A ubiquitin-coupled chimeric antigen receptor and immune cells

Abstract

This invention relates to the pharmaceutical field, and particularly to a ubiquitin-coupled chimeric antigen receptor and immune cells, wherein the ubiquitin is coupled to the C-terminus of the chimeric antigen receptor. The simple, easy-to-implement, and highly self-activated chimeric antigen receptor and immune cells provided by this invention, applicable to different targets, have advantages such as simple application, high reproducibility, good universality, and high optimization. They are suitable for various existing CAR-T cell production technologies, especially for CAR-T therapy products targeting solid tumors, providing a new solution to problems such as excessively high basal self-activation levels, poor proliferation capacity, excessive differentiation, and rapid depletion of CAR-T cells.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a ubiquitin-coupled chimeric antigen receptor and immune cells. Background Technology

[0002] Chimeric antigen receptors (CARs) are artificially engineered antigen receptors that target tumors. CAR-T therapy is an adoptive immunotherapy that involves expressing CARs on the patient's own T cells to specifically recognize antigens and transmit T cell signals, enabling them to recognize and kill tumor cells in the patient's body. The CAR mainly consists of: a single-chain antibody fragment (scFv) that recognizes and binds to specific tumor antigens, allowing for precise identification and "targeted attack" of tumor cells; the intracellular domain of the ζ chain of the T cell receptor (TCR), which effectively activates T cells after the scFv recognizes the tumor antigen. Activated T cells secrete large amounts of cytokines, rapidly proliferate, effectively kill tumor cells, and recruit other immune cells through secreted inflammatory factors; furthermore, adding signaling domains of co-stimulatory molecules such as CD28 or 41BB to the intracellular region of the CAR enhances its ability to activate T cells and improves the survival rate of CAR-T cells in vivo.

[0003] Currently, CAR-T therapy has achieved significant success in the clinical treatment of various types of tumors, especially hematologic malignancies. In 2017, the US FDA approved two commercially available CAR-T therapy products targeting the CD19 antigen for the treatment of relapsed / refractory B-cell leukemia and lymphoma, achieving significant efficacy. However, CAR-T therapy still faces many limitations in the treatment of solid tumors. In 2019, Robbie et al. summarized the problems encountered in the clinical application of CAR-T therapy and pointed out that one of the reasons limiting the function of CAR-T cells in vivo is the exhaustion of T cell function caused by factors such as the tumor microenvironment, excessive tumor target burden, and excessively high baseline T cell self-activation levels, thus failing to effectively kill tumors. Numerous studies have shown that reducing the impact of CAR on T cell self-activation can effectively alleviate the problem of CAR-T cell dysfunction caused by over-differentiation and premature exhaustion during the culture and proliferation of CAR-T cells, thereby effectively improving the efficacy of CAR-T therapy. This provides direction for optimizing CAR-T design.

[0004] Currently, existing optimization design strategies for CAR-T mainly include: reducing CAR signal by mutating and inactivating different combinations of the ITAM region in CD3ζ; reducing CD28 co-stimulatory signal by mutating the CD28 co-stimulatory domain; exploring different structural combinations to find the most suitable CAR structure with the lowest basal autoactivation level for different targets; and co-transferring transcription factors involved in regulating T cell proliferation and differentiation with CAR into T cells to reduce CAR-T cell autoactivation signals and inhibit T cell exhaustion. However, these designs often require trying CARs targeting different targets, consuming a lot of human and material resources, and have drawbacks such as large errors and low universality. Therefore, modifying the CAR structure itself in a more practical and universal way is a more optimized approach. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a ubiquitin-coupled chimeric antigen receptor and immune cells to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, the present invention first provides a method for modifying a chimeric antigen receptor, the method comprising coupling ubiquitin to the C-terminus of the chimeric antigen receptor.

[0007] The present invention provides a ubiquitin-modified chimeric antigen receptor, wherein the ubiquitin-modified chimeric antigen receptor comprises a transmembrane domain, an intracellular domain and an extracellular domain, wherein ubiquitin is coupled to the C-terminus of the intracellular domain.

[0008] The present invention provides an isolated polynucleotide comprising a nucleotide encoding the ubiquitin-modified chimeric antigen receptor.

[0009] The present invention provides a nucleic acid construct comprising the isolated polynucleotide.

[0010] The present invention provides an immune cell comprising the nucleic acid construct or the genome in which the exogenous polynucleotide is integrated, or a chimeric antigen receptor capable of expressing the ubiquitin-modified polynucleotide.

[0011] The immune cells are selected from CAR-T cells, CAR-NK cells, CAR-macrophages, or CAR-TIL cells.

[0012] The present invention also provides a method for preparing the immune cells, the method comprising introducing the nucleic acid construct or the polynucleotide into immune effector cells or stem cells that produce immune effector cells.

[0013] The immune effector cells are selected from T cells, NK cells, macrophages, or TIL cells.

[0014] The present invention also provides the use of the immune cells in the preparation of medicaments for treating cancer or immune diseases.

[0015] The present invention also provides a treatment method for tumors or immune diseases, the treatment method comprising administering a therapeutically effective amount of the immune cells to a subject.

[0016] As described above, the ubiquitin-coupled chimeric antigen receptor and immune cells of the present invention have the following beneficial effects: they provide a method for optimizing CAR-T cells, especially addressing the problem of poor CAR-T cell proliferation in solid tumors. The simple and easy-to-implement optimization scheme for high-self-activation-level CARs provided by the present invention, applicable to different targets, has advantages such as simple application, high reproducibility, good universality, high optimization degree, and good anti-tumor activity. It is applicable to various existing CAR-T cell production technologies, especially suitable for CAR-T therapy products developed targeting solid tumors, providing a new solution to problems such as excessively high CAR-T cell basal self-activation levels, poor proliferation capacity, excessive differentiation degree, and rapid depletion. Attached Figure Description

[0017] Figure 1 The figure shows the autoactivation levels of CAR-T cell lines targeting different targets constructed in the human T-lymphoma cell line Jurkat. The basal autoactivation levels of these cell lines were assessed by measuring the expression of indicator proteins of T-cell activation levels, such as CD69 and ICOS. The left figure shows the detected CD69 expression level, and the right figure shows the detected ICOS expression level. As shown, many CARs targeting different targets used in clinical and preclinical trials lead to high basal autoactivation levels of T cells. In particular, CARs targeting solid tumors such as GD2, Her2, CSPG4, EGFR, Meso, GPC3, and multiple targets (nucleotide sequences as shown in SEQ ID NO. 3-11, amino acid sequences as shown in SEQ ID NO. 19-27) exhibit relatively high basal autoactivation levels. Previous studies have shown that excessively high CAR-T cell autoactivation levels can lead to premature differentiation, decreased proliferation capacity, increased cell exhaustion, and a significant decrease in anti-tumor killing ability, which is positively correlated with poor clinical treatment efficacy.

[0018] Figures 2-1 to 2-3The diagram shows CD19-28 wtUb-CAR and CD19-28 MonoUb-CAR obtained by conjugating CD19-28 WT-CAR (nucleotide sequence as shown in SEQ ID NO.12, amino acid sequence as shown in SEQ ID NO.28) and a wild-type ubiquitin (wtUb) (nucleotide sequence as shown in SEQ ID NO.13, amino acid sequence as shown in SEQ ID NO.29) or monoubiquitin (monoUb) (nucleotide sequence as shown in SEQ ID NO.14, amino acid sequence as shown in SEQ ID NO.30) to the C-terminus of a CD19-targeting CAR, respectively, in the Jurkat T cell line. Figure 2-1 ) or primary human T cells ( Figure 2-2 In this study, changes in the expression levels of wtUb and wild-type CARs on the cell membrane were examined, as well as the effect of this modification on the tumor-killing ability of CAR-T cells. The results showed that conjugation of both wtUb and monoUb effectively downregulated the expression level of CARs on the cell membrane surface, with wtUb showing a more significant downregulation. The in vitro killing assay involved co-incubating CAR-T cells with CD19+ target cells at a specific ratio. After 18 hours, the tumor-killing ability of CAR-T cells was assessed by detecting the amount of target cells. Figure 2-3 The results showed that monoUb-modified CARs could still effectively mediate the killing effect of CAR-T cells on tumor target cells, while wtUb-modified CARs, due to their low expression levels, exhibited a significant decrease in tumor-killing ability. Therefore, subsequent research and optimization focused on CARs coupled with monoubiquitin.

[0019] Figures 3-1 to 3-3 This study demonstrates the effect of expressing multiple CD28 CARs conjugated with monoubiquitins targeting different targets in the Jurkat cell line on the basal autoactivation level of CAR-T cells, validating the generalizability of this approach. The study also examined the effect of CD19 (…) on… Figure 3-1 ), GD2 (nucleotide sequence as shown in SEQ ID NO.15, amino acid sequence as shown in SEQ ID NO.31, results as follows) Figure 3-2 GPC3 (nucleotide sequence as shown in SEQ ID NO.16, amino acid sequence as shown in SEQ ID NO.32, results as follows) Figure 3-3The expression levels of three target-conjugated monoubiquitin-based CARs on the cell membrane and the corresponding basal autoactivation signals of CAR-T cells were analyzed. The results, as shown in the figure, indicated that the expression levels of all three CARs were significantly decreased. Using the expression levels of T cell activation indicator proteins CD69 and ICOS to reflect the basal autoactivation signals of CAR-T cells, the basal autoactivation levels of all three CAR-T cells were significantly improved. This demonstrates the universality of this approach, effectively downregulating the autoactivation signals of CARs with different targets and high autoactivation levels.

[0020] Figures 4-1 to 4-3 This study, conducted under in vitro culture conditions, examined the differentiation of wild-type T cells targeting multiple targets (i.e., CD19-28 WT-CAR T cells, GD2-28 WT-CAR T cells, and GPC3-28 WT-CAR T cells) and monoubiquitin-conjugated CAR-T cells (i.e., CD19-28 MonoUb-CAR T cells, GD2-28 MonoUb-CAR T cells, and GPC3-28 MonoUb-CAR T cells). Flow cytometry analysis of CD45RA and CD62L expression levels categorized T cell differentiation into CD45RA+CD62L+ (Stem cell memory T cells, T cells...) SCM ),CD45RA-CD62L+(Central memory Tcell,T CM ),CD45RA-CD62L-(Effector memory T cell,T EM ),as well as

[0021] CD45RA+CD62L-(Effector T cell,T EFF The differentiation level increases sequentially, while the proliferation and differentiation capacity decrease sequentially. As shown in the figure, in CAR-T cells targeting three different targets ( Figure 4-1 For CAR-T cells targeting CD19, Figure 4-2 CAR-T cells targeting GD2, Figure 4-3 For CAR-T cells targeting GPC3 (the left image shows detected CD4, and the right image shows detected CD8), monoubiquitin-coupled modification can effectively slow down T cell differentiation, allowing more CAR-T cells to remain in the T cell stage. SCM This state also suggests that CAR-T cells coupled with monoubiquitin should have better sustained proliferation and tumor-killing capabilities.

[0022] Figure 5This study describes the in vitro detection of the proliferation of wild-type GD2-targeting CAR-T cells and monoubiquitin-conjugated CAR-T cells under continuous stimulation by target cells. First, equal amounts of GD2-28 WT-CAR and GD2-28 MonoUb-CAR T cells were stimulated with equal amounts of irradiated target cells. Subsequently, viable cell counts were performed every two days to record cell proliferation. Simultaneously, appropriate amounts of culture medium were added, and the cell density was adjusted to 1*10^6 / ml for continued culture. The results indicate that, in the presence of target cells and under continuous stimulation, monoubiquitin-conjugated CAR-T cells exhibited lower basal autoactivation levels and cell differentiation, as well as better sustained proliferation capacity.

[0023] Figures 6-1 to 6-2 This study, conducted in vivo on tumor-killing experiments in tumor-bearing mice, verified the functional differences between wild-type CD19-targeting CAR-T cells and monoubiquitin-conjugated CAR-T cells. Tumor-bearing mice were randomly divided into three groups, receiving equal amounts of untransfected normal T cells, CD19 WT CAR-T cells, and CD19 MonoUb CAR-T cells via tail vein injection, respectively. Tumor cell growth was monitored in mice using an in vivo imaging system (IVIS). With a smaller infusion of CAR-T cells, wild-type CAR-T cells showed limited tumor control, while monoubiquitin-conjugated CAR-T cells still effectively controlled tumor growth to some extent, achieving tumor clearance in a few mice. Figure 6-1 This effectively improved the survival rate of tumor-bearing mice. Figure 6-2 Its efficacy is significantly better than that of wild-type CAR-T cells.

[0024] Figures 7-1 to 7-2 This study examines the proliferation, differentiation, and depletion of CAR-T cells in tumor-bearing mice. The results show that, even under stimulation by tumor cells and other pathogens, CAR-T cells coupled with monoubiquitin still exhibit a higher proportion of stem cell memory T cells (T cells) with strong and sustained proliferative capacity. SCM ()( Figure 7-1 The left image shows CD4 CAR-T cells in the bone marrow, the middle image shows CD8 CAR-T cells in the bone marrow, and the right image shows the CAR-T cell count in the spleen. Correspondingly, the number of monoubiquitin-coupled CAR-T cells was significantly higher than that of wild-type CAR-T cells, and their cell exhaustion marker proteins (PD1, LAG3, TIM3, i.e., Figure 7-2The expression levels of CD4 CAR-T cells (left image shows CD4 CAR-T cells in bone marrow, right image shows CD8 CAR-T cells in bone marrow) were also significantly lower. This may be related to the weaker activation signal of CAR coupled with monoubiquitin under target cell stimulation conditions. A weaker activation signal is sufficient for T cells to perform normal tumor killing function. This also suggests that the excessively strong activation signal of wild-type CAR may lead to rapid differentiation and premature exhaustion of T cells, thereby limiting the anti-tumor efficacy of CAR-T cells. Detailed Implementation

[0025] This invention provides a simple and easy-to-implement optimized modification scheme for high-self-activation level CARs applicable to different targets. Specifically, it involves coupling a ubiquitin protein mutant (monoUb) to the C-terminus of CARs targeting different targets to obtain MonoUb-CARs. Therefore, this invention first provides a method for modifying chimeric antigen receptors, which includes coupling ubiquitin to the C-terminus of the chimeric antigen receptor.

[0026] Ubiquitin is a small protein found in all eukaryotes (most eukaryotic cells). It consists of 76 amino acids and has a molecular weight of approximately 8.451 kDa. Its primary function is to tag proteins that need to be broken down, allowing them to be degraded by the 26S proteasome. For membrane proteins, especially certain types of ubiquitin modifications, it may be involved in the downregulation, transport, and vesicle sorting of membrane proteins, thus participating in the regulation of their signal transduction. In this application, ubiquitin refers to any wild-type or mutant ubiquitin from any eukaryotic cell source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Ubiquitin is a highly conserved protein; almost all eukaryotic cells, starting with yeast, express ubiquitin proteins, and their amino acid sequences are essentially identical (yeast and human ubiquitin differ by only one amino acid).

[0027] The ubiquitin is either wild-type or mutant. Wild-type ubiquitin can be modified by endogenous ubiquitin within the cell but cannot be used as a substrate to modify other proteins, thus forming a ubiquitin chain. In a preferred embodiment, the ubiquitin is mutant ubiquitin. Because mutant ubiquitin cannot be modified by endogenous ubiquitin and cannot be used as a substrate to ubiquitinate other proteins, mutant ubiquitin is a monoubiquitin, and therefore can also be called mutant monoubiquitin or monoubiquitin.

[0028] The ubiquitin is coupled to the C-terminus of the chimeric antigen receptor via a linker peptide. The linker peptide described in this application can be any linker peptide commonly used in the art. Examples of such linker peptides include GSGGSG, GSGGSGG GSGGSGGG, GGGGSGGG, or GGSGGGSGGG or GGSGGGSGGGSAAA.

[0029] The present invention also provides a ubiquitin-modified chimeric antigen receptor, wherein the ubiquitin-modified chimeric antigen receptor comprises a transmembrane domain, an intracellular domain and an extracellular domain, wherein ubiquitin is coupled to the C-terminus of the intracellular domain.

[0030] The ubiquitin-modified chimeric antigen receptor was obtained through the aforementioned modification method.

[0031] In some embodiments of the present invention, the transmembrane domain may include transmembrane domains of protein molecules such as CD8α, CD28, and DAP 10. For example, the amino acid sequence of CD8α may include the following sequence: IYIWAPLAGTCGVLLLSLVITLYC. As another example, the sequence of CD8α can be found in NM_001145873, and the sequence of CD28 can be found in NM_006139.

[0032] In some embodiments of the present invention, the intracellular domain may include a co-stimulatory domain and / or a signaling domain. For example, the intracellular domain may include one or a combination of the following: signal transduction domains of protein molecules such as 4-1BB, CD28, OX40, ICOS, CD3ζ, and DAP10.

[0033] For example, the amino acid sequence of the 4-1BB includes the following:

[0034] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0035] The amino acid sequence of CD3ζ includes the following:

[0036] GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0037] For example, the sequence of 4-1BB can be referenced to NM_001561, the sequence of CD28 to NM_006139, the sequence of OX40 to NM_003327, the sequence of ICOS to NM_012092, the sequence of CD3ζ to NM_198053, and the sequence of DAP 10 to NM_014266. In one specific embodiment of the present invention, the intracellular domain includes CD28 and CD3ζ sequentially from the N-terminus to the C-terminus. In some embodiments of the present invention, the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain sequentially from the N-terminus to the C-terminus.

[0038] In some embodiments of the present invention, the chimeric antigen receptor comprises, from N-terminus to C-terminus, a single-chain antibody, a transmembrane domain, and an intracellular domain. In some specific embodiments of the present invention, the chimeric antigen receptor comprises, from N-terminus to C-terminus, a single-chain antibody, a CD8α transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In one specific embodiment of the present invention, the chimeric antigen receptor comprises, from N-terminus to C-terminus, a single-chain antibody, a CD28 transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ signaling domain. In another specific embodiment of the present invention, the chimeric antigen receptor comprises, from N-terminus to C-terminus, a single-chain antibody, a CD8α transmembrane region, an OX40 co-stimulatory domain, and a CD3ζ signaling domain. In yet another specific embodiment of the present invention, the chimeric antigen receptor comprises, from N-terminus to C-terminus, a single-chain antibody, a CD8α transmembrane region, an ICOS co-stimulatory domain, and a CD3ζ signaling domain. In another specific embodiment of the present invention, the chimeric antigen receptor, from N-terminus to C-terminus, sequentially comprises a single-chain antibody, a CD8α transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. In another specific embodiment of the present invention, the chimeric antigen receptor, from N-terminus to C-terminus, sequentially comprises a single-chain antibody, a CD28 transmembrane region, a CD28 co-stimulatory domain, an OX40 co-stimulatory domain, and a CD3ζ signaling domain. In another specific embodiment of the present invention, the chimeric antigen receptor, from N-terminus to C-terminus, sequentially comprises an extracellular domain composed of a CD8α signal peptide, a myc tag, scFv, and a CD8α hinge, a transmembrane domain of CD8α, and an intracellular domain composed of CD28 and CD3ζ in tandem. The ubiquitin-modified chimeric antigen receptor, i.e., any of the above-mentioned chimeric antigen receptors, has ubiquitin linked to its CD3ζ region via a linker peptide.

[0039] The single-chain antibodies described in this invention are not specifically limited and can be selected from any single-chain antibody. Examples of single-chain antibodies used in this invention include CD19 scFv, GD2 scFv, GPC3 scFv, Her2 scFv, CSPG4 scFv, EGFR scFv, Meso scFv, TRBC1 scFv, CD133 scFv, and BCMA scFv.

[0040] The ubiquitin referred to in this application is selected from wild-type ubiquitin or mutant ubiquitin.

[0041] The amino acid sequence of wild-type ubiquitin is shown in SEQ ID NO.17.

[0042] In a preferred embodiment, the ubiquitin is selected from mutant ubiquitin. The amino acid sequence of the mutant ubiquitin used in this invention is shown in SEQ ID NO.18.

[0043] The present invention provides an isolated polynucleotide comprising a nucleotide encoding the ubiquitin-modified chimeric antigen receptor.

[0044] The polynucleotides encoding the transmembrane domain, intracellular domain, and extracellular domain can all be selected from polynucleotides in the prior art.

[0045] In one specific embodiment of the present invention, the polynucleotide encoding the ubiquitin-modified chimeric antigen receptor includes, in sequence, a nucleotide encoding an extracellular domain consisting of a CD8α signal peptide, a myc tag, scFv, and a CD8α hinge, a nucleotide encoding a CD8α transmembrane domain, and a nucleotide encoding an intracellular domain consisting of CD28, CD3ζ, and ubiquitin tandem.

[0046] The nucleotide sequence of wild-type ubiquitin is shown in SEQ ID NO.1. In a preferred embodiment, the ubiquitin is selected from mutant ubiquitin. The polynucleotide sequence encoding mutant ubiquitin used in this invention is shown in SEQ ID NO.2.

[0047] The present invention provides a nucleic acid construct comprising the isolated polynucleotide.

[0048] The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into cells or tissues. The nucleic acid construct can be a non-viral vector or a viral vector. The viral vector can be a lentiviral vector, adenovirus vector, adeno-associated virus vector, or baculovirus vector. In some embodiments of the present invention, the nucleic acid construct is a lentiviral vector, which includes a vector backbone (empty vector) and an expression framework. The empty vector includes various elements controlling expression, including a promoter sequence, transcription initiation sequence, enhancer sequence, selection element, and reporter gene. Additionally, the vector may contain a replication initiation site. The empty vector is, for example, the pHR-hEF1α-IRES-EGFP empty vector. The expression framework is the isolated polynucleotide.

[0049] The term "vector" refers to a nucleic acid or polynucleotide fragment used to introduce or transfer one or more nucleic acids or polynucleotides into a target cell or tissue. Typically, a vector is used to introduce foreign DNA into another cell or tissue. A vector may contain a bacterial resistance gene for growth in bacteria and a promoter for expression of a target protein in an organism. The DNA can be produced in vitro by PCR or one or more suitable techniques known to those skilled in the art.

[0050] The present invention provides an immune cell comprising the nucleic acid construct or the genome in which the exogenous polynucleotide is integrated, or a chimeric antigen receptor capable of expressing the ubiquitin-modified antigen.

[0051] The immune cells are selected from CAR-T cells, CAR-NK cells, CAR-macrophages, or CAR-TIL cells.

[0052] The present invention also provides a method for preparing the immune cells, the method comprising introducing the nucleic acid construct or the polynucleotide into immune effector cells or stem cells that produce immune effector cells.

[0053] The immune effector cells are selected from T cells, NK cells, macrophages, or TIL cells.

[0054] The present invention also provides the use of the immune cells in the preparation of medicaments for treating cancer or immune diseases.

[0055] In this application, "cancer" refers to any medical condition mediated by the growth, proliferation, or metastasis of tumor or malignant cells, resulting in solid tumors and non-solid tumors such as leukemia. In this invention, "tumor" refers to the solid matter of tumors and / or malignant cells.

[0056] The cancers mentioned include, for example, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer; leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma and other hematologic malignancies such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocytic large B-cell lymphoma, EBV-positive and negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary exudative lymphoma, and Hodgkin lymphoma.

[0057] The immune diseases mentioned include, for example, systemic lupus erythematosus (SLE), autoimmune diabetes, psoriasis, vitiligo, scleroderma, and rheumatoid arthritis.

[0058] "Treatment" or "therapy" for a condition includes preventing or alleviating the condition, slowing the onset or progression of the condition, reducing the risk of developing the condition, preventing or delaying the development of symptoms associated with the condition, reducing or terminating symptoms associated with the condition, achieving complete or partial reversal of the condition, curing the condition, or a combination of the above. For cancer, "treatment" or "therapy" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" or "therapy" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination of the above.

[0059] The present invention also provides a treatment method for tumors or immune diseases, the treatment method comprising administering a therapeutically effective amount of the immune cells to a subject.

[0060] The cancers mentioned include, for example, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic cancer; leukemia, lymphoma, myeloma, mycosis fungoides, Merkel cell carcinoma and other hematologic malignancies such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell / histiocytic B-cell lymphoma, EBV-positive and negative PTLD and EBV-associated diffuse large B-cell lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK / T-cell lymphoma, nasopharyngeal carcinoma and HHV8-associated primary exudative lymphoma, and Hodgkin lymphoma.

[0061] The immune diseases mentioned include, for example, systemic lupus erythematosus (SLE), autoimmune diabetes, psoriasis, vitiligo, scleroderma, and rheumatoid arthritis.

[0062] In this invention, "therapeutic effective amount" or "effective dose" refers to the dose or concentration at which a certain drug is effective in treating antigen-related diseases or states associated with chimeric antigen receptors. For example, for the use of the antibody or its antigen-binding fragment disclosed in this invention, a therapeutic effective amount is at that dose or concentration at which the antibody or antigen conjugate can eliminate all or part of a tumor, inhibit or slow tumor growth, inhibit the growth or proliferation of cells mediating a cancerous state, inhibit tumor cell metastasis, alleviate any symptoms or markers associated with a tumor or cancerous state, prevent or delay the development of a tumor or cancerous state, or some combination thereof.

[0063] monoUb-CAR can effectively promote CAR endocytosis and degradation, significantly reducing CAR expression levels on cell surfaces. This leads to a substantial decrease in the basal auto-activation level of immune cells, such as CAR-T cells, and significantly reduces the tendency for immune cells, such as T cells, to differentiate too rapidly or become functionally exhausted. In some embodiments of this invention, monoUb-CAR significantly enhances the sustained proliferation capacity of CD28CAR-T cells under in vitro and in vivo target cell stimulation conditions, effectively improving the tumor-killing ability of CAR-T cells in tumor-bearing mice, thereby increasing mouse survival rates.

[0064] This invention compared the in vivo antitumor effects of CD28 monoUb CAR-T and CD28 WT CAR-T in a mouse tumor model. At the proliferation level, CD28 monoUb CAR-T exhibited a stronger proliferation response and more sustained proliferation capacity; regarding cell differentiation phenotype, the modified CAR-T cells accumulated more stem cell memory T cells (T cells) in the spleen, blood, and tumors. SCM It also reduces differentiation into terminal effector T cells. Therefore, the modified CAR-T cells can more effectively infiltrate tumor tissue for killing, and at the same T cell injection dose, the modified CAR-T cells can more effectively control tumor development.

[0065] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0066] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0067] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0068] Unless otherwise specified, the conventional technical operations such as molecular biology, microbiology, cell biology, biochemistry and immunology used in the implementation of this invention are all within the understanding and knowledge of those skilled in the art. These techniques are widely used and can be well illustrated in the following literature, such as: "Molecular cloning: A Laboratory Manual, Fourth Edition" (MR Green, et al. 2014); "Oligonucleotide Synthesis" (MJ Gait, et al. 1984); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting" (ME Babar, et al. 2011); "Short Protocols in Molecular Biology, Fifth Edition" (FMAusubel, et al. 2002); "Methods in Molecular Biology" (Humana Press); "Gene Transfer Vectors for Mammalian Cells" (JH Miller and M.P. Calos. 1987); "Culture of Animal Cell" (RI Freshney, et al. 2010); "Methods in Enzymology" (Academic Press, Inc.); "Using Antibodies: A Laboratory Manual" (E. Harlow and D. Lane. 1999); "Handbook of Experimental Immunology" (LA Herzenberg, et al. 1997); "Current Protocols in Immunology" (JEColigan, et al. 2002).

[0069] Example 1: Construction of CAR vectors

[0070] The antigen-specific single-chain antibody (scFv) sequences of CD19, GD2, and GPC3 CARs used in this invention are derived from clinically used FMC63, 14g2A, and GPC3 sequences, respectively. The extracellular segment of the CAR is composed of a CD8α signal peptide sequence, a myc tag sequence, an scFv sequence, and a CD8α hinge sequence tandemly; the transmembrane region sequence is the transmembrane region sequence of CD8α; and the intracellular segment structure is composed of a human CD28 intracellular segment sequence tandemly with a human CD3ζ intracellular segment sequence. The ubiquitin-modified chimeric antigen receptor connects ubiquitin to CD3ζ in the intracellular domain via a linker peptide. All scFv amino acid sequences were codon-optimized and converted into base sequences, and synthesized by a third-party company (genscript). All CAR base sequences in this invention were ultimately cloned into the pHR-hEF1α-IRES-EGFP vector (derived from addgene) via Gibson ligation.

[0071] Example 2: Human primary T cell culture and lentiviral infection

[0072] Primary T cells were obtained from healthy informed volunteers. Primary T cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin sulfate, 1 mM sodium pyruvate, non-essential amino acids, and 55 μM 2-mercaptoethanol (all reagents were purchased from Gibco). To maintain T cell proliferation, 100 U / ml hIL-2 (Sigma-Aldrich) was added to the medium.

[0073] Lentiviral preparation: Lenti-X 293T cells (TaKaRa#632180) were resuspended in DMEM medium (Gibco#11995-065) containing 10% fetal bovine serum and free of antibiotics, and cultured at 6.5 × 10⁻⁶ mg / L. 5 Cells were seeded at a density of 100 cells / well in 6-well cell culture plates (Corning#CLS3516) and cultured for 24 hours. Using a liposome transfection system (Mirus#2300), 500 ng of lentiviral packaging plasmid pCMVdR8.92 (Addgene#8455) and 50 ng of pMD2.G (Addgene#12259) were mixed with 500 ng of the lentiviral plasmid to be packaged, following the liposome transfection instructions, and then added to Lenti-X 293T cells. After 16-18 hours, the liposome-containing medium was discarded, and an appropriate amount of fresh medium was added. After 48 hours, the cell supernatant was collected, concentrated directly or by ultracentrifugation, and then frozen at -80°C for later use.

[0074] Lentiviral infection of primary T cells: T cells were activated using magnetic beads (LifeTechnologies #11132D) coated with anti-human CD3 and anti-human CD28 antibodies. T cells were mixed with magnetic beads at a 1:3 ratio and cultured for 24 hours before being inoculated with the prepared lentivirus. After 18 hours, the virus-containing culture medium was discarded and replaced with fresh complete T cell culture medium. Four to five days after stimulation with magnetic beads, the beads were removed, and the cell density was adjusted to 0.8-1 × 10^6 / ml with complete T cell culture medium. Fresh complete T cell culture medium was added every two days.

[0075] The steps for expressing CD28 CARs conjugated with monoubiquitins targeting different targets in the Jurkat cell line are the same as those for expressing them in T cells.

[0076] Example 3: Flow Cytometry Analysis

[0077] For staining of cell surface markers: Dilute the antibody in FACS buffer (phosphate-buffered saline PBS + 2% fetal bovine serum) at an appropriate ratio, resuspend the cells in an appropriate amount of antibody dilution, and incubate them together at 4°C in the dark for 25 minutes; wash the cells three times with FACS buffer, resuspend the cells in FACS buffer containing an appropriate concentration of DAPI, and then perform the detection.

[0078] Flow cytometry data were acquired using a BD LSRFortessa machine (BD Bioscience) and analyzed using FlowJo software (Tree Star). A list of antibodies used in the flow cytometry is provided below.

[0079]

[0080] Example 4: Detection of CAR-T cell killing function in vitro based on flow cytometry

[0081] Double-positive K562 target cells expressing CD19 and mCherry fluorescence were mixed with non-target K562 cells not expressing CD19 and mCherry fluorescence at a 1:1 ratio, and then mixed with CAR-T cells at a specific effector cell:target cell ratio and co-incubated for 24 hours. Cells were cultured in IL-2-free T cell complete medium. Flow cytometry analysis: A mixed K562 cell population without T cells was used as a control group, and the proportion of K562 target cells in the total K562 cell population was analyzed (CK%). In the experimental group containing T cells, T cells were distinguished from the K562 cell population by CD3ε staining, and then the proportion of CD19-K562 target cells in the total K562 cell population was analyzed (EX%). The T cell killing efficiency was calculated as (1-EX% / CK%) × 100%.

[0082] Example 5: Detection of CAR-T cell proliferation capacity in vitro

[0083] After counting CD19-28 WT-CAR and CD19-28 Mono-CAR T cells, they were mixed with irradiated target cells Nalm6 at a 3:1 ratio and resuspended in IL-2-free T cell complete culture medium until the cell density reached 1*10^6 / ml. Viable cell counts were performed every 2 days to calculate cell proliferation, and the cell density was adjusted to 1*10^6 / ml in IL-2-free T cell complete culture medium. When the cell density obtained from the viable cell count was less than or equal to 1*10^6 / ml, it indicated that the cells had stopped proliferating and the experiment was terminated.

[0084] Example 6: Mouse tumor model and CAR-T cell function detection

[0085] The in vivo experimental subjects were 5- to 8-week-old combined immunodeficient (NSG) mice. To compare the in vivo antitumor effect of CAR-T, NSG mice were first injected via the tail vein with 1×10⁻⁶ CAR-T cells. 6 Nalm6 B-cell lymphoma cells expressing the firefly luciferase gene were administered to NSG mice via tail vein injection after the target cells had grown in vivo for 4 days. 6 CAR-T cell therapy; weekly detection of firefly luciferase intensity carried by mouse tumor cells using a small animal in vivo imaging system to reflect tumor cell burden and track tumor development in vivo. The specific implementation of the small animal in vivo imaging system includes: intraperitoneal injection of firefly luciferase substrate (D-luciferin sodium salt) into tumor-bearing mice at a dosage of 0.15 mg / g mouse body weight; after 10 minutes, once the substrate has fully circulated throughout the mouse, the mice are anesthetized with 2.5%-3.5% isoflurane gas before imaging. Bioluminescence imaging is performed using an IVIS spectral imaging system (Perkin Elmer), and quantitative fluorescence data are obtained using in vivo imaging software (Perkin Elmer).

[0086] Example 7: Detection of CAR-T cell proliferation, differentiation and exhaustion levels in tumor-bearing mice

[0087] The in vivo experimental subjects were 5- to 8-week-old combined immunodeficient (NSG) mice. To compare the differences in in vivo proliferation, differentiation, and depletion levels of CAR-T, NSG mice were first injected via the tail vein with 2 × 10⁻⁶ CAR-T cells. 6 One 1000 B lymphoma cells (Nalm6); after the target cells had grown in vivo for 4 days, 2 × 10⁶ cells were administered to NSG mice via tail vein injection. 6Three mice inoculated with CD19-28 WT and CD19-28 MonoUb CAR-T cells were sacrificed at 7, 14, and 21 days. Their spleens and long bones of the hind limbs were harvested. The spleens were ground to obtain spleen cells. An appropriate amount of RPMI culture medium was drawn into a 5ml syringe with a 20-gauge needle. The two ends of the long bone were carefully cut off and the syringe needle was inserted into the bone marrow cavity to flush out the bone marrow cells. The spleen cells and bone marrow cells were treated with an appropriate amount of erythrocyte lysis buffer. The spleen cells were counted and divided into equal parts. The cells were incubated with different antibody dilutions for different test indicators and then subjected to flow cytometry.

[0088] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention. sequence list <110> ShanghaiTech University <120> A ubiquitin-coupled chimeric antigen receptor and immune cells <160> 32 <170> SIPOSequenceListing 1.0 <210> 1 <211> 222 <212> DNA <213> Artificial Sequence <400> 1 atgcagatct tcgtgaagac ccttaccggc aagaccatca cccttgaggt ggagcccagt 60 gacaccatcg aaaatgtgaa ggccaagatc caggataagg aaggcattcc ccccgaccag 120 cagaggctca tctttgcagg caagcagctg gaagatggcc gtactctttc tgactacaac 180 atccagaagg agtcgaccct gcacctggtc ctccgtctca ga 222 <210> 2 <211> 222 <212> DNA <213> Artificial Sequence <400> 2 atgcagatct tcgtgcgtac cctgactggt aggaccatca ctctcgaagt ggagccgagt 60 gacaccattg agaatgtcag ggcacgtatc caagacaggg aaggcatccc tcctgaccag 120 cagaggttga tctttgctgg gaggcagctg gaagatggac gcaccctgtc tgactacaac 180 atccagagag agtccaccct gcacctggtc ctccgtctca ga 222 <210> 3 <211> 2016 <212> DNA <213> Artificial Sequence <400> 3 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgcgat 60 gttgtcatga ctcaaacccc tttatctttg cccgtatccc ttggtgacca ggcttcaatt 120 tcgtgtcgta gtagccaatc tctcgtgcat cgcaatggca acacatatct acactggtac 180 ctgcagaaac caggacaatc cccgaagtta ttgatccata aagtttcaaa tcgattttcg 240 ggggtccctg atcggttcag tggtagcggc tctggaacgg actttactct taagatatcc 300 agagtagaag ccgaggatct cggggtgtat ttctgctcac agtcgaccca cgttccccca 360 ctaacatttg gtgcaggcac gaaactggaa ttaaagggtg gcggtggctc gggcggtggt 420 gggtcgggtg gcggtggatc tgaagttcaa ttattgcagt ctggtcctga gcttgaaaaa 480 cccggcgctt ccgtcatgat ttcatgtaag gcctcggggaa gtagctttac tgggtataat 540 atgaactggg tacgtcaaaa tatcggtaaa tctctcgaat ggataggcgc aattgatcca 600 tactatggag ggacctccta caaccagaag ttcaaaggtc gcgcgacact aacggtggac 660 aagtcatcga gtactgctta tatgcatctg aaaagcttaa cctctgaaga ttccgccgtt 720 tactattgcg tctcaggcat ggagtactgg ggacaaggga catcggtaac ggtgagtagc 780 gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc acctgtggca 840 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900 cctgaggtca cgtgcgtggt ggtggacgtg agccacgaag accccgaggt ccagttcaac 960 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccacggga ggagcagttc 1020 caaagcacgt tccgtgtggt cagcgtcctc accgttgtgc accaggactg gctgaacggc 1080 aaggagtca agtgcaaggt ctccaacaaa ggcctcccag cccccatcga gaaaccatc 1140 tccaaaacca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1260 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1380 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440 1500 gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1560 aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1620 acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1680 gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1740 aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1800 gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1860 ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1920 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1980 gacgcccttc acatgcaggc cctgccccct cgctaa 2016 <210> 4 <211> 1470 <212> DNA <213> Artificial Sequence <400> 4 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggatatac agatgacaca atctccatct agtctgtctg cctcagtcgg tgatcgcgtt 120 accatcactt gtagggcaag ccaggacgtg aatacagccg ttgcctggta tcagcagaaa 180 cctggaaagg ctcccaagct gctgatctat agcgccagtt tcctgtatag cggagttccc 240 tccagattca gtggtagcag gagtggcaca gatttcactc tcacaatcag cagcctccag 300 ccagaggact ttgctactta ctattgccaa cagcactata ccactcctcc cacatttggc 360 cagggcacca aagtcgagat taagcgcaca gggtctacaa gcggtagcgg aaagccagga 420 tcaggcgaag gcagcgaggt ccagctggtg gaatctggag gtggactggt gcaacccgga 480 ggatctctgc gcctctcatg tgccgcaagc gggttcaaca ttaaggacac ttacattcac 540 tgggtcaggc aggcacctgg gaagggactc gaatgggtgg ctaggatcta tccaaccaac 600 ggctacactc gctacgcaga ctcagtcaag ggtcgcttta ccatatcagc cgatacttct 660 aagaacaccg cctacctgca aatgaactca ctgagggctg aggacaccgc agtgtactac 720 tgctctaggt ggggtggaga tggcttctat gctatggatg tgtgggggca gggcaccctc 780 gtgaccgtca gtagtaccac cactcccgca ccccgccctc ctactcctgc ccctaccatt 840 gctagccaac cgcttagtct gagacctgag gcctgtaggc ccgctgctgg tggcgctgtg 900 cacacccgag gattggactt cgcttgcgac atctacatct gggcacctct ggctgggacc 960 tgcggcgtgt tgttgttgag cctggtgatt acgctgtact gtggatccag gagtaagagg 1020 agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080 aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1140 ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200 ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260 gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320 aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380 aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440 cttcacatgc aggccctgcc tcctcgctga 1470 <210> 5 <211> 2016 ​​​​​​​​​​​ aagaagactc caggaaaggg tttaaagtgg ctgggctgga taaacactgc gactggtgag 240 ccaacatatg cagatgactt caagggacgg tttgccatct ctttggaaac ctctgccagg 300 actgtctatt tgcagatcaa taatctcaga aatgaggaca cggctacata tttctgtttt 360 agttactacg actactgggg ccaaggcacc acggtcaccg tctcctcagg tgggggcggt 420 tcaggcggag gtggctctgg cggtggcgga ttggacatca agctcactca gtctccatcc 480 atcctgtctg tgactccagg tgaaacagtc agtctttcct gtagggccag ccagactatt 540 tacaagaacc tacactggta tcaacagaaa tcacatcggt ctcaaggct tctcatcaag 600 tatggttctg attccatctc tggcatcccc tccaggttca ctggcagtgg atcagggaca 660 gattacactc tcaatatcaa cagtgtgaag cccgaagatg aaggaatata ttactgtctt 720 caaggttaca gtacaccttg gacgttcggt ggagggacca agctggaaat aaaacgggag 780 cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 840 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900 cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 960 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 1020 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 1080 aaggagtca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaccatc 1140 tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1260 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1380 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440 1500 gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1560 aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1620 acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1680 gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1740 aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1800 gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 1860 ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 1920 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1980 gacgcccttc acatgcaggc cctgccccct cgctaa 2016 <210> 6 <211> 1506 <212> DNA <213> Artificial Sequence <400> 6 atggccctgc cagtgaccgc cctgctgctg ccactggccc tgctgctgca cgcagcacgg 60 ccagagcaga agctgatctc tgaggaggac ctggagatcc agctggtgca gtccggagca 120 gaggtgaaga agccaggcga gagcctgaga atctcctgca agggctctgg cttcaacatc 180 gaggattact atatccactg ggtgcggcag atgccaggca agggactgga gtggatggga 240 agaatcgacc ctgagaacga tgagacaaag tacggcccaa tcttccaggg ccacgtgacc 300 atcagcgccg acacctccat caatacagtg tatctgcagt ggagctccct gaaggccagc 360 gatacagcca tgtactattg cgcctttagg ggaggcgtgt actggggaca gggaaccaca 420 gtgaccgtgt ctagcggagg aggaggatcc ggaggaggag gatctggcgg cggcggctcc 480 ggcggcggcg gctccgacgt ggtcatgaca cagtctcctg atagcctggc cgtgagcctg 540 ggagagagag caaccatcaa ctgtaagtc tctcagtctc tgctggacag cgatggcaag 600 acatatctga attggctgca gcagaagcca ggacagccac ctaagaggct gatctccctg 660 gtgtctaagc tggactccgg cgtgcctgat cgcttctctg gaagcggatc cggaaccgac 720 tttaccctga caatcagctc cctgcaggcc gaggatgtgg ccgtgtacta ttgttggcag 780 ggcacccact tcccaggcac atttggcggc ggcaccaagg tggagatcaa gaccaccact 840 cccgcacccc gccctcctac tcctgcccct accattgcta gccaaccgct tagtctgaga 900 cctgaggcct gtaggcccgc tgctggtggc gctgtgcaca cccgaggatt ggacttcgct 960 tgcgacatct acatctgggc acctctggct gggacctgcg gcgtgttgtt gttgagcctg 10.20 gtgattacgc tgtactgtgg atccaggagt aagaggagca ggctcctgca cagtgactac 10.80 atgaacatga ctccccgccg ccccgggccc acccgcaagc attaccagcc ctatgcccca 11.40 ccacgcgact tcgcagccta tcgctccaga gtgaagttca gcaggagcgc agacgccccc 12.00 gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 12.60 tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 13.20 aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 13.80 agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 14.40 ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgcctcct 15.00 cgctga 15.06 <210> 7 <211> 1458 <212> DNA <213> Artificial Sequence <400> 7 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgcaggtac aactgcagca gtctgggcct gagctggaga agcctggcgc ttcagtgaag 120 atatcctgca aggcttctgg ttactcattc actggctaca ccatgaactg ggtgaagcag 180 agccatggaa agagccttga gtggattgga cttattactc cttacaatgg tgcttctagc 240 tacaaccaga agttcagggg caaggccaca ttaactgtag acaagtcatc cagcacagcc 300 tacatggacc tcctcagtct gacatctgaa gactctgcag tctatttctg tgcaaggggg 360 ggttacgacg ggaggggttt tgactactgg ggccaaggga ccacggtcac cgtctcctca 420 ggtggaggcg gttcaggcgg cggtggctct agcggtggtg gatcggacat cgagctcact 480 cagtctccag caatcatgtc tgcatctcca ggggagaagg tcaccatgac ctgcagtgcc 540 agctcaagtg taagttacat gcactggtac cagcagaagt caggcacctc ccccaaaaga 600 tggatttatg acacatccaa actggcttct ggagtcccag gtcgcttcag tggcagtggg 660 tctggaaact cttactctct cacaatcagc agcgtggagg ctgaagatga tgcaacttat 720 tactgccagc agtggagtaa gcaccctctc acgtacggtg ctgggacaaa gttggaaatc 780 aaaaccacca ctcccgcacc ccgccctcct actcctgccc ctaccattgc tagccaaccg 840 cttagtctga gacctgaggc ctgtaggccc gctgctggtg gcgctgtgca cacccgagga 900 ttggacttcg cttgcgacat ctacatctgg gcacctctgg ctgggacctg cggcgtgttg 960 ttgttgagcc tggtgattac gctgtactgt ggatccagga gtaagaggag caggctcctg 1020 cacagtgact acatgaacat gactccccgc cgccccgggc ccacccgcaa gcattaccag 1080 ccctatgccc caccacgcga cttcgcagcc tatcgctcca gagtgaagtt cagcaggagc 1140 gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1200 cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1260 aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320 gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380 ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440 gccctgcctc ctcgctga 1458 <210> 8 <211> 1467 <212> DNA <213> Artificial Sequence <400> 8 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggatgttg tgatgactca gtctccactc tccctgcccg tcacccctgg agagccggcc 120 tccatctcct gcagatctag tcagagcctt gtacacagta atgccaacac ctatttacat 180 tggtacctgc agaagccagg gcagtctcca cagctcctga tctataaagt ttccaaccga 240 ttttctgggg tccctgacag gttcagtggc agtggatcag gcacagattt tacactgaaa 300 atcagcagag tggaggctga ggatgttggg gtttattact gctctcaaaa tacacatgtt 360 cctcctacgt ttggccaggg gaccaagctg gagatcaaac gtggtggagg cggttcaggc 420 ggaggtggct ctggcggtgg cggatcgcag gtgcagctgg tgcagtctgg agctgaggtg 480 aagaagcctg gggcctcagt gaaggtctcc tgcaaggctt ctggatacac cttcaccgac 540 tatgaaatgc actgggtgcg acaggcccct ggacaagggc ttgagtggat gggagctctt 600 gatcctaaaa ctggtgatac tgcctacagt cagaagttca agggcagagt cacgctgacc 660 gcggacgaat ccacgagcac agcctacatg gagctgagca gcctgagatc tgaggacacg 720 gccgtgtatt actgtacaag attctactcc tatacttact ggggccaggg aaccctggtc 780 accgtctcct caaccaccac tcccgcaccc cgccctccta ctcctgcccc taccattgct 840 agccaaccgc ttagtctgag acctgaggcc tgtaggcccg ctgctggtgg cgctgtgcac 900 acccgaggat tggacttcgc ttgcgacatc tacatctggg cacctctggc tgggacctgc 960 ggcgtgttgt tgttgagcct ggtgattacg ctgtactgtg gatccaggag taagaggagc 1020 aggctcctgc acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag 1080 cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctccag agtgaagttc 1140 agcaggagcg cagacgcccc cgcgtaccag cagggccaga accagctcta taacgagctc 1200 aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260 atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1320 gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380 gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1440 cacatgcagg ccctgcctcc tcgctga 1467 <210> 9 <211> 1491 <212> DNA <213> Artificial Sequence <400> 9 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggtgaggc tgcagcagag cggccccgac ctgatcaagc ccggcgccag cgtgaagatg 120 agctgcaagg ccagcggcta caccttcacc ggctacgtga tgcactgggt gaagcagagg 180 cccggccagg gcctggagtg gatcggcttc atcaacccct acaacgacga catccagagc 240 aacgagaggt tcaggggcaa ggccaccctg accagcgaca agagcagcac caccgcctac 300 atggagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc caggggcgcc 360 ggctacaact tcgacggcgc ctacaggttc ttcgacttct ggggccaggg caccaccctg 420 accgtgagca gcggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagcgac 480 gtggtgatga cccagagccc cctgagcctg cccgtgagcc tgggcgacca ggccagcatc 540 agctgcagga gcagccagag gctggtgcac agcaacggca acacctacct gcactggtac 600 ctgcagaagc ccggccagag ccccaagctg ctgatctaca gggtgagcaa caggttcccc 660 ggcgtgcccg acaggttcag cggcagcggc agcggcaccg acttcaccct gaagatcagc 720 agggtggagg ccgaggacct gggcatctac ttctgcagcc agagcaccca cgtgccctac 780 accttcggcg gcggcaccaa gctggagatc aagaggacca ccactcccgc accccgccct 840 cctactcctg cccctacat tgctagccaa ccgcttagtc tgagacctga ggcctgtagg 900 cccgctgctg gtggcgctgt gcacacccga ggattggact tcgcttgcga catctacatc 960 tgggcacctc tgggctggac ctgcggcgtg ttgttgttga gcctggtgat tacgctgtac 1020 tgtggatcca ggagtaagag gagcaggctc ctgcacagtg actacatgaa catgactccc 1080 cgccgccccg ggcccacccg caagcattac cagccctatg ccccaccacg cgacttcgca 1140 gcctatcgct ccagagtgaa gttcagcagg agcgcagacg ccccgcgta ccagcagggc 1200 cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1260 aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1320 ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1380 aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1440 accaaggaca cctacgacgc ccttcacatg caggccctgc ctcctcgctg a 1491 <210> 10 <211> 1482 <212> DNA <213> Artificial Sequence <400> 10 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgcaggtcc agctgcagca gtctggagct gagctggtca gacccggcgc atcagtgaaa 120 ctgagctgca aggcttccgg ctatactttc tccgactttg agatgcactg ggtcaagcag 180 accccagtgc atggcctgga atggatcggg gacattgatc ccggcactgg ggacaccgcc 240 tataacctga agttcaaagg caaggctacc ctgaccacag ataagagctc ctctacagcc 300 tacatggagc tgaggtctct gactagtgaa gattcagcag tctactattg cacactgggg 360 gccttcgtgt actggggaca gggcacactg gtcaccgtga gcgccgctaa aactaccccc 420 aagctggagg aaggagagtt cagcgaagca agagtggacg tggtcgtgac ccagacaccc 480 ctgtctctgc ctgtcagttt tggcgatcag gtgagcatct cctgtaggag ttcacagtca 540 ctggccaaca gctacgggaa tacatatctg tcttggtacc tgcacaagcc aggacagagt 600 ccccagctgc tgatctatgg gatttccaat cgcttctctg gagtgcctga ccgattttct 660 gggagtggat caggcaccga tttcacactg aaaatcagca ccattaagcc cgaggacctg 720 ggcatgtact attgtctgca ggggacccat cagccttaca cttttggcgg gggaaccaaa 780 ctggagatca agcgagcaga cgcagcgacc accactcccg caccccgccc tcctactcct 840 gcccctacca ttgctagcca accgcttagt ctgagacctg aggcctgtag gcccgctgct 900 ggtggcgctg tgcacacccg aggattggac ttcgcttgcg acatctacat ctgggcacct 960 ctggctggga cctgcggcgt gttgttgttg agcctggtga ttacgctgta ctgtggatcc 1020 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 1080 gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 1140 tccagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 1200 ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1260 ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1320 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1380 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1440 acctacgacg cccttcacat gcaggccctg cctcctcgct ga 1482 <210> 11 <211> 1479 <212> DNA <213> Artificial Sequence <400> 11 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccgcaggtgc agctggtgca gagcggcgcc gaggtgaaga agcccggcgc cagcgtgaag 120 gtgagctgca aggccagcgg ctacagcttc cccgactact acatcaactg ggtgaggcag 180 gcccccggcc agggcctgga gtggatgggc tggatctact tcgccagcgg caacagcgag 240 tacaaccaga agttcaccgg cagggtgacc atgaccaggg acaccagcat caacaccgcc 300 tacatggagc tgagcagcct gaccagcgag gacaccgccg tgtacttctg cgccagcctg 360 tacgactacg actggtactt cgacgtgtgg ggccagggca ccatggtgac cgtgagcagc 420 ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgacat cgtgatgacc 480 cagacccccc tgagcctgag cgtgaccccc ggccagcccg ccagcatcag ctgcaagatc 540 agcagccaga gcctggtgca cagcaacggc aacacctacc tgcactggta cctgcagaag 600 cccggccaga gcccccagct gctgatctac aaggtgagca acaggttcag cggcgtgccc 660 gacaggttca gcggcagcgg cagcggcacc gacttcaccc tgaagatcag cagggtggag 720 gccgaggacg tgggcatcta ctactgcagc cagagcagca tctacccctg gaccttcggc 780 cagggcacca agctggagat caagaccacc actcccgcac cccgccctcc tactcctgcc 840 cctaccattg ctagccaacc gcttagtctg agacctgagg cctgtaggcc cgctgctggt 900 ggcgctgtgc acacccgagg attggacttc gcttgcgaca tctacatctg ggcacctctg 960 gctgggacct gcggcgtgtt gttgttgagc ctggtgatta cgctgtactg tggatccagg 1020 agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1080 cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1140 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 1200 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1260 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1320 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1380 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1440 tacgacgccc ttcacatgca ggccctgcct cctcgctga 1479 <210> 12 <211> 1464 <212> DNA <213> Artificial Sequence <400> 12 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggacatcc agatgacaca gactacatcc tccctgtctg cctctggg agacagagtc 120 accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180 ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240 tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300 caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360 ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420 ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480 ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540 cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600 tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660 gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720 cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780 gtctcctcaa ccaccactcc cgcaccccgc cctcctactc ctgcccctac cattgctagc 840 caaccgctta gtctgagacc tgaggcctgt aggcccgctg ctggtggcgc tgtgcacacc 900 cgaggattgg acttcgcttg cgacatctac atctgggcac ctctggctgg gacctgcggc 960 gtgttgttgt tgagcctggt gattacgctg tactgtggat ccaggagtaa gaggagcagg 1020 ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1080 taccagccct atgccccacc acgcgacttc gcagcctatc gctccagagt gaagttcagc 1140 aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1200 ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1260 gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1320 aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1380 cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1440 atgcaggccc tgcctcctcg ctga 1464 <210> 13 <211> 1758 <212> DNA <213> Artificial Sequence <400> 13 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggagcaga agctgatcag cgaggaggac ctggacatcc agatgacaca gactacatcc 120 tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 180 agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 240 catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 300 gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 360 cagggtaata cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc 420 ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg aggtgaaact gcaggagtca 480 ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc 540 tcattacccg actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg 600 ctgggagtaa tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg 660 accatcatca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 720 gatgacacag ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg 780 gactactggg gccaaggaac ctcagtcacc gtctcctcaa ccaccactcc cgcaccccgc 840 cctcctactc ctgcccctac cattgctagc caaccgctta gtctgagacc tgaggcctgt 900 aggcccgctg ctggtggcgc tgtgcacacc cgaggattgg acttcgcttg cgacatctac 960 atctgggcac ctctggctgg gacctgcggc gtgttgttgt tgagcctggt gattacgctg 1020 tactgtggat ccaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact 1080 ccccgccgcc ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc 1140 gcagcctatc gctccagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 1200 ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1260 gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1320 gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1380 atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1440 gccaccaagg acacctacga cgcccttcac atgcaggccc tgcctcctcg cggtggttca 1500 ggaggcggta gtggcggtgg gtcggcggcc gcaatgcaga tcttcgtgaa gacccttacc 1560 ggcaagacca tcacccttga ggtggagccc agtgacacca tcgaaaatgt gaaggccaag 1620 atccaggata aggaaggcat tccccccgac cagcagaggc tcatctttgc aggcaagcag 1680 ctggaagatg gccgtactct ttctgactac aacatccaga aggagtcgac cctgcacctg 1740 gtcctccgtc tcagatga 1758 <210> 14 <211> 1758 <212> DNA <213> Artificial Sequence <400> 14 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggagcaga agctgatcag cgaggaggac ctggacatcc agatgacaca gactacatcc 120 tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 180 agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 240 catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 300 gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 360 cagggtaata cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc 420 ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg aggtgaaact gcaggagtca 480 ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc 540 tcattacccg actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg 600 ctgggagtaa tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg 660 accatcatca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 720 gatgacacag ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg 780 gactactggg gccaaggaac ctcagtcacc gtctcctcaa ccaccactcc cgcaccccgc 840 cctcctactc ctgcccctac cattgctagc caaccgctta gtctgagacc tgaggcctgt 900 aggcccgctg ctggtggcgc tgtgcacacc cgaggattgg acttcgcttg cgacatctac 960 atctgggcac ctctggctgg gacctgcggc gtgttgttgt tgagcctggt gattacgctg 1020 tactgtggat ccaggagtaa gaggagcagg ctcctgcaca gtgactacat gaacatgact 1080 ccccgccgcc ccgggcccac ccgcaagcat taccagccct atgccccacc acgcgacttc 1140 gcagcctatc gctccagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 1200 ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1260 gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1320 gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 1380 atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 1440 gccaccaagg acacctacga cgcccttcac atgcaggccc tgcctcctcg cggtggttca 1500 ggaggcggta gtggcggtgg gtcggcggcc gcaatgcaga tcttcgtgcg taccctgact 1560 ggtaggacca tcactctcga agtggagccg agtgacacca ttgagaatgt cagggcacgt 1620 atccaagaca gggaaggcat ccctcctgac cagcagaggt tgatctttgc tgggaggcag 1680 ctggaagatg gacgcaccct gtctgactac aacatccaga gagagtccac cctgcacctg 1740 gtcctccgtc tcagatga 1758 <210> 15 <211> 2280 <212> DNA <213> Artificial Sequence <400> 15 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgcgat 60 gttgtcatga ctcaaacccc tttatctttg cccgtatccc ttggtgacca ggcttcaatt 120 tcgtgtcgta gtagccaatc tctcgtgcat cgcaatggca acacatatct acactggtac 180 ctgcagaaac caggacaatc cccgaagtta ttgatccata aagtttcaaa tcgattttcg 240 ggggtccctg atcggttcag tggtagcggc tctggaacgg actttactct taagatatcc 300 agagtagaag ccgaggatct cggggtgtat ttctgctcac agtcgaccca cgttccccca 360 ctaacatttg gtgcaggcac gaaactggaa ttaaagggtg gcggtggctc gggcggtggt 420 gggtcgggtg gcggcggatc tgaagttcaa ttattgcagt ctggtcctga gcttgaaaaa 480 cccggcgctt ccgtcatgat ttcatgtaag gcctcggggaa gtagctttac tgggtataat 540 atgaactggg tacgtcaaaa tatcggtaaa tctctcgaat ggataggcgc aattgatcca 600 tactatggag ggacctccta caaccagaag ttcaaaggtc gcgcgacact aacggtggac 660 aagtcatcga gtactgctta tatgcatctg aaaagcttaa cctctgaaga ttccgccgtt 720 tactattgcg tctcaggcat ggagtactgg ggacaaggga catcggtaac ggtgagtagc 780 gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc acctgtggca 840 ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 900 cctgaggtca cgtgcgtggt ggtggacgtg agccacgaag accccgaggt ccagttcaac 960 tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccacgggga ggagcagttc 1020 caaagcacgt tccgtgtggt cagcgtcctc accgttgtgc accaggactg gctgaacggc 1080 aaggagtaca agtgcaaggt ctccaacaaa ggcctcccag cccccatcga gaaaaccatc 1140 tccaaaacca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat 1200 gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac 1260 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1320 gtgctggact ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg 1380 tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1440 1500 gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1560 aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1620 acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaga 1680 gtgaagttca gcaggagcgc agacgcccc gcgtaccagc agggccagaa ccagctctat 1740 aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1800 gaccctgaga tgggggaaa gccgagaagg aagaaccctc aggaagcct gtacaatgaa 1860 ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaag cgagcgccgg 1920 aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 1980 gacgcccttc acatgcaggc cctgccccct cgcggtggtt caggaggcgg tagtggcggt 2040 gggtcggcgg ccgcaatgca gatcttcgtg cgtaccctga ctggtaggac catcactctc 2100 gaagtggagc cgagtgacac cattgagaat gtcagggcac gtatccaaga cagggaaggc 2160 atccctcctg accagcagag gttgatcttt gctgggaggc agctggaaga tggacgcacc 2220 ctgtctgact acaacatcca gagagagtcc accctgcacc tggtcctccg tctcagatga 2280 <210> 16 <211> 1761 <212> DNA <213> Artificial Sequence <400> 16 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccggagcaga agctgatcag cgaggaggac ctggatgttg tgatgactca gtctccactc 120 tccctgcccg tcacccctgg agagccggcc tccatctcct gcagatctag tcagagcctt 180 gtacacagta atgccaacac ctatttacat tggtacctgc agaagccagg gcagtctcca 240 cagctcctga tctataaagt ttccaaccga ttttctgggg tccctgacag gttcagtggc 300 agtggatcag gcacagattt tacactgaaa atcagcagag tggaggctga ggatgttggg 360 gtttattact gctctcaaaa tacacatgtt cctcctacgt ttggccaggg gaccaagctg 420 gagatcaaac gtggtggagg cggttcaggc ggaggtggct ctggcggtgg cggatcgcag 480 gtgcagctgg tgcagtctgg agctgaggtg aagaagcctg gggcctcagt gaaggtctcc 540 tgcaaggctt ctggatacac cttcaccgac tatgaaatgc actgggtgcg acaggcccct 600 ggacaagggc ttgagtggat gggagctctt gatcctaaaa ctggtgatac tgcctacagt 660 cagaagttca agggcagagt cacgctgacc gcggacgaat ccacgagcac agcctacatg 720 gagctgagca gcctgagatc tgaggacacg gccgtgtatt actgtacaag attctactcc 780 tatacttact ggggccaggg aaccctggtc accgtctcct caaccaccac tcccgcaccc 840 cgccctccta ctcctgcccc taccattgct agccaaccgc ttagtctgag acctgaggcc 900 tgtaggcccg ctgctggtgg cgctgtgcac acccgaggat tggacttcgc ttgcgacatc 960 tacatctggg cacctctggc tgggacctgc ggcgtgttgt tgttgagcct ggtgattacg 1020 ctgtactgtg gatccaggag taagaggagc aggctcctgc acagtgacta catgaacatg 1080 actccccgcc gccccgggcc cacccgcaag cattaccagc cctatgcccc accacgcgac 1140 ttcgcagcct atcgctccag agtgaagttc agcaggagcg cagacgcccc cgcgtaccag 1200 cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1260 ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1320 caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1380 gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1440 acagccacca aggacaccta cgacgccctt cacatgcagg ccctgcctcc tcgcggtggt 1500 tcaggaggcg gtagtggcgg tgggtcggcg gccgcaatgc agatcttcgt gcgtaccctg 1560 actggtagga ccatcactct cgaagtggag ccgagtgaca ccattgagaa tgtcagggca 1620 cgtatccaag acagggaagg catccctcct gaccagcaga ggttgatctt tgctgggagg 1680 cagctggaag atggacgcac cctgtctgac tacaacatcc agagagagtc caccctgcac 1740 ctggtcctcc gtctcagatg a 1761 <210> 17 <211> 74 <212> PRT <213> Artificial Sequence <400> 17 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg 65 70 <210> 18 <211> 74 <212> PRT <213> Artificial Sequence <400> 18 Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp 20 25 30 Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg 65 70 <210> 19 <211> 671 <212> PRT <213> Artificial Sequence <400> 19 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu 35 40 45 Val His Arg Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 100 105 110 Ser Gln Ser Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys 115 120 125 Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys 145 150 155 160 Pro Gly Ala Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe 165 170 175 Thr Gly Tyr Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu 180 185 190 Glu Trp Ile Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn 195 200 205 Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 210 215 220 Thr Ala Tyr Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val 225 230 235 240 Tyr Tyr Cys Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val 245 250 255 Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 260 265 270 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro 275 280 285 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 290 295 300 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn 305 310 315 320 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 325 330 335 Glu Glu Gln Phe Gln Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 340 345 350 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 355 360 365 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 370 375 380 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 385 390 395 400 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 405 410 415 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 420 425 430 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 435 440 445 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 450 455 460 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 465 470 475 480 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Phe Trp Val Leu Val 485 490 495 Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala 500 505 510 Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser 515 520 525 Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His 530 535 540 Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg 545 550 555 560 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 565 570 575 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 580 585 590 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 595 600 605 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 610 615 620 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 625 630 635 640 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 645 650 655 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 660 665 670 <210> 20 <211> 489 <212> PRT <213> Artificial Sequence <400> 20 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 20 25 30 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 35 40 45 Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala 50 55 60 Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile 85 90 95 Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His 100 105 110 Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120 125 Arg Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly 130 135 140 Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 145 150 155 160 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp 165 170 175 Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 180 185 190 Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser 195 200 205 Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 210 215 220 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 225 230 235 240 Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Val Trp Gly 245 250 255 Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg 260 265 270 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 275 280 285 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 290 295 300 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 305 310 315 320 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser 325 330 335 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 340 345 350 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 355 360 365 Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser 370 375 380 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 385 390 395 400 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 405 410 415 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln 420 425 430 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 435 440 445 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 450 455 460 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 465 470 475 480 Leu His Met Gln Ala Leu Pro Pro Arg 485 <210> 21 <211> 671 <212> PRT <213> Artificial Sequence <400> 21 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Ser Arg Met Ala Gln Val Lys Leu Lys Glu Ser Gly Pro 20 25 30 Glu Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser 35 40 45 [[ID=2i]]Gly Tyr Thr Phe Thr Asp Tyr Ser Met His Trp Val Lys Lys Thr Pro 50 55 60 Gly Lys Gly Leu Lys Trp Leu Gly Trp Ile Asn Thr Ala Thr Gly Glu 65 70 75 80 Pro Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Ile Ser Leu Glu 85 90 95 Thr Ser Ala Arg Thr Val Tyr Leu Gln Ile Asn Asn Leu Arg Asn Glu 100 105 110 Asp Thr Ala Thr Tyr Phe Cys Phe Ser Tyr Tyr Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140 Note: There seems to be a typo in line 21 where it should be "Gly Tyr Thr Phe Thr Asp Tyr Ser Met His Trp Val Lys Lys Thr Pro" instead of "Gly Tyr Thr Phe Thr Asp Tyr Ser Met His Trp Val Lys Lys Thr Pro" in the original text. I've translated it as it is but just for your reference. Gly Ser Gly Gly Gly Gly Leu Asp Ile Lys Leu Thr Gln Ser Pro Ser 145 150 155 160 Ile Leu Ser Val Thr Pro Gly Glu Thr Val Ser Leu Ser Cys Arg Ala 165 170 175 Ser Gln Thr Ile Tyr Lys Asn Leu His Trp Tyr Gln Gln Lys Ser His 180 185 190 Arg Ser Pro Arg Leu Leu Ile Lys Tyr Gly Ser Asp Ser Ile Ser Gly 195 200 205 Ile Pro Ser Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu 210 215 220 Asn Ile Asn Ser Val Lys Pro Glu Asp Glu Gly Ile Tyr Tyr Cys Leu 225 230 235 240 Gln Gly Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu 245 250 255 Ile Lys Arg Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 260 265 270 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 275 280 285 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 290 295 300 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 305 310 315 320 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 325 330 335 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 340 345 350 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 355 360 365 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 370 375 380 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 385 390 395 400 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 405 410 415 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 420 425 430 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 435 440 445 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 450 455 460 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 465 470 475 480 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Phe Trp Val Leu Val 485 490 495 Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala 500 505 510 Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser 515 520 525 Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His 530 535 540 Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg 545 550 555 560 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 565 570 575 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 580 585 590 Val Leu Asp Lys Arg Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 595 600 605 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 610 615 620 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 625 630 635 640 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 645 650 655 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 660 665 670 <210> 22 <211> 491 <212> PRT <213> Artificial Sequence <400> 22 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 Lys Lys Pro Gly Glu Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Phe 35 40 45 Asn Ile Glu Asp Tyr Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys 50 55 60 Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys 65 70 75 80 Tyr Gly Pro Ile Phe Gln Gly His Val Thr Ile Ser Ala Asp Thr Ser 85 90 95 Ile Asn Thr Val Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr 100 105 110 Ala Met Tyr Tyr Cys Ala Phe Arg Gly Gly Val Tyr Trp Gly Gln Gly 115 120 125 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr 145 150 155 160 Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile 165 170 175 Asn Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr 180 185 190 Leu Asn Trp Leu Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile 195 200 205 Ser Leu Val Ser Lys Leu Asp Ser Gly Val Pro Asp Arg Phe Ser Gly 210 215 220 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 225 230 235 240 Glu Asp Val Ala Val Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Gly 245 250 255 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala 260 265 270 Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 275 280 285 Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 290 295 300 Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 305 310 315 320 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys 325 330 335 Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn 340 345 350 Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr 355 360 365 Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser 370 375 380 Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr 385 390 395 400 Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415 Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425 430 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu 435 440 445 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly 450 455 460 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr 465 470 475 480 Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 23 <211> 485 <212> PRT <213> Artificial Sequence <400> 23 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu 20 25 30 Glu Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys 50 55 60 Ser Leu Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser 65 70 75 80 Tyr Asn Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp 115 120 125 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr 145 150 155 160 Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met 165 170 175 Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln 180 185 190 Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu 195 200 205 Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser 210 215 220 Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Trp Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr 245 250 255 Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 260 265 270 Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 275 280 285 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 290 295 300 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu 305 310 315 320 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser Arg Ser Lys Arg 325 330 335 Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro 340 345 350 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 355 360 365 Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro 370 375 380 Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly 385 390 395 400 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro 405 410 415 Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr 420 425 430 Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 435 440 445 Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 450 455 460 Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 465 470 475 480 Ala Leu Pro Pro Arg 485 <210> 24 <211> 488 <212> PRT <213> Artificial Sequence <400> 24 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu 20 25 30 Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 35 40 45 Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln 50 55 60 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg 65 70 75 80 Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 100 105 110 Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 145 150 155 160 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln 180 185 190 Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala 195 200 205 Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser 210 215 220 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln 245 250 255 Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro 260 265 270 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 275 280 285 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser Arg 325 330 335 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 340 345 350 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 355 360 365 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <210> 25 <211> 497 <212> PRT <213> Artificial Sequence <400> 25 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Arg Leu Gln Gln Ser Gly Pro Asp Leu 20 25 30 Ile Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Thr Gly Tyr Val Met His Trp Val Lys Gln Arg Pro Gly Gln 50 55 60 Gly Leu Glu Trp Ile Gly Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln<000!194>65 70 75 80 Ser Asn Glu Arg Phe Arg Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser 85 90 95 Ser Thr Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys Ala Arg Gly Ala Gly Tyr Asn Phe Asp Gly Ala 115 120 125 It should be noted that there seems to be a small error in the original text where "Gly Leu Glu Trp Ile Gly Phe Ile Asn Pro Tyr Asn Asp Asp Ile Gln" has an extra "!" in the tag "<000!194>". It is likely a typo and has been corrected in the translation as " ". Tyr Arg Phe Phe Asp Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly 165 170 175 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Arg Leu Val His Ser 180 185 190 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 195 200 205 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Pro Gly Val Pro 210 215 220 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 225 230 235 240 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser 245 250 255 Thr His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 260 265 270 Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 275 280 285 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 290 295 300 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 305 310 315 320 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 325 330 335 Val Ile Thr Leu Tyr Cys Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu 340 345 350 His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg 355 360 365 Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg 370 375 380 Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 385 390 395 400 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 405 410 415 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 420 425 430 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 435 440 445 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 450 455 460 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 465 470 475 480 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 485 490 495 Arg <210> 26 <211> 493 <212> PRT <213> Artificial Sequence <400> 26 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu 20 25 30 Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Thr Phe Ser Asp Phe Glu Met His Trp Val Lys Gln Thr Pro Val His 50 55 60 Gly Leu Glu Trp Ile Gly Asp Ile Asp Pro Gly Thr Gly Asp Thr Ala 65 70 75 80 Tyr Asn Leu Lys Phe Lys Gly Lys Ala Thr Leu Thr Thr Asp Lys Ser 85 90 95 Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser 100 105 110 Ala Val Tyr Tyr Cys Thr Leu Gly Ala Phe Val Tyr Trp Gly Gln Gly 115 120 125 Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Lys Leu Glu Glu 130 135 140 Gly Glu Phe Ser Glu Ala Arg Val Asp Val Val Val Thr Gln Thr Pro 145 150 155 160 Leu Ser Leu Pro Val Ser Phe Gly Asp Gln Val Ser Ile Ser Cys Arg 165 170 175 Ser Ser Gln Ser Leu Ala Asn Ser Tyr Gly Asn Thr Tyr Leu Ser Trp 180 185 190 Tyr Leu His Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gly Ile 195 200 205 Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser 210 215 220 Gly Thr Asp Phe Thr Leu Lys Ile Ser Thr Ile Lys Pro Glu Asp Leu 225 230 235 240 Gly Met Tyr Tyr Cys Leu Gln Gly Thr His Gln Pro Tyr Thr Phe Gly 245 250 255 Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Thr Thr Thr 260 265 270 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 275 280 285 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 290 295 300 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 305 310 315 320 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 325 330 335 Tyr Cys Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr 340 345 350 Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln 355 360 365 Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys 370 375 380 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 385 390 395 400 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 405 410 415 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 420 425 430 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 435 440 445 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 450 455 460 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 465 470 475 480 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 27 <211> 492 [[ID=3,0]]<212> PRT <213> Artificial Sequence <400> 27 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 20 25 30 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 35 40 45 Ser Phe Pro Asp Tyr Tyr Ile Asn Trp Val Arg Gln Ala Pro Gly Gln 50 55 60 Gly Leu Glu Trp Met Gly Trp Ile Tyr Phe Ala Ser Gly Asn Ser Glu 65 70 75 80 Tyr Asn Gln Lys Phe Thr Gly Arg Val Thr Met Thr Arg Asp Thr Ser 85 90 95 Ile Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr 100 105 110 Ala Val Tyr Phe Cys Ala Ser Leu Tyr Asp Tyr Asp Trp Tyr Phe Asp 115 120 125 Val Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly 130 135 140 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr 145 150 155 160 Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile 165 170 175 Ser Cys Lys Ile Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr 180 185 190 Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu 195 200 205 Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser 210 215 220 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu 225 230 235 240 Ala Glu Asp Val Gly Ile Tyr Tyr Cys Ser Gln Ser Ser Ile Tyr Pro 245 250 255 Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro 260 265 270 Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285 Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300 Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 305 310 315 320 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr 325 330 335 Cys Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 340 345 350 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 355 360 365 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe 370 375 380 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 385 390 395 400 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 405 410 415 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 420 425 430 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 435 440 445 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 450 455 460 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 465 470 475 480 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 28 <211> 487 <212> PRT <213> Artificial Sequence <400> 28 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu 20 25 30 Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln 35 40 45 Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr 50 55 60 Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro 65 70 75 80 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile 85 90 95 Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly 100 105 110 Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu 130 135 140 Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser 145 150 155 160 Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly 165 170 175 Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly 180 185 190 Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 195 200 205 Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys 210 215 220 Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys 225 230 235 240 His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly 245 250 255 Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro 260 265 270 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 275 280 285 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 290 295 300 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 305 310 315 320 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser Arg Ser 325 330 335 Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg 340 345 350 Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg 355 360 365 Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 420 425 430 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 435 440 445 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 450 455 460 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 465 470 475 480 Met Gln Ala Leu Pro Pro Arg 485 <210> 29 <211> 585 <212> PRT <213> Artificial Sequence <400> 29 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp 20 25 30 Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp 35 40 45 Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu 50 55 60 Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr 65 70 75 80 His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 85 90 95 Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu 100 105 110 Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 115 120 125 Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser 145 150 155 160 Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr 165 170 175 Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln 180 185 190 Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu 195 200 205 Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys 210 215 220 Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr 225 230 235 240 Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly 245 250 255 Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 260 265 270 Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 275 280 285 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 290 295 300 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 305 310 315 320 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 325 330 335 Val Ile Thr Leu Tyr Cys Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu 340 345 350 His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg 355 360 365 Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg 370 375 380 Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 385 390 395 400 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 405 410 415 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 420 425 430 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 435 440 445 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 450 455 460 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 465 470 475 480 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 485 490 495 Arg Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ala Ala Met 500 505 510 Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val 515 520 525 Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys 530 535 540 Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln 545 550 555 560 Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser 565 570 575 Thr Leu His Leu Val Leu Arg Leu Arg 580 585 <210> 30 <211> 585 <212> PRT <213> Artificial Sequence <400> 30 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asp 20 25 30 Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp 35 40 45 Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu 50 55 60 Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr 65 70 75 80 His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 85 90 95 Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu 100 105 110 Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr 115 120 125 Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser 145 150 155 160 Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr 165 170 175 Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln 180 185 190 Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu 195 200 205 Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys 210 215 220 Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr 225 230 235 240 Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly 245 250 255 Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser 260 265 270 Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 275 280 285 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 290 295 300 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 305 310 315 320 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 325 330 335 Val Ile Thr Leu Tyr Cys Gly Ser Arg Ser Lys Arg Ser Arg Leu Leu 340 345 350 His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg 355 360 365 Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg 370 375 380 Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln 385 390 395 400 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu 405 410 415 Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly 420 425 430 Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 435 440 445 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 450 455 460 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 465 470 475 480 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 485 490 495 Arg Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ala Ala Met 500 505 510 Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu Val 515 520 525 Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp Arg 530 535 540 Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg Gln 545 550 555 560 Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu Ser 565 570 575 Thr Leu His Leu Val Leu Arg Leu Arg 580 585 <210> 31 <211> 759 <212> PRT <213> Artificial Sequence <400> 31 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val 20 25 30 Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu 35 40 45 Val His Arg Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro 50 55 60 Gly Gln Ser Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser 65 70 75 80 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 85 90 95 Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys 100 105 110 Ser Gln Ser Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys 115 120 125 Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Glu Val Gln Leu Leu Gln Ser Gly Pro Glu Leu Glu Lys 145 150 155 160 Pro Gly Ala Ser Val Met Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe 165 170 175 Thr Gly Tyr Asn Met Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu 180 185 190 Glu Trp Ile Gly Ala Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn 195 200 205 Gln Lys Phe Lys Gly Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 210 215 220 Thr Ala Tyr Met His Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val 225 230 235 240 Tyr Tyr Cys Val Ser Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val 245 250 255 Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 260 265 270 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro 275 280 285 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 290 295 300 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn 305 310 315 320 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 325 330 335 Glu Glu Gln Phe Gln Ser Thr Phe Arg Val Val Ser Val Leu Thr Val 340 345 350 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 355 360 365 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 370 375 380 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 385 390 395 400 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 405 410 415 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 420 425 430 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 435 440 445 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 450 455 460 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 465 470 475 480 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Phe Trp Val Leu Val 485 490 495 Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala 500 505 510 Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser 515 520 525 Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His 530 535 540 Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg 545 550 555 560 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 565 570 575 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 580 585 590 Val Leu Asp Lys Arg Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 595 600 605 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 610 615 620 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 625 630 635 640 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 645 650 655 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Gly 660 665 670 Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ala Ala Met Gln Ile 675 680 685 Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu Val Glu Pro 690 695 700 Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp Arg Glu Gly 705 710 715 720 Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg Gln Leu Glu 725 730 735 Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu Ser Thr Leu 740 745 750 His Leu Val Leu Arg Leu Arg 755 <210> 32 <211> 576 <212> PRT <213> Artificial Sequence <400> 32 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu 20 25 30 Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln 35 40 45 Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln 50 55 60 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg 65 70 75 80 Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr 100 105 110 Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr 115 120 125 Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 145 150 155 160 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 165 170 175 Thr Phe Thr Asp Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln 180 185 190 Gly Leu Glu Trp Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala 195 200 205 Tyr Ser Gln Lys Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser 210 215 220 Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 225 230 235 240 Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln 245 250 255 Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro 260 265 270 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 275 280 285 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Gly Ser Arg 325 330 335 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 340 345 350 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 355 360 365 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 370 375 380 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 385 390 395 400 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 405 410 415 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg Gly Gly Ser Gly Gly Gly Ser Gly 485 490 495 Gly Gly Ser Ala Ala Ala Met Gln Ile Phe Val Arg Thr Leu Thr Gly 500 505 510 Arg Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 515 520 525 Arg Ala Arg Ile Gln Asp Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg 530 535 540 Leu Ile Phe Ala Gly Arg Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 545 550 555 560 Tyr Asn Ile Gln Arg Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg 565 570 575

Claims

1. A method of engineering a chimeric antigen receptor, comprising, The modification method comprises coupling ubiquitin to the C-terminus of a chimeric antigen receptor, the chimeric antigen receptor comprising a transmembrane domain, an intracellular domain and an extracellular domain, the intracellular domain being coupled with ubiquitin at the C-terminus, the ubiquitin being selected from a mutant ubiquitin, the amino acid sequence of the mutant ubiquitin being as shown in SEQ ID NO. 18, the ubiquitin being coupled to the C-terminus of the chimeric antigen receptor through a linker peptide.

2. A ubiquitin-modified chimeric antigen receptor, characterized in that, The ubiquitin-modified chimeric antigen receptor comprises a transmembrane domain, an intracellular domain and an extracellular domain, the intracellular domain being coupled with ubiquitin at the C-terminus, the ubiquitin being selected from a mutant ubiquitin, the amino acid sequence of the mutant ubiquitin being as shown in SEQ ID NO. 18, the ubiquitin being coupled to the C-terminus of the chimeric antigen receptor through a linker peptide.

3. The ubiquitin-modified chimeric antigen receptor of claim 2, wherein, Any one or more of the following features are also included: 1) the transmembrane domain is selected from CD8a, CD28 or DAP 10; 2) the intracellular domain comprises a costimulatory domain and / or a signaling domain.

4. The ubiquitin-modified chimeric antigen receptor of claim 3, wherein, The intracellular domain is selected from one or a combination of several of 4-1BB, CD28, OX40, ICOS, CD3zeta or DAP 10.

5. The ubiquitin-modified chimeric antigen receptor of claim 2, wherein, The chimeric antigen receptor comprises, in sequence, a CD8a signal peptide, a protein purification tag, a single-chain antibody, a CD8a hinge to form an extracellular domain, a CD8a transmembrane domain, and an intracellular domain comprising CD28 and CD3zeta in series.

6. The ubiquitin-modified chimeric antigen receptor of claim 5, wherein, The single-chain antibody is selected from GD2 scFv, GPC3 scFv, Her2 scFv, CSPG4 scFv, EGFR scFv, Meso scFv, TRBC1 scFv, CD133 scFv, BCMA scFv or CD19 scFv.

7. An isolated polynucleotide, comprising: The isolated polynucleotide comprises nucleotides encoding the ubiquitin-modified chimeric antigen receptor of any one of claims 2-6.

8. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the polynucleotide of claim 7.

9. An immune cell, characterized in that, The immune cell comprises the nucleic acid construct of claim 8 or has integrated into its genome the exogenous polynucleotide of claim 7, or is capable of expressing the ubiquitin-modified chimeric antigen receptor of any one of claims 2-6.

10. The immune cell of claim 9, wherein, The immune cell is selected from a CAR-T cell, a CAR-NK cell, a CAR-macrophage cell or a CAR-TIL cell.

11. A method of preparing an immune cell according to any one of claims 9-10, characterized in that, The method of preparation comprises introducing the nucleic acid construct of claim 8 or the polynucleotide of claim 7 into an immune effector cell or a stem cell that produces an immune effector cell.

12. The method of claim 11, wherein, The immune effector cell is selected from a T cell, a NK cell, a macrophage cell or a TIL cell.

13. Use of the immune cell of any one of claims 9-10 in the preparation of a medicament for treating cancer or an immunological disease.

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