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

Abstract

A complete human antibody and an antigen-binding portion thereof that binds to human 4-1BB and allows the binding of human 4-1BB to human 4-1BB ligands. On the one hand, the antibody is an IgG4 antibody. A method for treating a disease in a subject is also provided, comprising administering a therapeutically effective amount of an antibody to the subject.

Description

Fully human antibody against human 4-1BB (CD137)

technical field

The present invention relates to fully human antibodies, in particular to fully human antibodies to human 4-1BB (CD137).

Background technique

Multiple lines of evidence clearly demonstrate that a certain degree of anticancer immune response exists in humans and animals. In cancer patients, cellular components of the immune system can recognize antigens expressed by tumor cells, such as differentiated or mutated gene products of carcinoembryonic antigen (S. Rosenberg, Nature, 411:380-4 (2001); P. van der Bruggen et al. , Immunological Rev., 188:51-64 (2002)). A variety of clinical studies have shown that tumor-infiltrating lymphocytes have favorable prognostic significance (E. Halapi, Med. Oncol., 15 (4): 203-11 (1998); Y. Naito et al., Cancer Res., 58 (16 ): 3491-4 (1998); L. Zhang et al., N.E.J. Med., 348(3): 203-13 (2003)). Furthermore, therapy with immunomodulators such as cytokines or bacterial products, cancer vaccines or adaptive immunotherapy can lead to tumor regression in many patients (S. Rosenberg, Cancer J. Sci. Am. 6(S): 2 (2000); P. Bassi, Surg. Oncol., 11(1-2): 77-83 (2002); S. Antonia et al., Current Opinion in Immunol., 16: 130-6 (2004)). Despite these responses, immunity against cancer is often ineffective in eliminating tumor cells. The causes of such failures can be divided into three main groups: (i) Impaired tumor recognition by immune cells, resulting from variable expression of tumor antigens or reduced expression of major histocompatibility complex (MHC) class I (ii) an immunosuppressive tumor microenvironment, as a result of tumor cells secreting suppressive cytokines (eg, TGF-β); and (iii) tumor immunogens due to lack of expression of costimulatory molecules on tumor cells Poor sex, which results in tumor cells not being able to effectively stimulate T cells. Advances in our understanding of the need for tumor antigen recognition and immune effector function suggest that a possible strategy for enhancing anti-tumor immune responses is to provide co-stimulation through accessory molecules. Tumor antigen-specific T cells require co-stimulation to initiate and maintain effector functions. Therefore, therapies targeting co-stimulatory molecules can be used to modulate and enhance the immune response to tumors.

Current models of T cell activation assume that naive T cells require two signals for adequate activation: (i) a signal provided by the binding of processed antigens, which are presented to T cells by major histocompatibility complex (MHC) class I molecules receptors; and (ii) other signals provided by the interaction of co-stimulatory molecules on the surface of T cells with their ligands on antigen presenting cells. Antigen recognition by naive T cells is not, by itself, sufficient to elicit T cell activation. In the absence of co-stimulatory signals, T cells can be eliminated by death or induction of inactivity. Signaling through the costimulatory molecule CD28 appears to be key to the initiation of T cell responses. However, CD137(4-1BB) signaling has been shown to be important for maintaining and expanding immune responses to antigens, and for generating memory T cells.

CD137(4-1BB) is a member of the tumor necrosis factor receptor (TNF-R) gene family, which includes proteins involved in the regulation of cell proliferation, differentiation and programmed cell death. CD137 is a 30 kDa type I membrane glycoprotein expressed as a 55 kDa homodimer. The receptor was first described in mouse (B. Kwon et al., P.N.A.S. USA, 86: 1963-7 (1989)) and subsequently identified in humans (M. Alderson et al., Eur. J. Immunol., 24: 2219 -27 (1994); Z. Zhou et al., Immunol. Lett., 45:67 (1995)) (see also published PCT publications WO95/07984 and WO96/29348, and US Patent 6,569,997, incorporated herein by reference ( See, SEQ ID NO: 2.)). The human and mouse forms of CD137 are 60% identical at the amino acid level. Conserved sequences are present in the cytoplasmic domain as well as five other regions of the molecule, suggesting that these residues may be important for the function of the CD137 molecule (Z. Zhou et al., Immunol. Lett., 45:67 (1995)). CD137 is expressed mainly on lymphoid cell lines such as activated T-cells, activated natural killer (NK) cells, NKT-cells, CD4CD25 regulatory T-cells, but also on activated thymocytes and intradermal lymphocytes. In addition, CD137 is also expressed on myeloid-derived cells such as dendritic cells, monocytes, neutrophils, and eosinophils. Although CD137 expression is mainly restricted to immune/inflammatory cells, reports describe its expression on endothelial cells associated with a small number of tissues from inflammatory sites and tumors.

The functional activity of CD137 on T cells has been well characterized. Signaling through CD137 in the presence of suboptimal doses of anti-CD3 antibodies has been shown to induce T cell proliferation and cytokine synthesis (mainly IFN-γ), and to inhibit activated cell death. These effects have been observed using mouse and human T-cells (W.Shuford et al., J.Exp.Med., 186(1):47-55 (1997); D.Vinay et al., Semin.Immunol., 10( 6): 481-9 (1998); D. Laderach et al., Int. Immunol., 14(10): 1155-67 (2002)). In humans and mice, co-stimulation improves effector functions, such as IFN-γ production and cytotoxicity, by increasing antigen specificity and numbers of effector CD8+ T-cells. In the absence of anti-CD3 signaling, stimulation by CD137 did not alter T cell function, suggesting that CD137 is a co-stimulatory molecule.

The observed physiological events following CD137 stimulation of T cells are mediated by NF-κB and PI3K/ERK1/2 signaling with different physiological functions. NF-κB signaling stimulates the expression of an anti-apoptotic molecule, Bcl- _XL , resulting in increased survival, while PI3K and ERK1/2 signaling are specifically responsible for CD137-mediated cell cycle progression (H. Lee et al., J. Immunol ., 169(9):4882-8(2002)). The inhibitory effect of CD137 activation on activation-induced cell death was shown in vitro by Hurtado et al. (J. CD137 monoclonal (mab) was shown to produce long-term survival of superantigen-activated CD8+ T-cells by preventing clonal deletion (C. Takahashi et al., J. Immunol., 162:5037 (1999)). Subsequently, two reports showed that CD137 signaling regulates the clonal expansion and survival of CD8+ T-cells under different experimental conditions (D. Cooper et al., Eur. J. Immunol., 32 (2): 521-9 (2002 ); M. Maus et al., Nat. Biotechnol., 20: 143 (2002)). The decreased apoptosis observed after co-stimulation was associated with increased Bcl- _XL levels in CD8+ T-cells, while Bcl-2 expression was unchanged. Upregulation of the anti-apoptotic genes Bcl- _xL and bfl-1 via 4-1BB was shown to be mediated by NF-κB activation, as PDTC, an NF-κB-specific blocker, inhibits 4-1BB-mediated Bcl- _xL is upregulated (H. Lee et al., J. Immunol., 169(9):4882-8 (2002)). On the other hand, clonal expansion of activated T cells was shown to be mediated by increased expression of cyclins D2, D3, and E and downregulation of p27 ^kip1 protein. This effect occurs in an IL-2-dependent and -independent manner (H. Lee et al., J. Immunol., 169(9):4882-8 (2002)).

Altogether, CD137 stimulation resulted in enhanced expansion, survival, and effector function of newly primed CD8+ T-cells, in part acting directly on these cells. CD4+ and CD8+ T- cells have been shown to respond to CD137 stimulation, but the enhancement of T cell function was shown to be higher in CD8+ cells (W. Shuford et al., J. Exp. Med., 186(1):47-55 (1997 ); I. Gramaglia et al., Eur. J. Immunol., 30(2): 392-402 (2000); C. Takahashi et al., J. Immunol., 162: 5037 (1999)). Based on the essential role of CD137 stimulation in CD8+ T-cell function and survival, manipulation of the CD137/CD137L system offers a plausible approach to the treatment of tumor and viral antigens.

Recently, constitutive expression of CD137 on freshly isolated dendritic cells (DC) was detected in mice (R. Wilcox et al., J. Immunol., 169(8):4230-6 (2002); T. Futagawa et al., Int . Immunol., 14(3): 275-86 (2002)) and in humans (S. Pauly et al., J. Leukoc. Biol. 72(1): 35-42 (2002)). These reports show that stimulation of CD17 on DCs results in the secretion of IL-6 and IL-12 and, more importantly, enhances the ability of DCs to stimulate T cell responses to alloantigens. In addition, Pan et al. confirmed that CD137 signaling in DCs leads to upregulation of MHC class I molecules and co-stimulatory molecules, and enhances the ability of DCs to infiltrate tumors (P. Pan et al., J. Immunol., 172(8): 4779-89 (2004 )). Thus, CD137 co-stimulation on DCs appears to be a novel pathway for DC proliferation, maturation and migration.

Activated natural killer (NK) cells express CD137 after stimulation with cytokines (I. Melero et al., Cell Immunol., 190(2): 167-72 (1998); R.Wilcox et al., J.Immunol., 169( 8): 4230-6 (2002)). Several reports demonstrate that NK cells are important for anti-tumor autoimmunity induced by agonistic CD137 antibodies ((I. Melero et al., Cell Immunol., 190(2): 167-72 (1998); R. Miller et al. , J.Immunol., 169 (4): 1792-800 (2002); R.Wilcox et al., J.Immunol., 169 (8): 4230-6 (2002)). The depletion of NK cells significantly reduces the anti-CD137 Antitumor activity of mabs. Ligation of CD137 on NK cells induces proliferation and IFN-γ secretion, but does not affect their cytolytic activity. Clearly, in vitro, CD137-stimulated NK cells exhibit lytic effects on CD8+ cells The immunomodulatory or "helper" activity of NK cells leads to the expansion of activated T-cells. Thus, CD137 signaling on NK cells can regulate intrinsic immunity to tumors.

A possible effect was described for CD137 stimulation, where agonistic CD137 antibodies could induce suppression of humoral responses to T cell antigens in primates and in mouse models (H. Hong et al., J. Immunother., 23(6) : 613-21 (2000); R. Mittler et al., J. Exp. Med., 190(10): 1535-40 (1999)). Remarkably, CD137 agonistic antibodies were shown to result in significant relief of symptoms associated with antibody-dependent autoimmune diseases such as systemic lupus erythematosus and experimental autoimmune meningitis (J. Foell et al., N.Y. Acad. Sci., 987: 230-5(2003); Y. Sun et al., Nat. Med., 8(12):1405-13(2002)). Recently, Seo et al demonstrated that in a mouse model of rheumatoid arthritis, treatment with an agonistic anti-CD137 antibody prevented disease onset and significantly blocked disease progression (S.K.Seo et al., Nat.Med. 10;1099-94 (2004)). The mechanism leading to this effect is not well defined, but in a rheumatoid arthritis model it was shown that treatment with a CD137 agonistic antibody resulted in the expansion of IFN-γ producing CD11C-CD8+ T cells. IFN-γ in turn stimulates dendritic cells to produce indolamine-2,3-dioxygenase (IDO), which exhibits immunosuppressive activity. It has been postulated that CD137 signaling on antigen-activated CD4+ T-cells results in the induction of IFN-γ secretion, which activates macrophages. Activated macrophages can in turn signal B cell death. Continuous signaling through CD137 on CD4+ T-cells can then induce activation-induced cell death of these CD4+ activated T-cells. Thus, by depleting antigen-activated T-cells and B-cells, a reduced antibody response was observed, whereby a significant reduction in Th2-mediated inflammatory diseases was observed (B. Kwon et al., J. Immunol., 168(11) : 5483-90 (2002)). These studies suggest the use of agonistic CD137 antibodies to treat inflammatory or autoimmune diseases without inducing a general suppression of the immune system.

The natural ligand for CD137, CD137 ligand (CD137L), a 34 kDa glycoprotein member of the TNF superfamily, is detected primarily on activated antigen-presenting cells (APCs) such as B cells, macrophages, dendritic cells, and also in Murine B-cell lymphoma, activated T-cells, and human epithelial-derived cancer cell lines were detected (R.Goodwin et al., Eur.J.Immunol., 23(10):2631-41(1993); Z . Zhou et al., Immunol. Lett., 45: 67 (1995); H. Salih et al., J. Immunol., 165(5): 2903-10 (2000)). Human CD137L shares 36% homology with its murine counterpart (M. Alderson et al., Eur. J. Immunol., 24:2219-27 (1994)).

In addition to delivering signals to CD-137-expressing cells, the binding of CD137 to CD137L initiates bidirectional signaling that results in functional effects on CD137L-expressing cells. Langstein et al. confirmed that the binding of CD137-Ig fusion protein to CD137L on activated monocytes induces the production of IL-6, IL-8, and TNF-α, upregulates ICAM, and inhibits IL-10, resulting in increased adhesion (J . Langstein et al., J. Immunol., 160(5):2488-94 (1998)). Furthermore, proliferation of monocytes was shown to be accompanied by a high rate of apoptosis (J. Langstein et al., J. Leukoc. Biol., 65(6):829-33 (1999)). These observations were corroborated by studies by Ju et al. (S. Ju et al., Hybrid Hybridomics, 22(5):333-8 (2003)), which showed that functional anti-CD137L antibodies induced a high rate of proliferation of peripheral blood mononuclear cells. Blocking the ligand results in inhibition of T cell activation. Furthermore, soluble CD137L is found in the serum of patients with rheumatoid arthritis as well as hematological malignancies (H. Salih et al., J. Immunol., 167(7):4059-66 (2001)). Thus, the interaction of CD137 with CD137L influences and leads to functional effects on T-cells and APCs.

In another important aspect of T cell function, it was demonstrated that agonistic anti-CD137 antibodies rescue T cell responses to protein antigens in aged mice. The age-related reduction in immune response to antigens is well documented, a process known as immunosenescence (R. Miller, Science, 273:70-4 (1996); R. Miller, Vaccine, 18:1654-60 (2000); F. Hakim et al., Curr. Opinion Immunol., 16:151-156 (2004)). This phenomenon results from an altered balance between cell expansion and the extent to which cells survive or die. Bansal-Pakala et al. tested the hypothesis that secondary co-stimulation via CD137 could be used to enhance T-cell responses in situations where T-cells are not sufficiently stimulated due to reduced expression of CD3 or CD28, or reduced quality of the signal. Their studies showed that aged mice lacked in vitro recall responses compared to young mice (P. Bansal-Pakala et al., J. Immunol., 169(9):5005-9 (2002)). However, T cell proliferation and cytokine responses in aged mice treated with anti-CD137 mAb were the same as those observed in young mice. Although the exact mechanism responsible for this effect is unknown, enhanced expression of anti-apoptotic molecules such as Bcl- _XL and enhanced secretion of IL-2 in vivo are thought to play a role in rescuing defective T cell responses. These studies demonstrate the possibility of agonistic anti-CD137 antibodies to rescue weak T cell responses in elderly immunocompromised individuals and have profound implications for the application of anti-CD137 antibodies in cancer patients.

The role of CD137-targeted therapy in cancer treatment was demonstrated by in vivo efficacy studies in mice using an agonistic anti-mouse CD137 monoclonal antibody. In Melero et al., an agonistic anti-mouse CD137 antibody resulted in cure in P815 mastocytoma as well as in the poorly immunogenic tumor model Ag104 (I. Melero et al., Nat. Med., 3(6): 682-5 (1997)), CD4+, CD8+ T-cells and NK cells are required for anti-tumor effects, as selective in vivo depletion of each subset leads to a reduction or complete absence of anti-tumor effects. Multiple researchers have confirmed the effectiveness of this method for cancer therapy using anti-CD137 antibodies (J.Kim et al., Cancer Res., 61(5):2031-7(2001); O.Martinet et al., Gene Ther., 9( 12):786-92(2002); R.Miller et al., J.Immunol., 169(4):1792-800(2002); R.Wilcox et al., Cancer Res., 62(15):4413-8( 2002)).

In support of the antitumor efficacy data for agonistic CD137 antibodies, CD137L provided a signal that was shown to stimulate CTL activity and antitumor responses (M.DeBenedette et al., J.Immunol., 163(9):4833-41 (1999); B.Guinn et al., J. Immunol., 162(8):5003-10(1999)). Several reports demonstrated that gene transfer of CD137 ligands into mouse sarcomas resulted in tumor rejection, demonstrating the need for co-stimulation to generate an effective immune response (S. Mogi et al., Immunology, 101(4):541-7 (2000); I.Melero et al., Cell Immunol., 190 (2): 167-72 (1998); B.Guinn et al., J.Immunol., 162 (8): 5003-10 (1999)). Salih et al. reported that CD137L was in expression in human cancers and human cancer cell lines (H.Salih et al., J.Immunol., 165(5):2903-10(2000)), and demonstrated that tumor cells expressing the ligand were able to deliver Stimulatory signaling leads to the release of IFN-γ and IL-2, an effect that correlates with the level of CD137L on the tumor. Whether expressing CD137L in human tumors can render these tumors more sensitive to agonistic CD137 antibodies is unknown.

CD137L-/- mice have reduced the importance of the CD137/CD137L system in T cell responses to viruses and tumors (M. DeBenedette et al., J. Immunol., 163(9): 4833-41 (1999); J. Tan et al., J. Immunol., 164(5):2320-5 (2000); B. Kwon et al., J. Immunol., 168(11):5483-90 (2002)). Studies using CD137- and CD137L-deficient mice confirmed the importance of CD137 co-stimulation in graft-versus-host disease, antiviral cytolytic T-cell responses. T cell proliferation is enhanced in CD137-deficient mice, but cytokine production and toxic T cell activity are reduced (B.Kwon et al., J. Immunol., 168(11):5483-90 (2002); D.Vinay et al., Immunol . Cell Biol., 81(3):176-84(2003)). Recently, tumor metastasis was shown to be more frequent (4-fold) in knockout mice (CD137-/-) compared to control mice. These data suggest that restoration of CD137 signaling using agonistic anti-CD137 antibodies is a viable approach to enhance cellular immune responses to viral antigens and cancer.

In addition to data in mouse in vivo models that support the involvement of CD137 signaling in antitumor immune responses, studies in primary human tumor samples confirmed the role of CD137 in generating effector T cells. In patients with Ewing sarcoma, Zhang et al. showed that intratumoral effector T-cells presented a CD3+/CD8+/CD28-/CD137+ phenotype. Unexpectedly, the coexistence of progressive tumor growth and antitumor immunity (effector T cells) was observed. Ex vivo stimulation studies using patient cells demonstrated that co-stimulation with CD137L is required for tumor-induced T cell proliferation and activation. Stimulation of PBL with anti-CD3/CD137L, but not anti-CD3/anti-CD28, resulted in oncolytic effectors. These studies provide further evidence that CD137-mediated co-stimulation can lead to the expansion of tumor-reactive CTLs (H. Zhang et al., Cancer Biol. Ther., 2(5):579-86 (2003)). Furthermore, expression of CD137 demonstrates tumor infiltrating lymphocytes in hepatocellular carcinoma (HCC) (Y. Wan et al., World J. Gastroenterol, 10(2):195-9 (2004)). The detection rate of CD137 expression in HCC was 19/19 by RT-PCR and 13/19 by immunofluorescence staining. In contrast, CD137 was not detected in the peripheral mononuclear cells of the same patients. These studies did not intend to correlate clinical disease with CD137 expression. Thus, studies performed in Ewing sarcoma and in hepatocellular carcinoma revealed the presence of CD137-expressing TILs that accompany disease progression. In Ewing sarcoma, CD137+ TILs were demonstrated to kill ex vivo tumor cells, suggesting that the CD137 pathway is intact in these patients and that possible inhibitory factors in the tumor microenvironment inhibit their function. Therefore, it can be assumed that systemic administration of agonistic CD137 antibodies may provide the signal necessary to expand these effector T cells.

In addition to its role in the progression of cancer immunity, experimental data support the use of CD137 agonistic antibodies in the treatment of autoimmune and viral diseases (B.Kwon et al., Exp.Mol.Med., 35(1):8-16 (2003); H. Salih et al., J. Immunol., 167(7): 4059-66 (2001); E. Kwon et al., P.N.A.S. USA, 96: 15074-79 (1999); J. Foell et al., N.Y. Acad .Sci., 987:230-5(2003); Y.Sun et al., Nat.Med., 8(12):1405-13(2002) S.K.Seo et al, Nat.Med.10; 1099-94(2004 )).

Therefore, based on the role of 4-1BB in regulating immune responses, there is a need to generate anti-human 4-1BB antibodies with agonistic activity, which can be used to treat or prevent human diseases such as cancer, infectious diseases, and autoimmune diseases.

Brief description of the invention:

The present invention provides fully human antibodies that bind human 4-1BB (H4-1BB) and allow H4-1BB to bind human 4-1BB ligand (H4-1BBL). The present invention therefore relates to antibodies that bind H4-1BB and do not block the binding of H4-1BB to H4-1BBL, thereby allowing the antibodies of the present invention and the binding of H4-1BBL to H4-1BB. The present invention also provides antibodies having agonistic activity, wherein binding of the antibody to H4-1BB results in enhancement and stimulation of H4-1BBL-mediated immune responses. These antibodies can be used as immune enhancers for anti-tumor or anti-viral immune responses, or immune regulators for T cell-mediated autoimmune diseases. The antibodies can also be used as a diagnostic tool to detect H4-1BB in the blood or tissues of patients with cancer, autoimmune or other diseases.

In one aspect, the invention provides a monoclonal antibody or an antigen-binding portion thereof, which specifically binds to H4-1BB, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR1 as shown in Figure 4 (complementarity determining region 1), CDR2 (complementarity determining region 2), and CDR3 (complementarity determining region 3), the heavy chain variable region comprises CDR1 (complementarity determining region 1), CDR2 (complementarity determining region 1) as shown in Figure 3 or Figure 7 Determining region 2), and CDR3 (complementarity determining region 3). A monoclonal antibody (mab) can be, for example, an IgG4 antibody or an IgG1 antibody.

In another aspect, the invention provides a monoclonal antibody or an antigen-binding portion thereof, wherein the light chain comprises a variable region as shown in Figure 4 and the heavy chain comprises a variable region as shown in Figure 3 or Figure 7 .

In another aspect, the invention provides a monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises amino acid residues 21-236 of SEQ ID NO:6, and the heavy chain comprises amino acid residues 20-236 of SEQ ID NO:3. 467. In another aspect, the invention provides a monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises amino acid residues 21-236 of SEQ ID NO:6, and the heavy chain comprises amino acid residues 20-236 of SEQ ID NO:9. 470.

The antibodies of the invention have broad therapeutic utility as immunomodulators of diseases such as cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

The invention also provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of an antibody of the invention. In one aspect, the method further comprises administering the vaccine. Suitable vaccines include, for example, tumor cell vaccines, DNA vaccines, GM-CSF-modified tumor cell vaccines, or antigen-loaded dendritic cell vaccines. The cancer can be, for example, prostate cancer, melanoma, or epithelial cancer.

In another aspect, the invention provides methods for enhancing an immune response comprising administering an antibody of the invention and a SIV gag vaccine. In another aspect, the invention provides a method for enhancing an immune response comprising administering an antibody of the invention and a PSA vaccine. In another aspect, the invention provides methods for enhancing an immune response to a SIV gag vaccine comprising administering an antibody of the invention. In another aspect, the invention provides a method for enhancing an immune response to a PSA vaccine comprising administering an antibody of the invention.

The present invention also provides a pharmaceutical composition comprising an antibody of the present invention, or an antigen-binding portion thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition may be administered alone or in combination with other agents, for example, agents for the treatment of cancer such as chemotherapeutic agents or vaccines or other immunomodulators.

The present invention also provides an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) nucleotides encoding amino acid residues 20-467 of the amino acid sequence SEQ ID NO: 3; (b) a core Nucleotide, its encoded amino acid sequence SEQ ID NO: 3; (c) nucleotide, its encoded amino acid sequence SEQ ID NO: 6 amino acid residues 21-236; (d) nucleotide, its encoded amino acid sequence SEQ ID NO: 6; (e) nucleotides encoding amino acid residues 20-470 of the amino acid sequence SEQ ID NO: 9; (f) nucleotides encoding the amino acid sequence SEQ ID NO: 9; and (g) nucleotides, It encodes a fragment of the amino acid sequence (a)-(f), such as a variable region, a constant region, or one or more CDRs. The isolated polynucleotide of the invention also includes a nucleotide sequence encoding at least one CDR of FIG. 3 , at least one CDR of FIG. 4 , or at least one CDR of FIG. 7 . The invention provides an isolated polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 4, or SEQ ID NO: 7.

The present invention also provides an isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, and SEQ ID NO: 9. In another aspect, the invention provides an isolated polypeptide comprising amino acid residues 20-467 of the amino acid sequence of SEQ ID NO: 3, an isolated polypeptide comprising amino acid residues 21-236 of the amino acid sequence of SEQ ID NO: 6, an isolated polypeptide , which comprises amino acid residues 20-470 of the amino acid sequence SEQ ID NO:9. In another aspect, the invention provides an isolated polypeptide comprising the amino acid sequence of at least one CDR of Figure 3, Figure 4, or Figure 7, or at least the variable or constant region of Figure 3, Figure 4, or Figure 7.

The invention also includes immunoglobulins having binding specificity for H4-1BB, said immunoglobulins comprising an antigen binding region. In one aspect, the immunoglobulin is the Fab or F(ab') ₂ of an antibody of the invention.

The invention also includes cell lines that produce the antibodies of the invention or antigen-binding portions thereof, recombinant expression vectors that include the nucleotides of the invention, and methods of producing antibodies of the invention by culturing the antibody-producing cell lines.

Brief description of the drawings:

Figure 1 shows the plasmid map of pD17-20H4.9.h4a.

Figure 2 shows the plasmid map of pD16gate-20H4.9.LC.

Figure 3 (Fig. 3A-3H) shows the nucleotide sequence of plasmid pD17-20H4.9.h4a, including coding strand (SEQ ID NO: 1), complementary strand (SEQ ID NO: 2), and said coding strand encoding (leader peptide is amino acid residues 1-19 of SEQ ID NO: 3; heavy chain is amino acid residues 20-467 of SEQ ID NO: 3).

Figure 4 (Fig. 4A-4F) shows the nucleotide sequence of plasmid pD16gate-20H4.9.LC, including coding strand (SEQ ID NO: 4), complementary strand (SEQ ID NO: 5), and described coding strand encoding (leader peptide is amino acid residues 1-20 of SEQ ID NO:6; light chain is amino acid residues 21-236 of SEQ ID NO:6).

Figure 5 shows a schematic diagram of the 20H4.9-IgG1 heavy chain sequence construct.

Figure 6 shows a schematic diagram of the 20H4.9 light chain sequence construct.

Figure 7 (Figure 7A-7D) shows the nucleotide and amino acid sequences of the 20H4.9-IgG1 heavy chain construct, including the coding strand (SEQ ID NO: 7), the complementary strand (SEQ ID NO: 8), and all The amino acid sequence encoded by the coding chain (leader peptide is amino acid residues 1-19 of SEQ ID NO: 9; heavy chain is amino acid residues 20-470 of SEQ ID NO: 9).

Figure 8 (Figures 8A-8B) graphically illustrates ELISA results obtained from binding of mab 20H4.9-IgG1 to human CD137 (Figure 8A) and the effect of mab 20H4.9-IgG1 on CD137-CD137L interaction (Figure 8B).

Figure 9 (Figures 9A-9B) graphically depicts results obtained from binding of mab 20H4.9-IgG1 to PMA-ionomycin stimulated human or cynomolgus monkey cells. Human CEM (Fig. 9A) or monkey PBMC (Fig. 9B) were incubated with 20H4.9-IgG1 or human CD137L fusion protein.

Figure 10 (Figures 10A-10B) graphically depicts the results of induction of IFN-γ using anti-CD137 antibodies in co-stimulation studies expressed as fold increase in pg/ml relative to control. Data were normalized to the control treatment (=1) due to the varied background responses of the donors. Median IFN-γ baseline levels from human T-cells (Fig. 10A) or monkey PBMCs (Fig. 10B) stimulated with anti-CD3 alone were 592 pg/ml and 505 pg/ml, respectively.

Figure 11 provides plasmon resonance mapping of mab 20H4.9-IgG4 and mab 20H4.9-IgG1 binding to human CD137. 

Figure 12 illustrates the concentration-dependent binding of 20H4.9-IgG4 to human CEM cells stimulated with PMA ionomycin, but not to unstimulated CEM cells.

Figure 13 (Figure 13A-B) is a graph illustrating the induction of IFN-γ in costimulation studies using anti-CD137 antibodies. Results are expressed as fold increase in pg/ml relative to control. Data were normalized to the control treatment (=1 ) due to variability in background responses due to donors. Median baseline levels of IFN-γ in human T-cells (Fig. 13A) or monkey PBMCs (Fig. 13B) stimulated with anti-CD3 alone were 592 pg/ml and 505 pg/ml, respectively.

Figure 14 (Figure 14A-14B) shows the results of the dose-dependent enhancement of IFN-γ synthesis by mab 20H4.9-IgG4 (Figure 14A), and the addition of cross-linked anti-human IgG antibody (7 μg/ml) Effect of antibody cross-linking (Figure 14B).

Figure 15 illustrates the effect of mab 20H4.9-IgG4 on T-cell survival and cell cycle progression. Human T-cells were co-stimulated with anti-CD3 (lug/ml) ± mab20H4.9-IgG4 at the concentrations listed. Six days after the start of the assay, cells were harvested and stained with Annexin-V and propidium iodide to determine the number of viable cells (Annexin V/PI negative), or with PE-conjugated cyclin D2 to detect cells in cycle. cell. Results represent the mean (± SD) of 4 groups of mab 20H4.9-IgG4 tested in parallel.

Figure 16 (Figures 16A-16D) shows antigen-specific IFN-γ responses in rhesus monkeys following treatment with DNA vaccine ± anti-human 4-1BB antibody, as determined by ELISPOT. Animals were treated with SIV gag vaccine (days 0, 28, 56; Figure 16A), SIV gag vaccine (days 0, 28, 56) and mab20H4.9-IgG4 (days 12, 15, and 19; Figure 16B), or SIV gag vaccine (day 0, 28, 56) and hu39E3.G4 (day 12, 15 and 19; Figure 16C) treatments. One group of animals was untreated (Fig. 16D). At various times after treatment, blood was collected, PBMCs were isolated and assessed for their ability to secrete IFN-γ in the presence of antigenic stimulation.

Detailed description of the invention:

The present invention relates to the preparation and characterization of antibodies and antigen-binding fragments thereof (including fusion proteins comprising antigen-binding fragments of the antibodies of the invention) for use in the treatment of diseases such as cancer, infectious diseases, inflammatory diseases or autoimmune diseases. The cancer can be, for example, prostate cancer, melanoma, or epithelial cancer.

The antibody can bind to H4-1BB, and can exhibit high affinity to H4-1BB and effectively enhance T cell response. In one aspect, the antibody induces IFN-γ production in costimulation assays, but does not affect the binding of H4-1BB to its corresponding ligand, H4-1BBL, and does not fix complement.

Antibodies of the invention can be prepared by methods known in the art. In one aspect, the antibodies can be produced by expression in transfected cells such as immortalized eukaryotic cells such as myeloma or hybridoma cells

Antibodies of the invention can be used alone, or in combination with other therapeutic agents such as radiation therapy (including irradiation), hormone therapy, cytotoxic agents, vaccines, and other immunomodulatory agents, such as cytokines and biological response modifiers. These agents are particularly useful in the treatment of cancer and immunoproliferative diseases.

In one aspect, the invention provides monoclonal antibody (mab) 20H4.9-IgG4. Figures 1 and 2 provide plasmid maps of pD17-20H4.9.h4a and pD16gate-20H4.9.LC, respectively, which can be used to prepare mab 20H4.9-IgG4. Figure 3 (Fig. 3A-3H) provides the nucleotide sequence of plasmid pD17-20H4.9.h4a, including the coding strand (SEQ ID NO: 1), the complementary strand (SEQ ID NO: 2), and the coding strand encoding (leader peptide is amino acid residues 1-19 of SEQ ID NO: 3; heavy chain is amino acid residues 20-467 of SEQ ID NO: 3). Figure 4 (Fig. 4A-4F) shows the nucleotide sequence of plasmid pD16gate-20H4.9.LC, including coding strand (SEQ ID NO: 4), complementary strand (SEQ ID NO: 5), and described coding strand encoding (leader peptide is amino acid residues 1-20 of SEQ ID NO:6; light chain is amino acid residues 21-236 of SEQ ID NO:6).

In another aspect, the present invention provides monoclonal antibody (mab) 20H4.9-IgG1. Figure 5 is a schematic representation of the heavy chain sequence construct of mab20H4.9-IgG1. Figure 6 illustrates the light chain sequence constructs of mab 20H4.9 for mab 20H4.9-IgG4 and 20H4.9-IgG1. Figure 7 provides the nucleotide sequence (coding strand (SEQ ID NO: 7) and complementary strand (SEQ ID NO: 8)) of the heavy chain sequence construct of Fig. Amino acid residues 1-19 of ID NO:9; heavy chain is amino acid residues 20-470 of SEQ ID NO:9). The light chain of mab 20H4.9-IgG1 is identical to that of mab20H4.9-IgG4.

The invention also includes antibodies having conservative amino acid substitutions from the heavy and light chain amino acid sequences set forth in SEQ ID NOS: 3, 6, and 9 that have substantially no effect on H4-1BB binding. Conservative substitutions usually include the substitution of one amino acid for another amino acid with similar properties, such as substitutions in the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

A polynucleotide encoding a polypeptide of the invention typically also includes expression control sequences operably linked to the polypeptide coding sequence, including naturally linked and heterologous promoter regions. Preferably, the expression control sequences will be a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell, although control sequences for eukaryotic hosts may also be used. Once the vector has been incorporated into a suitable host and the host is maintained under conditions suitable for high level expression of the nucleotide sequence, as desired, the light chain, heavy chain, light/heavy chain dimer or light chain can then be harvested and purified. Whole antibodies, binding fragments or other immunoglobulin forms. (See, S. Beychok, Cells of Immunoglobin Synthesis, Academic Press, N.Y. (1979)). Single-chain antibodies or minibodies (single-chain antibodies fused to one or more CH domains) can also be prepared by ligating the nucleic acid sequences encoding the VL and VH regions disclosed herein with DNA encoding a polypeptide linker.

Prokaryotic hosts such as E. coli as well as other microorganisms such as yeast can be used to express the antibodies of the invention. In addition to organisms, mammalian tissue cell cultures can also be used to express and produce the antibodies of the invention. Eukaryotic cells are preferred because a variety of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including, for example, CHO (Chinese Hamster Ovary) cell lines, COS (Oreo monkey fibroblast cell line) cells line, HeLa cells, myeloma cell lines, and hybridomas. Expression vectors for these cells may include expression control sequences, such as promoters or enhancers, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences, All are known in the art.

Vectors containing DNA fragments of interest (eg, heavy and/or light chain coding sequences and expression control sequences) can be transferred into host cells by known methods, which depend on the type of cellular host. For example, calcium chloride transfection is commonly used in eukaryotic cells, while calcium phosphate treatment, lipofection, or electroporation can be used in other cellular hosts. (See, eg, T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1982)).

Once expressed, antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms, can be purified according to standard methods in the art, such as ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Substantially pure immunoglobulins of at least 90-95% homogeneity are desired, with 98-99% homogeneity or greater being more desirable.

Antibodies of the invention are useful for modulating T cell and antibody-mediated immune responses. Disease states generally amenable to treatment include cancer, infectious diseases, inflammatory diseases, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus and myasthenia gravis.

The invention also provides pharmaceutical compositions comprising at least one antibody of the invention and a pharmaceutically acceptable carrier. Said pharmaceutical compositions can be sterilized by conventional sterilization techniques known in the art. The pharmaceutical composition may also contain pharmaceutically acceptable auxiliary substances, which are required for suitable physiological conditions, such as pH adjustment and buffering agents, stability-enhancing agents such as mannitol or tween 80, toxicity regulators, etc., Examples include sodium acetate, sodium chloride, potassium chloride, calcium chloride or human serum albumin.

The antibodies and pharmaceutical compositions of the present invention are particularly useful for parenteral administration, including subcutaneous, intramuscular and intravenous administration. Pharmaceutical compositions for parenteral administration may comprise a solution of the antibody dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, all known in the art, such as water, buffered water, saline, glycine, and the like. These solutions are sterile and generally free of particulate matter. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of agent.

The pharmaceutical composition may further include other agents for the treatment of diseases. In one aspect, the pharmaceutical composition includes agents for the treatment of cancer, infectious diseases, inflammatory diseases and autoimmune diseases. Antibodies of the invention may also be co-administered or administered separately with another agent used to treat a disease.

Antibodies of the invention can be administered with other agents to enhance the immune response to cancerous cells in a patient. In one aspect, the antibodies are combined with immunogenic agents, such as cancerous cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), or cells transfected with genes encoding immunostimulatory cytokines and cell surface Antigen combination. In another aspect, the antibody is used in combination with vaccines such as tumor cell vaccines, DNA vaccines, gene-modified tumor cell vaccines such as GM-CSF-modified tumor cell vaccines, peptide vaccines, or antigen-loaded dendritic cell vaccines.

A number of experimental strategies for inoculation against tumors have been developed. In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation by tumor vaccination (Dranoff et al., P.N.A.S., 90:3539-43 (1993); E. Jafee et al., J. Clin. Oncol., 19:145-56 (2001); R. Salgia et al., J. Clin. Oncol., 21:624-30 (2003)).

Studies of gene expression and large-scale gene expression patterns in various tumors have led to the definition of so-called tumor-specific antigens (S. Rosenberg, Immunity 10:281-7 (1999)). In many cases these tumor-specific antigens are different antigens expressed in the tumor and in the tumor-producing cells, eg melanocyte antigen gp100, MAGE antigen, Trp-2. Many of these antigens are targets of tumor-specific T cells in the host. Antibodies of the present invention can be used in conjunction with various recombinant proteins and/or peptides expressed in tumors to amplify and direct Th1 immune responses to these antigens. These proteins are usually seen by the immune system as self-antigens and thus tolerated.

In one aspect of the invention, the antibodies and immunomodulators comprise SIV gag antigen (as a model HIV DNA vaccine) or prostate specific antigen (PSA), or comprise nucleotides encoding SIV gag antigen or prostate specific antigen (PSA) Sequenced DNA vaccine combination. PSA vaccines are described, for example, in M.Pavlenko et al., Br.J.Cancer, 91(4):688-94 (2004); J.Wolchok et al., Semin.Oncol., 30(5):659-66 (2003); J. . Kim et al., Clin. Cancer Res., 7(3 Suppl.): 882s-889s (2001). SIV gag vaccination is described, for example, in B. Makitalo et al., J. Gen. Virol., 85(Pt8): 2407-19 (2004); N. Letvin et al., J. Virol., 78(14): 7490-7 (2004); S. Mossman et al., AIDS Res. Hum. Retroviruses., 20(4): 425-34 (2004); F. Bertley et al., J. Immunol., 172(6): 3745-57 (2004) ; L Patterson et al., J.Virol., 78(5):2212-21(2004); E.O'Neill et al., AIDS Res.Hum.Retroviruses, 19(10):883-90(2003); Z.Bu et al. , Virology, 309(2):272-81 (2003).

Tumor antigens may include, for example, the protein telomerase, which is required for the synthesis of chromosomal telomerase and is expressed in over 85% of human cancers and only a limited number of somatic tissues (N. Kim et al., Science, 266, 2011 -2013(1994)). Tumor antigens are also "neo-antigens" expressed in cancer cells due to somatic mutations that alter the protein sequence and create fusion proteins between two unrelated sequences or idiotypes from B-cell tumors. Other tumor vaccines may also include proteins from viruses associated with human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's Herpes Sarcoma Virus (KHSV ). Another form of tumor-specific antigen that can be used with the antibodies of the invention is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins from tumor cells and these HSPs can be efficiently delivered to antigen-presenting cells to stimulate tumor immunity (R.Suot et al., Science 269:1585-1588 (1995); Y.Tamura et al., Science 278: 117-120 (1997)).

Antibodies of the invention may also be used to enhance vaccine immune responses to viral antigens, such as HIV or HCV. Antibodies of the invention can also be used to enhance immune responses to other immunomodulators, and to elicit anamnestic immune responses. Examples of these agents are cytokines such as GM-CSF, IL-2, IL-15, IL-12, F13 ligand, CD40 ligand, adjuvants such as CpG-oligodeoxyribonucleotides (bacterial DNA), or Anti-OX-40 or CTLA-4 antibodies.

The pharmaceutical composition of the present invention can be administered for prophylactic and/or curative treatment. In therapeutic applications, the pharmaceutical compositions are administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest or treat the disease. An amount suitable to achieve this result is defined as a "therapeutically effective amount". Amounts effective for this use will depend on the severity of the disease state and the patient (including, for example, the general state of the disease autoimmune system), and can be determined by one skilled in the art. In a specific application, the pharmaceutical composition is administered to a patient who is not already in a disease state, so as to enhance the patient's resistance to the disease state. Said amount is defined as "prophylactically effective amount". In such uses, the precise amount will depend on the state of the patient's health (including, for example, the general state of the patient's own immune system) and can be determined by one skilled in the art. In one aspect, the prophylactic use is for the prevention of tumor recurrence.

Example:

Embodiment 1: Preparation of antibody

Materials and methods

Fully human monoclonal antibody to human CD137(4-1BB) receptor in HuMAb-Mouse Produced in (Medarex, Inc., Princeton, New Jersey). HuMAb mice were immunized 5 times intraperitoneally (ip) and subcutaneously (sc) with 25 μg of human CD137 extracellular domain in RIBI adjuvant (Ribi Immunochemical). Prior to infusion, mice were boosted intravenously (iv) with the same amount of antigen. Splenocytes from immunized mice with appropriate anti-huCD137 antibody titers were fused to mouse myeloma cells according to standard methods.

Anti-human CD3 mab (Kolon: HIT3a), ELISA kits for human and monkey IFN-γ, cytometric bead array (CBA) kits, and all conjugates for flow cytometry Antibodies for the technique were purchased from BD Pharmingen (San Diego, California). Human IgG ₁ and human IgG ₁ κ were purchased from Sigma-Aldrich (St. Louis, Missouri). CEM cells (ATCC-CRL 2265) were purchased from ATCC, medium (RPMI), fetal bovine serum (FBS) were purchased from Mediatech Inc. (Herndon, Virginia). Sheep erythrocytes Alsevers were purchased from Colorado Serum Co. (Denver, Colorado).

Hybridoma Screening: Binding to huCD137 detected by ELISA: To identify hybridomas secreting anti-human CD137 antibody, ELISA plates (Nunc MaxiSorp) were coated with 1 μg/ml human CD137-mouse IgG _2b fusion protein PBS solution at 4C overnight . The plate was then washed three times with PBS containing 0.01% Tween-80 (PBS-T), and then blocked with PBS-T plus 1% bovine serum albumin (BSA) for 20 min at room temperature. 50 microliters of supernatant diluted in PBS-T was added to the plate and incubated for 1-2 hours at ambient temperature. Plates were then washed as previously described, and antibody binding was detected by incubation with alkaline phosphatase-conjugated goat F(ab') ₂ anti-human IgG antibody (Jackson Laboratories, West Grove, Pennsylvania). Plates were developed with pNPP and read at 405nm.

Blocking assay: The ability of the 26 antibody-secreting hybridomas recognizing huCD137 to allow a CD137-CD137L response was assessed by ELISA. These analyzes were initially performed in ELISA format. Plates were coated with human CD137-muIgG _2b , 0.2 μg/ml, 100 μl/well. Serial dilutions of mab 20H4.9-IgG1, or control antibody, in PBS-T and 1% bovine serum albumin were added to the plate. CD137L-CD8 fusion protein was added to the plate at a concentration of 0.2 μg/ml. Antibody binding was detected using a biotinylated anti-CD8 antibody (0.2 μg/ml, Ancell Corporation, Bayport, Minnesota). After several washes, streptavidin-alkaline phosphatase (1:2000) and pNPP were added for detection of bound antibody and the plate was read at 405 nm.

To confirm that the selected antibodies did not alter CD137-CD137L binding, purified antibodies were subsequently characterized by BIAcore analysis. All experiments were performed on a BIAcore 3000 device (BIAcore Inc., Piscataway, New Jersey). Human CD137 was covalently immobilized at high density on the carboxy-methylated dextran surface of a BIAcore sensorchip (BIAcore Inc., Piscataway, New Jersey). Injections were performed at 2 μg/mL in 10 mM acetate buffer, pH 5.0. Unoccupied active esters are then blocked by, for example, excess ethanolamine. Regeneration of the surface was performed using 10 mM glycine, pH 2.0.

Purified anti-CD137 antibody samples were diluted to a concentration of 200-1000 nM using HEPES buffered saline, pH 7.4 supplemented with 0.15M NaCl and 0.005% surfactant P20 (HBS-EP). Human CD137L-CD8 fusion protein (huCD137L) was used as a source of CD137 ligand. Experiments were performed in which huCD137L was injected before anti-CD137 antibody or vice versa. Injections can be performed at a flow rate of 5 μL/min. Bound ligand and antibody are removed by regeneration with 10 mM glycine buffer, pH 2.0.

Human T-cell purification: T-cells or PBMC were obtained from healthy human donors. Blood was collected in EDTA, suspended in elutriation buffer (RPMI, which contained 2.5mM EDTA, 10μg/ml polymyxin B), and the lower layer was Lymphocyte Separation Medium (LSM, Mediatech Inc., Herndon , Virginia), centrifuged at 1800 rpm for 25 minutes. The cell interface was collected and centrifuged at 1500 rpm for 10 minutes. Subsequently, the cell debris was resuspended in elutriation buffer and sheep red blood cells (SRBC, 1:10 dilution) were washed and incubated on ice for 1 hour. Cells were then placed on LSM and centrifuged at 2500 rpm for 25 minutes. The interface was removed and SRBCs were lysed with SRBC Lysis Buffer. Isolated T-cells were washed and resuspended in 10% FBS/RPMI.

Flow Cytometry: Binding of anti-human CD137 antibodies to CD137 expressed on cells was determined by flow cytometry. Human T-cell leukemia cell lines (CEM) or rhesus monkey peripheral blood mononuclear cells (PBMC) were used for these studies. These cells do not constitutively express CD137, but the receptor can be induced by stimulation with phorbol myristate (PMA, 10 ng/ml) and ionomycin (1 μM) for 18 hr. Cells were then washed and incubated with various concentrations of antibody in various staining buffers (phosphate buffered saline, PBS, plus 1% FCS, and 0.01% sodium azide). Antibody binding to stimulated or unstimulated cells was detected by luciferin (FITC) or phycoerithrin (PE) conjugated goat anti-human IgG (Jackson Immunoresearch, West Grove, Pennsylvania). To confirm the expression of CD137, a fusion protein composed of CD137 ligand extracellular domain and mouse CD8 (Ancell Corporation, Bayport, Minnesota) was used, followed by PE-conjugated anti-mouse CD8 (BDPharmingen, San Diego, California) insulation. Samples were fixed in 1% formalin, stored at 4°C, and read by flow cytometry.

Functional Assay: Primary human T-cells or monkey PBMCs obtained from healthy donors were stimulated with unfixed anti-CD3 antibody to provide the first signal of T cell activation and co-stimulated with human anti-human CD137 antibody. As a non-specific control, a humanized anti-cancer antibody (BR96) was used at the same antibody concentration. Plates were incubated overnight at 4°C with anti-CD3 antibody (0.5-1 μg/ml). The next day, T-cells or PBMCs were plated at a concentration of 1-1.5 x ¹⁰⁵ /well. After 72 hours of incubation at 37°C, IFN-γ synthesis was measured by cytometric bead array (CBA) or ELISA.

Cytokine assay

ELISA: After stimulating T-cells at various times, the plates were centrifuged and the medium was removed. Cytokine levels were detected by ELISA according to the manufacturer's instructions (BD Pharmingen, San Diego, California). Briefly, test samples and standards are added to anticytokine-coated 96-well plates. After 2 hours of incubation at ambient temperature, the plate was washed 3 times in PBS-T, then incubated first with working detection antibody and then substrate was added. Absorbance was read at 405 nm and concentrations were calculated based on standard curves.

Cytometry Bead Assay: The method used to measure cytokine production in vitro is flow cytometry utilizing the Cytometry Bead Array (CBA) from BD Pharmingen. IFN-γ, IL-2, IL-5, IL-4, IL-10, and TNF-α levels were determined in culture supernatants according to the manufacturer's instructions. Results were analyzed in flow cytometry using CBA analysis software.

result

Hybridoma-secreted antibodies binding to human CD137 were further expanded and subcloned. Secreted antibodies were purified and tested for their ability to bind huCD137 and allow CD137-CD137L interaction. Among the panel of anti-human CD137 antibodies evaluated, mab 20H4.9-IgG1 was selected for further evaluation based on its binding profile and non-binding properties. The 20H4.9-IgG1 antibody was IgG1 kappa as determined by ELISA using alkaline phosphatase anti-human IgG1, 2, 3, 4, and anti-kappa and lambda reagents (Southern Biotech, Birmingham, Alabama). Figure 8 (Figure 8A - Binding to human CD137 determined by ELISA; Figure 8B - Effect of mab 20H4.9-IgG1 on CD137-CD137L interaction) provides a preliminary characterization of mab 20H4.9-IgG1. Serial dilutions of mab 20H4.9-IgG1, 26G6 (blocking anti-CD137 antibody), or tetanus toxoid (TT, negative control) were assessed for their ability to alter CD137 binding to CD137L. Mab20H4.9-IgG1 at concentrations up to 10 μg/ml did not block CD137L binding, whereas mab 26G6 inhibited binding at concentrations >0.37 μg/ml.

Mab 20H4.9-IgG1 was also tested for reactivity to CD137 expressed on human T cells (CEM) and in rhesus monkey peripheral blood mononuclear cells (PBMC) stimulated with PMA and ionomycin. Previous studies determined that CD137 was upregulated on T-cells, which were then activated with PMA and ionomycin. Control molecules consisted of an irrelevant human IgG antibody (negative control) or CD137L-CD8 fusion protein (positive control, BD Pharmingen, San Diego, California). The results of these studies indicated that mab 20H4.9-IgG1 bound activated human CEM as well as PBMC from macaques, with minimal binding to unstimulated cells. Similar percentages of positive cells were detected with mab 20H4.9-IgG1 or CD137L. Figure 9 presents the results obtained demonstrating the binding of mab 20H4.9-IgG1 to PMA-ionomycin stimulated human or macaque cells. Human CEM (FIG. 9A) or monkey PBMC (FIG. 9B) were incubated with 20H4.9-IgG1 or human CD137L fusion protein. Add secondary antibodies and read samples by flow cytometry.

Subsequently, it was determined whether mab 20H4.9-IgG1 could induce an increase in IFN-γ in the presence of anti-CD3 stimulation in a co-stimulation assay, a key functional effect required for agonistic CD137 antibodies. The costimulatory activity of Mab 20H4.9-IgG1 was evaluated in functional studies in human and monkey lymphocytes. Based on initial data, anti-CD137 antibody (excess antibody) was used in these studies at a concentration of 20 ug/ml. Anti-CD3 antibodies were detected at 0.2-1 μg/ml, which resulted in 10-20% CD137-positive lymphocytes. The concentration of IFN-γ in the supernatant was measured after 72 hours of culture. As shown in Figure 10, mab 20H4.9-IgG1 increased IFN-γ synthesis in human and monkey co-stimulation assays to levels significantly higher than controls. The results of studies using T cells isolated from 8 healthy human donors showed that in 6 of them, mab 20H4.9-IgG1 increased IFN-γ synthesis by 2.2-4.3-fold relative to controls. One of the other two donors showed a 1.6-fold increase. The level of increase exceeds that observed with this humanized anti-CD137 antibody provided in hu39E3.G4 PCT application WO 04/010947 (incorporated herein by reference), which was shown in 5 out of 8 donors, IFN-γ increased and levels were lower than with mab 20H4.9-IgG1 (1.5-2-fold increase) (Fig. 10A). In monkey co-stimulation studies, mab 20H4.9-IgG1 also demonstrated enhanced functional activity leading to a clear increase in IFN-γ relative to controls ( FIG. 10B ). In the human study, the increase in IFN-γ was more consistent than with hu39E3.G4.

Induction of TNF-α synthesis above control levels was also observed in human cultures, but at much lower levels than IFN-γ. Anti-CD3 antibody alone induced TNF-[alpha] levels (baseline) approximately 20-50 fold lower than IFN-[gamma] baseline levels. Mab 20H4.9-IgG1 induced ~2 to 4.7-fold increases in 3 of 8 donors. hu39E3.G4 (run in parallel) here induced a ~2-fold increase in the same donor but at lower levels. The other cytokines tested, IL-2, IL-5, IL-10, and IL-4, were not significantly changed with each treatment.

Together, these studies demonstrate that mab 20H4.9-IgG1 exhibits the desired functional activity in humans and monkeys by inducing a Th1-type response. Clearly, these results support the selection of mab 20H4.9-IgG1 over isotype switching, as in vivo antitumor activity correlates with the ability of anti-CD137 antibodies to induce IFN-γ synthesis.

Example 2: In vitro characterization of mab 20H4.9-IgG4

Based on its binding kinetics, inability to block CD137-CD137L action, and functional effect on human T cells, mab 20H4.9-IgG1 was selected to convert to IgG4 form. The IgG4 form of mab 20H4.9-IgG1 is 20H4.9-IgG4 (shown graphically in Figures 3 and 4).

The second phase of the study involved comparing the in vitro properties of mab 20H4.9-IgG4 and mab 20H4.9-IgG1. In this section, the binding kinetic properties and functional effects of the antibodies in human and monkey lymphocytes are described.

binding kinetics

The kinetic properties of the anti-human CD137 antibody were evaluated using surface plasmon resonance using a BIAcore3000 device. Antigen, human CD137-mouse IgG _2a low-density covalent immobilization on the surface of CM5 sensor chip. Mab 20H4.9-IgG4 and mab 20H4.9-IgG1 were injected at a concentration of 25-200 nM. Figure 11 depicts the injection of mab 20H4.9-IgGlh mab 20H4.9-IgG4 at 100 nM. The data were calculated using BIAevaluation software (bivalent model, global curve fit analysis), and the results showed that the kinetic parameters of the two antibodies were similar (see Table 1). The dissociation constants K _D of mab 20H4.9-IgG1 and mab20H4.9-IgG4 were determined to be 11.2 and 16.6 nM, respectively. Under similar experimental conditions, mab 20H4.9-IgG4 did not bind murine 4-1BB.

Table 1 - Comparing the binding kinetics of mab 20H4.9-IgG4 to mab 20H4.9-IgG1

Antibody k _a1 (1/Ms) k _d1 (1/s) ka2(1/RUs) kd2(1/s) Rmax(RU) K _A 1 K _D 1(nM) 20H4.9-IgG1 3.43E+04 3.85E-04 2.30E-05 1.51E-03 262 8.91E+07 11.22 20H4.9-IgG4 3.92E+04 6.51E-04 0.0755 0.105 409 6.02E+07 16.61

Flow Cytometry

Binding of biotinylated mab 20H4.9-IgG4 to CEM cells±PMA-Ionomycin at concentrations ranging from 0.32 ng/ml to 5 μg/ml was detected. Mab20H4.9-IgG4 conjugated to PMA-ionomycin stimulated CEM cells in a concentration-dependent manner. Saturation is reached at 0.2 μg/ml. On the other hand, mab 20H4.9-IgG1, mab 20H4.9-IgG4 did not bind to CEM cells that were not stimulated with PMA-ionomycin as shown for their parental molecules (Figure 12). Concentration-dependent binding of mab 20H4-.9-IgG4 was demonstrated in PMA-ionomycin stimulated CEM cells (Figure 12). Samples were read by flow cytometry.

Cellular/functional assays

To confirm that the process of switching mab 20H4.9-IgG1 isotype does not alter antibody activity, an in vitro assay was performed to compare the activity of mab 20H4.9-IgG4 with the parental mab 20H4.9-IgG1 in monkey PBMCs and human T-cells. The effect of mab 20H4.9-IgG4 on human and monkey T-cells or PBMC was determined and compared to its parental molecule, mab 20H4.9-IgG1. Primary human T-cells or monkey PBMC obtained from healthy human donors were stimulated with anti-CD3 antibody (0.5 μg/ml-1 μg/ml) +/- anti-human CD137 antibody. IFN-[gamma] synthesis was determined after 72 hours of incubation at 37[deg.] C. using cytometric bead arrays (CBA) (for human samples) or by ELISA (for monkey samples). Antibodies were tested in co-stimulation assays in the presence of suboptimal concentrations of anti-CD3 antibody (1 μg/ml) or Concavalin A (1 μg/ml) (donors M5170 and 81 only). Results are expressed as fold increase in pg/ml relative to control. Due to the variable background response of the donors, the data were normalized to the control treatment (=1). Figure 13A provides human T-cell results and Figure 13B provides monkey PBMC results. As shown in Figures 13A-13B, mab20H4.9-IgG4 demonstrated co-stimulatory properties to generate high levels of IEN-γ in human and monkey cells compared to controls. Increased levels of IFN-[gamma] synthesis were compared to that of its parental molecule in human and monkey samples.

Subsequently, the effect of antibody cross-linking on the functional effects of mab 20H4.9-IgG4 was assessed. Cross-linking of antibodies was shown to result in an enhancement of their ability to generate signals. Thus, experiments were performed to determine the functional activity of several batches of mab20H4.9-IgG4 ± anti-human IgG antibodies. As shown in Figure 14A, in the presence of cross-linked antibody, a significant increase in IFN-γ synthesis was observed for all test groups, with a plateau at a concentration of 400 ng/ml. The promotion of IFNγ synthesis by mab 20H4.9-IgG4 was further enhanced by adding the anti-human IgG cross-linked antibody shown in Figure 14B. Different lots of mab 20H4.9-IgG4 had comparable cellular activities.

Cross-linking of mab 20H4.9-IgG4 thus results in an enhanced ability of the antibody to induce IFN-γ synthesis. Antibody cross-linking in vivo can occur through cellular receptors for the Fc portion of the immunoglobulin or through antibody dimerization. Mab 20H4.9-IgG4 is an IgG4 isotype that has a lower affinity for Fc receptors compared to other IgG isotypes. However, IgG4 can bind Fc RI (CD64) expressed on monocytes and neutrophils.

Two additional approaches were used to further characterize mab 20H4.9-IgG4: (i) effects on T-cell survival and (ii) effects on cyclin D2 expression. To determine whether mab 20H4.9-IgG4 stimulates signaling to human T-cells through CD137 and provides a co-stimulatory signal to T-cells leading to cell survival and expansion, anti-CD3 at concentrations known to induce IFN-γ synthesis Human T-cells stimulated with antibody +/- mab20H4.9-IgG4 were stained with Annexin-V and propidium iodide to detect viable cell numbers (Annexin V/propidium iodide negative) and stained for cyclins D2 to detect its effect on cell progression. Figure 15 shows the mean results of 4 groups of different mab 20H4.9-IgG4 on cyclin D2 expression and survival of T-cells. Concentrations of 0.4-10 μg/ml of mab 20H4.9-IgG4 resulted in an approximately 1.8-2-fold increase in the number of viable cells and a marked increase (2.5-3-fold) in the number of cyclin D2-expressing T-cells.

Example 3: In vivo evaluation of 4-1BB antibodies in a rhesus monkey pharmacodynamic model

This example illustrates the ability of mab 20H4.9-IgG4 and mab hu39E3.G4 to enhance antigen-specific immune responses elicited by DNA vaccines.

Materials and methods

Experimental Animal Group: Female and male rhesus monkeys (2.5-5.0 kg) used in this experiment were purchased from Charles River BRF (Houston, Texas) and housed in pairs. Each test group consisted of 4 males and 2 females, randomized by body weight. The test groups are as follows:

Group 1 - SIV gag and PSA DNA vaccine (2 mg each), on days 0, 28, 56, i.m., plus saline control, i.v., on days 12, 15 and 19;

Group 2 - SIV gag and PSA DNA vaccine (2mg each), on days 0, 28, 56, i.m., plus mab hu39E3.G4, i.v., on days 12, 15 and 19;

Group 3 - SIV gag and PSA DNA vaccine (2mg each), on days 0, 28, 56, i.m., plus mab 20H4.9-IgG4, i.v., on days 12, 15 and 19;

Group 4 - untreated control group.

Immunization and antibody therapy: PSA and SIV gag DNA vaccines were obtained from David B. Weiner, Department of Pathology and Laboratory of Medicine, University of Pennsylvania. (See, Kim et al., Oncogene 20, 4497-4506 (2001); Muthumani et al., Vaccine 21, 629-637 (2003).)

Monkeys were immunized intramuscularly with both PSA and SIV gag DNA constructs (2 mg/construct/immunization) followed by two boosts (days 0, 28, and 56) 4 weeks apart. On day 12 after the primary immunization, treatment with mab 20H4.9-IgG4 or mab hu39E3.G4 was started. Antibodies were administered at 50 mg/kg i.v. on days 12, 15 and 19 after the first immunization. This regimen was chosen because it suppressed the antibody response to mabhu39E3.G4.

Clinical and Clinical Pathology

All monkeys were physically examined by veterinarians throughout the study. Blood samples for hematology and serum chemistry analysis were collected before vaccination and on days 12, 42, 70, 97, 134, and 168 after immunization.

Immunoassay

To determine the effect on the immune responses induced by these treatment regimens, IFN-γ production by antigen-specifically stimulated lymphocytes was measured using an enzyme-linked immunospot assay (ELISPOT). Blood samples for ELISPOT analysis were collected before vaccination and at 12, 42, 70, 97, 134, and 168 days after immunization. Synthetic peptides corresponding to the complete sequences of SIVgag and PSA antigens were used to stimulate PBMCs ex vivo.

result

Antigen-specific IFN-γ secreting cells in response to PSA or SIV gag peptide were quantified by ELISPOT. Figure 16 (Figures 16A-16D) graphically illustrates the results for Groups 1-4, respectively. The level of response to PSA was very low in all groups, indicating that the vaccine itself did not induce a detectable and consistent immune response compared to non-vaccinated animals. On the other hand, SIV gag immunization alone increased over time in a large number of antigen-specific IFN-γ secreting cells (Fig. 16A). Untreated animals (non-vaccinated) displayed 100-1,000 spots per ¹⁰⁶ PBMCs throughout the study (Fig. 16D). These results established a threshold response to the vaccine; animals representing <1,000 points/ ¹⁰⁶ PBMC were considered non-responders. In the group of animals that received the vaccine, 5 out of 6 monkeys showed an increase in response over time, with the mean number of spots (day 70) after the third immunization being 1,727 spots/ ¹⁰⁶ PBMC (SD = 242, range = 1,403- 1,968 points/10 ⁶ PBMCs). One monkey was considered a non-responder (620 points/million PBMC). Since MHC typing was not performed in these studies. It is likely that some monkeys lack T cell immune responses to the vaccine due to MHC mismatches. Significantly, at day 70, 6 animals treated with SIV gag plus mab20H4.9-IgG4 had Only 4 showed a significantly higher number of IFN-γ spots (Fig. 16C). The mean number of spots after the third immunization in the mab 20H4.9-IgG4-treated group was 3,465 spots/10 ⁶ PBMC (SD=1,236, range=2,070-4,780 spots/10 ⁶ PBMC). Two monkeys in this group were unresponsive to the vaccine (<800 points/million PBMC). After the third immunization (day 70), treatment with mab hu39E3.G4 plus DNA vaccine resulted in 6 out of 6 animals considered responders, with a mean number of points of 2,348 points/ ¹⁰⁶ PBMC (SD = 588, range = 1,738-3,283) (Fig. 16B). For this group, the range of spot numbers was lower compared to those macaques treated with mab 20H4.9-IgG4.

Treatment with 20H4.9-IgG4 and mab hu9E3.G4 was well tolerated and did not result in significant changes in clinical symptoms, clinical chemistry or hematology parameters relative to control monkeys.

These data show that mab 20H4.9-IgG4 treatment in combination with a DNA vaccine elicits an in vivo enhancement of specific responses to the test antigen relative to control or treatment with mab hu39E3.G4, e.g. by antigen-specific IFN-γ -Secretary cell assay. Since only one dose level of antibody and one dosing regimen were used in these preliminary studies, it was not possible to induce a maximal response and further work is required to investigate optimal conditions. However, it is clear that even with these non-optimized protocols, the enhanced cellular response to the tested antigen can be achieved with mab 20H4.9-IgG4, suggesting that modulation of CD137 function is an attractive approach to increase the effectiveness of DNA vaccines.

While the invention has been described in detail by way of illustration and example, it will be apparent that certain changes and modifications may be made for clarity and understanding, which are intended to be within the scope of the appended claims.

sequence listing

<110> Bristol-Myers Squibb Company

  Jure-Kunkel, Maria

Hefta, Laura

Santoro, Marc

Ganguly, Subinay

the

<120> Fully human antibody against human 4-1BB (cD137)

the

<130>10060 PCT

the

<150>US 60/510193

<151>2003-10-10

the

<150>not yet assigned

<151>2004-10-08

the

<160>9

the

<170>PatentIn version 3.2

the

<210>1

<211>7057

<212>DNA

<213> Artificial

the

<220>

<223> Artificial sequence

the

<400>1

cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttat 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

accacctgcc cccatccccag gaggagatga ccaagaacca ggtcagcctg acctgcctgg 1860

tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg cagccggaga 1920

acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc ctctacagca 1980

ggctaaccgt ggacaagagc aggtggcagg agggaatgt cttctcatgc tccgtgatgc 2040

atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg ggtaaatgat 2100

ctagaggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc agcctcgact 2160

gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 2220

gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 2280

agtaggtgtc attctattct gggggtggg 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

atttttaa 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 tccccaacct ccccctgaac 4200

ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttaattgcagc 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 cacgaaccccc 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 ttatttatt 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

the

<210>2

<211>7057

<212>DNA

<213> Artificial

the

<220>

<223> Artificial sequence

the

<400>2

gctacatgcc cggtctatat gcgcaactgt aactaataac tgatcaataa ttatcattag 60

ttaatgcccc agtaatcaag tatcgggtat atacctcaag gcgcaatgta ttgaatgcca 120

tttaccgggc ggaccgactg gcgggttgct gggggcgggt aactgcagtt attackgcat 180

acaagggtat cattgcggtt atccctgaaa ggtaactgca gttacccacc tgataaatgc 240

catttgacgg gtgaaccgtc atgtagttca catagtatac ggttcatgcg ggggataact 300

gcagttactg ccattaccg 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 accccaggaca gggtccacgt tgatgtcgtc accccgcgtc 780

ctgacaactt cggaagcctc tgggacagggg agtggacgcg acagatacca cccaggaagt 840

caccaatgat gacctcgacc tatgcggtca gggtctctt 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 gagtcctga gatgaggggag 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 ccccccacccc 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 gcaccaagag 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 ttccattatt 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 gtttaatttcg taaaaaaagt 4500

gacgtaagat caacaccaaa caggtttgag tagttacata gaatagtaca gacatatggc 4560

agctggagat cgatctcgaa ccgcattagt accagtatcg acaaaggaca cactttaaca 4620

ataggcgagt gttaaggtgt gttgtatgct cggccttcgt atttcacatt tcggaccccca 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 catttatgcca ataggtgtct tagtccccta 4920

ttgcgtcctt tcttgtacac tcgttttccg gtcgttttcc ggtccttggc atttttccgg 4980

cgcaacgacc gcaaaaaggt atccgaggcg gggggactgc tcgtagtgtt tttagctgcg 5040

agttcagtct ccaccgcttt gggctgtcct gatatttcta tggtccgcaa aggggggacct 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 gggtcacga 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

the

<210>3

<211>467

<212>PRT

<213> Artificial

the

<220>

<223> Artificial sequence

the

<400>3

the

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 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 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

the

<210>4

<211>6435

<212> DNA

<213> Artificial

the

<220>

<223> Artificial sequence

the

<400>4

gacggatcgg gagatctgct agcccgggtg acctgaggcg cgccggcttc gaatagccag 60

agtaaccttt ttttttaatt 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 ttccatgac gtcaatgggt 540

ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 600

gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 660

cttatgggac tttccctactt 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 gccaggtca 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 tttttttat 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 ttttccccaga 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 aatttgtag 3840

aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga 3900

atgcaattgt tgttgttaac ttgtttatg cagcttataa tggttacaaa taaagcaata 3960

gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 4020

aactcatcaa tgtatcttat catgtctgga tcggctggat gatcctccag cgcggggatc 4080

tcatgctgga gttcttcgcc caccccaact tgtttatgc 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 agattatca 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 accactcgt 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

the

<210>5

<211>6435

<212>DNA

<213> Artificial

the

<220>

<223> Artificial sequence

the

<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 tgccattac 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 gggtcgagt cgaagagaag gaggacgatg agaccgaggg 1080

tctatggtgg cctctttaac acaactgtgt cagaggtcgg tgggacagaa acaggtcc 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 ttccattatt 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 aaagaccac 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

the

<210>6

<211>236

<212>PRT

<213> Artificial

the

<220>

<223> Artificial sequence

the

<400>6

the

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 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

the

<210>7

<211>1413

<212> DNA

<213> Artificial

the

<220>

<223> Artificial sequence

the

<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 accagtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggaca gagcaggtgg 1320

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380

cagaagagcc tctccctgtc cccgggtaaa tga 1413

the

<210>8

<211>1413

<212>DNA

<213> Artificial

the

<220>

<223> Artificial sequence

the

<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 acagagggt 420

cggaggtggt tcccgggtag ccagaagggg gaccgtggga ggaggttctc gtggagaccc 480

ccgtgtcgcc gggacccgac ggaccagttc ctgatgaagg ggcttggcca ctgccacagc 540

accttgagtc cgcggggactg 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 aggggacag gggcccattt act 1413

the

<210>9

<211>470

<212>PRT

<213> Artificial

the

<220>

<223> Artificial sequence

the

<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 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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