequence, such as regulating the sequence of duplicating (for example replication origin) and the selectable marker gene of carrier in host cell.But selectable marker gene can assist to pick out the host cell (referring to for example United States Patent (USP) 4,399,216,4,634,665 and 5,179,017, all being Axel etc.) that those carriers have imported.For example, but usually selectable marker gene make the host cell that has imported carrier produce resistance to medicine (such as G418, Totomycin or methotrexate).But preferred selectable marker gene comprises Tetrahydrofolate dehydrogenase (DHFR) gene (be used for the dhfr-host cell, select/increase with methotrexate) and neo gene (selecting with G418).

In order to express light chain and heavy chain, will encode the expression vector transfection of binding molecule heavy chain and light chain in host cell by standard technique.The various forms of term " transfection " is intended to contain the multiple technology that is usually used in foreign DNA is imported protokaryon or eukaryotic host cell, for example electroporation, calcium phosphate precipitation, the transfection of DEAE-dextran or the like.Binding molecule of the present invention can be expressed in protokaryon or eukaryotic host cell, most preferably in eukaryotic cell (most preferably being mammalian host cell), express binding molecule, because this class eukaryotic cell, especially mammalian cell, than prokaryotic cell prokaryocyte more can assemble and secrete correct folding, have an immunocompetent binding molecule.

Usually, expression vector contain selective marker (for example, ammonia benzyl resistance, hygromycin resistance, tetracyclin resistance or neomycin resistance) in case can detect the cell that transformed required dna sequence dna (referring to, Itakura etc. for example, United States Patent (USP) 4,704,362).

Intestinal bacteria are prokaryotic hosts that a clone who is particularly useful invents described polynucleotide (for example, dna sequence dna).Other microorganism host that is suitable for comprises genus bacillus (such as subtilis) and other enterobacteriaceae, such as Salmonellas, Serratia and various pseudomonas.In these prokaryotic hosts, also can prepare expression vector, they contain the expression regulation sequence compatible with host cell (for example, replication orgin) usually.In addition, any amount of well-known promotor can be arranged, such as lactose promoter systems, tryptophane (trp) promoter systems, β-Nei Xiananmei promoter systems or from the promoter systems of lambda particles phage.Promotor generally can be controlled the carrying out of expression, optional control and has that ribosome bind site sequence etc. is used to open the beginning and end is transcribed and translated with operon sequence.

Other microorganism also can be used for expressing such as yeast.Sugar yeast (Saccharomyces) is preferred yeast host, and it has the suitable carrier that carries expression regulation sequence (for example, promotor), replication orgin, terminator sequence etc. as required.Typical promotor comprises glycerol 3-phosphate acid kinase and other glycolytic ferment.The inducibility Yeast promoter comprises from promotor of the enzyme of ethanol dehydrogenase, different cell pigment C and responsible maltose and galactose utilization etc.

Except microorganism, the mammalian tissues cell culture also can be used for expression and produce polypeptide of the present invention (for example, the polypeptide of coding binding molecule).Referring to Winnacker, From Genes to Clones, VCH Publishers, N.Y.N.Y. (1987).In fact preferably eukaryotic cell, because this area set up in a large number can heterologous protein secretion (for example, complete binding molecule) suitable host clone comprises Chinese hamster ovary celI system, various Cos clone, HeLa cell, myeloma cell line or the B cell or the hybridoma that transform.Preferably, described cell is not people's cell.The expression vector that is used for these cells can comprise expression control sequenc, such as replication orgin, promotor and enhanser (Queen et al.Inmunol.Rev.89:49 (1986)), and necessary machining information site, such as ribosome bind site, RNA shearing site, poly-adenosine site and transcription termination sequence.Preferred expression regulation sequence is the promotor that derives from immunoglobulin gene, SV40, adenovirus, bovine papilloma virus, cytomegalovirus etc.Referring to Co et al.J.Immunol.148:1149 (1992).

Alternate, the sequence of coding binding molecule can be incorporated transgenosis into, is used to import the genome of transgenic animal, be expressed in subsequently in the milk of transgenic animal (referring to, Deboer et al.US5 for example, 741,957, Rosen, US 5,304,489 and Meade et al.US 5,849,992).The encoding sequence that suitable transgenosis comprises light chain and/or heavy chain operably connects together with promotor and enhanser from Mammals body of gland specific gene (such as casein or beta-lactoglobulin).

Be preferred for expressing the mammalian host cell of inventing described reorganization binding molecule and comprise that Chinese hamster ovary (Chinese hamster ovary celI) (comprises Urlaub and Chasin, (1980) the described dhfr-CHO cell of Proc.Natl.Acad.Sci.USA77:4216-4220, use with the DHFR selective marker, for example R.J.Kaufman and P.A.Shatp (1982) Mol.Biol.159:601-621 is described), NS0 myeloma cell, COS cell and SP2 cell.When the recombinant expression vector of coding binding molecule gene imports mammalian host cell, make the described binding molecule of host cell expression by host cell being cultivated the enough time, or more preferably, binding molecule is secreted in the substratum of host cell growth, produce binding molecule.Utilize conventional method of purifying protein can from substratum, reclaim binding molecule.

(for example, binding molecule heavy chain and light chain encoding sequence and expression regulation sequence) carrier can be transferred in the host cell by known method, and concrete grammar depends on the type of host cell to contain the herbicide-tolerant polynucleotide sequence.For example, the calcium chloride transfection is generally used for prokaryotic cell prokaryocyte, and calcium phosphate processing, electroporation, liposome transfection, gene gun technology or can be used for other host cell based on the transfection of virus (can be totally referring to Sambrook et al.Molecular Cloning:A Laboratory Manual (ColdSpring Harbor Press, 2nd ed.1989) (all incorporating this paper into) in the reference mode.Other method that is used for transformed mammalian cell comprises use, protoplastis fusion, liposome, electroporation and the microinjection (totally the same referring to Sambrook et al.) of polybrene.In order to produce transgenic animal, can with the transgenosis microinjection in the ovocyte of fertilization, perhaps be incorporated in the embryonic stem cell genome, the nucleus of these cells is transferred in the non-nucleus egg mother cell.

When heavy chain and light chain are cloned in the expression vector separately, with the carrier cotransfection so that express and assemble complete immunoglobulin (Ig).In case after expressing, whole binding molecule, its dimer, one light chain and heavy chain or other immunoglobulin (Ig) form of the present invention can be by this area standard method purifying, described method comprises that ammonium sulfate precipitation, affinity column, column chromatography, HPLC purifying, gel electrophoresis etc. (can be totally referring to Scopes, Protein Purification (Springer-Verlag, N.Y. (1982)).When being used for medicinal use, preferably at least about the pure basically binding molecule of 90-95% homogeneous, the binding molecule of 98-99% or above homogeneous most preferably.

Host cell also can be used to produce the part of complete binding molecule, such as Fab fragment or scFv molecule.The various variations that should be appreciated that above method also within the scope of the invention.For example, may wish with the light chain of code book invention binding molecule or the DNA transfection host cell of heavy chain (not being both whiles).Can also utilize recombinant DNA technology remove encoding those in conjunction with the optional light chain of GITR or heavy chain or both DNA partly or entirely.The molecule of being expressed by the dna molecular that blocks like this is also contained in the binding molecule scope of the present invention.In addition, can utilize conventional Chemical Crosslinking Methods, by binding molecule of the present invention and second binding molecule is crosslinked, make difunctional binding molecule, making one of them heavy chain and a light chain is binding molecule of the present invention, and another heavy chain and light chain are the antigen that is specific to except GITR.

As above-mentioned, the present invention relates to nucleic acid, carrier and the host cell composition that can be used to recombinant expressed binding molecule of the present invention on the other hand.The nucleotide sequence of coding 6C8 variable region of light chain is seen Figure 18 and SEQID NO:10.The CDR1 structural domain of VL is contained the Nucleotide 130-162 (SEQ IDNO:14) of SEQ ID NO:10, the CDR2 structural domain is contained the Nucleotide 208-228 (SEQ IDNO:15) among the SEQ ID NO:10, and the CDR3 structural domain is contained the Nucleotide 325-351 (SEQ IDNO:16) of SEQ ID NO:10.The nucleotide sequence of coding 6C8 variable region of heavy chain is seen Figure 18 and SEQ ID NO:9.The CDR1 structural domain of VH is contained the Nucleotide 133-168 (SEQ ID NO:11) of SEQ ID NO:9, the CDR2 structural domain is contained the Nucleotide 211-258 (SEQ ID NO:12) among the SEQ ID NO:9, and the CDR3 structural domain is contained the Nucleotide 355-381 (SEQ ID NO:13) among the SEQ ID NO:9.In one embodiment, the nucleotide sequence of coding VH CDR2 comprises SEQ ID NO:12.In another embodiment, the nucleotide sequence of coding VH CDR2 comprises SEQ ID NO:65 (CACATTTGGTGGGATGATGATAAGTACTAT CAACCATCCCTGAAGAGC).It will be understood by those skilled in the art that the nucleotide sequence that utilizes genetic code and conventional Protocols in Molecular Biology can obtain encoding the binding molecule relevant by the nucleotide sequence of coding 6C8 VL and VH with 6C8.

In one embodiment, the invention provides the isolated nucleic acid molecule of coded polypeptide sequence, described peptide sequence comprises 6C8 CDR, for example comprises the aminoacid sequence that is selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8.

In another embodiment, the invention provides the isolated nucleic acid molecule of coding binding molecule variable region of light chain, described variable region of light chain comprises the aminoacid sequence of SEQ ID NO:2, but those skilled in the art understand, the aminoacid sequence of SEQ IDNO:2 because the degeneracy of genetic code, other nucleic acid molecule also can be encoded.Described nucleic acid molecule can be the binding molecule constant region of light chain of only encoding VL or also can encode and being connected with the VL manipulative capability.In one embodiment, this nucleic acid molecule is in recombinant expression vector.

In another embodiment, the invention provides the isolated nucleic acid molecule of coding binding molecule variable region of heavy chain, described variable region of heavy chain comprises the aminoacid sequence of SEQ ID NO:1, but those skilled in the art understand, the aminoacid sequence of SEQ IDNO:1 because the degeneracy of genetic code, other nucleic acid molecule also can be encoded.In another embodiment, the invention provides the isolated nucleic acid molecule of coding binding molecule variable region of heavy chain, described variable region comprises the aminoacid sequence of SEQ ID NO:66, but those skilled in the art understand, the aminoacid sequence of SEQ ID NO:66 because the degeneracy of genetic code, other nucleic acid molecule also can be encoded.Described nucleic acid molecule can be the CH of only encoding VH or also can encode and being connected with the VH manipulative capability.In one embodiment, this nucleic acid molecule is in recombinant expression vector.

The present invention also provides the recombinant expression vector of coding binding molecule heavy chain and/or binding molecule light chain.For example, in one embodiment, recombinant expression vector coding provided by the invention:

A) binding molecule light chain, the variable region that this light chain contains comprises the aminoacid sequence of SEQ ID NO:2; With

B) binding molecule heavy chain, the variable region that this heavy chain contains comprises the aminoacid sequence of SEQ ID NO:1.

In another embodiment, recombinant expression vector coding provided by the invention:

A) binding molecule light chain, the variable region that this light chain contains comprises the aminoacid sequence of SEQ ID NO:2; With

B) binding molecule heavy chain, the variable region that this heavy chain contains comprises the aminoacid sequence of SEQ ID NO:66.

The present invention also provides the host cell that has imported one or more recombinant expression vector of the present invention.Preferably, described host cell is a mammalian host cell.

Further again, the invention provides synthetic method of inventing described reorganization binding molecule, be about to invent described host cell and cultivate in suitable medium until the described reorganization binding molecule of synthetic invention.This method can also also comprise and separate the reorganization binding molecule from substratum.

V. the purposes of binding molecule of the present invention

Consider their abilities in conjunction with GITR, binding molecule of the present invention can be used for the routine immunization detection method, survey GITR (for example, in resembling the such biological sample of serum or blood plasma) such as enzyme-linked immunosorbent assay (ELISA), radioimmunity detection (RIA) or histogenic immunity histological chemistry detection method.The invention provides the method for the hGITR in the detection of biological sample, this method comprises biological sample is contacted with binding molecule of the present invention, combine binding molecule or the unconjugated binding molecule of hGITR by detection, thus the hGITR in the detection of biological sample.Described method can be external or body in carry out.Detectable substance on the direct or indirect mark of binding molecule is detected combination or unconjugated binding molecule with assistance.Suitable detectable substance comprises various enzymes, prothetic group, fluorescent material, luminophore and radioactive substance.The example of suitable enzyme comprises horseradish peroxidase, alkaline phospholipase, beta-galactosidase enzymes or acetylcholinesterase; The example of suitable prothetic group comprises Streptavidin/vitamin H and avidin/biotin; The example of suitable fluorescent substance comprises Umbelliferone (umbelliferone), fluorescein, fluorescein isothiocyanate, rhodamine, dichloro three azine amine (dichlorotriazinylamine) fluoresceins, dansyl chloride (dansyl chloride) or phycoerythrin; An example of luminophore is luminol,3-aminophthalic acid cyclic hydrazide (luminal); And the example of suitable radioactive substance comprises ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Serve as a mark the substituting of binding molecule, the GITR standard and the unlabelled anti-hGITR binding molecule of detectable substance that can utilize mark comes hGITR in the detection of biological sample liquid by the competitive immunoassay method.In this analytical method, the GITR standard and the anti-hGITR binding molecule of biological sample, mark combined, determine to be attached to the amount of the mark GITR standard substance on the unmarked binding molecule.The amount of hGITR in the biological sample is inversely proportional to the amount that is attached to the mark GITR standard substance on the anti-hGITR binding molecule.

Anti-GITR binding molecule of the present invention can also be used for detecting the GITR of the sample of the species except that the people, the GITR of primates (for example, chimpanzee (chimpanzee), baboon (baboon), aye-aye (marmoset), cynomolgus monkey (cynomolgus) and macaque (rhesus)) particularly.

In another embodiment, the invention provides a kind of inhibiting method of eliminating regulatory T cells to T effector cell.The elimination regulatory T cells can have under the situation of T effector cell by for example measuring T effector cell's restraining effect, and the ability that described binding molecule strengthens T cytological effect subfunction detects.For example by measuring ³The H-thymidine mixes or FACS comes the generation (for example, IL-2 output) of the analysis of cells factor or cell proliferation (for example, the propagation of auxiliary T sexual cell).For example having under the situation of regulatory T cells, T effector cell's reaction or active low, even but after adding the GITR binding molecule regulatory T cells being arranged, they also can raise, and promptly the GITR binding molecule has been offset the restraining effect of regulatory T cells to T effector cell.

Binding molecule of the present invention can also be used for weakening the degraded of I-κ B at cell.For example, can handle cell, carry out the Western trace with anti-GITR binding molecule, and quantitative I-κ B, thereby the weakened condition that I-κ B degrades in the cell detected.

Numerous disease or pathological condition can be benefited from active enhancing of T effector cell and/or the active downward modulation of regulatory T cells, for example by offsetting the restraining effect of regulatory T cells to T effector cell.For example, immune effector cell often can not react with cancer cell effectively.Accordingly, when needs enhanced effector T cell or antibody response, method of the present invention can be used to dispose the experimenter who suffers from this class disorder.In one embodiment, these class methods comprise and give the experimenter with binding molecule of the present invention, thereby regulatory T cells is eliminated T effector cell's restraining effect, have therefore strengthened immune response.Preferably, described experimenter is people's study subject.Alternate, described study subject can be the Mammalss that can express with the GITR of binding molecule generation cross reaction of the present invention.Further, described experimenter can be the Mammals that has imported GITR (for example, by giving GITR or passing through to express the GITR transgenosis).Can be in order to treat or prevent purpose, with binding molecule administration of human study subject of the present invention.For example, described experimenter may be suffered from disease or disorder by diagnosis, perhaps easily suffers from this disease or to this disease susceptible.In addition, binding molecule of the present invention can be expressed the non-human mammal (for example, primate) with the GITR molecule of described binding molecule cross reaction, to realize animal doctor's purpose or as human disease's animal model.With regard to a back purpose, what such animal model can be used for estimating binding molecule of the present invention treats and/or prevents effect (for example, assessment dosage and/or drug delivery regimen).

The exemplary use of binding molecule of the present invention has more detailed discussion below:

Immunostimulatory compositions

As what describe among the embodiment that encloses hereinafter, binding molecule of the present invention can for example make up with antigen, improves the immune response of experimenter to target antigen (for example proteantigen) as immunostimulatory compositions (or vaccine).In other words, binding molecule of the present invention can be used as the adjuvant of enhancing immunity reaction.For example, in order (for example to stimulate to the antibody of target antigen or cell immune response, for the purpose of inoculating), can with described antigen with binding molecule administration of the present invention (for example, administration in identical or the composition that separates simultaneously) thus the enhanced immune response takes place.Target antigen and binding molecule can be mixed with same pharmaceutical composition, the composition that perhaps separates.In one embodiment, target antigen and binding molecule are given the experimenter simultaneously.Alternate in some cases, may wish to give earlier antigen, gives binding molecule then, perhaps opposite (for example, may give antigen separately earlier and induce reaction, be useful with binding molecule separately or with antigen then).In preferred embodiments, GITR binding molecule of the present invention promptly gives when giving antigen for the first time when carrying out sensitization with antigen.For example, the-3 ,-2 ,-1,0 ,+1 ,+2 ,+3 days.Particularly preferred a day of inventing described GITR binding molecule is to give antigen preceding one day.

Target antigen is, for example such antigen, and it can provide the protection of the attack of the contagium of originating at this antigen to the experimenter, perhaps can influence tumor growth and transfer in the mode that is of value to the experimenter.Therefore the exemplary example of target antigen comprises the antigen of those sources that come from infection, cancer cell etc., and can prevent or treat the disease that is caused by this source at this antigenic immune response this moment.This class antigen includes, but not limited to virus, bacterium, fungi or parasite protein matter, glycoprotein, lipoprotein, glycolipid or the like.Target antigen comprises that also those bring the antigen of benefit can for the dangerous experimenter who catches or suffered from tumour by diagnosis, may comprise for example special tumor associated antigen that express or that expression level improves in tumour cell of tumour cell.Described experimenter is Mammals preferably, most preferably is the people.

Be used for this paper, term " pathogenic bacteria " or " pathogenic agent " comprise and can infect or the microorganism of parasitic normal host (for example, animal (such as Mammals, preferred primates, for example people)).Be used for this paper, this term also comprises the opportunistic pathogenic agent, for example can infect or the microorganism of parasitic aberrant host, and for example because the host that treatment plan causes normal microflora to be upset, or the host of immune deficiency.Be used for this paper, this term comprises that also it is replicated among the experimenter is unwelcome microorganism, perhaps the toxicity molecule of these microorganisms (for example, toxin).

The non-limitative example of virus antigen includes but not limited to, the Gag protein of nucleoprotein of influenza virus (NP) and HIV.Other heterologous antigen includes, but are not limited to HIV Env albumen or its integral part: gp120 and gp41, HIV Nef albumen and HIV Pol albumen, reversed transcriptive enzyme and proteolytic enzyme.In addition, can also use other virus antigen, EBOV NP or glycoprotein (GP) (the Saliva Orthana district GP disappearance (Yang Z-Y, et al. (2000) Nat Med 6:886-9,2000) of total length or this molecule) such as Ebola virus (EBOV) antigen; Smallpox antigen; Hepatitis A, hepatitis B or hepatitis C virus; ERC group virus is such as 2 types and 14 types; Hsv; 2 types or 3 type polioviruses; Foot and mouth disease virus (FMDV); Rabies virus; Rotavirus; Influenza virus; Coxsackie virus; Human papillomavirus people (HPV), for example 16 type papilloma viruss, its E7 albumen, and the fragment that contains E7 albumen or its epi-position; And simian immunodeficiency virus (SIV).Target antigen does not need to be restricted to the antigen of viral source.Parasite antigen, in for example plasmodium antigens is also included within, and fungal antigen, bacterial antigens and tumour antigen also can be used for composition disclosed herein and method.The non-limitative example of bacterial antigens comprises: bordetella pertussis (Bordetella pertussis) (for example, P69 albumen and filamentous hemagglutinin (FHA) antigen), vibrio cholerae, Bacillus anthracis (Bacillus anhracis) and bacillus coli antigen, such as intestinal bacteria heat-labile toxin B subunit (LT-B), intestinal bacteria K88 antigen and enterotoxigenic escherichia coli antigen.Other antigenic example comprises Schistosoma mansoni (Schistosoma mansoni) P28 glutathione S-transferase antigen (P28 antigen) and fluke (f1uke), mycoplasma (mycoplasma), nematode (roundworm), tapeworm (tapeworm), chlamydia trachomatis (Chlamydia trachomatis) and malarial parasite (malaria parasite), the parasite of plasmodium (plasmodium) or burnt worm (babesia) for example, the for example antigen of plasmodium falciparum (Plasmodium falciparum), and the peptide that determines above-mentioned antigenic immunogenic epi-position.

Can be by giving infection that vaccine of the present invention treats or prevent, disease or disorderly infection, disease or the disorder that comprises that any meeting causes host immune response and prevents.Can be by giving the disease that immunostimulatory compositions of the present invention is treated or prevented, disorderly or infection comprises, but be not limited to, any by fungi, parasite, viral or bacterial or relevant infection with them, disease or disorder, cause or the disease relevant by following various pathogenic agent with them, disorder or infection, comprise and be used for that bio-terrorism attacks, the listeria spp disease, Ebola virus, SARS, smallpox, hepatitis A, hepatitis B, third liver or viral hepatitis type E, ERC group virus, HIV (for example, HIV-1 and HIV-2), and AIDS, bleb, poliomyelitis, foot and mouth disease, rabies, rotavirus, influenza, Coxsackie virus, human papillomavirus, SIV, plasmodium, cancer, tumour for example, the herpes virus hominis, cytomegalovirus (particularly human cytomegalic inclusion disease virus), Epstein-Barr virus, varicella zoster virus, hepatitis virus is (such as hepatitis B virus, hepatitis A virus (HAV), hepatitis C virus), paramyxovirus: respiratory syncytial virus, parainfluenza virus, Measles virus, mumps virus, human papillomavirus (HPV6 for example, 11,16,18 or the like), flavivirus (yellow fever virus (Yellow Fever Virus) for example, dengue virus (Dengue Virus), tick-brone encephalitis virus (Tick-borne encephalitis virus), japanese encephalitis virus (Japanese Encephalitis Virus)) or influenza virus, influenza A virus (for example, hemagglutinin (H) and neuraminidase (N) hypotype) for example, Influenza B virus and influenza virus C; And, comprise relevant disease or disorder that Gram-positive and gram negative bacterium cause by bacterium living beings.Example includes, but are not limited to Neisseria gonorrhoeae, comprises gonococcus (N.gonorrhea) and Neisseria meningitidis (N.meningitidis); Suis comprises streptococcus pneumoniae (S.pneumoniae), micrococcus scarlatinae (S.pyogenes), streptococcus agalactiae (S.agalactiae), streptococcus mutans (S.mutans); Hemophilic bacterium species (Haemophilus spp) comprise second type influenza virus hemophilic bacterium (H.influenzae type B), non-somatotype Haemophilus influenzae (non typeable H.influenzae), Roger Ducret hemophilic bacterium (H.ducreyi); Some kinds of Moraxella comprise moraxelle catarrhalis (M catarrhalis), are called branhamella catarrhalis (Branhamella catarrhalis) again; Bordetella comprises bordetella pertussis, parapertussis bacillus (B.parapertussis) and bacillus bronchisepticus (B.bronchiseptica); Mycobacterium comprises mycobacterium tuberculosis (M.tuberculosis), Mycobacterium bovis (M.bovis), leprosy bacillus (M.leprae), mycobacterium avium (M.avium), mycobacterium paratuberculosis (M.paratuberculosis), M. smegmatics (M.smegmatis); Legionella comprises legionella pneumophilia (L.pneumophila); Escherichia comprises enterotoxigenic E.Coli (enterotoxicE.coli), enterohemorrhagic Escherichia coli (enterohemorragic E.coli), enteropathogenic Escherichia coli (enteropathogenic E.coli); Vibrio (Vibrio) bacterium comprises vibrio cholerae (V.cholera); Shigellae species (Shigella spp) comprise Song Shi shigella (S.sonnei), shigella dysenteriae (S.dysenteriae), shigella flexneri (S.flexnerii); Yersinia's genus comprises Yersinia enterocolitica (Y.enterocolitica), plague bacillus (Y. pestis), yersinia pseudotuberculosis (Y.pseudotuberculosis); Crooked fungus kind (Campylobacter spp) comprises the crooked bacterium (C.coli) of campylobacter jejuni (C.jejuni) and large intestine; Salmonella comprises Salmonella typhi (S.typhi), bacillus paratyphosus (S.paratyphi), Salmonella choleraesuls (S.choleraesuis), Salmonella enteritidis (S.enteritidis); Listeria comprises monocyte hyperplasia listeria spp (L.monocytogenes); Helicobacterium comprises helicobacter pylori (H.pylori); Rhodopseudomonas comprises Pseudomonas aeruginosa (P.aeruginosa); Staphylococcus comprises streptococcus aureus (S.aureus), staphylococcus epidermidis (S.epidermidis); Enterococcus spp comprises enterococcus faecalis (E.faecalis), faecium (E.faecium); Fusobacterium comprises clostridium tetani (C.tetani), Clostridium botulinum (C. botulinum), clostridium difficile (C.difficile); Bacillus comprises Bacillus anthracis; Coryneform bacteria comprises diphtheria corynebacterium (C.diphtheriae); Borrelia comprises B. burgdorferi (B.burgdorferi), loud, high-pitched sound borrelia burgdorferi (B.garinii), Ah's borrelia burgdorferi (B.afzelii), B.andersonii, He Shi tick burgdorferi (B.hermsii); The ehrlichiosis body belongs to, and comprises horse ehrlichiosis body (E.equi) and people's ehrlichiosis body (Human Granulocytic Ehrlichiosis); Dermacentroxenus comprises Rickettsia rickettsii (R.rickettsii); Chlamydiaceae comprises chlamydia trachomatis (C.trachomatis), Chlamydia pneumoniae (C.pneumoniae), chlamydia psittaci (C.psittac); Leptospira comprises leptospira interrogans (L.interrogans); Treponema comprises Treponoma palladium (T.pallidum), treponema denticola (T.enticola), swine dysentery treponema (T.hyodysenteriae).Preferred bacterium comprises, but be not limited to listeria spp, mycobacterium (for example mycobacterium tuberculosis), anthrax-bacilus, Salmonellas and monocyte hyperplasia listeria spp, bordetella pertussis, vibrio cholerae, fluke, chlamydozoan, nematode, tapeworm, chlamydia trachomatis and malarial parasite.

Be used for this paper, term " bacterial infection " comprises by various infectation of bacteria.In another embodiment, can remove the T cell from the patient, contact at external and anti-GITR binding molecule, optional have activation signals (for example, antigen adds APC or polyclonal antibody) also to import again in patient's body simultaneously.

Regulatory T cells is by suppressing the immune response to autoimmune disorder and cancer, plays an important role keeping self-immunotolerance invention.Accordingly, in one embodiment, offset regulatory T cells and will help strengthening immune response in the cancer T effector cell's restraining effect.Therefore, binding molecule of the present invention can be used to dispose malignant tumour, suppresses tumor growth or transfer.Described binding molecule can be systematically or is administered to tumor sites partly.

In one embodiment, regulate the GITR function and may be used for the induced tumor immunity, promptly be used to dispose the experimenter who suffers from ND or cancer.In one embodiment, binding molecule of the present invention has reduced the tumour size, suppressed tumor growth and/or has prolonged the survival time of being with the knurl experimenter.The GITR binding molecule can be had the patient of tumour cell (for example, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, cancer (carcinoma)) so that overcome experimenter's tumour-specific tolerance.

Term " tumor associated antigen " is used for this paper and represents that host living beings influences the antigen of tumor growth or transfer.Tumor associated antigen can be the antigen of tumor cells expression, or the antigen of non-tumor cell expression, but in the latter event, can promote the growth or the transfer of tumour cell when it is expressed.The type of tumour antigen and tumor associated antigen comprises any known or unknown up to now tumour antigen, includes but not limited to the bcr/abl antigen in the leukemia; HPVE6 of the oncovirus relevant and E7 antigen with cervical cancer; MAGE1 in the melanoma or relevant with melanoma and MZ2-E antigen; And MVC-1 in the mammary cancer or relevant with mammary cancer and HER-2 antigen.

Be used for this paper, the characteristics of term " ND " are malignant growth or are in optimum propagation and proliferative cell is the morbid state of characteristics.The general medicine implication of term " tumorigenesis " is meant and causes " the new cell growth " that normal growth control is lost reaction, for example tumorigenesis cell growth.

Be used for this paper, term " hyperplasia ", " outgrowth ", " virulent " and " neoplastic " can exchange use, are meant that being in fast breeding or tumorigenesis is those cells of the error state (ERST) or the situation of feature.These term intentions comprise all types of hyperplasia growths, hyperplasia, canceration growth or oncogenic process, branch problem or malignant conversioning cell, tissue or organ, and no matter the histopathology type or the stage of infecting.But, being used for this paper, term tumorigenesis and hyperplasia can exchange use, resemble their name prompting, all are the cells of general reference experience abnormal cell growth speed.Tumorigenesis and hyperplasia comprise " tumour ", can be benign, precancerous lesion or virulent.

Term " tumorigenesis ", " hyperplasia " and " tumour " all are called as " cancer " usually, and it is more than 100 a kind of general name that is grown to the disease of feature with uncontrolled, unusual cell.The example of cancer includes but not limited to: mammary cancer; Colorectal carcinoma; Nonsmall-cell lung cancer, head and neck cancer; Large bowel cancer; Lung cancer; Prostate cancer; Ovarian cancer; Kidney; Melanoma; And gastrointestinal cancer (for example, carcinoma of the pancreas and cancer of the stomach); And osteosarcoma.

In one embodiment, described cancer is selected from: the cancer of carcinoma of the pancreas, melanoma, mammary cancer, lung cancer, bronchogenic carcinoma, colorectal carcinoma, prostate cancer, cancer of the stomach, ovarian cancer, Urinary Bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, the esophageal carcinoma, cervical cancer, uterus or carcinoma of endometrium, oral carcinoma or pharynx cancer, liver cancer, kidney, carcinoma of testis, cholangiocarcinoma, small intestine or adnexal carcinoma, salivary-gland carcinoma, thyroid carcinoma, adrenal carcinoma, osteosarcoma, chondrosarcoma and hemopoietic tissue.

Accordingly, the invention still further relates to ND or method for cancer among the treatment experimenter (preferably people or other animal), this method is to give experimenter or animal by the binding molecule of the present invention with significant quantity.Those skilled in the art can be identified for disposing the purpose of ND or cancer by normal experiment, and how many polypeptide of significant quantity is.For example, the treatment significant quantity of binding molecule of the present invention may change according to multiple factor, such as disease stage (for example, the I phase is with respect to the IV phase), age, sex, medical science syndrome (for example, immunosuppressive condition or disease) and experimenter's body weight and binding molecule causes required reaction in the experimenter ability.Can regulate dosage the best reaction that treats and/or prevents is provided.For example, the dosage that can give to separate several times every day, perhaps according to the treatment situation the corresponding minimizing dosage of emergency situation.But in general, effective dose estimates in about 0.05 to the 1 00 milligram scope of per kilogram of body weight every day, more preferably every day about 0.5 to 10 milligram of per kilogram of body weight.

The method of enhancing immunity reaction

Described binding molecule can also be used to improve immunoreactive method.Raise immune response and can take to improve existing immunoreactive form, perhaps cause initial immunoreactive form.For example, improving immune response by adjusting GITR can be useful in the situation of virus infection.Because anti-GITR binding molecule can improve immune response, they need or remove more up hill and dale in the situation of pathogenic agent (for example bacterium and virus) that at those medical use is arranged faster.Accordingly, anti-GITR binding molecule of the present invention can be used for separately or be used in combination to treat with antigen or other immunostimulant to suffer from disease or disorderly experimenter, such as those infectious diseases or the cancer enumerated previously.

Anti-GITR binding molecule can also be used in the various pathogenic agent of prevention in the vaccine.Can be by viral protein and GITR binding molecule being inoculated together the immunizing power (as mentioned above) of inducing anti-disease substance (for example virus).Alternate can use the expression carrier of coding pathogen antigen and GITR binding molecule to inoculate, and for example is used for expressing the nucleic acid of coding viral protein and the vaccinia virus expression vector of the nucleic acid of coding GITR binding molecule by through engineering approaches.The pathogenic agent that vaccine comes in handy comprises, for example hepatitis B, third liver, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, influenza, pulmonary tuberculosis, plasmodium and schistosomicide.

The invention further relates to therapy based on described binding molecule, this therapy comprises and gives animal patient with binding molecule of the present invention, preferably Mammals most preferably is the people, so that one or more in disease, disorder or the situation disposing, detect and/or prevent to mention.Therapeutic composition of the present invention includes but not limited to, binding molecule of the present invention (comprising analogue and the derivative described in the literary composition) and antiidiotype binding molecule described herein.Binding molecule of the present invention can be used for disposing, diagnose, suppress or prevention is relevant with the GITR abnormal activity disease, disorder or situation; include but not limited to; in the disease of describing in the literary composition, disorder or the situation one or more (for example, binding molecule of the present invention can be provided as the pharmaceutically acceptable composition of describing in known in the art or civilian).

Binding molecule of the present invention can be advantageously with other mono-clonal or chimeric binding molecule or lymphokine or hematopoietic cytokine (such as, IL-2 for example, IL-3 and IL-7) unite use, for example can improve with the interactional effector cell's of binding molecule quantity or active those.

Binding molecule of the present invention can be separately or with methods of treatment (for example, radiotherapy, chemotherapy, hormonotherapy, immunotherapy) and antineoplastic agent, the microbiotic of other type, unite use at the therapy (such as the immunotherapy or the chemotherapeutics that can effectively resist viral pathogen or bacterial antigens) and the immunostimulant of pathogenic agent.Can also be with binding molecule of the present invention and antigen combined administration, described antigen is exactly the immunoreactive antigen that wish to improve it, for example from the vaccine of pathogenic agent or antigen (or the virus of attenuation form or bacterium) or from the antigen of tumour described above.In one embodiment, the experimenter who binding molecule of the present invention is suffered from infection with independent or conjoint therapy.In another embodiment, binding molecule of the present invention is by separately or unite the experimenter that chronic viral infection is arranged.Also have in the embodiment, binding molecule of the present invention is by separately or unite the experimenter who suffers from cancer.

Usually, the binding molecule that preferably will derive from the something kind gives the patient of same species.Therefore, in preferred embodiments, human binding molecules, its derivative, analogue or nucleic acid are given people patient and are used for the treatment of or prevent.

VI. pharmaceutical composition

Binding molecule of the present invention can be imported into the pharmaceutical composition that is fit to give the experimenter.Usually, described pharmaceutical composition comprises binding molecule of the present invention and pharmaceutically acceptable carrier.When being used for this paper, saying " pharmaceutically acceptable carrier " comprises the solvent compatible with pharmaceutical composition, dispersion medium, dressing, antibacterial agent and anti-mycotic agent, isotonic agent and absorption delay agent etc.It is well-known in the art that these media and reagent are used for pharmaceutically active substance.Unless the medium of any routine or reagent and active compound are inconsistent, otherwise the present invention relates to their application in composition.The active compound of complementarity also can be incorporated in the composition.

Pharmaceutical composition of the present invention is formulated into the formulation compatible with its expection route of administration.The example of route of administration comprises parenteral, for example intravenously, intracutaneous, subcutaneous, oral cavity (for example sucking), through skin (surface local), through mucous membrane and rectal administration.The solution or the suspension that are used for parenteral, intracutaneous or subcutaneous use can comprise following composition: sterile diluent, such as water for injection, salt brine solution, fixed oil (fixedoil), polyoxyethylene glycol, glycerine, propylene glycol or other synthetic; Antibacterial agent is such as phenylcarbinol or methyl p-hydroxybenzoate (methyl parabens); Antioxidant is such as xitix or sodium bisulfite; Sequestrant is such as ethylenediamine tetraacetic acid (EDTA); Damping fluid such as acetate, Citrate trianion or phosphoric acid salt and the reagent of regulating osmotic pressure such as sodium-chlor or glucose.Can regulate pH with acid or alkali, such as hydrochloric acid or sodium hydroxide.The parenteral with medicament can be enclosed in ampoule, disposable syringe or the multiple dose vials that glass or plastics make.

The pharmaceutical composition that is suitable for injecting purposes comprises aseptic aqueous solution (when water soluble) or suspension and is used for preparing immediately the sterilized powder of aseptic injection with liquid or suspension.The carrier that is fit to intravenous administration comprises physiological saline, bacteriostatic water, Cremophor EL ^TM(BASF, Parsippany, NJ) or phosphate buffered saline buffer (PBS).In all these situations, described composition must be aseptic, and its flowability should reach the level of carrying out the needle tubing injection easily.It must be stable under manufacturing and storage condition, must preserve under the condition that prevents microorganism (such as bacterium and fungi) pollution.Carrier can be to contain for example water, ethanol, polyvalent alcohol (for example, ethylene glycol, propylene glycol and liquid polyethylene glycol etc.) solvent or dispersion medium, and their suitable mixture.By for example using dressing (such as Yelkin TTS), in the situation of suspension, keep required granular size and using tensio-active agent to keep suitable flowability.Can utilize various antibacterial agents and anti-mycotic agent to prevent microbial contamination, for example p-Hydroxybenzoate (parabens), butylene-chlorohydrin (chlorobutanol), phenol (phenol), xitix, Thiomersalate etc.In many situations, preferably in composition, comprise isotonic agent, for example sugar (sugar), polyvalent alcohol (such as N.F,USP MANNITOL, sorbyl alcohol) and sodium-chlor.The absorption that postpones Injectable composition can be by comprising the reagent that can postpone absorption in composition, for example aluminum monostearate and gelatin are realized.

Aseptic injectable solution can be by introducing requirement in suitable solvent active compound and in the above-named composition one or more make through sterile filtration.Usually, suspension is to introduce active compound to prepare in the sterile carrier that contains basic dispersion medium and required above-mentioned other composition.Situation at the sterilized powder that is used for preparing aseptic parenteral solution, preferred manufacturing procedure is vacuum-drying and lyophilize, thereby produce the powder of activeconstituents and any other required composition, described other required composition shifts to an earlier date the solution of sterile filtration from this composition.

Oral compositions generally comprises inert diluent or edible carrier.Can be sealed in them in the gelatine capsule or be pressed into tablet.For per os treatment administration, active compound and vehicle one can be used from tablet, lozenge (troche) or capsular form.Can also prepare oral compositions as collutory with liquid vehicle, wherein be formulated in the compound dosage forms for oral administration in the liquid carrier, spue after gargling or swallow.Pharmacy consistency tackiness agent and/or auxiliary component can be used as the part of composition.Tablet, pill, capsule, lozenge etc. can contain any following composition, or the compound of similar characteristics: tackiness agent, such as Microcrystalline Cellulose, tragakanta (gum tragacanth) or gelatin; Vehicle is such as starch or lactose; Disintegrating agent is such as Lalgine, Primogel or W-Gum; Lubricant is such as Magnesium Stearate or Sterotes; Glidant is such as colloid silica; Sweeting agent is such as sucrose or asccharin; Perhaps odorant is such as Mentha arvensis L. syn.M.haplocalyxBrig, methyl salicylate or the agent (orange flavoring) of orange flavor.

For by inhalation, from pressurizing vessel or contain the decollator of suitable propelling agent (for example such gas of carbonic acid gas), perhaps send described compound with the aerosol spray form in the atomizer.

Be administered systemically and pass through through mucous membrane or through the skin approach.Be used for when mucous membrane or percutaneous dosing, in preparation, use the permeate agent that is suitable for barrier to be penetrated.This class permeate agent is well known in the art, when mucosal, comprises for example tensio-active agent, cholate and fusidic acid (fusidic acid) derivative.Mucosal can be realized by using nasal spray or suppository.For percutaneous dosing, active compound can be mixed with ointment well known in the art (ointment), ointment (salve), gel or creme.

Form (for example, with conventional suppository substrate such as Oleum Cocois and other glyceryl ester) or retention enema that described composition can also be mixed with suppository come per rectum to send.

In one embodiment, the carrier that binding molecule of the present invention is not removed in the body fast with the protection compound prepares, and described carrier comprises implant and micro-capsule delivery system such as the controlled release preparaton.Can use biodegradable, biocompatible polymer, such as ethylene vinyl acetate copolymer, polyanhydride, polyglycolic acid, collagen protein, poe and poly(lactic acid).The method for preparing these preparations it will be apparent to those skilled in the art that.These materials also can be from Alza Corporation and NovaPharmaceuticals, and Inc buys.Liposome suspension also can be used as pharmaceutically acceptable carrier.Can resemble United States Patent (USP) 4,522 according to method known to those skilled in the art, that describes in 811 prepares these.

Especially advantageously oral the or parenteral composition that is mixed with unit dosage form is to make things convenient for administration and to reach the unity of dosage.Unit dosage form is used for this paper and represents physically independent unit, is applicable to as the single dosage of waiting to dispose the experimenter; Each unit contains with the combination of required pharmaceutical carrier, has pre-determined the active compound of consumption, can produce required result of treatment through calculating.The characteristic of dosage unit form of the present invention is directly to be decided by the peculiar property of described active compound and the concrete result of treatment that will reach, and this active compound of combination is used for the individual limitation of disposing in the prior art.

The toxicity of these compounds and treatment are renderd a service and can be determined in cell culture or laboratory animal by the pharmacy program of standard, for example determine LD50 (to colony's 50% lethal dosage) and ED50 (to 50% medicable dosage of colony).Dosage rate between toxicity and the result of treatment is a therapeutic index, can be expressed as ratio LD50/ED50.Preferably show the compound of higher therapeutic index.Though can use the compound that shows toxic side effects, the design of being careful can decide this compound the delivery system of target to affected tissue, so that reduce as far as possible to may the destroying of uninfluenced cell, thus the minimizing side effect.

The data that obtained by cell culture and experimentation on animals can be used to prepare the dosage of human.The dosage of these compounds preferably is in the scope of such circulation composition, promptly comprises ED50 and has seldom or do not have toxicity.Can in this scope, do change according to the dosage form and the used route of administration that adopt to dosage.For any compound that is used to invent described method, treat effective dosage and can at first determine by cell culture assays.Can in animal model, determine dosage, comprise the IC50 (promptly reaching concentration) that determines by cell cultures the testing compound of maximum half that suppresses of symptom to reach the circulating plasma concentration range.Can determine to be used for people's dosage with this category information more accurately.Concentration in the blood plasma can be measured by for example efficient liquid chromatography.

Described pharmaceutical composition can be included in container, packing or the decollator with medication instruction.

VII. administration binding molecule of the present invention

Binding molecule of the present invention is contacted with the bio-capacitivity form in external or body with cell from the experimenter." bio-capacitivity form " means the form of the reagent that will give, and wherein any toxic effect has all been offset by the result of treatment of described binding molecule.

In one embodiment, described composition is given the experimenter.The pharmaceutical composition of the present invention that gives the pharmaceutical activity amount is meant, reaches required result's effective level and each dosage within a certain period of time.For example, the pharmaceutical activity amount of binding molecule may change along with some factors, and such as disease stage, age, sex and whose body weight, and this binding molecule causes the ability of anticipation reaction in individuality.Can regulate dosage maximum medical treatment reaction is provided.For example, the dosage that can give to separate several times every day is perhaps according to the promptly corresponding minimizing dosage whether of treatment situation.

Pharmaceutical composition of the present invention can comprise the binding molecule of the present invention of " treatment significant quantity " or " prevention significant quantity "." treatment significant quantity " is meant effective level and the each dosage that reaches the goal treatment result in required time.The treatment significant quantity of binding molecule may change along with some factors, and such as disease stage, age, sex and whose body weight, and this binding molecule causes the ability of anticipation reaction in individuality.The treatment significant quantity also is that any toxicity or harmful effect of wherein binding molecule all treated the transcendental consumption of beneficial effect by it." prevention significant quantity " is meant effective level and the each dosage that reaches target prevention result in required time.Usually because preventive dose be premorbid or disease be used for the experimenter in early days, the prevention significant quantity can be lower than the treatment significant quantity.

Can regulate dosage and realize best anticipation reaction (for example, treatment or prophylactic response).For example, can give single heavy dose, give several dosage that separate in for some time or promptly whether correspondingly reduce or increase dosage according to the treatment situation.Especially advantageously the parenteral composition that is mixed with unit dosage form is to make things convenient for administration and to reach the unity of dosage.Unit dosage form is used for this paper and represents physically independent unit, is applicable to as the single dosage of waiting to dispose mammalian subject; Each unit contains and required pharmaceutical carrier active compound combination, predetermined volume, can produce required result of treatment through calculating.The characteristic of dosage unit form of the present invention directly is decided by peculiar property of (a) described active compound and the concrete result of treatment that will reach, and (b) makes up the limitation that this active compound is used for the individual susceptibility of disposing in the prior art.

The treatment of binding molecule of the present invention or the prevention significant quantity exemplary, non-limiting scope for example is, about 0.1-25mg/kg, about 1.0-10mg/kg, about 0.5-2.5mg/kg, about 5-25mg/kg, about 1-400mg/kg.Need to prove that dose value may alleviate the type of situation and severity along with desire and change.To further understand; for any concrete experimenter; should be according to individuality required and give or concrete dosage is adjusted in the professional judgement of supervising the people who gives described composition at any time; dosage range given here only is an example, the scope of claimed without limits composition or the intention of use.In addition, the non-limiting scope of the treatment of binding molecule of the present invention or prevention significant quantity is about 0.0001 to 100mg/kg, with about 0.01 to 5mg/kg (for example, 0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg etc.) experimenter's body weight.For example, dosage can be 1mg/kg body weight or 10mg/kg body weight or in the scope of 1-10mg/kg, preferably 1mg/kg at least.Be in above-mentioned scope intermediary dosage also within the scope of the invention.

The table At All Other Times of such dosage every day, every other day, weekly or rule of thumb decision can be come administration.Exemplary disposal need for example be carried out administration with a plurality of dosage at least six months in for some time.Other exemplary disposal method stipulates that per two weeks or every month are once or per 3 to 6 months single administrations.Exemplary dosage comprises and gives 1-10mg/kg or 15mg/kg continuous every day, gives 30mg/kg or give 60mg/kg weekly every other day.

Binding molecule of the present invention can repeatedly give.Interval between single dosage for example can be, every day, weekly, every month or annual.By measuring the blood levels of binding molecule in patient's body, also can determine it is erratic at interval.

Optional with binding molecule of the present invention and other disorder or situation effective agents Combined Preparation to required disposal (for example, prevention or treatment).Preferred other reagent is the prior art approval, and routine gives concrete those disorderly reagent.

Can give binding molecule in mode easily, such as injection (subcutaneous, intravenously etc.), oral, suction, applied dermally or colon administration.According to route of administration, active compound can be coated in certain material so that protect this compound not to be subjected to enzyme, acid and other may make the effect of the natural situation that compound is inactivated.For example, when approach gives reagent outside by parenteral, may wish the pack quilt, perhaps with the material administration together that can prevent that it is inactivated.

Binding molecule of the present invention can come administration by several different methods known in the art, although use for many therapeutic, preferred route of administration/pattern is intravenous injection or instillation.It will be understood by those skilled in the art that route of administration and/or pattern can change according to expected results.In some embodiments, active compound and the carrier that can make it avoid being fast released can be prepared into together such as controlled release preparation, comprise implant, through skin patch and microencapsulation delivery system.Can use biodegradable, biocompatible polymer, such as ethylene vinyl acetate copolymer, polyanhydride, polyglycolic acid, collagen protein, poe and poly(lactic acid).The many methods that prepare this class preparation be subjected to patent protection or those skilled in the art generally all know.Referring to for example, Sustained and ControlledRelease Drug Delivery Systems, J.R.Robinson, ed.Marcel Dekker, Inc.NewYork, 1978.

In some embodiments, binding molecule of the present invention can be that for example maybe can to assimilate edible carrier with inert diluent oral.Compound (and other composition, can also be sealed in the hard or soft gelatine capsule if necessary), be squeezed into tablet, perhaps directly mix in experimenter's the diet.Be used for the administration of per os therapeutic, compound and excipient composition can be used with decomposable tablet, lozenge, plaster, capsule, elixir, suspension, syrup, instant forms such as (wafer).With compound of the present invention during, have and necessary described compound bag is prevented to be inactivated by last certain material or with certain material with the outer administration of parenteral.

Can or in suitable carriers (such as liposome), give together binding molecule and enzyme inhibitors.Pharmaceutically acceptable diluent comprises salt solution and water damping fluid.Can use broadest adjuvant, comprise that any immune-stimulating compound is such as Interferon, rabbit.Here Yu Qi adjuvant comprises resorcin (resorcinol), nonionogenic tenside, such as polyoxyethylene oleyl ether (polyoxyethylene oleyl ether) and n-hexadecyl polyvinyl ether (n-hexadecyl polyethylene ether).Enzyme inhibitors comprise trypsin inhibitor, diisopropylfluorophosphate (diisopropylfluorophosphate, DEEP) and Trypsin inhibitor,Trasylol (trasylol).Liposome comprises water-in-oil-in-water emulsion and conventional liposome (Sterna et al. (1984) J.Neuroimmunol.7:27).

Active compound can also give through parenteral or intraperitoneal.Dispersion can also prepare in glycerine, lipid polyoxyethylene glycol and their mixture and oil.Under common preservation and working conditions, these goods can contain sanitas so that prevent microbial growth.

Have suitable protection, binding molecule or can assimilate edibility carrier per os like that to give when the active ingredient image is above-described with for example inert diluent.

The complementarity active compound also can add in the composition.In some embodiments, other therapeutical agent of binding molecule of the present invention and one or more becomes agent and/or co-administered jointly.For example, anti-GITR binding molecule of the present invention can become agent and/or co-administered jointly with one or more other antibody in conjunction with other target molecule, and wherein said antibody is for example can be in conjunction with the antibody of other cytokine or cell surface binding molecule.This conjoint therapy can advantageously use low dose therapy reagent, thereby avoids toxicity or the syndrome relevant with various independent therapies.

The binding molecule of the present invention has further been contained coupling diagnosis or therapeutical agent.Binding molecule can be used for to diagnostic, for example as the part of clinical trial, and monitoring tumor development or the effect of given treatment plan for confirmation of making progress.Can be by assisting to survey to detectable substance on the antibody coupling.The example of detectable substance comprises various enzymes, prothetic group, fluorescent substance, luminophore, noclilucence material, radioactive substance, with the metal of various positron radiation x-ray tomography art positron radiations, and on-radiation paramagnetic metal ion.Described detectable substance can directly or utilize technology known in the art by intermediate (such as, joint for example known in the art) indirectly with the binding molecule coupling or put together.Referring to for example, United States Patent (USP) 4,741,900 metal ion can be puted together with binding molecule and be used as diagnostic reagent.The example of suitable enzyme comprises horseradish peroxidase, alkaline phospholipase, beta-galactosidase enzymes or acetylcholinesterase; The example of suitable prothetic group comprises Streptavidin/vitamin H and avidin/biotin; The example of suitable fluorescent substance comprises Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichloro three azine group amine fluoresceins, dansyl chloride or phycoerythrin; An example of luminophore is a luminol,3-aminophthalic acid cyclic hydrazide; The noclilucence examples of substances comprises luciferase, fluorescein and aequorin; The example of suitable radioactive substance comprises I ¹²⁵, I ¹³¹, I ¹¹¹, In ^99Tc

Further, binding molecule can coupling on a treatment part, such as cytotoxin, for example cytostatic agent or kill cellularity reagent, therapeutical agent, (for example, αFang Sheyuan resembles radioactive metal ion ²¹³Bi), biotoxin, prodrug, peptide, protein, enzyme, virus, lipid, biological respinse amendment, pharmaceutical agent, immunocompetence part (for example, lymphokine and other antibody).In another embodiment, binding molecule of the present invention can suppress the molecule that tumor vessel forms in the coupling.In other embodiments, the binding molecule of the present invention of composition disclosed herein medicine that can comprise coupling or prodrug.Some embodiments more of the present invention have comprised coupling such as Ricin, have set the purposes of these special biotoxins of toxalbumin (gelonin), Pseudomonas exotoxin or diphtheria toxin or their the segmental binding molecule of cytotoxicity in vain.Select to use which type of coupling or not the coupling binding molecule depend on cancer type and stage, whether use assisting therapy (for example, chemotherapy or external beam radiotherapy) and patient's situation.We think that those skilled in the art can easily make one's options with reference to the instruction of this paper.

Cytotoxin or cytotoxic agent comprise the deleterious reagent of any pair cell.Example comprises taxol (paclitaxol), cytochalasin (cytochalasin) B, linear gramicidins (gramicidin) D, ethidium bromide, Hemometine (emetine), mitomycin (mitomycin), Etoposide (etoposide), teniposide (tenoposide), vincristin (vincristine), vincaleucoblastine (vinblastine), colchicine (colchicin), Dx (doxorubicin), daunorubicin (daunorubicin), dihydroxyl anthracin diketone (dihydroxy anthracin dione), mitoxantrone (mitoxantrone), Plicamycin (mithramycin), actinomycin (actinomycin) D, 1-boldenone (dehydrotestosterone), glucocorticosteroid (glucocorticoids), PROCAINE HCL, PHARMA GRADE (procaine), tetracaine (tetracaine), lignocaine (lidocaine), Proprasylyte (propranolol) and tetracycline (puromycin) and their analogue or homologue.Therapeutical agent includes but not limited to, metabolic antagonist (for example, methotrexate (methotrexate), Ismipur (6-mercaptopurine), 6-Tioguanine (6-thioguanine), cytosine arabinoside (cytarabine), 5 FU 5 fluorouracil (5-fluorouracil), Dacarbazine (dacarbazine)), alkylating agent (for example, mustargen (mechlorethamine), thio-tepa (thiotepa), Amboclorin (chlorambucil), melphalan (melphalan), carnustine (BSNU) and lomustine (lomustine, CCNU), endoxan (cyclophosphamide), busulfan (busulfan), mitobronitol (dibromomannitol), streptozotocin (streptozotocin), ametycin and cis dichloro two ammino platinum (cis-dichlorodiamine platinum) are (DDP (II), cis-platinum (cisplatin)), anthracycline drug (anthracycline) (for example, daunorubicin (being called daunomycin in the past) and Zorubicin), microbiotic (for example, dactinomycin (dactinomycin, be called actinomycin in the past), bleomycin (bleomycin), Plicamycin and anthramycin (anthramycin, AMC), and antimitotic reagent (for example, vincristin and vincaleucoblastine).

The present invention further sets forth by following examples, and these embodiment should not be understood that the restriction to invention.The full content of all reference of mentioning among the application, patent and disclosed patent application, and accompanying drawing is all incorporated this paper by reference into.

Embodiment

Following material and method are used for some embodiment:

Method

Cultivate T clone

In all sorts of ways from the cell that derives from human cord blood or peripheral blood CD4+CD45RA+ originally the T cell produce the clone of differentiation, described method comprises that flow cytometer separates with magnetic bead.Initial colony purity＞95%.Cell stimulates the cell type that produces differentiation with CD3 and CD28 antibody in RPMI 1640 then, and described substratum contains 10%FCS and 1% people AB serum, and the prescribed mixt of cytokine and antibacterial agent neutralizing antibody.Cultivate with IL12 (62U/ml) and anti-IL4 (0.2 μ g/ml), produce the Th1 cell; With IL4 (145U/ml) and anti-IL12 (10ug/ml) and anti-IFN γ (10ug/ml) cultivation, produce the Th2 cell; Cultivate with TGF β (32U/ml), IL9 (42U/ml), anti-IL4 (10ug/ml) and anti-IL12 (10ug/ml) and anti-IFN γ (10ug/ml), produce regulatory T cells.(annotating: all do not use anti-IL12 in all experiments).All cultures have all replenished IL2 (65U/ml) and IL15 (4500U/ml).According to fissional needs cell is assigned in the bigger culture plate.

Embodiment 1: separate and purifying 6C8

6C8 antibody is IgG2b, the κ type.Purified existence two heavy chains (Fig. 1) that show of this antibody.This can be owing to getting one of two kinds of glycosylations or having polluted due to the another kind of antibody (Ab).Size exclusion chromatography only shows a peak (Fig. 2).

Following purifying 6C8 antibody:

1. the dPBS with 5 column volumes washes 20ml Protein G (Pharmacia HR10/30)

2. go up sample 1L (the 1st takes turns) or 2L (the 2nd takes turns) hGITR (6C8) supernatant liquor

3. the dPBS with 10 column volumes washs

4. use the 100mM Citrate trianion, pH 2.8 directly is eluted to 1M Tris (20-25%v; V)

5. peel off (strip) with 100mM Citrate trianion (pH 2.8), 0.3M NaCl

Embodiment 2: identify 6C8

6C8 antibody with transfection the cell of GITR-L-M (Fig. 3) and activatory PBL (Fig. 4) combine.The saturation curve of biotin labeled anti-GITR prompting on the activated lymphocytes, relative affinity good (Fig. 5).

6C8 antibody is for utilizing the inferior anti-CD3 activated T lymphocyte of optimizing to have collaborative stimulating activity (Fig. 6).The collaborative stimulation of this antibody does not reach the level the same with CD28, still with anti-GITR commodity (R﹠amp; D) be suitable.

6C8 antibody is not induced activated lymphocytes apoptosis (Fig. 7).Use the PHA activated lymphocyte, add antibody after 3 days.Compare with YTH 655 (the known anti-people CD2 that can induce the activated lymphocytes apoptosis), 6C8 does not increase the lymphocytic apoptosis of activated T.

6C8 antibody is not blocked elementary mixed lymphocyte reacion (MLR) (Fig. 8).Make the positive control of described MLR with TRX1 (anti-people CD4).

Embodiment 3:6C8 antibody is offset the restraining effect of regulatory T cells to T effector cell

6C8 antibody can be blocked the restraining effect (Fig. 9) that regulatory T cells brings out.The CD4+/CD25+ cell adds the CD4+/CD25-cell with different ratios.Described cell stimulates with the anti-CD3 and the anti-CD28 that are combined on the microwell plate.When ratio was 1: 1, the CD4+/CD25+ cell can be eliminated the propagation of CD4+/CD25-cell.In culture, add 6C8 and can block this restraining effect in dosage dependence mode.

When only by anti-CD3 (being not to work in coordination with stimulation) when stimulating the T cell with anti-CD28, the CD4+/CD25+ cell is added in the CD4+/CD25-cell, do not observe restraining effect, in fact, described anti-GITR antibody has faint collaborative effect of stimulation (Figure 10) in these situations.

Embodiment 4:6C8 antibody is regulated the signal conduction of carrying out via NF-kB

The T cell causes activating I-κ B signal transduction path through CD3 or CD3+CD28 activation, and this can assess bright by phosphorylation (Fig. 12 and 1 4) and the degraded subsequently (Figure 11 and 13) of I-κ B.

As shown in figure 11, under part activatory situation, conduction has tangible influence to anti-GITR to I-κ B signal, and the time-dependent manner degraded of I-kB has confirmed this point.In the situation that the GITR binding molecule is arranged, at all time points of being analyzed, degraded obviously weakens.The decline of above-mentioned variation and I-κ B phosphorylation has good dependency (Figure 12).

What is interesting is, the response intensity (magnitude of response) of TH2 and Treg is compared the big of TH1.In addition, in parallel laboratory test, compare the expression of GITR as if higher on the TH1 cell (drawing) through MCF (average channel fluorescence) evaluation with the Treg cell with TH2.Lost their reactivities by crosslinked CD3 and CD28 by activated T cell fully, but kept the I-κ b activation carried out via TNF-α fully at anti-GITR.

The reaction of embodiment 5:6C8 antibody enhancing immunity

B16 melanoma tumor model is a kind of aggressiveness (aggressive) melanoma model that has, and it once was used to study the effect of regulatory T cells in cancer.The processing that the mouse that exhausts anti-CD 25 antibody or anti-CTLA-4 is carried out has demonstrated the result who gets a good chance of in this model.In two kinds of situations, described processing all can postpone the generation of tumour and the growth of tumour size.Because GITR expresses on the CD25+ cell, and may participate in offsetting the restraining effect of regulatory T cells, thereby disposes the B16 tumor-bearing mice with anti-GITR binding molecule, so that determine whether tumour generation or tumour size influential.Mouse is disposed with anti-GITR binding molecule behind a day of injection tumour, causes tumour generation and size increases to be delayed (Figure 17).In addition, there are some mouse not occur tumour at last yet in the GITR treatment group in experiment.

The 0th day, all animals were injected 104 B16 melanoma cells on right side.The GITR group has been accepted 2 milligrams, 1 milligram, 0.5 milligram or 0.2 milligram of anti-GITR binding molecule at the 1st day.The tumour that can measure appearred since the 16th day.

Embodiment 6: send anti-GITR and antigen simultaneously and cause adjuvant effect

Further studied anti-mGITR antibody to the adjuvant effect in the humoral response of antiovalbumin (Ova) or hemagglutinin (HA).Mouse was disposed with the YAML (isotype contrast) of no antibody, 0.4mg/ days or 0.4mg/ days 2F8 (rat-anti-mGITR) at the-1,0 and 1 day.For the importance in the mechanism of action that participates in described binding molecule of estimating the Fc acceptor, an other treated animal is disposed at the-1,0 and 1 day with 6mg/ days 2F8 F (ab ') 2.This dosage is to select than complete antibody weak point according to the half life of F (ab ') 2.The 0th day, mouse was used Ova (100 μ g) or HA (10 μ g) immunity.Attack with 100 μ g Ova at the 14th day through the mouse that Ova disposes, got blood then at the 21st and 28 day, obtain serum and carry out elisa assay.Attack with 5 μ g HA at the 14th day through the mouse that HA disposes, also got blood at the 21st and 28 day.

The serum-concentration of monitoring 2F8 and 2F8 F (ab ') 2 is estimated the change of pharmacokinetics of binding molecule.The 1st day, in the mouse of disposing through 2F8 or 2F8 F (ab ') 2 fragments, the serum level of binding molecule was suitable.In the mouse that 2F8 disposes, the situation that detects binding molecule continued until the 9th day, and only lasted till the 3rd day through the situation that the mouse that 2F8 F (ab ') 2 fragments are disposed detects binding molecule, though its dosage is 15 times of 2F8.

These results show, in the HA of this research branch, and the 21st day and 28 days, to compare with the mouse of disposing without antibody through the mouse that 2F8 disposes, anti-HA antibody increases by 4 and 5 times respectively; Compare with the mouse of disposing through YAML, anti-HA antibody increases by 18 and 20 times (Figure 19) respectively.Observed anti-HA titre when being adjuvant with anti-mGITR antibody, with HA and Freund's incomplete adjuvant (IFA) when giving observed titre suitable.This prompting is suitable with one of the most potent adjuvant of using always in observed reaction of described anti-mGITR antibody and the immunology research.

In the Ova of this research branch, the 21st day and 28 days, to compare with the mouse of disposing without antibody through the mouse that 2F8 disposes, anti-Ova antibody increases by 13 and 6 times respectively; Compare with the mouse of disposing through YAML, anti-Ova antibody increases by 17 and 8 times (Figure 20) respectively.2F8 antibody is suitable with observed influence in the reaction at HA at the influence in the reaction of Ova.The 21st day and 28 days, to compare with the mouse of disposing without antibody through the mouse that 2F8 F (ab ') 2 disposes, anti-Ova antibody increases by 4 and 3 times respectively; Compare with the mouse of disposing through YAML, anti-Ova antibody increases by 6 and 5 times (Figure 20) respectively.F (ab ') 2 dosage and different change of pharmacokinetics than complete antibody, can explain decline with anti-Ova reaction when the mouse of 2F8 disposal is compared.

In a word, these data presentation, 2F8 antibody is in the F of mainly giving the credit to this antibody at the influence in the antigenic humoral response (ab ') 2 parts, may and nonessentially have the Fc acceptor to participate in when described anti-mGITR antibody performance adjuvant effect.

Embodiment 7: preparation inosculating antibody GITR binding molecule

Adopt conventional Protocols in Molecular Biology that the variable light chain of 6C8 district is transplanted on people's constant region of light chain.What use is the IgG1 constant region of light chain.The amino acid of complete chimeric light chain GITR binding molecule is as follows:

DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ?ID?NO：22)。

Adopt conventional Protocols in Molecular Biology, also the 6C8 variable heavy chain is transplanted on people's CH.What use is the IgG1 CH.The aminoacid sequence of complete chimeric heavy chain GITR binding molecule (being called " Gly " again) as follows:

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ?ID?NO：23)。

Because aminoacid sequence NX (S/T) is the glycosylation site consensus sequence of a supposition, this sequence may have influence on the generation of binding molecule, and the IgG1 constant region of 6C8 heavy chain contains sequence NST, second version that we have prepared CH is with the conservative l-asparagine (runic, and be added with underscore) that is substituted by of the glutamine of amino-acid residue 299 among the SEQ ID NO:23.Accordingly, second human constant region is transplanted on the 6C8 variable region of heavy chain.The aminoacid sequence of complete chimeric heavy chain GITR binding molecule (being called " Agly " again) as follows:

QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY ASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ?ID?NO：24)。

Embodiment 8: the anti-GITR binding molecule of 6C8 of preparation humanization form

Adopt the strategy of describing among Hwang et al. (2005) Methods (36) 35-42 to come humanization 6C8 based on the CDR homology.With public database heavy chain and light-chain amino acid sequence are retrieved, the result shows that 6C8 has a 3-1 heavy chain norm structure and a 2-1-1 light chain norm structure.Thus, be that K chain V gene and 6C8 antibody sequence compare with all kinds that possess the 2-1-1 norm structure in the IMGT database.Same all 3-1 kinds are that heavy chain V gene and 6C8 aminoacid sequence compare.Only compared the CDR sequence, and which kind to be that sequence has maximum couplings to select framework in CDR according to.(referring to following sequence alignment).

For light chain, 3-15 ^*01 sequence has 14 couplings in CDR, selected this sequence.Because CDR 3 ends are leucine and Threonine, we have used Jk4 J gene fragment order.

The light chain V gene that possesses the 2-1-1 norm structure

IMGT

Gene title CDR1 CDR2 CDR3 IDs

IGKV1-5 RASQSISSWLA......DASSLES.......QQYNSYS..11

IGKV1-6 RASQGIRNDLG......AASSLSQ.......LQDYNYP..9

IGKV1-9 RASQGISSYLA......AASTLQS.......QQLNSYP..11

IGKV1-12 RASQGISSWLA......AASSLQS.......QQANSFP..11

IGKV1-16 RASQGISSWLA......AASSLQS.......QQYNSYP..12

IGKV1D-16 RARQGISSWLA......AASSLQS.......QQYNSYP..11

IGKV1-17 RASQGIRNDLG......AASSLQS.......LQHNSYP..9

IGKV1-27 RASQGISNYLA......AASTLQS.......QKYNSAP..11

IGKV1-33 QASQDISNYLN......DASNLET.......QQYDNLP..9

IGKV1-39 RASQSISSYLN......AASSLQS.......QQSYSTP..9

IGKV1D-43 WASQGISSYLA......YASSLQS.......QQYYSTP..11

IGKV3-11 RASQSVSSYLA......DASNRAT.......QQRSNWP..11

IGKV3D-11 RASQGVSSYLA......DASNRAT.......QQRSNWH..10

IGKV3-15 RASQSVSSNLA......GASTRAT.......QQYNNWP..14

6C8 KASQNVGTNVA......SASYRYS.......QQYNTDP

With all kinds that possess the 2-1-1 norm structure in the IMGT database is that light chain κ chain V gene and 6C8 antibody sequence compare.Be that heavy chain V gene and 6C8 aminoacid sequence compare equally with all 3-1 kinds.

Utilize this method to prepare a version of light chain:

EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRLLIYSASYRYS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNTDPLTFGGGTKVEIK (SEQ ID NO:44) (CDR represents with italic)

Concerning heavy chain, sequence 2-05 ^*01 has 17 couplings.But, the sequence different with 6C8 (YYCAR vs.YYCAHR) around the CDR3.Because existing proof shows that CDR 3 is most important for the recognition capability of CDR, should make this zone reach coupling fully as best one can.Sequence 2-70*01 has 16 couplings in CDR, the sequence of adjacent CDR3 front and the coupling fully of 6C8, so we have selected 2-70 ^*01.

For the J gene fragment of heavy chain, JH4 has maximum couplings, and is therefore selected.Aminoacid sequence is translated by counter then, the primer of corresponding target nucleotide sequence available from IDT (Coralville, IA).

The heavy chain V gene that has the 3-1 norm structure

IMGT

Gene title CDR1 CDR2 IDs

IGHV2-5 TSGVGVG.....LIYWNDDKRYSPSLKS 17

IGHV2-26 NARMGVS.....HIFSNDEKSYSTSLKS 12

IGHV2-70 TSGMCVS.....LIDWDDDKYYSTSLKT 16

IGHV4-30-2 SGGYSWS.....YIYHSGSTYYNPSLKS 10

IGHV4-30-4 SGDYYWS.....YIYYSGSTYYNPSLKS 9

IGHV4-31 SGGYYWS.....YIYYSGSTYYNPSLKS 9

IGHV4-39 SSSYYWG.....SIYYSGSTYYNPSLKS 10

IGHV4-61 SGSYYWS.....YIYYSGSTNYNPSLKS 8

6C8 TSGMGVG.....HIWWDDDKYYNPSLKS

Utilize this method, prepared a version of heavy chain:

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWD DDKYY NPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVS S (SEQ ID NO:53) (being called " N " again).

Because aminoacid sequence NX (S/T) is the glycosylation site consensus sequence of a supposition, may have influence on the generation of binding molecule, and the CDR2 of 6C8 heavy chain contains sequence NPS, second version that we have prepared heavy chain is with the conservative l-asparagine (runic, and be added with underscore) that is substituted by of the glutamine of amino-acid residue 62 among the SEQ ID NO:53.Accordingly, second version that has prepared heavy chain:

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMG VGWIRQPPGKALEWLAHIWWDDDKYY QPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVS S (SEQ ID NO:54) (being called " Q " again).

We also have been 6C8 variable region of light chain and 3-15 ^*01 kind is CLUSTAL W (1.82) the multiple sequence comparison (utilize Blosum scoring array, gap penalty is 10) of sequence of light chain.The result is as follows:

6C8 DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPD

3-15 ^*01 EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPA

:****** :*.*?*:*.:::*:***.*.:*:*********:*:?***.**?*?:*:*

6C8 RFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK

3-15*01 RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP------------

**:******:*****..::***:* *:*****.*

Analyze based on CLUSTAL W, identifying some amino-acid residues in people's framework may replace with the amino-acid residue corresponding with the 6C8 framework residue of humanization 6C8 light chain.Specifically, be the E in site 1, the P in site 8, the A in site 9, the T in site 10, the L in site 11, the V in site 13, the P in site 15, the E in site 17, the A in site 19, the T in site 20, the L in site 21, the S in site 22, the A in site 43, the R in site 45, the L in site 46, the I in site 58, the A in site 60, the S in site 63, the E in site 70, the S in site 76, the S in site 77, the L in site 78, the Q in site 79, the F in site 83, the V in site 85, the Y in site 87, the G in site 100, and the V in site 104.

Similarly, also to the 6C8 variable region of heavy chain with contain 2-70 ^*The kind of 01 aminoacid sequence is that heavy chain protein matter has been carried out CLUSTAL W (1.82) multiple sequence comparison (with the Blosum array of marking, gap penalty is 10).The result is as follows:

6C8 QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKY

2-70*01 QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCVSWIRQPPGKALEWLALIDWDDDKY

****:****.::**:***:***:***********?*.*****.**.*****?*?******

6C8 YNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSS

2-70*01 YSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARI-------------------

*..***::********:***.*.:*.:*..*:*******

Analyze based on CLUSTAL W, identifying some amino-acid residues in people's framework may replace with the amino-acid residue corresponding with the 6C8 framework residue of humanization 6C8 heavy chain.Specifically, be the R in site 5, the A in site 10, the L in site 11, the V of site 12, the T in site 15, site 19 T, the T in site 23, the P in site 43, the A in site 46, the R in site 68, the K in site 77, the V in site 81, the T in site 83, the M in site 84, the N in site 86, the M in site 87, the P in site 89, the V in site 90, and/or in the site 92 T.

Prepared 4 kinds of humanization total length 6C8 binding molecules with following humanization heavy chain and light chain array mode:

Total length version 1 (HuN6C8-Gly)-humanization (Hu) 6C8 light chain (L)/humanization heavy chain has N (" N ") and comprises the constant region (" Gly ") with N among the CDR2

Total length version 2 (HuN6C8-Agly)-humanization (Hu) 6C8 light chain (L)/humanized heavy chain has N (" N ") and comprises the constant region (" Agly ") with A among the CDR2

The total length version 3-(HuQ6C8-Gly)-humanization (Hu) 6C8 light chain (L)/humanized heavy chain, Q (" Q ") is arranged among the CDR2 and comprise constant region (" Gly ") with N

Q (" Q ") is arranged and comprise constant region (" Agly ") among total length edition 4-(HuQ6C8-Agly)-humanization (Hu) 6C8 light chain (L)/humanized heavy chain, the CDR2 with A

Glycosylation IgG1 CH amino acid sequence for generation of the total length binding molecule is as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:55).

Deglycosylation IgG1 CH amino acid sequence for generation of the total length binding molecule is as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:56).

The IgG1 constant region of light chain aminoacid sequence that is used to prepare the total length binding molecule is as follows: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:57).

The complete amino acid sequence of humanization 6C8 light chain is as follows: EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRLLIYSASYRYS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNTDPLTFGGGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:58).

Also can comprise homing sequence METQSQVFVYMLLWLSGVDG (SEQ ID NO:59).

The complete amino acid sequence of humanization 6C8 heavy chain version HuN6C8-Agly, HuQ6C8-Gly and HuQ6C8-Agly is as follows:

HuN6C8-Gly

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ?ID?NO：60)；

HuN6C8-Agly

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVS?SASTKGPSVFPLAPS?SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS?SGLYSLS?SVVTVPS?S?SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ?ID?NO：61)；

HuQ6C8-Gly

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYQPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO：62)； And

HuQ6C8-Agly

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYQPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ?ID?NO：63).

Also can comprise homing sequence MDRLTFSFLLLIVPAYVLS (SEQ ID NO:64).

Equivalent

Those skilled in the art only need use normal experiment just can recognize, perhaps confirm many equivalents of the specific embodiments of the present invention described in the literary composition.Following claim is intended to contain these equivalents.

Sequence table

＜110〉Tolerrx Inc. (TolerRx, Inc.)

＜120〉GITR binding molecule and uses thereof

<130>TLN-029PC

<140>

<141>

<150>60/665,322

<151>2005-03-25

<150>60/687,265

<151>2005-06-03

<160>68

<170>PatentIn?Ver.3.3

<210>1

<211>138

<212>PRT

＜213〉mouse (Mus musculus)

<400>1

Met?Asp?Arg?Leu?Thr?Phe?Ser?Phe?Leu?Leu?Leu?Ile?Val?Pro?Ala?Tyr

1 5 10 15

Val?Leu?Ser?Gln?Val?Thr?Leu?Lys?Glu?Ser?Gly?Pro?Gly?Ile?Leu?Lys

20 25 30

Pro?Ser?Gln?Thr?Leu?Ser?Leu?Thr?Cys?Ser?Phe?Ser?Gly?Phe?Ser?Leu

35 40 45

Ser?Thr?Ser?Gly?Met?Gly?Val?Gly?Trp?Ile?Arg?Gln?Pro?Ser?Gly?Lys

50 55 60

Gly?Leu?Glu?Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr

65 70 75 80

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

85 90 95

Asn?Gln?Val?Phe?Leu?Lys?Ile?Thr?Ser?Val?Asp?Thr?Ala?Asp?Ala?Ala

100 105 110

Thr?Tyr?Tyr?Cys?Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp

115 120 125

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

130 135

<210>2

<211>127

<212>PRT

＜213〉mouse (Mus musculus)

<400>2

Met?Glu?Thr?Gln?Ser?Gln?Val?Phe?Val?Tyr?Met?Leu?Leu?Trp?Leu?Ser

1 5 10 15

Gly?Val?Asp?Gly?Asp?Ile?Val?Met?Thr?Gln?Ser?Gln?Lys?Phe?Met?Ser

20 25 30

Thr?Ser?Val?Gly?Asp?Arg?Val?Ser?Val?Thr?Cys?Lys?Ala?Ser?Gln?Asn

35 40 45

Val?Gly?Thr?Asn?Val?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ser?Pro

50 55 60

Lys?Ala?Leu?Ile?Tyr?Ser?Ala?Ser?Tyr?Arg?Tyr?Ser?Gly?Val?Pro?Asp

65 70 75 80

Arg?Phe?Thr?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Asn

85 90 95

Asn?Val?His?Ser?Glu?Asp?Leu?Ala?Glu?Tyr?Phe?Cys?Gln?Gln?Tyr?Asn

100 105 110

Thr?Asp?Pro?Leu?Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu?Ile?Lys

115 120 125

<210>3

<211>12

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>3

Gly?Phe?Ser?Leu?Ser?Thr?Ser?Gly?Met?Gly?Val?Gly

1 5 10

<210>4

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>4

His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser?Leu?Lys?Ser

1 5 10 15

<210>5

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>5

Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr

1 5

<210>6

<211>11

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>6

Lys?Ala?Ser?Gln?Asn?Val?Gly?Thr?Asn?Val?Ala

1 5 10

<210>7

<211>7

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>7

Ser?Ala?Ser?Tyr?Arg?Tyr?Ser

1 5

<210>8

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>8

Gln?Gln?Tyr?Asn?Thr?Asp?Pro?Leu?Thr

1 5

<210>9

<211>414

<212>DNA

＜213〉mouse (Mus musculus)

<400>9

atggacagac?ttacattctc?attcctgctg?ctgattgtcc?ctgcatatgt?cttgtcccaa?60

gttactctaa?aagagtctgg?ccctgggata?ttgaagccct?cacagaccct?cagtctgact?120

tgttctttct?ctgggttttc?actgagcact?tctggtatgg?gtgtaggctg?gattcgtcag?180

ccttcaggga?agggtctgga?gtggctggcg?cacatttggt?gggatgatga?taagtactat?240

aatccatccc?tgaagagcca?gctcacaatc?tccaaggata?cctccagaaa?ccaggtattc?300

ctcaagatca?ccagtgtgga?cactgcagat?gctgccactt?actactgtgc?tcgaactagg?360

aggtacttcc?cctttgctta?ctggggccaa?gggacactag?tcacagtctc?ctca 414

<210>10

<211>381

<212>DNA

＜213〉mouse (Mus musculus)

<400>10

atggagacac?agtctcaggt?ctttgtatac?atgttgctgt?ggttgtctgg?tgttgatgga?60

gacattgtga?tgacccagtc?tcaaaaattc?atgtccacat?cagtaggaga?cagggtcagc?120

gtcacctgca?aggccagtca?gaatgtgggt?actaatgtag?cctggtatca?acagaaacca?180

gggcaatctc?ctaaagcact?gatttactcg?gcatcctacc?ggtacagtgg?agtccctgat?240

cgcttcacag?gcagtggatc?tgggacagat?ttcactctca?ccatcaacaa?tgtgcactct?300

gaagacttgg?cagagtattt?ctgtcaacaa?tataacaccg?atccgctcac?gttcggagct?360

gggaccaagc?tggaaatcaa?a 381

<210>11

<211>36

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>11

gggttttcac?tgagcacttc?tggtatgggt?gtaggc 36

<210>12

<211>48

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>12

cacatttggt?gggatgatga?taagtactat?aatccatccc?tgaagagc 48

<210>13

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>13

actaggaggt?acttcccctt?tgcttac 27

<210>14

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>14

aaggccagtc?agaatgtggg?tactaatgta?gcc 33

<210>15

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>15

tcggcatcct?accggtacag?t 21

<210>16

<211>27

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>16

caacaatata?acaccgatcc?gctcacg 27

<210>17

<211>1214

<212>DNA

＜213〉people (Homo sapiens)

<400>17

gtctacaccc?cctcctcaca?cgcacttcac?ctgggtcggg?attctcaggt?catgaacggt?60

cccagccacc?tccgggcagg?gcgggtgagg?acggggacgg?ggcgtgtcca?actggctgtg?120

ggctcttgaa?acccgagcat?ggcacagcac?ggggcgatgg?gcgcgtttcg?ggccctgtgc?180

ggcctggcgc?tgctgtgcgc?gctcagcctg?ggtcagcgcc?ccaccggggg?tcccgggtgc?240

ggccctgggc?gcctcctgct?tgggacggga?acggacgcgc?gctgctgccg?ggttcacacg?300

acgcgctgct?gccgcgatta?cccgggcgag?gagtgctgtt?ccgagtggga?ctgcatgtgt?360

gtccagcctg?aattccactg?cggagaccct?tgctgcacga?cctgccggca?ccacccttgt?420

cccccaggcc?agggggtaca?gtcccagggg?aaattcagtt?ttggcttcca?gtgtatcgac?480

tgtgcctcgg?ggaccttctc?cgggggccac?gaaggccact?gcaaaccttg?gacagactgc?540

acccagttcg?ggtttctcac?tgtgttccct?gggaacaaga?cccacaacgc?tgtgtgcgtc?600

ccagggtccc?cgccggcaga?gccgcttggg?tggctgaccg?tcgtcctcct?ggccgtggcc?660

gcctgcgtcc?tcctcctgac?ctcggcccag?cttggactgc?acatctggca?gctgaggagt?720

cagtgcatgt?ggccccgaga?gacccagctg?ctgctggagg?tgccgccgtc?gaccgaagac?780

gccagaagct?gccagttccc?cgaggaagag?cggggcgagc?gatcggcaga?ggagaagggg?840

cggctgggag?acctgtgggt?gtgagcctgg?ccgtcctccg?gggccaccga?ccgcagccag?900

cccctcccca?ggagctcccc?aggccgcagg?ggctctgcgt?tctgctctgg?gccgggccct?960

gctcccctgg?cagcagaagt?gggtgcagga?aggtggcagt?gaccagcgcc?ctggaccatg?1020

cagttcggcg?gccgcggctg?ggccctgcag?gagggagaga?gagacacagt?catggccccc?1080

ttcctccctt?gctggccctg?atggggtggg?gtcttaggac?gggaggctgt?gtccgtgggt?1140

gtgcagtgcc?cagcacggga?cccggctgca?ggggaccttc?aataaacact?tgtccagtga?1200

aaaaaaaaaa?aaaa 1214

<210>18

<211>241

<212>PRT

＜213〉people (Homo sapiens)

<400>18

Met?Ala?Gln?His?Gly?Ala?Met?Gly?Ala?Phe?Arg?Ala?Leu?Cys?Gly?Leu

1 5 10 15

Ala?Leu?Leu?Cys?Ala?Leu?Ser?Leu?Gly?Gln?Arg?Pro?Thr?Gly?Gly?Pro

20 25 30

Gly?Cys?Gly?Pro?Gly?Arg?Leu?Leu?Leu?Gly?Thr?Gly?Thr?Asp?Ala?Arg

35 40 45

Cys?Cys?Arg?Val?His?Thr?Thr?Arg?Cys?Cys?Arg?Asp?Tyr?Pro?Gly?Glu

50 55 60

Glu?Cys?Cys?Ser?Glu?Trp?Asp?Cys?Met?Cys?Val?Gln?Pro?Glu?Phe?His

65 70 75 80

Cys?Gly?Asp?Pro?Cys?Cys?Thr?Thr?Cys?Arg?His?His?Pro?Cys?Pro?Pro

85 90 95

Gly?Gln?Gly?Val?Gln?Ser?Gln?Gly?Lys?Phe?Ser?Phe?Gly?Phe?Gln?Cys

100 105 110

Ile?Asp?Cys?Ala?Ser?Gly?Thr?Phe?Ser?Gly?Gly?His?Glu?Gly?His?Cys

115 120 125

Lys?Pro?Trp?Thr?Asp?Cys?Thr?Gln?Phe?Gly?Phe?Leu?Thr?Val?Phe?Pro

130 135 140

Gly?Asn?Lys?Thr?His?Asn?Ala?Val?Cys?Val?Pro?Gly?Ser?Pro?Pro?Ala

145 150 155 160

Glu?Pro?Leu?Gly?Trp?Leu?Thr?Val?Val?Leu?Leu?Ala?Val?Ala?Ala?Cys

165 170 175

Val?Leu?Leu?Leu?Thr?Ser?Ala?Gln?Leu?Gly?Leu?His?Ile?Trp?Gln?Leu

180 185 190

Arg?Ser?Gln?Cys?Met?Trp?Pro?Arg?Glu?Thr?Gln?Leu?Leu?Leu?Glu?Val

195 200 205

Pro?Pro?Ser?Thr?Glu?Asp?Ala?Arg?Ser?Cys?Gln?Phe?Pro?Glu?Glu?Glu

210 215 220

Arg?Gly?Glu?Arg?Ser?Ala?Glu?Glu?Lys?Gly?Arg?Leu?Gly?Asp?Leu?Trp

225 230 235 240

Val

<210>19

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>19

His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Gln?Pro?Ser?Leu?Lys?Ser

1 5 10 15

<210>20

<211>105

<212>PRT

＜213〉mouse (Mus musculus)

<400>20

Ala?Asp?Ala?Ala?Pro?Thr?Val?Ser?Ile?Phe?Pro?Pro?Ser?Ser?Glu?Gln

1 5 10 15

Leu?Thr?Ser?Gly?Gly?Ala?Ser?Val?Val?Cys?Phe?Leu?Asn?Asn?Phe?Tyr

20 25 30

Pro?Lys?Asp?Ile?Asn?Val?Lys?Trp?Lys?Ile?Asp?Gly?Ser?Glu?Arg?Gln

35 40 45

Asn?Gly?Val?Leu?Asn?Ser?Trp?Thr?Asp?Gln?Asp?Ser?Lys?Asp?Ser?Thr

50 55 60

Tyr?Ser?Met?Ser?Ser?Thr?Leu?Thr?Leu?Thr?Lys?Asp?Glu?Tyr?Glu?Arg

65 70 75 80

His?Asn?Ser?Tyr?Thr?Cys?Glu?Ala?Thr?His?Lys?Thr?Ser?Thr?Ser?Pro

85 90 95

Ile?Val?Lys?Ser?Phe?Asn?Arg?Asn?Glu

100 105

<210>21

<211>334

<212>PRT

＜213〉mouse (Mus musculus)

<400>21

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

1 5 10 15

Asp?Thr?Thr?Gly?Ser?Ser?Val?Thr?Leu?Gly?Cys?Leu?Val?Lys?Gly?Tyr

20 25 30

Phe?Pro?Glu?Ser?Val?Thr?Val?Thr?Trp?Asn?Ser?Gly?Ser?Leu?Ser?Ser

35 40 45

Ser?Val?His?Thr?Phe?Pro?Ala?Leu?Leu?Gln?Ser?Gly?Leu?Tyr?Thr?Met

50 55 60

Ser?Ser?Ser?Val?Thr?Val?Pro?Ser?Ser?Thr?Trp?Pro?Ser?Gln?Thr?Val

65 70 75 80

Thr?Cys?Ser?Val?Ala?His?Pro?Ala?Ser?Ser?Thr?Thr?Val?Asp?Lys?Lys

85 90 95

Leu?Glu?Pro?Ser?Gly?Pro?Ile?Ser?Thr?Ile?Asn?Pro?Cys?Pro?Pro?Cys

100 105 110

Lys?Glu?Cys?Lys?Cys?Pro?Ala?Pro?Asn?Leu?Glu?Gly?Gly?Pro?Ser?Val

115 120 125

Phe?Ile?Phe?Pro?Pro?Asn?Ile?Lys?Asp?Val?Leu?Met?Ile?Ser?Leu?Thr

130 135 140

Pro?Lys?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?Glu?Asp?Asp?Pro?Asp

145 150 155 160

Val?Gln?Ile?Ser?Trp?Phe?Val?Asn?Asn?Val?Glu?Val?His?Thr?Ala?Gln

165 170 175

Thr?Gln?Thr?His?Arg?Glu?Asp?Tyr?Asn?Ser?Thr?Ile?Arg?Val?Val?Ser

180 185 190

Thr?Leu?Pro?Ile?Gln?His?Gln?Asp?Trp?Met?Ser?Gly?Lys?Glu?Phe?Lys

195 200 205

Cys?Lys?Val?Asn?Asn?Lys?Asp?Leu?Pro?Ser?Pro?Ile?Glu?Arg?Thr?Ile

210 215 220

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

225 230 235 240

Pro?Ala?Glu?Gln?Leu?Ser?Arg?Lys?Asp?Val?Ser?Leu?Thr?Cys?Leu?Val

245 250 255

Val?Gly?Phe?Asn?Pro?Gly?Asp?Ile?Ser?Val?Glu?Trp?Thr?Ser?Asn?Gly

260 265 270

His?Thr?Glu?Glu?Asn?Tyr?Lys?Asp?Thr?Ala?Pro?Val?Leu?Asp?Ser?Asp

275 280 285

Gly?Ser?Tyr?Phe?Ile?Tyr?Ser?Lys?Leu?Asn?Met?Lys?Thr?Ser?Lys?Trp

290 295 300

Glu?Lys?Thr?Asp?Ser?Phe?Ser?Cys?Asn?Val?Arg?His?Glu?Gly?Leu?Lys

305 310 315 320

Asn?Tyr?Tyr?Leu?Lys?Lys?Thr?Ile?Ser?Arg?Ser?Pro?Gly?Lys

325 330

<210>22

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic mouse/people's light chain construct

<400>22

Asp?Ile?Val?Met?Thr?Gln?Ser?Gln?Lys?Phe?Met?Ser?Thr?Ser?Val?Gly

1 5 10 15

Asp?Arg?Val?Ser?Val?Thr?Cys?Lys?Ala?Ser?Gln?Asn?Val?Gly?Thr?Asn

20 25 30

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

35 40 45

Tyr?Ser?Ala?Ser?Tyr?Arg?Tyr?Ser?Gly?Val?Pro?Asp?Arg?Phe?Thr?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Asn?Asn?Val?His?Ser

65 70 75 80

Glu?Asp?Leu?Ala?Glu?Tyr?Phe?Cys?Gln?Gln?Tyr?Asn?Thr?Asp?Pro?Leu

85 90 95

Thr?Phe?Gly?Ala?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala

100 105 110

Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly

115 120 125

Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala

130 135 140

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

145 150 155 160

Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser

165 170 175

Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr

180 185 190

Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser

195 200 205

Phe?Asn?Arg?Gly?Glu?Cys

210

<210>23

<211>449

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic mouse/people's heavy chain construct

<400>23

Gln?Val?Thr?Leu?Lys?Glu?Ser?Gly?Pro?Gly?Ile?Leu?Lys?Pro?Ser?Gln

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Ser?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys?Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp?Gly?Gln?Gly

100 105 110

Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe

115 120 125

Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu

130 135 140

Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp

145 150 155 160

Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu

165 170 175

Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser

180 185 190

Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro

195 200 205

Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys

210 215 220

Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro

225 230 235 240

Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser

245 250 255

Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp

260 265 270

Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn

275 280 285

Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val

290 295 300

Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu

305 310 315 320

Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys

325 330 335

Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr

340 345 350

Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr

355 360 365

Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu

370 375 380

Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu

385 390 395 400

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

405 410 415

Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu

420 425 430

Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly

435 440 445

Lys

<210>24

<211>449

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the complete chimeric heavy chain of synthetic mouse/people

<400>24

Gln?Val?Thr?Leu?Lys?Glu?Ser?Gly?Pro?Gly?Ile?Leu?Lys?Pro?Ser?Gln

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Ser?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys?Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp?Gly?Gln?Gly

100 105 110

Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe

115 120 125

Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu

130 135 140

Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp

145 150 155 160

Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu

165 170 175

Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser

180 185 190

Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro

195 200 205

Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys

210 215 220

Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro

225 230 235 240

Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser

245 250 255

Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp

260 265 270

Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn

275 280 285

Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Ala?Ser?Thr?Tyr?Arg?Val

290 295 300

Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu

305 310 315 320

Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys

325 330 335

Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr

340 345 350

Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr

355 360 365

Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu

370 375 380

Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu

385 390 395 400

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

405 410 415

Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu

420 425 430

Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly

435 440 445

Lys

<210>25

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>25

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

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Asn

20 25 30

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

35 40 45

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

50 55 60

Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Asn?Trp?Pro

85 90 95

<210>26

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>26

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

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Gly?Val?Ser?Ser?Tyr

20 25 30

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

35 40 45

Tyr?Asp?Ala?Ser?Asn?Arg?Ala?Thr?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly

50 55 60

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

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Arg?Ser?Asn?Trp?His

85 90 95

<210>27

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>27

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

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Tyr

20 25 30

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

35 40 45

Tyr?Asp?Ala?Ser?Asn?Arg?Ala?Thr?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly

50 55 60

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

65 70 75 80

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

85 90 95

<210>28

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>28

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

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Tyr

20 25 30

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

35 40 45

Tyr?Asp?Ala?Ser?Asn?Arg?Ala?Thr?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly

50 55 60

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

65 70 75 80

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

85 90 95

<210>29

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>29

Ala?Ile?Arg?Met?Thr?Gln?Ser?Pro?Phe?Ser?Leu?Ser?Ala?Ser?Val?Gly

1 5 10 15

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

20 25 30

Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Ala?Lys?Ala?Pro?Lys?Leu?Phe?Ile

35 40 45

Tyr?Tyr?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Tyr?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Tyr?Ser?Thr?Pro

85 90 95

<210>30

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>30

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

1 5 10 15

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

20 25 30

Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Tyr?Ser?Thr?Pro

85 90 95

<210>31

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>31

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

1 5 10 15

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

20 25 30

Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Cys?Gly?Tyr?Ser?Thr?Pro

85 90 95

<210>32

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>32

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Gln?Ala?Ser?Gln?Asp?Ile?Ser?Asn?Tyr

20 25 30

Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Tyr?Asp?Ala?Ser?Asn?Leu?Glu?Thr?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Ile?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asp?Asn?Leu?Pro

85 90 95

<210>33

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>33

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr?Ala?Ala?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Val?Ala?Thr?Tyr?Tyr?Cys?Gln?Lys?Tyr?Asn?Ser?Ala?Pro

85 90 95

<210>34

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>34

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Arg?Asn?Asp

20 25 30

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

35 40 45

Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Leu?Gln?His?Asn?Ser?Tyr?Pro

85 90 95

<210>35

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>35

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Arg?Asn?Asp

20 25 30

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

35 40 45

Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr?Leu?Thr?Ile?Ser?Asn?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Leu?Gln?His?Asn?Ser?Tyr?Pro

85 90 95

<210>36

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>36

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Trp

20 25 30

Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Glu?Lys?Ala?Pro?Lys?Ser?Leu?Ile

35 40 45

Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Ser?Tyr?Pro

85 90 95

<210>37

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>37

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Arg?Gln?Gly?Ile?Ser?Ser?Trp

20 25 30

Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Glu?Lys?Ala?Pro?Lys?Ser?Leu?Ile

35 40 45

Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Ser?Tyr?Pro

85 90 95

<210>38

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>38

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

1 5 10 15

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

20 25 30

Leu?Ala?Trp?Phe?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Ser?Leu?Ile

35 40 45

Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Ser?Tyr?Pro

85 90 95

<210>39

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>39

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Trp

20 25 30

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

35 40 45

Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ala?Asn?Ser?Phe?Pro

85 90 95

<210>40

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>40

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Ser?Ser?Trp

20 25 30

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

35 40 45

Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ala?Asn?Ser?Phe?Pro

85 90 95

<210>41

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>41

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

1 5 10 15

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

20 25 30

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

35 40 45

Tyr?Ala?Ala?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Leu?Asn?Ser?Tyr?Pro

85 90 95

<210>42

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>42

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Gly?Ile?Arg?Asn?Asp

20 25 30

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

35 40 45

Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Leu?Gln?Asp?Tyr?Asn?Tyr?Pro

85 90 95

<210>43

<211>95

<212>PRT

＜213〉people (Homo sapiens)

<400>43

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

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Ser?Trp

20 25 30

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

35 40 45

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

50 55 60

Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro

65 70 75 80

Asp?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Ser?Tyr?Ser

85 90 95

<210>44

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>44

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

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Lys?Ala?Ser?Gln?Asn?Val?Gly?Thr?Asn

20 25 30

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

35 40 45

Tyr?Ser?Ala?Ser?Tyr?Arg?Tyr?Ser?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Thr?Asp?Pro?Leu

85 90 95

Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys

100 105

<210>45

<211>96

<212>PRT

＜213〉people (Homo sapiens)

<400>45

Gln?Ile?Thr?Leu?Lys?Glu?Ser?Gly?Pro?Thr?Leu?Val?Lys?Pro?Thr?Gln

1 5 10 15

Thr?Leu?Thr?Leu?Thr?Cys?Thr?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

Gly?Val?Gly?Val?Gly?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Ala?Leu?Glu

35 40 45

Trp?Leu?Ala?Leu?Ile?Tyr?Trp?Asn?Asp?Asp?Lys?Arg?Tyr?Ser?Pro?Ser

50 55 60

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

65 70 75 80

Val?Leu?Thr?Met?Thr?Asn?Met?Asp?Pro?Val?Asp?Thr?Ala?Thr?Tyr?Tyr

85 90 95

<210>46

<211>100

<212>PRT

＜213〉people (Homo sapiens)

<400>46

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

1 5 10 15

Thr?Leu?Thr?Leu?Thr?Cys?Thr?Val?Ser?Gly?Phe?Ser?Leu?Ser?Asn?Ala

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Phe?Ser?Asn?Asp?Glu?Lys?Ser?Tyr?Ser?Thr?Ser

50 55 60

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

65 70 75 80

Val?Leu?Thr?Met?Thr?Asn?Met?Asp?Pro?Val?Asp?Thr?Ala?Thr?Tyr?Tyr

85 90 95

Cys?Ala?Arg?Ile

100

<210>47

<211>100

<212>PRT

＜213〉people (Homo sapiens)

<400>47

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

1 5 10 15

Thr?Leu?Thr?Leu?Thr?Cys?Thr?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?Leu?Ile?Asp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Ser?Thr?Ser

50 55 60

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

65 70 75 80

Val?Leu?Thr?Met?Thr?Asn?Met?Asp?Pro?Val?Asp?Thr?Ala?Thr?Tyr?Tyr

85 90 95

Cys?Ala?Arg?Ile

100

<210>48

<211>99

<212>PRT

＜213〉people (Homo sapiens)

<400>48

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

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Ala?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Gly

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys?Ala?Arg

<210>49

<211>99

<212>PRT

＜213〉people (Homo sapiens)

<400>49

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

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Gly

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys?Ala?Arg

<210>50

<211>99

<212>PRT

＜213〉people (Homo sapiens)

<400>50

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

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Gly

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys?Ala?Arg

<210>51

<211>99

<212>PRT

＜213〉people (Homo sapiens)

<400>51

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

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Ile?Ser?Ser?Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys?Ala?Arg

<210>52

<211>99

<212>PRT

＜213〉people (Homo sapiens)

<400>52

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

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Gly?Gly?Ser?Val?Ser?Ser?Gly

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Cys?Ala?Arg

<210>53

<211>119

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>53

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

1 5 10 15

Thr?Leu?Thr?Leu?Thr?Cys?Thr?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser

50 55 60

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

65 70 75 80

Val?Leu?Thr?Met?Thr?Asn?Met?Asp?Pro?Val?Asp?Thr?Ala?Thr?Tyr?Tyr

85 90 95

Cys?Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp?Gly?Gln?Gly

100 105 110

Thr?Leu?Val?Thr?Val?Ser?Ser

115

<210>54

<211>119

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>54

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

1 5 10 15

Thr?Leu?Thr?Leu?Thr?Cys?Thr?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Gln?Pro?Ser

50 55 60

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

65 70 75 80

Val?Leu?Thr?Met?Thr?Asn?Met?Asp?Pro?Val?Asp?Thr?Ala?Thr?Tyr?Tyr

85 90 95

Cys?Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp?Gly?Gln?Gly

100 105 110

Thr?Leu?Val?Thr?Val?Ser?Ser

115

<210>55

<211>330

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>55

Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys

1 5 10 15

Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr

20 25 30

Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser

35 40 45

Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser

50 55 60

Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr

65 70 75 80

Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys

85 90 95

Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys

100 105 110

Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro

115 120 125

Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys

130 135 140

Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp

145 150 155 160

Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu

165 170 175

Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu

180 185 190

His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn

195 200 205

Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly

210 215 220

Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu

225 230 235 240

Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr

245 250 255

Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn

260 265 270

Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe

275 280 285

Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn

290 295 300

Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr

305 310 315 320

Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

325 330

<210>56

<211>330

<212>PRT

＜213〉artificial sequence

<220>

＜223〉retouching of artificial sequence reaches: synthetic albumen construct

<400>56

Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys

1 5 10 15

Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr

20 25 30

Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser

35 40 45

Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser

50 55 60

Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr

65 70 75 80

Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys

85 90 95

Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys

100 105 110

Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro

115 120 125

Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys

130 135 140

Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp

145 150 155 160

Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu

165 170 175

Glu?Gln?Tyr?Ala?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu

180 185 190

His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn

195 200 205

Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly

210 215 220

Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu

225 230 235 240

Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr

245 250 255

Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn

260 265 270

Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe

275 280 285

Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn

290 295 300

Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr

305 310 315 320

Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys

325 330

<210>57

<211>107

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>57

Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu

1 5 10 15

Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe

20 25 30

Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln

35 40 45

Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser

50 55 60

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

65 70 75 80

Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser

85 90 95

Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys

100 105

<210>58

<211>214

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>58

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

1 5 10 15

Glu?Arg?Ala?Thr?Leu?Ser?Cys?Lys?Ala?Ser?Gln?Asn?Val?Gly?Thr?Asn

20 25 30

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

35 40 45

Tyr?Ser?Ala?Ser?Tyr?Arg?Tyr?Ser?Gly?Ile?Pro?Ala?Arg?Phe?Ser?Gly

50 55 60

Ser?Gly?Ser?Gly?Thr?Glu?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Ser

65 70 75 80

Glu?Asp?Phe?Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Asn?Thr?Asp?Pro?Leu

85 90 95

Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala

100 105 110

Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly

115 120 125

Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala

130 135 140

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

145 150 155 160

Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser

165 170 175

Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr

180 185 190

Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser

195 200 205

Phe?Asn?Arg?Gly?Glu?Cys

210

<210>59

<211>20

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic amino acid homing sequence

<400>59

Met?Glu?Thr?Gln?Ser?Gln?Val?Phe?Val?Tyr?Met?Leu?Leu?Trp?Leu?Ser

1 5 10 15

Gly?Val?Asp?Gly

20

<210>60

<211>449

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>60

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

1 5 10 15

Thr?Leu?Thr?Leu?Thr?Cys?Thr?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser

50 55 60

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

65 70 75 80

Val?Leu?Thr?Met?Thr?Asn?Met?Asp?Pro?Val?Asp?Thr?Ala?Thr?Tyr?Tyr

85 90 95

Cys?Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp?Gly?Gln?Gly

100 105 110

Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe

115 120 125

Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu

130 135 140

Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp

145 150 155 160

Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu

165 170 175

Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser

180 185 190

Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro

195 200 205

Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys

210 215 220

Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro

225 230 235 240

Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser

245 250 255

Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp

260 265 270

Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn

275 280 285

Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val

290 295 300

Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu

305 310 315 320

Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys

325 330 335

Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr

340 345 350

Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr

355 360 365

Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu

370 375 380

Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu

385 390 395 400

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

405 410 415

Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu

420 425 430

Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly

435 440 445

Lys

<210>61

<211>449

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>61

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

1 5 10 15

Thr?Leu?Thr?Leu?Thr?Cys?Thr?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Asn?Pro?Ser

50 55 60

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

65 70 75 80

Val?Leu?Thr?Met?Thr?Asn?Met?Asp?Pro?Val?Asp?Thr?Ala?Thr?Tyr?Tyr

85 90 95

Cys?Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp?Gly?Gln?Gly

100 105 110

Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe

115 120 125

Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu

130 135 140

Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp

145 150 155 160

Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu

165 170 175

Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser

180 185 190

Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro

195 200 205

Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys

210 215 220

Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro

225 230 235 240

Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser

245 250 255

Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp

260 265 270

Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn

275 280 285

Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Ala?Ser?Thr?Tyr?Arg?Val

290 295 300

Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu

305 310 315 320

Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys

325 330 335

Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr

340 345 350

Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr

355 360 365

Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu

370 375 380

Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu

385 390 395 400

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

405 410 415

Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu

420 425 430

Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly

435 440 445

Lys

<210>62

<211>449

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>62

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

1 5 10 15

Thr?Leu?Thr?Leu?Thr?Cys?Thr?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Gln?Pro?Ser

50 55 60

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

65 70 75 80

Val?Leu?Thr?Met?Thr?Asn?Met?Asp?Pro?Val?Asp?Thr?Ala?Thr?Tyr?Tyr

85 90 95

Cys?Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp?Gly?Gln?Gly

100 105 110

Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe

115 120 125

Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu

130 135 140

Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp

145 150 155 160

Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu

165 170 175

Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser

180 185 190

Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro

195 200 205

Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys

210 215 220

Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro

225 230 235 240

Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser

245 250 255

Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp

260 265 270

Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn

275 280 285

Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val

290 295 300

Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu

305 310 315 320

Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys

325 330 335

Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr

340 345 350

Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr

355 360 365

Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu

370 375 380

Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu

385 390 395 400

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

405 410 415

Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu

420 425 430

Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly

435 440 445

Lys

<210>63

<211>449

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>63

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

1 5 10 15

Thr?Leu?Thr?Leu?Thr?Cys?Thr?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr?Gln?Pro?Ser

50 55 60

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

65 70 75 80

Val?Leu?Thr?Met?Thr?Asn?Met?Asp?Pro?Val?Asp?Thr?Ala?Thr?Tyr?Tyr

85 90 95

Cys?Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp?Gly?Gln?Gly

100 105 110

Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe

115 120 125

Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu

130 135 140

Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp

145 150 155 160

Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu

165 170 175

Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser

180 185 190

Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro

195 200 205

Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys?Asp?Lys

210 215 220

Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro

225 230 235 240

Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser

245 250 255

Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp

260 265 270

Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn

275 280 285

Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Ala?Ser?Thr?Tyr?Arg?Val

290 295 300

Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu

305 310 315 320

Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys

325 330 335

Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr

340 345 350

Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr

355 360 365

Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu

370 375 380

Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu

385 390 395 400

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

405 410 415

Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu

420 425 430

Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly

435 440 445

Lys

<210>64

<211>19

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic homing sequence peptide

<400>64

Met?Asp?Arg?Leu?Thr?Phe?Ser?Phe?Leu?Leu?Leu?Ile?Val?Pro?Ala?Tyr

1 5 10 15

Val?Leu?Ser

<210>65

<211>50

<212>DNA

<220>

＜223〉description of artificial sequence: synthetic oligonucleotide

<400>65

cacatttggt?gggatgatga?taagtactat?caaccatccc?tgaagagcca 50

<210>66

<211>138

<212>PRT

＜213〉mouse (Mus musculus)

<400>66

Met?Asp?Arg?Leu?Thr?Phe?Ser?Phe?Leu?Leu?Leu?Ile?Val?Pro?Ala?Tyr

1 5 10 15

Val?Leu?Ser?Gln?Val?Thr?Leu?Lys?Glu?Ser?Gly?Pro?Gly?Ile?Leu?Lys

20 25 30

Pro?Ser?Gln?Thr?Leu?Ser?Leu?Thr?Cys?Ser?Phe?Ser?Gly?Phe?Ser?Leu

35 40 45

Ser?Thr?Ser?Gly?Met?Gly?Val?Gly?Trp?Ile?Arg?Gln?Pro?Ser?Gly?Lys

50 55 60

Gly?Leu?Glu?Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Tyr

65 70 75 80

Gln?Pro?Ser?Leu?Lys?Ser?Gln?Leu?Thr?Ile?Ser?Lys?Asp?Thr?Ser?Arg

85 90 95

Asn?Gln?Val?Phe?Leu?Lys?Ile?Thr?Ser?Val?Asp?Thr?Ala?Asp?Ala?Ala

100 105 110

Thr?Tyr?Tyr?Cys?Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp

115 120 125

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

130 135

<210>67

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>67

Gln?Val?Thr?Leu?Lys?Glu?Ser?Gly?Pro?Gly?Ile?Leu?Gln?Pro?Ser?Gln

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Ser?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Asn?Pro?Ser?Leu

50 55 60

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

65 70 75 80

Leu?Lys?Ile?Thr?Ser?Val?Asp?Thr?Arg?Asp?Thr?Ala?Thr?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp?Gly?Glu?Gly?Thr

100 105 110

Ser?Val?Thr?Val?Thr?Ser

115

<210>68

<211>118

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic albumen construct

<400>68

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

1 5 10 15

Thr?Leu?Thr?Leu?Thr?Cys?Thr?Phe?Ser?Gly?Phe?Ser?Leu?Ser?Thr?Ser

20 25 30

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

35 40 45

Trp?Leu?Ala?His?Ile?Trp?Trp?Asp?Asp?Asp?Lys?Tyr?Asn?Pro?Ser?Leu

50 55 60

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

65 70 75 80

Leu?Thr?Met?Thr?Asn?Met?Asp?Pro?Val?Asp?Thr?Ala?Thr?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Thr?Arg?Arg?Tyr?Phe?Pro?Phe?Ala?Tyr?Trp?Gly?Gln?Gly?Thr

100 105 110

Leu?Val?Thr?Val?Ser?Ser

115

Claims (57)

1.GITR binding molecule, it comprises amino-acid residue 20-138 among the SEQ ID NO.1 or the amino-acid residue 20-138 among the SEQ ID NO:66.

2.GITR binding molecule, it comprises the amino-acid residue 21-127 among the SEQ ID NO.2.

3.GITR binding molecule, it comprises at least one and is selected from SEQ ID NO.3, complementary determining region (CDR) aminoacid sequence of SEQ ID NO.4 or SEQ ID NO:19 and SEQ ID NO.5.

4. the GITR binding molecule of claim 3, it comprises at least two CDR.

5. the GITR binding molecule of claim 3, it comprises three CDR.

6.GITR binding molecule, it comprises at least one cdr amino acid sequence that is selected from SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

7. the GITR binding molecule of claim 6, it comprises at least two CDR.

8. the GITR binding molecule of claim 6, it comprises three CDR.

9.GITR binding molecule, it comprises the CDR shown in the SEQ ID NO:3,4 or 19,5,6,7 or 8.

10.GITR binding molecule, it comprises variable region of heavy chain and variable region of light chain, described variable region of heavy chain contains amino-acid residue 20-138 among the SEQ ID NO.1 or the amino-acid residue 20-138 among the SEQ ID NO:66, and described variable region of light chain contains the amino-acid residue 21-127 among the SEQ ID NO:2.

11. each GITR binding molecule of claim 3-10, it comprises ethnic group is heavy chain and light chain framework region.

12. the GITR binding molecule of claim 11, one or more people's framework amino acid residue is wherein become corresponding mouse amino-acid residue by reverse mutation.

13. the GITR binding molecule of claim 9, wherein constant region comprises the IgG2b CH.

14. the GITR binding molecule of claim 9, wherein said binding molecule is in conjunction with people GITR.

15. the GITR binding molecule of claim 9, wherein said binding molecule does not bring out apoptosis.

16. the GITR binding molecule of claim 9, wherein said binding molecule is not blocked elementary mixed lymphocyte reacion.

17. the GITR binding molecule of claim 9, wherein said binding molecule are offset the restraining effect of regulatory T cells to T effector cell.

18. the GITR binding molecule of claim 9, wherein said binding molecule promotes T effector cell's propagation.

19. the GITR binding molecule of claim 9, wherein said binding molecule derives from mouse.

20. the GITR binding molecule of claim 19, wherein said binding molecule comprise mouse IgG2a heavy chain.

21. the GITR binding molecule of claim 9, the activity of wherein said binding molecule mediator GITR.

22. the GITR binding molecule of claim 9, wherein said binding molecule weaken the degraded of I-κ B in the T cell.

23. the GITR binding molecule of claim 11, wherein said binding molecule is a chimeric antibody.

24.GITR binding molecule, this molecule are in conjunction with the GITR on human T-cell and the human dendritic cell, and binding constant (Kd) is 1 * 10 ^-9Or it is lower.

25. the GITR binding molecule of claim 24, wherein said binding molecule are offset the restraining effect of regulatory T cells to T effector cell.

26. the GITR binding molecule of claim 24, wherein said binding molecule is a humanized antibody.

27. composition, it comprises claim 1-10 or 13-26 each described GITR binding molecule and pharmaceutically acceptable carrier.

28. the composition of claim 27 also comprises at least a other therapeutical agent that is used for the treatment of experimenter's cancer.

29. the composition of claim 27 also comprises the therapeutical agent of at least a other virus infection that is used for the treatment of the experimenter.

30. the composition of claim 27 also comprises the tumour antigen of at least a treatment experimenter cancer.

31. the composition of claim 27 also comprises the pathogen antigen of at least a treatment experimenter's virus infection.

32. inhibiting method of eliminating regulatory T cells to T effector cell, this method comprises people's immunocyte is contacted with claim 1-10 or 13 each described GITR binding molecules, thereby eliminates the restraining effect of regulatory T cells to T effector cell.

33. regulate the method for conducting through TXi Baoshouti inductive signal among the T effector cell for one kind, this method comprises cell is contacted with claim 1-10 or 13 each described GITR binding molecules, thereby makes among the T effector cell adjusted through the conduction of TXi Baoshouti inductive signal.

34. the method for claim 33, the degraded of wherein said I-κ B is regulated.

35. the method for claim 33, wherein said T cell is the Th1 cell.

36. the method for claim 35, wherein said T cell is the CD4+ cell.

37. the method for claim 35, wherein said T cell is the CD8+ cell.

38. the immunoreactive method of enhancing experimenter, this method comprise cell is contacted with claim 1-10 or each described GITR binding molecule of 13-26, thereby experimenter's immune response is strengthened.

39. a method for the treatment of experimenter's cancer, this method comprise cell is contacted with claim 1-10 or each described GITR binding molecule of 13-26, thereby experimenter's cancer is obtained medical treatment.

40. the method for claim 39, the type of wherein said cancer are selected from the cancer of carcinoma of the pancreas, melanoma, mammary cancer, lung cancer, bronchogenic carcinoma, colorectal cancer, prostate cancer, cancer of the stomach, ovarian cancer, Urinary Bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, the esophageal carcinoma, cervical cancer, uterus or carcinoma of endometrium, oral carcinoma or pharynx cancer, liver cancer, kidney, carcinoma of testis, cholangiocarcinoma, small intestine or adnexal carcinoma, salivary-gland carcinoma, thyroid carcinoma, adrenal carcinoma, osteosarcoma, chondrosarcoma and hemopoietic tissue.

41. treat infection or the disorderly method that causes by pathogenic agent in the subject for one kind, this method comprises the described GITR binding molecule of cell and claim 1 is contacted, thereby the disease, disorder or the situation that are caused by pathogen in the subject are obtained medical treatment.

42. the method for claim 41, wherein said pathogenic agent are to be selected from following virus: HIV, herpes virus hominis, cytomegalovirus, rotavirus, Epstein-Barr virus, varicella zoster virus, hepatitis virus (such as hepatitis B virus, hepatitis A virus (HAV), hepatitis C virus and viral hepatitis type E virus), paramyxovirus: respiratory syncytial virus, parainfluenza virus, Measles virus, mumps virus, human papillomavirus, flavivirus and influenza virus.

43. the method for claim 41, wherein said pathogenic agent are to be selected from following bacterium: the Neisseria gonorrhoeae species, the suis species, Streptococcus mutans, the hemophilic bacterium species, the catarrhalis species, the Bordetella species, mycobacterium species, the legionella species, the Escherichia species, the vibrios species, the Yersinia species, crooked fungus kind, the Salmonellas species, the listeria spp species, the Helicobacter pylori species, the pseudomonas species, the staphylococcus species, the faecalis species, the clostridium species, bacillus species, rod bacillus species, the burgdorferi species, ehrlichiosis body species, the Rickettsiae species, the chlamydozoan species, the Leptospira species, the treponema species.

44. a method of regulating the GITR function, this method are included under the situation of stimulant, people GITR is contacted with claim 1-10 or each described GITR binding molecule of 13-26, thereby make the GITR function obtain adjusting.

45. an isolated nucleic acid molecule, it comprises the nucleotide sequence of encoding heavy chain variable region, and described sequence contains the Nucleotide 58-414 among the SEQ ID NO:9.

46. an isolated nucleic acid molecule, it comprises the nucleotide sequence of encoded light chain variable region, and described sequence contains the Nucleotide 61-381 among the SEQ ID NO:10.

47. an isolated nucleic acid molecule, it comprises the nucleotide sequence of at least one CDR that encodes, and described sequence is selected from SEQ ID NO.11, SEQ ID NO.12 or SEQ ID NO:65 and SEQ IDNO.13.

48. the isolated nucleic acid molecule of claim 47, it comprises the nucleotide sequence of at least two CDR of coding.

49. the isolated nucleic acid molecule of claim 47, it comprises the nucleotide sequence of three CDR that encode.

50. an isolated nucleic acid molecule, it comprises the nucleotide sequence of at least one CDR that encodes, and described sequence is selected from SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16.

51. the isolated nucleic acid molecule of claim 50, it comprises the nucleotide sequence of at least two CDR of coding.

52. the isolated nucleic acid molecule of claim 50, it comprises the nucleotide sequence of three CDR that encode.

53. an isolated nucleic acid molecule, it comprises the nucleotide sequence shown in the SEQ ID NO:11,12 or 65,13,14,15 and 16.

54. recombinant expression vector comprises each described nucleic acid molecule of claim 45-53.

55. recombinant expression vector, it comprises nucleic acid molecule, and described molecule has the nucleotide sequence of the described binding molecule of coding claim 24.

56. host cell has imported the described recombinant expression vector of claim 55.

57. a method for preparing in conjunction with the GITR binding molecule of people GITR, this method are included in the host cell of cultivating claim 56 in the substratum, until the binding molecule of described cell generation in conjunction with people GITR.

Images