CN1867585B - Fully human antibodies against human 4-1BB (CD137) - Google Patents

Abstract

Fully human antibodies and antigen-binding portions thereof that bind to human 4-1BB and that allow binding of human 4-1BB to a human 4-1BB ligand. In one aspect, the antibody is an IgG4 antibody. Also provided is a method for treating a disease in a subject comprising administering a therapeutically effective amount of the antibody to said subject.

Description

The fully human antibodies of anti-people 4-1BB (CD137)

Technical field

The present invention relates to fully human antibodies, be specifically related to complete (fully) people antibody of people 4-1BB (CD137).

Background technology

Multiple evidence proves that clearly antitumor immune reaction to a certain degree exists in the humans and animals.Among the cancer patients, the antigen that immune cellular component can tumor cell be expressed is such as gene product (S.Rosenberg, Nature, the 411:380-4 (2001) of the differentiation or the sudden change of carcinomebryonic antigen; P.van derBruggen etc., Immunological Rev., 188:51-64 (2002)).Various clinical studies show that the favourable prognosis meaning of having of tumor infiltrating lymphocyte (E. Halapi, Med.Oncol., 15 (4): 203-11 (1998); Y.Naito etc., Cancer Res., 58 (16): 3491-4 (1998); L.Zhang etc., N.E.J.Med., 348 (3): 203-13 (2003)).In addition, utilize immunomodulator such as cytokine or bacterial product, the therapy of cancer vaccine or acquired immunity treatment can cause regression (S.Rosenberg, the Cancer J.Sci.Am.6 (S): 2 (2000) of tumour in many patients; P.Bassi, Surg.Oncol., 11 (1-2): 77-83 (2002); S.Antonia etc., Current Opinion in Immunol., 16:130-6 (2004)).Although have these reactions, often can not effectively eliminate tumour cell at the immunizing power of cancer.Cause the reason of described failure can be divided into three groups of main types: (i) identification of the tumour of immunocyte is impaired, is caused by the variable expression of tumour antigen or the expression reduction institute of I class major histocompatibility complex (MHC); (ii) inhibitive ability of immunity tumor microenvironment is as the result (for example, TGF-β) of the tumor cell secretion SC factor; (iii) poor owing to lacking the immunogenicity of tumor that the expression of costimulatory molecules on tumour cell produce, it causes the tumour cell can not effective stimulus T cell.We show that for the progress of the understanding of the needs of tumour antigen identification and immunoeffectors function the possible strategy that is used to strengthen anti tumor immune response is to provide common stimulation by accessory molecule.Specific for tumour antigen T cell need stimulate altogether to start and the maintenance effector function.Therefore, the therapy of target costimulatory molecules can be used for regulating and strengthens immune response to tumour.

At present the model assumption T cells of T cell activation needs two kinds of signals with abundant activation: (i) signal that provides through the antigenic combination of processing, and it is by major histocompatibility complex (MHC) I quasi-molecule passs TXi Baoshouti; (ii) other signal of providing of the interaction of the costimulatory molecules of T cell surface and its part on the antigen presenting cell.Is insufficient by T cells identification antigen itself for the activated t cell activation.Do not have costimulatory signal, the T cell can or induce non-activity to eliminate by death.Signal by the CD28 costimulatory molecules is the key that starts of t cell responses seemingly.Yet CD137 (4-1BB) signal shows for keeping and enlarge antigenic immune response, and is important for producing memory T cell.

CD137 (4-1BB) is the member of Tumor Necrosis Factor Receptors (TNF-R) gene family, and it comprises the albumen that participates in regulating cell proliferation, differentiation and apoptosis.CD137 is the I type membrane glycoprotein of 30kDa, and its homodimer as 55 kDa is expressed.Described acceptor is at first described (B.Kwon etc., P.N.A.S.USA, 86:1963-7 (1989)) in mouse, identify (M.Alderson etc., Eur.J.Immunol., 24:2219-27 (1994) subsequently in the people; Z.Zhou etc., Immunol.Lett., 45:67 (1995)) (, be included in this paper and (see SEQ ID NO:2.) as a reference) also referring to disclosed PCT publication WO95/07984 and WO96/29348 and United States Patent (USP) 6,569,997.The CD137 of people and mouse form has 60% identity at amino acid levels.Conservative sequence exists in 5 other zones of tenuigenin structural domain and molecule, shows that these residues may be important (Z.Zhou etc., Immunol.Lett., 45:67 (1995)) for the function of CD137 molecule.The expression of CD137 mainly at lymphocyte series such as activated T-cell, activatory NK cell (NK) cell, NKT-cell, CD4CD25 are regulated on the T-cell, also on activatory thymocyte and intracutaneous lymphocyte.In addition, CD137 also at the bone marrow derived cell such as dendritic cell, monocyte, neutrophil leucocyte, and expressing on the eosinophil.Although CD137 expresses major limitation at immunity/inflammatory cell, have report describe its with the minority endotheliocyte relevant from the tissue of inflammatory site and tumour on express.

The functionally active of CD137 on the T cell fully characterizes.Show inducing T cell propagation and cytokine synthetic (mainly being IFN-γ) by the signal of CD137 under the anti-cd 3 antibodies that has suboptimal dosage, and suppressed the activatory necrocytosis.These effects have utilized mouse and people T-cell observation to (W.Shuford etc., J.Exp.Med., 186 (1): 47-55 (1997); D.Vinay etc., Semin.Immunol., 10 (6): 481-9 (1998); D.Laderach etc., Int.Immunol., 14 (10): 1155-67 (2002)).In people and mouse, stimulate improved effect thing function altogether, generate and cell toxicant such as IFN-γ, the person realizes by the quantity that improves antigen-specific and effect CD8+T-cell.Under the situation that lacks anti--CD3 signal, the stimulation by CD137 does not change the T cell function, shows that CD137 is a costimulatory molecules.

It is to be mediated by NF-κ B with various physiological functions and PI3K/ERK1/2 signal that CD137 stimulates the later observed physiological event of T cell.NF-κ B signal excitation Bcl- _XLThe expression of this anti-apoptosis molecule cause survival rate to improve thus, and PI3K and ERK1/2 signal is specifically responsible for cell cycle progress (H.Lee etc., J.Immunol., 169 (9): 4882-8 (2002)) of CD137-mediation.CD137 activation to the restraining effect of activation inductive necrocytosis external by (J.Hurtado etc. such as Hurtado, J.Immunol., 158 (6): 2600-9 (1997)) show, and also in vivo in the system, wherein anti-CD137 mono-clonal (mab) shows by preventing that clonal deletion from producing the long-term surviving (C.Takahashi etc. of superantigen activatory CD8+T-cell, J.Immunol., 162:5037 (1999)).Subsequently, two report demonstrations, under the different tests condition, the clonal expansion of CD137 Signal Regulation CD8+T-cell and survival (D.Cooper etc., Eur.J.Immunol., 32 (2): 521-9 (2002); M.Maus etc., Nat.Biotechnol., 20:143 (2002)).Observe the Bcl-that increases in the apoptosis of reduction and the CD8+T-cell after stimulating altogether _XLLevel is relevant, and the Bcl-2 expression is constant.Anti-apoptotic genes expression Bcl- _XLRaise demonstration by the mediation of NF-kB activation with bfl-1 via 4-1BB, this is because this NF-κ of PDTC B-specific inhibition thing suppresses the Bcl-of 4-1BB-mediation _XLRaise (H.Lee etc., J.Immunol., 169 (9): 4882-8 (2002)).On the other hand, the clonal expansion of activated T cell shows that by cyclin D2 the expression of D3 and E increases and p27 ^Kip1Proteic downward modulation mediation.This effect (H.Lee etc., J.Immunol., 169 (9): 4882-8 (2002)) occur in the mode with non-dependence that IL-2 relies on.

Generally speaking, CD137 stimulates the CD8+T-cell amplification, survival and the effector function that cause newly exciting to strengthen, and part directly acts on these cells.CD4+ and CD8+T-cell have shown the irritant reaction to CD137, but show enhancing higher in the CD8+ cell (W.Shuford etc., J.Exp.Med., 186 (1): 47-55 (1997) of T cell function; I.Gramaglia etc., Eur.J.Immunol., 30 (2): 392-402 (2000); C.Takahashi etc., J.Immunol., 162:5037 (1999)).As if based on the vital role of CD137 stimulation in CD8+T-cell function and survival, the operation of CD137/CD137L system provides the reasonable method of treatment tumour and virus antigen.

Recently, the formation type of CD137 on the dendritic cell (DC) of fresh separated is expressed in mouse (R.Wilcox etc., J.Immunol., 169 (8): 4230-6 (2002); T.Futagawa etc., Int.Immunol., 14 (3): 275-86 (2002)) and among the people (S.Pauly etc., J.Leukoc.Biol.72 (1): 35-42 (2002)) confirm.These reports show that the CD17 that stimulates on the DC causes the secretion of IL-6 and IL-12, and the more important thing is, it has strengthened the ability that DC stimulates the T cell that allogenic antigen is reacted.In addition, the CD137 signal among the confirmation DC such as Pan causes MHC I quasi-molecule and costimulatory molecules to raise, and makes the ability that DC soaks into tumour strengthen (P.Pan etc., J.Immunol., 172 (8): 4779-89 (2004)).Therefore, the CD137 on the DC stimulates the new way of DC propagation, maturation and migration seemingly altogether.

Activatory NK cell (NK) cell is stimulating the back to express CD137 (I.Melero etc., Cell Immunol., 190 (2): 167-72 (1998) with cytokine; R.Wilcox etc., J.Immunol., 169 (8): 4230-6 (2002)).Several pieces of reports confirm that the NK cell is for be important ((I.Melero etc., Cell Immunol., 190 (2): 167-72 (1998) for the antitumor autoimmunization of excitability CD137 antibody induction; R.Miller etc., J.Immunol., 169 (4): 1792-800 (2002); R.Wilcox etc., J.Immunol., 169 (8): 4230-6 (2002)).The exhaustion of NK cell obviously reduces the anti-tumor activity of anti--CD137 mabs.Propagation and IFN-γ secretion are induced in the connection of CD137 on the NK cell, but do not influence their cell lysis activity.Significantly, external, the amplification that the NK cell performance that CD137 stimulates causes activated T-cell for immunomodulatory or " subsidiary " activity of CD8+ cytolysis type T-cell.Therefore, the CD137 signal on the NK cell can be regulated the innate immune power to tumour.

Possible effect has been described in stimulation for CD137, and wherein excitability CD137 antibody can be induced inhibition (H.Hong etc., J.Immunother., 23 (6): 613-21 (2000) to the humoral response of T cell antigen in primates and in the mouse model; R.Mittler etc., J.Exp.Med., 190 (10): 1535-40 (1999)).Significantly, the CD137 agonistic antibody shows and causes and antibody dependent autoimmune disease such as systemic lupus erythematous and the related indication obvious alleviation (J.Foell etc. of tentative autoimmunity meningitis, N.Y.Acad.Sci., 987:230-5 (2003); Y. Sun etc., Nat.Med., 8 (12): 1405-13 (2002)).Recently, confirmations such as Seo, in the mouse model of rheumatoid arthritis, utilize excitability anti--treatment of CD137 antibody prevented the generation of disease, and obviously blocked progress (S.K.Seo etc., the Nat.Med.10 of disease; 1099-94 (2004)).Cause the mechanism of this effect not have finely to determine, but in the rheumatoid arthritis model, show that the treatment that utilizes the CD137 agonistic antibody causes producing the amplification of the CD11C-CD8+T cell of IFN-γ.IFN-γ stimulates dendritic cell to produce indoles amine (indolamine)-2 conversely, 3-dioxygenase (IDO), and it shows immunosuppressive activity.The CD137 signal of having inferred on antigen-activatory CD4+T-cell causes IFN-γ excretory to induce its activated macrophage.The activatory scavenger cell can produce B necrocytosis signal conversely.Can induce the activation inductive necrocytosis of these CD4+ activated T-cells subsequently by the continuous signal of the CD137 on the CD4+T-cell.Therefore, by eliminating antigen activated T-cell and B cell, observe the antibody response of reduction, the inflammatory diseases of observing the Th2-mediation thus obviously alleviates (B.Kwon etc., J.Immunol., 168 (11): 5483-90 (2002)).These research promptings utilize the purposes of excitability CD137 Antybody therapy inflammation or autoimmune disease, and need not to induce to immune comprehensive inhibition.

The native ligand of CD137, CD137 part (CD137L), the 34kDa glycoprotein member of TNF superfamily, mainly at activatory antigen presenting cell (APC) such as B cell, scavenger cell, detect on the dendritic cell, and, detect (R.Goodwin etc. in the cancerous cell line of activated T-cell and people's epithelial origin also at mouse B-cell lymphoma, Eur.J.Immunol., 23 (10): 2631-41 (1993); Z.Zhou etc., Immunol.Lett., 45:67 (1995); H.Salih etc., J.Immunol., 165 (5): 2903-10 (2000)).People CD137L and its mouse counterpart are shared 36% homology (M.Alderson etc., Eur.J.Immunol., 24:2219-27 (1994)).

Except giving CD-137-expression type cell with signal delivery, CD137 starts two-way signaling with combining of CD137L, causes the functional effect to CD137L-expression type cell.Langstein etc. confirm the zygotic induction IL-6 of the CD137L on CD137-Ig fusion rotein and the activatory monocyte, the generation of IL-8 and TNF-α, raise ICAM, and suppress IL-10, cause adhering to increase (J.Langstein etc., J.Immunol., 160 (5): 2488-94 (1998)).In addition, a high proportion of apoptosis (J.Langstein etc., J.Leukoc.Biol., 65 (6): 829-33 (1999)) are followed in monocytic propagation demonstration.These are observed, and (22 (5): studies confirm that 333-8 (2003)), its display functionality is anti--the two-forty propagation of CD137L antibody induction peripheral blood lymphocytes for S.Ju etc., Hybrid Hybridomics by Ju etc.Block the inhibition that described part causes the T cell activation.In addition, solubility CD137L sees (H.Salih etc., J.Immunol., 167 (7): 4059-66 (2001)) in rheumatoid arthritis and blood malignant disease patient's the serum.Thus, the interaction of CD137 and CD137L influence and cause functional effect to T-cell and APC.

In another importance of T cell function, confirm excitability anti--rescue of CD137 antibody (rescue) to the t cell responses of the proteantigen in the Aged Mice.On the books about antigenic immunoreactive age related is occurred reducing in a lot of documents, this is process (R.Miller, Science, the 273:70-4 (1996) that is known as immunosenescence; R.Miller, Vaccine, 18:1654-60 (2000); F.Hakim etc., Curr.Opinion Immunol., 16:151-156 (2004)).This phenomenon is because the balance change between the degree of cell amplification and cell survival or death causes.Bansal-Pakala etc. have detected this hypothesis, and secondary the stimulation altogether by CD137 is used in because the expression of CD3 or CD28 reduces, or quality of signals reduces the T-cell that causes and do not accept to strengthen under the situation of enough stimulations the T-cell response.Their studies show that with young mice compared, and Aged Mice lacks external anamnestic reaction (P.Bansal-Pakala etc., J.Immunol., 169 (9): 5005-9 (2002)).But in the Aged Mice with anti--CD137 mab processing, the propagation of T cell is identical with observed situation in young mice with the cytokine reaction.Though cause the concrete mechanism of this effect not clear, think for example Bcl-of anti-apoptosis molecule _XLExpression strengthen and promote the body internal secretion of IL-2 in rescue defective t cell responses, to work.These studies confirm that the anti-CD137 antibody of excitability saves the possibility of the weak t cell responses in the individuality of old immunocompromised host, and for the meaningful far-reaching enlightenment of the application of anti-CD137 antibody in the cancer patients.

The effect of CD137 targeted therapies in cancer therapy shows by effect research in the mouse body that utilizes the anti-mouse CD137 of excitability monoclonal antibody.In the document of Melero etc., excitability is anti--and mouse CD137 antibody causes curing (I.Melero etc. in P815 mastocytoma (mastocytoma) and reduced immunogenicity tumor model Ag104, Nat.Med., 3 (6): the Anti-tumor effect needs CD4+, CD8+T-cell and NK cell 682-5 (1997)), and this is because the selection gonosome in-fighting of every kind of subgroup exhausts the minimizing that causes anti-tumour effect or lacks fully.A plurality of investigators use anti-CD137 antibody to confirm validity (J.Kim etc., Cancer Res., 61 (5): 2031-7 (2001) of this method for cancer therapy; O.Martinet etc., Gene Ther., 9 (12): 786-92 (2002); R.Miller etc., J.Immunol., 169 (4): 1792-800 (2002); R.Wilcox etc., Cancer Res., 62 (15): 4413-8 (2002)).

For supporting the antitumor efficacy data of excitability CD137 antibody, the signal demonstration that CD137L provides excites CTL active and antitumor reaction (M.DeBenedette etc., J.Immunol., 163 (9): 4833-41 (1999); B.Guinn etc., J.Immunol., 162 (8): 5003-10 (1999)).Several reports confirm that the transgenosis of CD137 part in murine sarcoma causes tumor rejection, need to have confirmed to stimulate altogether to produce effective immune response (S.Mogi etc., Immunology, 101 (4): 541-7 (2000); I.Melero etc., Cell Immunol., 190 (2): 167-72 (1998); B.Guinn etc., J.Immunol., 162 (8): 5003-10 (1999)) .Salih etc. has reported the expression (H.Salih etc. of CD137L in human cancer and human carcinoma cell line, J.Immunol., 165 (5): 2903-10 (2000)), and confirmed that the tumour cell of expressing described part can send stimulus signal to the T cell, and causing discharging IFN-γ and IL-2, the level of CD137L is relevant on this effect and the tumour.Whether expressing CD137L in people's tumour, can to make these tumours more responsive for excitability CD137 antibody be unknown.

CD137L-/-mouse reduced the CD137/CD137L system at the T cell to importance (M.DeBenedette etc., J.Immunol., 163 (9): 4833-41 (1999) in virus and the tumor response; J.Tan etc., J.Immunol., 164 (5): 2320-5 (2000); B.Kwon etc., J.Immunol., 168 (11): 5483-90 (2002)).Utilize the CD137 that studies confirm that of CD137-and CD137L-deficient mice to stimulate at graft versus host disease the importance in the antiviral molten cell t cell responses altogether.The propagation of T cell strengthens in the CD137 deficient mice, but cytokine produces and the toxicity T cytoactive reduces (B.Kwon etc., J.Immunol., 168 (11): 5483-90 (2002); D.Vinay etc., Immunol.Cell Biol., 81 (3): 176-84 (2003)).Recently, the metastases that shows knock-out mice (CD137-/-) is compared frequency higher (4-doubly) with control mice.These Notes of Key Datas utilize excitability anti--to recover the CD137 signal be the feasible method that strengthens the cell immune response of virus antigen and cancer to CD137 antibody.

Except the data in the mouse body inner model (it supports the participation of CD137 signal in anti tumor immune response), the effect of CD137 in producing effector T cell of in former generation people tumor sample, carrying out that studies confirm that.In the patient of Ewing sarcoma (Ewing sarcoma), demonstration tumour internal effect T-cells such as Zhang present the CD3+/CD8+/CD28-/CD137+ phenotype.Unexpectedly, observe co-existing in of progressivity tumor growth and antineoplastic immune (effector T cell).Stripped stimulation study utilizes patient's cell to confirm the T cell proliferation of tumor inducing and the common stimulation that activation need utilize CD137L.Utilize anti--CD3/CD137L, but not anti--CD3/ anti--CD28 stimulates PBL to cause molten tumor effect thing.These researchs provide further evidence, amplification (H.Zhang etc., cancer Biol.Ther., 2 (5): 579-86 (2003)) that the common stimulation of CD137 mediation can cause tumor response CTL.In addition, the expression of CD137 confirms tumor infiltrating lymphocyte (Y.Wan etc., World J.Gastroenterol, 10 (2): 195-9 (2004)) in the hepatocellular carcinoma (HCC).The recall rate of expressing by RT-PCR CD137 in HCC is 19/19, and the recall rate by immunofluorescence dyeing is 13/19.Otherwise CD137 does not detect in the peripheral mononuclear cells of same patient.These researchs are not intended to express relevant with CD137 clinical disease.Therefore, the existence of in Ewing sarcoma, carrying out that studies show that the TIL that expresses CD137, accompanying diseases progress with hepatocellular carcinoma.In Ewing sarcoma, confirm that CD137+TIL can kill stripped tumour cell, prompting CD137 approach is complete in these patients, and the possible inhibition factor suppresses their function in tumor microenvironment.Therefore, can infer being administered systemically of excitability CD137 antibody can provide these effector T cells of amplification essential signal.

Except its effect in cancer immunity power progress, testing data has supported to utilize purposes (B.Kwon etc., Exp.Mol.Med., 35 (1): 8-16 (2003) in CD137 agonistic antibody treatment autoimmunization and the virus disease; H.Salih etc., J.Immunol., 167 (7): 4059-66 (2001); E.Kwon etc., P.N.A.S.USA, 96:15074-79 (1999); J.Foell etc., N.Y.Acad.Sci., 987:230-5 (2003); Y.Sun etc., Nat.Med., 8 (12): 1405-13 (2002) S.K.Seo et al, Nat.Med.10; 1099-94 (2004)).

Therefore, based on the effect of 4-1BB in regulating immune response, need to produce the Anti-Human 4-1BB antibody with agonist activity, it can be used for treatment or prevents the human disease such as cancer, catch, and autoimmune disease.

The invention summary:

The invention provides fully human antibodies, it is in conjunction with people 4-1BB (H4-1BB) and allow H4-1BB in conjunction with people 4-1BB part (H4-1BBL).Therefore the present invention relates to not block H4-1BB and H4-1BBL bonded antibody, allow combining of antibody of the present invention and H4-1BBL and H4-1BB thus in conjunction with H4-1BB.The present invention also provides has the active antibody of excitability, and wherein antibody and combining of H4-1BB cause the immunoreactive enhancing and the stimulation of H4-1BBL mediation.These antibody can be used as the immunostimulant thing of the antitumor or antiviral immunity of immunostimulant thing reaction, or the immunomodifier of the cell-mediated autoimmune disease of T.Described antibody also can be used as diagnostic tool and detects patient's the blood of cancer, autoimmunization or other disease or the H4-1BB in the tissue.

On the one hand, the invention provides monoclonal antibody or its antigen-binding portion thereof, its specificity is in conjunction with H4-1BB, comprise variable region of light chain and variable region of heavy chain, wherein variable region of light chain comprises CDR1 (complementary determining region 1) as shown in Figure 4, CDR2 (complementary determining region 2), and CDR3 (complementary determining region 3), variable region of heavy chain comprises as Fig. 3 or CDR1 (complementary determining region 1) shown in Figure 7, CDR2 (complementary determining region 2), and CDR3 (complementary determining region 3).Monoclonal antibody (mab) can be, for example, and IgG4 antibody or IgG1 antibody.

On the other hand, the invention provides monoclonal antibody or its antigen-binding portion thereof, wherein light chain comprises variable region as shown in Figure 4, and heavy chain comprises as Fig. 3 or variable region shown in Figure 7.

On the other hand, the invention provides monoclonal antibody, it comprises light chain and heavy chain, and wherein light chain comprises the amino-acid residue 21-236 of SEQ ID NO:6, and heavy chain comprises the amino-acid residue 20-467 of SEQ ID NO:3.On the other hand, the invention provides monoclonal antibody, it comprises light chain and heavy chain, and wherein light chain comprises the amino-acid residue 21-236 of SEQ ID NO:6, and heavy chain comprises the amino-acid residue 20-470 of SEQ ID NO:9.

Antibody of the present invention has the wide range of therapeutic purposes as the immunomodifier of disease, described diseases such as cancer, autoimmune disease, inflammatory diseases and catching.

The present invention also provides the method for cancer among the treatment experimenter, comprises that the administration experimenter treats the antibody of the present invention of significant quantity.On the one hand, this method also comprises administration of vaccines.Suitable vaccine comprises, for example, and tumour-cell vaccine, dna vaccination, the tumour-cell vaccine that GM-CSF-modifies, or the dendritic cell vaccine of antigen loaded.Described cancer can be, for example, and prostate cancer, melanoma, or epithelial cancer.

On the other hand, the invention provides the method that is used for the enhancing immunity reaction, comprise administration antibody of the present invention and SIV gag vaccine.On the other hand, the invention provides the method that is used for the enhancing immunity reaction, comprise administration antibody of the present invention and PSA vaccine.On the other hand, the invention provides the immunoreactive method that is used to strengthen to SIV gag vaccine, comprise administration antibody of the present invention.On the other hand, the invention provides the immunoreactive method that is used to strengthen to the PSA vaccine, comprise administration antibody of the present invention.

The present invention also provides pharmaceutical composition, and it comprises antibody of the present invention, or its antigen-binding portion thereof, and pharmaceutically acceptable carrier.Described pharmaceutical composition can be separately or with other medicament coupling administration, for example, medicament such as chemotherapeutics or vaccine or other immunomodulator of treatment cancer.

The present invention also provides isolating polynucleotide, and it comprises the nucleotide sequence that is selected from down group: (a) Nucleotide, the amino-acid residue 20-467 of its encoding amino acid sequence SEQ ID NO:3; (b) Nucleotide, its encoding amino acid sequence SEQ ID NO:3; (c) Nucleotide, the amino-acid residue 21-236 of its encoding amino acid sequence SEQ IDNO:6; (d) its encoding amino acid sequence of Nucleotide SEQ ID NO:6; (e) Nucleotide, the amino-acid residue 20-470 of its encoding amino acid sequence SEQ ID NO:9; (f) its encoding amino acid sequence of Nucleotide SEQ ID NO:9; (g) Nucleotide, the fragment of its encoding amino acid sequence (a)-(f), such as the variable region, constant region, or one or more CDR.The isolating polynucleotide of the present invention also comprise at least one CDR of code pattern 3, at least one CDR of Fig. 4, or the nucleotide sequence of at least one CDR of Fig. 7.The invention provides isolating polynucleotide, it comprises nucleotide sequence SEQID NO:1, SEQ ID NO:4, or SEQ ID NO:7.

The present invention also provides isolated polypeptide, comprises to be selected from SEQ ID NO:3 the aminoacid sequence of the group that SEQ ID NO:6 and SEQ ID NO:9 form.On the other hand, the invention provides the amino-acid residue 20-467 that isolated polypeptide comprises aminoacid sequence SEQ ID NO:3, isolated polypeptide, it comprises the amino-acid residue 21-236 of aminoacid sequence SEQ ID NO:6, isolated polypeptide, it comprises the amino-acid residue 20-470 of aminoacid sequence SEQ ID NO:9.On the other hand, the invention provides isolated polypeptide, it comprises Fig. 3, Fig. 4, or the aminoacid sequence of at least one CDR of Fig. 7, or Fig. 3, Fig. 4, or the variable at least or constant region of Fig. 7.

The present invention also comprises immunoglobulin (Ig), and it has binding specificity H4-1BB, and described immunoglobulin (Ig) comprises antigen binding domain.In the aspect, described immunoglobulin (Ig) is the Fab of antibody of the present invention or F (ab ') ₂

The present invention also comprises the clone that produces antibody of the present invention or its antigen-binding portion thereof, comprises the recombinant expression vector of Nucleotide of the present invention, and the method that produces antibody of the present invention by the clone of cultivating generation antibody.

The accompanying drawing summary:

Fig. 1 shows the plasmid figure of pD17-20H4.9.h4a.

Fig. 2 shows the plasmid figure of pD16gate-20H4.9.LC.

Fig. 3 (Fig. 3 A-3H) shows the nucleotide sequence of plasmid pD17-20H4.9.h4a, comprise coding strand (SEQ ID NO:1), (leading peptide is the amino-acid residue 1-19 of SEQ ID NO:3 for complementary strand (SEQ ID NO:2) and described coding strand amino acid sequence coded; Heavy chain is the amino-acid residue 20-467 of SEQ ID NO:3).

Fig. 4 (Fig. 4 A-4F) shows the nucleotide sequence of plasmid pD16gate-20H4.9.LC, comprise coding strand (SEQ ID NO:4), (leading peptide is the amino-acid residue 1-20 of SEQ ID NO:6 for complementary strand (SEQ ID NO:5) and described coding strand amino acid sequence coded; Light chain is the amino-acid residue 21-236 of SEQ ID NO:6).

Fig. 5 shows the synoptic diagram of 20H4.9-IgG1 sequence of heavy chain construct.

Fig. 6 shows the synoptic diagram of 20H4.9 sequence of light chain construct.

Fig. 7 (Fig. 7 A-7D) shows the nucleotide sequence and the aminoacid sequence of 20H4.9-IgG1 heavy chain construct, comprise coding strand (SEQ ID NO:7), (leading peptide is the amino-acid residue 1-19 of SEQ ID NO:9 for complementary strand (SEQ ID NO:8) and described coding strand amino acid sequence coded; Heavy chain is the amino-acid residue 20-470 of SEQ IDNO:9).

Fig. 8 (Fig. 8 A-8B) diagram available from the bonded ELISA result (Fig. 8 A) of mab 20H4.9-IgG1 and people CD137 and mab 20H4.9-IgG1 to the interactional influence of CD137-CD137L (Fig. 8 B).

Fig. 9 (Fig. 9 A-9B) diagram is available from mab 20H4.9-IgG1 and the plain people who stimulates of her room promise of PMA-mould (ionomycin) or the bonded result of macaque (cynomolgus monkey) cell.People CEM (Fig. 9 A) or monkey PBMC (Fig. 9 B) are incubated with 20H4.9-IgG1 or people CD137L fusion rotein.

Figure 10 (Figure 10 A-10B) diagram utilizes anti--CD137 antibody to induce the result of IFN-γ gained in being total to stimulation study, and its pg/ml that is expressed as with respect to contrast increases multiple.Because the background response of donor is different, data are with respect to control treatment (=1) stdn.Be respectively 592pg/ml and 505pg/ml with independent anti--CD3 stimulation from the intermediate value IFN-γ baseline values of people T-cell (Figure 10 A) or monkey PBMC (Figure 10 B).

Figure 11 provides mab 20H4.9-IgG4 and mab 20H4.9-IgG1 and the resonance of people CD137 bonded cytoplasmic mass to draw.

Figure 12 illustrates 20H4.9-IgG4 and combines with the concentration dependent of people's cem cell of PMA ionomycin stimulation, but debond is without the cem cell that stimulates.

Figure 13 (Figure 13 A-B) illustrates inducing IFN-γ in the common stimulation study that utilizes anti--CD137 antibody.The pg/ml that the result is expressed as with respect to contrast increases multiple.Because because the background response of donor is different, data are with respect to control treatment (=1) stdn.Be respectively 592pg/ml and 505pg/ml with independent anti--people T-cell (Figure 13 A) of CD3 stimulation or the intermediate value IFN-γ baseline values of monkey PBMC (Figure 13 B).

Figure 14 (Figure 14 A-14B) diagram is passed through mab 20H4.9-IgG4 to IFN-γ synthetic dose dependent enhanced results (Figure 14 A), and the antibody linked effect (Figure 14 B) that adds cross-linking type Anti-Human's IgG antibody (7 μ g/ml).

Figure 15 illustrates the influence of mab 20H4.9-IgG4 to T-cell survival and cell cycle progress.People T-cell carries out common stimulation with anti--CD3 (1ug/ml) ± mab20H4.9-IgG4 in listed concentration.After 6 days on-tests, collecting cell and with the dyeing of annexin-V and propidium iodide determining survivaling cell number (Annexin V/PI feminine gender), or be in cell in the cycle with the dyeing detection with PE-link coupled cyclin D2.The result represent parallel detection mab 20H4.9-IgG4 4 groups mean value (± SD).

Figure 16 (Figure 16 A-16D) demonstration utilizes after dna vaccination ± Anti-Human 4-1BB antibody treatment, antigen-specific IFN-gamma reaction in the macaque, as utilize the ELISPOT mensuration.Animal is used SIV gag vaccine (the 0th, 28,56 days; Figure 16 A), SIV gag vaccine (the 0th, 28,56 days) and mab20H4.9-IgG4 are (the 12nd, 15 and 19 day; Or SIV gag vaccine (the 0th, 28,56 days) and hu39E3.G4 (the 12nd, 15 and 19 day Figure 16 B); Figure 16 C) handles.One treated animal unprocessed (Figure 16 D).Handle each time of back, collect blood, separate PBMC and assess the ability of their secretion of gamma-IFN in the presence of antigenic stimulation.

Detailed Description Of The Invention:

The present invention relates to preparation and qualitative (the comprising that fusion comprises the Fab of antibody of the present invention) of antibody and Fab thereof, it is used for the treatment of diseases such as cancer, infectious diseases, inflammatory disease or autoimmunity disease. Described cancer for example can be, prostate cancer, melanoma, or epithelioma.

Described antibody capable is in conjunction with H4-1BB, and can show high-affinity and effectively strengthen t cell responses H4-1BB. On the one hand, described antibody induction stimulates the generation of IFN-γ in the determination method altogether, but does not affect the combination of H4-1BB and its respective ligand H4-1BBL, and complement-fixing not.

Antibody of the present invention can prepare by methods known in the art. On the one hand, described antibody can prepare by expressing in such as myeloma or hybridoma at cell such as the Immortalized eukaryotic of transfection

Antibody of the present invention can use separately, or with other treatment agent such as radiotherapy (comprising irradiation), hormone therapy, cytotoxic agent, vaccine, and other immunomodulator use together such as cell factor and biological respinse trim. These medicaments specifically can be used for treating cancer and immunoproliferation disease.

On the one hand, the invention provides monoclonal antibody (mab) 20H4.9-IgG4. Fig. 1 and 2 provides respectively the plasmid figure of pD17-20H4.9.h4a and pD16gate-20H4.9.LC, and described plasmid can be used for preparing mab 20H4.9-IgG4. Fig. 3 (Fig. 3 A-3H) provides the nucleotide sequence of plasmid pD17-20H4.9.h4a, comprise coding strand (SEQ ID NO:1), (leader peptide is the amino acid residue 1-19 of SEQ ID NO:3 to the amino acid sequence of complementary strand (SEQ ID NO:2) and described coding strand coding; Heavy chain is the amino acid residue 20-467 of SEQ ID NO:3). Fig. 4 (Fig. 4 A-4F) shows the nucleotide sequence of plasmid pD16gate-20H4.9.LC, comprise coding strand (SEQ ID NO:4), (leader peptide is the amino acid residue 1-20 of SEQ ID NO:6 to the amino acid sequence of complementary strand (SEQ ID NO:5) and described coding strand coding; Light chain is the amino acid residue 21-236 of SEQ ID NO:6).

On the other hand, the invention provides monoclonal antibody (mab) 20H4.9-IgG1. Fig. 5 illustrates the sequence of heavy chain construct of mab20H4.9-IgG1. Fig. 6 illustrates the sequence of light chain construct of mab 20H4.9, is used for mab 20H4.9-IgG4 and 20 H4.9-IgG1. Fig. 7 provides the nucleotide sequence (coding strand (SEQ ID NO:7) and complementary strand (SEQ ID NO:8)) of the sequence of heavy chain construct of Fig. 5, and the amino acid sequence of coding strand coding (leader peptide is the amino acid residue 1-19 of SEQ ID NO:9; Heavy chain is the amino acid residue 20-470 of SEQ ID NO:9). The light chain of mab 20H4.9-IgG1 is identical with the light chain of mab20H4.9-IgG4.

The present invention also comprises having the NOS:3 from SEQ ID, the antibody that the conserved amino acid of heavy chain and light-chain amino acid sequence shown in 6 and 9 replaces, and it does not affect in conjunction with having H4-1BB basically. Conservative replacement generally includes a seed amino acid to the amino acid whose replacement of another kind of similar performance, for example the replacement in the lower group: valine, glycine; Glycine, alanine; Valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; Lysine, arginine; And phenylalanine, tyrosine.

The polynucleotides of code book invention polypeptide also comprise expression control sequenc usually, and it is operatively connected in polypeptid coding sequence, comprise natural connection with the allogeneic promoter district. Preferably, described expression control sequenc will be the eukaryotic promoter system in the carrier, and it can transform or the transfection eukaryotic host cell, but also can use for the control sequence of eucaryon host. In case carrier is impregnated in suitable host, the host keeps under the condition that is suitable for the described nucleotide sequence of high level expression, as required, can collect then and the purifying light chain, heavy chain, light/heavy chain homodimer or complete antibody, binding fragment or other immunoglobulin (Ig) form. (seeing S.Beychok, Cells of Immunoglobin Synthesis, Academic Press, N.Y. (1979)). Single-chain antibody or miniantibody (being blended in the single-chain antibody of one or more CH domain) also can link to each other to prepare with the DNA of coded polypeptide joint by the nucleotide sequence with coding VL disclosed by the invention and VH district.

Prokaryotic hosts can be used for expressing antibody of the present invention such as Escherichia coli and other microorganism such as yeast. Except organism, the mammalian tissues cell culture also can be used for expressing and preparing antibody of the present invention. Eukaryotic is preferred, because can secrete the multiple suitable host cell of complete immunoglobulin (Ig) is to be developed, comprise, for example, CHO (Chinese hamster ovary) clone, COS (cercopithecus aethiops fibroblast clone) clone, HeLa cell, myeloma cell line, and hybridoma. The expression vector of these cells can comprise expression control sequenc, such as promoter or enhancer and necessary machining information site, and such as ribosome bind site, the RNA splice site, polyadenylic acid site, and transcription terminator, all are known in the art.

The carrier (for example, heavy chain and/or light chain coded sequence and expression control sequenc) that contains target DNA fragment can change host cell over to by known method, and it depends on the type of cell host. For example, the calcium chloride transfection is usually used in eukaryotic, and calcium phosphate is processed, and lipofection or electroporation can be used for other cell host. (see, for example, T.Maniatis etc., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Press (1982)).

In case express, antibody, their dimer, single light and heavy chain, or other immunoglobulin (Ig) form can be according to this area standard method purifying, such as ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis etc. The homogeneity at least substantially pure immunoglobulin (Ig) of 90-95% is required, homogeneity 98-99% or higher be more desirable.

Antibody of the present invention can be used for regulating T cell and antibody-mediated immune response. Usually the morbid state that is suitable for treating comprises cancer, catches, and inflammatory disease and autoimmunity disease be such as multiple sclerosis, rheumatoid arthritis, systemic loupus erythematosus and myasthenia gravis.

The present invention also provides pharmaceutical composition to comprise at least a antibody of the present invention and pharmaceutically acceptable carrier. and described pharmaceutical composition can be by conventional sterilising technology sterilization known in the art.Described pharmaceutical composition also can contain pharmaceutically acceptable auxiliary substance, and this is that suitable physiological condition is required, such as pH regulator and buffer reagent, stability enhancers such as N.F,USP MANNITOL or tween (tween) 80, toxicity conditioning agent etc., for example sodium acetate, sodium-chlor, Repone K, calcium chloride or human serum albumin.

Antibody of the present invention and pharmaceutical composition specifically can be used for parenteral admin, comprise through subcutaneous intramuscular and intravenous administration.The pharmaceutical composition of parenteral admin can comprise and is dissolved in the antibody-solutions that can accept the preferred aqueous carrier of carrier.Multiple aqueous carrier can use, and all is known in the art water for example, buffered water, salt solution, glycine etc.These solution are the aseptic particulate matters that do not have usually.Especially advantageously the parenteral composition of preparing unit dosage form is used for the homogeneity of facilitation administration and reagent.

Described pharmaceutical composition can comprise further that other medicament is used for the treatment of disease.On the one hand, described pharmaceutical composition comprises and is used for the treatment of cancer, infectious diseases, the medicament of inflammatory diseases and autoimmune disease.Antibody of the present invention also can be total to administration or be used for the treatment of the respectively administration of medicament of disease with another kind.

Antibody of the present invention can with other medicament together administration to strengthen among the patient immune response to cancerous cells.On the one hand, described antibody and immunogenicity medicament, such as cancerous cells, the tumour antigen of purifying (comprising recombinant protein, peptide and carbohydrate molecule), or with the cell and the cell-surface antigens coupling of the gene transfection of coding immunostimulating cytokine.On the other hand, described antibody and vaccine be such as tumour-cell vaccine, dna vaccination, and the tumour-cell vaccine of gene-modification is such as the tumour-cell vaccine that GM-CSF-modifies, peptide vaccine, or the dendritic cell vaccine coupling of antigen loaded.

Many test strategies of inoculating at tumour are developed.In the middle of one of these strategies, the vaccine utilization is from body or the preparation of allogene tumour cell.These cell vaccines have shown when tumour cell is transduceed with expression of GM-CSF the most effective.GM-CSF shows effective activator (Dranoff etc., P.N.A.S., the 90:3539-43 (1993) of the antigen presentation that is tumor inoculation; E.Jafee etc., J.Clin.Oncol., 19:145-56 (2001); R.Salgia etc., J.Clin.Oncol., 21:624-30 (2003)).

The research of genetic expression and extensive gene expression pattern causes defining so-called tumour specific antigen (S.Rosenberg, Immunity 10:281-7 (1999)) in the various tumours.In many situations, these tumour specific antigens are in tumour and produce the not synantigen of expressing in the cell of tumour, melanocyte antigen gp100 for example, MAGE antigen, Trp-2.The target that tumour-specific T cell among many hosts of being is arranged in these antigens.Antibody of the present invention can and guide these antigenic Th1 immune responses with amplification with multiple recombinant protein of expressing in tumour and/or peptide coupling.These albumen are observed by immunity system as self antigen and usually thus to its tolerance.

One aspect of the present invention, described antibody and immunomodulator comprise SIV gag antigen (as model HIV dna vaccination) or prostate specific antigen (PSA), or comprise the dna vaccination coupling of the nucleotide sequence of coding SIV gag antigen or prostate specific antigen (PSA).The PSA vaccine for example is described in, M.Pavlenko etc., B r.J.Cancer, 91 (4): 688-94 (2004); J.Wolchok etc., Semin.Oncol., 30 (5): 659-66 (2003); J.Kim etc., Clin.Cancer Res., 7 (3 Suppl.): 882s-889s (2001).SIV gag epidemic disease is described in, for example, and B.Makitalo etc., J.Gen.Virol., 85 (Pt8): 2407-19 (2004); N.Letvin etc., J.Virol., 78 (14): 7490-7 (2004); S.Mossman etc., AIDS Res.Hum.Retroviruses., 20 (4): 425-34 (2004); F.Bertley etc., J.Immunol., 172 (6): 3745-57 (2004); L Patterson etc., J.Virol., 78 (5): 2212-21 (2004); E.O ' Neill etc., AIDS Res.Hum.Retroviruses, 19 (10): 883-90 (2003); Z.Bu etc., Virology, 309 (2): 272-81 (2003).

Tumour antigen can comprise, albumen Telomerase for example, and it is that synthetic fringes of chromosome enzyme is required, and expression (N.Kim etc., Science, 266,2011-2013 (1994)) in surpassing 85% human cancer and only a limited number of idiosoma organization.Tumour antigen is " the neo-antigen " for expressing in the cancer cells also, and its reason is to change protein sequence and produce the somatic mutation of two kinds of fusion roteins between the irrelevant sequence or from the idiotype of B cell tumour.Other tumor vaccine also can comprise the protein from the virus relevant with human cancer, and such as human papillomavirus (HPV), hepatitis virus (HBV and HCV) and Ka Boqi bleb sarcoma virus (Kaposi ' s Herpes Sarcoma Virus) are (KHSV).The tumour specific antigen of the another kind of form that can use with antibody of the present invention is the heat shock protein(HSP) (HSP) that separates from the purifying of tumor tissues itself.These heat shock protein(HSP)s contain from the proteic fragment of tumour cell and these HSP can efficiently be delivered to antigen presenting cell to excite tumour immunity (R.Suot etc., Science 269:1585-1588 (1995); Y.Tamura etc., Science 278:117-120 (1997)).

Antibody of the present invention also can be used for strengthening the immune response of vaccine to virus antigen, and described virus antigen is such as HIV or HCV.Antibody of the present invention also can be used for strengthening the immune response to other immunomodulator, and excites the memory immune response.The example of these medicaments be cytokine such as GM-CSF, IL-2, IL-15, IL-12, the Fl3 part, CD40 part, adjuvant are such as CpG-oligodeoxynucleotide (DNA of bacteria), or anti-OX-40 or CTLA-4 antibody.

Pharmaceutical composition of the present invention can be administered for preventative and/or the treatment of curing the disease property.In the application of curing the disease property, described pharmaceutical composition is to be enough to cure or give to suffer to the amount of small part retardance or treatment disease the patient of disease.Suitable this result's the amount that realizes is defined as " treatment significant quantity ".The significant quantity of this purposes will depend on the severity of morbid state and patient (comprise, for example, the overall status of disease autoimmunization system), and can determine by those skilled in the art.In the concrete application, be not the patient who has been in the morbid state, to strengthen the resistibility of patient to morbid state with described pharmaceutical composition.Described amount is defined as " prevention significant quantity ".In this purposes, accurate amount depends on patient's state of health (comprise, for example, the overall status of patient's autoimmunization system), and can be determined by those skilled in the art.On the one hand, described prophylactic applications is to be used to prevent tumor recurrence.

Embodiment:

Embodiment 1: the preparation of antibody

Material and method

The complete human monoclonal antibodies of people CD137 (4-1BB) acceptor is at HuMAb-Mouse Middle produce (Medarex, Inc., Princeton, New Jersey).The 25 μ g people CD137 extracellular domains immunity of HuMAb mouse in abdominal cavity (i.p.) and subcutaneous (s.c.) usefulness RIBI adjuvant (Ribi Immunochemical) 5 times.Before the infusion, mouse is strengthened with the antigen of same amount through intravenously (i.v.).Be blended in murine myeloma cell from splenocyte according to standard method through immune mouse with suitable anti-huCD137 antibody titers.

Anti-Human CD3 mab (can swell: HIT3a), the ELISA test kit that is used for people and monkey IFN-γ, raji cell assay Raji pearl array (cytometric bead array) is test kit (CBA), the antibody that is used for flow cytometry with all couplings available from BD Pharmingen (San Diego, California).Human IgG ₁And human IgG ₁κ available from Sigma-Aldrich (St.Louis, Missouri).Cem cell (ATCC-CRL 2265) is available from ATCC, substratum (RPMI), foetal calf serum (FBS) available from Mediatech Inc. (Herndon, Virginia).Sheep red blood cell (SRBC) Alsevers available from Colorado Serum Co. (Denver, Colorado).

Hybridoma screening: detect and the combining of huCD137 by ELISA: for identifying the hybridoma of secreting Anti-Human CD137 antibody, with 1 μ g/ml people CD137-mouse IgG _2bFusion rotein PBS solution is spent the night by elisa plate (Nunc MaxiSorp) at the 4C bag.This plate adds 1% bovine serum albumin (BSA) at room temperature sealing 20min with PBS-T then subsequently with the PBS washing that contains 0.01% tween-80 (PBS-T) three times.The 50 microlitre supernatants that will dilute in PBS-T add entering plate envrionment temperature insulation 1-2 hour.Wash plate as described above then, the combination of antibody by with alkaline phosphatase link coupled goat F (ab ') ₂(Pennsylvania) insulation detects Anti-Human IgG antibody for Jackson Laboratories, West Grove.Plate is read plate with the pNPP colour developing and at 405nm.

Blocking test: the ability of assessing the hybridoma permission CD137-CD137L reaction of 26 kinds of secretory antibodies discerning huCD137 by ELISA.These analyses are carried out in the ELISA pattern at first.Plate personnel selection CD137-muIgG _2b, 0.2 μ g/ml, 100 μ l/ holes bags quilt.With mab 20H4.9-IgG1, or control antibodies, serial dilution in PBS-T and 1% bovine serum albumin are added into plate.The CD137L-CD8 fusion rotein is added entering plate with concentration 0.2 μ g/ml.The combination of antibody utilizes biotinylated resisting-CD8 antibody, and (Bayport Minnesota) detects for 0.2 μ g/ml, Ancell Corporation.After the washing for several times, add streptavidin-alkaline phosphatase (1: 2000) and pNPP and be used to detect bonded antibody, read plate at 405nm.

For confirming that selected antibody does not change the CD137-CD137L combination, antibody purified is analyzed qualitative by BIAcore subsequently.All tests are all carried out on BIAcore 3000 devices (BIAcore Inc., Piscataway, New Jersey).People CD137 is with the carboxyl-methylated dextran surface of high-density Covalent Immobilization at BIAcore sensing chip (sensorchip) (BIAcore Inc., Piscataway, New Jersey).Injection, is carried out among the pH 5.0 at the 10mM acetate buffer with 2 μ g/mL.The active ester that does not occupy is subsequently by sealing such as excess ethyl alcohol amine.The regeneration 10mM glycine on surface, pH 2.0 carries out.

Anti--CD137 antibody the sample of purifying utilizes the HEPES buffered saline, and pH 7.4 is diluted to concentration 200-1000nM, has replenished 0.15M NaCl and 0.005% tensio-active agent P20 (HBS-EP) in the described salt solution.People CD137L-CD8 fusion rotein (huCD137L) is as the source of CD137 part.Test, on the contrary wherein huCD137L before anti--CD137 antibody, inject or.Injection can be carried out by flow velocity 5 μ L/min.Bonded part and antibody are by utilizing 10 mM glycine buffers, and pH 2.0 regeneration remove.

People T-cell purification: T-cell or PBMC are available from healthy people's donor.Blood collecting is in EDTA, be suspended in and eluriate damping fluid (RPMI, wherein contain 2.5mM EDTA, 10 μ g/ml PXB), lower floor is lymphocyte separating medium (Lymphocyte Separaion Medium) (LSM, Mediatech Inc., Herndon, Virginia), 1800rpm is centrifugal 25 minutes.The collecting cell interface, centrifugal 10 minutes of 1500rpm.Subsequently, cell debris is resuspended in the elutriation damping fluid and washs sheep red blood cell (SRBC) (SRBC, 1: 10 extent of dilution), be incubated 1 hour on ice.Cell is with being placed on the LSM centrifugal 25 minutes of 2500rpm.Remove the interface and use SRBC lysis buffer cracking SRBC.Wash isolating T-cell and be resuspended among the 10%FBS/RPMI.

Flow cytometry: the combining by flow cytometry of CD137 of expressing on Anti-Human CD137 antibody and the cell measured.People T-chronic myeloid leukemia clone (CEM) or macaque peripheral blood lymphocytes (PBMC) are used for these research.These cells not formation type are expressed CD137, but acceptor can by with phorbol myristate (ester) (PMA, 10ng/ml) and ionomycin (1 μ M) stimulation 18hr induce.Washed cell is also with the insulation in various dyeing damping fluids (salt solution of phosphate buffered, PBS add 1%FCS and 0.01% sodiumazide) of various concentration antibody subsequently.Antibody and stimulation or not stimulated cells combine that (Jackson Immunoresearch, West Grove Pennsylvania) detect by luciferin (FITC) or phycoerithrin (PE) link coupled goat Anti-Human IgG.For confirming the expression of CD137, the fusion rotein (AncellCorporation, the Bayport that use CD137 part extracellular domain and mouse CD8 to form, Minnesota), resist-mouse CD8 (BDPharmingen, San Diego, California) insulation then with the PE-link coupled.Sample is fixing in 1% formalin, is kept at 4 ℃, by the flow cytometry reading.

Functional examination method: stimulate so that first signal of T cell activation to be provided with loose anti-CD 3 antibodies available from the former generation people T-cell of healthy donors or monkey PBMC, and personnel selection Anti-Human CD137 antibody stimulates altogether.As non-specific contrast, humanization anticancrin (BR96) is used with identical antibody concentration.Plate uses anti-CD 3 antibodies (0.5-1 μ g/ml) 4 ℃ of incubated overnight.Second day, T-cell or PBMC were with 1-1.5 * 10 ⁵The concentration bed board in/hole.Cultivate after 72 hours for 37 ℃, the synthetic of IFN-γ measured by cytometer beads array (CBA) or ELISA.

Cytokine assay

ELISA: after each time stimulated the T-cell, centrifugal plate was also removed substratum.(BD Pharmingen, San Diego California) detect cytokine levels according to manufacturer's explanation by ELISA.In brief, add given the test agent and standard substance in 96 orifice plates of antibacterial agent bag quilt.After 2 hours, in PBS-T, wash plate 3 times in the envrionment temperature insulation, at first add substrate then then with the insulation of operation detection antibody.405nm reads absorbancy, based on the typical curve calculating concentration.

Cytometer beads assay method: being used to measure the method that the cell in vitro factor generates is flow cytometry, and it utilizes the cell pearl array (CBA) of BD Pharmingen.IFN-γ, IL-2, IL-5, IL-4, IL-10 and TNF-alpha levels illustrate mensuration according to the manufacturer in culture supernatant.The result utilizes the CBA analysis software in flow cytometry.

The result

Further increase and subclone with people CD137 bonded hybridoma secretor type antibody.Excretory antibody is purified and detects it in conjunction with huCD137 and the interactional ability of permission CD137-CD137L.In the Anti-Human CD137 antibody group of assessment, select mab 20H4.9-IgG1 further to assess in conjunction with collection of illustrative plates and non-binding character based on it.20H4.9-IgG1 antibody is IgG1 kappa, as utilizing alkaline phosphatase Anti-Human IgG1 by ELISA, 2,3,4 and anti--kappa and lambda reagent (Southern Biotech, Birmingham Alabama) measure.Fig. 8 (Fig. 8 A-ELISA measure with the combining of people CD137; Fig. 8 B-mab 20H4.9-IgG1 is to the interactional influence of CD137-CD137L) tentatively qualitative to mab 20H4.9-IgG1 be provided.Assessment mab 20H4.9-IgG1,26G6 (blocking-up type resists-CD137 antibody), or the serial dilution thing of Toxoid,tetanus (tetanus toxoid) (TT, negative control) changes CD137 and CD137L bonded ability.The Mab20H4.9-IgG1 that concentration reaches 10 μ g/ml does not block the CD137L combination, suppresses combination during and mab 26G6 concentration＞0.37 μ g/ml.

Also detect the reactive of CD137 that Mab 20H4.9-IgG1 go up to express human T-cell (CEM) and with in the macaque peripheral blood lymphocytes (PBMC) of PMA and ionomycin stimulation to the reactivity of CD137.Research is in the past measured, and CD137 raises on the T-cell, then with PMA and ionomycin activation.(San Diego California) forms the contrast molecule for positive control, BD Pharmingen for have nothing to do human IgG antibody's (negative control) or CD137L-CD8 fusion rotein.The result of these researchs shows that mab 20H4.9-IgG1 is in conjunction with activatory people CEM and from the PBMC of macaque, with minimum without combining of stimulated cells.The similar per-cent of positive cell utilizes mab 20H4.9-IgG1 or CD137L to detect.Fig. 9 provides the gained result to confirm mab 20H4.9-IgG1 and the people of PMA-ionomycin stimulation or combining of macaque cell.People CEM (Fig. 9 A) or monkey PBMC (Fig. 9 B) and 20H4.9-IgG1 or the insulation of people CD137L fusion rotein.Add secondary antibodies and read sample by flow cytometry.

Can subsequently, measure mab 20H4.9-IgG1 and induce IFN-γ to increase under the condition that stimulates assay method existing anti--CD3 to stimulate altogether, this be the required key function effect of excitability CD137 antibody.The common stimulating activity of assessment Mab 20H4.9-IgG1 in people and the lymphocytic functional study of monkey.Based on primary data, anti--CD137 antibody (excessive antibodies) of concentration 20ug/ml is used for these research.Detect the anti-CD 3 antibodies of 0.2-1 μ g/ml, it causes the 10-20%CD137-positive lymphocyte.IFN-γ concentration in the supernatant is measured after cultivating 72h.As shown in figure 10, mab 20H4.9-IgG1 makes synthetic being increased to apparently higher than the level of contrast of IFN-γ in people and the common irritant test of monkey.The result of study of utilize separating from the T of 8 healthy people's donors cell shows, in therein 6, mab 20H4.9-IgG1 improves IFN-γ Synthetic 2 .2-4.3-doubly with respect to contrast.One of other two donors show 1.6-increase doubly.This humanization that the horizontal exceeding that increases utilizes hu39E3.G4 PCT application WO 04/010947 (being included in this paper as a reference) to provide resists-the observed result of CD137 antibody, it is presented among in 8 donors 5, and IFN-γ increases and level is lower than the situation (1.5-2-doubly increases) (Figure 10 A) of utilizing mab 20H4.9-IgG1.Be total in the stimulation study monkey, mab 20H4.9-IgG1 confirms that also the enhanced functionally active causes the obvious increase (Figure 10 B) of IFN-γ with respect to contrast.In people's research, the increase consistence of IFN-γ is higher than the situation of utilizing hu39E3.G4.

TNF-α synthetic is induced above control level and is also observed in people's culture, but level is more much lower than IFN-γ.Independent anti-CD 3 antibodies inductive TNF-alpha levels (baseline) than the low about 20-50 of IFN-γ baseline values doubly.Mab 20H4.9-IgG1 induces 3 in 8 donors～2 to 4.7-times increase to occur.Hu39E3.G4 (parallel detection) this induction phase with in the donor～2-doubly increases but level is lower.Other subject cell factor, IL-2, IL-5, IL-10 and IL-4 obviously do not change under the situation of every kind of processing.

The common mab 20H4.9-IgG1 that confirms of these researchs is by inducing required functionally active in Th1 type reaction performance people and the monkey.Significantly since anti-tumor in vivo active and anti--CD1 37 antibody induction IFN-γ synthetic abilities are relevant, these results have supported the selection that mab 20H4.9-IgG1 changes with respect to isotype.

Embodiment 2:mab 20H4.9-IgG4's is external qualitative

Based on its binding kinetics, can not block the CD137-CD137L effect, and, select mab 20H4.9-IgG1 to convert the IgG4 form to human T-cell's function.The IgG4 form of mab 20H4.9-IgG1 is 20H4.9-IgG4 (being illustrated in Fig. 3 and 4).

The subordinate phase of this research relates to the body outer property of comparison mab 20H4.9-IgG4 and mab 20H4.9-IgG1.In this part, binding kinetics character and the functional effect of antibody in people and monkey lymphocyte described.

Binding kinetics

The dynamics of Anti-Human CD137 antibody utilizes surface plasmon resonance, utilizes the assessment of BIAcore3000 device.Antigen, people CD137-mouse IgG _2aThe low density Covalent Immobilization is on CM5 sensing chip surface.Mab 20H4.9-IgG4 and mab 20H4.9-IgG1 inject with the concentration of 25-200nM.Figure 11 describes the injection of mab 20H4.9-IgG1h mab 20H4.9-IgG4 with 100nM.Utilize BIAevaluation software (bivalent model, global curve fit analysis) data calculated, the result shows the kinetic parameter of two kinds of antibody similar (seeing Table 1).The dissociation constant K of mab 20H4.9-IgG1 and mab20H4.9-IgG4 _DBe determined as 11.2and 16.6nM respectively.Under the similar test condition, mab 20H4.9-IgG4 debond mouse 4-1BB.

Table 1-compares the binding kinetics of mab 20H4.9-IgG4 and mab 20H4.9-IgG1

Antibody

k a1(1/Ms)

?k d1(1/s)

?ka2?(1/RUs)

kd2(1/s)

Rmax(RU)

K A1

K D1(nM)

?20H4.9-I?gG1

3.43E+04

?3.85E-0?4

?2.30E-0?5

1.51E-03

262

8.91E+07

11.22

?20H4.9-I?gG4

3.92E+04

?6.51E-0?4

?0.0755

0.105

409

6.02E+07

16.61

Flow cytometry

Detectable level scope 0.32ng/ml-5 μ g/ml, biotinylated mab 20H4.9-IgG4 combines with cem cell ± PMA-ionomycin.The Mab20H4.9-IgG4 that is incorporated into the PMA-ionomycin stimulates cem cell in the mode that concentration relies on.Reach saturated at 0.2 μ g/ml.On the other hand, as to shown in its parent's molecule like that, mab 20H4.9-IgG1, the cem cell (Figure 12) that mab 20H4.9-IgG4 debond does not stimulate through the PMA-ionomycin.The concentration dependent of mab 20H4-.9-IgG4 is combined in the cem cell that the PMA-ionomycin stimulates and confirms (Figure 12).Sample is by the flow cytometry reading.

Cell/functional examination method

For the process that confirms conversion mab 20H4.9-IgG1 isotype does not change antibody activity, carry out in vitro tests to compare mab 20H4.9-IgG4 and the activity of parent mab 20H4.9-IgG1 in monkey PBMC and people T-cell.Measure mab 20H4.9-IgG4 to the influence of people and monkey T-cell or PBMC and with its parent's molecule, mab 20H4.9-IgG1 is relatively.Available from the former generation people T-cell of healthy people's donor or monkey PBMC with anti-CD 3 antibodies (0.5 μ g/ml-1 μ g/ml)+/-Anti-Human CD137 antibody stimulates.Synthesizing at 37 ℃ of IFN-γ utilizes cytometer beads array (cytometric beadarray) (CBA) (to the human sample) or measure by ELISA (to the monkey sample) after cultivating 72 hours.Antibody in irritant test altogether, exist under the condition of the anti-CD 3 antibodies (1 μ g/ml) of suboptimal concentrations or Concavalin A (1 μ g/ml) and detecting (only for donor M5170 and 81).Results expression is the pg/ml increase multiple with respect to contrast.Because the background response of donor is variable, data are with respect to contrast treatment (=1) stdn.Figure 13 A provides people T-cell result, and Figure 13 B provides monkey PBMC result.Shown in Figure 13 A-13B, mab20H4.9-IgG4 confirms that compared with the control stimulus quality produces high-level IEN-γ in people and MC altogether.The raising of the synthetic level of IFN-γ is compared with the situation of its parent's molecule in people and monkey sample.

Subsequently, the influence of the antibody linked functional effect to mab 20H4.9-IgG4 of assessment.Show the crosslinked enhancing that causes their generation signal capabilities of antibody.Thus, test to measure the functionally active of several crowdes of mab20H4.9-IgG4 ± Anti-Human IgG antibody.Shown in Figure 14 A, in the presence of cross-linking type antibody, be subjected to the examination group to observe the synthetic obviously increase of IFN-γ for all, platform appears in 400ng/ml in concentration.Mab 20H4.9-IgG4 promotes further to strengthen by adding the Anti-Human IgG cross-linking type antibody shown in Figure 14 B to IFN γ synthetic.The mab 20H4.9-IgG4 of different batches has comparable cytoactive.

The crosslinked antibody induction IFN-γ synthetic ability that causes of mab 20H4.9-IgG4 strengthens thus.Crosslinked in the antibody body by the cell receptor of immunoglobulin Fc part or by the appearance of antibody dimerization.Mab 20H4.9-IgG4 is the IgG4 isotype, and it is compared with other IgG isotype, and is lower to the avidity of Fc acceptor.Yet IgG4 can be in conjunction with the Fc RI (CD64) that expresses on monocyte and the neutrophil leucocyte.

Adopt two kinds of further qualitative mab 20H4.9-IgG4:(i of other method) to the influence of T-cell survival and the (ii) influence of cell cycle protein D 2 expression.Whether excite the signal to people T-cell for measuring mab 20H4.9-IgG4 by CD137, and provide costimulatory signal to cause cell survival and amplification to the T-cell, known anti-CD 3 antibodies of inducing IFN-γ synthetic concentration+/-detecting viable count (annexin V/propidium iodide feminine gender), and staining cell cyclin D2 is to detect the influence of its pair cell progress with annexin-V and propidium iodide dyeing for people T-cell that mab20H4.9-IgG4 stimulates.Figure 15 shows that 4 groups of different mab 20H4.9-IgG4 cell cycle protein D 2 are expressed and the average result of the survival of T-cell.The mab 20H4.9-IgG4 of concentration 0.4-10 μ g/ml causes viable count to increase about 1.8-2 doubly, and cyclin D2-expression type T-cell count obviously increases (2.5-3 doubly).

Embodiment 3: assess in the body to 4-1BB antibody in the macaque pharmacodynamics model

The ability of the antigen specific immune reaction that present embodiment diagram mab 20H4.9-IgG4 and mab hu39E3.G4 enhancing dna vaccination excite.

Material and method

The experimental animal group: the female and male rhesus macaque (2.5-5.0kg) that is used for this test is available from CharlesRiver BRF (Houston, Texas) also stable breeding in pairs.Every test group is male and 2 female compositions by 4, and it is by the body weight random packet.Test is grouped as follows:

Group 1-SIV gag and PSA dna vaccination (every of 2mg), the 0th, 28,56 days, i.m. added saline control, and i.v. was at the 12nd, 15 and 19 day;

Group 2-SIV gag and PSA dna vaccination (every of 2mg), the 0th, 28,56 days, i.m. added mab hu39E3.G4, and i.v. was at the 12nd, 15 and 19 day;

Group 3-SIV gag and PSA dna vaccination (every of 2mg), the 0th, 28,56 days, i.m. added mab 20H4.9-IgG4, and i.v. was at the 12nd, 15 and 19 day;

The untreated control group of group 4-.

Immunity and Antybody therapy: PSA and SIV gag dna vaccination be available from David B.Weiner, Department of Pathology and Laboratory of Medicine, University ofPennsylvania.(see, Kim etc., Oncogene 20,4497-4506 (2001); Muthumani etc., Vaccine21,629-637 (2003) .)

Monkey uses PSA and SIV gag DNA construct (2mg/ construct/immunity) through the immunity of intramuscular approach simultaneously, carries out twice enhancing (the 0th, 28 and 56 day) in 4 weeks at interval then.Behind the initial immunity the 12nd day, begin with mab 20H4.9-IgG4 or mab hu39E3.G4 treatment.After the first immunisation the 12nd, 15 and 19 day, antibody was with 50mg/kg i.v administration.Select this scheme to be because it suppresses the antibody response to mabhu39E3.G4.

Clinical and clinical pathology

In the whole research process, all monkeys are carried out physical examination by the animal doctor.The blood sample that hematology and serum chemistry are analyzed was collected before inoculation and immunity back the 12nd, 42,70,97 in 134 and 168 days.

Immunoassay

For measuring the immunoreactive effect of these treatment plan inductive, the lymphocytic IFN-γ that utilizes enzyme linked immunological point assay method (ELISPOT) to detect antigen-differential stimulus produces.The blood sample that ELISPOT analyzes is before inoculation and immunity back 12,42,70,97,134, and and collected in 168 days.The synthetic peptide of corresponding SIVgag and the antigenic complete sequence of PSA is used for the stripped PBMC of stimulation.

The result

The antigen-specific IFN-γ secretor type cell that responds to PSA or SIV gag peptide is quantitative by ELISPOT.Figure 16 (Figure 16 A-16D) is the result of diagram group 1-4 respectively.Reaction level to PSA is very low in all groups, shows with the animal without immunization and compare that vaccine itself is not induced and can be detected and fixed (consistent) immune response.On the other hand, independent SIV gag immunization strengthens (Figure 16 A) in time in a large amount of antigens-specificity IFN-γ secretor type cell.In the whole research process, untreated animal (nonvaccinated) shows 100-1,000 point/10 ⁶PBMC (Figure 16 D).These results have determined the thresholding reaction to vaccine; Representative＜1,000 point/10 ⁶The animal of PBMC is considered to the nonresponder.Accept in the animal groups of vaccine, have 5 to show that reaction strengthens in time in 6 monkeys, the average number of immunity back point (the day 70) is 1,727 point/10 for the third time ⁶PBMC (SD=242, scope=1,403-1,968 point/10 ⁶PBMC).A monkey is considered to nonresponder's (620 points/1,000,000 PBMC).Owing in these researchs, do not carry out the MHC somatotype.Some monkeys are owing to the MHC mispairing lacks t cell immune response to vaccine probably.Significantly, at the 70th day, compare with control animal (Figure 16 D) and with compare (Figure 16 A) with the macaque of independent dna vaccination immunity, adding in 6 animals that mab20H4.9-IgG4 handles with SIV gag has the obviously IFN-γ points (Figure 16 C) of higher numbers of 4 performances.The group that mab 20H4.9-IgG4-handles is 3,465 point/10 through the average number of point after the immunity for the third time ⁶PBMC (SD=1,236, scope=2,070-4,780 point/10 ⁶PBMC).Two monkeys in this group are to vaccine reactionless (＜800 points/1,000,000 PBMC).After the immunity (the 70th day), the treatment that utilizes mab hu39E3.G4 to add dna vaccination causes having 6 to be considered to reactor in 6 animals for the third time, and the equalization point number is 2,348 point/10 ⁶PBMC (SD=588, scope=1,738-3,283) (Figure 16 B).For this group, the scope of counting out is compared low with those macaques for the treatment of with mab 20H4.9-IgG4.

Utilize the treatment of 20H4.9-IgG4 and mab hu9E3.G4 better to be tolerated the clinical disease that does not cause with respect to the contrast monkey, the obvious change of clinical chemistry or hematologic parameter.

These data presentation mab 20H4.9-IgG4 treatment coupling dna vaccination excite with respect to contrast or utilize for the treatment of mab hu39E3.G4, at strengthening in the body that is tried antigenic specific reaction, for example by antigen-specific IFN-gamma-secretase raji cell assay Raji.Owing to only use antibody and a dosage of a dosage level in these preliminary study, can not induce maximum reaction, and need further work research top condition.Yet, clearly,, also can be strengthened and utilize mab 20H4.9-IgG4 to realize trying antigenic cell response even utilize these schemes of not optimizing, the adjusting of prompting CD137 function is the attracting method that improves the validity of dna vaccination.

Although the present invention describes in detail with embodiment by way of example, in order to be aware and understand, clearly can carry out some changes and modification, it is included within the claim scope of the present invention.

Sequence table

＜110〉Squibb Bristol Myers Co.

Jure-Kunkel，Maria

Hefta，Laura

Santoro，Marc

Ganguly，Subinay

 

＜120〉fully human antibodies of anti-people 4-1BB (cD137)

 

<130>10060?PCT

 

<150>US?60/510193

<151>2003-10-10

 

<150>not?yet?assigned

<151>2004-10-08

 

<160>9

 

<170>PatentIn?version?3.2

 

<210>1

<211>7057

<212>DNA

＜213〉artificial

 

<220>

＜223〉artificial sequence

 

<400>1

cgatgtacgg?gccagatata?cgcgttgaca?ttgattattg?actagttatt?aatagtaatc 60

aattacgggg?tcattagttc?atagcccata?tatggagttc?cgcgttacat?aacttacggt 120

aaatggcccg?cctggctgac?cgcccaacga?cccccgccca?ttgacgtcaa?taatgacgta 180

tgttcccata?gtaacgccaa?tagggacttt?ccattgacgt?caatgggtgg?actatttacg 240

gtaaactgcc?cacttggcag?tacatcaagt?gtatcatatg?ccaagtacgc?cccctattga 300

cgtcaatgac?ggtaaatggc?ccgcctggca?ttatgcccag?tacatgacct?tatgggactt 360

tcctacttgg?cagtacatct?acgtattagt?catcgctatt?accatggtga?tgcggttttg 420

gcagtacatc?aatgggcgtg?gatagcggtt?tgactcacgg?ggatttccaa?gtctccaccc 480

cattgacgtc?aatgggagtt?tgttttggca?ccaaaatcaa?cgggactttc?caaaatgtcg 540

taacaactcc?gccccattga?cgcaaatggg?cggtaggcgt?gtacggtggg?aggtctatat 600

aagcagagct?ctctggctaa?ctagagaacc?cactgcttac?tggcttatcg?aaattaatac 660

gactcactat?agggagaccc?aagcttggta?ccgccatgaa?acacctgtgg?ttcttcctcc 720

tcctggtggc?agctcccaga?tgggtcctgt?cccaggtgca?actacagcag?tggggcgcag 780

gactgttgaa?gccttcggag?accctgtccc?tcacctgcgc?tgtctatggt?gggtccttca 840

gtggttacta?ctggagctgg?atacgccagt?ccccagagaa?ggggctggag?tggattgggg 900

aaatcaatca?tggtggatac?gtcacctaca?atccgtccct?cgagagtcga?gtcaccatat 960

cagtagacac?gtccaagaac?cagttctccc?tgaagctgag?ctctgtgacc?gccgcggaca 1020

cggctgtata?ttactgtgcg?agggactatg?gtccggggaa?ttatgactgg?tacttcgatc 1080

tctggggccg?tggcaccctg?gtcactgtct?cctcagctag?caccaagggc?ccatccgtct 1140

tccccctggc?gccctgctcc?aggagcacct?ccgagagcac?agccgccctg?ggctgcctgg 1200

tcaaggacta?cttccccgaa?ccggtgacgg?tgtcgtggaa?ctcaggcgcc?ctgaccagcg 1260

gcgtgcacac?cttcccggct?gtcctacagt?cctcaggact?ctactccctc?agcagcgtgg 1320

tgaccgtgcc?ctccagcagc?ttgggcacga?agacctacac?ctgcaacgta?gatcacaagc 1380

ccagcaacac?caaggtggac?aagagagttg?agtccaaata?tggtccacct?tgcccacctt 1440

gcccagcacc?tgagttcctg?gggggaccat?cagtcttcct?gttcccccca?aaacccaagg 1500

acactctcat?gatctcccgg?acccctgagg?tcacgtgcgt?ggtggtggac?gtgagccagg 1560

aagaccccga?ggtccagttc?aactggtacg?tggatggcgt?ggaggtgcat?aatgccaaga 1620

caaagccgcg?ggaggagcag?ttcaacagca?cgtaccgtgt?ggtcagcgtc?ctcaccgtcc 1680

tgcaccagga?ctggctgaac?ggcaaggagt?acaagtgcaa?ggtctccaac?aaaggcctcc 1740

cgtcctccat?cgagaaaacc?atctccaaag?ccaaagggca?gccccgagag?ccacaggtgt 1800

acaccctgcc?cccatcccag?gaggagatga?ccaagaacca?ggtcagcctg?acctgcctgg 1860

tcaaaggctt?ctaccccagc?gacatcgccg?tggagtggga?gagcaatggg?cagccggaga 1920

acaactacaa?gaccacgcct?cccgtgctgg?actccgacgg?ctccttcttc?ctctacagca 1980

ggctaaccgt?ggacaagagc?aggtggcagg?aggggaatgt?cttctcatgc?tccgtgatgc 2040

atgaggctct?gcacaaccac?tacacacaga?agagcctctc?cctgtctctg?ggtaaatgat 2100

ctagagggcc?ctattctata?gtgtcaccta?aatgctagag?ctcgctgatc?agcctcgact 2160

gtgccttcta?gttgccagcc?atctgttgtt?tgcccctccc?ccgtgccttc?cttgaccctg 2220

gaaggtgcca?ctcccactgt?cctttcctaa?taaaatgagg?aaattgcatc?gcattgtctg 2280

agtaggtgtc?attctattct?ggggggtggg?gtggggcagg?acagcaaggg?ggaggattgg 2340

gaagacaata?gcaggcatgc?tggggatgcg?gtgggctcta?tggcttctga?ggcggaaaga 2400

accagctggg?gctctagggg?gtatccccac?gcgccctgta?gcggcgcatt?aagcgcggcg 2460

ggtgtggtgg?ttacgcgcag?cgtgaccgct?acacttgcca?gcgccctagc?gcccgctcct 2520

ttcgctttct?tcccttcctt?tctcgccacg?ttcgccgggc?ctctcaaaaa?agggaaaaaa 2580

agcatgcatc?tcaattagtc?agcaaccata?gtcccgcccc?taactccgcc?catcccgccc 2640

ctaactccgc?ccagttccgc?ccattctccg?ccccatggct?gactaatttt?ttttatttat 2700

gcagaggccg?aggccgcctc?ggcctctgag?ctattccaga?agtagtgagg?aggctttttt 2760

ggaggcctag?gcttttgcaa?aaagcttgga?cagctcaggg?ctgcgatttc?gcgccaaact 2820

tgacggcaat?cctagcgtga?aggctggtag?gattttatcc?ccgctgccat?catggttcga 2880

ccattgaact?gcatcgtcgc?cgtgtcccaa?aatatgggga?ttggcaagaa?cggagaccta 2940

ccctggcctc?cgctcaggaa?cgagttcaag?tacttccaaa?gaatgaccac?aacctcttca 3000

gtggaaggta?aacagaatct?ggtgattatg?ggtaggaaaa?cctggttctc?cattcctgag 3060

aagaatcgac?ctttaaagga?cagaattaat?atagttctca?gtagagaact?caaagaacca 3120

ccacgaggag?ctcattttct?tgccaaaagt?ttggatgatg?ccttaagact?tattgaacaa 3180

ccggaattgg?caagtaaagt?agacatggtt?tggatagtcg?gaggcagttc?tgtttaccag 3240

gaagccatga?atcaaccagg?ccaccttaga?ctctttgtga?caaggatcat?gcaggaattt 3300

gaaagtgaca?cgtttttccc?agaaattgat?ttggggaaat?ataaacttct?cccagaatac 3360

ccaggcgtcc?tctctgaggt?ccaggaggaa?aaaggcatca?agtataagtt?tgaagtctac 3420

gagaagaaag?actaacagga?agatgctttc?aagttctctg?ctcccctcct?aaagctatgc 3480

atttttataa?gaccatggga?cttttgctgg?ctttagatct?ctttgtgaag?gaaccttact 3540

tctgtggtgt?gacataattg?gacaaactac?ctacagagat?ttaaagctct?aaggtaaata 3600

taaaattttt?aagtgtataa?tgtgttaaac?tactgattct?aattgtttgt?gtattttaga 3660

ttccaaccta?tggaactgat?gaatgggagc?agtggtggaa?tgcctttaat?gaggaaaacc 3720

tgttttgctc?agaagaaatg?ccatctagtg?atgatgaggc?tactgctgac?tctcaacatt 3780

ctactcctcc?aaaaaagaag?agaaaggtag?aagaccccaa?ggactttcct?tcagaattgc 3840

taagtttttt?gagtcatgct?gtgtttagta?atagaactct?tgcttgcttt?gctatttaca 3900

ccacaaagga?aaaagctgca?ctgctataca?agaaaattat?ggaaaaatat?tctgtaacct 3960

ttataagtag?gcataacagt?tataatcata?acatactgtt?ttttcttact?ccacacaggc 4020

atagagtgtc?tgctattaat?aactatgctc?aaaaattgtg?tacctttagc?tttttaattt 4080

gtaaaggggt?taataaggaa?tatttgatgt?atagtgcctt?gactagagat?cataatcagc 4140

cataccacat?ttgtagaggt?tttacttgct?ttaaaaaacc?tcccacacct?ccccctgaac 4200

ctgaaacata?aaatgaatgc?aattgttgtt?gttaacttgt?ttattgcagc?ttataatggt 4260

tacaaataaa?gcaatagcat?cacaaatttc?acaaataaag?catttttttc?actgcattct 4320

agttgtggtt?tgtccaaact?catcaatgta?tcttatcatg?tctggatcgg?ctggatgatc 4380

ctccagcgcg?gggatctcat?gctggagttc?ttcgcccacc?ccaacttgtt?tattgcagct 4440

tataatggtt?acaaataaag?caatagcatc?acaaatttca?caaataaagc?atttttttca 4500

ctgcattcta?gttgtggttt?gtccaaactc?atcaatgtat?cttatcatgt?ctgtataccg 4560

tcgacctcta?gctagagctt?ggcgtaatca?tggtcatagc?tgtttcctgt?gtgaaattgt 4620

tatccgctca?caattccaca?caacatacga?gccggaagca?taaagtgtaa?agcctggggt 4680

gcctaatgag?tgagctaact?cacattaatt?gcgttgcgct?cactgcccgc?tttccagtcg 4740

ggaaacctgt?cgtgccagct?gcattaatga?atcggccaac?gcgcggggag?aggcggtttg 4800

cgtattgggc?gctcttccgc?ttcctcgctc?actgactcgc?tgcgctcggt?cgttcggctg 4860

cggcgagcgg?tatcagctca?ctcaaaggcg?gtaatacggt?tatccacaga?atcaggggat 4920

aacgcaggaa?agaacatgtg?agcaaaaggc?cagcaaaagg?ccaggaaccg?taaaaaggcc 4980

gcgttgctgg?cgtttttcca?taggctccgc?ccccctgacg?agcatcacaa?aaatcgacgc 5040

tcaagtcaga?ggtggcgaaa?cccgacagga?ctataaagat?accaggcgtt?tccccctgga 5100

agctccctcg?tgcgctctcc?tgttccgacc?ctgccgctta?ccggatacct?gtccgccttt 5160

ctcccttcgg?gaagcgtggc?gctttctcaa?tgctcacgct?gtaggtatct?cagttcggtg 5220

taggtcgttc?gctccaagct?gggctgtgtg?cacgaacccc?ccgttcagcc?cgaccgctgc 5280

gccttatccg?gtaactatcg?tcttgagtcc?aacccggtaa?gacacgactt?atcgccactg 5340

gcagcagcca?ctggtaacag?gattagcaga?gcgaggtatg?taggcggtgc?tacagagttc 5400

ttgaagtggt?ggcctaacta?cggctacact?agaaggacag?tatttggtat?ctgcgctctg 5460

ctgaagccag?ttaccttcgg?aaaaagagtt?ggtagctctt?gatccggcaa?acaaaccacc 5520

gctggtagcg?gtggtttttt?tgtttgcaag?cagcagatta?cgcgcagaaa?aaaaggatct 5580

caagaagatc?ctttgatctt?ttctacgggg?tctgacgctc?agtggaacga?aaactcacgt 5640

taagggattt?tggtcatgag?attatcaaaa?aggatcttca?cctagatcct?tttaaattaa 5700

aaatgaagtt?ttaaatcaat?ctaaagtata?tatgagtaaa?cttggtctga?cagttaccaa 5760

tgcttaatca?gtgaggcacc?tatctcagcg?atctgtctat?ttcgttcatc?catagttgcc 5820

tgactccccg?tcgtgtagat?aactacgata?cgggagggct?taccatctgg?ccccagtgct 5880

gcaatgatac?cgcgagaccc?acgctcaccg?gctccagatt?tatcagcaat?aaaccagcca 5940

gccggaaggg?ccgagcgcag?aagtggtcct?gcaactttat?ccgcctccat?ccagtctatt 6000

aattgttgcc?gggaagctag?agtaagtagt?tcgccagtta?atagtttgcg?caacgttgtt 6060

gccattgcta?caggcatcgt?ggtgtcacgc?tcgtcgtttg?gtatggcttc?attcagctcc 6120

ggttcccaac?gatcaaggcg?agttacatga?tcccccatgt?tgtgcaaaaa?agcggttagc 6180

tccttcggtc?ctccgatcgt?tgtcagaagt?aagttggccg?cagtgttatc?actcatggtt 6240

atggcagcac?tgcataattc?tcttactgtc?atgccatccg?taagatgctt?ttctgtgact 6300

ggtgagtact?caaccaagtc?attctgagaa?tagtgtatgc?ggcgaccgag?ttgctcttgc 6360

ccggcgtcaa?tacgggataa?taccgcgcca?catagcagaa?ctttaaaagt?gctcatcatt 6420

ggaaaacgtt?cttcggggcg?aaaactctca?aggatcttac?cgctgttgag?atccagttcg 6480

atgtaaccca?ctcgtgcacc?caactgatct?tcagcatctt?ttactttcac?cagcgtttct 6540

gggtgagcaa?aaacaggaag?gcaaaatgcc?gcaaaaaagg?gaataagggc?gacacggaaa 6600

tgttgaatac?tcatactctt?cctttttcaa?tattattgaa?gcatttatca?gggttattgt 6660

ctcatgagcg?gatacatatt?tgaatgtatt?tagaaaaata?aacaaatagg?ggttccgcgc 6720

acatttcccc?gaaaagtgcc?acctgacgtc?gacggatcgg?gagatctgct?aggtgacctg 6780

aggcgcgccg?gcttcgaata?gccagagtaa?cctttttttt?taattttatt?ttattttatt 6840

tttgagatgg?agtttggcgc?cgatctcccg?atcccctatg?gtcgactctc?agtacaatct 6900

gctctgatgc?cgcatagtta?agccagtatc?tgctccctgc?ttgtgtgttg?gaggtcgctg 6960

agtagtgcgc?gagcaaaatt?taagctacaa?caaggcaagg?cttgaccgac?aattgcatga 7020

agaatctgct?tagggttagg?cgttttgcgc?tgcttcg 7057

 

<210>2

<211>7057

<212>DNA

＜213〉artificial

 

<220>

＜223〉artificial sequence

 

<400>2

gctacatgcc?cggtctatat?gcgcaactgt?aactaataac?tgatcaataa?ttatcattag 60

ttaatgcccc?agtaatcaag?tatcgggtat?atacctcaag?gcgcaatgta?ttgaatgcca 120

tttaccgggc?ggaccgactg?gcgggttgct?gggggcgggt?aactgcagtt?attactgcat 180

acaagggtat?cattgcggtt?atccctgaaa?ggtaactgca?gttacccacc?tgataaatgc 240

catttgacgg?gtgaaccgtc?atgtagttca?catagtatac?ggttcatgcg?ggggataact 300

gcagttactg?ccatttaccg?ggcggaccgt?aatacgggtc?atgtactgga?ataccctgaa 360

aggatgaacc?gtcatgtaga?tgcataatca?gtagcgataa?tggtaccact?acgccaaaac 420

cgtcatgtag?ttacccgcac?ctatcgccaa?actgagtgcc?cctaaaggtt?cagaggtggg 480

gtaactgcag?ttaccctcaa?acaaaaccgt?ggttttagtt?gccctgaaag?gttttacagc 540

attgttgagg?cggggtaact?gcgtttaccc?gccatccgca?catgccaccc?tccagatata 600

ttcgtctcga?gagaccgatt?gatctcttgg?gtgacgaatg?accgaatagc?tttaattatg 660

ctgagtgata?tccctctggg?ttcgaaccat?ggcggtactt?tgtggacacc?aagaaggagg 720

aggaccaccg?tcgagggtct?acccaggaca?gggtccacgt?tgatgtcgtc?accccgcgtc 780

ctgacaactt?cggaagcctc?tgggacaggg?agtggacgcg?acagatacca?cccaggaagt 840

caccaatgat?gacctcgacc?tatgcggtca?ggggtctctt?ccccgacctc?acctaacccc 900

tttagttagt?accacctatg?cagtggatgt?taggcaggga?gctctcagct?cagtggtata 960

gtcatctgtg?caggttcttg?gtcaagaggg?acttcgactc?gagacactgg?cggcgcctgt 1020

gccgacatat?aatgacacgc?tccctgatac?caggcccctt?aatactgacc?atgaagctag 1080

agaccccggc?accgtgggac?cagtgacaga?ggagtcgatc?gtggttcccg?ggtaggcaga 1140

agggggaccg?cgggacgagg?tcctcgtgga?ggctctcgtg?tcggcgggac?ccgacggacc 1200

agttcctgat?gaaggggctt?ggccactgcc?acagcacctt?gagtccgcgg?gactggtcgc 1260

cgcacgtgtg?gaagggccga?caggatgtca?ggagtcctga?gatgagggag?tcgtcgcacc 1320

actggcacgg?gaggtcgtcg?aacccgtgct?tctggatgtg?gacgttgcat?ctagtgttcg 1380

ggtcgttgtg?gttccacctg?ttctctcaac?tcaggtttat?accaggtgga?acgggtggaa 1440

cgggtcgtgg?actcaaggac?ccccctggta?gtcagaagga?caaggggggt?tttgggttcc 1500

tgtgagagta?ctagagggcc?tggggactcc?agtgcacgca?ccaccacctg?cactcggtcc 1560

ttctggggct?ccaggtcaag?ttgaccatgc?acctaccgca?cctccacgta?ttacggttct 1620

gtttcggcgc?cctcctcgtc?aagttgtcgt?gcatggcaca?ccagtcgcag?gagtggcagg 1680

acgtggtcct?gaccgacttg?ccgttcctca?tgttcacgtt?ccagaggttg?tttccggagg 1740

gcaggaggta?gctcttttgg?tagaggtttc?ggtttcccgt?cggggctctc?ggtgtccaca 1800

tgtgggacgg?gggtagggtc?ctcctctact?ggttcttggt?ccagtcggac?tggacggacc 1860

agtttccgaa?gatggggtcg?ctgtagcggc?acctcaccct?ctcgttaccc?gtcggcctct 1920

tgttgatgtt?ctggtgcgga?gggcacgacc?tgaggctgcc?gaggaagaag?gagatgtcgt 1980

ccgattggca?cctgttctcg?tccaccgtcc?tccccttaca?gaagagtacg?aggcactacg 2040

tactccgaga?cgtgttggtg?atgtgtgtct?tctcggagag?ggacagagac?ccatttacta 2100

gatctcccgg?gataagatat?cacagtggat?ttacgatctc?gagcgactag?tcggagctga 2160

cacggaagat?caacggtcgg?tagacaacaa?acggggaggg?ggcacggaag?gaactgggac 2220

cttccacggt?gagggtgaca?ggaaaggatt?attttactcc?tttaacgtag?cgtaacagac 2280

tcatccacag?taagataaga?ccccccaccc?caccccgtcc?tgtcgttccc?cctcctaacc 2340

cttctgttat?cgtccgtacg?acccctacgc?cacccgagat?accgaagact?ccgcctttct 2400

tggtcgaccc?cgagatcccc?cataggggtg?cgcgggacat?cgccgcgtaa?ttcgcgccgc 2460

ccacaccacc?aatgcgcgtc?gcactggcga?tgtgaacggt?cgcgggatcg?cgggcgagga 2520

aagcgaaaga?agggaaggaa?agagcggtgc?aagcggcccg?gagagttttt?tccctttttt 2580

tcgtacgtag?agttaatcag?tcgttggtat?cagggcgggg?attgaggcgg?gtagggcggg 2640

gattgaggcg?ggtcaaggcg?ggtaagaggc?ggggtaccga?ctgattaaaa?aaaataaata 2700

cgtctccggc?tccggcggag?ccggagactc?gataaggtct?tcatcactcc?tccgaaaaaa 2760

cctccggatc?cgaaaacgtt?tttcgaacct?gtcgagtccc?gacgctaaag?cgcggtttga 2820

actgccgtta?ggatcgcact?tccgaccatc?ctaaaatagg?ggcgacggta?gtaccaagct 2880

ggtaacttga?cgtagcagcg?gcacagggtt?ttatacccct?aaccgttctt?gcctctggat 2940

gggaccggag?gcgagtcctt?gctcaagttc?atgaaggttt?cttactggtg?ttggagaagt 3000

caccttccat?ttgtcttaga?ccactaatac?ccatcctttt?ggaccaagag?gtaaggactc 3060

ttcttagctg?gaaatttcct?gtcttaatta?tatcaagagt?catctcttga?gtttcttggt 3120

ggtgctcctc?gagtaaaaga?acggttttca?aacctactac?ggaattctga?ataacttgtt 3180

ggccttaacc?gttcatttca?tctgtaccaa?acctatcagc?ctccgtcaag?acaaatggtc 3240

cttcggtact?tagttggtcc?ggtggaatct?gagaaacact?gttcctagta?cgtccttaaa 3300

ctttcactgt?gcaaaaaggg?tctttaacta?aaccccttta?tatttgaaga?gggtcttatg 3360

ggtccgcagg?agagactcca?ggtcctcctt?tttccgtagt?tcatattcaa?acttcagatg 3420

ctcttctttc?tgattgtcct?tctacgaaag?ttcaagagac?gaggggagga?tttcgatacg 3480

taaaaatatt?ctggtaccct?gaaaacgacc?gaaatctaga?gaaacacttc?cttggaatga 3540

agacaccaca?ctgtattaac?ctgtttgatg?gatgtctcta?aatttcgaga?ttccatttat 3600

attttaaaaa?ttcacatatt?acacaatttg?atgactaaga?ttaacaaaca?cataaaatct 3660

aaggttggat?accttgacta?cttaccctcg?tcaccacctt?acggaaatta?ctccttttgg 3720

acaaaacgag?tcttctttac?ggtagatcac?tactactccg?atgacgactg?agagttgtaa 3780

gatgaggagg?ttttttcttc?tctttccatc?ttctggggtt?cctgaaagga?agtcttaacg 3840

attcaaaaaa?ctcagtacga?cacaaatcat?tatcttgaga?acgaacgaaa?cgataaatgt 3900

ggtgtttcct?ttttcgacgt?gacgatatgt?tcttttaata?cctttttata?agacattgga 3960

aatattcatc?cgtattgtca?atattagtat?tgtatgacaa?aaaagaatga?ggtgtgtccg 4020

tatctcacag?acgataatta?ttgatacgag?tttttaacac?atggaaatcg?aaaaattaaa 4080

catttcccca?attattcctt?ataaactaca?tatcacggaa?ctgatctcta?gtattagtcg 4140

gtatggtgta?aacatctcca?aaatgaacga?aattttttgg?agggtgtgga?gggggacttg 4200

gactttgtat?tttacttacg?ttaacaacaa?caattgaaca?aataacgtcg?aatattacca 4260

atgtttattt?cgttatcgta?gtgtttaaag?tgtttatttc?gtaaaaaaag?tgacgtaaga 4320

tcaacaccaa?acaggtttga?gtagttacat?agaatagtac?agacctagcc?gacctactag 4380

gaggtcgcgc?ccctagagta?cgacctcaag?aagcgggtgg?ggttgaacaa?ataacgtcga 4440

atattaccaa?tgtttatttc?gttatcgtag?tgtttaaagt?gtttatttcg?taaaaaaagt 4500

gacgtaagat?caacaccaaa?caggtttgag?tagttacata?gaatagtaca?gacatatggc 4560

agctggagat?cgatctcgaa?ccgcattagt?accagtatcg?acaaaggaca?cactttaaca 4620

ataggcgagt?gttaaggtgt?gttgtatgct?cggccttcgt?atttcacatt?tcggacccca 4680

cggattactc?actcgattga?gtgtaattaa?cgcaacgcga?gtgacgggcg?aaaggtcagc 4740

cctttggaca?gcacggtcga?cgtaattact?tagccggttg?cgcgcccctc?tccgccaaac 4800

gcataacccg?cgagaaggcg?aaggagcgag?tgactgagcg?acgcgagcca?gcaagccgac 4860

gccgctcgcc?atagtcgagt?gagtttccgc?cattatgcca?ataggtgtct?tagtccccta 4920

ttgcgtcctt?tcttgtacac?tcgttttccg?gtcgttttcc?ggtccttggc?atttttccgg 4980

cgcaacgacc?gcaaaaaggt?atccgaggcg?gggggactgc?tcgtagtgtt?tttagctgcg 5040

agttcagtct?ccaccgcttt?gggctgtcct?gatatttcta?tggtccgcaa?agggggacct 5100

tcgagggagc?acgcgagagg?acaaggctgg?gacggcgaat?ggcctatgga?caggcggaaa 5160

gagggaagcc?cttcgcaccg?cgaaagagtt?acgagtgcga?catccataga?gtcaagccac 5220

atccagcaag?cgaggttcga?cccgacacac?gtgcttgggg?ggcaagtcgg?gctggcgacg 5280

cggaataggc?cattgatagc?agaactcagg?ttgggccatt?ctgtgctgaa?tagcggtgac 5340

cgtcgtcggt?gaccattgtc?ctaatcgtct?cgctccatac?atccgccacg?atgtctcaag 5400

aacttcacca?ccggattgat?gccgatgtga?tcttcctgtc?ataaaccata?gacgcgagac 5460

gacttcggtc?aatggaagcc?tttttctcaa?ccatcgagaa?ctaggccgtt?tgtttggtgg 5520

cgaccatcgc?caccaaaaaa?acaaacgttc?gtcgtctaat?gcgcgtcttt?ttttcctaga 5580

gttcttctag?gaaactagaa?aagatgcccc?agactgcgag?tcaccttgct?tttgagtgca 5640

attccctaaa?accagtactc?taatagtttt?tcctagaagt?ggatctagga?aaatttaatt 5700

tttacttcaa?aatttagtta?gatttcatat?atactcattt?gaaccagact?gtcaatggtt 5760

acgaattagt?cactccgtgg?atagagtcgc?tagacagata?aagcaagtag?gtatcaacgg 5820

actgaggggc?agcacatcta?ttgatgctat?gccctcccga?atggtagacc?ggggtcacga 5880

cgttactatg?gcgctctggg?tgcgagtggc?cgaggtctaa?atagtcgtta?tttggtcggt 5940

cggccttccc?ggctcgcgtc?ttcaccagga?cgttgaaata?ggcggaggta?ggtcagataa 6000

ttaacaacgg?cccttcgatc?tcattcatca?agcggtcaat?tatcaaacgc?gttgcaacaa 6060

cggtaacgat?gtccgtagca?ccacagtgcg?agcagcaaac?cataccgaag?taagtcgagg 6120

ccaagggttg?ctagttccgc?tcaatgtact?agggggtaca?acacgttttt?tcgccaatcg 6180

aggaagccag?gaggctagca?acagtcttca?ttcaaccggc?gtcacaatag?tgagtaccaa 6240

taccgtcgtg?acgtattaag?agaatgacag?tacggtaggc?attctacgaa?aagacactga 6300

ccactcatga?gttggttcag?taagactctt?atcacatacg?ccgctggctc?aacgagaacg 6360

ggccgcagtt?atgccctatt?atggcgcggt?gtatcgtctt?gaaattttca?cgagtagtaa 6420

ccttttgcaa?gaagccccgc?ttttgagagt?tcctagaatg?gcgacaactc?taggtcaagc 6480

tacattgggt?gagcacgtgg?gttgactaga?agtcgtagaa?aatgaaagtg?gtcgcaaaga 6540

cccactcgtt?tttgtccttc?cgttttacgg?cgttttttcc?cttattcccg?ctgtgccttt 6600

acaacttatg?agtatgagaa?ggaaaaagtt?ataataactt?cgtaaatagt?cccaataaca 6660

gagtactcgc?ctatgtataa?acttacataa?atctttttat?ttgtttatcc?ccaaggcgcg 6720

tgtaaagggg?cttttcacgg?tggactgcag?ctgcctagcc?ctctagacga?tccactggac 6780

tccgcgcggc?cgaagcttat?cggtctcatt?ggaaaaaaaa?attaaaataa?aataaaataa 6840

aaactctacc?tcaaaccgcg?gctagagggc?taggggatac?cagctgagag?tcatgttaga 6900

cgagactacg?gcgtatcaat?tcggtcatag?acgagggacg?aacacacaac?ctccagcgac 6960

tcatcacgcg?ctcgttttaa?attcgatgtt?gttccgttcc?gaactggctg?ttaacgtact 7020

tcttagacga?atcccaatcc?gcaaaacgcg?acgaagc 7057

 

<210>3

<211>467

<212>PRT

＜213〉artificial

 

<220>

＜223〉artificial sequence

 

<400>3

 

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr?Cys?Ala?Arg?Asp?Tyr?Gly?Pro?Gly?Asn?Tyr?Asp?Trp?Tyr?Phe?Asp

115 120 125

Leu?Trp?Gly?Arg?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys

130 135 140

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

145 150 155 160

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

165 170 175

Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr

180 185 190

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

195 200 205

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

210 215 220

Val?Asp?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Arg?Val?Glu?Ser

225 230 235 240

Lys?Tyr?Gly?Pro?Pro?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Phe?Leu?Gly

245 250 255

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

260 265 270

Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?Gln

275 280 285

Glu?Asp?Pro?Glu?Val?Gln?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val

290 295 300

His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Phe?Asn?Ser?Thr?Tyr

305 310 315 320

Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro

405 410 415

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

420 425 430

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

435 440 445

His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser

450 455 460

Leu?Gly?Lys

465

 

<210>4

<211>6435

<212>DNA

＜213〉artificial

 

<220>

＜223〉artificial sequence

 

<400>4

gacggatcgg?gagatctgct?agcccgggtg?acctgaggcg?cgccggcttc?gaatagccag 60

agtaaccttt?ttttttaatt?ttattttatt?ttatttttga?gatggagttt?ggcgccgatc 120

tcccgatccc?ctatggtcga?ctctcagtac?aatctgctct?gatgccgcat?agttaagcca 180

gtatctgctc?cctgcttgtg?tgttggaggt?cgctgagtag?tgcgcgagca?aaatttaagc 240

tacaacaagg?caaggcttga?ccgacaattg?catgaagaat?ctgcttaggg?ttaggcgttt 300

tgcgctgctt?cgcgatgtac?gggccagata?tacgcgttga?cattgattat?tgactagtta 360

ttaatagtaa?tcaattacgg?ggtcattagt?tcatagccca?tatatggagt?tccgcgttac 420

ataacttacg?gtaaatggcc?cgcctggctg?accgcccaac?gacccccgcc?cattgacgtc 480

aataatgacg?tatgttccca?tagtaacgcc?aatagggact?ttccattgac?gtcaatgggt 540

ggagtattta?cggtaaactg?cccacttggc?agtacatcaa?gtgtatcata?tgccaagtac 600

gccccctatt?gacgtcaatg?acggtaaatg?gcccgcctgg?cattatgccc?agtacatgac 660

cttatgggac?tttcctactt?ggcagtacat?ctacgtatta?gtcatcgcta?ttaccatggt 720

gatgcggttt?tggcagtaca?tcaatgggcg?tggatagcgg?tttgactcac?ggggatttcc 780

aagtctccac?cccattgacg?tcaatgggag?tttgttttgg?caccaaaatc?aacgggactt 840

tccaaaatgt?cgtaacaact?ccgccccatt?gacgcaaatg?ggcggtaggc?gtgtacggtg 900

ggaggtctat?ataagcagag?ctctctggct?aactagagaa?cccactgctt?actggcttat 960

cgaaattaat?acgactcact?atagggagac?ccaagcttat?caacaagttt?gtacaaaaaa 1020

gcaggctggt?accatggaag?ccccagctca?gcttctcttc?ctcctgctac?tctggctccc 1080

agataccacc?ggagaaattg?tgttgacaca?gtctccagcc?accctgtctt?tgtctccagg 1140

ggaaagagcc?accctctcct?gcagggccag?tcagagtgtt?agcagctact?tagcctggta 1200

ccaacagaaa?cctggccagg?ctcccaggct?cctcatctat?gatgcatcca?acagggccac 1260

tggcatccca?gccaggttca?gtggcagtgg?gtctgggaca?gacttcactc?tcaccatcag 1320

cagcctagag?cctgaagatt?ttgcagttta?ttactgtcag?cagcgtagca?actggcctcc 1380

ggcgctcact?ttcggcggag?ggaccaaggt?ggagatcaaa?cgtacggtgg?ctgcaccatc 1440

tgtcttcatc?ttcccgccat?ctgatgagca?gttgaaatct?ggaactgcct?ctgttgtgtg 1500

cctgctgaat?aacttctatc?ccagagaggc?caaagtacag?tggaaggtgg?ataacgccct 1560

ccaatcgggt?aactcccagg?agagtgtcac?agagcaggac?agcaaggaca?gcacctacag 1620

cctcagcagc?accctgacgc?tgagcaaagc?agactacgag?aaacacaaag?tctacgcctg 1680

cgaagtcacc?catcagggcc?tgagctcgcc?cgtcacaaag?agcttcaaca?ggggagagtg 1740

ttagacccag?ctttcttgta?caaagtggtt?gatctagagg?gccctattct?atagtgtcac 1800

ctaaatgcta?gagctcgctg?atcagcctcg?actgtgcctt?ctagttgcca?gccatctgtt 1860

gtttgcccct?cccccgtgcc?ttccttgacc?ctggaaggtg?ccactcccac?tgtcctttcc 1920

taataaaatg?aggaaattgc?atcgcattgt?ctgagtaggt?gtcattctat?tctggggggt 1980

ggggtggggc?aggacagcaa?gggggaggat?tgggaagaca?atagcaggca?tgctggggat 2040

gcggtgggct?ctatggcttc?tgaggcggaa?agaaccagct?ggggctctag?ggggtatccc 2100

cacgcgccct?gtagcggcgc?attaagcgcg?gcgggtgtgg?tggttacgcg?cagcgtgacc 2160

gctacacttg?ccagcgccct?agcgcccgct?cctttcgctt?tcttcccttc?ctttctcgcc 2220

acgttcgccg?ggcctctcaa?aaaagggaaa?aaaagcatgc?atctcaatta?gtcagcaacc 2280

atagtcccgc?ccctaactcc?gcccatcccg?cccctaactc?cgcccagttc?cgcccattct 2340

ccgccccatg?gctgactaat?tttttttatt?tatgcagagg?ccgaggccgc?ctcggcctct 2400

gagctattcc?agaagtagtg?aggaggcttt?tttggaggcc?taggcttttg?caaaaagctt 2460

ggggggacag?ctcagggctg?cgatttcgcg?ccaaacttga?cggcaatcct?agcgtgaagg 2520

ctggtaggat?tttatccccg?ctgccatcat?ggttcgacca?ttgaactgca?tcgtcgccgt 2580

gtcccaaaat?atggggattg?gcaagaacgg?agacctaccc?tggcctccgc?tcaggaacga 2640

gttcaagtac?ttccaaagaa?tgaccacaac?ctcttcagtg?gaaggtaaac?agaatctggt 2700

gattatgggt?aggaaaacct?ggttctccat?tcctgagaag?aatcgacctt?taaaggacag 2760

aattaatata?gttctcagta?gagaactcaa?agaaccacca?cgaggagctc?attttcttgc 2820

caaaagtttg?gatgatgcct?taagacttat?tgaacaaccg?gaattggcaa?gtaaagtaga 2880

catggtttgg?atagtcggag?gcagttctgt?ttaccaggaa?gccatgaatc?aaccaggcca 2940

cctcagactc?tttgtgacaa?ggatcatgca?ggaatttgaa?agtgacacgt?ttttcccaga 3000

aattgatttg?gggaaatata?aacttctccc?agaataccca?ggcgtcctct?ctgaggtcca 3060

ggaggaaaaa?ggcatcaagt?ataagtttga?agtctacgag?aagaaagact?aacaggaaga 3120

tgctttcaag?ttctctgctc?ccctcctaaa?gctatgcatt?tttataagac?catgggactt 3180

ttgctggctt?tagatctgat?ctttgtgaag?gaaccttact?tctgtggtgt?gacataattg 3240

gacaaactac?ctacagagat?ttaaagctct?aaggtaaata?taaaattttt?aagtgtataa 3300

tgtgttaaac?tactgattct?aattgtttgt?gtattttaga?ttccaaccta?tggaactgat 3360

gaatgggagc?agtggtggaa?tgcctttaat?gaggaaaacc?tgttttgctc?agaagaaatg 3420

ccatctagtg?atgatgaggc?tactgctgac?tctcaacatt?ctactcctcc?aaaaaagaag 3480

agaaaggtag?aagaccccaa?ggactttcct?tcagaattgc?taagtttttt?gagtcatgct 3540

gtgtttagta?atagaactct?tgcttgcttt?gctatttaca?ccacaaagga?aaaagctgca 3600

ctgctataca?agaaaattat?ggaaaaatat?tctgtaacct?ttataagtag?gcataacagt 3660

tataatcata?acatactgtt?ttttcttact?ccacacaggc?atagagtgtc?tgctattaat 3720

aactatgctc?aaaaattgtg?tacctttagc?tttttaattt?gtaaaggggt?taataaggaa 3780

tatttgatgt?atagtgcctt?gactagagat?cgatcataat?cagccatacc?acatttgtag 3840

aggttttact?tgctttaaaa?aacctcccac?acctccccct?gaacctgaaa?cataaaatga 3900

atgcaattgt?tgttgttaac?ttgtttattg?cagcttataa?tggttacaaa?taaagcaata 3960

gcatcacaaa?tttcacaaat?aaagcatttt?tttcactgca?ttctagttgt?ggtttgtcca 4020

aactcatcaa?tgtatcttat?catgtctgga?tcggctggat?gatcctccag?cgcggggatc 4080

tcatgctgga?gttcttcgcc?caccccaact?tgtttattgc?agcttataat?ggttacaaat 4140

aaagcaatag?catcacaaat?ttcacaaata?aagcattttt?ttcactgcat?tctagttgtg 4200

gtttgtccaa?actcatcaat?gtatcttatc?atgtctgtat?accgtcgacc?tctagctaga 4260

gcttggcgta?atcatggtca?tagctgtttc?ctgtgtgaaa?ttgttatccg?ctcacaattc 4320

cacacaacat?acgagccgga?agcataaagt?gtaaagcctg?gggtgcctaa?tgagtgagct 4380

aactcacatt?aattgcgttg?cgctcactgc?ccgctttcca?gtcgggaaac?ctgtcgtgcc 4440

agctgcatta?atgaatcggc?caacgcgcgg?ggagaggcgg?tttgcgtatt?gggcgctctt 4500

ccgcttcctc?gctcactgac?tcgctgcgct?cggtcgttcg?gctgcggcga?gcggtatcag 4560

ctcactcaaa?ggcggtaata?cggttatcca?cagaatcagg?ggataacgca?ggaaagaaca 4620

tgtgagcaaa?aggccagcaa?aaggccagga?accgtaaaaa?ggccgcgttg?ctggcgtttt 4680

tccataggct?ccgcccccct?gacgagcatc?acaaaaatcg?acgctcaagt?cagaggtggc 4740

gaaacccgac?aggactataa?agataccagg?cgtttccccc?tggaagctcc?ctcgtgcgct 4800

ctcctgttcc?gaccctgccg?cttaccggat?acctgtccgc?ctttctccct?tcgggaagcg 4860

tggcgctttc?tcatagctca?cgctgtaggt?atctcagttc?ggtgtaggtc?gttcgctcca 4920

agctgggctg?tgtgcacgaa?ccccccgttc?agcccgaccg?ctgcgcctta?tccggtaact 4980

atcgtcttga?gtccaacccg?gtaagacacg?acttatcgcc?actggcagca?gccactggta 5040

acaggattag?cagagcgagg?tatgtaggcg?gtgctacaga?gttcttgaag?tggtggccta 5100

actacggcta?cactagaagg?aacagtattt?ggtatctgcg?ctctgctgaa?gccagttacc 5160

ttcggaaaaa?gagttggtag?ctcttgatcc?ggcaaacaaa?ccaccgctgg?tagcggtggt 5220

ttttttgttt?gcaagcagca?gattacgcgc?agaaaaaaag?gatctcaaga?agatcctttg 5280

atcttttcta?cggggtctga?cgctcagtgg?aacgaaaact?cacgttaagg?gattttggtc 5340

atgagattat?caaaaaggat?cttcacctag?atccttttaa?attaaaaatg?aagttttaaa 5400

tcaatctaaa?gtatatatga?gtaaacttgg?tctgacagtt?accaatgctt?aatcagtgag 5460

gcacctatct?cagcgatctg?tctatttcgt?tcatccatag?ttgcctgact?ccccgtcgtg 5520

tagataacta?cgatacggga?gggcttacca?tctggcccca?gtgctgcaat?gataccgcga 5580

gacccacgct?caccggctcc?agatttatca?gcaataaacc?agccagccgg?aagggccgag 5640

cgcagaagtg?gtcctgcaac?tttatccgcc?tccatccagt?ctattaattg?ttgccgggaa 5700

gctagagtaa?gtagttcgcc?agttaatagt?ttgcgcaacg?ttgttgccat?tgctacaggc 5760

atcgtggtgt?cacgctcgtc?gtttggtatg?gcttcattca?gctccggttc?ccaacgatca 5820

aggcgagtta?catgatcccc?catgttgtgc?aaaaaagcgg?ttagctcctt?cggtcctccg 5880

atcgttgtca?gaagtaagtt?ggccgcagtg?ttatcactca?tggttatggc?agcactgcat 5940

aattctctta?ctgtcatgcc?atccgtaaga?tgcttttctg?tgactggtga?gtactcaacc 6000

aagtcattct?gagaatagtg?tatgcggcga?ccgagttgct?cttgcccggc?gtcaatacgg 6060

gataataccg?cgccacatag?cagaacttta?aaagtgctca?tcattggaaa?acgttcttcg 6120

gggcgaaaac?tctcaaggat?cttaccgctg?ttgagatcca?gttcgatgta?acccactcgt 6180

gcacccaact?gatcttcagc?atcttttact?ttcaccagcg?tttctgggtg?agcaaaaaca 6240

ggaaggcaaa?atgccgcaaa?aaagggaata?agggcgacac?ggaaatgttg?aatactcata 6300

ctcttccttt?ttcaatatta?ttgaagcatt?tatcagggtt?attgtctcat?gagcggatac 6360

atatttgaat?gtatttagaa?aaataaacaa?ataggggttc?cgcgcacatt?tccccgaaaa 6420

gtgccacctg?acgtc 6435

 

<210>5

<211>6435

<212>DNA

＜213〉artificial

 

<220>

＜223〉artificial sequence

 

<400>5

ctgcctagcc?ctctagacga?tcgggcccac?tggactccgc?gcggccgaag?cttatcggtc 60

tcattggaaa?aaaaaattaa?aataaaataa?aataaaaact?ctacctcaaa?ccgcggctag 120

agggctaggg?gataccagct?gagagtcatg?ttagacgaga?ctacggcgta?tcaattcggt 180

catagacgag?ggacgaacac?acaacctcca?gcgactcatc?acgcgctcgt?tttaaattcg 240

atgttgttcc?gttccgaact?ggctgttaac?gtacttctta?gacgaatccc?aatccgcaaa 300

acgcgacgaa?gcgctacatg?cccggtctat?atgcgcaact?gtaactaata?actgatcaat 360

aattatcatt?agttaatgcc?ccagtaatca?agtatcgggt?atatacctca?aggcgcaatg 420

tattgaatgc?catttaccgg?gcggaccgac?tggcgggttg?ctgggggcgg?gtaactgcag 480

ttattactgc?atacaagggt?atcattgcgg?ttatccctga?aaggtaactg?cagttaccca 540

cctcataaat?gccatttgac?gggtgaaccg?tcatgtagtt?cacatagtat?acggttcatg 600

cgggggataa?ctgcagttac?tgccatttac?cgggcggacc?gtaatacggg?tcatgtactg 660

gaataccctg?aaaggatgaa?ccgtcatgta?gatgcataat?cagtagcgat?aatggtacca 720

ctacgccaaa?accgtcatgt?agttacccgc?acctatcgcc?aaactgagtg?cccctaaagg 780

ttcagaggtg?gggtaactgc?agttaccctc?aaacaaaacc?gtggttttag?ttgccctgaa 840

aggttttaca?gcattgttga?ggcggggtaa?ctgcgtttac?ccgccatccg?cacatgccac 900

cctccagata?tattcgtctc?gagagaccga?ttgatctctt?gggtgacgaa?tgaccgaata 960

gctttaatta?tgctgagtga?tatccctctg?ggttcgaata?gttgttcaaa?catgtttttt 1020

cgtccgacca?tggtaccttc?ggggtcgagt?cgaagagaag?gaggacgatg?agaccgaggg 1080

tctatggtgg?cctctttaac?acaactgtgt?cagaggtcgg?tgggacagaa?acagaggtcc 1140

cctttctcgg?tgggagagga?cgtcccggtc?agtctcacaa?tcgtcgatga?atcggaccat 1200

ggttgtcttt?ggaccggtcc?gagggtccga?ggagtagata?ctacgtaggt?tgtcccggtg 1260

accgtagggt?cggtccaagt?caccgtcacc?cagaccctgt?ctgaagtgag?agtggtagtc 1320

gtcggatctc?ggacttctaa?aacgtcaaat?aatgacagtc?gtcgcatcgt?tgaccggagg 1380

ccgcgagtga?aagccgcctc?cctggttcca?cctctagttt?gcatgccacc?gacgtggtag 1440

acagaagtag?aagggcggta?gactactcgt?caactttaga?ccttgacgga?gacaacacac 1500

ggacgactta?ttgaagatag?ggtctctccg?gtttcatgtc?accttccacc?tattgcggga 1560

ggttagccca?ttgagggtcc?tctcacagtg?tctcgtcctg?tcgttcctgt?cgtggatgtc 1620

ggagtcgtcg?tgggactgcg?actcgtttcg?tctgatgctc?tttgtgtttc?agatgcggac 1680

gcttcagtgg?gtagtcccgg?actcgagcgg?gcagtgtttc?tcgaagttgt?cccctctcac 1740

aatctgggtc?gaaagaacat?gtttcaccaa?ctagatctcc?cgggataaga?tatcacagtg 1800

gatttacgat?ctcgagcgac?tagtcggagc?tgacacggaa?gatcaacggt?cggtagacaa 1860

caaacgggga?gggggcacgg?aaggaactgg?gaccttccac?ggtgagggtg?acaggaaagg 1920

attattttac?tcctttaacg?tagcgtaaca?gactcatcca?cagtaagata?agacccccca 1980

ccccaccccg?tcctgtcgtt?ccccctccta?acccttctgt?tatcgtccgt?acgaccccta 2040

cgccacccga?gataccgaag?actccgcctt?tcttggtcga?ccccgagatc?ccccataggg 2100

gtgcgcggga?catcgccgcg?taattcgcgc?cgcccacacc?accaatgcgc?gtcgcactgg 2160

cgatgtgaac?ggtcgcggga?tcgcgggcga?ggaaagcgaa?agaagggaag?gaaagagcgg 2220

tgcaagcggc?ccggagagtt?ttttcccttt?ttttcgtacg?tagagttaat?cagtcgttgg 2280

tatcagggcg?gggattgagg?cgggtagggc?ggggattgag?gcgggtcaag?gcgggtaaga 2340

ggcggggtac?cgactgatta?aaaaaaataa?atacgtctcc?ggctccggcg?gagccggaga 2400

ctcgataagg?tcttcatcac?tcctccgaaa?aaacctccgg?atccgaaaac?gtttttcgaa 2460

cccccctgtc?gagtcccgac?gctaaagcgc?ggtttgaact?gccgttagga?tcgcacttcc 2520

gaccatccta?aaataggggc?gacggtagta?ccaagctggt?aacttgacgt?agcagcggca 2580

cagggtttta?tacccctaac?cgttcttgcc?tctggatggg?accggaggcg?agtccttgct 2640

caagttcatg?aaggtttctt?actggtgttg?gagaagtcac?cttccatttg?tcttagacca 2700

ctaataccca?tccttttgga?ccaagaggta?aggactcttc?ttagctggaa?atttcctgtc 2760

ttaattatat?caagagtcat?ctcttgagtt?tcttggtggt?gctcctcgag?taaaagaacg 2820

gttttcaaac?ctactacgga?attctgaata?acttgttggc?cttaaccgtt?catttcatct 2880

gtaccaaacc?tatcagcctc?cgtcaagaca?aatggtcctt?cggtacttag?ttggtccggt 2940

ggagtctgag?aaacactgtt?cctagtacgt?ccttaaactt?tcactgtgca?aaaagggtct 3000

ttaactaaac?ccctttatat?ttgaagaggg?tcttatgggt?ccgcaggaga?gactccaggt 3060

cctccttttt?ccgtagttca?tattcaaact?tcagatgctc?ttctttctga?ttgtccttct 3120

acgaaagttc?aagagacgag?gggaggattt?cgatacgtaa?aaatattctg?gtaccctgaa 3180

aacgaccgaa?atctagacta?gaaacacttc?cttggaatga?agacaccaca?ctgtattaac 3240

ctgtttgatg?gatgtctcta?aatttcgaga?ttccatttat?attttaaaaa?ttcacatatt 3300

acacaatttg?atgactaaga?ttaacaaaca?cataaaatct?aaggttggat?accttgacta 3360

cttaccctcg?tcaccacctt?acggaaatta?ctccttttgg?acaaaacgag?tcttctttac 3420

ggtagatcac?tactactccg?atgacgactg?agagttgtaa?gatgaggagg?ttttttcttc 3480

tctttccatc?ttctggggtt?cctgaaagga?agtcttaacg?attcaaaaaa?ctcagtacga 3540

cacaaatcat?tatcttgaga?acgaacgaaa?cgataaatgt?ggtgtttcct?ttttcgacgt 3600

gacgatatgt?tcttttaata?cctttttata?agacattgga?aatattcatc?cgtattgtca 3660

atattagtat?tgtatgacaa?aaaagaatga?ggtgtgtccg?tatctcacag?acgataatta 3720

ttgatacgag?tttttaacac?atggaaatcg?aaaaattaaa?catttcccca?attattcctt 3780

ataaactaca?tatcacggaa?ctgatctcta?gctagtatta?gtcggtatgg?tgtaaacatc 3840

tccaaaatga?acgaaatttt?ttggagggtg?tggaggggga?cttggacttt?gtattttact 3900

tacgttaaca?acaacaattg?aacaaataac?gtcgaatatt?accaatgttt?atttcgttat 3960

cgtagtgttt?aaagtgttta?tttcgtaaaa?aaagtgacgt?aagatcaaca?ccaaacaggt 4020

ttgagtagtt?acatagaata?gtacagacct?agccgaccta?ctaggaggtc?gcgcccctag 4080

agtacgacct?caagaagcgg?gtggggttga?acaaataacg?tcgaatatta?ccaatgttta 4140

tttcgttatc?gtagtgttta?aagtgtttat?ttcgtaaaaa?aagtgacgta?agatcaacac 4200

caaacaggtt?tgagtagtta?catagaatag?tacagacata?tggcagctgg?agatcgatct 4260

cgaaccgcat?tagtaccagt?atcgacaaag?gacacacttt?aacaataggc?gagtgttaag 4320

gtgtgttgta?tgctcggcct?tcgtatttca?catttcggac?cccacggatt?actcactcga 4380

ttgagtgtaa?ttaacgcaac?gcgagtgacg?ggcgaaaggt?cagccctttg?gacagcacgg 4440

tcgacgtaat?tacttagccg?gttgcgcgcc?cctctccgcc?aaacgcataa?cccgcgagaa 4500

ggcgaaggag?cgagtgactg?agcgacgcga?gccagcaagc?cgacgccgct?cgccatagtc 4560

gagtgagttt?ccgccattat?gccaataggt?gtcttagtcc?cctattgcgt?cctttcttgt 4620

acactcgttt?tccggtcgtt?ttccggtcct?tggcattttt?ccggcgcaac?gaccgcaaaa 4680

aggtatccga?ggcgggggga?ctgctcgtag?tgtttttagc?tgcgagttca?gtctccaccg 4740

ctttgggctg?tcctgatatt?tctatggtcc?gcaaaggggg?accttcgagg?gagcacgcga 4800

gaggacaagg?ctgggacggc?gaatggccta?tggacaggcg?gaaagaggga?agcccttcgc 4860

accgcgaaag?agtatcgagt?gcgacatcca?tagagtcaag?ccacatccag?caagcgaggt 4920

tcgacccgac?acacgtgctt?ggggggcaag?tcgggctggc?gacgcggaat?aggccattga 4980

tagcagaact?caggttgggc?cattctgtgc?tgaatagcgg?tgaccgtcgt?cggtgaccat 5040

tgtcctaatc?gtctcgctcc?atacatccgc?cacgatgtct?caagaacttc?accaccggat 5100

tgatgccgat?gtgatcttcc?ttgtcataaa?ccatagacgc?gagacgactt?cggtcaatgg 5160

aagccttttt?ctcaaccatc?gagaactagg?ccgtttgttt?ggtggcgacc?atcgccacca 5220

aaaaaacaaa?cgttcgtcgt?ctaatgcgcg?tctttttttc?ctagagttct?tctaggaaac 5280

tagaaaagat?gccccagact?gcgagtcacc?ttgcttttga?gtgcaattcc?ctaaaaccag 5340

tactctaata?gtttttccta?gaagtggatc?taggaaaatt?taatttttac?ttcaaaattt 5400

agttagattt?catatatact?catttgaacc?agactgtcaa?tggttacgaa?ttagtcactc 5460

cgtggataga?gtcgctagac?agataaagca?agtaggtatc?aacggactga?ggggcagcac 5520

atctattgat?gctatgccct?cccgaatggt?agaccggggt?cacgacgtta?ctatggcgct 5580

ctgggtgcga?gtggccgagg?tctaaatagt?cgttatttgg?tcggtcggcc?ttcccggctc 5640

gcgtcttcac?caggacgttg?aaataggcgg?aggtaggtca?gataattaac?aacggccctt 5700

cgatctcatt?catcaagcgg?tcaattatca?aacgcgttgc?aacaacggta?acgatgtccg 5760

tagcaccaca?gtgcgagcag?caaaccatac?cgaagtaagt?cgaggccaag?ggttgctagt 5820

tccgctcaat?gtactagggg?gtacaacacg?ttttttcgcc?aatcgaggaa?gccaggaggc 5880

tagcaacagt?cttcattcaa?ccggcgtcac?aatagtgagt?accaataccg?tcgtgacgta 5940

ttaagagaat?gacagtacgg?taggcattct?acgaaaagac?actgaccact?catgagttgg 6000

ttcagtaaga?ctcttatcac?atacgccgct?ggctcaacga?gaacgggccg?cagttatgcc 6060

ctattatggc?gcggtgtatc?gtcttgaaat?tttcacgagt?agtaaccttt?tgcaagaagc 6120

cccgcttttg?agagttccta?gaatggcgac?aactctaggt?caagctacat?tgggtgagca 6180

cgtgggttga?ctagaagtcg?tagaaaatga?aagtggtcgc?aaagacccac?tcgtttttgt 6240

ccttccgttt?tacggcgttt?tttcccttat?tcccgctgtg?cctttacaac?ttatgagtat 6300

gagaaggaaa?aagttataat?aacttcgtaa?atagtcccaa?taacagagta?ctcgcctatg 6360

tataaactta?cataaatctt?tttatttgtt?tatccccaag?gcgcgtgtaa?aggggctttt 6420

cacggtggac?tgcag 6435

 

<210>6

<211>236

<212>PRT

＜213〉artificial

 

<220>

＜223〉artificial sequence

 

<400>6

 

Met?Glu?Ala?Pro?Ala?Gln?Leu?Leu?Phe?Leu?Leu?Leu?Leu?Trp?Leu?Pro

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Asn?Trp?Pro?Pro?Ala?Leu?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile

115 120 125

Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp

130 135 140

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

145 150 155 160

Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu

165 170 175

Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp

180 185 190

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

195 200 205

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

210 215 220

Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys

225 230 235

 

<210>7

<211>1413

<212>DNA

＜213〉artificial

 

<220>

＜223〉artificial sequence

 

<400>7

atgaaacacc?tgtggttctt?cctcctcctg?gtggcagctc?ccagatgggt?cctgtcccag 60

gtgcaactac?agcagtgggg?cgcaggactg?ttgaagcctt?cggagaccct?gtccctcacc 120

tgcgctgtct?atggtgggtc?cttcagtggt?tactactgga?gctggatacg?ccagtcccca 180

gagaaggggc?tggagtggat?tggggaaatc?aatcatggtg?gatacgtcac?ctacaatccg 240

tccctcgaga?gtcgagtcac?catatcagta?gacacgtcca?agaaccagtt?ctccctgaag 300

ctgagctctg?tgaccgccgc?ggacacggct?gtatattact?gtgcgaggga?ctatggtccg 360

gggaattatg?actggtactt?cgatctctgg?ggccgtggca?ccctggtcac?tgtctcctca 420

gcctccacca?agggcccatc?ggtcttcccc?ctggcaccct?cctccaagag?cacctctggg 480

ggcacagcgg?ccctgggctg?cctggtcaag?gactacttcc?ccgaaccggt?gacggtgtcg 540

tggaactcag?gcgccctgac?cagcggcgtg?cacaccttcc?cggctgtcct?acagtcctca 600

ggactctact?ccctcagcag?cgtggtgacc?gtgccctcca?gcagcttggg?cacccagacc 660

tacatctgca?acgtgaatca?caagcccagc?aacaccaagg?tggacaagag?agttgagccc 720

aaatcttgtg?acaaaactca?cacatgccca?ccgtgcccag?cacctgaact?cctgggggga 780

ccgtcagtct?tcctcttccc?cccaaaaccc?aaggacaccc?tcatgatctc?ccggacccct 840

gaggtcacat?gcgtggtggt?ggacgtgagc?cacgaagacc?ctgaggtcaa?gttcaactgg 900

tacgtggacg?gcgtggaggt?gcataatgcc?aagacaaagc?cgcgggagga?gcagtacaac 960

agcacgtacc?gtgtggtcag?cgtcctcacc?gtcctgcacc?aggactggct?gaatggcaag 1020

gagtacaagt?gcaaggtctc?caacaaagcc?ctcccagccc?ccatcgagaa?aaccatctcc 1080

aaagccaaag?ggcagccccg?agaaccacag?gtgtacaccc?tgcccccatc?ccgggatgag 1140

ctgaccaaga?accaggtcag?cctgacctgc?ctggtcaaag?gcttctatcc?cagcgacatc 1200

gccgtggagt?gggagagcaa?tgggcagccg?gagaacaact?acaagaccac?gcctcccgtg 1260

ctggactccg?acggctcctt?cttcctctac?agcaagctca?ccgtggacaa?gagcaggtgg 1320

cagcagggga?acgtcttctc?atgctccgtg?atgcatgagg?ctctgcacaa?ccactacacg 1380

cagaagagcc?tctccctgtc?cccgggtaaa?tga 1413

 

<210>8

<211>1413

<212>DNA

＜213〉artificial

 

<220>

＜223〉artificial sequence

 

<400>8

tactttgtgg?acaccaagaa?ggaggaggac?caccgtcgag?ggtctaccca?ggacagggtc 60

cacgttgatg?tcgtcacccc?gcgtcctgac?aacttcggaa?gcctctggga?cagggagtgg 120

acgcgacaga?taccacccag?gaagtcacca?atgatgacct?cgacctatgc?ggtcaggggt 180

ctcttccccg?acctcaccta?acccctttag?ttagtaccac?ctatgcagtg?gatgttaggc 240

agggagctct?cagctcagtg?gtatagtcat?ctgtgcaggt?tcttggtcaa?gagggacttc 300

gactcgagac?actggcggcg?cctgtgccga?catataatga?cacgctccct?gataccaggc 360

cccttaatac?tgaccatgaa?gctagagacc?ccggcaccgt?gggaccagtg?acagaggagt 420

cggaggtggt?tcccgggtag?ccagaagggg?gaccgtggga?ggaggttctc?gtggagaccc 480

ccgtgtcgcc?gggacccgac?ggaccagttc?ctgatgaagg?ggcttggcca?ctgccacagc 540

accttgagtc?cgcgggactg?gtcgccgcac?gtgtggaagg?gccgacagga?tgtcaggagt 600

cctgagatga?gggagtcgtc?gcaccactgg?cacgggaggt?cgtcgaaccc?gtgggtctgg 660

atgtagacgt?tgcacttagt?gttcgggtcg?ttgtggttcc?acctgttctc?tcaactcggg 720

tttagaacac?tgttttgagt?gtgtacgggt?ggcacgggtc?gtggacttga?ggacccccct 780

ggcagtcaga?aggagaaggg?gggttttggg?ttcctgtggg?agtactagag?ggcctgggga 840

ctccagtgta?cgcaccacca?cctgcactcg?gtgcttctgg?gactccagtt?caagttgacc 900

atgcacctgc?cgcacctcca?cgtattacgg?ttctgtttcg?gcgccctcct?cgtcatgttg 960

tcgtgcatgg?cacaccagtc?gcaggagtgg?caggacgtgg?tcctgaccga?cttaccgttc 1020

ctcatgttca?cgttccagag?gttgtttcgg?gagggtcggg?ggtagctctt?ttggtagagg 1080

tttcggtttc?ccgtcggggc?tcttggtgtc?cacatgtggg?acgggggtag?ggccctactc 1140

gactggttct?tggtccagtc?ggactggacg?gaccagtttc?cgaagatagg?gtcgctgtag 1200

cggcacctca?ccctctcgtt?acccgtcggc?ctcttgttga?tgttctggtg?cggagggcac 1260

gacctgaggc?tgccgaggaa?gaaggagatg?tcgttcgagt?ggcacctgtt?ctcgtccacc 1320

gtcgtcccct?tgcagaagag?tacgaggcac?tacgtactcc?gagacgtgtt?ggtgatgtgc 1380

gtcttctcgg?agagggacag?gggcccattt?act 1413

 

<210>9

<211>470

<212>PRT

＜213〉artificial

 

<220>

＜223〉artificial sequence

 

<400>9

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr?Cys?Ala?Arg?Asp?Tyr?Gly?Pro?Gly?Asn?Tyr?Asp?Trp?Tyr?Phe?Asp

115 120 125

Leu?Trp?Gly?Arg?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys

130 135 140

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

145 150 155 160

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

165 170 175

Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr

180 185 190

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

195 200 205

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

210 215 220

Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Arg?Val?Glu?Pro

225 230 235 240

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

245 250 255

Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile

385 390 395 400

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

405 410 415

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

420 425 430

Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys

435 440 445

Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu

450 455 460

Ser?Leu?Ser?Pro?Gly?Lys

465 470

Claims (10)

1. specificity is in conjunction with monoclonal antibody or its antigen-binding portion thereof of 4-1BB, and it comprises variable region of light chain and variable region of heavy chain, wherein:

Described variable region of light chain comprises the CDR1 that the amino acid 44-54 by SEQ ID NO:6 forms, CDR2 that is made up of the amino acid 70-76 of SEQ ID NO:6 and the CDR3 that is made up of the amino acid/11 09-119 of SEQ ID NO:6; With

Described variable region of heavy chain comprises the CDR1 that the amino acid 50-54 by SEQ ID NO:3 forms, CDR2 that is made up of the amino acid 69-84 of SEQ ID NO:3 and the CDR3 that is made up of the amino acid/11 17-129 of SEQ ID NO:3.

2. the monoclonal antibody of claim 1 or its antigen-binding portion thereof, wherein:

Described light chain comprises the variable region that the amino acid 21-129 by SEQ ID NO:6 forms; With

Described heavy chain comprises the variable region that the amino acid 20-140 by SEQ ID NO:3 forms.

3. the monoclonal antibody that comprises light chain and heavy chain, wherein said light chain are made up of the amino-acid residue 21-236 of SEQ ID NO:6 and described heavy chain is made up of the amino-acid residue 20-467 of SEQ ID NO:3.

4. pharmaceutical composition comprises:

The monoclonal antibody of claim 1 or its antigen-binding portion thereof; With

Pharmaceutically acceptable carrier.

5. pharmaceutical composition comprises:

The monoclonal antibody of claim 3; With

Pharmaceutically acceptable carrier.

6. the monoclonal antibody of claim 1 or its antigen-binding portion thereof are used for the treatment of purposes in the medicine of the cancer among the experimenter in preparation.

7. isolating polynucleotide, it comprises the nucleotide sequence of the amino-acid residue 20-467 of encoding amino acid sequence SEQ ID NO:3.

8. the polynucleotide of claim 7, it comprises nucleotide sequence SEQ ID NO:1.

9. isolating polynucleotide, it comprises the nucleotide sequence of the amino-acid residue 21-236 of encoding amino acid sequence SEQ ID NO:6.

10. the polynucleotide of claim 9, it comprises nucleotide sequence SEQ ID NO:4.

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