Artificial immune surveillance chimeric antigen receptor (AI-CAR) and cells expressing same

文档序号：491497 发布日期：2022-01-04 浏览：14次中文

阅读说明：本技术 人工免疫监视嵌合抗原受体(ai-car)及其表达细胞 (Artificial immune surveillance chimeric antigen receptor (AI-CAR) and cells expressing same ) 是由陆满晴唐纳德·E·斯汤顿约翰·M·哈兰于 2020-02-21 设计创作，主要内容包括：本申请提供了非病毒载体,其包含侧翼为两个转座子或病毒末端重复序列(IR)的人工免疫监视嵌合抗原受体(AI-CAR)表达盒,其中所述AI-CAR表达盒包含诱导型基因表达单元和CAR表达单元。(The present application provides a non-viral vector comprising an artificial immune surveillance chimeric antigen receptor (AI-CAR) expression cassette flanked by two transposons or viral terminal repeats (IR), wherein the AI-CAR expression cassette comprises an inducible gene expression unit and a CAR expression unit.)

1. A chimeric antigen receptor complex comprising:

a first protein comprising a first extracellular domain linked to a first intercellular domain by a first linker, wherein the first extracellular domain comprises a first scFv having affinity for a first tumor epitope, and wherein the first intercellular domain comprises a JAK1 binding domain, and

a second protein comprising a second extracellular domain linked to a second intercellular domain by a second linker, wherein the second extracellular domain comprises a second scFv having affinity for a second tumor epitope, and wherein the second intercellular domain comprises a JAK3 binding domain;

wherein the first tumor epitope is on a first tumor antigen, and wherein the second tumor epitope is on a second tumor antigen.

2. The chimeric antigen receptor complex according to claim 1, wherein the first intracellular domain comprises IL7R a (CD 127).

3. The chimeric antigen receptor complex of claim 1, wherein the first intracellular domain comprises an intracellular domain of IL15R β (CD122), IL21R a (CD360), or a combination thereof.

4. The chimeric antigen receptor complex according to claim 1, wherein the first cell further comprises within the first cell a first cytotoxic signaling domain linked to a JAK1 binding domain.

5. The chimeric antigen receptor complex of claim 4, wherein the first cytotoxic signaling domain comprises CD28, CD3 ζ, CD137, OX40, CD27, ICOS, or a combination thereof.

6. The chimeric antigen receptor complex of claim 1, wherein the first scFv domain or the second scFv domain independently has affinity for CD19 or CD 22.

7. The chimeric antigen receptor complex of claim 1, wherein the first scFv domain has affinity for CD 19.

8. The chimeric antigen receptor complex of claim 1, wherein the second scFv domain has affinity for CD 22.

9. The chimeric antigen receptor complex according to claim 1, wherein the second intracellular domain comprises γ (CD 132).

10. The chimeric antigen receptor complex according to claim 1, wherein the second intracellular domain further comprises a second cytotoxic signaling domain linked to a JAK3 binding domain.

11. The chimeric antigen receptor complex of claim 1, wherein the second cytotoxic domain comprises CD28, CD3 ζ, CD137, OX40, CD27, ICOS, or a combination thereof.

12. The chimeric antigen receptor complex of claim 1, wherein the second intracellular domain comprises γ (CD132), JAK3 binding domain, CD28, and CD3 ζ in tandem.

13. The chimeric antigen receptor complex of claim 1, wherein the first intracellular domain is configured to dimerize with the second intracellular domain.

14. The chimeric antigen receptor complex according to claim 1, wherein the first and second linkers independently comprise CD 8.

15. The chimeric antigen receptor complex of claim 1, wherein the first and second linkers independently comprise a stem and a transmembrane domain.

16. The chimeric antigen receptor complex of claim 13, wherein the stem comprises CD8, an Fc hinge, Fc CH2-CH3, TCR α, TCR β, truncated IL7R α (CD127), truncated IL15R β (CD122), IL15R α (CD215), truncated γ (CD132), truncated IL21R α (CD360), or a combination thereof.

17. The chimeric antigen receptor complex of claim 13, wherein the transmembrane domain comprises CD8, CD28, CD3 ζ, CD3 ε, CD3 δ, CD3 γ, CD3 ζ, TCR α, TCR β, IL15R β (CD122), γ (CD132), IL7R α (CD127), IL21R α (CD360), IL15R α (CD215), or a combination thereof.

18. The chimeric antigen receptor complex according to claim 1, wherein the tumor antigen comprises CDH17, TROP2, CD19, CD22, CD37, BCMA, CD48, EGFR, HER2, EpCAM, CEACAM5, PSMA, GD2, GPC3, or a combination thereof.

19. An Open Reading Frame (ORF) comprising, in order, a CD19 scFv, a stem transmembrane region, and an IL7 α endodomain.

20. An Open Reading Frame (ORF) comprising, in order, a CD22 scFv, a stem transmembrane region, a γ intrachain domain, a CD28 endodomain, and a CD3 zeta endodomain.

21. An Open Reading Frame (ORF) comprising, in order, PD-1scFv, CCL21, and IL 7.

22. A biomolecule complex, comprising:

a first tumor antigen, and

(ii) a second tumor antigen which is,

wherein the first tumor epitope binds to a first tumor antigen, wherein the second tumor epitope binds to the tumor antigen.

23. The biomolecule complex of claim 30, wherein the first intracellular domain is dimerized with the second intracellular domain.

24. The biomolecule complex of claim 30, wherein JAK1 dimerizes JAK 3.

25. A non-viral DNA construct comprising, in order, from 5 'to 3',

followed by an inducible promoter of a first ORF, wherein said first ORF comprises anti-PD-1 scFv, CLL21 and IL7, each leading with a single peptide and ending with a ribosome skipping peptide,

a second ORF comprising at least one constitutive chimeric antigen receptor, and

followed by a third promoter for at least one RNA sequence.

26. A chimeric antigen receptor comprising, in order:

a cytokine domain;

the joint is connected with the power supply device,

a truncated CD8 domain, and

a signaling endodomain.

27. The chimeric antigen receptor according to claim 28, wherein the cytokine domain comprises IL7, IL12, IL21, or a combination thereof.

28. The chimeric antigen receptor according to claim 28, wherein the truncated CD8 domain comprises at least a portion of a hinge, a transmembrane domain, and a cytoplasmic domain.

29. The chimeric antigen receptor of claim 30, wherein the cytoplasmic domain comprises CD28/CD170, CD3 ζ, or a combination thereof.

30. The chimeric antigen receptor according to claim 28, further comprising a tumor antigen domain intermediate the cytokine domain and the truncated CD8 domain.

31. A non-viral vector comprising an artificial immune surveillance chimeric antigen receptor (AI-CAR) expression cassette flanked by two transposons or viral terminal repeats (IR), wherein the AI-CAR expression cassette comprises an inducible gene expression unit and a CAR expression unit.

32. The non-viral vector of claim 33, wherein the inducible gene expression unit comprises a STAT, NFAT or NF- κ B inducible promoter, a coding region of one or more genes linked by an IRES or self-cleaving ribosomal skip peptide, followed by a first polyA signal sequence.

33. The non-viral vector of claim 34, wherein the self-cleaving ribosomal skip peptide comprises TA 2.

34. The non-viral vector of claim 33, wherein the inducible gene expression unit comprises genes for expressing at least two different cytokine receptors.

35. The non-viral vector of claim 33, wherein the inducible gene expression unit comprises a gene for expression of an antigen binding protein.

36. The non-viral vector of claim 33, wherein the inducible gene expression unit comprises a gene for expression of anti-PD 1 scFv.

37. The non-viral vector of claim 33, wherein the inducible gene expression unit comprises a gene for expressing CCL 21.

38. The non-viral vector of claim 33, wherein the inducible gene expression unit comprises a gene for expression of IL 7.

39. The non-viral vector of claim 33, wherein the CAR expression unit comprises genes for expression of anti-CDH 17 scFv, anti-TROP 2 scFv, and CAR.

40. The non-viral vector of claim 33, wherein the CAR expression unit comprises a promoter, one or two CAR genes, followed by a second polyA signal sequence.

41. The non-viral vector of claim 33, wherein the CAR expression unit further comprises a gene for expressing a safety switch.

42. The non-viral vector of claim 43, wherein the safety switch comprises truncated EGFR (tEGFR) or truncated CD 20.

43. The non-viral vector of claim 33, wherein the AI-CAR expression cassette is configured to express an shRNA, wherein the shRNA is configured to inhibit the endogenous TCR.

Technical Field

The present invention relates to a technique for improving the expansion, manufacture, survival and efficacy of Chimeric Antigen Receptor (CAR) -T cells or NK cells.

Background

Unless otherwise indicated herein, the materials described in this section are not prior art to the claims in this application and are not admitted to be prior art by inclusion in this section.

Manufacturing CAR cells is laborious and can be complicated by the need for artificial antigen presenting cells (aapcs), antibody stimulation of the TCR and co-stimulation of receptors and/or multiple cytokines to expand autologous or heterologous CAR cells prior to administration. For example, when electroporation conditions are set to high percentages of CAR expression, electroporation of T cells, NK cells, PBLs, or PBMCs with the DNA vector of the CAR typically results in cell death of a majority of the cells. Thus, after electroporation, the cells can be co-cultured with the irradiated aapcs, antibodies, and/or growth factors, and the CAR cell population can be specifically expanded multiple times to produce a single dose for therapeutic use. Currently, standard CAR vectors (CARs) do not express genes capable of inducing anti-tumor immune responses in patients that are typically required in immunosuppressive tumor microenvironments.

Ideally, the efficacy of CAR cells after administration should be correlated with T cells having an undifferentiated memory phenotype characterized by persistence in vivo and maximal therapeutic potential. To selectively expand CAR cells with this phenotype and/or prevent terminal differentiation, several cytokines have been utilized, including IL15, IL7, and IL 21. IL15 and IL7 are known to be critical for the generation and support of early memory T cells due to their ability to direct the generation of human memory stem cells from naive precursors (cirei et al, 2013; Boyman et al, 2012; Gattinoni L et al, 2011). IL15 and IL7 may be tools for de-differentiating T cells (e.g., human CD8+ memory T cell subpopulations) in response to antigens or homeostatic cytokines (Geginat 2003). IL15 is required for innate T cell immune surveillance (Dadi S et al, 2016). Soluble and transported IL15/IL-15 Ra was able to maintain IL-15 activity and contribute to the long-term survival of CD8 memory T cells (Sato et al, 2007). Thus, CART cells with an undifferentiated memory phenotype exhibit maximum in vivo persistence and therapeutic efficacy.

However, due to the safety assessment of each component, all components for manufacturing and maintaining such CAR cells with high therapeutic potential cannot be co-administered. However, constructing these components into CAR vectors with constitutive expression may not be a viable practice because of the high likelihood of toxicity over time. Alternatively, if the CAR cell expansion and signaling pathways of the undifferentiated memory phenotype are under the control of a single molecule (e.g., a target tumor antigen), then it would not only greatly reduce costs but also enable point-of-care therapy. In this way, CAR signaling can be sufficient to support expansion of CAR cells without terminal differentiation.

From a clinical perspective, a variety of factors lead to treatment relapse, including CAR cell persistence deficiency (depletion or host anti-CAR), loss of target antigen, lack of induction of host anti-tumor response, and/or inability to effectively localize to lymphoma/solid tumor. Almost all forms of CARs address the loss of tumor antigens addressed by targeting 2 or more tumor antigens. Therefore, it is highly desirable to have one or more CARs and a durable host response by including additional genes in the CAR vector/RNA.

Disclosure of Invention

In one aspect, the present application provides chimeric antigen receptor complexes. In one embodiment, the chimeric antigen receptor complex comprises a first protein comprising a first extracellular domain linked to a first intercellular domain by a first linker, wherein the first extracellular domain comprises a first scFv having affinity for a first tumor epitope, and wherein the first intercellular domain comprises a JAK1 binding domain, and a second protein comprising a second extracellular domain linked to a second intercellular domain by a second linker, wherein the second extracellular domain comprises a second scFv having affinity for a second tumor epitope, and wherein the second intercellular domain comprises a JAK3 binding domain. The first tumor epitope is on a first tumor antigen. The second tumor epitope is on a second tumor antigen.

In one embodiment, the first intracellular domain comprises IL7R a (CD 127). In one embodiment, the first intracellular domain comprises an intracellular domain of IL15R β (CD122), IL21R α (CD360), or a combination thereof. In one embodiment, the first cell further comprises within the first cell a first cytotoxic signaling domain linked to a JAK1 binding domain.

In one embodiment, the first cytotoxic signaling domain comprises CD28, CD3 ζ, CD137, OX40, CD27, ICOS, or a combination thereof.

In one embodiment, the first scFv domain or the second scFv domain independently has affinity for CD19 or CD 22. In one embodiment, the first scFv domain has affinity for CD 19. In one embodiment, the second scFV domain has affinity for CD 22. In one embodiment, the second intracellular domain comprises γ (CD 132).

In one embodiment, the second intracellular domain further comprises a second cytotoxic signaling domain linked to a JAK3 binding domain. In one embodiment, the second cytotoxic domain comprises CD28, CD3 ζ, CD137, OX40, CD27, ICOS, or a combination thereof. In one embodiment, the second intracellular domain comprises the tandem γ (CD132), JAK3 binding domain, CD28, and CD3 ζ. In one embodiment, the first intracellular domain is configured to dimerize with the second intracellular domain.

In one embodiment, the first and second linkers independently comprise CD 8. In one embodiment, the first and second linkers independently comprise a stem and a transmembrane domain.

In one embodiment, the stem comprises CD8, an Fc hinge, Fc CH2-CH3, TCR α, TCR β, truncated IL7R α (CD127), truncated IL15R β (CD122), IL15R α (CD215), truncated γ (CD132), truncated IL21R α (CD360), or a combination thereof.

In one embodiment, the transmembrane domain comprises CD8, CD28, CD3 ζ, CD3 ε, CD3 δ, CD3 γ, CD3 ζ, TCR α, TCR β, IL15R β (CD122), γ (CD132), IL7R α (CD127), IL21R α (CD360), IL15R α (CD215), or a combination thereof.

In one embodiment, the tumor antigen comprises CDH17, TROP2, CD19, CD22, CD37, BCMA, CD48, EGFR, HER2, EpCAM, CEACAM5, PSMA, GD2, GPC3, or a combination thereof.

In another aspect, the present application provides an Open Reading Frame (ORF). In one embodiment, the Open Reading Frame (ORF) comprises, in order, CD19 scFv, a stem transmembrane region, and an IL7 α endodomain. In one embodiment, the Open Reading Frame (ORF) comprises, in order, CD22 scFv, a stem transmembrane region, a γ chain endodomain, a CD28 endodomain, and a CD3 zeta endodomain. In one embodiment, the Open Reading Frame (ORF) comprises, in order, PD-1scFv, CCL21, and IL 7.

In another aspect, the present application provides a biomolecule complex. In one embodiment, the biomolecule complex comprises a first protein comprising a first extracellular domain linked to a first intercellular domain by a first linker, wherein the first extracellular domain comprises a first scFv having affinity for a first tumor epitope, and wherein the first intercellular domain comprises a JAK1 binding domain, a second protein comprising a second extracellular domain linked to a second intercellular domain by a second linker, wherein the second extracellular domain comprises a second scFv having affinity for a second tumor epitope, and wherein the second intercellular domain comprises a JAK3 domain, a first tumor antigen, and a second tumor antigen. The first tumor epitope binds a first tumor antigen. The second tumor epitope binds to the tumor antigen.

In one embodiment, the first intracellular domain dimerizes with the second intracellular domain. In one embodiment, JAK1 dimerizes JAK 3.

In another aspect, the present application provides a non-viral DNA construct. In one embodiment, the non-viral DNA construct comprises, in order, an inducible promoter from 5 'to 3', followed by a first ORF, wherein the first ORF comprises an anti-PD-1 scFV, CLL21, and IL7, each directed with a single peptide and ending with a ribosome skipping peptide, a second ORF comprising at least one constitutive chimeric antigen receptor, and a third promoter, followed by at least one RNA sequence.

In another aspect, the present application provides chimeric antigen receptors. In one embodiment, the chimeric antigen receptor comprises, in order, a cytokine domain, a linker, a truncated CD8 domain, and a signaling endodomain.

In one embodiment, the cytokine domain comprises IL7, IL12, IL21, or a combination thereof. In one embodiment, the truncated CD8 domain comprises at least a portion of the hinge, transmembrane domain, and cytoplasmic domain. In one embodiment, the cytoplasmic domain comprises CD28/CD170, CD3 zeta, or a combination thereof.

In one embodiment, the chimeric antigen receptor further comprises a tumor antigen domain intermediate the cytokine domain and the truncated CD8 domain.

In one embodiment, the present application provides a biomolecule complex comprising a chimeric antigen receptor as disclosed herein bound to a tumor antigen.

In another aspect, the present application provides a non-viral vector comprising an artificial immune surveillance chimeric antigen receptor (AI-CAR) expression cassette flanked by two transposons or viral terminal repeats (IR), wherein the AI-CAR expression cassette comprises an inducible gene expression unit and a CAR expression unit.

In one embodiment, the inducible gene expression unit comprises a STAT, NFAT or NF- κ B inducible promoter, a coding region of one or more genes linked by an IRES or self-cleaving ribosomal skip peptide, followed by a first polyA signal sequence. In one embodiment, the self-cleaving ribosome skipping peptide comprises TA 2. In one embodiment, the inducible gene expression unit comprises genes for expressing at least two different cytokine receptors. In one embodiment, the inducible gene expression unit comprises a gene for expressing an antigen binding protein. In one embodiment, the inducible gene expression unit comprises a gene for expression of anti-PD 1 scFv. In one embodiment, the inducible gene expression unit comprises a gene for expressing CCL 21. In one embodiment, the inducible gene expression unit comprises a gene for expression of IL 7.

In one embodiment, the CAR expression unit comprises genes for expressing the anti-CDH 17 scFv, the anti-TROP 2 scFv, and the CAR. In one embodiment, the CAR expression unit comprises a promoter, one or two CAR genes, followed by a second polyA signal sequence. In one embodiment, the CAR expression unit further comprises a gene for expressing a safety switch. In one embodiment, the safety switch comprises truncated egfr (tfegfr) or truncated CD 20. In one embodiment, the AI-CAR expression cassette is configured to express an shRNA, wherein the shRNA is configured to inhibit the endogenous TCR.

In another aspect, the present application provides isolated nucleic acids encoding the biomolecule complexes, biomolecules, antigens and proteins as disclosed herein.

In another aspect, the present application provides an expression vector comprising an isolated nucleic acid as disclosed herein. In one embodiment, the expression vector comprises an ORF as disclosed therein. In one embodiment, the expression vector comprises a non-viral DNA construct as disclosed herein. The expression vector may be viral or non-viral. The vector may be expressed in a cell.

In further embodiments, the present application provides a host cell. In one embodiment, the host cell comprises an isolated nucleic acid and/or an expression vector as disclosed herein. In one embodiment, the host cell comprises a non-viral DNA construct as disclosed herein. In one embodiment, the host cell comprises a non-viral vector as disclosed herein.

In another embodiment, the present application provides a mammalian cell comprising a chimeric antigen receptor complex, a biomolecule, an antigen and a protein as disclosed herein. In one embodiment, the mammalian cell comprises a chimeric antigen receptor as disclosed herein. In one embodiment, the mammalian cell comprises a biomolecule complex as disclosed herein.

In further embodiments, the application provides CAR-T or CAR-NK cells. In one embodiment, the CAR-T or CAR-NK cell expresses a chimeric antigen receptor complex as disclosed herein. In one embodiment, the CAR-T or CAR-NK cell expresses a chimeric antigen receptor as disclosed herein.

In another embodiment, the application provides a method for treating a tumor in a subject comprising administering to the subject a sufficient amount of a CAR-T or CAR-NK cell as disclosed herein.

In another aspect, the present application provides a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a vector, non-viral vector, CAR-T or CAR-NK cell, protein, biomolecule, or biomolecule complex as disclosed herein. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle.

Drawings

The foregoing and other features of the present disclosure will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. Understanding that these drawings depict only several embodiments arranged in accordance with the disclosure and are not therefore to be considered to be limiting of its scope, the disclosure will be described with additional specificity and detail through use of the accompanying drawings in which:

FIG. 1 depicts an AI-CAR gene expression cassette comprising an inducible gene expression unit and a CAR expression unit in a non-viral vector (pPI) for constitutive expression of one or two CARs to induce gene expression for a host anti-tumor response;

FIG. 2 shows the general concept of AI-CAR;

fig. 3 depicts an AI-CAR expression vector encoding a constitutively expressed dual CAR targeting CDH17 and TROP2 and the anti-PD 1scFv, CCL21, and IL17 gene cassettes under an inducible promoter in a non-viral vector (pPI) for CAR-induced host anti-tumor response; the AI-CAR expression vector encodes a constitutively expressed dual CAR that targets CDH17 and TROP2 and the anti-PD 1scFv, CCL21, and IL17 gene cassettes under an inducible promoter in a non-viral vector (pPI) for CAR-induced host anti-tumor response;

FIG. 4 shows tumor antigen induction of the integrated pPI anti-CDH 17 AI-CAR vector gene; tumor antigen induction of the integrated pPI-anti-CDH 17-AI-CAR vector gene. (A) Measuring the expression of the integrated ppii-anti-CDH 17-AI-CAR vector by GFP levels in T cells (Jurkat) in response to different concentrations of CDH 17; (B) induction with recombinant CDH17 in colon cancer cells (SW 480); and (C) cytotoxicity of ppii-anti-CDH 17-AI-CAR integrated T cells against SW480 cells expressing CDH 17;

FIG. 5 shows the expression and binding specificity of pPI-anti-CDH 17-TROP2 AI-CAR; and is

Fig. 6 depicts the iPro variant supporting AI-CAR proliferation and persistence. iPro variants that support AI-CAR proliferation and persistence. (A) Examples of iPro7 expression; (B) induction of proliferation of CD 25T cell population; and (C) increasing T cell survival.

Detailed Description

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like reference numerals generally identify like components unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

The present disclosure provides, inter alia, isolated antibodies, methods of making such antibodies, bispecific or multispecific molecules, antibody-drug conjugates and/or immunoconjugates comprised of such antibodies or antigen-binding fragments, pharmaceutical compositions containing the antibodies, bispecific or multispecific molecules, antibody-drug conjugates and/or immunoconjugates, methods of making the molecules and compositions, and methods of treating cancer using the molecules and compositions disclosed herein.

The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist and antagonist antibodies), antibody compositions having polyepitopic specificity, and antibody fragments (e.g., Fab, F (ab')₂And Fv) as long as they exhibit the desired biological activity. In some embodiments, the antibody can be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a single chain antibody, a bispecific or diabody, a mimobody, a human antibody, and a humanized antibody and active fragments thereofAnd (4) section. Examples of active fragments of molecules that bind to known antigens include Fab, F (ab')₂scFv and Fv fragments, including products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the above antibodies and fragments. In some embodiments, antibodies may include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain a binding site that immunospecifically binds to an antigen. The immunoglobulin may be of any class (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules. In one embodiment, the antibody can be a whole antibody and any antigen-binding fragment derived from a whole antibody. A typical antibody refers to a heterotetrameric protein that typically comprises two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated VH) and a heavy chain constant domain. Each light chain consists of a light chain variable domain (abbreviated VL) and a light chain constant domain. The VH and VL regions can be further subdivided into domains of hypervariable Complementarity Determining Regions (CDRs) and more conserved regions known as Framework Regions (FRs). Each variable domain (VH or VL) is typically composed of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxy terminus. Within the variable domains of the light and heavy chains are binding regions that interact with antigens.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are synthesized by hybridoma cultures and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present disclosure can be prepared by the hybridoma method first described by Kohler & Milstein, Nature, 256:495(1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

Monoclonal antibodies may include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851-6855[1984 ]).

Monoclonal antibodies can be produced using a variety of methods, including mouse hybridomas or phage display (see review by siegel. transfus. clin. biol.9:15-22 (2002)) or molecular cloning of antibodies directly from primary B cells (see tiller. new biotechnol.28:453-7 (2011)). In the present disclosure, antibodies are generated by immunizing rabbits with human PD-L1 protein and cells that transiently express human PD-L1 on the cell surface. Rabbits are known to produce antibodies of high affinity, diversity and specificity (Weber et al, exp. mol. med.49: e 305). B cells from immunized animals were cultured in vitro and screened for production of anti-PD-L1 antibodies. Antibody variable genes are isolated using recombinant DNA techniques and the resulting antibodies are recombinantly expressed and further screened for desirable characteristics, such as the ability to inhibit PD-L1 binding to PD-1, ability to bind to non-primate PD-L1, and ability to enhance human T cell activation. The general method of discovery of this antibody is similar to that described by Seeber et al, PLOS one.9: e86184 (2014).

The term "antigen or epitope binding portion or fragment" refers to an antibody fragment that is capable of binding an antigen (in this case CD 19). These fragments may have antigen binding functions as well as other functions of the intact antibody. Examples of binding fragments include, but are not limited to, single chain Fv fragments (scFv) consisting of VL and VH domains of a single arm of an antibody linked in a single polypeptide chain by a synthetic linker, or Fab fragments, which are monovalent fragments consisting of VL, constant light Chain (CL), VH, and constant heavy chain 1(CH1) domains. Antibody fragments may be even smaller subfragments and may consist of domains as small as single CDR domains, in particular CDR3 regions from VL and/or VH domains (see e.g. Beiboer et al, j.mol.biol.296:833-49(2000)) using conventional methods known to those skilled in the art to generate antibody fragments. Antibody fragments can be screened for utility using the same techniques used for intact antibodies.

An "antigen-or epitope-binding fragment" can be derived from an antibody of the present disclosure by a number of techniques known in the art. For example, purified monoclonal antibodies can be cleaved with enzymes such as pepsin and subjected to HPLC gel filtration. The appropriate fraction containing the Fab fragment is then collected and concentrated by membrane filtration or the like. For a further description of the general techniques for isolating active fragments of antibodies, see, e.g., Khaw, B.A. et al, J.Nucl.Med.23:1011-1019 (1982); rousseaux et al, Methods Enzymology, 121:663-69, Academic Press, 1986.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produces F (ab') which has two antigen binding sites and is still capable of cross-linking the antigen₂And (3) fragment.

The Fab fragment may contain the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is understood herein to mean Fab' in which the cysteine residues of the constant domains carry a free thiol group. F (ab')₂Antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Chemical coupling of other antibody fragments is also known.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. This region consists of dimers of one heavy and one light chain variable domain in close, non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. The 6 CDRs collectively confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

The "light chain" of an antibody (immunoglobulin) from any vertebrate species can be assigned to one of two distinctly different types, termed kappa and lambda, based on the amino acid sequence of its constant domain.

Immunoglobulins can be classified into different classes according to the amino acid sequence of the constant domain of the heavy chain of the immunoglobulin. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

"humanized antibodies" refers to a class of engineered antibodies in which the CDRs are derived from a non-human donor immunoglobulin and the remaining immunoglobulin-derived portions of the molecule are derived from one (or more) human immunoglobulins. In addition, framework support residues can be altered to maintain binding affinity. Methods for obtaining "humanized antibodies" are well known to those skilled in the art. (see, e.g., Queen et al, Proc. Natl Acad Sci USA, 86: 10029-.

As used herein, the terms "polypeptide," "peptide," and "protein" are interchangeable and defined to mean a biomolecule composed of amino acids linked by peptide bonds.

The terms "a", "an", and "the" as used herein are defined to mean "one or more" and include the plural unless the context is not appropriate.

"isolated" refers to a biomolecule that is free of at least some of its naturally occurring components. "isolated" when used to describe various polypeptides disclosed herein means a polypeptide that has been identified and isolated and/or recovered from the cell or cell culture in which it is expressed. Typically, an isolated polypeptide will be prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen binding specificities.

By "recombinant" is meant the production of an antibody in a foreign host cell using recombinant nucleic acid techniques.

The term "antigen" refers to an entity or fragment thereof that can induce an immune response in an organism, particularly an animal, more particularly a mammal (including a human). The term includes immunogens and regions thereof that are responsible for antigenicity or antigenic determinants.

Furthermore, as used herein, the term "immunogenic" refers to a substance that elicits or enhances the production of antibodies, T-cells, or other reactive immune cells to an immunogenic agent and contributes to an immune response in a human or animal. An immune response occurs when an individual produces sufficient antibodies, T-cells, and other reactive immune cells against an administered immunogenic composition of the present disclosure to alleviate or alleviate the condition to be treated.

"specific binding" or "specifically binds to" or "specific for" a particular antigen or epitope refers to binding that is measurably distinct from non-specific interactions. Specific binding can be measured, for example, by determining the binding of the molecule to a control molecule, which is typically a similarly structured molecule that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

For example, specific binding to a particular antigen or epitope can be by having at least about 10 for the antigen or epitope^-4M, at least about 10^-5M, at least about 10^-6M, at least about 10^-7M, at least about 10^-8M, at least about 10^-9M, or at least about 10^-10M, at least about 10^-11M, at least about 10^-12M or greaterKD, where KD refers to the off-rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds an antigen will have a KD for a control molecule that is 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000-, or more fold relative to the antigen or epitope.

In addition, specific binding to a particular antigen or epitope can be exhibited, for example, by an antibody to the antigen or epitope having at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000-, or more times KA or KA relative to a control for the epitope, where KA or KA refers to the binding rate of a particular antibody-antigen interaction.

The "homology" between two sequences is determined by sequence identity. If the two sequences to be compared differ in length, sequence identity preferably refers to the percentage of nucleotide residues of the shorter sequence that are identical to the nucleotide residues of the longer sequence. Sequence identity can be routinely determined using computer programs. Deviations that occur in comparisons between a given sequence and the above-described sequences of the present disclosure can result from, for example, additions, deletions, substitutions, insertions, or recombinations.

The disclosure may be understood more readily by reference to the following detailed description of specific embodiments and the examples included herein. While the present disclosure has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the disclosure.

In previous disclosures, CAR depletion was addressed by cytokine signaling pathways to drive expansion without terminal differentiation. The compositions and methods of use disclosed herein relate to AI-CAR vectors for artificial immune monitoring of chimeric antigen receptors. Advances in this AI-CAR technology are intended to replace standard CAR manufacturing and achieve effective "combination" and point-of-care treatment. While other CAR technologies may require multiple administrations of soluble cytokine growth factor expression and/or sustained activity to generate a complete and sustained response, AI-CAR vectors incorporate these activities and are capable of generating effective and sustained CAR cells in the absence of multiple administrations of CAR that proliferate, constitutively active repellent or sustained anti-tumor response.

Second, AI-CAR signaling increases the efficiency of CAR cell production while maintaining the same function as standard CARs. AI-CARs may require only one target antigen in vitro and in vivo to achieve full proliferation and cytotoxic activity. Herein, AI-CARs can significantly reduce manufacturing costs as the expansion of standard CAR-T cells typically requires the use of a combination of growth factors and aapcs for manufacturing.

Third, expression of several anti-tumor genes encoded by the integrated AI-CAR vector can be induced after AI-CAR conjugation of tumor cells in vivo. Expression of these endogenous genes can lead to patients developing anti-tumor responses that more broadly target different tumor antigens, such as neoantigens. For example, the STAT5 reporter system was used to induce STAT5 responsive genes in human T cells (Kanai et al, 2014; Zeng et al, 2016; Bednorz et al, 2011; and Fang et al, 2008). This feature is unique in that standard CAR constructs are generally unable to induce gene expression. Along with these cofactors, AI-CARs will become a platform technology that provides a practical, economical and effective solution for point-of-care treatment of cancer. Many forms of cancer may exist in the context of immunosuppressed tumors. AI-CARs would be highly desirable because AI-CAR vectors are designed to express additional anti-cancer genes that can reduce tumor immunosuppression and activate the patient's anti-tumor immune response. One of the unique features of AI-CARs is their ability to modulate the expression of the relevant anti-tumor genes at the tumor site, rather than to make them constitutively expressed, which may be toxic. Another feature is that the AI-CAR is designed to have a single dose when administered, followed by its long-term activity and greater efficacy. With these advantageous features, AI-CARs are a better solution to the unmet challenge in the market, which is predictive of efficacy in treating most, if not all, types of cancer.

With respect to CAR therapy for the treatment of hematologic cancers, targets include CD19 and CD22, CD20, CD9, CD38, which can be targeted by dual bispecific AI-CARs using non-viral DNA vectors or RNA-CARs (transient). In this way, the AI-CAR can be used as a transient therapeutic bridge for transplantation or more persistence. In addition, AI-CARs can be used as point-of-care therapies to induce host anti-tumor immune responses against neoantigens.

In addition, for certain solid tumors, an RNA encoding an AI-CAR combination therapy may be applied. For aggressive tumors that fail other therapies, ready-made RNA CARs can be efficiently and rapidly manufactured due to the high electroporation efficiency of RNA. Multiple anti-tumor mechanisms, such as induced AI-CAR vector genes, transiently expressed by RNA AI-CARs should enable more effective and safer anti-tumor activity and potentially induce immune responses in patients. Multiple dose RNA AI-CARs can be used as a bridge to determine efficacy prior to treatment with certain persistent AI-CAR cells.

Examples

Example 1 construction of AI-CAR vectors for continuous and combination therapy

The aim of AI-CAR cells is to improve the efficacy of cancer immunotherapy by sustained, long-term immune surveillance in a quiescent state until stimulated by tumor cells. After tumor stimulation, the inducible AI-CAR gene enables locally safe and more effective "combination" therapy involving other mechanisms of anti-tumor activity. For a higher frequency of complete and sustained response, it is expected to stimulate the patient's anti-tumor response.

Typically, AI-CAR constructs consist of a non-viral vector (pPI) such as the AI-CAR expression cassette in transposon-based integration systems (Ivics and Izsv k, 2010; Z Cooper et al, US9,629,877; Uckert et al, US20190071484A 1). As shown in fig. 1, the AI-CAR vector comprises an inducible gene expression unit and a CAR expression unit, i.e., an AI-CAR expression cassette flanked by transposon terminal Inverted Repeats (IR). AI-CAR expression cassettes can be constructed with STAT, NFAT or NF-. kappa.B inducible promoters by IRES or self-cleaving ribosomal skip peptides such as P2A or the coding region of one or more genes linked by T2A (e.g., SEQ ID 18-21), followed by a polyA signal sequence. This may be followed by another promoter for constitutive expression of one or both CARs, which is followed by another polyA signal. See SEQ ID1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10 for examples of AI-CAR chain pairs. The two coding regions may be located between two transposons or viral terminal repeats (IR) for integration. Alternatively, the coding region of the AI-CAR construct can be integrated at a specific genomic site (Eyquem2017) using zinc fingers, TALENs, or CRISPR/Cas9 nucleases.

As shown in figure 2, constitutive expression of one or two CARs on the surface of T cells binds to tumor associated antigens at the tumor. The AI-CAR induces expression of inducible genes, achieving a safe and multiple mechanism of anti-tumor activity and efficient stimulation of the host anti-tumor response. Ideally, expression of these genes will be induced upon engagement of the AI-CAR with a tumor antigen or an antigen within the Tumor Microenvironment (TME). To extend the repertoire of these genes, a second AI-CAR chain can be expressed from a second vector.

The composition of AI-CAR non-viral vectors has at least some flexibility, e.g., piggyBac, Tol2, and Sleeping Beauty (Ivics and Izsv a k, 2010). Additionally, AI-CARs may express a safety switch, such as truncated egfr (tfegfr), which may be targeted for elimination by the FDA-approved antibody cetuximab. Alternatively, the safe target may be truncated CD20, which may be targeted by rituximab for CAR cell elimination. The AI-CAR vector can express shRNA that, for example, inhibits endogenous TCRs to enable generation of universal AI-CAR cells.

As an alternative to an inducible promoter, the AI-CAR may include a weak promoter, such as a modified PGK promoter, to safely express other anti-tumor activity mechanisms.

Example 2 Single and Dual AI-CAR design

Single or dual AI-CARs were designed to signal through cytokine receptor pathways to achieve greater CAR cell persistence and to induce vector-encoded genes for additional anti-tumor mechanisms and enhanced efficacy. Furthermore, simple conjugation of AI-CARs may promote efficient AI-CAR amplification and may be used in manufacturing to simplify in vitro amplification prior to administration.

Dual AI-CARs were constructed with the in vivo domain of IL12, IL7, IL21, or IL15 cytokine receptors to enable persistence and induction of the vector gene for additional anti-tumor mechanisms. As listed in table 2, the cytokine endodomain is fused to one or more TCR or TCR costimulatory cytoplasmic regions (also referred to as costimulatory domains), such as CD3 ζ, CD28, CD137, CD27, OX40, and ICOS. For example, a dual AIL-CAR can be constructed with one CAR consisting of a tumor antigen-specific scFv fused to the stem and transmembrane domain as well as to the intracellular (endo) domain of IL12 β 1 chain and a fragment of CD3 ζ. The second CAR may consist of a tumor antigen-specific scFv fused to the stem and transmembrane domains, a fragment of the common β 2 chain endodomain and a CD137 costimulatory endodomain.

AI-CAR can induce gene expression by transcription factors such as STAT 4. The IL12 β 1 chain has a Ty2k binding site, while the β 2 chain has a JAK2 binding site. Binding of the two chains can be stabilized by binding to two different but adjacent epitopes that are adjacent to the target antigen or to the same tumor target antigen. When the CAR binds to tumor antigens, Ty2k and JAK2 will phosphorylate the beta chain and eventually STAT4, which STAT4 then dimerizes and translocates to the nucleus. STAT4 may then bind to promoter Transcription Factor (TF) responsive elements and facilitate induction of gene expression. Promoters and downstream genes that are regulated by STAT4 can be incorporated into CAR transposons or viral vectors. Once integrated, these genes can be induced after the CAR cells engage the tumor or TME antigen. STAT 4-induced endogenous genes also supported CAR cell persistence (DeRenzo 2019).

Most dual AI-CAR components are independent functional units, i.e., anti-tumor scFv, stem, transmembrane and endodomain, as well as different fragments of IL15, IL7 and IL21 chain, CD3 ζ and costimulatory proteins, and are replaced for many specific purposes. As examples shown in tables 1 and 2, there may be many combinations for making a particular AI-CAR.

The monospecific AI-CAR can be constructed such that CAR cell persistence is supported by an inducible gene encoded within the CAR vector. The AI-CAR may consist of an scFv, stem, transmembrane domain and costimulatory CD137 and CD3 zeta endodomains that target tumor or TME-associated antigens. Upon engagement of NFAT with tumor antigen activity, NFAT will be produced that binds to a response element within the integrated CAR vector that induces expression of one or more genes that support persistence (e.g., IL15, IL12, and IL7) as well as other mechanisms of anti-tumor activity.

Table 2 shows additional genes encoding proteins or mirnas with persistent or anti-tumor activity, such as anti-Immune Checkpoint Inhibitors (ICI), OX40 agonists, TLR agonists, cytokines, bispecific antibodies, ipros, chemokines and chemokine receptors, which can be placed under the control of inducible promoters. Alternatively, mrnas encoding these proteins (e.g., SEQ ID18-20) can be co-transfected with AI-CAR vectors for transient expression. Alternatively, mRNAs encoding the CAR and these proteins can be used as transient, safe therapeutic AI-CAR (SEQ ID 11-17). Several doses of AI-CARs transiently expressing these genes may be sufficient to reduce immunosuppression in the tumor microenvironment and activate the patient's anti-tumor immune response.

Example 3 encoding bispecific anti-CDH 17 and anti-TROP 2 AI-CAR of CAR and use for persistence and enhancement Of an antitumor response

As shown in fig. 3, AI-CAR vectors can be constructed with NFAT inducible promoters, coding regions for one or more genes such as anti-PD-1, CCL21, and IL7 linked by ribosomal skipping peptides such as T2A followed by polyA signal sequences. This may be followed by the promoter of a single bispecific CAR targeting CDH17 and TROP2 with CD137 and CD3 zeta endodomains, followed by T2A, signal peptide, tfegfr and polyA signals. The two coding regions may be located between transposons or viral terminal repeats (IR) for integration. Alternatively, the AI-CAR expression cassette can be integrated at a specific genomic site using TALEN or CRISPR/Cas9(Eyquem 2017).

Example 4 AI-CAR Gene Induction of tumor target antigens

Recombinant and cellular tumor target antigens are used to induce the AI-CAR vector gene upon integration into a T cell line. The pPI-anti-CDH 17-AI-CAR construct expressing GFP under the control of an NFAT-inducible promoter was electroporated into the Jurkat T cell line. The pBac transposon construct was co-electroporated with a transposase expression vector for AI-CAR vector integration. The T cell line with integrated pPI anti-CDH 17 AI-CAR vector (Jurkat) was incubated (37 ℃, 5% CO2) for 2 or 14 hours in microtiter wells coated with 0, 1.25, 2.5, 5, 10 or 20 μ g/ml CDH 17-Fc. The pPI anti-CDH 17 AI-CAR vector contains GFP regulated by the NFAT-inducible promoter. At 14 hours, GFP expression levels were determined by flow cytofluorimetry. The level of GFP expression was relative to the level induced maximally at 14 h of treatment with the immune cultures (anti-CD 3, CD28 and CD 2; stem cells). CDH17 was exposed for only 2 hours and low levels of GFP were detected by flow cytometry as shown in figure 4A. After 14 hours, GFP increased to 70% in a concentration-dependent manner after CDH17 exposure relative to the immune cultures. There was an acceptable level of GFP in unstimulated cells. CDH17 was expressed at different levels in SW480 cells by electroporation with 0, 1.25, 5, 10 or 20 μ g CDH17 RNA (per 10^7 cells). The expression of CDH17 was determined by standard flow cytofluorimetry, as shown in figure 4B. Jurkat line integrated with the pPI anti-CDH 17 AI-CAR vector was incubated with a monolayer of SW480 expressing different levels of CDH17 for 2 or 14 hours. At 14 hours, GFP expression levels were determined by flow cytofluorimetry. GFP expression levels were relative to the maximum induction level at 14 hours of immune culture. As shown in fig. 4C, similar levels of GFP induction (50-80%) were detected after exposure of AI-CAR to Sw480 expressing CDH 172 or 14 hours. Thus, tumor cells expressing the target antigen appear to efficiently induce AI-CAR vector gene expression. Thus, the AI-CAR constructs can be used to express proteins with anti-tumor activity at the tumor site to safely and enhance tumor killing.

Example 5 expression of Dual AI-CAR

The expression and binding activity of the double-stranded dual AI-CAR is shown. Dual AI-CARs were expressed in CHO cells, one with the IL15 β and CD28 endodomain, and the other with the IL15 γ and CD3 ζ endodomain (SEQ ID1 and 2). And as shown in figure 5, expression of single strands, either pSh3C15b28 and pSh3A4C15g3, or both strands, was determined by staining with biotinylated protein-L (for scFv). Binding to tumor antigens, CDH17-Fc or TROP2-Fc was also determined by flow cytometry. Expression and tumor antigen binding were determined using streptavidin-phycoerythrin (for protein-L) and anti-human IgG-Alexa647 (for CDH17 and TROP 2). These results indicate that the transfection, expression, and ligand binding functions of a dual CAR can be easily achieved.

Example 6 iPro design and expression to support proliferation and persistence of AI-CAR

Proliferation inducers (iPro) can be induced in a single AI-CAR to support proliferation and persistence. iPro can be constructed with an N-terminal cytokine or scFv that binds to the TME antigen, followed by a linker, stem, transmembrane domain, and an internal domain with JAK family and STAT binding sites, as shown in figure 6A. Upon engagement of the N-terminal domain with a cytokine receptor or tumor antigen, iPro can be signaled by STAT to induce expression of T or NK cell proteins that supportAnd survival and maintenance of the Tcm stem phenotype. Having an N-terminal cytokine domain, e.g., IL7, IL15, or IL 12. iPro can support bi-directional inside-out and outside-in stimulation, which activates patient T cells and NK cells in addition to AI-CAR cells. In addition to enhancing AI-CAR activity in vivo, expression of iPro can facilitate intron amplification of AI-CAR for manufacturing.

iPro (iPro 7; SEQ ID 19) with N-terminal IL7 transiently expressed in PBMC by electroporated mRNA transcribed in vitro was detected by flow cytometry at 24 hours instead of 48 hours (FIG. 6B, left panel). On day 2, inducible expression of IL2 receptor CD25 was demonstrated by flow cytometry (fig. 6C, middle panel). Without further stimulation, T cell counts remained stable over 10 days, whereas mock control T cell counts were significantly reduced (fig. 6D, right panel). These results indicate that variants of AI-CAR inducing genes (e.g., iPro) can be designed to support AI-CAR proliferation and persistence.

Reference to the literature

Rodríguez-Galán A,Fernández-Messina L,Sánchez-Madrid F.Control of Immunoregulatory Molecules by miRNAs in T Cell Activation.Front Immunol.2018Sep 25；9:2148.

Lykken EA,Li QJ.The MicroRNA miR-191Supports T Cell Survival Following CommonγChain Signaling.J Biol Chem.2016Nov 4；291(45):23532-23544.Epub 2016Sep 15.

Ivics,Z.,Izsvák,Z.The expanding universe of transposon technologies for gene and cell engineering.Mobile DNA 1,25(2010).

Cooper Lj,Torikai H,Zhang L,Huls H,Wang-Johanning F,Hurton L,Olivares S,Krishnamurthy J.Human application of engineered chimeric antigen receptor(CAR)T-cells.US9629877B2.Uckert W,Bunse M,Clauss J,Izsvák Z.A transposon-based transfection system for primary cells US20190071484A1.

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Table 1 lists examples of AI-CAR compositions. AI-CARs can be constructed using fragments of different genes encoding different functional fragments, including anti-Tumor Associated Antigen (TAA) scFv, stem, transmembrane domain, and endodomain. Different kinds of endodomains can play a role, for example, in signaling cytokine receptor proliferation and survival responses or tumor cytotoxic responses. The cytokine receptor, CD3 ζ and costimulatory endodomain may be fused in various tandem arrangements.

Table 2 lists examples of inducible genes that can be incorporated into AI-CAR vectors. These genes can be selected to enhance CAR localization to tumors (e.g., lymphoma), reverse tumor immunosuppression and stimulate host immune response or for direct anti-tumor cell activity. The gene can be placed downstream of the STAT5 inducible promoter to avoid any toxicity that may occur with long-term constitutive expression. Alternatively, certain chemokine and chemokine receptor genes and cytokines, such as IL7, may be located downstream of a weak promoter. Low levels of expression may avoid toxicity.

Sequence listing

SEQ ID NO:1

TROP 2-specific AI-CAR (pSh3A4C15g3)

MEFGLSWVFLVALLRGVQCQVQLVQSGAEVKKPGASVKVSCKASGYTFTDFYMNWVRQAPGQGLEWMGRVNPSNGDTNYNQKFKGRVTSTRDTSISTAYMELSRLRSDDTAVYYCARERIYYGISWYFDVWDTGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASGNIHNYLAWYQQKPGKAPKLLLYNAKTLAEGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCHHYYSTPPTFGQGTKLEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:2

CDH 17-specific AI-CAR (pSh3C15b28)

SEQ ID NO:3

h2FM19AIg3z (CD 19-specific AI-CAR-g3z)

MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQHPGKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGGAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPYTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFAADAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:4

hAT38AIa7(CD 38-specific, AI-CAR-a7)

MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVRPSQTLSLTCTVSGFTFTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFAADPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

SEQ ID NO:5

h81AIaI7(CD 81-specific, AI-CAR-a7)

MKHLWFFLLLVAAPRWVLSQVQLVQSGAEVKKPGASVKVSCKASGYTFSSSWMNWVRQAPGQRLEWMGRIYSGDGDAIYNGKFKGRVTITADTSASTAYMELSSLRSEDTAVYYCAREGKTGDLLLRSWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCRASKSVSTSIYSYMHWYQQKPGQPPKLLIYYASYLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCEHSREFPFTFGQGTKLEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFAADPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQSEQ ID NO:6

hRF22AIgC7(CD 22-specific, AI-CAR-gC7)

MKHLWFFLLLVAAPRWVLSEVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTVSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPWTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFAADIPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFRSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSP

SEQ ID NO:7

h2FM19b1AI3z (CD19 specific AI-CAR-IL 12b1 CD137-CD3z)

MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQHPGKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGGAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPYTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFAADWLIFFASLGSFLSILLVGVLGYLGLNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKMKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:8

hAT38bAI2(CD38 specificity, AI-CAR-IL 12b2)

MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVRPSQTLSLTCTVSGFTFTSYGVHWVRQPPGRGLEWIGVMWRGGSTDYNAAFMSRVTMLVDTSKNQFSLRLSSVTAADTAVYYCAKSMITTGFVMDSWGQGSLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASEDIYNRLTWYQQKPGKAPKLLISGATSLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYWSNPYTFGQGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFAADWMAFVAPSICIAIIMVGIFSTHYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEPLVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLVDLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQHISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML

SEQ ID NO:9

h5AIg137.3z (CDH 17-specific AI-CAR γ/CD137/CD3z)

MEFGLSWVFLVALLRGVQCQVQLVQSGAEVKKPGASVKVSCKVSAYAFSSSWMNWVRQAPGKGLEWMGRIYPRDGDTNYNGKFKGRVTMTADTSTDTAYMELSSLRSEDTAVYYCAREGDGYYWYFDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSIRNYLHWYQQKPGEAPRLLIYYASQSISGIPARFSGSGSGTDFTLTISSLETEDFAMYYCQHSNSWPLTFGQGTKLEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFAADAVVISVGSMGLIISLLCVYFWLERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPETKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:10

h10AIa7(CDH17 specificity a7)

Examples of RNA AI-CAR

SEQ ID NO:11

h10C7-P2A-IL18(CDH17 CAR-IL18)

SEQ ID NO:12

h10C7-P2A-INFg(CDH17 CAR-INFγ)

-MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQTPGKGLEWVAVIDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGGAIKRLIYTTSTLDSGVPKRFSGSGSGTDFTLTISSLQSEDFATYYCLQYASSPFTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRASGSGATNFSLLKQAGDVEENPGPMKHLWFFLLLVAAPRWVLSQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ

SEQ ID NO:13

h10C7-P2A-IL18-pro-CCL21(CDH17 CAR-IL18-proCCL21)

SEQ ID NO:14

h10C7-P2A-h8C7-P2A-IL18(CDH17 CAR-TROP2CAR-IL18)

MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQTPGKGLEWVAV IDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGGAIKRLIYTTSTLDSGVPKRFSGSGSGTDFTLTISSLQSEDFATYYCLQYASSPFTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRASGSGATNFSLLKQAGDVEENPGPMEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMSWVRQTPAKGLVWVSTINSDGYNIYYSDSMKGRFTISRDNAKYTLYLQMNSLRAEDTAMYYCARCSYYSYDYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASENIDNYLAWYQQKQGKVPKLLIYAATNLADGMPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHYYSNQLTFGQGTKLEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL YCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRASGSGATNFSLLKQAGDVEENPGPMEFGLSWVFLVALLRGVQCYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

SEQ ID NO:15

h10C7-P2A-h3U1(CDH17 CAR-bispecific antibody anti-CDH 17xCD3)

MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQTPGKGLEWVAV IDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGGAIKRLIYTTSTLDSGVPKRFSGSGSGTDFTLTISSLQSEDFATYYCLQYASSPFTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRASGSGATNFSLLKQAGDVEENPGPMEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSAGGGGSGGGGSGGGGSGDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK

SEQ ID NO:16

h10C7-P2A-h3U1-pro-IL18(CDH17 CAR-bispecific antibody anti-CDH 17xCD3-pro-IL18)

MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQTPGKGLEWVAV IDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGGAIKRLIYTTSTLDSGVPKRFSGSGSGTDFTLTISSLQSEDFATYYCLQYASSPFTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRASGSGATNFSLLKQAGDVEENPGPMEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSAGGGGSGGGGSGGGGSGDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKPAGGGTKTESSSRGYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

SEQ ID NO:17

h10C7-P2A-h3U1-pro-IL18-pro-XCL1(CDH17 CAR-bispecific antibody anti-CDH 17xCD3-proIL18-proXCL1)

MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQTPGKGLEWVAV IDSNGGSTYYPDTVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCSSYTNLGAYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISGYLNWLQQKPGGAIKRLIYTTSTLDSGVPKRFSGSGSGTDFTLTISSLQSEDFATYYCLQYASSPFTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRASGSGATNFSLLKQAGDVEENPGPMEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSAGGGGSGGGGSGGGGSGDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKPAGGGTKTESSSRGYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDSGGGTKTESSSRGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCA

DPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG

Examples of AI-CAR inducible Gene combinations

SEQ ID NO:18

h3U 1-P2A-IL 18 (bispecific antibody anti-CDH 17xCD3-IL18)

MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSAGGGGSGGGGSGGGGSGDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKPAGGGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKASGSGATNFSLLKQAGDVEENPGPMEFGLSWVFLVALLRGVQCYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

SEQ ID NO:19

iPro7 (iPro with n-terminal IL7)

SEQ ID NO:20

iPro7-P2A-h3U 1-P2A-IL 18(iProIL 7-bispecific antibody anti-CDH 17xCD3-IL18)

MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEHGGGSGGGSGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKKPAGRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLNTDAYLSLQELQGQDPASGSGATNFSLLKQAGDVEENPGPQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYYMYWVRQAPGKGLEWVASISFDGTYTYYTDRVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRPAWFPYWGQGTLVTVSAGGGGSGGGGSGGGGSGDIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGAGTKLELKPAGSGATNFSLLKQAGDVEENPGPMEFGLSWVFLVALLRGVQCYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

SEQ ID NO:21

iPro7-P2A-h3U 1-P2A-IL 18-pro-CCL21(iProIL 7-bispecific antibody anti-CDH 17xCD3-IL18-proCCL21)

Sequence listing

<110> Eibele pharmaceutical technology Co., Ltd

<120> Artificial immune monitoring chimeric antigen receptor (AI-CAR) and cell expressing the same

<130> ARTI917PCT

<141> 2020-02-21

<150> 62/808,815

<151> 2019-02-21

<150> 62/808,823

<151> 2019-02-21

<150> 62/808,833

<151> 2019-02-21

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 532

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 1

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

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

20 25 30

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

35 40 45

Thr Asp Phe Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Arg Val Asn Pro Ser Asn Gly Asp Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Ser Thr Arg Asp Thr Ser Ile Ser

85 90 95

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

100 105 110

Tyr Tyr Cys Ala Arg Glu Arg Ile Tyr Tyr Gly Ile Ser Trp Tyr Phe

115 120 125

Asp Val Trp Asp Thr Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

145 150 155 160

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

165 170 175

Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu Tyr Asn Ala Lys

195 200 205

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

210 215 220

Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

225 230 235 240

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

245 250 255

Gly Thr Lys Leu Glu Ile Lys Gly Ala Pro Thr Thr Thr Pro Ala Pro

260 265 270

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

275 280 285

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

290 295 300

Gly Leu Asp Phe Ala Cys Asp Ala Val Val Ile Ser Val Gly Ser Met

305 310 315 320

Gly Leu Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu Glu Arg Thr

325 330 335

Met Pro Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val Thr Glu

340 345 350

Tyr His Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Ala

355 360 365

Glu Ser Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu

370 375 380

Ile Pro Pro Lys Gly Gly Ala Leu Gly Glu Gly Pro Gly Ala Ser Pro

385 390 395 400

Cys Asn Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys

405 410 415

Pro Glu Thr Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

420 425 430

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

435 440 445

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

450 455 460

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

465 470 475 480

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

485 490 495

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

500 505 510

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

515 520 525

Leu Pro Pro Arg

530

<210> 2

<211> 496

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 2

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ser Ile Ser Phe Asp Gly Thr Tyr Thr Tyr Tyr Thr

65 70 75 80

Asp Arg Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Arg Pro Ala Trp Phe Pro Tyr Trp Gly Gln

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp

180 185 190

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

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 Arg Val Glu Ala Glu Asp Val

225 230 235 240

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

245 250 255

Ala Gly Thr Lys Leu Glu Leu Lys Gly Ala Pro 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 Pro Trp Leu Gly His Leu Leu

305 310 315 320

Val Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu

325 330 335

Ile Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys

340 345 350

Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His

355 360 365

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

370 375 380

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

385 390 395 400

Glu Arg Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro

405 410 415

Glu Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr

420 425 430

Asn Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu

435 440 445

Ala Cys Gln Val Tyr Phe Arg Ser Lys Arg Ser Arg Gly Gly His Ser

450 455 460

Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His

465 470 475 480

Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Pro

485 490 495

<210> 3

<211> 530

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 3

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

1 5 10 15

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

20 25 30

Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu

35 40 45

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu

50 55 60

Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Cys Ala Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

115 120 125

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

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln

145 150 155 160

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

165 170 175

Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln

180 185 190

Lys Pro Gly Gly Ala Pro Lys Leu Leu Ile Tyr His Thr Ser Arg Leu

195 200 205

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

210 215 220

Phe Thr Phe Thr Ile Ser Ser Leu Gln Gln Glu Asp Ile Ala Thr Tyr

225 230 235 240

Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr

245 250 255

Lys Val Glu Ile Lys Gly Ala Pro 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 Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

290 295 300

Asp Phe Ala Ala Asp Ala Val Val Ile Ser Val Gly Ser Met Gly Leu

305 310 315 320

Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu Glu Arg Thr Met Pro

325 330 335

Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His

340 345 350

Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser

355 360 365

Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro

370 375 380

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

385 390 395 400

Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu

405 410 415

Thr Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

420 425 430

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

435 440 445

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

450 455 460

Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

465 470 475 480

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

485 490 495

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

500 505 510

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

515 520 525

Pro Arg

530

<210> 4

<211> 528

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu

50 55 60

Glu Trp Ile Gly Val Met Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala

65 70 75 80

Ala Phe Met Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln

85 90 95

Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Lys Ser Met Ile Thr Thr Gly Phe Val Met Asp Ser Trp

115 120 125

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

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

145 150 155 160

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

165 170 175

Lys Ala Ser Glu Asp Ile Tyr Asn Arg Leu Thr Trp Tyr Gln Gln Lys

180 185 190

Pro Gly Lys Ala Pro Lys Leu Leu Ile Ser Gly Ala Thr Ser Leu Glu

195 200 205

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

210 215 220

Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr

225 230 235 240

Cys Gln Gln Tyr Trp Ser Asn Pro Tyr Thr Phe Gly Gln Gly Thr Lys

245 250 255

Val Glu Ile Lys Gly Ala Pro 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 Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Ala Asp Pro Ile Leu Leu Thr Ile Ser Ile Leu Ser Phe Phe

305 310 315 320

Ser Val Ala Leu Leu Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg

325 330 335

Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu

340 345 350

Glu His Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn

355 360 365

Pro Glu Ser Phe Leu Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln

370 375 380

Ala Arg Asp Glu Val Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln

385 390 395 400

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

405 410 415

Asn Cys Pro Ser Glu Asp Val Val Ile Thr Pro Glu Ser Phe Gly Arg

420 425 430

Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala

435 440 445

Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys

450 455 460

Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr

465 470 475 480

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

485 490 495

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

500 505 510

Asn Gln Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln

515 520 525

<210> 5

<211> 533

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 5

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

1 5 10 15

Val Leu Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

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

35 40 45

Ser Ser Ser Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu

50 55 60

Glu Trp Met Gly Arg Ile Tyr Ser Gly Asp Gly Asp Ala Ile Tyr Asn

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Tyr Cys Ala Arg Glu Gly Lys Thr Gly Asp Leu Leu Leu Arg Ser

115 120 125

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

130 135 140

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

145 150 155 160

Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn

165 170 175

Cys Arg Ala Ser Lys Ser Val Ser Thr Ser Ile Tyr Ser Tyr Met His

180 185 190

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

195 200 205

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

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp

225 230 235 240

Val Ala Val Tyr Tyr Cys Glu His Ser Arg Glu Phe Pro Phe Thr Phe

245 250 255

Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Ala Pro 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 Ser Arg Pro Ala Ala Gly Gly Ala Val His

290 295 300

Thr Arg Gly Leu Asp Phe Ala Ala Asp Pro Ile Leu Leu Thr Ile Ser

305 310 315 320

Ile Leu Ser Phe Phe Ser Val Ala Leu Leu Val Ile Leu Ala Cys Val

325 330 335

Leu Trp Lys Lys Arg Ile Lys Pro Ile Val Trp Pro Ser Leu Pro Asp

340 345 350

His Lys Lys Thr Leu Glu His Leu Cys Lys Lys Pro Arg Lys Asn Leu

355 360 365

Asn Val Ser Phe Asn Pro Glu Ser Phe Leu Asp Cys Gln Ile His Arg

370 375 380

Val Asp Asp Ile Gln Ala Arg Asp Glu Val Glu Gly Phe Leu Gln Asp

385 390 395 400

Thr Phe Pro Gln Gln Leu Glu Glu Ser Glu Lys Gln Arg Leu Gly Gly

405 410 415

Asp Val Gln Ser Pro Asn Cys Pro Ser Glu Asp Val Val Ile Thr Pro

420 425 430

Glu Ser Phe Gly Arg Asp Ser Ser Leu Thr Cys Leu Ala Gly Asn Val

435 440 445

Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser Ser Arg Ser Leu Asp Cys

450 455 460

Arg Glu Ser Gly Lys Asn Gly Pro His Val Tyr Gln Asp Leu Leu Leu

465 470 475 480

Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro Pro Pro Phe Ser Leu Gln

485 490 495

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

500 505 510

Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala Tyr Val Thr Met Ser Ser

515 520 525

Phe Tyr Gln Asn Gln

530

<210> 6

<211> 495

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 6

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

1 5 10 15

Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe

35 40 45

Ser Ile Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro

65 70 75 80

Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg His Ser Gly Tyr Gly Ser Ser Tyr Gly Val Leu

115 120 125

Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

145 150 155 160

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

165 170 175

Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser

195 200 205

Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Gln Glu Asp Ile Ala

225 230 235 240

Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly

245 250 255

Gly Thr Lys Val Glu Ile Lys Gly Ala Pro Thr Thr Thr Pro Ala Pro

260 265 270

Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu

275 280 285

Arg Pro Glu Ala Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

290 295 300

Gly Leu Asp Phe Ala Ala Asp Ile Pro Trp Leu Gly His Leu Leu Val

305 310 315 320

Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu Val Tyr Leu Leu Ile

325 330 335

Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys Val Leu Lys Cys Asn

340 345 350

Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser Ser Glu His Gly

355 360 365

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

370 375 380

Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu Glu Val Leu Glu

385 390 395 400

Arg Asp Lys Val Thr Gln Leu Leu Leu Gln Gln Asp Lys Val Pro Glu

405 410 415

Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr Ser Cys Phe Thr Asn

420 425 430

Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala Leu Glu Ile Glu Ala

435 440 445

Cys Gln Val Tyr Phe Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp

450 455 460

Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr

465 470 475 480

Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Pro

485 490 495

<210> 7

<211> 581

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 7

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

1 5 10 15

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

20 25 30

Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu

35 40 45

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu

50 55 60

Glu Trp Ile Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Cys Ala Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

115 120 125

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

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln

145 150 155 160

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

165 170 175

Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln

180 185 190

Lys Pro Gly Gly Ala Pro Lys Leu Leu Ile Tyr His Thr Ser Arg Leu

195 200 205

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

210 215 220

Phe Thr Phe Thr Ile Ser Ser Leu Gln Gln Glu Asp Ile Ala Thr Tyr

225 230 235 240

Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr

245 250 255

Lys Val Glu Ile Lys Gly Ala Pro 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 Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

290 295 300

Asp Phe Ala Ala Asp Trp Leu Ile Phe Phe Ala Ser Leu Gly Ser Phe

305 310 315 320

Leu Ser Ile Leu Leu Val Gly Val Leu Gly Tyr Leu Gly Leu Asn Arg

325 330 335

Ala Ala Arg His Leu Cys Pro Pro Leu Pro Thr Pro Cys Ala Ser Ser

340 345 350

Ala Ile Glu Phe Pro Gly Gly Lys Glu Thr Trp Gln Trp Ile Asn Pro

355 360 365

Val Asp Phe Gln Glu Glu Ala Ser Leu Gln Glu Ala Leu Val Val Glu

370 375 380

Met Ser Trp Asp Lys Gly Glu Arg Thr Glu Pro Leu Glu Lys Thr Glu

385 390 395 400

Leu Pro Glu Gly Ala Pro Glu Leu Ala Leu Asp Thr Glu Leu Ser Leu

405 410 415

Glu Asp Gly Asp Arg Cys Lys Ala Lys Met Lys Arg Gly Arg Lys Lys

420 425 430

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr

435 440 445

Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly

450 455 460

Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala

465 470 475 480

Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg

485 490 495

Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu

500 505 510

Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

515 520 525

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

530 535 540

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

545 550 555 560

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

565 570 575

Ala Leu Pro Pro Arg

580

<210> 8

<211> 548

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Arg Gly Leu

50 55 60

Glu Trp Ile Gly Val Met Trp Arg Gly Gly Ser Thr Asp Tyr Asn Ala

65 70 75 80

Ala Phe Met Ser Arg Val Thr Met Leu Val Asp Thr Ser Lys Asn Gln

85 90 95

Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Lys Ser Met Ile Thr Thr Gly Phe Val Met Asp Ser Trp

115 120 125

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

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

145 150 155 160

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

165 170 175

Lys Ala Ser Glu Asp Ile Tyr Asn Arg Leu Thr Trp Tyr Gln Gln Lys

180 185 190

Pro Gly Lys Ala Pro Lys Leu Leu Ile Ser Gly Ala Thr Ser Leu Glu

195 200 205

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

210 215 220

Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr

225 230 235 240

Cys Gln Gln Tyr Trp Ser Asn Pro Tyr Thr Phe Gly Gln Gly Thr Lys

245 250 255

Val Glu Ile Lys Gly Ala Pro 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 Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

290 295 300

Phe Ala Ala Asp Trp Met Ala Phe Val Ala Pro Ser Ile Cys Ile Ala

305 310 315 320

Ile Ile Met Val Gly Ile Phe Ser Thr His Tyr Phe Gln Gln Lys Val

325 330 335

Phe Val Leu Leu Ala Ala Leu Arg Pro Gln Trp Cys Ser Arg Glu Ile

340 345 350

Pro Asp Pro Ala Asn Ser Thr Cys Ala Lys Lys Tyr Pro Ile Ala Glu

355 360 365

Glu Lys Thr Gln Leu Pro Leu Asp Arg Leu Leu Ile Asp Trp Pro Thr

370 375 380

Pro Glu Asp Pro Glu Pro Leu Val Ile Ser Glu Val Leu His Gln Val

385 390 395 400

Thr Pro Val Phe Arg His Pro Pro Cys Ser Asn Trp Pro Gln Arg Glu

405 410 415

Lys Gly Ile Gln Gly His Gln Ala Ser Glu Lys Asp Met Met His Ser

420 425 430

Ala Ser Ser Pro Pro Pro Pro Arg Ala Leu Gln Ala Glu Ser Arg Gln

435 440 445

Leu Val Asp Leu Tyr Lys Val Leu Glu Ser Arg Gly Ser Asp Pro Lys

450 455 460

Pro Glu Asn Pro Ala Cys Pro Trp Thr Val Leu Pro Ala Gly Asp Leu

465 470 475 480

Pro Thr His Asp Gly Tyr Leu Pro Ser Asn Ile Asp Asp Leu Pro Ser

485 490 495

His Glu Ala Pro Leu Ala Asp Ser Leu Glu Glu Leu Glu Pro Gln His

500 505 510

Ile Ser Leu Ser Val Phe Pro Ser Ser Ser Leu His Pro Leu Thr Phe

515 520 525

Ser Cys Gly Asp Lys Leu Thr Leu Asp Gln Leu Lys Met Arg Cys Asp

530 535 540

Ser Leu Met Leu

545

<210> 9

<211> 572

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 9

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ser Ser Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Met Gly Arg Ile Tyr Pro Arg Asp Gly Asp Thr Asn Tyr Asn

65 70 75 80

Gly Lys Phe Lys Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Asp

85 90 95

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

100 105 110

Tyr Tyr Cys Ala Arg Glu Gly Asp Gly Tyr Tyr Trp Tyr Phe Asp Val

115 120 125

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

130 135 140

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln

145 150 155 160

Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser

165 170 175

Cys Arg Ala Ser Gln Ser Ile Arg Asn Tyr Leu His Trp Tyr Gln Gln

180 185 190

Lys Pro Gly Glu Ala Pro Arg Leu Leu Ile Tyr Tyr Ala Ser Gln Ser

195 200 205

Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

210 215 220

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

225 230 235 240

Tyr Cys Gln His Ser Asn Ser Trp Pro Leu Thr Phe Gly Gln Gly Thr

245 250 255

Lys Leu Glu Ile Lys Gly Ala Pro 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 Ser Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

290 295 300

Asp Phe Ala Ala Asp Ala Val Val Ile Ser Val Gly Ser Met Gly Leu

305 310 315 320

Ile Ile Ser Leu Leu Cys Val Tyr Phe Trp Leu Glu Arg Thr Met Pro

325 330 335

Arg Ile Pro Thr Leu Lys Asn Leu Glu Asp Leu Val Thr Glu Tyr His

340 345 350

Gly Asn Phe Ser Ala Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser

355 360 365

Leu Gln Pro Asp Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro

370 375 380

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

385 390 395 400

Gln His Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu

405 410 415

Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe

420 425 430

Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg

435 440 445

Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser

450 455 460

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

465 470 475 480

Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys

485 490 495

Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys

500 505 510

Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

515 520 525

Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys

530 535 540

Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr

545 550 555 560

Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

565 570

<210> 10

<211> 526

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 10

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Asp Ser Asn Gly Gly Ser Thr Tyr Tyr Pro

65 70 75 80

Asp Thr Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ser Ser Tyr Thr Asn Leu Gly Ala Tyr Trp Gly Gln Gly

115 120 125

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

130 135 140

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

145 150 155 160

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

165 170 175

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

180 185 190

Ala Ile Lys Arg Leu Ile Tyr Thr Thr Ser Thr Leu Asp Ser Gly Val

195 200 205

Pro Lys Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

210 215 220

Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

225 230 235 240

Tyr Ala Ser Ser Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

245 250 255

Lys Gly Ala Pro Pro 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 Ser

275 280 285

Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala

290 295 300

Ala Asp Pro Ile Leu Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val

305 310 315 320

Ala Leu Leu Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys

325 330 335

Pro Ile Val Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His

340 345 350

Leu Cys Lys Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu

355 360 365

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

370 375 380

Asp Glu Val Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu

385 390 395 400

Glu Ser Glu Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn Cys

405 410 415

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

420 425 430

Ser Leu Thr Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro Ile

435 440 445

Leu Ser Ser Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly

450 455 460

Pro His Val Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser

465 470 475 480

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

485 490 495

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

500 505 510

Glu Glu Ala Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln

515 520 525

<210> 11

<211> 684

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 11

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Asp Ser Asn Gly Gly Ser Thr Tyr Tyr Pro

65 70 75 80

Asp Thr Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ser Ser Tyr Thr Asn Leu Gly Ala Tyr Trp Gly Gln Gly

115 120 125

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

130 135 140

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

145 150 155 160

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

165 170 175

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

180 185 190

Ala Ile Lys Arg Leu Ile Tyr Thr Thr Ser Thr Leu Asp Ser Gly Val

195 200 205

Pro Lys Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

210 215 220

Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

225 230 235 240

Tyr Ala Ser Ser Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

245 250 255

Lys Gly Ala Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala

260 265 270

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg

275 280 285

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys

290 295 300

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

305 310 315 320

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

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

355 360 365

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

370 375 380

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

385 390 395 400

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

405 410 415

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

420 425 430

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

435 440 445

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

450 455 460

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

465 470 475 480

Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu

485 490 495

Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly

500 505 510

Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val Gln Cys Tyr

515 520 525

Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn Asp

530 535 540

Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp Met

545 550 555 560

Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile Ile

565 570 575

Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile Ser

580 585 590

Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile Ile

595 600 605

Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys Ser

610 615 620

Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys Met

625 630 635 640

Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu Lys

645 650 655

Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu Gly

660 665 670

Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

675 680

<210> 12

<211> 670

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 12

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Asp Ser Asn Gly Gly Ser Thr Tyr Tyr Pro

65 70 75 80

Asp Thr Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ser Ser Tyr Thr Asn Leu Gly Ala Tyr Trp Gly Gln Gly

115 120 125

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

130 135 140

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

145 150 155 160

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

165 170 175

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

180 185 190

Ala Ile Lys Arg Leu Ile Tyr Thr Thr Ser Thr Leu Asp Ser Gly Val

195 200 205

Pro Lys Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

210 215 220

Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

225 230 235 240

Tyr Ala Ser Ser Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

245 250 255

Lys Gly Ala Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala

260 265 270

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg

275 280 285

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys

290 295 300

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

305 310 315 320

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

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

355 360 365

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

370 375 380

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

385 390 395 400

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

405 410 415

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

420 425 430

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

435 440 445

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

450 455 460

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

465 470 475 480

Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu

485 490 495

Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Lys His Leu

500 505 510

Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp Val Leu Ser Gln

515 520 525

Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn Ala

530 535 540

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

545 550 555 560

Lys Asn Trp Lys Glu Glu Ser Asp Arg Lys Ile Met Gln Ser Gln Ile

565 570 575

Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln Ser

580 585 590

Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys Phe

595 600 605

Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr Asn

610 615 620

Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu Leu

625 630 635 640

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

645 650 655

Lys Arg Ser Gln Met Leu Phe Arg Gly Arg Arg Ala Ser Gln

660 665 670

<210> 13

<211> 808

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 13

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Asp Ser Asn Gly Gly Ser Thr Tyr Tyr Pro

65 70 75 80

Asp Thr Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ser Ser Tyr Thr Asn Leu Gly Ala Tyr Trp Gly Gln Gly

115 120 125

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

130 135 140

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

145 150 155 160

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

165 170 175

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

180 185 190

Ala Ile Lys Arg Leu Ile Tyr Thr Thr Ser Thr Leu Asp Ser Gly Val

195 200 205

Pro Lys Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

210 215 220

Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

225 230 235 240

Tyr Ala Ser Ser Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

245 250 255

Lys Gly Ala Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala

260 265 270

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg

275 280 285

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys

290 295 300

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

305 310 315 320

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

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

355 360 365

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

370 375 380

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

385 390 395 400

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

405 410 415

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

420 425 430

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

435 440 445

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

450 455 460

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

465 470 475 480

Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu

485 490 495

Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly

500 505 510

Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val Gln Cys Tyr

515 520 525

Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn Asp

530 535 540

Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp Met

545 550 555 560

Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile Ile

565 570 575

Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile Ser

580 585 590

Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile Ile

595 600 605

Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys Ser

610 615 620

Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys Met

625 630 635 640

Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu Lys

645 650 655

Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu Gly

660 665 670

Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp Pro Ala Gly Gly

675 680 685

Gly Thr Lys Thr Glu Ser Ser Ser Arg Ser Asp Gly Gly Ala Gln Asp

690 695 700

Cys Cys Leu Lys Tyr Ser Gln Arg Lys Ile Pro Ala Lys Val Val Arg

705 710 715 720

Ser Tyr Arg Lys Gln Glu Pro Ser Leu Gly Cys Ser Ile Pro Ala Ile

725 730 735

Leu Phe Leu Pro Arg Lys Arg Ser Gln Ala Glu Leu Cys Ala Asp Pro

740 745 750

Lys Glu Leu Trp Val Gln Gln Leu Met Gln His Leu Asp Lys Thr Pro

755 760 765

Ser Pro Gln Lys Pro Ala Gln Gly Cys Arg Lys Asp Arg Gly Ala Ser

770 775 780

Lys Thr Gly Lys Lys Gly Lys Gly Ser Lys Gly Cys Lys Arg Thr Glu

785 790 795 800

Arg Ser Gln Thr Pro Lys Gly Pro

805

<210> 14

<211> 1119

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 14

Tyr Tyr Pro Asp Thr Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys Ser Ser Tyr Thr Asn Leu Gly Ala Tyr Trp

35 40 45

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly

50 55 60

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

65 70 75 80

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

85 90 95

Arg Ala Ser Gln Asp Ile Ser Gly Tyr Leu Asn Trp Leu Gln Gln Lys

100 105 110

Pro Gly Gly Ala Ile Lys Arg Leu Ile Tyr Thr Thr Ser Thr Leu Asp

115 120 125

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

130 135 140

Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr

145 150 155 160

Cys Leu Gln Tyr Ala Ser Ser Pro Phe Thr Phe Gly Gly Gly Thr Lys

165 170 175

Val Glu Ile Lys Gly Ala Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro

180 185 190

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

195 200 205

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

210 215 220

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

225 230 235 240

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

245 250 255

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

260 265 270

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

275 280 285

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

290 295 300

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

305 310 315 320

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

325 330 335

Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly

340 345 350

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

355 360 365

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

370 375 380

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

385 390 395 400

Met Gln Ala Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn Phe

405 410 415

Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met

420 425 430

Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val

435 440 445

Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

450 455 460

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser

465 470 475 480

Thr Tyr Thr Met Ser Trp Val Arg Gln Thr Pro Ala Lys Gly Leu Val

485 490 495

Trp Val Ser Thr Ile Asn Ser Asp Gly Tyr Asn Ile Tyr Tyr Ser Asp

500 505 510

Ser Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Tyr Thr

515 520 525

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr

530 535 540

Tyr Cys Ala Arg Cys Ser Tyr Tyr Ser Tyr Asp Tyr Phe Asp Tyr Trp

545 550 555 560

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

565 570 575

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

580 585 590

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

595 600 605

Arg Ala Ser Glu Asn Ile Asp Asn Tyr Leu Ala Trp Tyr Gln Gln Lys

610 615 620

Gln Gly Lys Val Pro Lys Leu Leu Ile Tyr Ala Ala Thr Asn Leu Ala

625 630 635 640

Asp Gly Met Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

645 650 655

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr

660 665 670

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

675 680 685

Leu Glu Ile Lys Gly Ala Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro

690 695 700

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

705 710 715 720

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

725 730 735

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

740 745 750

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

755 760 765

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

770 775 780

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

785 790 795 800

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

805 810 815

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

820 825 830

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

835 840 845

Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly

850 855 860

Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu

865 870 875 880

Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu

885 890 895

Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His

900 905 910

Met Gln Ala Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn Phe

915 920 925

Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met

930 935 940

Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val

945 950 955 960

Gln Cys Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn

965 970 975

Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe

980 985 990

Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile

995 1000 1005

Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val

1010 1015 1020

Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn

1025 1030 1035 1040

Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp

1045 1050 1055

Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp

1060 1065 1070

Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala

1075 1080 1085

Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp

1090 1095 1100

Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

1105 1110 1115

<210> 15

<211> 1024

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 15

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Asp Ser Asn Gly Gly Ser Thr Tyr Tyr Pro

65 70 75 80

Asp Thr Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ser Ser Tyr Thr Asn Leu Gly Ala Tyr Trp Gly Gln Gly

115 120 125

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

130 135 140

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

145 150 155 160

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

165 170 175

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

180 185 190

Ala Ile Lys Arg Leu Ile Tyr Thr Thr Ser Thr Leu Asp Ser Gly Val

195 200 205

Pro Lys Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

210 215 220

Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

225 230 235 240

Tyr Ala Ser Ser Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

245 250 255

Lys Gly Ala Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala

260 265 270

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg

275 280 285

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys

290 295 300

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

305 310 315 320

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

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

355 360 365

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

370 375 380

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

385 390 395 400

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

405 410 415

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

420 425 430

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

435 440 445

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

450 455 460

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

465 470 475 480

Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu

485 490 495

Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly

500 505 510

Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val Gln Cys Gln

515 520 525

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

530 535 540

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr

545 550 555 560

Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

565 570 575

Ser Ile Ser Phe Asp Gly Thr Tyr Thr Tyr Tyr Thr Asp Arg Val Lys

580 585 590

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

595 600 605

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

610 615 620

Arg Asp Arg Pro Ala Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val

625 630 635 640

Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

645 650 655

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

660 665 670

Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Cys Phe Gln Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys

755 760 765

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

770 775 780

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

785 790 795 800

Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp

805 810 815

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

820 825 830

Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe

835 840 845

Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

945 950 955 960

Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro

965 970 975

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

980 985 990

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

995 1000 1005

Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

1010 1015 1020

<210> 16

<211> 1195

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 16

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Asp Ser Asn Gly Gly Ser Thr Tyr Tyr Pro

65 70 75 80

Asp Thr Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ser Ser Tyr Thr Asn Leu Gly Ala Tyr Trp Gly Gln Gly

115 120 125

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

130 135 140

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

145 150 155 160

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

165 170 175

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

180 185 190

Ala Ile Lys Arg Leu Ile Tyr Thr Thr Ser Thr Leu Asp Ser Gly Val

195 200 205

Pro Lys Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

210 215 220

Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

225 230 235 240

Tyr Ala Ser Ser Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

245 250 255

Lys Gly Ala Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala

260 265 270

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg

275 280 285

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys

290 295 300

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

305 310 315 320

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

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

355 360 365

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

370 375 380

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

385 390 395 400

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

405 410 415

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

420 425 430

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

435 440 445

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

450 455 460

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

465 470 475 480

Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu

485 490 495

Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly

500 505 510

Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val Gln Cys Gln

515 520 525

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

530 535 540

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr

545 550 555 560

Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

565 570 575

Ser Ile Ser Phe Asp Gly Thr Tyr Thr Tyr Tyr Thr Asp Arg Val Lys

580 585 590

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

595 600 605

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

610 615 620

Arg Asp Arg Pro Ala Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val

625 630 635 640

Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

645 650 655

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

660 665 670

Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Cys Phe Gln Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys

755 760 765

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

770 775 780

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

785 790 795 800

Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp

805 810 815

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

820 825 830

Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe

835 840 845

Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

945 950 955 960

Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro

965 970 975

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

980 985 990

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

995 1000 1005

Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

1010 1015 1020

Pro Ala Gly Gly Gly Thr Lys Thr Glu Ser Ser Ser Arg Gly Tyr Phe

1025 1030 1035 1040

Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn Asp Gln

1045 1050 1055

Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp Met Thr

1060 1065 1070

Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile Ile Ser

1075 1080 1085

Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile Ser Val

1090 1095 1100

Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile Ile Ser

1105 1110 1115 1120

Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys Ser Asp

1125 1130 1135

Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys Met Gln

1140 1145 1150

Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu Lys Glu

1155 1160 1165

Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu Gly Asp

1170 1175 1180

Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

1185 1190 1195

<210> 17

<211> 1301

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 17

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Val Ile Asp Ser Asn Gly Gly Ser Thr Tyr Tyr Pro

65 70 75 80

Asp Thr Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ser Ser Tyr Thr Asn Leu Gly Ala Tyr Trp Gly Gln Gly

115 120 125

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

130 135 140

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

145 150 155 160

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

165 170 175

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

180 185 190

Ala Ile Lys Arg Leu Ile Tyr Thr Thr Ser Thr Leu Asp Ser Gly Val

195 200 205

Pro Lys Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

210 215 220

Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln

225 230 235 240

Tyr Ala Ser Ser Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

245 250 255

Lys Gly Ala Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala

260 265 270

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg

275 280 285

Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys

290 295 300

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

305 310 315 320

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

325 330 335

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

340 345 350

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

355 360 365

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

370 375 380

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

385 390 395 400

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

405 410 415

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

420 425 430

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

435 440 445

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

450 455 460

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

465 470 475 480

Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu

485 490 495

Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly

500 505 510

Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val Gln Cys Gln

515 520 525

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

530 535 540

Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr Tyr

545 550 555 560

Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

565 570 575

Ser Ile Ser Phe Asp Gly Thr Tyr Thr Tyr Tyr Thr Asp Arg Val Lys

580 585 590

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

595 600 605

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

610 615 620

Arg Asp Arg Pro Ala Trp Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val

625 630 635 640

Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

645 650 655

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

660 665 670

Val Thr Pro Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

Cys Phe Gln Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys

755 760 765

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

770 775 780

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

785 790 795 800

Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp

805 810 815

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

820 825 830

Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe

835 840 845

Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

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

930 935 940

Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

945 950 955 960

Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro

965 970 975

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

980 985 990

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

995 1000 1005

Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

1010 1015 1020

Pro Ala Gly Gly Gly Thr Lys Thr Glu Ser Ser Ser Arg Gly Tyr Phe

1025 1030 1035 1040

Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn Asp Gln

1045 1050 1055

Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp Met Thr

1060 1065 1070

Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile Ile Ser

1075 1080 1085

Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile Ser Val

1090 1095 1100

Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile Ile Ser

1105 1110 1115 1120

Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys Ser Asp

1125 1130 1135

Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys Met Gln

1140 1145 1150

Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu Lys Glu

1155 1160 1165

Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu Gly Asp

1170 1175 1180

Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp Ser Gly Gly Gly Thr

1185 1190 1195 1200

Lys Thr Glu Ser Ser Ser Arg Gly Val Gly Ser Glu Val Ser Asp Lys

1205 1210 1215

Arg Thr Cys Val Ser Leu Thr Thr Gln Arg Leu Pro Val Ser Arg Ile

1220 1225 1230

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

1235 1240 1245

Thr Lys Arg Gly Leu Lys Val Cys Ala Asp Pro Gln Ala Thr Trp Val

1250 1255 1260

Arg Asp Val Val Arg Ser Met Asp Arg Lys Ser Asn Thr Arg Asn Asn

1265 1270 1275 1280

Met Ile Gln Thr Lys Pro Thr Gly Thr Gln Gln Ser Thr Asn Thr Ala

1285 1290 1295

Val Thr Leu Thr Gly

1300

<210> 18

<211> 716

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 18

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ser Ile Ser Phe Asp Gly Thr Tyr Thr Tyr Tyr Thr

65 70 75 80

Asp Arg Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Arg Pro Ala Trp Phe Pro Tyr Trp Gly Gln

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp

180 185 190

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

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 Arg Val Glu Ala Glu Asp Val

225 230 235 240

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

245 250 255

Ala Gly Thr Lys Leu Glu Leu Lys Pro Ala Gly Gly Gly Gly Gly Ser

260 265 270

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

275 280 285

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

290 295 300

Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

305 310 315 320

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

325 330 335

Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr

340 345 350

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

355 360 365

Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

370 375 380

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

385 390 395 400

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser

405 410 415

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

420 425 430

Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys

435 440 445

Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu

450 455 460

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

465 470 475 480

Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr

485 490 495

Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys

500 505 510

Val Glu Ile Lys Ala Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu

515 520 525

Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly

530 535 540

Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val Gln Cys Tyr

545 550 555 560

Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn Asp

565 570 575

Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp Met

580 585 590

Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile Ile

595 600 605

Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile Ser

610 615 620

Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile Ile

625 630 635 640

Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys Ser

645 650 655

Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys Met

660 665 670

Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu Lys

675 680 685

Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu Gly

690 695 700

Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

705 710 715

<210> 19

<211> 349

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 19

Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile

1 5 10 15

Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys

20 25 30

Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu

35 40 45

Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe

50 55 60

Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe

65 70 75 80

Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser

85 90 95

Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr

100 105 110

Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala

115 120 125

Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu

130 135 140

Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu

145 150 155 160

Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu

165 170 175

His Gly Gly Gly Ser Gly Gly Gly Ser Gly Ala Pro Thr Thr Thr Pro

180 185 190

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

195 200 205

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

210 215 220

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

225 230 235 240

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

245 250 255

Cys Lys Lys Pro Ala Gly Arg Asn Thr Gly Pro Trp Leu Lys Lys Val

260 265 270

Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser

275 280 285

Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro

290 295 300

Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu

305 310 315 320

Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu Leu Leu Asn Thr Asp

325 330 335

Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro

340 345

<210> 20

<211> 818

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 20

Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile

1 5 10 15

Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys

20 25 30

Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu

35 40 45

Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe

50 55 60

Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe

65 70 75 80

Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser

85 90 95

Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr

100 105 110

Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala

115 120 125

Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu

130 135 140

Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu

145 150 155 160

Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu

165 170 175

His Gly Gly Gly Ser Gly Gly Gly Ser Gly Ala Pro Thr Thr Thr Pro

180 185 190

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

195 200 205

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

210 215 220

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

225 230 235 240

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

245 250 255

Cys Lys Lys Pro Ala Gly Arg Asn Thr Gly Pro Trp Leu Lys Lys Val

260 265 270

Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser

275 280 285

Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro

290 295 300

Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu

305 310 315 320

Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu Leu Leu Asn Thr Asp

325 330 335

Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Ala Ser Gly

340 345 350

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

355 360 365

Glu Asn Pro Gly Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val

370 375 380

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

385 390 395 400

Thr Phe Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys

405 410 415

Gly Leu Glu Trp Val Ala Ser Ile Ser Phe Asp Gly Thr Tyr Thr Tyr

420 425 430

Tyr Thr Asp Arg Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

435 440 445

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

450 455 460

Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ala Trp Phe Pro Tyr Trp

465 470 475 480

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro

625 630 635 640

Gly Pro Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu

645 650 655

Arg Gly Val Gln Cys Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val

660 665 670

Ile Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg

675 680 685

Pro Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro

690 695 700

Arg Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly

705 710 715 720

Met Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser

725 730 735

Cys Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn

740 745 750

Ile Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro

755 760 765

Gly His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr

770 775 780

Phe Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys

785 790 795 800

Lys Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn

805 810 815

Glu Asp

<210> 21

<211> 940

<212> PRT

<213> Artificial Sequence

<220>

<223> synthesized

<400> 21

Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile

1 5 10 15

Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys

20 25 30

Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu

35 40 45

Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe

50 55 60

Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe

65 70 75 80

Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser

85 90 95

Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr

100 105 110

Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala

115 120 125

Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu

130 135 140

Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu

145 150 155 160

Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu

165 170 175

His Gly Gly Gly Ser Gly Gly Gly Ser Gly Ala Pro Thr Thr Thr Pro

180 185 190

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

195 200 205

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

210 215 220

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

225 230 235 240

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

245 250 255

Cys Lys Lys Pro Ala Gly Arg Asn Thr Gly Pro Trp Leu Lys Lys Val

260 265 270

Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu Ser

275 280 285

Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe Pro

290 295 300

Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro Leu

305 310 315 320

Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu Leu Leu Asn Thr Asp

325 330 335

Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Ala Ser Gly

340 345 350

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

355 360 365

Glu Asn Pro Gly Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val

370 375 380

Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

385 390 395 400

Thr Phe Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Lys

405 410 415

Gly Leu Glu Trp Val Ala Ser Ile Ser Phe Asp Gly Thr Tyr Thr Tyr

420 425 430

Tyr Thr Asp Arg Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

435 440 445

Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr

450 455 460

Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ala Trp Phe Pro Tyr Trp

465 470 475 480

Gly Gln Gly Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro

625 630 635 640

Gly Pro Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu

645 650 655

Arg Gly Val Gln Cys Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val

660 665 670

Ile Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg

675 680 685

Pro Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro

690 695 700

Arg Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly

705 710 715 720

Met Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser

725 730 735

Cys Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn

740 745 750

Ile Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro

755 760 765

Gly His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr

770 775 780

Phe Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys

785 790 795 800

Lys Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn

805 810 815

Glu Asp Gly Gly Gly Thr Lys Thr Glu Ser Ser Ser Arg Ser Asp Gly

820 825 830

Gly Ala Gln Asp Cys Cys Leu Lys Tyr Ser Gln Arg Lys Ile Pro Ala

835 840 845

Lys Val Val Arg Ser Tyr Arg Lys Gln Glu Pro Ser Leu Gly Cys Ser

850 855 860

Ile Pro Ala Ile Leu Phe Leu Pro Arg Lys Arg Ser Gln Ala Glu Leu

865 870 875 880

Cys Ala Asp Pro Lys Glu Leu Trp Val Gln Gln Leu Met Gln His Leu

885 890 895

Asp Lys Thr Pro Ser Pro Gln Lys Pro Ala Gln Gly Cys Arg Lys Asp

900 905 910

Arg Gly Ala Ser Lys Thr Gly Lys Lys Gly Lys Gly Ser Lys Gly Cys

915 920 925

Lys Arg Thr Glu Arg Ser Gln Thr Pro Lys Gly Pro

930 935 940

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