Artificial immune surveillance chimeric antigen receptor and its expressing cells

Abstract

A non-viral vector comprising, from 5' to 3', a first coding region flanked by two transposon terminal inverted repeat sequences, an inducible promoter, a specific combination of genes whose expression is localized to the tumor microenvironment and whose function is to mediate CAR persistence and anti-tumor mechanisms to stimulate or enhance the patient's anti-tumor response; a second promoter and coding region expressing one or more artificial immune surveillance chimeric antigen receptors (AI-CAR), and a truncated CD20 or truncated EGFR safety target. The polycistronic mRNA consists of a specific combination of transiently expressed anti-tumor mechanisms designed to stimulate the patient's anti-tumor response, the same as the AI-CAR vector.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 808,815 filed on February 21, 2019, U.S. Provisional Application Serial No. 62 / 808,823 filed on February 21, 2019, U.S. Provisional Application Serial No. 62 / 808,830 filed on February 21, 2019, and U.S. Provisional Application Serial No. 62 / 808,833 filed on February 21, 2019 under 35 U.S.C. 119(e), the entire disclosures of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to a technology for improving the expansion, production, survival and efficacy of chimeric antigen receptor (CAR)-T cells or NK cells. Background Art

[0004] Chimeric antigen receptors (CARs) are a central part of immunotherapy, which directly targets antibodies into cellular immunity to treat cancer. The resulting CAR has a tumor antigen binding domain connected to the intracellular domain of the TCR and TCR co-stimulatory proteins. After the patient's T or NK cells are transformed to produce CARs and infused back into the patient, T cells expressing CARs are equipped to kill cancer cells expressing tumor antigens. The first two FDA-approved CART cell therapies both target CD19, a pan-B-cell surface molecule on many types of B-cell cancers. Kymriah (tisagenlecleucel) is approved for the treatment of relapsed / refractory B-cell precursor acute lymphoblastic leukemia (ALL), while Yescarta (axicabtageneciloleucel) is approved for the treatment of relapsed / refractory diffuse large B-cell lymphoma (DLBCL). In clinical trials, results after Kymriah treatment showed a remission rate of 83% for all types of B-cell ALL after 3 months. However, 49% of ALL patients suffer from cytokine release syndrome (CRS), a serious side effect that has caused multiple deaths in clinical trials. In addition, at least 10% of patients relapse due to loss of the targeted CD19 epitope. In the CLL trial, Kymriah treatment resulted in a 57% overall response rate (Boyiadzis 2018).

[0005] A variety of factors may lead to relapse of CAR-T therapy, such as insufficient persistence of CAR T cells (due to exhaustion or host anti-CAR T response), loss of target antigen, lack of induction of effective host anti-tumor response, and inability to effectively locate lymphoma / solid tumors. Loss of CD19 expression occurs in B-cell tumors and after immunotherapy targeting CD19 (Masir 2006, Kimura 2007, Yu 2017). The loss of tumor antigens can be solved by targeting a second pan-B cell / tumor antigen, such as CD20 and CD22. In the case of CD22, it is widely expressed on ALL embryonic cells and has been successfully targeted with immunoconjugates and monospecific CAR T cells (Boyiadzis 2018). One strategy to significantly reduce the chance of immune escape is to use bispecific CAR to target CD22 as a second antigen.

[0006] Several clinical trials of anti-CD19 x anti-CD22 bispecific CARs are ongoing (Boyiadzis 2018). The configuration of these CARs is based on FDA-approved second-generation CARs, which in common have a single chain with a CD3ζ endodomain. Although the terms dual and bispecific are used, they are all bispecific CARs. If the side effect of CRS is due to the infusion of the CAR, the addition of the anti-CD22 scFv will not make a big difference. Still, it is unclear how many patients will experience CRS in these trials.

[0007] Non-viral bispecific CAR expression constructs are needed, which constitute a combination therapy that leads to a more complete and lasting response. In this way, cytokines are provided not only for enhancing CAR cytotoxicity, survival and proliferation, but also for inducing host anti-tumor responses. In addition, cytokine responses are local and transient, thus avoiding the toxicity observed when cytokines are administered systemically. In addition to considerations of systemic cytokine therapy and tumor antigen loss, the problems of CAR cell survival, persistence, exhaustion and manufacturing are also relevant. In order to establish a complete and lasting response, there are methods for supplementing soluble cytokine growth factors, multiple administrations or activating cytokine signaling pathways to drive amplification without terminal differentiation. Cytokine treatment can enhance the in vivo amplification and activity of CAR, and the production of CAR cells can be amplified using a variety of cytokines. These problems can be solved by a selection combination of cytokines produced in response to target antigens. Summary of the invention

[0008] In one aspect, the present application provides a bispecific chimeric antigen receptor. In one embodiment, the receptor comprises an extracellular domain connected to an intercellular domain by a linker, wherein the extracellular domain comprises a first scFv connected to a second scFv, wherein the first scFv domain and the second scFv domain each independently have affinity for CD19 or CD22, wherein the first scFv domain and the second scFv domain have affinity for different antigens, and wherein the intracellular domain comprises a co-stimulatory internal domain and a CD3ξ domain.

[0009] In a second aspect, the present application provides a bispecific chimeric antigen receptor complex. In one embodiment, such a complex comprises a first protein, the first protein comprising a first extracellular domain connected to a first intercellular domain via a first joint, wherein the first extracellular domain comprises a first scFv with affinity for CD19 or CD22, and wherein the first intercellular domain comprises a JAK1 binding domain, and a second protein, the second protein comprising a second extracellular domain connected to a second intercellular domain via a second joint, wherein the second extracellular domain comprises a second scFv with affinity for CD19 or CD22, and wherein the second intercellular domain comprises a JAK3 binding domain. The first scFv domain and the second scFv domain have affinity for different tumor antigens.

[0010] In one embodiment, the first intracellular domain comprises IL7Rα (CD127). In one embodiment, the first intracellular domain comprises the intracellular domain of IL15Rβ (CD122), IL21Rα (CD360), or a combination thereof. In one embodiment, the first intracellular domain further comprises a first cytotoxic signaling domain connected to a JAK1 binding domain. In one embodiment, the first cytotoxic signaling domain comprises CD28, CD3ζ, CD137, OX40, CD27, ICOS, or a combination thereof.

[0011] In one embodiment, the first scFv domain has affinity for CD19.

[0012] In one embodiment, the second scFv domain has affinity for CD22.

[0013] In one embodiment, the first intracellular domain is configured to dimerize with the second intracellular domain.

[0014] In one embodiment, the second intracellular domain comprises γ (CD132). In one embodiment, the second intracellular domain further comprises a second cytotoxic signaling domain connected to a JAK3 binding domain. In one embodiment, the second intracellular domain comprises a tandem γ (CD132), a JAK3 binding domain, CD28, and CD3ζ.

[0015] In one embodiment, the second cytotoxic domain comprises CD28, CD3ζ, CD137, OX40, CD27, ICOS, or a combination thereof.

[0016] In one embodiment, the first and second linkers independently comprise CD8. In one embodiment, the first and second linkers independently comprise a stem and a transmembrane domain.

[0017] In one embodiment, the stem comprises CD8, Fc hinge, Fc CH2-CH3, TCRα, TCRβ, truncated IL7Rα (CD127), truncated IL15Rβ (CD122), IL15Rα (CD215), truncated γ (CD132), truncated IL21Rα (CD360), or a combination thereof.

[0018] In one embodiment, the transmembrane domain comprises CD8, CD28, CD3ζ, CD3ε, CD3δ, CD3γ, CD3ζ, TCRα, TCRβ, IL15Rβ (CD122), γ (CD132), IL7Rα (CD127), IL21Rα (CD360), IL15Rα (CD215), or a combination thereof.

[0019] On the other hand, the application provides an open reading frame (ORF). In one embodiment, the open reading frame (ORF) sequentially comprises a nucleic acid encoding a protein disclosed therein, a nucleic acid encoding a ribosomal skipping sequence, and a nucleic acid encoding a protein disclosed therein. In one embodiment, the open reading frame (ORF) sequentially comprises a CD22 scFv, a linker, a CD22 scFv, and a chimeric antigen receptor domain.

[0020] In another aspect, the present application provides a biomolecule complex. In one embodiment, such a complex comprises a bispecific or dual chimeric antigen receptor as disclosed therein that binds to a CD19 antigen or a CD22 antigen. In one embodiment, the first intracellular domain dimerizes with the second intracellular domain. In one embodiment, JAK1 dimerizes with JAK3.

[0021] On the other hand, the present application provides a non-viral vector. In one embodiment, the non-viral vector comprises, 5' to 3' flanked by two transposons, a promoter, a first coding region comprising a gene for expressing a first artificial immune surveillance chimeric antigen receptor (AI-CAR), a fourth coding region comprising a gene expressing a truncated CD20 or a truncated EGFR safety target, followed by a polyA signal sequence.

[0022] In one embodiment, the first promoter comprises STAT, NFAT or NF-κB inducible promoter. In one embodiment, the first coding region and the fourth coding region are connected by IRES. In one embodiment, the first AI-CAR comprises CD19 CAR or CD22 CAR.

[0023] In one embodiment, the non-viral vector further includes a second coding region, and the second coding region includes a gene for expressing a second AI-CAR, between the first coding region and the fourth coding region. In one embodiment, the first CAR includes CD19 CAR and wherein the second region includes CD22 CAR or CD20 CAR.

[0024] In one embodiment, the non-viral vector further comprises a third coding region, the third coding region comprising a gene for expressing a third AI-CAR, based on the second coding region and the fourth coding region. In one embodiment, the first CAR comprises CD19 scFv, wherein the second CAR comprises CD22 scFv or CD20 scFv, and wherein the first CAR is connected to the second CAR via a linker.

[0025] In another aspect, the present application provides an isolated nucleic acid. In one embodiment, the isolated nucleic acid encodes a protein, a receptor, a biomolecule, or a biomolecule complex thereof.

[0026] In a further aspect, the present application provides an expression vector. In one embodiment, the expression vector comprises an isolated nucleic acid as disclosed therein. In one embodiment, the expression vector comprises a nucleic acid encoding cytokine expression and its disclosed ORF. In one embodiment, the vector can be expressed in a cell.

[0027] In a further aspect, the present application provides a host cell. In one embodiment, the host cell comprises an isolated nucleic acid or expression vector as disclosed therein. In one embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0028] In another aspect, the present application provides CAR-T or CAR-NK cells. In one embodiment, such cells express a chimeric antigen receptor or a chimeric antigen receptor complex as disclosed therein.

[0029] In another aspect, the present application provides mammalian cells. In one embodiment, the mammalian cells contain a chimeric antigen receptor or a chimeric antigen receptor complex as disclosed therein. In one embodiment, the mammalian cells contain a biomolecule complex as disclosed therein.

[0030] In another aspect, the present application provides a method for treating a tumor in a subject. In one embodiment, the method comprises administering to the subject a sufficient amount of a CAR-T or CAR-NK cell or a non-viral vector as disclosed.

[0031] On the other hand, the present application provides a pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of a vector, a non-viral vector, a CAR-T or CAR-NK cell, a protein, a biomolecule, or a biomolecule complex as disclosed therein. In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable vehicle. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] 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 present disclosure and are therefore not to be considered limiting of its scope, the present disclosure will be described with additional specificity and detail through use of the accompanying drawings, in which:

[0033] Figure 1 AI-CAR expression cassettes (ACs) in non-viral DNA constructs encoding monospecific, dual, or bispecific CARs targeting CD19, CD22, and / or CD20 are shown, which are also capable of inducing expression of proteins encoded within the AI-CAR vector that support CAR cell persistence and additional anti-tumor activity mechanisms;

[0034] Figure 2 In vitro transcribed mRNA encoding a monospecific, dual, or bispecific CAR targeting CD19, CD22, and / or CD20 and an additional therapeutic protein having anti-tumor activity is depicted;

[0035] Figure 3 Described are mechanisms by which dual and bispecific AI-CARs targeting CD19 and CD22 or CD20 in non-viral vectors induce expression of genes (e.g., cytokines and / or bispecific antibodies) encoded by the same integrated AI-CAR expression cassette;

[0036] Figure 4Flow cytometric analysis of SW480 cells expressing both CD19 and CD22 for specific binding of humanized anti-CD19 scFv-Fc and anti-CD22 scFv-Fc on day 1 after electroporation is shown;

[0037] Figure 5 Flow cytometric analysis results of CD19 and CD22 bispecific and bispecific CAR-T cells combined with recombinant human anti-CD19-Fc and anti-CD22-Fc are shown. Bispecific CAR cells were generated by electroporation of a mixture of plasmids or as separate plasmids, and then monospecific CAR cells were pooled;

[0038] Figure 6 Shown is a summary of dual and bispecific CAR expression on IL-15 expanded T cells, one day after electroporation (protein-L; A), and in combination with rhCD19hFc (B) and rhCD22hFc (C);

[0039] Figure 7 showing cytotoxicity of CAR-T cells expressing single, dual, or bispecific anti-CD19 and / or anti-CD22 CARs as measured by viability of SW480 colon cancer cells expressing CD19, CD22, or both in a 24-hour T cell-dependent cytotoxicity (TDCC) assay; and

[0040] Figure 8 Cytotoxicity of CAR-T cells expressing dual or bispecific anti-CD19 and anti-CD22 CARs is shown, as measured by the viability of colon cancer cells SW480 expressing CD19, CD22, or both in a 24-hour T cell-dependent cytotoxicity (TDCC) assay. DETAILED DESCRIPTION

[0041] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components unless the context indicates 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 herein. It will be readily understood that the aspects of the present disclosure as generally described herein and illustrated in the accompanying drawings may be arranged, substituted, combined, separated, and designed into a variety of different configurations, all of which are expressly contemplated herein.

[0042] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components unless the context indicates 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 herein. It will be readily understood that the aspects of the present disclosure as generally described herein and illustrated in the accompanying drawings may be arranged, substituted, combined, separated, and designed into a variety of different configurations, all of which are expressly contemplated herein.

[0043] The present disclosure provides, inter alia, isolated antibodies, methods of making such antibodies, bispecific or multispecific molecules, antibody-drug conjugates and / or immunoconjugates composed 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.

[0044] The term "antibody" is used in the broadest sense and specifically covers single monoclonal antibodies (including agonist antibodies and antagonist antibodies), antibody compositions with multiple epitope specificities, and antibody fragments (e.g., Fab, F(ab')2, and Fv), as long as they exhibit the desired biological activity. In some embodiments, the antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a single-chain antibody, a bispecific antibody or a bi-effective antibody, a mimetic antibody, a human antibody, and a humanized antibody and active fragments thereof. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab')2, scFv, 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 binding sites that immunospecifically bind to antigens. Immunoglobulins can be of any type (IgG, IgM, IgD, IgE, IgA and IgY) or class (IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecules. In one embodiment, antibodies can be whole antibodies and any antigen-binding fragments derived from whole antibodies. Typical antibodies refer to heterotetrameric proteins that usually contain two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain consists of a light chain variable domain (abbreviated as VL) and a light chain constant domain. The VH and VL regions can be further subdivided into domains of hypervariable complementary determining regions (CDRs) and more conservative regions called framework regions (FRs). Each variable domain (VH or VL) is usually composed of three CDRs and four FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from amino terminus to carboxyl terminus. Within the variable domains of the light chain and heavy chain are binding regions that interact with antigens.

[0045] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody group, that is, each antibody comprising the group is identical, except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, in contrast to conventional (polyclonal) antibody preparations that 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 without being contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the characteristic of an antibody is obtained from a substantially homogeneous antibody group, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibodies used according to 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. Patent No. 4,816,567).

[0046] Monoclonal antibodies can include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical 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, as 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 hybridoma or phage display (see Siegel. Transfus. Clin. Biol. 9: 15-22 (2002) for review) or molecular cloning of antibodies directly from primary B cells (see Tiller. New Biotechnol. 28: 453-7 (2011)).

[0047] The term "antigen or epitope binding part or fragment" refers to an antibody fragment that is capable of binding to an antigen (here CD19, CD20 and CD22). These fragments may have antigen binding function and other functions of a complete antibody. Examples of binding fragments include, but are not limited to, single-chain Fv fragments (scFv) consisting of the VL and VH domains of a single arm of an antibody connected 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 a single CDR domain, particularly CDR3 regions from VL and / or VH domains (see, e.g., Beiboer et al., J. Mol. Biol. 296: 833-49 (2000)). Antibody fragments are produced using conventional methods known to those skilled in the art. The utility of antibody fragments may be screened using the same techniques used for complete antibodies.

[0048] "Antigen- or epitope-binding fragments" can be derived from antibodies of the present disclosure by a number of techniques known in the art. For example, purified monoclonal antibodies can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Appropriate fractions containing Fab fragments are then collected and concentrated by membrane filtration or the like. For further description of general techniques for isolating active fragments of antibodies, see, e.g., Khaw, BA et al., J. Nucl. Med. 23: 1011-1019 (1982); Rousseaux et al., Methods Enzymology, 121: 663-69, Academic Press, 1986.

[0049] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0050] The Fab fragment may contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that a few residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH herein refers to Fab' in which the cysteine ​​residues of the constant domains bear free sulfhydryl groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0051] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a heavy chain and a light chain variable domain in a tight, non-covalently associated dimer. 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 six CDRs together give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than that of the entire binding site.

[0052] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

[0053] According to the amino acid sequence of the constant domain of the immunoglobulin heavy chain, immunoglobulins can be divided into different classes. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses (isotypes), for example, 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 respectively called α, δ, ε, γ and μ. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known.

[0054] "Humanized antibodies" refer to a class of engineered antibodies whose CDRs are derived from non-human donor immunoglobulins and the remaining immunoglobulin-derived portions of the molecule are derived from one (or more) human immunoglobulins. In addition, framework support residues may 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: 10029-10032 (1989), Hodgson et al., Bio / Technology, 9: 421 (1991)).

[0055] As used herein, the terms "polypeptide," "peptide," and "protein" are interchangeable and are defined to mean a biological molecule composed of amino acids linked by peptide bonds.

[0056] The terms "a," "an," and "the" as used herein are defined to mean "one or more" and include the plural unless the context is inappropriate.

[0057] "Isolated" refers to a biological molecule that is free of at least some of its naturally occurring components. "Isolated," when used to describe the various polypeptides disclosed herein, means a polypeptide that has been identified and separated 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 with different antigen-binding specificities.

[0058] "Recombinant" means that the antibody is produced in a foreign host cell using recombinant nucleic acid technology.

[0059] 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.

[0060] In addition, as used herein, the term "immunogenic" refers to a substance that causes 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 to reduce or alleviate the condition to be treated in response to the administered immunogenic composition of the present disclosure.

[0061] "Specific binding" or "specifically binds to" or "specific for" a particular antigen or epitope refers to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule to a control molecule, which is typically a molecule of similar structure 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.

[0062] For example, specific binding to a particular antigen or epitope can be achieved by having at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 24 or greater, 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 of 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000-, or more times greater than a control molecule relative to the antigen or epitope.

[0063] In addition, specific binding to a particular antigen or epitope can be demonstrated, for example, by an antibody having a KA or Ka for the antigen or epitope that is at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000-, or more times that of a control, where KA or Ka refers to the association rate of a specific antibody-antigen interaction.

[0064] "Homology" between two sequences is determined by sequence identity. If the two sequences to be compared are of different lengths, 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 a comparison between a given sequence and the above sequences of the present disclosure may be caused, for example, by additions, deletions, substitutions, insertions or recombination.

[0065] The present disclosure may be more readily understood by reference to the following detailed description of specific embodiments and examples included herein.While the present disclosure has been described with reference to specific details of certain embodiments of the present disclosure, it is not intended that these details should be construed as limiting the scope of the present disclosure.

[0066] In order to solve the problem of cytokine signaling pathways in CAR amplification, terminal differentiation and exhaustion, the present application discloses compositions and methods for regulating host responses to CAR using artificial immune monitoring chimeric antigen receptors (AI-CAR). The advancement of the AI-CAR technology is intended to replace standard CAR manufacturing and achieve effective care treatment points. Although other CAR technologies may require the expression of soluble cytokine growth factors and / or multiple administrations of sustained activity to produce a complete and lasting response, AI-CAR is able to produce CAR cells in the absence of constitutively active drivers for proliferation or multiple CAR administrations for persistent anti-tumor responses.

[0067] Compared with standard CAR, AI-CAR increases the efficiency of manufacturing CAR cells. AI-CAR may only require one target antigen in vitro and in vivo to obtain full proliferation and cytotoxic activity. In this article, since the expansion of standard CAR-T cells usually requires the use of a combination of growth factors and aAPCs for manufacturing, AI-CAR can significantly reduce manufacturing costs.

[0068] Subsequently, after AI-CAR engagement of tumor cells in vivo, the expression of several anti-tumor genes encoded by the integrated AI-CAR vector can be induced. The expression of these endogenous genes can enable patients to produce anti-tumor responses that target different tumor antigens such as new antigens more widely. For example, the STAT5 reporter system is 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 because standard CAR constructs are generally unable to induce gene expression. Together with these auxiliary factors, AI-CAR will become a platform technology that provides practical, economical and effective solutions for cancer point-of-care treatment.

[0069] Many forms of cancer can be present in an immunosuppressive tumor environment. AI-CAR will be highly desirable because the AI-CAR vector is 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-CAR is its ability to regulate the expression of related anti-tumor genes at the tumor site without constitutively expressing it. Another feature is that AI-CAR is designed to have a single dose when administered, followed by its long-term activity and greater efficacy. With these favorable features, AI-CAR is a better solution for unmet challenges on the market, which predicts the efficacy of treating most (if not all) types of cancer.

[0070] Regarding CAR therapy for the treatment of hematological cancer, there are multiple targets, such as CD19, CD20 and CD22, CD9 and CD38. Targeting strategies may involve the use of dual, bispecific AI-CARs encoded in non-viral DNA vectors or transiently expressed RNA-CARs. In this way, AI-CAR can be used for greater persistence or as a transient treatment bridge for transplantation. Or it can be used as a point-of-care treatment for inducing host anti-tumor immune responses against new antigens. Due to strong in vivo amplification, fewer DNA vector AI-CAR cells can be administered. In this article, non-viral cassette constructs encoding one or two CARs and multiple inducible genes are new methods for point-of-care treatment and stimulation of persistent host anti-tumor responses. This configuration of CAR enables the production of CAR in T cells, NK cells, and other immune cells without the presence of potentially toxic constitutively active drivers for proliferation or inflammatory cytokines, and without multiple CAR administration for persistent anti-tumor responses. The bispecific CAR in the present application not only targets CD19 and CD22 to prevent tumor escape via antigen loss, but is also capable of inducing responses, such as cytokines, chemokines, or bispecific antibodies, that are localized or restricted to tumor cell engagement.

[0071] Example

[0072] Example 1. AI-CAR constructs

[0073] CAR vectors with inducible promoters can be constructed to induce the expression of additional proteins after tumor engagement, which have different anti-tumor activity mechanisms to enhance CAR activity. Therefore, AI-CAR constitutes a combination therapy. Monospecific AI-CARs can be constructed by fusing signal peptides, anti-CD19 CAR scFv, CD8 extracellular domain stem and transmembrane domain, CD137 and CD3z internal domains to P2A ribosomal skipping sequence or IRES and then to signal peptides, targets for CAR elimination such as truncated EGFR or truncated CD20 and polyA signals. Figure 1 A, SEQ ID1-5). To generate a bispecific AI-CAR, a second complete CAR sequence can be inserted between the first CAR and the safety target, such as Figure 1 B; SEQ ID 8. Bispecific AI-CARs can be generated by fusing the coding region of the signal peptide to the CD19 binding scFv and linker peptide, CD22 or CD20 binding scFv, CD8 hinge and transmembrane domains, and CD137 and CD3z endodomains, followed by fusion of IRES or P2A sequence signal sequence safety target sequence and pol A signal sequence. Figure 1 C, SEQ ID 6 and 7). In addition, the AI-CAR vector comprises an inducible promoter with a transcription factor response element (TF-RE) of a transcription factor such as STAT, NFAT or NF-κB, followed by the coding region of one or more genes (e.g., SEQ ID 9, 10, 16, 17) connected by an IRES or a self-cleaving ribosomal skipping peptide such as T2A or P2A, followed by a Figure 2 The polyA signal sequence shown in AC. The two coding regions can be located between transposons or viral terminal repeats (IR) for integration into T or NK cells. Alternatively, the coding region of the AI-CAR construct can be integrated at a specific genomic site using zinc fingers, TALEN or CRISPR / Cas9 nuclease (Eyquem 2017).

[0074] Example 2. mRNA CARs for transient expression of other mechanisms of anti-tumor activity

[0075] Alternatively, transiently expressed AI-CARs with other mechanisms of anti-tumor activity can be expressed from in vitro transcribed (IVT) RNA ( Figure 2 AC, SEQ ID 11-15). The coding region of the monospecific, dual or bispecific CAR is as follows Figure 1As shown in the construction. mRNA AI-CAR is expressed horizontally, so there is no need for the target of CAR cell elimination. Proteins for transient expression of additional anti-tumor activity include 3' fusions of IRES or P2A sequences, such as the coding region of cytokines or bispecific antibodies (SEQID 11-15), which are followed by polyA tails. mRNA AI-CAR is constructed in a vector for in vitro transcription (IVT). Therefore, the coding region is flanked by T7 promoter and polyA tail. The mRNA is transcribed with T7 polymerase, purified and electroporated into T or NK cells using standard methods. Additional proteins can also be co-expressed by co-transfecting their mRNA to enhance anti-tumor activity.

[0076] Example 3. Example of AI-CAR design

[0077] CAR T cells with an undifferentiated memory phenotype are characterized by in vivo persistence and greatest therapeutic potential. Several cytokines, including IL15, IL7, and IL21, have been used to selectively expand CAR cells with this phenotype and prevent terminal differentiation. These cytokines can also promote T cell rejection of lymphomas (Markley 2010). Other proinflammatory cytokines can enhance the efficacy of CAR cells to eradicate tumors such as IL18 or IL12 (Chmielewski 2017, Kueberuwa 2018). Incorporating a combination of these cytokines into a CAR vector with expression regulated by CAR-bound tumor cells has several advantages, including easy manufacture of CAR cells (i.e., in vitro expansion), enabling point-of-care treatment, enhancing the activity and persistence of CAR cells, and potentially inducing or enhancing anti-tumor responses in patients.

[0078] Two types of CARs targeting CD19 and CD22 or CD20 are designed as Figure 3 The bispecific CAR complex contains two independently configured receptors targeting CD19 and CD22, respectively. Figure 3 A). The bispecific CAR can be a classic second generation CAR, but contains two scFvs that bind to CD19 and CD22 or CD20 ( Figure 3 B).

[0079] After binding to the tumor target antigen, AI-CAR can activate the transcription factor bound to the inducible promoter of the integrated CAR vector to induce the expression of genes that support AI-CAR cell persistence, undifferentiated memory phenotype and efficacy through additional anti-tumor mechanisms (SEQ ID 9, 10, 16, 17). For example, these activities can be provided by certain cytokines and by cytotoxic bispecific antibodies such as anti-CD20xNKG2D to target NK cell toxicity to tumor cells. Relative to constitutive expression, the transient expression of additional cytotoxic mechanisms reduces the risk of, for example, extra-tumor cytotoxicity. Alternatively, other mechanisms of anti-tumor activity can also be transiently expressed using mRNA AI-CAR (SEQ ID 11-17).

[0080] Example 4. Humanized anti-CD19 scFv-Fc and anti-CD22 scFv-Fc specifically bind to target antigens

[0081] To demonstrate the functionality of humanized anti-CD19 scFv and anti-CD22 scFv for AI-CAR construction, scFv was fused to Fc and expressed in HEK-293 cells. Anti-CD19 and anti-CD22 scFv specifically bind to SW480 cells that have been transfected with CD19 or CD20 mRNA. scFv does not bind to mock transfectants or cells transfected with CD20 mRNA. Binding to CD19, CD22, and CD20 mRNA was determined 1 day after SW480 electroporation. Binding was determined by standard flow cytometry.

[0082] Example 5. CD19 and CD22 binding to CAR T cells

[0083] Humanized dual and bispecific CARs were expressed in T cells to demonstrate their binding to CD19 and CD22. T cells were electroporated with mRNA encoding different CARs. A total of 50 or 100 μg of RNA was used per 50 × 10^6 cells (double), and the combination of CD19 and CD22 CAR RNA was co-electroporated into T cells. CD19 and CD22 CAR RNA were electroporated into T cells individually using 25 or 50 μg of each RNA per 50 × 10^6 cells. Individual or monospecific CAR cells (double individuals) were then pooled. One day after electroporation, CAR expression was confirmed by standard flow cytometry to determine the binding of protein-L. Soluble recombinant CD19-Fc and CD22-Fc specifically bind to dual and bispecific CARs without simulating transfected T cells. The data show that a greater percentage of dual or co-electroporated T cells bind CD19 and CD22 relative to electroporated and pooled CAR T cells alone.

[0084] Example 6. Summary of CAR expression on IL-15-expanded T cells on day 1 after electroporation

[0085] Figure 6The percentage and level (median fluorescence intensity) of CD19-Fc and CD22-Fc binding to T cells expressing humanized CD19 and CD22 RNA CARs are summarized one day after electroporation. T cells were electroporated with different amounts of CAR RNA as shown. The binding of protein-L determined by standard flow cytometry demonstrated CAR expression.

[0086] Example 7. Monospecific, Dual and Bispecific CD19 and CD22 CAR T Cell Cytotoxicity

[0087] The cytotoxic activity of monospecific, dual and bispecific CD19 and CD22 CAR cells was determined. T cells were electroporated with CAR RNA, and SW480 expressing luciferase was electroporated with CD19, CD22 or a transmembrane form of both. CD20 is also expressed in SW480 as a negative control. One day later, the CAR T cells were co-incubated with target cells at an effector cell to target cell ratio of 0:1 to 20:1. Cytotoxicity was determined at 24 hours by measuring residual luciferase activity using a multimode plate reader. The monospecific CD19 CAR showed the most effective killing, followed by dual CD19, CD22 CAR cells. However, dual CD19, CD22 CAR cells showed the greatest background killing of mock and CD20-expressing targets. This nonspecific activity may reflect the higher level of input RNA in co-electroporation, which is twice the amount used for other electroporations (a total of 100 μg per 50×10^6 T cells). This was resolved in subsequent cytotoxicity assays ( Figure 8 ).

[0088] Example 8. Dual and Bispecific CD19 and CD22 CAR T Cell Cytotoxicity

[0089] The cytotoxic activity of dual and bispecific CD19 and CD22 CAR cells was determined. T cells were electroporated with CAR RNA, and SW480 expressing luciferase was electroporated with CD19, CD22, or a transmembrane form of both. CD20 was also expressed as a negative control. One day later, the CAR T cells were co-incubated with target cells at an effector cell to target cell ratio of 0: 1 to 20: 1. The residual luciferase activity was determined using a multimode plate reader, and cytotoxicity was determined at 24 hours. The data show that dual CAR co-electroporated with 25 μg CD19 RNA and 25 μg CD22 RNA (per 50×10^6 cells) has a greater specific activity than dual CAR electroporated with 2× more RNA. In addition, the dual CAR format exhibits greater activity relative to the bispecific CAR.

[0090] Sequence Listing

[0091] 1. Humanized CD19 CAR sequence. Humanized CD19 scFv from FMC63 mouse antibody ((Nicholson 1997)) was fused to CD8 stem / hinge, CD8 transmembrane domain, CD137 endodomain and CD3ζ endodomain. A safe target was fused using P2A peptide.

[0092] SEQ ID NO:1

[0093] h1FM19C7tER

[0094] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSETLSLTCTVSGGSIPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHYYYGG SYAMDYWGQGTSLTVSSGGGGSGGGGSGGGSGDIQMTQSPSSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ GNTLPYTFGGGTKVEITGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGPMNAKVVVVLVLVLTALCLSDGRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR

[0095] SEQ ID NO:2

[0096] h2FM19C7tER

[0097] MKHLWFFLLLVAAPRWVLSDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGGAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQHPGKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHYYYGGSYAMDYWGQGTLVTVSSGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGPMNAKVVVVLVLVLTALCLSDGRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR

[0098] SEQ ID NO:3

[0099] h3FM19C7tER

[0100] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSLTCTVSGVSIPDYGVSWIRQHPGKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPYTFGGGTKVEIK GAP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGP MNAKVVVVLVLVLTALCLSDGRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKII SNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR

[0101] 2. Humanized CD22 CAR sequence. A humanized CD22-specific scFv from the RFB4 mouse antibody (Mansfeild 1997) was fused to the CD8 stem / hinge, CD8 transmembrane domain, CD137 endodomain, and CD3ζ endodomain.

[0102] SEQ ID NO:4

[0103] h1RF22C7tER

[0104] MKHLWFFLLLVAAPRWVLSEVQLVESGGGLVQPGGSLRLSCAASGFAFSIYDMSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTVSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPWTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGP MNAKVVVVLVLVLTALCLSDGRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR

[0105] SEQ ID NO:5

[0106] h2RF22C7tER

[0107] MKHLWFFLLLVAAPRWVLSEVQLVESGGGLVQPGGSLRLSCAASGFTFSIYDMSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQQEDFATYYCQQGNTLPWTFGGGTKVEIK GAP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGP MNAKVVVVLVLVLTALCLSDGRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR

[0108] 3. Humanized CD19 and CD22 bispecific CAR sequences. A bispecific CAR targeting CD19 and CD22 can be generated by fusing a CD19 scFv (with a leader) to a linker sequence, followed by fusing a CD22 scFv, a CD8 stem / hinge, a CD8 transmembrane domain, a CD137 endodomain, and a CD3ζ endodomain.

[0109] SEQ ID NO:6

[0110] hRC19hRF22C7tER

[0111] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQHPGKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSSLSASVGDRVTITCRASQDISKYLNWYQQKPGGAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPYTFGGGTKVEIKPAGGGEPKSSDKTHTGGASEVQLVESGGGLVQPGGSLRLSCAASGFAF sI MSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTVSGGGGSGGGGSGGGGSDIQMT QSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPWTFGGGTKVEIKGAPTTTPA PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR ASGSGATNFSLLKQAGDVEEN PGP MNAKVVVVLVLVLTALCLSDGRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR

[0112] SEQ ID NO:7

[0113] h3FM19h2RF22C7tER

[0114] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSLTCTVSGVSIPDYGVSWIRQHPGKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGKAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPYTFGGGTKVEIKPAGGGEPKSSDKTHTGGASEVQLVESGGGLVQPGGSLRLSCAASGFTFSIYDMSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQQEDFATYYCQQGNTLPWTFGGGTKVEIK GAP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGPMNAKVVVVLVLVLTALCLSDGRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKII SNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR

[0115] 4. Humanized CD19 and CD22 dual CAR sequences. Dual CARs targeting CD19 and CD22 can be generated by fusing CD19 CAR (with leader) to a ribosomal skipping peptide, such as T2A or P2A, followed by fusing CD22 CAR.

[0116] SEQ ID NO:8

[0117] h2FM19C7xh2RF22C7tER

[0118] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSLTCTVSGVSLPDYGVSWIRQHPGKGLEWIGVIWGSETTYYNSALKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISKYLNWYQQKPGGAPKLLIYHTSRLHSGVPSRFSGSGSGTDFTFTISSLQQEDIATYYCQQGNTLPYTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPEVQLVESGGGLVQPGGSLRLSCAASGFTFSIYDMSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQQEDFATYYCQQGNTLPWTFGGGTKVEIK GAP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGP MNAKVVVVLVLVLTALCLSDGRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKII SNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRR

[0119] 5. Examples of combined expression of cytokines in CAR cells to enhance anti-tumor activity.

[0120] The combination of IL7, IL15 or IL21 can be transiently expressed in CAR cells to activate CAR cells or host T and NK cells to induce proliferation and anti-tumor cytotoxicity. The combination sequence of IL7, IL15 and IL21 can be composed of a signal peptide, a cytokine, a ribosomal skipping peptide such as P2A, and another cytokine.

[0121] SEQ ID NO:9

[0122] IL7 x IL21

[0123] MKHLWFFLLLVAAPRWVLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNNKILMGTKEH PAGGGTKTESSSRGQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0124] SEQ ID NO:10

[0125] IL7 x IL15

[0126] MKHLWFFLLLVAAPRWVLSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH PAG GGTKTESSSRGGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0127] 6. Examples of RNA CAR:

[0128] SEQ ID NO:11

[0129] <h2 style=";text-align:left;direction:ltr">h5RM19C7-P2A-h2RF22U1(<h2 style=";text-align:left;direction:ltr"> Figure 2 <h2 style=";text-align:left;direction:ltr"> A)

[0130] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKMSSVTAADTAVYYCA K HYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC RASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISSLQPEDIATYFC QQGNTLPYT FGGGTKVEIK GAP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGP MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSIYDMSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQQEDFATYYCQQGNTLPWTFGGGTKVEIK PAG GGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKP

[0131] SEQ ID NO:12

[0132] <h2 style=";text-align:left;direction:ltr">h5RM19C7-P2A-IL18(<h2 style=";text-align:left;direction:ltr"> Figure 2 <h2 style=";text-align:left;direction:ltr"> A)

[0133] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKMSSVTAADTAVYYCA K HYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC RASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISSLQPEDIATYFC QQGNTLPYT FGGGTKVEIK GAP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGP MEFGLSWVFLVALLRGVQCYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0134] SEQ ID NO:13

[0135] <h2 style=";text-align:left;direction:ltr">h5RM19C7-P2A-h2RF22C7-P2A-IL18(<h2 style=";text-align:left;direction:ltr"> Figure 2 <h2 style=";text-align:left;direction:ltr"> B)

[0136] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKMSSVTAADTAVYYCA KHYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC RASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISSLQPEDIATYFC QQGNTLPYT FGGGTKVEIK GAP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGP MKHLWFFLLLVAAPRWVLSEVQLVESGGGLVQPGGSLRLSCAASGFTFSIYDMSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQQEDFATYYCQQGNTLPWTFGGGTKVEIKPAGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR GS GATNFSLLKQAGDVEEN PGPMEFGLSWVFLVALLRGVQCYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0137] SEQ ID NO:14

[0138] <h2 style=";text-align:left;direction:ltr">h5RM19C7-P2A-h2RF22C7-P2A-IL12B-IL12A(<h2 style=";text-align:left;direction:ltr"> Figure 2 <h2 style=";text-align:left;direction:ltr"> B)

[0139] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKMSSVTAADTAVYYCA K HYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC RASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISSLQPEDIATYFC QQGNTLPYT FGGGTKVEIK GAP TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR AS GSGATNFSLLKQAGDVEEN PGPMKHLWFFLLLVAAPRWVLSEVQLVESGGGLVQPGGSLRLSCAASGFTFSIYDMSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQQEDFATYYCQQGNTLPWTFGGGTKVEIKPAGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR GS GATNFSLLKQAGDVEEN PGPMEFGLSWVFLVALLRGVQCIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNY SGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYSTSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYF SLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSSEWASVPCSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACL PLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

[0140] SEQ ID NO:15

[0141] h5RM19scFv-linker-h2RF22C7-P2A-IL18( Figure 2 C)

[0142] MKHLWFFLLLVAAPRWVLSQVQLQESGPGLVKPSQTLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTISKDNSKSQVSLKMSSVTAADTAVYYCA K HYYYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITC RASQDISKYLN WYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISSLQPEDIATYFC QQGNTLPYTFGGGTKVEIKPAGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSIYDMSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSILHSGVPSRFSGSGSGTDFTLTISSLQQEDFATYYCQQGNTLPWTFGGGTKVEIKGAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR GS GATNFSLLKQAGDVEEN PGP MEFGLSWVFLVALLRGVQCYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0143] 7. Examples of additional inducible genes:

[0144] SEQ ID NO:16

[0145] h2RF22U1-P2A-IL18( Figure 1 ; inducible gene; anti-CD19xCD3 bispecific antibody and IL18)

[0146] MEFGLSWVFLVALLRGVQCEVQLVESGGGLVQPGGSLRLSCAASGFTFSIYDMSWVRQAPGKGLEWVSYISSGGGTTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHSGYGSSYGVLFAYWGQGTLVTGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLILIYTSILHSGVPSRFSGSGTDFTLTISSLQQEDFATYYCQQGNTLPWTFGGGTKVEIK PAG GGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESPSGVRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKP GS GATNFSLLKQAGDVEEN PGP MEFGLSWVFLVALLRGVQCYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0147] SEQ ID NO:17

[0148] IL12B-Connector-IL12A( Figure 1 ; Inducible gene; Fusion of IL12B and IL12A chains)

[0149] MKHLWFFLLLVAAPRWVLSIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNY SGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYF SLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACL PLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS Sequence Listing <110> Abeile Pharmaceutical Technology Co., Ltd. <120> Artificial immune surveillance chimeric antigen receptor and its expressing cells <130> AVAR1901PCT <141> 2020-02-21 <150> 62 / 808,815 <151> 2019-02-21 <150> 62 / 808,823 <151> 2019-02-21 <150> 62 / 808,830 <151> 2019-02-21 <150> 62 / 808,833 <151> 2019-02-21 <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> 488 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 1 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 Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro 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 Ser Leu Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly 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 Lys 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 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe 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 Thr 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 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <210> 2 <211> 488 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 2 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile 35 40 45 Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Ala Pro Lys 50 55 60 Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 85 90 95 Leu Gln Gln Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr 100 105 110 Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln 130 135 140 Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser 145 150 155 160 Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser 165 170 175 Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Val Ile 180 185 190 Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Val 195 200 205 Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser 210 215 220 Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Tyr 225 230 235 240 Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 245 250 255 Val Thr Val Ser Ser 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 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <210> 3 <211> 488 <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 Ile 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 Lys 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 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <210> 4 <211> 490 <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 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 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 290 295 300 Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 305 310 315 320 Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys 325 330 335 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 340 345 350 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro 355 360 365 Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser 370 375 380 Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu 385 390 395 400 Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg 405 410 415 Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro 420 425 430 Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 435 440 445 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His 450 455 460 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 465 470 475 480 Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 5 <211> 488 <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 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 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 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 Asn Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Ile 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 Leu Thr Ile Ser Ser Leu Gln Gln Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 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 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg 485 <210> 6 <211> 751 <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 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 Pro Ala Gly Gly Gly Glu Pro Lys Ser Ser Asp 260 265 270 Lys Thr His Thr Gly Gly Ala Ser Glu Val Gln Leu Val Glu Ser Gly 275 280 285 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 290 295 300 Ser Gly Phe Ala Phe Ser Ile Tyr Asp Met Ser Trp Val Arg Gln Ala 305 310 315 320 Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Ser Gly Gly Gly 325 330 335 Thr Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg 340 345 350 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 355 360 365 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Ser Gly Tyr Gly Ser 370 375 380 Ser Tyr Gly Val Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 385 390 395 400 Val Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 405 410 415 Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 420 425 430 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn 435 440 445 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 450 455 460 Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser 465 470 475 480 Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln 485 490 495 Gln Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 500 505 510 Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ala Pro Thr 515 520 525 Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser 530 535 540 Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly 545 550 555 560 Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp 565 570 575 Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile 580 585 590 Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 595 600 605 Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys 610 615 620 Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val 625 630 635 640 Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn 645 650 655 Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val 660 665 670 Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln 675 680 685 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 690 695 700 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 705 710 715 720 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 725 730 735 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 740 745 750 <210> 7 <211> 749 <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 Ile 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 Lys 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 Pro Ala Gly Gly Gly Glu Pro Lys Ser Ser Asp 260 265 270 Lys Thr His Thr Gly Gly Ala Ser Glu Val Gln Leu Val Glu Ser Gly 275 280 285 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 290 295 300 Ser Gly Phe Thr Phe Ser Ile Tyr Asp Met Ser Trp Val Arg Gln Ala 305 310 315 320 Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser Ser Gly Gly Gly 325 330 335 Thr Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg 340 345 350 Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 355 360 365 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Ser Gly Tyr Gly Ser 370 375 380 Ser Tyr Gly Val Leu Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 385 390 395 400 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 405 410 415 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp 420 425 430 Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu 435 440 445 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 450 455 460 Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 465 470 475 480 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Gln Glu 485 490 495 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr 500 505 510 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ala Pro Thr Thr Thr 515 520 525 Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 530 535 540 Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 545 550 555 560 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro 565 570 575 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu 580 585 590 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 595 600 605 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 610 615 620 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 625 630 635 640 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 645 650 655 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 660 665 670 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg 675 680 685 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 690 695 700 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 705 710 715 720 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 725 730 735 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 740 745 <210> 8 <211> 979 <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 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 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser 485 490 495 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Glu Val 500 505 510 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 515 520 525 Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr Asp Met 530 535 540 Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr 545 550 555 560 Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Asp Thr Val Lys Gly 565 570 575 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln 580 585 590 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 595 600 605 His Ser Gly Tyr Gly Ser Ser Tyr Gly Val Leu Phe Ala Tyr Trp Gly 610 615 620 Gln Gly Thr Leu Val Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 625 630 635 640 Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu 645 650 655 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln 660 665 670 Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala 675 680 685 Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro 690 695 700 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 705 710 715 720 Ser Ser Leu Gln Gln Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly 725 730 735 Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 740 745 750 Gly Ala Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 755 760 765 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 770 775 780 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 785 790 795 800 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 805 810 815 Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu 820 825 830 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 835 840 845 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 850 855 860 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 865 870 875 880 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 885 890 895 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 900 905 910 Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 915 920 925 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 930 935 940 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 945 950 955 960 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 965 970 975 Pro Pro Arg <210> 9 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 9 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu 20 25 30 Ser Val Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu 35 40 45 Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His 50 55 60 Ile Cys Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg 65 70 75 80 Lys Leu Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu 85 90 95 His Leu Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr 100 105 110 Gly Gln Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro 115 120 125 Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu 130 135 140 Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys 145 150 155 160 Trp Asn Lys Ile Leu Met Gly Thr Lys Glu His Gly Ser Gly Ala Thr 165 170 175 Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly 180 185 190 Pro Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp 195 200 205 Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe 210 215 220 Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe 225 230 235 240 Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn 245 250 255 Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro 260 265 270 Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser 275 280 285 Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe 290 295 300 Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr 305 310 315 320 His Gly Ser Glu Asp Ser 325 <210> 10 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 10 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu 20 25 30 Ser Val Leu Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu 35 40 45 Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His 50 55 60 Ile Cys Asp Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg 65 70 75 80 Lys Leu Arg Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu 85 90 95 His Leu Leu Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr 100 105 110 Gly Gln Val Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro 115 120 125 Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu 130 135 140 Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys 145 150 155 160 Trp Asn Lys Ile Leu Met Gly Thr Lys Glu His Gly Ser Gly Ala Thr 165 170 175 Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly 180 185 190 Pro Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro 195 200 205 Lys Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile 210 215 220 Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu 225 230 235 240 Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu 245 250 255 Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His 260 265 270 Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser 275 280 285 Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu 290 295 300 Glu Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln 305 310 315 320 Met Phe Ile Asn Thr Ser 325 <210> 11 <211> 1026 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 11 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 Val Thr Cys Thr Val Ser Gly Val Ser Leu 35 40 45 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 65 70 75 80 Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Ser Leu Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Lys 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 Asp Gly Thr Val 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 Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr 225 230 235 240 Phe 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 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn 485 490 495 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 500 505 510 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 515 520 525 Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 530 535 540 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 545 550 555 560 Ser Ile Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 565 570 575 Glu Trp Val Ser Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro 580 585 590 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 595 600 605 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 610 615 620 Tyr Tyr Cys Ala Arg His Ser Gly Tyr Gly Ser Ser Tyr Gly Val Leu 625 630 635 640 Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Gly Gly Gly Gly Ser 645 650 655 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 660 665 670 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 675 680 685 Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln 690 695 700 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Ile Leu 705 710 715 720 His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 725 730 735 Phe Thr Leu Thr Ile Ser Ser Leu Gln Gln Glu Asp Phe Ala Thr Tyr 740 745 750 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr 755 760 765 Lys Val Glu Ile Lys Pro Ala Gly Gly Gly Gly Gly Ser Glu Val Gln 770 775 780 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 785 790 795 800 Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn 805 810 815 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile 820 825 830 Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg 835 840 845 Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met 850 855 860 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser 865 870 875 880 Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly 885 890 895 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 900 905 910 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 915 920 925 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 930 935 940 Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 945 950 955 960 Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val 965 970 975 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr 980 985 990 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 995 1000 1005 Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 1010 1015 1020 Lys Pro 1025 <210> 12 <211> 688 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 12 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 Val Thr Cys Thr Val Ser Gly Val Ser Leu 35 40 45 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 65 70 75 80 Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Ser Leu Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Lys 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 Asp Gly Thr Val 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 Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr 225 230 235 240 Phe 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 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn 485 490 495 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 500 505 510 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 515 520 525 Val Gln Cys Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg 530 535 540 Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu 545 550 555 560 Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr 565 570 575 Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala 580 585 590 Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu 595 600 605 Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys 610 615 620 Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His 625 630 635 640 Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu 645 650 655 Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu 660 665 670 Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp 675 680 685 <210> 13 <211> 1198 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 13 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 Val Thr Cys Thr Val Ser Gly Val Ser Leu 35 40 45 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 65 70 75 80 Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Ser Leu Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Lys 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 Asp Gly Thr Val 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 Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr 225 230 235 240 Phe 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 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn 485 490 495 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 500 505 510 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 515 520 525 Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 530 535 540 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 545 550 555 560 Ser Ile Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 565 570 575 Glu Trp Val Ser Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro 580 585 590 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 595 600 605 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 610 615 620 Tyr Tyr Cys Ala Arg His Ser Gly Tyr Gly Ser Ser Tyr Gly Val Leu 625 630 635 640 Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Gly Gly Gly Gly Ser 645 650 655 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 660 665 670 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 675 680 685 Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln 690 695 700 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Ile Leu 705 710 715 720 His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 725 730 735 Phe Thr Leu Thr Ile Ser Ser Leu Gln Gln Glu Asp Phe Ala Thr Tyr 740 745 750 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr 755 760 765 Lys Val Glu Ile Lys Pro Ala Gly Thr Thr Thr Pro Ala Pro Arg Pro 770 775 780 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 785 790 795 800 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 805 810 815 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 820 825 830 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 835 840 845 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 850 855 860 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 865 870 875 880 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 885 890 895 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 900 905 910 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 915 920 925 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 930 935 940 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 945 950 955 960 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 965 970 975 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 980 985 990 His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser 995 1000 1005 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu 1010 1015 1020 Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val Gln 1025 1030 1035 1040 Cys Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu 1045 1050 1055 Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu 1060 1065 1070 Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe 1075 1080 1085 Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr 1090 1095 1100 Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys 1105 1110 1115 1120 Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr 1125 1130 1135 Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn 1140 1145 1150 Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys 1155 1160 1165 Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu 1170 1175 1180 Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp 1185 1190 1195 <210> 14 <211> 1554 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 14 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 Val Thr Cys Thr Val Ser Gly Val Ser Leu 35 40 45 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 65 70 75 80 Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Ser Leu Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Lys 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 Asp Gly Thr Val 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 Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr 225 230 235 240 Phe 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 Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 290 295 300 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 305 310 315 320 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 325 330 335 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 340 345 350 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 355 360 365 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 370 375 380 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 385 390 395 400 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 405 410 415 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 420 425 430 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 435 440 445 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 450 455 460 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 465 470 475 480 His Met Gln Ala Leu Pro Pro Arg Ala Ser Gly Ser Gly Ala Thr Asn 485 490 495 Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 500 505 510 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 515 520 525 Val Leu Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 530 535 540 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 545 550 555 560 Ser Ile Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 565 570 575 Glu Trp Val Ser Tyr Ile Ser Ser Gly Gly Gly Thr Thr Tyr Tyr Pro 580 585 590 Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 595 600 605 Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 610 615 620 Tyr Tyr Cys Ala Arg His Ser Gly Tyr Gly Ser Ser Tyr Gly Val Leu 625 630 635 640 Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Gly Gly Gly Gly Ser 645 650 655 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 660 665 670 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 675 680 685 Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln 690 695 700 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Ile Leu 705 710 715 720 His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 725 730 735 Phe Thr Leu Thr Ile Ser Ser Leu Gln Gln Glu Asp Phe Ala Thr Tyr 740 745 750 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr 755 760 765 Lys Val Glu Ile Lys Pro Ala Gly Thr Thr Thr Pro Ala Pro Arg Pro 770 775 780 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 785 790 795 800 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 805 810 815 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 820 825 830 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 835 840 845 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 850 855 860 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 865 870 875 880 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp 885 890 895 Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 900 905 910 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 915 920 925 Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu 930 935 940 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 945 950 955 960 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 965 970 975 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 980 985 990 His Met Gln Ala Leu Pro Pro Arg Gly Ser Gly Ala Thr Asn Phe Ser 995 1000 1005 Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu 1010 1015 1020 Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly Val Gln 1025 1030 1035 1040 Cys Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu Asp Trp 1045 1050 1055 Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp Thr Pro 1060 1065 1070 Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu Val Leu 1075 1080 1085 Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly Asp Ala 1090 1095 1100 Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His Ser Leu 1105 1110 1115 1120 Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu 1125 1130 1135 Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala 1140 1145 1150 Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser 1155 1160 1165 Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser Asp Pro 1170 1175 1180 Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg Val Arg 1185 1190 1195 1200 Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser 1205 1210 1215 Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met Val Asp 1220 1225 1230 Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile 1235 1240 1245 Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu Lys Pro 1250 1255 1260 Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro Asp Thr 1265 1270 1275 1280 Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val Gln Val 1285 1290 1295 Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr Asp Lys 1300 1305 1310 Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser Val Arg 1315 1320 1325 Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala Ser Val 1330 1335 1340 Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Asn Leu 1345 1350 1355 1360 Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His His Ser 1365 1370 1375 Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln 1380 1385 1390 Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp 1395 1400 1405 Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu 1410 1415 1420 Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile 1425 1430 1435 1440 Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala 1445 1450 1455 Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu 1460 1465 1470 Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile 1475 1480 1485 Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala 1490 1495 1500 Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu 1505 1510 1515 1520 Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala 1525 1530 1535 Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr Leu Asn 1540 1545 1550 Ala Ser <210> 15 <211> 935 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 15 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 Val Thr Cys Thr Val Ser Gly Val Ser Leu 35 40 45 Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 50 55 60 Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 65 70 75 80 Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln 85 90 95 Val Ser Leu Lys Met Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Lys 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 Asp Gly Thr Val 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 Tyr Ser Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr 225 230 235 240 Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr 245 250 255 Lys Val Glu Ile Lys Pro Ala Gly Gly Gly Gly Ser Glu Val Gln Leu 260 265 270 Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu 275 280 285 Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr Asp Met Ser Trp 290 295 300 Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Tyr Ile Ser 305 310 315 320 Ser Gly Gly Gly Thr Thr Tyr Tyr Pro Asp Thr Val Lys Gly Arg Phe 325 330 335 Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn 340 345 350 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg His Ser 355 360 365 Gly Tyr Gly Ser Ser Tyr Gly Val Leu Phe Ala Tyr Trp Gly Gln Gly 370 375 380 Thr Leu Val Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 385 390 395 400 Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala 405 410 415 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile 420 425 430 Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 435 440 445 Leu Leu Ile Tyr Tyr Thr Ser Ile Leu His Ser Gly Val Pro Ser Arg 450 455 460 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 465 470 475 480 Leu Gln Gln Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr 485 490 495 Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly Ala 500 505 510 Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile 515 520 525 Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala 530 535 540 Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr 545 550 555 560 Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu 565 570 575 Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile 580 585 590 Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp 595 600 605 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 610 615 620 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 625 630 635 640 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 645 650 655 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 660 665 670 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 675 680 685 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 690 695 700 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 705 710 715 720 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 725 730 735 Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp 740 745 750 Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly Leu Ser Trp Val Phe 755 760 765 Leu Val Ala Leu Leu Arg Gly Val Gln Cys Tyr Phe Gly Lys Leu Glu 770 775 780 Ser Lys Leu Ser Val Ile Arg Asn Leu Asn Asp Gln Val Leu Phe Ile 785 790 795 800 Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp Met Thr Asp Ser Asp Cys 805 810 815 Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp 820 825 830 Ser Gln Pro Arg Gly Met Ala Val Thr Ile Ser Val Lys Cys Glu Lys 835 840 845 Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile Ile Ser Phe Lys Glu Met 850 855 860 Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe 865 870 875 880 Gln Arg Ser Val Pro Gly His Asp Asn Lys Met Gln Phe Glu Ser Ser 885 890 895 Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe 900 905 910 Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu Gly Asp Arg Ser Ile Met 915 920 925 Phe Thr Val Gln Asn Glu Asp 930 935 <210> 16 <211> 712 <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 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 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 Asn Tyr Leu Asn Trp Tyr Gln Gln 180 185 190 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Ile 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 Leu Thr Ile Ser Ser Leu Gln Gln Glu Asp Phe Ala Thr Tyr 225 230 235 240 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr 245 250 255 Lys Val Glu Ile Lys Pro Ala Gly Gly Gly Gly Gly Ser Glu Val Gln 260 265 270 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 275 280 285 Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn 290 295 300 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile 305 310 315 320 Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg 325 330 335 Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met 340 345 350 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser 355 360 365 Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly 370 375 380 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 385 390 395 400 Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 405 410 415 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 420 425 430 Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 435 440 445 Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val 450 455 460 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr 465 470 475 480 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 485 490 495 Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 500 505 510 Lys Pro Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly 515 520 525 Asp Val Glu Glu Asn Pro Gly Pro Met Glu Phe Gly Leu Ser Trp Val 530 535 540 Phe Leu Val Ala Leu Leu Arg Gly Val Gln Cys Tyr Phe Gly Lys Leu 545 550 555 560 Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn Asp Gln Val Leu Phe 565 570 575 Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp Met Thr Asp Ser Asp 580 585 590 Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile Ile Ser Met Tyr Lys 595 600 605 Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile Ser Val Lys Cys Glu 610 615 620 Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile Ile Ser Phe Lys Glu 625 630 635 640 Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys Ser Asp Ile Ile Phe 645 650 655 Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys Met Gln Phe Glu Ser 660 665 670 Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu Lys Glu Arg Asp Leu 675 680 685 Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu Gly Asp Arg Ser Ile 690 695 700 Met Phe Thr Val Gln Asn Glu Asp 705 710 <210> 17 <211> 532 <212> PRT <213> Artificial Sequence <220> <223> Synthesized <400> 17 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Ile Trp Glu Leu Lys Lys Asp Val Tyr Val Val Glu Leu 20 25 30 Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu Thr Cys Asp 35 40 45 Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln Ser Ser Glu 50 55 60 Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys Glu Phe Gly 65 70 75 80 Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val Leu Ser His 85 90 95 Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp Ser Thr Asp 100 105 110 Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe Leu Arg Cys 115 120 125 Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp Leu Thr Thr 130 135 140 Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg Gly Ser Ser 145 150 155 160 Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser Ala Glu Arg 165 170 175 Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu Cys Gln Glu 180 185 190 Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile Glu Val Met 195 200 205 Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr Ser Ser Phe 210 215 220 Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln Leu 225 230 235 240 Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp Glu Tyr Pro 245 250 255 Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr Phe Cys Val 260 265 270 Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg Val Phe Thr 275 280 285 Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala Ser Ile Ser 290 295 300 Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser Glu Trp Ala 305 310 315 320 Ser Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg 325 330 335 Asn Leu Pro Val Ala Thr Pro Asp Pro Gly Met Phe Pro Cys Leu His 340 345 350 His Ser Gln Asn Leu Leu Arg Ala Val Ser Asn Met Leu Gln Lys Ala 355 360 365 Arg Gln Thr Leu Glu Phe Tyr Pro Cys Thr Ser Glu Glu Ile Asp His 370 375 380 Glu Asp Ile Thr Lys Asp Lys Thr Ser Thr Val Glu Ala Cys Leu Pro 385 390 395 400 Leu Glu Leu Thr Lys Asn Glu Ser Cys Leu Asn Ser Arg Glu Thr Ser 405 410 415 Phe Ile Thr Asn Gly Ser Cys Leu Ala Ser Arg Lys Thr Ser Phe Met 420 425 430 Met Ala Leu Cys Leu Ser Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln 435 440 445 Val Glu Phe Lys Thr Met Asn Ala Lys Leu Leu Met Asp Pro Lys Arg 450 455 460 Gln Ile Phe Leu Asp Gln Asn Met Leu Ala Val Ile Asp Glu Leu Met 465 470 475 480 Gln Ala Leu Asn Phe Asn Ser Glu Thr Val Pro Gln Lys Ser Ser Leu 485 490 495 Glu Glu Pro Asp Phe Tyr Lys Thr Lys Ile Lys Leu Cys Ile Leu Leu 500 505 510 His Ala Phe Arg Ile Arg Ala Val Thr Ile Asp Arg Val Met Ser Tyr 515 520 525 Leo Asn Ala Ser 530

Claims

1. A bispecific chimeric antigen receptor comprising an extracellular domain connected to an intercellular domain by a linker, wherein the extracellular domain comprises a first scFv connected to a second scFv, wherein the first scFv domain and the second scFv domain each independently have affinity for CD19 or CD22, wherein the first scFv domain and the second scFv domain have affinity for different antigens, and wherein the intercellular domain comprises a co-stimulatory endodomain and a CD3ξ domain, wherein the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8.

2. A biomolecular complex comprising the bispecific chimeric antigen receptor of claim 1 bound to a CD19 antigen and a CD22 antigen.

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