Chimeric antigen receptors and methods of use thereof

Abstract

To provide a heterodimeric conditionally active chimeric antigen receptor (CAR), a nucleic acid comprising a nucleotide sequence encoding CAR, and a genetically modified cell to produce the CAR.SOLUTION: Disclosed herein is a heterodimeric, conditionally active chimeric antigen receptor (CAR) comprising: a first polypeptide comprising a first member of a specific binding pair, a first modulatory domain; a first member of a dimerization pair, and a transmembrane domain interposed between the first member of a specific binding pair and the first modulatory domain; and a second polypeptide comprising a transmembrane domain, a second modulatory domain, a second member of the dimerization pair, and an intracellular signaling domain, or a CAR comprising: a first polypeptide comprising a first member of a specific binding pair, a modulatory domain, a first member of a dimerization pair, a transmembrane domain interposed between the first member of a specific binding pair and the modulatory domain; and a second polypeptide comprising a second member of the dimerization pair and an intracellular signaling domain.SELECTED DRAWING: None

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 61 / 765,585, filed Feb. 15, 2013, which is incorporated by reference in its entirety.

[0002] Statement on Federally Sponsored Research This invention was made with Government support under Grant Nos. EY016546 and GM101782 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Incorporation by reference of sequence listings provided as text files The sequence listing is provided herein as a text file "UCSF-464WO SeqList_ST25.txt," created on February 13, 2014, and having a size of 153 KB. The contents of the text file are incorporated herein by reference in their entirety.

[0004] Introduction In cell-based adoptive immunotherapy, immune cells isolated from a patient can be modified to express synthetic proteins that allow the cells to perform new therapeutic functions after they are later transferred back to the patient. An example of such a synthetic protein is a chimeric antigen receptor (CAR). An example of a CAR currently used is a fusion of an extracellular recognition domain (e.g., an antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. Upon antigen engagement, the intracellular signaling portion of the CAR can initiate an activation-related response in the immune cell, such as the release of cytolytic molecules to induce the death of tumor cells. However, such CARs do not have the ability to be pharmacologically controlled. There is a need in the art for conditionally activatable CARs that can be pharmacologically controlled. Summary of the Invention

[0005] The present disclosure provides a heterodimeric conditionally active chimeric antigen receptor (CAR), and a nucleic acid comprising a nucleotide sequence encoding the CAR. The present disclosure provides a cell genetically modified to produce the CAR. The CAR of the present disclosure can be used in a variety of methods, which are also disclosed. [The present invention 1001] A heterodimeric conditionally active chimeric antigen receptor (CAR), a) a first polypeptide, i) a first member of a specific binding pair; ii) a first regulatory domain; and iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between said first member of a specific binding pair and said first regulatory domain; a first polypeptide comprising: b) a second polypeptide, i) a transmembrane domain; ii) a second regulatory domain; and iii) a second member of the dimerization pair; and iv) intracellular signaling domains; The second polypeptide comprising: Contains or a) a first polypeptide, i) a first member of a specific binding pair; ii) a regulatory domain; and iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between said first member of a specific binding pair and said regulatory domain; a first polypeptide comprising: b) a second polypeptide, i) a second member of the dimerization pair; and ii) intracellular signaling domains; A second polypeptide comprising: The heterodimeric conditionally active chimeric antigen receptor (CAR). [The present invention 1002] The heterodimeric conditionally active CAR of the present invention 1001, wherein the first polypeptide comprises a hinge region interposed between the first member of the specific binding pair and the transmembrane domain. [The present invention 1003] The heterodimeric conditionally active CAR of the present invention 1001, wherein said first member of said specific binding pair is an antibody or antibody fragment, a ligand, or a receptor. [The present invention 1004] The heterodimeric conditionally active CAR of the present invention 1002, wherein the hinge region is an immunoglobulin IgG hinge region or a hinge derived from CD8. [The present invention 1005] The heterodimeric conditionally active CAR of the present invention 1001, wherein the first and second regulatory domains are selected from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD27, CD30, GITR, HVEM, DAP10, DAP12, and CD28. [The present invention 1006] The heterodimeric conditionally active CAR of the present invention 1001, wherein said intracellular signaling domain is selected from ZAP70 and CD3-zeta. [The present invention 1007] The heterodimeric, conditionally active CAR of the present invention, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). [The present invention 1008] The heterodimeric conditionally active CAR of the present invention 1001, wherein said first and second members of said dimerization pair form a homodimer in the presence of a small molecule dimerizer. [The present invention 1009] The heterodimeric conditionally active CAR of the present invention 1001, wherein said first and second members of said dimerization pair form a heterodimer in the presence of a small molecule dimerizer. [The present invention 1010] the first and second members of the dimerization pair being a) FK506 binding protein (FKBP) and FKBP, b) FKBP and calcineurin catalytic subunit A (CnA); c) FKBP and cyclophilin, d) FKBP and FKBP-rapamycin-related protein (FRB); e) gyrase B (GyrB) and GryB; f) dihydrofolate reductase (DHFR) and DHFR; g) DmrB and DmrB, h) PYL and ABI; i) Cry2 and CIP, j) GAI and GID1 1001. A heterodimeric conditionally active CAR of the present invention selected from: [The present invention 1011] i) the first and second regulatory domains are derived from 4-1BB; ii) the first and second members of the dimerization pair are FKBP and FRB, and ii) the signaling domain comprises an ITAM; A heterodimeric conditionally active CAR of the present invention 1001. [The present invention 1012] The heterodimeric conditionally active CAR of the present invention, wherein said first member of said specific binding pair is a single-chain Fv. [The present invention 1013] The heterodimeric, conditionally active CAR of the present invention 1001, wherein said first member of said specific binding pair binds to an epitope present on a cell, on a solid surface, or on a lipid bilayer. [The present invention 1014] The heterodimeric conditionally active CAR of the present invention 1013, wherein the cell is a cancer cell. [The present invention 1015] Mammalian cells genetically engineered to produce the heterodimeric, conditionally active CAR of the present invention. [The present invention 1016] The cell of the present invention 1015, which is a stem cell, a progenitor cell, or a cell derived from a stem cell or a progenitor cell. [The present invention 1017] The cell of the present invention, which is a T lymphocyte or a NK cell. [The present invention 1018] A nucleic acid comprising a nucleotide sequence encoding a heterodimeric conditionally active CAR of the present invention. [The present invention 1019] The nucleic acid of the invention, wherein the nucleotide sequence is operably linked to a T lymphocyte-specific promoter or a NK cell-specific promoter. [The present invention 1020] A nucleic acid of the invention which is an in vitro transcribed RNA. [The present invention 1021] A recombinant expression vector comprising a nucleic acid of the present invention. [The present invention 1022] A method of activating a T lymphocyte comprising contacting the T lymphocyte with a dimerizing agent and a second member of a specific binding pair, wherein the T lymphocyte is genetically modified to produce a heterodimeric, conditionally active CAR of the present invention, and in the presence of the dimerizing agent and the second member of a specific binding pair, the heterodimeric, conditionally active CAR dimerizes and activates the T lymphocyte, thereby producing an activated T lymphocyte. [The present invention 1023] The method of claim 1022, wherein said second member of the specific binding pair is an antigen. [The present invention 1024] The method of claim 1022, wherein said contacting occurs in vivo. [The present invention 1025] The method of claim 1022, wherein said activated T lymphocytes mediate the killing of target cells. [The present invention 1026] The method of claim 1022, wherein said activated T lymphocytes produce IL-2 and / or IFN-γ. [The present invention 1027] The method of claim 1025, wherein said target cell is a cancer cell. [The present invention 1028] The method of claim 1022, wherein the first member of the specific binding pair of the heterodimeric conditionally active CAR is an antibody specific for an epitope on a cancer cell. [The present invention 1029] A method for producing a cell of the invention 1015, comprising genetically modifying a mammalian cell with an expression vector comprising a nucleotide sequence encoding a heterodimeric conditionally active CAR of the invention 1001, or genetically modifying a mammalian cell with an RNA comprising a nucleotide sequence encoding a heterodimeric conditionally active CAR of the invention 1001. [The present invention 1030] The method of claim 1029, wherein said genetic modification is carried out ex vivo. [The present invention 1031] The method of claim 1029, wherein said cell is a T lymphocyte, a stem cell, a NK cell, a progenitor cell, a cell derived from a stem cell, or a cell derived from a progenitor cell. [The present invention 1032] 1. A method of treating cancer in an individual, comprising: i) genetically modifying T lymphocytes obtained from said individual with an expression vector comprising a nucleotide sequence encoding a heterodimeric conditionally active CAR of the present invention, wherein the antigen-binding domain of said heterodimeric conditionally active CAR is specific for an epitope on cancer cells in said individual, and said genetic modification is performed ex vivo; ii) introducing the genetically modified T lymphocytes into the individual; and iii) administering to the individual an effective amount of a dimerizing agent, wherein the dimerizing agent induces dimerization of the heterodimeric conditionally active CAR, the dimerization providing activation of the genetically modified T lymphocytes and killing of the cancer cells, thereby treating the cancer. The method comprising: [The present invention 1033] The method of claim 1032, wherein the dimerizer is a rapalog. [The present invention 1034] A method for regulating an activity of a host cell, comprising contacting the host cell with a dimerizing agent and a second member of a specific binding pair, wherein a T lymphocyte is genetically modified to produce a heterodimeric conditionally active CAR of the present invention, and in the presence of the dimerizing agent and the second member of the specific binding pair, the heterodimeric conditionally active CAR dimerizes to regulate at least one activity of the host cell. [The present invention 1035] The method of claim 1034, wherein said activity is proliferation, cell survival, apoptosis, gene expression, or immune activation. [The present invention 1036] The method of claim 1034, wherein said second member of the specific binding pair is an antigen. [Brief description of the drawings]

[0006] [Figure 1A] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 122. [Figure 1B] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 122. [Figure 2A] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 123. [Figure 2B] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 123. [Figure 3A] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 125. [Figure 3B] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 125. [Figure 4] The nucleotide and amino acid sequences of the domain of construct no. 126 are provided. [Figure 5A] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 168. [Figure 5B] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 168. [Figure 6A]1 provides the nucleotide and amino acid sequences of the domain of construct no. 169. [Figure 6B] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 169. [Figure 6C] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 169. [Figure 7A] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 170. [Figure 7B] 1 provides the nucleotide and amino acid sequences of the domain of construct no. 170. [Figure 8A] The nucleotide and amino acid sequences of the domain of construct number 197 are provided. [Figure 8B] The nucleotide and amino acid sequences of the domain of construct number 197 are provided. [Figure 9A] The nucleotide and amino acid sequences of the domain of construct number 206 are provided. [Figure 9B] The nucleotide and amino acid sequences of the domain of construct number 206 are provided. [Figure 9C] The nucleotide and amino acid sequences of the domain of construct number 206 are provided. [Figure 10A] The nucleotide and amino acid sequences of the domain of construct number 207 are provided. [Figure 10B] The nucleotide and amino acid sequences of the domain of construct number 207 are provided. [Figure 11A] The nucleotide and amino acid sequences of the domain of construct number 199 are provided. [Figure 11B] The nucleotide and amino acid sequences of the domain of construct number 199 are provided. [Figure 11C] The nucleotide and amino acid sequences of the domain of construct number 199 are provided. [Figure 12] Depicting IL-2 production driven by five on-switch CAR mutants. [Figure 13] IL-2 production by a control Jurkat strain is depicted. [Figure 14] A comparison between CAR constructs "122+206" and "197+206" is depicted. [Figure 15] Cytotoxicity data is depicted using the on-switch CAR "197+206". [Figure 16] 1 depicts T cell activation data using CAR constructs "122+199", "197+199", and "122+168". [Figure 17] 1 is a schematic diagram of an exemplary on-switch CAR. [Figure 18A] 1 depicts various exemplary on-switch CARs. [Figure 18B] 1 depicts various exemplary on-switch CARs. [Figure 19A] Depicts IL-2 production driven by three different on-switch CAR mutants that recognize human mesothelin. [Figure 19B] Depicts IL-2 production driven by three different on-switch CAR mutants that recognize human mesothelin. [Figure 19C] Depicts IL-2 production driven by three different on-switch CAR mutants that recognize human mesothelin. [Figure 19D] Depicts IL-2 production driven by three different on-switch CAR mutants that recognize human mesothelin. [Figure 19E] Depicts IL-2 production driven by three different on-switch CAR mutants that recognize human mesothelin. [Figure 19F] Depicts IL-2 production driven by three different on-switch CAR mutants that recognize human mesothelin. [Figure 19G] Depicts IL-2 production driven by three different on-switch CAR mutants that recognize human mesothelin. [Figure 20A] 1 depicts IL-2 production triggered by an on-switch CAR mutant with a gibberellic acid-responsive dimerization pair. [Figure 20B]1 depicts IL-2 production triggered by an on-switch CAR mutant with a gibberellic acid-responsive dimerization pair. [Figure 20C] 1 depicts IL-2 production triggered by an on-switch CAR mutant with a gibberellic acid-responsive dimerization pair. [Figure 21A] 1 depicts exemplary on-switch and conventional CARs with various costimulatory domains. [Figure 21B] 1 depicts exemplary on-switch and conventional CARs with various costimulatory domains. [Figure 21C] 1 depicts exemplary on-switch and conventional CARs with various costimulatory domains. [Figure 21D] 1 depicts exemplary on-switch and conventional CARs with various costimulatory domains. [Figure 22A] The nucleotide and amino acid sequences of the domain of construct number 270 are provided. [Figure 22B] The nucleotide and amino acid sequences of the domain of construct number 270 are provided. [Figure 23A] The nucleotide and amino acid sequences of the domain of construct number 300 are provided. [Figure 23B] The nucleotide and amino acid sequences of the domain of construct number 300 are provided. [Figure 24A] The nucleotide and amino acid sequences of the domain of construct number 336 are provided. [Figure 24B] The nucleotide and amino acid sequences of the domain of construct number 336 are provided. [Figure 25A] 1 provides the nucleotide and amino acid sequences of the domain of construct number 337. [Figure 25B] 1 provides the nucleotide and amino acid sequences of the domain of construct number 337. [Figure 26A] 1 provides the nucleotide and amino acid sequences of the domain of construct number 357. [Figure 26B]1 provides the nucleotide and amino acid sequences of the domain of construct number 357. [Figure 27A] The nucleotide and amino acid sequences of the domain of construct no. 365 are provided. [Figure 27B] The nucleotide and amino acid sequences of the domain of construct no. 365 are provided. [Figure 28A] The nucleotide and amino acid sequences of the domain of construct no. 366 are provided. [Figure 28B] The nucleotide and amino acid sequences of the domain of construct no. 366 are provided. [Figure 29A] The nucleotide and amino acid sequences of the domain of construct no. 367 are provided. [Figure 29B] The nucleotide and amino acid sequences of the domain of construct no. 367 are provided. [Figure 30A] The nucleotide and amino acid sequences of the domain of construct number 398 are provided. [Figure 30B] The nucleotide and amino acid sequences of the domain of construct number 398 are provided. [Figure 31A] The nucleotide and amino acid sequences of the domain of construct number 399 are provided. [Figure 31B] The nucleotide and amino acid sequences of the domain of construct number 399 are provided. [Figure 32A] The nucleotide and amino acid sequences of the domain of construct no. 400 are provided. [Figure 32B] The nucleotide and amino acid sequences of the domain of construct no. 400 are provided. [Figure 33A] 1 provides the nucleotide and amino acid sequences of the domain of construct number 358. [Figure 33B] 1 provides the nucleotide and amino acid sequences of the domain of construct number 358. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] definition The terms "polynucleotide" and "nucleic acid," as used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0008] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and fusion proteins comprising antigen-binding portions of antibodies and non-antibody proteins.

[0009] "Antibody fragments" include portions of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bispecific antibodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062(1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields F(ab')2, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0010] "Single-chain Fv" or "sFv" antibody fragments are fragments of the V H and V L In some embodiments, an Fv polypeptide comprises a V domain, and these domains are present in a single polypeptide chain. H Domain and V LIt further comprises a polypeptide linker between the domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0011] As used herein, the term "affinity" refers to the equilibrium constant of reversible binding of two substances, expressed as the dissociation constant (Kd). The affinity can be at least 1-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 20-fold higher, at least 30-fold higher, at least 40-fold higher, at least 50-fold higher, at least 60-fold higher, at least 70-fold higher, at least 80-fold higher, at least 90-fold higher, at least 100-fold higher, or at least 1000-fold higher, or more, than the affinity of the antibody to an unrelated amino acid sequence. The affinity of the antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more substances to dissociation upon dilution. The terms "immunoreactive" and "preferential binding" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0012] The term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. Nonspecific binding is approximately 10 -7 Binding with an affinity of less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 It may refer to binding with an affinity such as M.

[0013] As used herein, the term "hinge region" refers to a flexible polypeptide connector region (also referred to herein as a "hinge" or "spacer") that provides structural flexibility and spacing to adjacent polypeptide regions and may be composed of natural or synthetic polypeptides. A "hinge region" from an immunoglobulin (e.g., IgG1) is generally derived from the Glu of human IgG1. 216 From Pro 230 (Burton (1985) Molec. Immunol., 22:161-206). Hinge regions of other IgG isotypes may be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide (SS) bond in the same positions. The hinge region may be naturally occurring or non-naturally occurring, including but not limited to modified hinge regions described in U.S. Pat. No. 5,677,425. The hinge region may include a complete hinge region derived from a class or subclass different from that of the CH1 domain. The term "hinge region" may also include regions derived from CD8 and other receptors that provide a similar function in providing flexibility and spacing to adjacent regions.

[0014] An "isolated" polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminating components of its natural environment are substances that may interfere with diagnostic or therapeutic uses for the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide may be purified (1) to greater than 90%, greater than 95%, or greater than 98%, e.g., greater than 99%, by weight of the antibody as determined by the Lowry method, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. Since at least one component of the polypeptide's natural environment may not be present, an isolated polypeptide includes the polypeptide in situ in a recombinant cell. In some examples, an isolated polypeptide may be prepared by at least one purification step.

[0015] As used herein, the term "immune cells" includes white blood cells (leukocytes), which are generally derived from hematopoietic stem cells (HSCs) generated in the bone marrow. "Immune cells" include, for example, lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells).

[0016] "T cells" are T helper cells (CD4 + cells), cytotoxic T cells (CD8 + These include all types of immune cells that express CD3, including T-cells, T regulatory cells (Tregs), and gamma delta T cells.

[0017] "Cytotoxic cells" are CD8 + These cells include T cells, natural killer (NK) cells, and neutrophils, and these cells can mediate cytotoxic responses.

[0018] As used herein, the term "stem cells" generally includes pluripotent or multipotent stem cells. "Stem cells" include, for example, embryonic stem cells (ES), mesenchymal stem cells (MSC), induced pluripotent stem cells (iPS), and committed progenitor cells (hematopoietic stem cells (HSC), bone marrow derived cells, etc.).

[0019] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic, in that it partially or completely treats the disease and / or the adverse effects attributable to the disease. "Treatment", as used herein, encompasses any treatment of a mammalian, e.g., human, disease, and includes (a) preventing the disease from developing in a patient who may be susceptible to the disease but has not yet been diagnosed as having it, (b) inhibiting the disease, i.e., arresting its progression, and (c) relieving the disease, i.e., causing the disease to recede.

[0020] The terms "individual," "subject," "host," and "patient," as used interchangeably herein, refer to mammals, including, but not limited to, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, canines, felines, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0021] A "therapeutically effective amount" or "effective amount" refers to an amount of a substance, or a combined amount of two substances, that when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" can vary depending on the substance(s), the disease and its severity, as well as the age, weight, etc., of the subject being treated.

[0022] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0023] Where a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, as well as any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded value in the stated range. Where a stated range includes one or both of its upper and lower limits, ranges excluding either or both of those included upper and lower limits are also encompassed within the invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0025] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "chimeric antigen receptor" includes a plurality of such chimeric antigen receptors, a reference to a "dimerizer-binding pair" includes one or more dimerizer-binding pairs and equivalents thereof known to those skilled in the art, and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate to the use of exclusive language such as "solely," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claim elements.

[0026] For clarity, it is understood that certain features of the invention that are described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention that are described in the context of a single embodiment may be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination was individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are specifically embraced by the present invention and are disclosed herein as if each and every such subcombination was individually and expressly disclosed herein.

[0027] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0028] Detailed Description The present disclosure provides a heterodimeric conditionally active chimeric antigen receptor (CAR), and a nucleic acid comprising a nucleotide sequence encoding the CAR. The present disclosure provides a cell genetically modified to produce the CAR. The CAR of the present disclosure can be used in a variety of methods, which are also disclosed.

[0029] Heterodimeric, conditionally active chimeric antigen receptors The present disclosure provides heterodimeric, conditionally active chimeric antigen receptors, which for simplicity are referred to herein as "CARs."

[0030] In some embodiments, a CAR of the present disclosure comprises a) a first polypeptide comprising i) a member of a specific binding pair (e.g., an antigen-binding domain), ii) a first regulatory domain, iii) a first member of a dimerization pair, and iv) a transmembrane domain interposed between the member of the specific binding pair (e.g., the antigen-binding domain) and the first regulatory domain, and b) a second polypeptide comprising i) a transmembrane domain, ii) a second regulatory domain, iii) a second member of the dimerization pair, and iv) an intracellular signaling domain. The regulatory domain can be a costimulatory domain.

[0031] In some embodiments, a CAR of the disclosure comprises a) a first polypeptide comprising i) a member of a specific binding pair (e.g., an antigen-binding domain), ii) a first costimulatory domain, iii) a first member of a dimerization pair (e.g., a dimerizer-binding pair), and iv) a transmembrane domain interposed between the member of the specific binding pair (e.g., the antigen-binding domain) and the first costimulatory domain, and b) a second polypeptide comprising i) a transmembrane domain, ii) a second costimulatory domain, iii) a second member of the dimerization pair (e.g., a dimerizer-binding pair), and iv) an intracellular signaling domain.

[0032] In some embodiments, a CAR of the disclosure comprises a) a first polypeptide comprising i) a member of a specific binding pair (e.g., an antigen-binding domain), ii) a regulatory domain, iii) a first member of a dimerization pair (e.g., a dimerizer-binding pair), and iv) a transmembrane domain interposed between the member of the specific binding pair (e.g., the antigen-binding domain) and the regulatory domain, and b) a second polypeptide comprising i) a second member of the dimerization pair (e.g., a dimerizer-binding pair), and ii) an intracellular signaling domain. The regulatory domain can be a costimulatory domain.

[0033] In some embodiments, a CAR of the disclosure comprises a) a first polypeptide comprising i) a member of a specific binding pair (e.g., an antigen binding domain), ii) a costimulatory domain, iii) a first member of a dimerization pair (e.g., a dimerizer binding pair), and iv) a transmembrane domain interposed between the member of the specific binding pair (e.g., the antigen binding domain) and the costimulatory domain, and b) a second polypeptide comprising i) the second member of the dimerization pair (e.g., a dimerizer binding pair), and ii) an intracellular signaling domain.

[0034] An example of a CAR of the present application is depicted diagrammatically in FIG. 17. A CAR of the present disclosure may reside within the plasma membrane of a eukaryotic cell, such as a mammalian cell, suitable mammalian cells including, but not limited to, cytotoxic cells, T lymphocytes, stem cells, progeny of stem cells, progenitor cells, progeny of progenitor cells, and NK cells. When present within the plasma membrane of a eukaryotic cell, a CAR of the present disclosure is active in the presence of: 1) a dimerizer that binds to the first and second members of a dimerizer binding pair in the CAR or otherwise induces dimerization of the first and second members of the dimer, and 2) an agent that binds a member of a specific binding pair (e.g., an antigen binding domain), such as an antigen that binds to the antigen binding domain of the CAR. The agent that binds to the member of the specific binding pair is the second member of the specific binding pair. The second member of the specific binding pair may be a soluble (e.g., not bound to a cell) agent, an agent that is present on the surface of a cell, such as a target cell, an agent provided on a solid surface, an agent that is present within a lipid bilayer, and the like. Where a member of the specific binding pair is an antibody and the second member of the specific binding pair is an antigen, the antigen can be a soluble (e.g., not cell bound) antigen, an antigen present on the surface of a cell, such as a target cell, an antigen provided on a solid surface, an antigen present within a lipid bilayer, etc.

[0035] In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and activated by a second member of the specific binding pair (e.g., an antigen that binds to the antigen binding domain of the CAR) that binds to a member of the specific binding pair of the CAR, and a dimerization agent, increases expression of at least one nucleic acid in the cell. For example, in some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and activated by an antigen that binds to the antigen binding domain of the CAR, and a dimerization agent, increases expression of at least one nucleic acid in the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the transcription level of the nucleic acid in the absence of the antigen and / or dimerization agent.

[0036] For example, the second polypeptide of a CAR of the present disclosure can include an immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptide, in which case the CAR of the present disclosure increases nuclear factor of activated T cells (NFAT)-dependent transcription when present in the plasma membrane of a eukaryotic cell and when activated by antigen and a dimerizer that binds to the antigen-binding domain of the CAR. NFAT-dependent transcription includes transcription induced by any member of the NFAT family, including, for example, NFATc1, NFATc2, NFATc3, NFATc4, NFAT5; AP-1; Sp1; NKκB; and the like.

[0037] A CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and activated by an antigen and a dimerizer that binds to the antigen binding domain of the CAR, can, in some cases, result in increased production of one or more cytokines by the cell. For example, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and activated by an antigen and a dimerizer that binds to the antigen binding domain of the CAR, can increase cytokine production by the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the amount of cytokine produced by the cell in the absence of antigen and / or dimerizer. Cytokines whose production can be increased include, but are not limited to, interferons, such as IL-2, interferon gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), IL-15, IL-12, IL-4, IL-5, IL-10; chemokines; growth factors, and the like.

[0038] In some cases, a CAR of the present disclosure, when present within the plasma membrane of a eukaryotic cell and activated by an antigen and a dimerization agent that binds to the antigen binding domain of the CAR, can result in both increased transcription of nucleic acids within the cell and increased production of cytokines by the cell.

[0039] In some cases, a CAR of the present disclosure, when present in the plasma membrane of a eukaryotic cell and activated by a dimerizing agent, results in cytotoxic activity by the cell against a target cell expressing on its cell surface an antigen to which the antigen-binding domain of the first polypeptide of the CAR binds. For example, when the eukaryotic cell is a cytotoxic cell (e.g., an NK cell or a cytotoxic T lymphocyte), a CAR of the present disclosure, when present in the plasma membrane of the cell and activated by a dimerizing agent, increases the cytotoxic activity of the cell against a target cell expressing on its cell surface an antigen to which the antigen-binding domain of the first polypeptide of the CAR binds. For example, when the eukaryotic cell is a NK cell or a T lymphocyte, the CAR of the disclosure, when present within the plasma membrane of the cell and activated by a dimerizing agent, increases the cytotoxicity of the cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the cytotoxic activity of the cell in the absence of the dimerizing agent.

[0040] In some cases, the CARs of the present disclosure, when present within the plasma membrane of a eukaryotic cell and activated by antigen and dimerization agents that bind to the antigen-binding domain of the CAR, can result in other CAR activation-related events, such as proliferation and expansion (either by increased cell division or an anti-apoptotic response).

[0041] In some cases, the CAR of the present disclosure, when present within the plasma membrane of a eukaryotic cell and activated by antigen and dimerization agents that bind to the antigen-binding domain of the CAR, can result in other CAR activation-related events, such as intracellular signaling modulation, cell differentiation, or cell death.

[0042] The CAR of the present disclosure may exist in a eukaryotic cell membrane, and the first and second polypeptides of the CAR are not covalently linked to each other. The CAR of the present disclosure may exist in a eukaryotic cell membrane as a single heterodimer that is not covalently linked to any other polypeptide in the membrane. Alternatively, the first CAR of the present disclosure may exist in a eukaryotic cell membrane as a heterodimer that is covalently or non-covalently linked to the second CAR of the present disclosure. In some cases, the first and second CARs are covalently linked via disulfide bonds formed between cysteines present in the hinge region present in both the first polypeptide of the first CAR and the first polypeptide of the second CAR.

[0043] In some cases, the CAR of the present disclosure can be present in a eukaryotic cell membrane, and upon dimerization, the first polypeptide of the CAR comprises an antibody fragment and the second polypeptide of the CAR comprises a signaling domain derived from a cytokine receptor, such that the CAR can represent a heterodimeric signalobody CAR, e.g., a signalobody composed of at least two dependent polypeptides. A "signalobody," as known to those skilled in the art, is a single chimeric macromolecule composed of an antibody fragment and a signaling domain derived from a cytokine receptor. In certain examples, the heterodimeric signalobody CAR of the present disclosure can be dimerized by a dimerizing agent and activated by an antigen, e.g., a multimerizing antigen, to induce multimerization of the heterodimeric signalobody CAR when present in the cell membrane of a eukaryotic cell. Such ligand-induced multimerization of a heterodimeric signalobody CAR can activate, e.g., increase, or perpetuate, e.g., maintain, signal transduction, e.g., ligand-induced multimerization of a heterodimeric signalobody CAR can transmit a signal that elicits a cellular response. In some cases, multiple heterodimeric signalobody CARs can be utilized in combination to elicit a desired cellular response.

[0044] Members of a specific binding pair The CARs of the present disclosure comprise members of specific binding pairs, including, but not limited to, antigen-antibody binding pairs, ligand-receptor binding pairs, etc. Thus, members of specific binding pairs suitable for use in the CARs of the present disclosure include antigens, antibodies, ligands, and ligand-binding receptors.

[0045] Antigen-binding domain The antigen binding domain suitable for use in the CAR of the present disclosure can be any antigen binding polypeptide, various types of which are known to those of skill in the art. In some examples, the antigen binding domain is a single chain Fv (scFv). Other antibody-based recognition domains (cAb VHH (camelid antibody variable domain) and humanized versions, IgNAR VH (shark antibody variable domain) and humanized versions, sdAb VH (single domain antibody variable domain) and "camelized" antibody variable domains are suitable for use. In some examples, T cell receptor (TCR)-based recognition domains such as single chain TCR (scTv, VαVβ-containing single chain two domain TCR) are also suitable for use.

[0046] The antigen binding domain suitable for use in the CAR of the present disclosure may have various antigen binding specificities. In some cases, the antigen binding domain is specific for an epitope present in an antigen expressed (synthesized) by a cancer cell, i.e., a cancer cell-associated antigen. The cancer cell-associated antigen may be, for example, an antigen associated with breast cancer cells, B cell lymphoma, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer cells, mesothelioma, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin's B cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer cells, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, gliomas, glioblastomas, medulloblastomas, colon cancer cells, etc. The cancer cell-associated antigen may also be expressed by non-cancer cells.

[0047] Non-limiting examples of antigens to which the antigen binding domain of the CAR of the present application can bind include, for example, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like.

[0048] Ligand In some cases, a member of a specific binding pair suitable for use in the CAR of the present application is a ligand for a receptor, including, but not limited to, cytokines (e.g., IL-13, etc.), growth factors (e.g., heregulin, vascular endothelial growth factor (VEGF), etc.), integrin-binding peptides (e.g., peptides containing the sequence Arg-Gly-Asp), etc.

[0049] When the member of the specific binding pair in the CAR of the present application is a ligand, the CAR can be activated in the presence of both a dimerizer agent and a second member of the specific binding pair, and the second member of the specific binding pair is a receptor for the ligand.For example, when the ligand is VEGF, the second member of the specific binding pair can be a VEGF receptor, including a soluble VEGF receptor.In another example, when the ligand is heregulin, the second member of the specific binding pair can be Her2.

[0050] Receptor As mentioned above, in some cases, the member of the specific binding pair included in the CAR of the present application is a receptor, e.g., a receptor for a ligand, a co-receptor. The receptor can be a ligand-binding fragment of a receptor. Suitable receptors include, but are not limited to, growth factor receptors (e.g., VEGF receptor); killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptide (receptor for MICA, MICB, and ULB6); cytokine receptors (e.g., IL-13 receptor, IL-2 receptor, etc.); Her2; CD27; natural cytotoxicity receptors (NCRs) (e.g., NKP30 (NCR3 / CD337) polypeptide (receptor for HLA-B associated transcript 3 (BAT3) and B7-H6); etc.); and the like.

[0051] Hinge Area In some cases, the first polypeptide of the CAR of the present application comprises a hinge region (also referred to herein as a "spacer"), which is interposed between the antigen-binding domain and the transmembrane domain. In some cases, the hinge region is an immunoglobulin heavy chain hinge region. In some cases, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region).

[0052] The hinge region can have a length of about 4 amino acids to about 50 amino acids, for example, about 4 amino acids to about 10 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 20 amino acids, about 20 amino acids to about 25 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 40 amino acids, or about 40 amino acids to about 50 amino acids.

[0053] A suitable spacer can be readily selected and can be any of a number of suitable lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0054] An exemplary spacer is a glycine polymer (G). n , glycine-serine polymers (e.g., (GS) n , (GSGGS) n (SEQ ID NO:37), and (GGGS) n(SEQ ID NO:38), where n is an integer of at least 1, inclusive), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known to those of skill in the art. Glycine and glycine-serine polymers can be used, where both Gly and Ser are relatively unstructured and can therefore act as intermediate tethers between components. Glycine-polymers can be used, where glycine has significantly more access to the phi-psi interval than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary spacers are: The amino acid sequences may include, but are not limited to, TIFF0007676476000001.tif20156, etc.

[0055] In some cases, the hinge region in the first polypeptide of the CAR of the present application comprises at least one cysteine. For example, in some cases, the hinge region may comprise the sequence Cys-Pro-Pro-Cys. If present, the cysteine ​​in the hinge region of the first CAR may be available to form a disulfide bond with the hinge region in the second CAR.

[0056] Immunoglobulin hinge region amino acid sequences are known to those of skill in the art, see, e.g., Tan et al. (1990) Proc. Natl. Acad. Sci. USA 87:162; and Huck et al. (1986) Nucl. Acids Res. 14:1779. As a non-limiting example, an immunoglobulin hinge region may have the following amino acid sequence: TIFF0007676476000002.tif6160 (see, e.g., Glaser et al. (2005) J. Biol. Chem. 280:41494); TIFF0007676476000003.tif35157, etc.

[0057] The hinge region may comprise the amino acid sequence of a human IgG1, IgG2, IgG3, or IgG4 hinge region. The hinge region may contain one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, the His 229 can be replaced with Tyr, such that the hinge region has the sequence Includes TIFF0007676476000004.tif5128; see, e.g., Yan et al. (2012) J. Biol. Chem. 287:5891.

[0058] The hinge region can comprise an amino acid sequence derived from human CD8, for example, the hinge region comprises the amino acid sequence TIFF0007676476000005.tif5153 or variants thereof.

[0059] Transmembrane domain The first and second polypeptides of the CAR of the present disclosure comprise a transmembrane domain for insertion into a eukaryotic cell membrane. The transmembrane domain of the first polypeptide is interposed between the antigen-binding domain and the costimulatory domain. If the first polypeptide comprises a hinge region, the transmembrane domain is interposed between the hinge region and the costimulatory domain such that the first polypeptide comprises, in order from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), an antigen-binding domain, a hinge region, a transmembrane domain, a first costimulatory domain, and a first member of a dimerizer binding pair.

[0060] The transmembrane domain of the second polypeptide is at or near the N-terminus of the polypeptide, such that the second polypeptide comprises, in order from N-terminus to C-terminus, a transmembrane domain, a second costimulatory domain, a second member of the dimerizer binding pair, and an intracellular signaling domain.

[0061] Any transmembrane (TM) domain that provides for insertion of a polypeptide into the cell membrane of a eukaryotic (e.g., mammalian) cell is suitable for use. As one non-limiting example, the TM sequence TIFF0007676476000006.tif5128 can be used. Additional non-limiting examples of suitable TM sequences include: a) from CD8 beta: TIFF0007676476000007.tif5128;b)CD4 origin: TIFF0007676476000008.tif6128;c) CD3 Zeta derived: TIFF0007676476000009.tif5128;d) CD28 derived: TIFF0007676476000010.tif5128; e) CD134 (OX40) origin: TIFF0007676476000011.tif5128; and f) from CD7: TIFF0007676476000012.tif5128 is an example.

[0062] Linker In some cases, the first polypeptide of the CAR of the present application comprises a linker between any two adjacent domains. For example, the linker can be placed between the transmembrane domain and the first costimulatory domain of the first polypeptide. As another example, the linker can be placed between the first costimulatory domain and the first member of the dimerizer binding pair of the first polypeptide. As another example, the linker can be placed between the transmembrane domain and the second costimulatory domain of the second polypeptide. As another example, the linker can be placed between the second costimulatory domain and the second member of the dimerizer binding pair of the second polypeptide. As another example, the linker can be placed between the second member of the dimerizer binding pair and the intracellular signaling domain of the second polypeptide.

[0063] The linker peptide may have any of a variety of amino acid sequences. The proteins may be linked by spacer peptides, generally of a flexible nature, although other chemical linkages are not excluded. The linker may be a peptide between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length. These linkers may be generated by using synthetic, linker-encoded oligonucleotides to link the proteins. Peptide linkers having some degree of flexibility may be used. It is noted that suitable linkers may generally have sequences that result in flexible peptides, and the linking peptide may have virtually any amino acid sequence. The use of small amino acids such as granine and alanine is useful in creating flexible peptides. The creation of such sequences is routine to one of skill in the art.

[0064] Suitable linkers can be easily selected and can be any suitable of different lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0065] An exemplary flexible linker is a glycine polymer (G) n , glycine-serine polymers (e.g. (GS) n , G.S.G.S.G.S. n (SEQ ID NO:37), and GGGS n(SEQ ID NO:38), inclusive, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known to those of skill in the art. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and therefore may act as intermediate tethers between components. Glycine polymers are of particular interest because glycine has significantly more access to the phi-psi interval than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexible linkers are: TIFF0007676476000013.tif19155 and the like. One of skill in the art will recognize that the design of peptides to be attached to any of the above elements can include linkers that are all or partially flexible, such that the linker can include flexible linkers as well as one or more moieties that result in a less flexible structure.

[0066] Regulatory domains Regulatory domains suitable for use in the CARs of the present disclosure include costimulatory domains.

[0067] In some cases, the regulatory domain of the first polypeptide of the CAR of the present application has substantially the same amino acid sequence as the regulatory domain of the second polypeptide of the CAR. For example, in some cases, the regulatory domain of the first polypeptide of the CAR comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of the regulatory domain of the second polypeptide of the CAR. The regulatory domain of the first polypeptide of the CAR of the present application can have substantially the same length as the regulatory domain of the second polypeptide of the CAR of the present application, for example, the first and second regulatory domains can differ in length from each other by less than 10 amino acids or less than 5 amino acids. In some cases, the first and second regulatory domains have the same length.

[0068] Regulatory domains suitable for inclusion in the first and second polypeptides of the CAR of the present application can have a length of about 30 amino acids to about 70 amino acids (aa), for example, the regulatory domain can have a length of about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 65 amino acids, or about 65 amino acids to about 70 amino acids. In other cases, the regulatory domain can have a length of about 70 amino acids to about 100 amino acids, about 100 amino acids to about 200 amino acids, or more than 200 amino acids.

[0069] The costimulatory domain suitable for use in the CAR of the present disclosure is generally a polypeptide derived from a receptor. In some embodiments, the costimulatory domain homodimerizes. The costimulatory domain of the present application can be the intracellular part of a transmembrane protein (i.e., the costimulatory domain can be derived from a transmembrane protein). Non-limiting examples of suitable costimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0070] In some cases, the costimulatory domain of the first polypeptide of the CAR of the present application has substantially the same amino acid sequence as the costimulatory domain of the second polypeptide of the CAR. For example, in some cases, the costimulatory domain of the first polypeptide of the CAR comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence of the costimulatory domain of the second polypeptide of the CAR. The costimulatory domain of the first polypeptide of the CAR of the present application can have substantially the same length as the costimulatory domain of the second polypeptide of the CAR of the present application, for example, the first and second costimulatory domains can differ in length from each other by less than 10 amino acids or less than 5 amino acids. In some cases, the first and second costimulatory domains have the same length.

[0071] Costimulatory domains suitable for inclusion in the first and second polypeptides of the CAR of the present application can have a length of about 30 amino acids to about 70 amino acids (aa), for example, the costimulatory domain can have a length of about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 65 amino acids, or about 65 amino acids to about 70 amino acids. In other cases, the costimulatory domain can have a length of about 70 amino acids to about 100 amino acids, about 100 amino acids to about 200 amino acids, or more than 200 amino acids.

[0072] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; 4-1BB; CDw137; ILA, etc.). For example, a suitable costimulatory domain has the following amino acid sequence: TIFF0007676476000014.tif5146. In some of these embodiments, the costimulatory domains of both the first and second polypeptides have a length of about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 65 amino acids, or about 65 amino acids to about 70 amino acids.

[0073] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a suitable costimulatory domain has the following amino acid sequence: TIFF0007676476000015.tif5156. In some of these embodiments, the costimulatory domain of both the first and second polypeptides has a length of about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 65 amino acids, or about 65 amino acids to about 70 amino acids.

[0074] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein ICOS (also known as AILIM, CD278, and CVID1). For example, a suitable costimulatory domain has the following amino acid sequence: TIFF0007676476000016.tif5132. In some of these embodiments, the costimulatory domain of both the first and second polypeptides has a length of about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 65 amino acids, or about 65 amino acids to about 70 amino acids.

[0075] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). For example, a suitable costimulatory domain has the following amino acid sequence: TIFF0007676476000017.tif5132. In some of these embodiments, the costimulatory domains of both the first and second polypeptides have a length of about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 65 amino acids, or about 65 amino acids to about 70 amino acids.

[0076] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). For example, a suitable costimulatory domain has the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity with TIFF0007676476000018.tif20158.

[0077] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, Tp55). For example, a suitable costimulatory domain has the following amino acid sequence: TIFF0007676476000019.tif13149. In some of these embodiments, the costimulatory domain of both the first and second polypeptides has a length of about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 65 amino acids, or about 65 amino acids to about 70 amino acids.

[0078] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). For example, a suitable costimulatory domain has the following amino acid sequence: The amino acid sequence may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 amino acids to about 115 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 130 amino acids, about 130 amino acids to about 140 amino acids, about 140 amino acids to about 150 amino acids, about 150 amino acids to about 160 amino acids, or about 160 amino acids to about 185 amino acids of TIFF0007676476000020.tif27159.

[0079] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitable costimulatory domain has the following amino acid sequence: TIFF0007676476000021.tif13152. In some of these embodiments, the costimulatory domains of both the first and second polypeptides have a length of about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 65 amino acids, or about 65 amino acids to about 70 amino acids.

[0080] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a suitable costimulatory domain has the following amino acid sequence: TIFF0007676476000022.tif13158. In some of these embodiments, the costimulatory domains of both the first and second polypeptides have a length of about 30 amino acids to about 35 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 65 amino acids, or about 65 amino acids to about 70 amino acids.

[0081] dimer pair Dimerization pairs suitable for use in the CARs of the present application include dimerization agent binding pairs. Dimerization agent binding pairs suitable for use in the CARs of the present disclosure are, in some embodiments, polypeptides (referred to herein as "dimerization agents") that bind to different sites of the same molecule. In the presence of a dimerization agent, both members of the dimerization agent binding pair bind to different sites of the dimerization agent and are thus in close proximity to each other. In some embodiments, the binding to the dimerization agent is reversible. In some embodiments, the binding to the dimerization agent is irreversible. In some embodiments, the binding to the dimerization agent is non-covalent. In some embodiments, the binding to the dimerization agent is covalent.

[0082] Other dimer pairs suitable for use include dimerizer-binding pairs that dimerize upon binding of a first member of the dimer pair to a dimerizer, where the dimerizer induces a conformational change in the first member of the dimer pair, which conformational change allows the first member of the dimer pair to bind (covalently or non-covalently) to a second member of the dimer pair.

[0083] Other dimer pairs suitable for use include those in which exposure to light (eg, blue light) induces dimerization of the dimer pair.

[0084] Regardless of mechanism, the dimerization pair dimerizes upon exposure to a dimerization-inducing agent, which in some cases is a small molecule and in other cases is light, and thus, for simplicity, the following discussion referring to a "dimerizer-binding pair" includes dimerization pairs that dimerize regardless of mechanism.

[0085] Non-limiting examples of suitable dimers (eg, dimerizer binding pairs) include, but are not limited to, the following: a) FK506 binding protein (FKBP) and FKBP, b) FKBP and calcineurin catalytic subunit A (CnA); c) FKBP and cyclophilin, d) FKBP and FKBP-rapamycin-related protein (FRB); e) gyrase B (GyrB) and GryB; f) dihydrofolate reductase (DHFR) and DHFR; g) DmrB and DmrB, h) PYL and ABI; i) Cry2 and CIB1, and j) GAI and GID1.

[0086] The first or second member of a dimer (e.g., a dimerizer binding pair) of the CAR of the present application can have a length of from about 50 amino acids to about 300 amino acids or more, for example, the first or second member of a dimer (e.g., a dimerizer binding pair) of the CAR of the present application can have a length of from about 50 amino acids to about 100 amino acids, from about 100 amino acids to about 150 amino acids, from about 150 amino acids to about 200 amino acids, from about 200 amino acids to about 250 amino acids, from about 250 amino acids to about 300 amino acids, or more than 300 amino acids.

[0087] In some cases, a member of the dimer (e.g., dimerizer binding pair) of the CAR of the present application is derived from FKBP. For example, a suitable dimerizer binding pair member has the following amino acid sequence: The amino acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity with TIFF0007676476000023.tif19158.

[0088] In some cases, a member of the dimerizer binding pair of the CAR of the present application is derived from calcineurin catalytic subunit A (also known as PPP3CA; CALN; CALNA; CALNA1; CCN1; CNA1; PPP2B; CAM-PRP catalytic subunit; calcineurin A alpha; calmodulin-dependent calcineurin A subunit alpha isoform; protein phosphatase 2B, catalytic subunit, alpha isoform; etc.). For example, a suitable dimerizer binding pair member has the following amino acid sequence (PP2Ac domain): The amino acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity with TIFF0007676476000024.tif42158.

[0089] In some cases, a member of the dimer (e.g., dimerizer binding pair) is derived from a cyclophilin (also known as cyclophilin A, PPIA, CYPA, CYPH, PPIaseA, etc.). For example, a suitable dimerizer binding pair member has the following amino acid sequence: The amino acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity with TIFF0007676476000025.tif27158.

[0090] In some cases, a member of the dimer (e.g., dimerizer binding pair) is derived from MTOR (also known as FKBP-rapamycin-related protein; FK506-binding protein 12-rapamycin-related protein 1; FK506-binding protein 12-rapamycin-related protein 2; FK506-binding protein 12-rapamycin complex-associated protein 1; FRAP; FRAP1; FRAP2; RAFT1; and RAPT1). For example, a suitable dimerizer binding pair member has the following amino acid sequence (also known as "Frb": Fkbp-rapamycin binding domain): It may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity with TIFF0007676476000026.tif13157.

[0091] In some cases, a member of the dimer (e.g., dimerizer binding pair) is derived from GyrB, also known as DNA gyrase subunit B. For example, a suitable dimerizer binding pair member has the following amino acid sequence of GyrB from E. coli: The nucleic acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 amino acids to about 200 amino acids (aa), about 200 amino acids to about 300 amino acids, about 300 amino acids to about 400 amino acids, about 400 amino acids to about 500 amino acids, about 500 amino acids to about 600 amino acids, about 600 amino acids to about 700 amino acids, or about 700 amino acids to about 800 amino acids of TIFF0007676476000027.tif107158 (or a DNA gyrase subunit B sequence from any organism). In some cases, a member of the dimerizer binding pair comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to amino acids 1-220 of the GyrB amino acid sequence from E. coli described above.

[0092] In some cases, a member of the dimer (e.g., dimerizer binding pair) is derived from DHFR (dihydrofolate reductase, also known as DHFRP1, and DYR). For example, a suitable dimerizer binding pair member has the following amino acid sequence: It may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity with TIFF0007676476000028.tif28158.

[0093] In some cases, a member of a dimer (e.g., a dimerizer binding pair) is derived from a DmrB binding domain (i.e., a DmrB homodimerization domain). For example, a suitable member of a dimerizer binding pair has the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity with TIFF0007676476000029.tif20158.

[0094] In some cases, a member of a dimer (e.g., a dimerizer binding pair) is derived from a PYL protein (also known as an abscisic acid receptor and also as RCAR). For example, a member of a dimerizer binding pair of the present application can be derived from a protein such as those of Arabidopsis thaliana: PYR1, RCAR1 (PYL9), PYL1, PYL2, PYL3, PYL4, PYL5, PYL6, PYL7, PYL8 (RCAR3), PYL10, PYL11, PYL12, PYL13. For example, a suitable dimerizer binding pair member can include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any of the following amino acid sequences: TIFF0007676476000030.tif210158TIFF0007676476000031.tif239158TIFF0007676476000032.tif71158

[0095] In some cases, a member of a dimer (e.g., a dimerizer-binding pair) is derived from an ABI protein (also known as abscisic acid insensitive). For example, a member of a dimerizer-binding pair of the present application can be derived from a protein such as that of Arabidopsis thaliana: ABI1 (also known as abscisic acid insensitive 1, protein phosphatase 2C 56, AtPP2C56, P2C56, and PP2C ABI1) and / or ABI2 (also known as P2C77, protein phosphatase 2C 77, AtPP2C77, abscisic acid insensitive 2, protein phosphatase 2C ABI2, and PP2C ABI2). For example, suitable dimerizer binding pair members may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 amino acids, about 115 to about 120 amino acids, about 120 to about 130 amino acids, about 130 to about 140 amino acids, about 140 to about 150 amino acids, about 150 to about 160 amino acids, about 160 to about 170 amino acids, about 170 to about 180 amino acids, about 180 to about 190 amino acids, or about 190 to about 200 amino acids of any of the following amino acid sequences: TIFF0007676476000033.tif64158TIFF0007676476000034.tif63158

[0096] In some cases, a member of the dimer (e.g., dimerizer-binding pair) is derived from a Cry2 protein (also known as cryptochrome 2). For example, a member of the dimer (e.g., dimerizer-binding pair) of the present application can be derived from a Cry2 protein from any organism (e.g., a plant), such as, but not limited to, that of Arabidopsis thaliana. For example, a suitable dimerizer binding pair member may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 amino acids, about 115 to about 120 amino acids, about 120 to about 130 amino acids, about 130 to about 140 amino acids, about 140 to about 150 amino acids, about 150 to about 160 amino acids, about 160 to about 170 amino acids, about 170 to about 180 amino acids, about 180 to about 190 amino acids, or about 190 to about 200 amino acids of any of the following amino acid sequences: Cry2 (Arabidopsis thaliana) TIFF0007676476000035.tif86158

[0097] In some cases, a member of the dimer (e.g., dimerizer binding pair) is derived from the CIB1 Arabidopsis thaliana protein (also known as transcription factor bHLH63). For example, a suitable dimer (e.g., dimerizer binding pair) member has the following amino acid sequence: The amino acid sequence may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 amino acids, about 115 to about 120 amino acids, about 120 to about 130 amino acids, about 130 to about 140 amino acids, about 140 to about 150 amino acids, about 150 to about 160 amino acids, about 160 to about 170 amino acids, about 170 to about 180 amino acids, about 180 to about 190 amino acids, or about 190 to about 200 amino acids of TIFF0007676476000036.tif50158.

[0098] In some cases, a member of the dimer (e.g., dimerizer binding pair) is derived from the GAI Arabidopsis thaliana protein (also known as gibberellic acid insensitive, and DELLA protein GAI). For example, a suitable dimerizer binding pair member has the following amino acid sequence: The amino acid sequence may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 amino acids to about 115 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 130 amino acids, about 130 amino acids to about 140 amino acids, about 140 amino acids to about 150 amino acids, about 150 amino acids to about 160 amino acids, about 160 amino acids to about 170 amino acids, about 170 amino acids to about 180 amino acids, about 180 amino acids to about 190 amino acids, or about 190 amino acids to about 200 amino acids of TIFF0007676476000037.tif72158.

[0099] In some cases, a member of the dimer (eg, a dimerizer-binding pair) is derived from the GID1 Arabidopsis thaliana protein (also known as the gibberellin receptor GID1). For example, suitable dimer members may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 amino acids, about 115 to about 120 amino acids, about 120 to about 130 amino acids, about 130 to about 140 amino acids, about 140 to about 150 amino acids, about 150 to about 160 amino acids, about 160 to about 170 amino acids, about 170 to about 180 amino acids, about 180 to about 190 amino acids, or about 190 to about 200 amino acids of any of the following amino acid sequences: TIFF0007676476000038.tif173158

[0100] Dimerizing Agent Dimerizers ("dimerizing agents") that can provide for dimerization of a first member of a dimerizer binding pair and a second member of the dimerizer binding pair include, for example, the following (the dimerizer is in parentheses following the dimerizer binding pair): a) FKBP and FKBP (rapamycin); b) FKBP and CnA (rapamycin); c) FKBP and cyclophilin (rapamycin); d) FKBP and FRG (rapamycin); e) GyrB and GyrB (coumermycin); f) DHFR and DHFR (methotrexate); g) DmrB and DmrB (AP20187); h) PYL and ABI (abscisic acid); i) Cry2 and CIB1 (blue light); and j) GAI and GID1 (gibberellins).

[0101] As mentioned above, rapamycin may act as a dimerizer. Alternatively, rapamycin derivatives or analogs may be used. See, for example, WO96 / 41865, WO99 / 36553, WO01 / 14387, and Ye et al (1999) Science 283:88-91. For example, analogs, homologs, derivatives, and other compounds structurally related to rapamycin ("rapalogs") include, inter alia, variants of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, removal, or replacement of methoxy at C7, C42, and / or C29; removal, derivatization, or replacement of hydroxy at C13, C43, and / or C28; reduction, removal, or derivatization of ketone at C14, C24, and / or C30; replacement of a 5-membered prolyl ring with a 6-membered pipecolate ring; alternative substitutions on the cyclohexyl ring or replacement of a cyclopentyl ring with a cyclohexyl ring. Additional information is provided, for example, in U.S. Patent Nos. 5,525,610, 5,310,903, 5,362,718, and 5,527,907. Selective epimerization of the C-28 hydroxyl group has been described, see, for example, WO 01 / 14387. Additional synthetic dimerizers suitable for use as replacements for rapamycin include those described in U.S. Patent Application Publication No. 2012 / 0130076.

[0102] Rapamycin has the following structure: TIFF0007676476000039.tif58128

[0103] Suitable rapalogs include, for example: TIFF0007676476000040.tif68128

[0104] Further suitable as rapalogs are compounds of the formula: TIFF0007676476000041.tif56128, where n is 1 or 2; R 28 and R 43is independently H or a substituted or unsubstituted aliphatic or acyl moiety; R 7a and R 7b One of them is H and the other is halo, R A , OR A , S.R. A , -OC(O)R A , -OC(O)NR A R B , -NR A R B , -NR B C(OR)R A , N.R. B C(O)OR A , -NR B SO2R A , or NR B SO2NR A R B' or R 7a and R 7b taken together are the H's in the tetraene moiety of: TIFF0007676476000042.tif18128In formula, R A is H or a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, or heteroaryl moiety, and wherein R B and R B' is independently H, OH, or a substituted or unsubstituted aliphatic, heteroaliphatic, aryl, or heteroaryl moiety.

[0105] As mentioned above, coumermycin may act as a dimerizer. Alternatively, coumermycin analogs may be used. See, e.g., Farrar et al. (1996) Nature 383:178-181, and U.S. Patent No. 6,916,846.

[0106] As noted above, in some cases, the dimerizer is methotrexate, e.g., a non-cytotoxic homobifunctional methotrexate dimer, see, e.g., U.S. Patent No. 8,236,925.

[0107] Intracellular signaling domains Intracellular signaling domains suitable for use in the CARs of the present disclosure include any desired signaling domain that provides a clear, detectable signal (e.g., increased production of one or more cytokines by the cell; a change in transcription of a target gene; a change in activity of a protein; a change in cellular behavior, such as cell death; cell proliferation; cell differentiation; cell survival; modulation of a cell signaling response, etc.) in response to activation of the CAR (i.e., activated by an antigen and a dimerization agent). In some embodiments, the intracellular signaling domain comprises at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif, as described below. In some embodiments, the intracellular signaling domain comprises a signaling chain of the DAP10 / CD28 type. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane-bound CAR, but instead is diffused within the cytoplasm.

[0108] ITAM Intracellular signaling domains suitable for use in the CARs of the present disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. The ITAM motif is YX1X2L / I, where X1 and X2 are independently any amino acid (SEQ ID NO:130). In some cases, the intracellular signaling domain of the CAR of the present application comprises one, two, three, four, or five ITAM motifs. In some cases, the ITAM motif is repeated twice within the intracellular signaling domain, and the first and second instances of the ITAM motif are separated from each other by 6-8 amino acids, e.g., (YX1X2L / I)(X3). n (YX1X2L / I), where n is an integer from 6 to 8, and each of 6 to 8X3 can be any amino acid (SEQ ID NO:131). In some cases, the intracellular signaling domain of the CAR of the present application comprises three ITAM motifs.

[0109] A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain does not need to include the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12; FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3Z (CD3 zeta); and CD79A (antigen receptor complex-associated protein alpha chain).

[0110] In some cases, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR associated protein; TYRO protein tyrosine kinase binding protein; killer activating receptor associated protein; killer activating receptor associated protein; etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence (four isoforms): TIFF0007676476000043.tif79158, wherein the ITAM motif is in bold and underlined.

[0111] Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion of the full-length DAP12 amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide may comprise the following amino acid sequence: The amino acid sequence may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007676476000044.tif5128, where the ITAM motif is in bold and underlined.

[0112] In some cases, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; high affinity gamma chain of the immunoglobulin E receptor; etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007676476000045.tif13157, where the ITAM motif is in bold and underlined.

[0113] Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion of the full-length FCER1G amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide may comprise the following amino acid sequence: The amino acid sequence may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007676476000046.tif5128, where the ITAM motif is in bold and underlined.

[0114] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-delta; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence (two isoforms): TIFF0007676476000047.tif49158, and may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any contiguous stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 amino acids, about 115 to about 120 amino acids, about 120 to about 130 amino acids, about 130 to about 140 amino acids, about 140 to about 150 amino acids, or about 150 to about 170 amino acids in TIFF0007676476000047.tif49158, and the ITAM motif is underlined in bold.

[0115] Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion of the full-length CD3 delta amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide may comprise the following amino acid sequence: The amino acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007676476000048.tif5128, where the ITAM motif is in bold and underlined.

[0116] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence: and may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 amino acids, about 115 to about 120 amino acids, about 120 to about 130 amino acids, about 130 to about 140 amino acids, about 140 to about 150 amino acids, or about 150 to about 205 amino acids of TIFF0007676476000049.tif27158, and the ITAM motif is underlined in bold.

[0117] Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion of the full-length CD3 epsilon amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide may comprise the following amino acid sequence: The amino acid sequence may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007676476000050.tif5128, where the ITAM motif is in bold and underlined.

[0118] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-gamma, T3G, gamma polypeptide (TiT3 complex), etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence: and may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 amino acids, about 115 to about 120 amino acids, about 120 to about 130 amino acids, about 130 to about 140 amino acids, about 140 to about 150 amino acids, or about 150 to about 180 amino acids of TIFF0007676476000051.tif27158, and the ITAM motif is underlined in bold.

[0119] Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion of the full-length CD3 gamma amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide may comprise the following amino acid sequence: The amino acid sequence may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007676476000052.tif5128, where the ITAM motif is in bold and underlined.

[0120] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-zeta, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence (two isoforms): TIFF0007676476000053.tif56158, and may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any contiguous stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 amino acids, about 115 to about 120 amino acids, about 120 to about 130 amino acids, about 130 to about 140 amino acids, about 140 to about 150 amino acids, or about 150 to about 160 amino acids in TIFF0007676476000053.tif56158, and the ITAM motif is underlined in bold.

[0121] Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion of the full-length CD3 zeta amino acid sequence. Thus, the intracellular signaling domain polypeptide may comprise the following amino acid sequence: TIFF0007676476000054.tif42158, wherein the ITAM motif is in bold and underlined.

[0122] In some cases, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-associated immunoglobulin-associated protein; surface IgM-associated protein; etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence (two isoforms): TIFF0007676476000055.tif65159, and may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any contiguous stretch of about 100 to about 110 amino acids (aa), about 110 to about 115 amino acids, about 115 to about 120 amino acids, about 120 to about 130 amino acids, about 130 to about 150 amino acids, about 150 to about 200 amino acids, or about 200 to about 220 amino acids in TIFF0007676476000055.tif65159, and the ITAM motif is underlined in bold.

[0123] Similarly, a suitable intracellular signaling domain polypeptide may comprise an ITAM motif-containing portion of the full-length CD79A amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide may comprise the following amino acid sequence: The amino acid sequence may have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007676476000056.tif5128, where the ITAM motif is in bold and underlined.

[0124] DAP10 / CD28 Intracellular signaling domains suitable for use in the CARs of the present disclosure include DAP10 / CD28-type signaling chains.

[0125] An example of the DAP10 signaling chain is the amino acid sequence: TIFF0007676476000057.tif5128. In some embodiments, a suitable intracellular signaling domain has the amino acid sequence: It comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to the full length of TIFF0007676476000058.tif5128.

[0126] An example of the CD28 signaling chain is the amino acid sequence: TIFF0007676476000059.tif13158. In some embodiments, a suitable intracellular signaling domain has the amino acid sequence: It comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to the full length of TIFF0007676476000060.tif13157.

[0127] ZAP70 An intracellular signaling domain suitable for use in a CAR of the present disclosure is a ZAP70 polypeptide, such as the following amino acid sequence: The present invention also includes polypeptides comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a contiguous stretch of about 300 amino acids to about 400 amino acids, about 400 amino acids to about 500 amino acids, or about 500 amino acids to 619 amino acids of TIFF0007676476000061.tif86158.

[0128] Additional Arrays The first and / or second polypeptides of the CAR of the present application may further comprise one or more additional polypeptide domains, including, but not limited to, signal sequences, epitope tags, affinity domains, and polypeptides that generate a detectable signal.

[0129] Signal sequence Suitable signal sequences for use in a CAR of the present application, e.g., in the first polypeptide of a CAR of the present application, include any eukaryotic signal sequence, including naturally occurring signal sequences, synthetic (e.g., artificial) signal sequences, and the like.

[0130] Epitope tag Suitable epitope tags include, but are not limited to, hemagglutinin (HA; e.g., YPYDVPDYA (SEQ ID NO:122); FLAG (e.g., DYKDDDDK (SEQ ID NO:123); c-myc (e.g., EQKLISEEDL; SEQ ID NO:4), and the like.

[0131] Affinity Domains Affinity domains include peptide sequences that can interact with a binding partner, such as those that can be immobilized on a solid support useful for identification or purification. DNA sequences that code for multiple consecutive single amino acids, such as histidine, when fused to an expressed protein can be used for one-step purification of recombinant proteins by high affinity binding to resin columns such as nickel sepharose. Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO:124), HisX6 (HHHHHH) (SEQ ID NO:125), C-myc (EQKLISEEDL) (SEQ ID NO:4), Flag (DYKDDDDK) (SEQ ID NO:123), Strep tag (WSHPQFEK) (SEQ ID NO:126), hemagglutinin, e.g., HA tag (YPYDVPDYA) (SEQ ID NO:122), GST, thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:127), Phe-His-His-Thr (SEQ ID NO:128), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag, TIFF0007676476000062.tif5128; metal binding domains, such as zinc-binding domains or calcium-binding domains, such as those from calcium-binding proteins, e.g., calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large subunit, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin; intein, biotin, streptavidin, MyoD, Id, leucine zipper sequences, and maltose binding protein.

[0132] Detectable Signal-Generating Polypeptides Suitable detectable signal-generating proteins include, for example, fluorescent proteins, enzymes that catalyze a reaction that produces a detectable signal as a product, and the like.

[0133] Suitable fluorescent proteins include green fluorescent protein (GFP) or variants thereof, blue fluorescent variants of GFP (BFP), cyan fluorescent variants of GFP (CFP), yellow fluorescent variants of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed monomer, J-Red, dimer2, t-dimer2 (12), mRFP1, pocilloporin, Renilla GFP, Monster. Examples of fluorescent proteins include, but are not limited to, GFP, paGFP, Kaede and kindling proteins, phycobiliproteins, and phycobiliprotein conjugates, including B-phycoerythrin, R-phycoerythrin, and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of the various fluorescent and colored proteins from anthozoan species, for example, as described in Matz et al. (1999) Nature Biotechnol. 17:969-973, are suitable for use.

[0134] Suitable enzymes include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), and the like.

[0135] Sequence recombination In certain examples, the polypeptide of a CAR, for example, the sequence of a CAR domain, can be rearranged or deleted in a cell through the use of site-specific recombination techniques. In certain embodiments, the cell activation-associated response to a particular CAR can be altered by site-specific recombination, for example, a first intracellular signaling domain of a CAR that induces a first activation-associated response can be replaced with a second intracellular signaling domain that induces a second activation-associated response. In certain examples, the response of a CAR to a particular dimerizer can be altered by site-specific recombination, for example, a first dimerizer binding pair that causes CAR dimerization in the presence of a first dimerizer can be replaced with a second dimerizer binding pair that causes CAR dimerization in the presence of a second dimerizer. As will be clear to one of skill in the art, site-specific recombination can be used in a cell to exchange any domain or sequence of a CAR for any other domain or sequence disclosed herein. Furthermore, as will be apparent to those skilled in the art, site-specific recombination can be used in cells to delete any domain or sequence of CAR. Such exchanges and excisions of sequences and domains are known to those skilled in the art, see, for example, domain switching in signalobodies as described in Tone et al. (2013) Biotechnology and Bioengineering, 3219-3226, the disclosure of which is incorporated herein by reference. The mechanisms and requirements for performing site-specific recombination in vivo are known to those skilled in the art, see, for example, Grindley et al. (2006) Annual Review of Biochemistry, 567-605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, MA), the disclosure of which is incorporated herein by reference.

[0136] nucleic acid The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a first and / or second polypeptide of a heterodimeric conditionally active CAR of the present disclosure. The nucleic acid comprising a nucleotide sequence encoding a first and / or second polypeptide of a heterodimeric conditionally active CAR of the present disclosure can be DNA, in some embodiments, including, for example, a recombinant expression vector. The nucleic acid comprising a nucleotide sequence encoding a first and / or second polypeptide of a heterodimeric conditionally active CAR of the present disclosure can be RNA, for example, in vitro synthesized RNA, in some embodiments.

[0137] In some cases, the nucleic acid of the present disclosure comprises a nucleotide sequence that encodes only the first polypeptide (and not the second polypeptide) of a heterodimeric conditionally active CAR of the present disclosure. In some cases, the nucleic acid of the present disclosure comprises a nucleotide sequence that encodes only the second polypeptide (and not the first polypeptide) of a heterodimeric conditionally active CAR of the present disclosure. In some cases, the nucleic acid of the present disclosure comprises a nucleotide sequence that encodes both the first and second polypeptides of a heterodimeric conditionally active CAR of the present disclosure.

[0138] In some cases, the nucleic acid of the present application provides for the production of a CAR of the present disclosure, e.g., in a mammalian cell. In other cases, the nucleic acid of the present application provides for the amplification of a CAR-encoding nucleic acid.

[0139] The nucleotide sequence encoding the first and / or second polypeptide of the CAR of the disclosure can be operably linked to a transcriptional control element, e.g., a promoter, enhancer, etc.

[0140] Suitable promoters and enhancer elements are known to those skilled in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P, and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in long terminal repeats from retroviruses; mouse metallothionein-I promoter; and various tissue-specific promoters known to those skilled in the art.

[0141] Suitable reversible promoters, including reversibly inducible promoters, are known to those skilled in the art. Such reversible promoters can be isolated and derived from many organisms, such as eukaryotes and prokaryotes. Modification of a reversible promoter from a first organism for use in a second organism, such as a first prokaryote and a second eukaryote, a second eukaryote and a second prokaryote, etc., is known to those skilled in the art. Such reversible promoters, and systems based on such reversible promoters but also including additional regulatory proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator protein (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including Tet activators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0142] In some examples, the locus or construct or transgene containing a suitable promoter is irreversibly switched through the induction of an inducible system.Suitable systems for the introduction of irreversible switching are known to those skilled in the art, for example, the induction of irreversible switching can utilize Cre-lox mediated recombination (see, for example, Fuhrmann-Benzakein et al., PNAS (2000) 28: e99, the disclosure of which is incorporated herein by reference).Any suitable combination of recombinase, endonuclease, ligase, recombination site, etc., known to those skilled in the art, can be used in generating irreversibly switchable promoters. The methods, mechanisms, and requirements for performing site-specific recombination described elsewhere herein find use in generating irreversibly switched promoters and are known to those of skill in the art, see, e.g., Grindley et al. (2006) Annual Review of Biochemistry, 567-605 and Tropp (2012) Molecular Biology (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0143] In some cases, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK-specific promoter. For example, the CD4 gene promoter can be used, see, for example, Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739, and Marodon et al. (2003) Blood 101:3416. As another example, the CD8 gene promoter can be used. NK cell-specific expression can be achieved by using the Ncr1 (p46) promoter, see, for example, Eckelhart et al. (2011) Blood 117:1565.

[0144] In some embodiments, for example, for expression in yeast cells, suitable promoters are constitutive promoters such as the ADH1 promoter, the PGK1 promoter, the ENO promoter, the PYK1 promoter, or regulatable promoters such as the GAL1 promoter, the GAL10 promoter, the ADH2 promoter, the PHO5 promoter, the CUP1 promoter, the GAL7 promoter, the MET25 promoter, the MET3 promoter, the CYC1 promoter, the HIS3 promoter, the ADH1 promoter, the PGK promoter, the GAPDH promoter, the ADC1 promoter, the TRP1 promoter, the URA3 promoter, the LEU2 promoter, the ENO promoter, the TP1 promoter, and the AOX1 (e.g., for use in Pichia). Selection of appropriate vectors and promoters is well within the level of ordinary skill in the art.

[0145] Suitable promoters for use in prokaryotic host cells include the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lactose operon promoter; hybrid promoters, such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, and the like; the araBAD promoter; the ssaG promoter or related promoters (see, e.g., U.S. Patent Publication No. 20040131637), the pagC promoter (Pulkkinen and Miller, J. Bacteriol, 1991:173(1):86-93; Alpuche-Aranda et al., PNAS, 1992;89(21):10079-83), the nirB promoter (Harborne et al. (1992) Mol. Micro. 6:2805-2813), and the like (see, e.g., Dunstan et al. in vivo regulated promoters such as, for example, the sigma 70 promoter, e.g., the consensus sigma 70 promoter (see, for example, GenBank Accession Nos. AX798980, AX798961, and AX798183); stationary phase promoters such as the dps promoter, the spv promoter, and the like; promoters from pathogenicity island SPI-2 (see, for example, WO 96 / 17951); the actA promoter (see, for example, Shetron-Rama et al. (2002) Infect. Immun. 70:1087-1096); the rpsM promoter (see, for example, Valdivia and Falkow (1996). Mol. Microbiol. 22:367); the tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U.(eds.) Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (e.g., Melton et al. (1984) Nucl. Acids Res. 12:7035), and the like. Strong promoters suitable for use in prokaryotes such as E. coli include Trc, Tac, T5, T7, and P. ラムダ Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation upon contact with lactose, thereby preventing the LacI repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator, and in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, e.g., deBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21-25).

[0146] The nucleotide sequence encoding the CAR of the present application can be present in an expression vector and / or a cloning vector. When the CAR of the present application comprises two separate polypeptides, the nucleotide sequences encoding the two polypeptides can be cloned in the same or separate vectors. The expression vector can include a selection marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like.

[0147] A number of suitable vectors and promoters are known to those of skill in the art, and many are commercially available for generating the recombinant constructs of the present application. The following vectors are provided by way of example: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0148] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operable in the expression host may be present.Suitable expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984, and WO95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., J. Immunol. 1999, ... al.,PNAS 94:6916 6921,1997;Bennett et al.,Invest Opthalmol Vis Sci 38:2857 2863,1997;Jomary et al.,Gene Ther 4:683 690,1997,Rolling et al.,Hum Gene Ther 10:641 648,1999;Ali et al. al., Hum Mol Genet 5:591 594,1996; Srivastava in WO93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al. herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vectors (e.g., viral vectors based on murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0149] As described above, in some embodiments, the nucleic acid comprising a nucleotide sequence encoding the first and / or second polypeptide of the heterodimeric conditionally active CAR of the present disclosure can be RNA, e.g., in vitro synthesized RNA, in some embodiments. Methods for in vitro synthesis of RNA are known to those of skill in the art, and any known method can be used to synthesize RNA comprising nucleotides encoding the first and / or second polypeptide of the heterodimeric conditionally active CAR of the present disclosure. Methods for introducing RNA into a host cell are known to those of skill in the art. See, for example, Zhao et al. (2010) Cancer Res. 15:9053. Introducing RNA comprising a nucleotide sequence encoding the first and / or second polypeptide of the heterodimeric conditionally active CAR of the present disclosure into a host cell can be performed in vitro or ex vivo or in vivo. For example, a host cell (e.g., a NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated with RNA having a nucleotide sequence encoding the first and / or second polypeptide of the heterodimeric conditionally active CAR of the present disclosure.

[0150] cell The present disclosure provides mammalian cells genetically engineered to produce the heterodimeric, conditionally active CARs of the present disclosure.

[0151] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH These include, but are not limited to, 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0152] In some instances, the cells are not immortalized cell lines, but instead are cells (e.g., primary cells) obtained from an individual. For example, in some instances, the cells are immune cells obtained from an individual. For example, the cells are T lymphocytes obtained from an individual. In another example, the cells are cytotoxic cells obtained from an individual. In another example, the cells are stem or progenitor cells obtained from an individual.

[0153] How to activate immune cells The present disclosure provides a method of activating immune cells in vitro, in vivo, or ex vivo. The method generally includes contacting an immune cell (in vitro, in vivo, or ex vivo) with a dimerizing agent and an antigen, where the immune cell is genetically modified to generate a heterodimeric conditionally active CAR of the present disclosure. In the presence of the dimerizing agent and the antigen, the heterodimeric conditionally active CAR dimerizes and activates the immune cell, thereby generating an activated immune cell. The immune cell may be, for example, a cytotoxic T lymphocyte, a NK cell, a CD4 + Including T cells, T regulatory (Treg) cells, etc.

[0154] Contacting the genetically modified immune cells (e.g., T lymphocytes, NK cells) with a dimerizer and a second member of a specific binding pair (e.g., antigen, ligand, receptor) may increase cytokine production by the immune cells by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the amount of cytokine produced by the immune cells in the absence of the second member of the specific binding pair and / or the dimerizer. Cytokines whose production may be increased include, but are not limited to, IL-2 and IFN-γ.

[0155] Contacting the genetically modified immune cells (e.g., T lymphocytes, NK cells) with a dimerizing agent and an antigen may increase cytokine production by the immune cells by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the amount of cytokine produced by the immune cells in the absence of the antigen and / or dimerizing agent. Cytokines whose production may be increased include, but are not limited to, IL-2 and IFN-γ.

[0156] Contacting a genetically modified cytotoxic cell (e.g., a cytotoxic T lymphocyte) with a dimerizing agent and a second member of the specific binding pair (e.g., an antigen, ligand, receptor) may increase the cytotoxic activity of the cytotoxic cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the cytotoxic activity of the cytotoxic cell in the absence of the dimerizing agent.

[0157] Contacting a genetically modified cytotoxic cell (e.g., a cytotoxic T lymphocyte) with a dimerizing agent and an antigen may increase the cytotoxic activity of the cytotoxic cell by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the cytotoxic activity of the cytotoxic cell in the absence of the dimerizing agent.

[0158] In other embodiments, for example, depending on the host immune cells, contacting the genetically modified host cells with a dimerizer and an antigen may increase or decrease cell proliferation, cell survival, cell death, etc.

[0159] Methods for generating conditionally activatable cells The present disclosure provides a method for generating a conditionally activatable cell. The method generally includes genetically modifying a mammalian cell with an expression vector or RNA (e.g., in vitro transcribed RNA) that includes a nucleotide sequence encoding a heterodimeric conditionally active CAR of the present disclosure. The genetically modified multicellular cell is conditionally activatable in the presence of a) an antigen to which the first polypeptide of the CAR binds, and b) a dimerizer (dimerizing agent). The genetic modification can be performed in vivo, in vitro, or ex vivo. The cell can be an immune cell (e.g., T lymphocyte or NK cell), stem cell, progenitor cell, etc.

[0160] In some cases, the genetic modification is performed ex vivo. For example, T lymphocytes, stem cells, or NK cells are obtained from an individual, and the cells obtained from the individual are genetically modified to express the CAR of the present disclosure. The genetically modified multicellular cells can be conditionally activated in the presence of a) an antigen to which the first polypeptide of the CAR binds, and b) a dimerizing agent. In some cases, the genetically modified multicellular cells are activated ex vivo. In other cases, the genetically modified multicellular cells are introduced into an individual (e.g., the individual from which the cells were obtained), and the genetically modified multicellular cells are activated in vivo, for example, by administering a dimerizing agent to the individual. For example, if the antigen is present on the surface of a cell in the individual, it is not necessary to administer the antigen. The genetically modified multicellular cells are contacted with an antigen present on the surface of a cell in the individual, and upon administration of the dimerizing agent to the individual, the genetically modified cells are activated. For example, if the genetically modified cells are T lymphocytes, the genetically modified cells can exhibit cytotoxicity against cells that present an antigen on their surface to which the CAR binds.

[0161] Treatment method The present disclosure provides various therapeutic methods using the CARs of the present application.

[0162] Cytotoxicity method When present in T lymphocyte or NK cell, the CAR of the present disclosure can mediate cytotoxicity to target cell.The CAR of the present disclosure binds to the antigen present on target cell, thereby mediating the killing of target cell by the T lymphocyte or NK cell genetically modified to generate CAR.The antigen binding domain of CAR binds to the antigen present on the surface of target cell.

[0163] Target cells include, but are not limited to, cancer cells. Thus, the present disclosure provides a method of killing or inhibiting the proliferation of a target cancer cell, comprising contacting a cytotoxic immune effector cell (e.g., a cytotoxic T cell, or a NK cell) genetically engineered to produce a CAR of the present application, such that the T lymphocyte or NK cell recognizes an antigen present on the surface of the target cancer cell and mediates the killing of the target cell.

[0164] The present disclosure provides a method of treating cancer in an individual having cancer, the method comprising: i) genetically modifying T lymphocytes obtained from the individual with an expression vector comprising a nucleotide sequence encoding a heterodimeric conditionally active CAR of the present disclosure, wherein the antigen binding domain of the heterodimeric conditionally active CAR is specific for an epitope on a cancer cell in the individual, and the genetic modification is performed ex vivo; ii) introducing the genetically modified T lymphocytes into the individual; and iii) administering to the individual an effective amount of a dimerization agent, wherein the dimerization induces dimerization of the heterodimeric conditionally active CAR, which provides for activation of the genetically modified T lymphocytes and killing of the cancer cells, thereby treating the cancer.

[0165] Carcinomas that may be amenable to therapy according to the methods disclosed herein include, but are not limited to, esophageal carcinoma, stem cell carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma including transitional cell carcinoma (malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma including small cell carcinoma and non-small cell carcinoma of the lung, adrenal cortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0166] Sarcomas that may be amenable to therapy according to the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovium, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0167] Other solid tumors that may be amenable to therapy according to the methods disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0168] Leukemias that may be amenable to therapy according to the methods disclosed herein include, but are not limited to, a) chronic myeloproliferative syndromes (neoplastic disorders of pluripotent hematopoietic stem cells); b) acute myeloid leukemia (neoplastic transformation of pluripotent hematopoietic stem cells or hematopoietic cells of restricted lineage potential; c) chronic lymphocytic leukemia (CLL; clonal proliferation of immunologically immature and dysfunctional small lymphocytes), including B-cell CLL, T-cell CLL, prolymphocytic leukemia, and hairy cell leukemia; and d) acute lymphoblastic leukemia (characterized by accumulation of lymphoblasts). Lymphomas that may be treated using the methods of the present application include, but are not limited to, B-cell lymphomas (e.g., Burkitt's lymphoma), Hodgkin's lymphoma, non-Hodgkin's lymphoma, and the like.

[0169] Other cancers that may be amenable to treatment in accordance with the methods disclosed herein include atypical meningioma (brain), islet cell carcinoma (pancreas), medullary carcinoma (thyroid), mesenchymoma (intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver), clear cell carcinoma (kidney), and neurofibroma mediastinalis.

[0170] immunomodulation method The method of the present application can also be used to treat inflammatory conditions and autoimmune diseases.The CAR of the present application is expressed in T helper cells or Tregs for use in immunomodulatory methods.Immunomodulatory methods include, for example, enhancing immune response to pathogens in mammalian subjects, enhancing immune response in immunocompromised subjects, reducing inflammatory response, for example, reducing immune response to self-antigens in mammalian subjects to treat autoimmune diseases, reducing immune response to transplanted organs or tissues in mammalian subjects to reduce organ or tissue rejection.

[0171] When the method involves reducing the immune response to a self-antigen, the antigen used to activate the CAR is a self-antigen.When the method involves reducing the immune response to a transplanted organ or tissue, the antigen used to activate the CAR is an antigen specific to the transplanted organ.

[0172] Formulation, Dosage, and Route of Administration As noted above, the therapeutic methods of the present disclosure include administration of an effective amount of a dimerizer to an individual in need thereof, and may also include administration of an antigen.

[0173] An "effective amount" of a dimerizer is, in some cases, an amount that, when administered in one or more doses to an individual in need thereof, increases the level of cytotoxic activity of a T lymphocyte expressing a CAR of the present application by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the cytotoxic activity of the T lymphocyte in the absence of the dimerizer.

[0174] An "effective amount" of a dimerizer is, in some cases, an amount that, when administered in one or more doses to an individual in need thereof, increases the level of cytotoxic activity of an NK cell expressing a CAR of the present application by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, or more than 10-fold, compared to the cytotoxic activity of the NK cell in the absence of the dimerizer.

[0175] An "effective amount" of a dimerizer is, in some cases, an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of cancer cells in the individual and / or reduces the tumor burden in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or more than 75% compared to the number of cancer cells and / or tumor burden in the absence of the dimerizer.

[0176] In some embodiments, an effective amount of a dimerizer is an amount that, when administered in one or more doses alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents (e.g., in combination therapy), is effective to reduce one or more of tumor growth rate, cancer cell number, tumor mass by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, compared to the tumor growth rate, cancer cell number, or tumor mass in the absence of treatment with the dimerizer.

[0177] formulation In the method of the present application, the dimerizer can be administered to the host using any convenient means that can produce the desired therapeutic or diagnostic effect. Thus, the dimerizer can be incorporated into various formulations for therapeutic administration. More specifically, the dimerizer can be formulated into a pharmaceutical composition by combining with a suitable pharma- ceutically acceptable carrier or diluent, and can be formulated into a solid, semi-solid, liquid, or gaseous form of preparation, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

[0178] In pharmaceutical dosage forms, the dimerizers may be administered in the form of their pharma- ceutically acceptable salts, or they may be used alone or, in appropriate association, in combination with other pharma- ceutically active compounds. The following methods and excipients are merely illustrative and in no way limiting.

[0179] Suitable excipient vehicles are, for example, water, saline, glucose, glycerin, ethanol, etc., and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents or pH buffering agents. Actual methods for preparing such dosage forms will be known or apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any event, contain a sufficient amount of dimerizer to achieve the desired state in the subject being treated.

[0180] Pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers, or diluents, are readily available to the public. Moreover, pharma- ceutically acceptable auxiliary agents, such as pH adjusting and buffering agents, isostatic agents, stabilizers, wetting agents, and the like, are readily available to the public.

[0181] For oral preparations, the dimerizer may be used alone or in combination with suitable excipients, e.g., with conventional excipients such as lactose, mannitol, corn starch, or potato starch; with binders such as microcrystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; with disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; with lubricants such as talc or magnesium stearate; and, if desired, with diluents, buffers, wetting agents, preservatives, and flavorings to produce tablets, powders, granules, or capsules.

[0182] The dimerizing agents can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents such as vegetable oils or other similar oils, synthetic fatty acid glycerides, esters of higher fatty acids, or propylene glycol, and, if desired, with conventional additives such as solubilizing agents, isocratic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.

[0183] Pharmaceutical compositions containing a dimerizer are prepared by mixing the dimerizer having a desired degree of purity with any physiologically acceptable carrier, excipient, stabilizer, surfactant, buffer, and / or isoformizer. Acceptable carriers, excipients, and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, phosphoric acid, citric acid, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine, and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propyl paraben, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or buffers such as non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0184] The pharmaceutical composition may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, and lyophilized preparations should be reconstituted in a sterile environment before administration.The standard procedure for reconstituting a lyophilized composition is to add back an amount of pure water (typically equal to the amount removed during lyophilization), however, solutions containing antibacterial agents may be used to produce pharmaceutical compositions for parenteral administration, see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0185] The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined amount of dimerizer calculated in association with a pharma- ceutically acceptable diluent, carrier, or vehicle in an amount sufficient to produce a desired effect. The specifications for a given dimerizer may depend on the particular dimerizer employed and the effect to be achieved, as well as the pharmacodynamics associated with each dimerizer in the host.

[0186] In some embodiments, the dimerizer is formulated into a controlled release formulation. The sustained release preparation can be prepared using methods known to those skilled in the art. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers in contact with the dimerizer, where the matrix is ​​a shaped article, such as a film or a microcapsule. Examples of sustained release matrices include polyesters, copolymers of L-glutamic acid and ethyl-L-glutamic acid, non-degradable ethylene vinyl acetate, hydrogels, polylactides, degradable lactic acid-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyric acid. Potential loss of biological activity can be prevented by using appropriate additives, by controlling water content, and by developing specific polymer matrix compositions.

[0187] dose The appropriate dose can be determined by the attending physician or other qualified medical practitioner based on various clinical factors. As known to those skilled in the art of medicine, the dose for any one patient depends on many factors, including the patient's size, body surface area, age, the specific dimerizer administered, the patient's sex, the time and route of administration, health status, and other drugs being administered concomitantly. The dimerizer can be administered in an amount of 1 ng / kg body weight to 20 mg / kg body weight, e.g., 0.1 mg / kg body weight to 10 mg / kg body weight, e.g., 0.5 mg / kg body weight to 5 mg / kg body weight per administration, however, doses below or above this exemplary range are contemplated, especially considering the aforementioned factors. If the administration regimen is a continuous infusion, this can also be in the range of 1 μg to 10 mg per kilogram of body weight per minute.

[0188] One of skill in the art will readily understand that dosage levels can vary as a function of the specific dimerizer, the severity of the symptoms, and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

[0189] Route of administration The dimerizer is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, and systemic and local routes of administration.

[0190] Conventional pharmaceutical acceptable routes of administration include intratumoral, peritumoral, intramuscular, intratracheal, intracranial, subcutaneous, intradermal, topical, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined or, if desired, adjusted depending on the dimerizer and / or the desired effect. The dimerizer may be administered in a single dose or multiple doses. In some embodiments, the dimerizer is administered orally. In some embodiments, the dimerizer is administered via an inhalation route. In some embodiments, the dimerizer is administered intranasally. In some embodiments, the dimerizer is administered topically. In some embodiments, the dimerizer is administered intratumorally. In some embodiments, the dimerizer is administered peritumorally. In some embodiments, the dimerizer is administered intracranially. In some embodiments, the dimerizer is administered intravenously.

[0191] The dimerizer may be administered to a host using any available conventional method and route suitable for delivery of conventional drugs, including systemic or local routes. In general, routes of administration contemplated by the present invention include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

[0192] Parenteral administration routes other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intrathecal, intrasternal, intratumoral, peritumoral, and intravenous routes, i.e., any route of administration other than through the digestive tract. Parenteral administration can proceed to effect systemic or local delivery of the dimerizer. When systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

[0193] The dimerizer may also be delivered to a subject by enteral administration, including, but not necessarily limited to, oral and rectal (e.g., using a suppository) delivery.

[0194] By treatment is intended at least ameliorating the symptoms associated with a pathological condition affecting the host, where amelioration is used in a broad sense to refer to at least reducing a parameter, e.g., the intensity of a symptom, associated with the pathological condition being treated, such as cancer. Thus, treatment also includes situations in which the pathological condition or at least the symptoms associated therewith are completely suppressed, e.g., prevented from developing, or arrested, e.g., terminated, such that the host no longer suffers from the pathological condition or at least the symptoms characterizing the pathological condition.

[0195] In some embodiments, the dimerizer is administered by injection and / or delivery to a site in a cerebral artery or directly to brain tissue. The dimerizer can be administered directly to the target site, for example, by direct injection, by implantation of a drug delivery device such as an osmotic pump or slow release particles, by biolistic delivery to the target site, etc.

[0196] Combination therapy In some embodiments, the dimerizer is administered as an adjuvant therapy to standard cancer therapies, including surgery (e.g., surgical removal of cancerous tissue), radiation therapy, bone marrow transplantation, chemotherapy treatment, antibody therapy, biological response modifier therapy, and certain combinations of the foregoing.

[0197] Radiation therapy includes, but is not limited to, X-rays or gamma rays delivered from an externally applied source such as a beam or by implantation of small radioactive sources.

[0198] Suitable antibodies for use in cancer therapy include naked antibodies, such as trastuzumab (Herceptin), bevacizumab (Avastin™), cetuximab (Erbitux™), panitumumab (Vectibix™), ipilimumab (Yervoy™), rituximab (Rituxan), alemtuzumab (Lemtrada™), ofatumumab (Arzerra™), oregovomab (OvaRex™), lambrolizumab (MK-3475), pertuzumab (Perjeta™), ranibizumab (Lucentis™), and the like, and conjugated antibodies, such as gemtuzumab ozogamicin (Mylortarg™), brentuximab vedotin (Adcetris™), 90 Y-labeled ibritumomab tiuxetan (Zevalin™), 131 Antibodies suitable for use in cancer therapy include, but are not limited to, antibodies raised against tumor-associated antigens. Such antigens include, but are not limited to, CD20, CD30, CD33, CD52, EpCAM, CEA, gpA33, mucin, TAG-72, CAIX, PSMA, folate binding protein, gangliosides (e.g., GD2, GD3, GM2, etc.), Le y , VEGF, VEGFR, integrin alpha-V-beta-3, integrin alpha-5-beta-1, EGFR, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, tenascin, and the like.

[0199] Biological response modifiers suitable for use in connection with the methods of the present disclosure include, but are not limited to, (1) inhibitors of tyrosine kinase (RTK) activity, (2) inhibitors of serine / threonine kinase activity, (3) tumor-associated antigen antagonists, such as antibodies that specifically bind to tumor antigens, (4) apoptosis receptor agonists, (5) interleukin-2, (6) interferon-α, (7) interferon-γ, (8) colony-stimulating factors, (9) inhibitors of angiogenesis; and (10) tumor necrosis factor antagonists.

[0200] Chemotherapeutic agents are non-peptidic (i.e., non-proteinaceous) compounds that reduce the proliferation of cancer cells and include cytotoxic agents and cytostatic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, and steroid hormones.

[0201] Substances that act to reduce cell proliferation are known to those skilled in the art and are widely used. Such substances include alkylating agents such as nitrogen mustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, and triazenes, including, but not limited to, mechlorethamine, cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide.

[0202] Antimetabolites include folate analogs, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors, including, but not limited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP), pentostatin, 5-fluorouracil (5-FU), methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolate (DDATHF), leucovorin, fludarabine phosphate, pentostatin, and gemcitabine.

[0203] Suitable natural products and their derivatives (e.g., vinca alkaloids, antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins) include Ara-C, paclitaxel (Taxol®), docetaxel (Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine; brequinar; alkaloids, e.g., vincristine, vinblastine, vinorelbine, vindesine, etc.; podophyllotoxins, e.g., etoposide, teniposide, etc.; antibiotics, e.g., anthracycline, daunorubicin hydrochloride, etc. These include, but are not limited to, phenoxyzolidinone cyclopeptides such as dactinomycin; basic glycopeptides such as bleomycin; anthraquinone glycosides such as plicamycin (mithramycin); anthracenediones such as mitoxantrone; azirinopyrroloindole diones such as mitomycin; macrocyclic immunosuppressants such as cyclosporine, FK-506 (tacrolimus, prograf), rapamycin, and the like; and the like.

[0204] Other antiproliferative cytotoxic agents are navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

[0205] Also suitable for use are microtubule affecting agents with antiproliferative activity, including, but not limited to, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine (NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC 376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel (Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC 361792), trityl cysteine, vinblastine sulfate, vincristine sulfate; natural and synthetic epothilones, including, but not limited to, epothilone A, epothilone B, discodermolide; estramustine, nocodazole, and the like.

[0206] Hormonal regulators and steroids (including synthetic analogs) suitable for use include, but are not limited to, corticosteroids such as prednisone, dexamethasone, and the like; estrogens and pregestins such as medroxyprogesterone acetate, megestrol acetate, estradiol, clomiphene, tamoxifen, and the like; and corticosteroids such as aminoglutethimide; 17α-ethynyl estradiol; diethylstilbestrol, testosterone, fluoxymesterone, dromostanolone propionate, testolactone, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide (Drogenil), toremifene (Fareston), and Zoladex®. Estrogen mimics proliferation and differentiation, therefore compounds that bind to the estrogen receptor are used to inhibit this activity Corticosteroids can suppress T cell proliferation.

[0207] Other chemotherapeutic agents include metal complexes, such as cisplatin (cis-DDP), carboplatin, and the like; ureas, such as hydroxyurea; hydrazines, such as N-methylhydrazine; epodophyllotoxins; topoisomerase inhibitors; procarbazine; mitoxantrone; leucovorin; tegafur; etc. Other antiproliferative agents of interest include, for example, mycophenolic acid, thalidomide, desoxyspergualin, azasporine, leflunomide, mizoribine, azaspirane (SKF105685); Iressa® (ZD 1839, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline); and the like.

[0208] "Taxane" includes paclitaxel, as well as any active taxane derivative or prodrug. "Paclitaxel" (which should be understood herein to include analogs, formulations, and derivatives, such as, for example, docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), the 10-desacetyl analog of paclitaxel, and the 3'N-desbenzoyl-3'Nt-butoxycarbonyl analog of paclitaxel) can be readily prepared using techniques known to those of skill in the art (WO 94 / 07882, WO Nos. 5,294,637, 5,283,253, 5,279,949, 5,274,137, 5,202,448, 5,200,534, 5,229,529, and EP 590,267), or from a variety of commercially available sources, including, for example, Sigma Chemical Co. of St. Louis, Missouri (T7402 from European yew, T-1912 from Taxus yannanensis).

[0209] It should be understood that paclitaxel refers not only to the common, chemically available forms of paclitaxel, but also to analogues and derivatives (e.g., Taxotere™ docetaxel, as described above) and paclitaxel conjugates (e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

[0210] In addition, various known derivatives, including both hydrophilic and hydrophobic derivatives, are also included in the term "taxane". Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application WO99 / 18113; the piperazino and other derivatives described in WO99 / 14209; the taxane derivatives described in WO99 / 09021, WO98 / 22451, and U.S. Patent No. 5,869,680; the 6-thio derivatives described in WO98 / 28288; the sulfenamide derivatives described in U.S. Patent No. 5,821,263; and the taxol derivatives described in U.S. Patent No. 5,415,869. This further includes prodrugs of paclitaxel, including, but not limited to, those described in WO98 / 58927, WO98 / 13059, and U.S. Patent No. 5,824,701.

[0211] Suitable subjects for treatment A variety of subjects are suitable for treatment with the present methods of treating cancer. Suitable subjects include any individual, e.g., a human or non-human animal, who has cancer, has been diagnosed with cancer, is at risk of developing cancer, has had cancer and is at risk of cancer recurrence, has been treated with an agent other than a dimerizer for cancer and has failed to respond to such treatment, or has been treated with an agent other than a dimerizer for cancer and has relapsed after an initial response to such treatment.

[0212] Subjects suitable for treatment with the immunomodulatory methods of the present application include individuals with autoimmune disorders, immunocompromised individuals, such as individuals who are organ or tissue transplant recipients, and individuals infected with a pathogen. EXAMPLES

[0213] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Abbreviations such as bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); im intramuscular; ip intraperitoneal; sc subcutaneous; iv intravenous; and the like may be used.

[0214] Example 1: Generation of CARs Materials and Methods Through the design optimization process, anti-human CD19scFV was selected as the antigen recognition domain in the CAR. Figures 18A and 18B summarize the molecular structure of each CAR, which consists of two polypeptides that are numerically identified. All membrane anchor polypeptides are disulfide-bonded homodimers. The membrane anchor polypeptides are depicted as monomers to simplify the drawings.

[0215] Generation of CAR constructs The non-human CD19scFv coding sequence was cloned from one construct. The human 4-1BB costimulatory and CD3 zeta ITAM signaling chains were cloned from cDNA provided by Open Biosystems. The FKBP and FRB coding sequences were cloned from plasmids provided by Addgene.

[0216] Standard molecular cloning techniques (polymerase chain reaction (PCR), restriction digestion, ligation, etc.) were applied to generate lentiviral expression plasmids.

[0217] Culture conditions of effector and target cells Human primary CD8+ T cells were isolated from the blood of an anonymous donor after apheresis (Trima residuals from Blood Centers of the Pacific, San Francisco, CA) by negative selection using RosetteSep Human CD8+ T Cell Enrichment Cocktail (STEMCELL Technologies, #15063) as approved by the University Institutional Review Board. Cells were cultured in human T cell medium consisting of X-VIVO15 (Lonza, #04-418Q), 5% human AB serum (Valley Biomedical Inc., #HP1022), 10 mM N-acetyl-L-cysteine ​​(Sigma-Aldrich, #A9165), and 100 IU / mL recombinant human IL-2 (NCI / BRB Preclinical Repository). The Jurkat cell line, which expresses green fluorescent protein (GFP) upon NFAT activation, was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin. K562 target cells from U. Penn were cultured in IMDM supplemented with 10% FBS.

[0218] Engineering effector and target cells using lentiviruses Pantropic VSV-G pseudotyped lentiviruses were generated from Lenti-X293T cells (Clontech Laboratories, #632180) co-transfected with pHR'SIN:CSW transgene expression vector, viral packaging plasmids, pCMVdR8.91, and pMD2.G using Lipofectamine LTX (Life Technologies, #15338). Infection medium supernatants were harvested 48 hours post-transfection and used directly for transduction.

[0219] 24 hours prior to viral transduction, primary human T cells were activated using human T-Activator CD3 / CD28 Dynabeads (Life Technologies, no. 111-31D) at a cell:bead ratio of 1:3. Jurkat and K562 cells were split 1-2 days in advance to ensure that the cultures would be in log phase at the time of transduction. Transduced Jurkat and K562 cells were cultured for at least 7 days before performing the experiments. Primary T cells were maintained at approximately 10^6 / mL in human T cell medium for approximately 2 weeks until the cells returned to quiescent phase. Expression levels of the CAR encoded in the lentiviral construct were quantified using a flow cytometer by detecting either fluorophore-conjugated antibodies or fluorescent reporter proteins.

[0220] Quantification of IL-2 production and NFAT activity Jurkat CD4+ T cells expressing CAR were mixed with cognate or non-cognate K562 target cells from U.Penn at an effector:target ratio of 1:2. Rapalog A / C Heterodimerizer (Clontech Laboratories, no. 635055) was serially diluted in medium and added to the reaction mixture. After 20–24 h of incubation, medium supernatants were harvested and analyzed with a BD OptEIA Human IL-2 ELISA Set (BD Biosciences, no. 555190). Flow cytometry was performed to quantify NFAT-dependent GFP reporter expression in Jurkat cells as a separate indicator of CAR activity.

[0221] Flow cytometry-based re-directed cytotoxicity assay Cognate and non-cognate K562 target cells were engineered to express distinct fluorescent proteins so that both types of cells in the mixture could be quantified simultaneously by flow cytometry. Target cell types were mixed in a 1:1 ratio and co-incubated with human primary CD8+ effector T cells at an effector:target ratio of 5:2. 100 IU / mL human IL-2 and various amounts of rapalog (Clontech Laboratories, no. 635055) were added to the reaction mixture. After 24 hours of incubation, samples were centrifuged at 400 g for 5 minutes. Pelleted cells were resuspended in wash buffer (PBS + 0.5% BSA + 0.1% sodium azide) and fixed with an equivalent volume of BD Cytofix (BD cat, no. 554655) prior to flow cytometry. To enumerate the redirected cytotoxic activity of effector cells, the ratio of surviving cognate to non-cognate target cells was calculated for each sample.

[0222] result IL-2 production elicited by various CAR constructs was evaluated and the data is provided in FIG.

[0223] Figure 12: IL-2 production driven by five on-switch CAR mutants. Effector = human CD4+ Jurkat T cells engineered with CAR. Target = K562 cell line with or without the cognate CD19 antigen. The amount of IL-2 secreted by effector cells was quantified by enzyme-linked immunosorbent assay (ELISA).

[0224] Figure 13: IL-2 production by a control Jurkat line in the same experiment as described in Figure 12. Construct "125" represents the conventional control currently used in clinical trials.

[0225] FIG. 14: Comparison between “122+206” and “197+206” in a separate experiment under conditions identical to those described in FIG.

[0226] Figure 15 shows the pharmacologically titratable cytotoxicity conferred by the on-switch CAR "197+206". In the presence of a small molecule rapalog, the CAR effectively mediates redirected cytotoxicity against cognate target cells. At high doses of the rapalog, this on-switch CAR can signal as potently as the "125" conventional CAR. Effectors = human primary CD8+ T cells engineered with CAR or control vector. Targets = fluorescent derivatives of K562 cell lines expressing either the cognate human CD19 antigen or the non-cognate human mesothelin antigen.

[0227] FIG. 16 shows data for a CAR constructed with the cytoplasmic tyrosine kinase Zap70 from the T cell receptor pathway as the intracellular signaling domain.

[0228] FIG. 16 shows data from Jurkat cells engineered with several variants of the on-switch CAR. Engineered Jurkat cells were co-incubated with K562 target cells with or without the cognate antigen (CD19) and the indicated concentrations of rapalogs. As a CAR component, Zap70 kinase (first and second structures from the left, labeled "199") was as effective as ITAM (third structure from the left, labeled "168") in activating NFAT function. Addition of the 4-1BB signaling domain increased surface expression of the antigen recognition portion of the receptor, resulting in stronger signaling by "197+199". A non-signaling CAR (far right) was included as a negative control.

[0229] Example 2: Mesothelin-targeted CAR Materials and Methods Several chimeric antigen receptor constructs have been made and tested. The constructs shown here encode three different anti-human mesothelin scFvs as antigen recognition domains. Figures 19A, 19B, and 19C summarize the molecular structure of each anti-human mesothelin CAR, where each CAR contains two polypeptides. The intracellular portion of each anti-human mesothelin CAR contains two 4-1BB costimulatory domains, an FKBP and FRB dimerizer binding pair, and an ITAM intracellular signaling domain. The three different antigen recognition domains shown here are anti-mesothelin HN1scFv, SS1scFv, and m912scFv. All membrane anchor polypeptides are disulfide-linked homodimers.

[0230] Generation of CAR constructs Anti-mesothelin coding sequences were cloned from constructs or synthesized via gene assembly by PCR. Human 4-1BB costimulatory and CD3 zeta ITAM signaling chains were cloned from cDNA provided by Open Biosystems. HN1scFv, SS1scFv, and m912scFv coding sequences were synthesized by PCR and, in some cases, codon-optimized. FKBP and FRB coding sequences were cloned from Addgene plasmids.

[0231] Standard molecular cloning techniques (polymerase chain reaction (PCR), restriction digestion, ligation, etc.) were applied to generate lentiviral expression plasmids.

[0232] Culture conditions of effector and target cells The Jurkat cell line, which expresses GFP upon NFAT activation, was maintained in RPMI-1640 medium supplemented with 10% FBS, penicillin, and streptomycin. K562 target cells were cultured in IMDM supplemented with 10% fetal bovine serum (FBS).

[0233] Engineering effector and target cells using lentiviruses Pantropic VSV-G pseudotyped lentiviruses were generated from Lenti-X293T cells (Clontech Laboratories, #632180) co-transfected with pHR'SIN:CSW transgene expression vector, viral packaging plasmids, pCMVdR8.91, and pMD2.G using Lipofectamine LTX (Life Technologies, #15338). Infection medium supernatants were harvested 48 hours post-transfection and used directly for transduction.

[0234] Jurkat and K562 cells were split 1-2 days in advance to ensure that the cultures would be in log phase at the time of transduction. Transduced Jurkat and K562 cells were cultured for at least 7 days before performing experiments. Expression levels of CAR encoded in the lentiviral construct were quantified using a flow cytometer by detecting either a fluorophore-conjugated antibody or a fluorescent reporter protein.

[0235] Quantification of IL-2 production Jurkat CD4+ T cells expressing CAR were mixed with cognate or non-cognate K562 target cells at an effector:target ratio of 1:2. Rapalog A / C Heterodimerizer (Clontech Laboratories, no. 635055) was serially diluted in medium and added to the reaction mixture. After 20–24 h of incubation, medium supernatants were harvested and analyzed with BD OptEIA Human IL-2 ELISA Set (BD Biosciences, no. 555190).

[0236] result IL-2 production elicited by the anti-mesothelin CAR construct was assessed, and the data are provided in Figures 19D-F.

[0237] Figure 19: IL-2 production driven by HN1scFv (Figure 19D), SS1scFv (Figure 19E), and m912scFv (Figure 19F) on-switch CAR mutants. IL-2 production by conventional CAR (Figure 19G, construct number 358) was measured and included for comparison with on-switch CAR (Figure 19D). Effector = human CD4+ Jurkat T cells engineered with CAR. Target = K562 cell line with or without cognate mesothelin antigen. The amount of IL-2 secreted by effector cells was quantified by enzyme-linked immunosorbent assay (ELISA).

[0238] Example 3: Gibberellic Acid as a Dimerizer of On-Switch CAR Materials and Methods Figure 20A summarizes the molecular structure of the present gibberellic acid dimerizer CAR. The antigen-binding portion contains anti-human CD19scFv. The intracellular portion contains two 4-1BB costimulatory domains, the GID1 and GAI dimerizer binding pair, and the ITAM intracellular signaling domain. All membrane-anchored polypeptides are disulfide-bonded homodimers.

[0239] Generation of CAR constructs The sequence encoding the gibberellic acid dimerizer CAR was cloned from the construct. Anti-CD19 scFv was cloned from a plasmid. Human 4-1BB costimulatory and CD3 zeta ITAM signaling chains were cloned from cDNA provided by Open Biosystems. GID1 and GAI coding sequences were cloned from Addgene plasmids. Standard molecular cloning techniques (polymerase chain reaction (PCR), restriction digestion, ligation, etc.) were applied to generate lentiviral expression plasmids.

[0240] Culture conditions of effector and target cells The Jurkat cell line, which expresses GFP upon NFAT activation, was maintained in RPMI-1640 medium supplemented with 10% FBS, penicillin, and streptomycin. K562 target cells were cultured in IMDM supplemented with 10% fetal bovine serum (FBS).

[0241] Engineering effector and target cells using lentiviruses Pantropic VSV-G pseudotyped lentiviruses were generated from Lenti-X293T cells (Clontech Laboratories, #632180) co-transfected with pHR'SIN:CSW transgene expression vector, viral packaging plasmids, pCMVdR8.91, and pMD2.G using Lipofectamine LTX (Life Technologies, #15338). Infection medium supernatants were harvested 48 hours post-transfection and used directly for transduction.

[0242] Jurkat and K562 cells were split 1-2 days in advance to ensure that the cultures would be in log phase at the time of transduction. Transduced Jurkat and K562 cells were cultured for at least 7 days before performing experiments. Expression levels of CAR encoded in the lentiviral construct were quantified using a flow cytometer by detecting either a fluorophore-conjugated antibody or a fluorescent reporter protein.

[0243] Quantification of IL-2 production Jurkat CD4+ T cells expressing CAR were mixed with cognate or non-cognate K562 target cells at an effector:target ratio of 1:2. Gibberellic acid-3 acetoxymethyl ester (gibberellic acid-3AM) pre-dissolved in ethanol (Toronto Research Chemicals, no. G377500) was diluted in growth medium and added to the reaction mixture. Gibberellic acid (gibberellic acid-3AM) was used at 10 mM. After 20–24 h of incubation, culture supernatants were harvested and analyzed with the BD OptEIA Human IL-2 ELISA Set (BD Biosciences, no. 555190).

[0244] result IL-2 production elicited by the gibberellic acid dimerizer CAR construct was evaluated, and the data are provided in FIG.

[0245] Figure 20: IL-2 production driven by gibberellic acid dimerizer CAR mutants (Figure 20B). IL-2 production by a conventional CAR (Figure 20C, construct "125") was measured and included for comparison with the on-switch CAR. Effector = human CD4+ Jurkat T cells engineered with CAR. Target = K562 cell line with or without the cognate CD19 antigen. The amount of IL-2 secreted by effector cells was quantified by enzyme-linked immunosorbent assay (ELISA).

[0246] Example 4: On-switch CARs with various costimulatory domains Materials and Methods Multiple chimeric antigen receptor constructs were generated essentially as described for Example 1, except that the 4-1BB costimulatory domain was replaced with various other costimulatory domains. Figures 21A and 21B summarize the molecular structures of the CARs described herein.

[0247] Generation of CAR constructs The sequence encoding the anti-human CD19scFv was cloned from a plasmid. The human CD3 zeta ITAM signaling chain, and the human costimulatory domains CD28 and OX-40 coding sequences were cloned from cDNA provided by Open Biosystems. The FKBP and FRB coding sequences were cloned from plasmids from Addgene.

[0248] Standard molecular cloning techniques (polymerase chain reaction (PCR), restriction digestion, ligation, etc.) were applied to generate lentiviral expression plasmids.

[0249] Testing of CAR constructs Effector and target cells were cultured and transfected according to Example 1 using the described on-switch CAR constructs containing CD28 and OX-40 costimulatory domains (Figures 21A-B, constructs "365+367" and "399+400", respectively), as well as the corresponding conventional CAR counterparts (Figures 21C-D, constructs "366" and "398", respectively). IL-2 production, NFAT activity assays, and flow cytometry-based assays can also be performed using CD28 costimulatory domain containing constructs and OX-40 costimulatory domain containing constructs, as described for Example 1. Alternatively, subunits of on-switch CAR constructs containing CD28 and OX-40 costimulatory domains can be paired with subunits of constructs from Example 1 (e.g., "197+367", "365+206", "197+400", "399+206", etc.).

[0250] Example 5: In vivo evaluation of on-switch CARs On-switch CARs can be evaluated for their ability to mediate target tumor cell killing in vivo. The killing of tumor cells in vivo elicited by injection of T cells expressing the on-switch CAR is evaluated. CD8 T cells confirmed to express the cognate antigen in vitro and expressing the corresponding CAR are used. +Use a tumor cell line that can be killed by T cells. Tumor cells engineered to express either firefly or Renilla luciferase can be used to allow for quantification of tumor burden in vivo by bioluminescence imaging. Tumor cells are injected into immunocompromised mice (e.g., 6-10 week old female NOD severe combined immunodeficiency gamma (NSG) mice) either subcutaneously for subcutaneous tumor models or intravenously for systemic tumor models. The method of tumor implantation and the optimal number of tumor cells to implant can be based on the optimal conditions for the tumor cell line used. Tumor burden can be monitored twice weekly by bioluminescence imaging and, if applicable, by caliper measurements. As soon as tumor burden is detectable, a total of 0.5-2.5x10^7 T cells expressing the on-switch CAR (1:1 CD4 + :CD8 + ) is injected intravenously into mice to initiate treatment. Dimerizing small molecule drugs (e.g., rapalogs) are administered intraperitoneally in a vehicle formulation. T cells expressing the on-switch CAR can be repeatedly injected during the experiment to enhance the anti-tumor effect. Interleukin-2 (IL-2) can be administered to enhance the anti-tumor effect.

[0251] Although the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention. Also, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

[0252] Sequence information SEQUENCE LISTING <110> THE REGENTS OF THE UNIVERSITY OF CALIFORNIA <120> CHIMERIC ANTIGEN RECEPTOR AND METHODS OF USE THEREOF <150> US 61 / 765,585 <151> 2013-02-15 <160> 145 <170> PatentIn version 3.5 <210> 1 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 1 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 2 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 2 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 3 gaggagaagc tgatcaggcga gaggacctg 30 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 4 Leu Glu Gln Lys Is The Leu Glu Glu Asp 1 5 10 <210> 5 <211> 726 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 5 gataccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gggcaagtca ggacattagt aaatatta attggtatca gcagaaacca 120 gatgaactg ttaaactcct gatctaccat acatcagat tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattctca ccattagcaa cctggagcaa 240 gaagatattg catcattactt gttcggaggg ggtaatacgc tccgtacac gttcggaggg 300 gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360 ggactgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420 tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480 cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540 tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600 ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660 tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720 tcctca 726 <210> 6 <211> 242 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 6 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr and Ser Cys Arg Ala Ser Gln Asp and Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140 Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg 145 150 155 160 Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175 Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190 Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205 Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220 Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 225 230 235 240 Ser Ser <210> 7 <211> 207 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 7 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180 ctgtcactgg ttatcaccct ttactgc 207 <210> 8 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 8 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 35 40 45 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 50 55 60 Ile Thr Leu Tyr Cys 65 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 9 tccctaggaa gcgggtccgg tagcggatct 30 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 10 Ser Leu Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <210> 11 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 11 atgggagtcc aggtggaaac catctcccca ggagacgggc gcaccttccc caagcgcggc 60 cagacctgcg tggtgcacta caccgggatg cttgaagatg gaaagaaatt tgattcctcc 120 cgggacagaa acaagccctt taagtttatg ctaggcaagc aggaggtgat ccgaggctgg 180 gaagaagggg ttgcccagat gagtgtgggt cagagagcca aactgactat atctccagat 240 tatgcctatg gtgccactgg gcacccaggc atcatcccac cacatgccac tctcgtcttc 300 gatgtggagc ttctaaaact ggaa 324 <210> 12 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 12 Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe 1 5 10 15 Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu 20 25 30 Asp Gly Lys Lys Phe Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys 35 40 45 Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val 50 55 60 Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp 65 70 75 80 Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala 85 90 95 Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 100 105 <210> 13 <211> 282 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 13 atgatcctct ggcatgagat gtggcatgaa ggctggaag agcatctcg tttgtacttt 60 ggggaaagga acgtgaaagg catgtttgag gtgctggagc ccttgcatgc tatgatggaa 120 cggggccccc agactctga ggaaacatcc tttaatcagg cctatgtcg agatttaatg 180 gaggcccaag agtggtgcag gaagtacatg aaatcaggga atgtcagga cctcctccaa 240 gcctgggacc tctattatca tgtgttccga cgaatctcaa ag 282 <210> 14 <211> 94 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 14 Met Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser 1 5 10 15 Arg Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu 20 25 30 Glu Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu 35 40 45 Thr Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu 50 55 60 Trp Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln 65 70 75 80 Ala Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys 85 90 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 15 ggaagcggtt ccggtagcgg atctcccta 30 <210> 16 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 16 Gly Ser Gly Ser Gly Ser Gly Ser Leu 1 5 10 <210> 17 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 17 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgagagag gagtacgatg tttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagatagat ggcggaggcc tacagtgaga ttgggatga aggcgagcgc 240 cggaggggca aggggcacga tggctttac cagggtctca gtacagccac caggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgc 336 <210> 18 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 18 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 19 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 19 tcgcgaggaa gcgggtccgg tagcggatct 30 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 20 Ser Arg Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 <210> 21 <211> 708 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 21 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480 gcctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaag 708 <210> 22 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 22 Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe 1 5 10 15 Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe 20 25 30 Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr 35 40 45 Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp 50 55 60 Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His 65 70 75 80 Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe 85 90 95 Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val 100 105 110 Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys 115 120 125 Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys 130 135 140 Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly 145 150 155 160 Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly 165 170 175 His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val 180 185 190 Gln Leu Pro Gly Only Tyr Asn Val Asn With Lys Leu Asp With Thr Ser 195 200 205 His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly 210 215 220 Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 23 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 23 aaacggggca gaagaaact cctgtatata ttcaacaac catttatgag accagtacaa 60 actactcaag aggagatgg ctgtagctgc cgatttccag agagaga aggagatgt 120 failure 126 <210> 24 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 24 Lys Arg Gly Arg Lys Leu Leu Tyr Ile Phe Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 25 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 25 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgcct cctcgc 336 <210> 26 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 26 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 27 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 27 atgatccatc tgggtcacat cctcttcctg cttttgctcc cagtggctgc agctcagacg 60 actccaggag agagatcatc actccctgcc ttttaccctg gcacttcagg ctcttgttcc 120 ggatgtgggt ccctctctct gccg 144 <210> 28 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 28 Met Ile His Leu Gly His Ile Leu Phe Leu Leu Leu Leu Pro Val Ala 1 5 10 15 Ala Ala Gln Thr Thr Pro Gly Glu Arg Ser Ser Leu Pro Ala Phe Tyr 20 25 30 Pro Gly Thr Ser Gly Ser Cys Ser Gly Cys Gly Ser Leu Ser Leu Pro 35 40 45 <210> 29 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 29 atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60 accctttact gc 72 <210> 30 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 30 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 31 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 31 ggttccggca gcggatctgg tagcggaagc gggtccggta gcggatct 48 <210> 32 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 32 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 15 <210> 33 <211> 279 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 33 atcctctggc atgagatgtg gcatgaaggc ctggaagagg catctcgttt gtactttggg 60 gaaaggaacg tgaaaggcat gtttgaggtg ctggagccct tgcatgctat gatggaacgg 120 ggcccccaga ctctgaagga aacatccttt aatcaggcct atggtcgaga tttaatggag 180 gcccaagagt ggtgcaggaa gtacatgaaa tcagggaatg tcaaggacct cctccaagcc 240 tgggacctct attatcatgt gttccgacga atctcaaag 279 <210> 34 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 34 Ile Leu Trp His Glu Met Trp His Glu Gly Leu Glu Glu Ala Ser Arg 1 5 10 15 Leu Tyr Phe Gly Glu Arg Asn Val Lys Gly Met Phe Glu Val Leu Glu 20 25 30 Pro Leu His Ala Met Met Glu Arg Gly Pro Gln Thr Leu Lys Glu Thr 35 40 45 Ser Phe Asn Gln Ala Tyr Gly Arg Asp Leu Met Glu Ala Gln Glu Trp 50 55 60 Cys Arg Lys Tyr Met Lys Ser Gly Asn Val Lys Asp Leu Leu Gln Ala 65 70 75 80 Trp Asp Leu Tyr Tyr His Val Phe Arg Arg Ile Ser Lys 85 90 <210> 35 <211> 1857 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 35 atgccagacc ccgcggcgca tctgcccttc ttctacggca gcatctcgcg tgccgaggcc 60 gaggagcacc tgaagctggc gggcatggcg gacgggctct tctgctgcg ccagtgcctg 120 cgctcgctg gcggctatgt gctgcgctc gtgcacgatg tgcgcttcca cactttccc 180 atcgagcgcc agctcaacgg cacctacgcc attgccggcg gcaaagcgca ctgtggaccg 240 gcagagctct gcgagttcta ctcgcgcgac ccgacgggc tgccctgcaa cctgcgcaag 300 ccgtgcaacc ggccgtcggg cctcgagccg cagccggggg tcttcgactg cctgcgagac 360 gccatggtgc gtgactacgt gcgccagacg tggaagctgg agggcgaggc cctggagcag 420 gccatcatca gccaggcccc gcaagtggag aagctcattg ctacgacggc ccacgagcgg 480 atgccctggt accacagcag cctgacgcgt gaggaggccg agcgcaaact ttactctggg 540 gcgcagaccg acggcaagtt cctgctgagg ccgcggaagg agcagggcac atacgccctg 600 tccctcatct atgggaagac ggtgtaccac tacctcatca gccaagacaa ggcgggcaag 660 tactgcattc ccgagggcac caagtttgac acgctctggc agctggtgga gtatctgaag 720 ctgaaggcgg acgggctcat ctactgcctg aaagggcct gccccaacag cagtgccagc 780 aacgcctcag gggctgctgc tcccacatc ccagcccacc catccacgtt gactcatcct 840 cagagagaa tcgacaccct caactcagat ggatacaccc ctgagccagc acgcataacg 900 tccccagaca aaccgcggcc gatgcccatg gacacgagcg tgtatgagag cccctacagc 960 gacccagagg agctcaagga aagaagctc ttcctgaagc gcgataacct cctcatagct 1020 gacattgaac ttggctgcgg caactttggc tcagtgcgcc agggcgtgta ccgcatgcgc 1080 aagaagcaga tcgacgtggc catcaaggtg ctgaagcagg gcacggagaa ggcagacacg 1140 gaagagatga tgcgcgaggc gcagatcatg caccagctgg acaaccccta catcgtgcgg 1200 ctcattggcg tctgccaggc cgaggccctc atgctggtca tggagatggc tgggggcggg 1260 ccgctgcaca agttcctggt cggcaagagg gaggagatcc ctgtgagcaa tgtggccgag 1320 ctgctgcacc aggtgtccat ggggatgaag tacctggagg agaagaactt tgtgcaccgt 1380 gacctggcgg cccgcaacgt cctgctggtt aaccggcact acgccaagat cagcgacttt 1440 ggcctctcca aagcactggg tgccgacgac agctactaca ctgcccgctc agcagggaag 1500 tggccgctca agtggtacgc acccgaatgc atcaacttcc gcaagttctc cagccgcagc 1560 gatgtctgga gctatggggt caccatgtgg gaggccttgt cctacggcca gaagccctac 1620 aagaagatga aagggccgga ggtcatggcc ttcatcgagc agggcaagcg gatggagtgc 1680 ccaccagagt gtccacccga actgtacgca ctcatgagtg actgctggat ctacaagtgg 1740 gaggatcgcc ccgacttcct gaccgtggag cagcgcatgc gagcctgtta ctacagcctg 1800 gccagcaagg tggaagggcc cccaggcagc acacagaagg ctgaggctgc ctgtgcc 1857 <210> 36 <211> 619 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 36 Met Pro Asp Pro Ala Ala His Leu Pro Phe Phe Tyr Gly Ser Ile Ser 1 5 10 15 Arg Ala Glu Ala Glu Glu His Leu Lys Leu Ala Gly Met Ala Asp Gly 20 25 30 Leu Phe Leu Leu Arg Gln Cys Leu Arg Ser Leu Gly Gly Tyr Val Leu 35 40 45 Ser Leu Val His Asp Val Arg Phe His His Phe Pro Ile Glu Arg Gln 50 55 60 Leu Asn Gly Thr Tyr Ala Ile Ala Gly Gly Lys Ala His Cys Gly Pro 65 70 75 80 Ala Glu Leu Cys Glu Phe Tyr Ser Arg Asp Pro Asp Gly Leu Pro Cys 85 90 95 Asn Leu Arg Lys Pro Cys Asn Arg Pro Ser Gly Leu Glu Pro Gln Pro 100 105 110 Gly Val Phe Asp Cys Leu Arg Asp Ala Met Val Arg Asp Tyr Val Arg 115 120 125 Gln Thr Trp Lys Leu Glu Gly Glu Ala Leu Glu Gln Ala Ile Ile Ser 130 135 140 Gln Ala Pro Gln Val Glu Lys Leu Ile Ala Thr Thr Ala His Glu Arg 145 150 155 160 Met Pro Trp Tyr His Ser Ser Leu Thr Arg Glu Glu Ala Glu Arg Lys 165 170 175 Leu Tyr Ser Gly Ala Gln Thr Asp Gly Lys Phe Leu Leu Arg Pro Arg 180 185 190 Lys Glu Gln Gly Thr Tyr Ala Leu Ser Leu Ile Tyr Gly Lys Thr Val 195 200 205 Tyr His Tyr Leu Ile Ser Gln Asp Lys Ala Gly Lys Tyr Cys Ile Pro 210 215 220 Glu Gly Thr Lys Phe Asp Thr Leu Trp Gln Leu Val Glu Tyr Leu Lys 225 230 235 240 Leu Lys Ala Asp Gly Leu Ile Tyr Cys Leu Lys Glu Ala Cys Pro Asn 245 250 255 Ser Ser Ala Ser Asn Ala Ser Gly Ala Ala Ala Pro Thr Leu Pro Ala 260 265 270 His Pro Ser Thr Leu Thr His Pro Gln Arg Arg Ile Asp Thr Leu Asn 275 280 285 Ser Asp Gly Tyr Thr Pro Glu Pro Ala Arg Ile Thr Ser Pro Asp Lys 290 295 300 Pro Arg Pro Met Pro Met Asp Thr Ser Val Tyr Glu Ser Pro Tyr Ser 305 310 315 320 Asp Pro Glu Glu Leu Lys Asp Lys Lys Leu Phe Leu Lys Arg Asp Asn 325 330 335 Leu Leu Ile Ala Asp Ile Glu Leu Gly Cys Gly Asn Phe Gly Ser Val 340 345 350 Arg Gln Gly Val Tyr Arg Met Arg Lys Lys Gln Ile Asp Val Ala Ile 355 360 365 Lys Val Leu Lys Gln Gly Thr Glu Lys Ala Asp Thr Glu Glu Met Met 370 375 380 Arg Glu Ala Gln Ile Met His Gln Leu Asp Asn Pro Tyr Ile Val Arg 385 390 395 400 Leu Ile Gly Val Cys Gln Ala Glu Ala Leu Met Leu Val Met Glu Met 405 410 415 Ala Gly Gly Gly Pro Leu His Lys Phe Leu Val Gly Lys Arg Glu Glu 420 425 430 Ile Pro Val Ser Asn Val Ala Glu Leu Leu His Gln Val Ser Met Gly 435 440 445 Met Lys Tyr Leu Glu Glu Lys Asn Phe Val His Arg Asp Leu Ala Ala 450 455 460 Arg Asn Val Leu Leu Val Asn Arg His Tyr Ala Lys Ile Ser Asp Phe 465 470 475 480 Gly Leu Ser Lys Ala Leu Gly Ala Asp Asp Ser Tyr Tyr Thr Ala Arg 485 490 495 Ser Ala Gly Lys Trp Pro Leu Lys Trp Tyr Ala Pro Glu Cys Ile Asn 500 505 510 Phe Arg Lys Phe Ser Ser Arg Ser Asp Val Trp Ser Tyr Gly Val Thr 515 520 525 Met Trp Glu Ala Leu Ser Tyr Gly Gln Lys Pro Tyr Lys Lys Met Lys 530 535 540 Gly Pro Glu Val Met Ala Phe Ile Glu Gln Gly Lys Arg Met Glu Cys 545 550 555 560 Pro Pro Glu Cys Pro Pro Glu Leu Tyr Ala Leu Met Ser Asp Cys Trp 565 570 575 Ile Tyr Lys Trp Glu Asp Arg Pro Asp Phe Leu Thr Val Glu Gln Arg 580 585 590 Met Arg Ala Cys Tyr Tyr Ser Leu Ala Ser Lys Val Glu Gly Pro Pro 595 600 605 Gly Ser Thr Gln Lys Ala Glu Ala Ala Cys Ala 610 615 <210> 37 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <220> <221> REPEAT <222> (1)..(5) <223> the amino acids in this region can be repeated n times, where n is an integer of at least one <400> 37 Gly Ser Gly Gly Ser 1 5 <210> 38 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <220> <221> REPEAT <222> (1)..(4) <223> the amino acids in this region can be repeated n times, where n is an integer of at least one <400> 38 Gly Gly Gly Ser 1 <210> 39 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 39 Gly Gly Ser Gly 1 <210> 40 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 40 Gly Gly Ser Gly Gly 1 5 <210> 41 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 41 Gly Ser Gly Ser Gly 1 5 <210> 42 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 42 Gly Ser Gly Gly Gly 1 5 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 43 Gly Gly Gly Ser Gly 1 5 <210> 44 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 44 Gly Ser Ser Ser Gly 1 5 <210> 45 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 45 Asp Lys Thr His Thr 1 5 <210> 46 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 46 Cys Pro Pro Cys 1 <210> 47 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 47 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 1 5 10 15 <210> 48 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 48 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr 1 5 10 <210> 49 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 49 Lys Ser Cys Asp Lys Thr His Thr Cys Pro 1 5 10 <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 50 Lys Cys Cys Val Asp Cys Pro 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 51 Lys Tyr Gly Pro Pro Cys Pro 1 5 <210> 52 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 52 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 53 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 53 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 1 5 10 <210> 54 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 54 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys 1 5 10 15 Pro <210> 55 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 55 Ser Pro Asn Met Val Pro His Ala His His Ala Gln 1 5 10 <210> 56 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 56 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 57 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 57 Leu Gly Leu Leu Val Ala Gly Val Leu Val Leu Leu Val Ser Leu Gly 1 5 10 15 Val Ala Ile His Leu Cys Cys 20 <210> 58 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 58 Ala Leu Ile Val Leu Gly Gly Val Ala Gly Leu Leu Leu Phe Ile Gly 1 5 10 15 Leu Gly Ile Phe Phe Cys Val Arg Cys 20 25 <210> 59 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 59 Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu 1 5 10 15 Thr Ala Leu Phe Leu Arg Val 20 <210> 60 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 60 Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu 1 5 10 15 Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 61 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 61 Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly Leu Leu Gly Pro 1 5 10 15 Leo Ala Ile Leo Leo Ala Leo Tyr Leo Leo 20 25 <210> 62 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 62 Ala Leu Pro Ala Ala Leu Ala Val Ile Ser Phe Leu Leu Gly Leu Gly 1 5 10 15 Leu Gly Val Ala Cys Val Leu Ala 20 <210> 63 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 63 Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met 1 5 10 15 Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro 20 25 30 Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 64 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 64 Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr 1 5 10 15 Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp 20 25 30 Val Thr Leu 35 <210> 65 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 65 Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys Pro Pro Gly Gly 1 5 10 15 Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln Ala Asp Ala His Ser 20 25 30 Thr Leu Ala Lys Ile 35 <210> 66 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 66 Cys Cys Leu Arg Arg His Gln Gly Lys Gln Asn Glu Leu Ser Asp Thr 1 5 10 15 Ala Gly Arg Glu Ile Asn Leu Val Asp Ala His Leu Lys Ser Glu Gln 20 25 30 Thr Glu Ala Ser Thr Arg Gln Asn Ser Gln Val Leu Leu Ser Glu Thr 35 40 45 Gly Ile Tyr Asp Asn Asp Pro Asp Leu Cys Phe Arg Met Gln Glu Gly 50 55 60 Ser Glu Val Tyr Ser Asn Pro Cys Leu Glu Glu Asn Lys Pro Gly Ile 65 70 75 80 Val Tyr Ala Ser Leu Asn His Ser Val Ile Gly Pro Asn Ser Arg Leu 85 90 95 Ala Arg Asn Val Lys Glu Ala Pro Thr Glu Tyr Ala Ser Ile Cys Val 100 105 110 Arg Ser <210> 67 <211> 49 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 67 His Gln Arg Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu 1 5 10 15 Pro Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser 20 25 30 Thr Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser 35 40 45 Pro <210> 68 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 68 Arg Arg Ala Cys Arg Lys Arg Ile Arg Gln Lys Leu His Leu Cys Tyr 1 5 10 15 Pro Val Gln Thr Ser Gln Pro Lys Leu Glu Leu Val Asp Ser Arg Pro 20 25 30 Arg Arg Ser Ser Thr Gln Leu Arg Ser Gly Ala Ser Val Thr Glu Pro 35 40 45 Val Ala Glu Glu Arg Gly Leu Met Ser Gln Pro Leu Met Glu Thr Cys 50 55 60 His Ser Val Gly Ala Ala Tyr Leu Glu Ser Leu Pro Leu Gln Asp Ala 65 70 75 80 Ser Pro Ala Gly Gly Pro Ser Ser Pro Arg Asp Leu Pro Glu Pro Arg 85 90 95 Val Ser Thr Glu His Thr Asn Asn Lys Ile Glu Lys Ile Tyr Ile Met 100 105 110 Lys Ala Asp Thr Val Ile Val Gly Thr Val Lys Ala Glu Leu Pro Glu 115 120 125 Gly Arg Gly Leu Ala Gly Pro Ala Glu Pro Glu Leu Glu Glu Glu Leu 130 135 140 Glu Ala Asp His Thr Pro His Tyr Pro Glu Gln Glu Thr Glu Pro Pro 145 150 155 160 Leu Gly Ser Cys Ser Asp Val Met Leu Ser Val Glu Glu Glu Gly Lys 165 170 175 Glu Asp Pro Leu Pro Thr Ala Ala Ser Gly Lys 180 185 <210> 69 <211> 54 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 69 His Ile Trp Gln Leu Arg Ser Gln Cys Met Trp Pro Arg Glu Thr Gln 1 5 10 15 Leu Leu Leu Glu Val Pro Pro Ser Thr Glu Asp Ala Arg Ser Cys Gln 20 25 30 Phe Pro Glu Glu Glu Arg Gly Glu Arg Ser Ala Glu Glu Lys Gly Arg 35 40 45 Leu Gly Asp Leu Trp Val 50 <210> 70 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 70 Cys Val Lys Arg Arg Lys Pro Arg Gly Asp Val Val Lys Val Ile Val 1 5 10 15 Ser Val Gln Arg Lys Arg Gln Glu Ala Glu Gly Glu Ala Thr Val Ile 20 25 30 Glu Ala Leu Gln Ala Pro Pro Asp Val Thr Thr Val Ala Val Glu Glu 35 40 45 Thr Ile Pro Ser Phe Thr Gly Arg Ser Pro Asn His 50 55 60 <210> 71 <211> 292 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 71 Leu Glu Glu Ser Val Ala Leu Arg Ile Ile Thr Glu Gly Ala Ser Ile 1 5 10 15 Leu Arg Gln Glu Lys Asn Leu Leu Asp Ile Asp Ala Pro Val Thr Val 20 25 30 Cys Gly Asp Ile His Gly Gln Phe Phe Asp Leu Met Lys Leu Phe Glu 35 40 45 Val Gly Gly Ser Pro Ala Asn Thr Arg Tyr Leu Phe Leu Gly Asp Tyr 50 55 60 Val Asp Arg Gly Tyr Phe Ser Ile Glu Cys Val Leu Tyr Leu Trp Ala 65 70 75 80 Leu Lys Ile Leu Tyr Pro Lys Thr Leu Phe Leu Leu Arg Gly Asn His 85 90 95 Glu Cys Arg His Leu Thr Glu Tyr Phe Thr Phe Lys Gln Glu Cys Lys 100 105 110 Ile Lys Tyr Ser Glu Arg Val Tyr Asp Ala Cys Met Asp Ala Phe Asp 115 120 125 Cys Leu Pro Leu Ala Ala Leu Met Asn Gln Gln Phe Leu Cys Val His 130 135 140 Gly Gly Leu Ser Pro Glu Ile Asn Thr Leu Asp Asp Ile Arg Lys Leu 145 150 155 160 Asp Arg Phe Lys Glu Pro Pro Ala Tyr Gly Pro Met Cys Asp Ile Leu 165 170 175 Trp Ser Asp Pro Leu Glu Asp Phe Gly Asn Glu Lys Thr Gln Glu His 180 185 190 Phe Thr His Asn Thr Val Arg Gly Cys Ser Tyr Phe Tyr Ser Tyr Pro 195 200 205 Ala Val Cys Glu Phe Leu Gln His Asn Asn Leu Leu Ser Ile Leu Arg 210 215 220 Ala His Glu Ala Gln Asp Ala Gly Tyr Arg Met Tyr Arg Lys Ser Gln 225 230 235 240 Thr Thr Gly Phe Pro Ser Leu Ile Thr Ile Phe Ser Ala Pro Asn Tyr 245 250 255 Leu Asp Val Tyr Asn Asn Lys Ala Ala Val Leu Lys Tyr Glu Asn Asn 260 265 270 Val Met Asn Ile Arg Gln Phe Asn Cys Ser Pro His Pro Tyr Trp Leu 275 280 285 Pro Asn Phe Met 290 <210> 72 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 72 Met Val Asn Pro Thr Val Phe Phe Asp Ile Ala Val Asp Gly Glu Pro 1 5 10 15 Leu Gly Arg Val Ser Phe Glu Leu Phe Ala Asp Lys Val Pro Lys Thr 20 25 30 Ala Glu Asn Phe Arg Ala Leu Ser Thr Gly Glu Lys Gly Phe Gly Tyr 35 40 45 Lys Gly Ser Cys Phe His Arg Ile Ile Pro Gly Phe Met Cys Gln Gly 50 55 60 Gly Asp Phe Thr Arg His Asn Gly Thr Gly Gly Lys Ser Ile Tyr Gly 65 70 75 80 Glu Lys Phe Glu Asp Glu Asn Phe Ile Leu Lys His Thr Gly Pro Gly 85 90 95 Ile Leu Ser Met Ala Asn Ala Gly Pro Asn Thr Asn Gly Ser Gln Phe 100 105 110 Phe Ile Cys Thr Ala Lys Thr Glu Trp Leu Asp Gly Lys His Val Val 115 120 125 Phe Gly Lys Val Lys Glu Gly Met Asn Ile Val Glu Ala Met Glu Arg 130 135 140 Phe Gly Ser Arg Asn Gly Lys Thr Ser Lys Lys Ile Thr Ile Ala Asp 145 150 155 160 Cys Gly Gln Leu Glu 165 <210> 73 <211> 804 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 73 Met Ser Asn Ser Tyr Asp Ser Ser Ser Ile Lys Val Leu Lys Gly Leu 1 5 10 15 Asp Ala Val Arg Lys Arg Pro Gly Met Tyr Ile Gly Asp Thr Asp Asp 20 25 30 Gly Thr Gly Leu His His Met Val Phe Glu Val Val Asp Asn Ala Ile 35 40 45 Asp Glu Ala Leu Ala Gly His Cys Lys Glu Ile Ile Val Thr Ile His 50 55 60 Ala Asp Asn Ser Val Ser Val Gln Asp Asp Gly Arg Gly Ile Pro Thr 65 70 75 80 Gly Ile His Pro Glu Glu Gly Val Ser Ala Ala Glu Val Ile Met Thr 85 90 95 Val Leu His Ala Gly Gly Lys Phe Asp Asp Asn Ser Tyr Lys Val Ser 100 105 110 Gly Gly Leu His Gly Val Gly Val Ser Val Val Asn Ala Leu Ser Gln 115 120 125 Lys Leu Glu Leu Val Ile Gln Arg Glu Gly Lys Ile His Arg Gln Ile 130 135 140 Tyr Glu His Gly Val Pro Gln Ala Pro Leu Ala Val Thr Gly Glu Thr 145 150 155 160 Glu Lys Thr Gly Thr Met Val Arg Phe Trp Pro Ser Leu Glu Thr Phe 165 170 175 Thr Asn Val Thr Glu Phe Glu Tyr Glu Ile Leu Ala Lys Arg Leu Arg 180 185 190 Glu Leu Ser Phe Leu Asn Ser Gly Val Ser Ile Arg Leu Arg Asp Lys 195 200 205 Arg Asp Gly Lys Glu Asp His Phe His Tyr Glu Gly Gly Ile Lys Ala 210 215 220 Phe Val Glu Tyr Leu Asn Lys Asn Lys Thr Pro Ile His Pro Asn Ile 225 230 235 240 Phe Tyr Phe Ser Thr Glu Lys Asp Gly Ile Gly Val Glu Val Ala Leu 245 250 255 Gln Trp Asn Asp Gly Phe Gln Glu Asn Ile Tyr Cys Phe Thr Asn Asn 260 265 270 Ile Pro Gln Arg Asp Gly Gly Thr His Leu Ala Gly Phe Arg Ala Ala 275 280 285 Met Thr Arg Thr Leu Asn Ala Tyr Met Asp Lys Glu Gly Tyr Ser Lys 290 295 300 Lys Ala Lys Val Ser Ala Thr Gly Asp Asp Ala Arg Glu Gly Leu Ile 305 310 315 320 Ala Val Val Ser Val Lys Val Pro Asp Pro Lys Phe Ser Ser Gln Thr 325 330 335 Lys Asp Lys Leu Val Ser Ser Glu Val Lys Ser Ala Val Glu Gln Gln 340 345 350 Met Asn Glu Leu Leu Ala Glu Tyr Leu Leu Glu Asn Pro Thr Asp Ala 355 360 365 Lys Ile Val Val Gly Lys Ile Ile Asp Ala Ala Arg Ala Arg Glu Ala 370 375 380 Ala Arg Arg Ala Arg Glu Met Thr Arg Arg Lys Gly Ala Leu Asp Leu 385 390 395 400 Ala Gly Leu Pro Gly Lys Leu Ala Asp Cys Gln Glu Arg Asp Pro Ala 405 410 415 Leu Ser Glu Leu Tyr Leu Val Glu Gly Asp Ser Ala Gly Gly Ser Ala 420 425 430 Lys Gln Gly Arg Asn Arg Lys Asn Gln Ala Ile Leu Pro Leu Lys Gly 435 440 445 Lys Ile Leu Asn Val Glu Lys Ala Arg Phe Asp Lys Met Leu Ser Ser 450 455 460 Gln Glu Val Ala Thr Leu Ile Thr Ala Leu Gly Cys Gly Ile Gly Arg 465 470 475 480 Asp Glu Tyr Asn Pro Asp Lys Leu Arg Tyr His Ser Ile Ile Ile Met 485 490 495 Thr Asp Ala Asp Val Asp Gly Ser His Ile Arg Thr Leu Leu Leu Thr 500 505 510 Phe Phe Tyr Arg Gln Met Pro Glu Ile Val Glu Arg Gly His Val Tyr 515 520 525 Ile Ala Gln Pro Pro Leu Tyr Lys Val Lys Lys Gly Lys Gln Glu Gln 530 535 540 Tyr Ile Lys Asp Asp Glu Ala Met Asp Gln Tyr Gln Ile Ser Ile Ala 545 550 555 560 Leu Asp Gly Ala Thr Leu His Thr Asn Ala Ser Ala Pro Ala Leu Ala 565 570 575 Gly Glu Ala Leu Glu Lys Leu Val Ser Glu Tyr Asn Ala Thr Gln Lys 580 585 590 Met Ile Asn Arg Met Glu Arg Arg Tyr Pro Lys Ala Met Leu Lys Glu 595 600 605 Leu Ile Tyr Gln Pro Thr Leu Thr Glu Ala Asp Leu Ser Asp Glu Gln 610 615 620 Thr Val Thr Arg Trp Val Asn Ala Leu Val Ser Glu Leu Asn Asp Lys 625 630 635 640 Glu Gln His Gly Ser Gln Trp Lys Phe Asp Val His Thr Asn Ala Glu 645 650 655 Gln Asn Leu Phe Glu Pro Ile Val Arg Val Arg Thr His Gly Val Asp 660 665 670 Thr Asp Tyr Pro Leu Asp His Glu Phe Ile Thr Gly Gly Glu Tyr Arg 675 680 685 Arg Ile Cys Thr Leu Gly Glu Lys Leu Arg Gly Leu Leu Glu Glu Asp 690 695 700 Ala Phe Ile Glu Arg Gly Glu Arg Arg Gln Pro Val Ala Ser Phe Glu 705 710 715 720 Gln Ala Leu Asp Trp Leu Val Lys Glu Ser Arg Arg Gly Leu Ser Ile 725 730 735 Gln Arg Tyr Lys Gly Leu Gly Glu Met Asn Pro Glu Gln Leu Trp Glu 740 745 750 Thr Thr Met Asp Pro Glu Ser Arg Arg Met Leu Arg Val Thr Val Lys 755 760 765 Asp Ala Ile Ala Ala Asp Gln Leu Phe Thr Thr Leu Met Gly Asp Ala 770 775 780 Val Glu Pro Arg Arg Ala Phe Ile Glu Glu Asn Ala Leu Lys Ala Ala 785 790 795 800 Asn Ile Asp Ile <210> 74 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 74 Met Val Gly Ser Leu Asn Cys Ile Val Ala Val Ser Gln Asn Met Gly 1 5 10 15 Ile Gly Lys Asn Gly Asp Leu Pro Trp Pro Pro Leu Arg Asn Glu Phe 20 25 30 Arg Tyr Phe Gln Arg Met Thr Thr Thr Ser Ser Val Glu Gly Lys Gln 35 40 45 Asn Leu Val Ile Met Gly Lys Lys Thr Trp Phe Ser Ile Pro Glu Lys 50 55 60 Asn Arg Pro Leu Lys Gly Arg Ile Asn Leu Val Leu Ser Arg Glu Leu 65 70 75 80 Lys Glu Pro Pro Gln Gly Ala His Phe Leu Ser Arg Ser Leu Asp Asp 85 90 95 Ala Leu Lys Leu Thr Glu Gln Pro Glu Leu Ala Asn Lys Val Asp Met 100 105 110 Val Trp Ile Val Gly Gly Ser Ser Val Tyr Lys Glu Ala Met Asn His 115 120 125 Pro Gly His Leu Lys Leu Phe Val Thr Arg Ile Met Gln Asp Phe Glu 130 135 140 Ser Asp Thr Phe Phe Pro Glu Ile Asp Leu Glu Lys Tyr Lys Leu Leu 145 150 155 160 Pro Glu Tyr Pro Gly Val Leu Ser Asp Val Gln Glu Glu Lys Gly Ile 165 170 175 Lys Tyr Lys Phe Glu Val Tyr Glu Lys Asn Asp 180 185 <210> 75 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 75 Met Ala Ser Arg Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly 1 5 10 15 Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly 20 25 30 Met Leu Glu Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys 35 40 45 Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu 50 55 60 Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile 65 70 75 80 Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro 85 90 95 Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu 100 105 110 <210> 76 <211> 183 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 76 Met Asn Gly Asp Glu Thr Lys Lys Val Glu Ser Glu Tyr Ile Lys Lys 1 5 10 15 His His Arg His Glu Leu Val Glu Ser Gln Cys Ser Ser Thr Leu Val 20 25 30 Lys His Ile Lys Ala Pro Leu His Leu Val Trp Ser Ile Val Arg Arg 35 40 45 Phe Asp Glu Pro Gln Lys Tyr Lys Pro Phe Ile Ser Arg Cys Val Val 50 55 60 Gln Gly Lys Lys Leu Glu Val Gly Ser Val Arg Glu Val Asp Leu Lys 65 70 75 80 Ser Gly Leu Pro Ala Thr Lys Ser Thr Glu Val Leu Glu Ile Leu Asp 85 90 95 Asp Asn Glu His Ile Leu Gly Ile Arg Ile Val Gly Gly Asp His Arg 100 105 110 Leu Lys Asn Tyr Ser Ser Thr Ile Ser Leu His Ser Glu Thr Ile Asp 115 120 125 Gly Lys Thr Gly Thr Leu Ala Ile Glu Ser Phe Val Val Asp Val Pro 130 135 140 Glu Gly Asn Thr Lys Glu Glu Thr Cys Phe Phe Val Glu Ala Leu Ile 145 150 155 160 Gln Cys Asn Leu Asn Ser Leu Ala Asp Val Thr Glu Arg Leu Gln Ala 165 170 175 Glu Ser Met Glu Lys Lys Ile 180 <210> 77 <211> 161 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 77 Met Glu Thr Ser Gln Lys Tyr His Thr Cys Gly Ser Thr Leu Val Gln 1 5 10 15 Thr Ile Asp Ala Pro Leu Ser Leu Val Trp Ser Ile Leu Arg Arg Phe 20 25 30 Asp Asn Pro Gln Ala Tyr Lys Gln Phe Val Lys Thr Cys Asn Leu Ser 35 40 45 Ser Gly Asp Gly Gly Glu Gly Ser Val Arg Glu Val Thr Val Val Ser 50 55 60 Gly Leu Pro Ala Glu Phe Ser Arg Glu Arg Leu Asp Glu Leu Asp Asp 65 70 75 80 Glu Ser His Val Met Met Ile Ser Ile Ile Gly Gly Asp His Arg Leu 85 90 95 Val Asn Tyr Arg Ser Lys Thr Met Ala Phe Val Ala Ala Asp Thr Glu 100 105 110 Glu Lys Thr Val Val Val Glu Ser Tyr Val Val Asp Val Pro Glu Gly 115 120 125 Asn Ser Glu Glu Glu Thr Thr Ser Phe Ala Asp Thr Ile Val Gly Phe 130 135 140 Asn Leu Lys Ser Leu Ala Lys Leu Ser Glu Arg Val Ala His Leu Lys 145 150 155 160 Leu <210> 78 <211> 159 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 78 Met Lys Thr Ser Gln Glu Gln His Val Cys Gly Ser Thr Val Val Gln 1 5 10 15 Thr Ile Asn Ala Pro Leu Pro Leu Val Trp Ser Ile Leu Arg Arg Phe 20 25 30 Asp Asn Pro Lys Thr Phe Lys His Phe Val Lys Thr Cys Lys Leu Arg 35 40 45 Ser Gly Asp Gly Gly Glu Gly Ser Val Arg Glu Val Thr Val Val Ser 50 55 60 Asp Leu Pro Ala Ser Phe Ser Leu Glu Arg Leu Asp Glu Leu Asp Asp 65 70 75 80 Glu Ser His Val Met Val Ile Ser Ile Ile Gly Gly Asp His Arg Leu 85 90 95 Val Asn Tyr Gln Ser Lys Thr Thr Val Phe Val Ala Ala Glu Glu Glu 100 105 110 Lys Thr Val Val Val Glu Ser Tyr Val Val Asp Val Pro Glu Gly Asn 115 120 125 Thr Glu Glu Glu Thr Thr Leu Phe Ala Asp Thr Ile Val Gly Cys Asn 130 135 140 Leu Arg Ser Leu Ala Lys Leu Ser Glu Lys Met Met Glu Leu Thr 145 150 155 <210> 79 <211> 164 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 79 Met Glu Ser Ser Lys Gln Lys Arg Cys Arg Ser Ser Val Val Glu Thr 1 5 10 15 Ile Glu Ala Pro Leu Pro Leu Val Trp Ser Ile Leu Arg Ser Phe Asp 20 25 30 Lys Pro Gln Ala Tyr Gln Arg Phe Val Lys Ser Cys Thr Met Arg Ser 35 40 45 Gly Gly Gly Gly Gly Lys Gly Gly Glu Gly Lys Gly Ser Val Arg Asp 50 55 60 Val Thr Leu Val Ser Gly Phe Pro Ala Asp Phe Ser Thr Glu Arg Leu 65 70 75 80 Glu Glu Leu Asp Asp Glu Ser His Val Met Val Val Ser Ile Ile Gly 85 90 95 Gly Asn His Arg Leu Val Asn Tyr Lys Ser Lys Thr Lys Val Val Ala 100 105 110 Ser Pro Glu Asp Met Ala Lys Lys Thr Val Val Val Glu Ser Tyr Val 115 120 125 Val Asp Val Pro Glu Gly Thr Ser Glu Glu Asp Thr Ile Phe Phe Val 130 135 140 Asp Asn Ile Ile Arg Tyr Asn Leu Thr Ser Leu Ala Lys Leu Thr Lys 145 150 155 160 Lys Met Met Lys <210> 80 <211> 221 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 80 Met Ala Asn Ser Glu Ser Ser Ser Ser Pro Val Asn Glu Glu Glu Asn 1 5 10 15 Ser Gln Arg Ile Ser Thr Leu His His Gln Thr Met Pro Ser Asp Leu 20 25 30 Thr Gln Asp Glu Phe Thr Gln Leu Ser Gln Ser Ile Ala Glu Phe His 35 40 45 Thr Tyr Gln Leu Gly Asn Gly Arg Cys Ser Ser Leu Leu Ala Gln Arg 50 55 60 Ile His Ala Pro Pro Glu Thr Val Trp Ser Val Val Arg Arg Phe Asp 65 70 75 80 Arg Pro Gln Ile Tyr Lys His Phe Ile Lys Ser Cys Asn Val Ser Glu 85 90 95 Asp Phe Glu Met Arg Val Gly Cys Thr Arg Asp Val Asn Val Ile Ser 100 105 110 Gly Leu Pro Ala Asn Thr Ser Arg Glu Arg Leu Asp Leu Leu Asp Asp 115 120 125 Asp Arg Arg Val Thr Gly Phe Ser Ile Thr Gly Gly Glu His Arg Leu 130 135 140 Arg Asn Tyr Lys Ser Val Thr Thr Val His Arg Phe Glu Lys Glu Glu 145 150 155 160 Glu Glu Glu Arg Ile Trp Thr Val Val Leu Glu Ser Tyr Val Val Asp 165 170 175 Val Pro Glu Gly Asn Ser Glu Glu Asp Thr Arg Leu Phe Ala Asp Thr 180 185 190 Val Ile Arg Leu Asn Leu Gln Lys Leu Ala Ser Ile Thr Glu Ala Met 195 200 205 Asn Arg Asn Asn Asn Asn Asn Asn Ser Ser Gln Val Arg 210 215 220 <210> 81 <211> 190 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 81 Met Ser Ser Ser Pro Ala Val Lys Gly Leu Thr Asp Glu Glu Gln Lys 1 5 10 15 Thr Leu Glu Pro Val Ile Lys Thr Tyr His Gln Phe Glu Pro Asp Pro 20 25 30 Thr Thr Cys Thr Ser Leu Ile Thr Gln Arg Ile His Ala Pro Ala Ser 35 40 45 Val Val Trp Pro Leu Ile Arg Arg Phe Asp Asn Pro Glu Arg Tyr Lys 50 55 60 His Phe Val Lys Arg Cys Arg Leu Ile Ser Gly Asp Gly Asp Val Gly 65 70 75 80 Ser Val Arg Glu Val Thr Val Ile Ser Gly Leu Pro Ala Ser Thr Ser 85 90 95 Thr Glu Arg Leu Glu Phe Val Asp Asp Asp His Arg Val Leu Ser Phe 100 105 110 Arg Val Val Gly Gly Glu His Arg Leu Lys Asn Tyr Lys Ser Val Thr 115 120 125 Ser Val Asn Glu Phe Leu Asn Gln Asp Ser Gly Lys Val Tyr Thr Val 130 135 140 Val Leu Glu Ser Tyr Thr Val Asp Ile Pro Glu Gly Asn Thr Glu Glu 145 150 155 160 Asp Thr Lys Met Phe Val Asp Thr Val Val Lys Leu Asn Leu Gln Lys 165 170 175 Leu Gly Val Ala Ala Thr Ser Ala Pro Met His Asp Asp Glu 180 185 190 <210> 82 <211> 209 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 82 Met Asn Leu Ala Pro Ile His Asp Pro Ser Ser Ser Ser Thr Thr Thr 1 5 10 15 Thr Ser Ser Ser Thr Pro Tyr Gly Leu Thr Lys Asp Glu Phe Ser Thr 20 25 30 Leu Asp Ser Ile Ile Arg Thr His His Thr Phe Pro Arg Ser Pro Asn 35 40 45 Thr Cys Thr Ser Leu Ile Ala His Arg Val Asp Ala Pro Ala His Ala 50 55 60 Ile Trp Arg Phe Val Arg Asp Phe Ala Asn Pro Asn Lys Tyr Lys His 65 70 75 80 Phe Ile Lys Ser Cys Thr Ile Arg Val Asn Gly Asn Gly Ile Lys Glu 85 90 95 Ile Lys Val Gly Thr Ile Arg Glu Val Ser Val Val Ser Gly Leu Pro 100 105 110 Ala Ser Thr Ser Val Glu Ile Leu Glu Val Leu Asp Glu Glu Lys Arg 115 120 125 Ile Leu Ser Phe Arg Val Leu Gly Gly Glu His Arg Leu Asn Asn Tyr 130 135 140 Arg Ser Val Thr Ser Val Asn Glu Phe Val Val Leu Glu Lys Asp Lys 145 150 155 160 Lys Lys Arg Val Tyr Ser Val Val Leu Glu Ser Tyr Ile Val Asp Ile 165 170 175 Pro Gln Gly Asn Thr Glu Glu Asp Thr Arg Met Phe Val Asp Thr Val 180 185 190 Val Lys Ser Asn Leu Gln Asn Leu Ala Val Ile Ser Thr Ala Ser Pro 195 200 205 Thr <210> 83 <211> 207 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 83 Met Leu Ala Val His Arg Pro Ser Ser Ala Val Ser Asp Gly Asp Ser 1 5 10 15 Val Gln Ile Pro Met Met Ile Ala Ser Phe Gln Lys Arg Phe Pro Ser 20 25 30 Leu Ser Arg Asp Ser Thr Ala Ala Arg Phe His Thr His Glu Val Gly 35 40 45 Pro Asn Gln Cys Cys Ser Ala Val Ile Gln Glu Ile Ser Ala Pro Ile 50 55 60 Ser Thr Val Trp Ser Val Val Arg Arg Phe Asp Asn Pro Gln Ala Tyr 65 70 75 80 Lys His Phe Leu Lys Ser Cys Ser Val Ile Gly Gly Asp Gly Asp Asn 85 90 95 Val Gly Ser Leu Arg Gln Val His Val Val Ser Gly Leu Pro Ala Ala 100 105 110 Ser Ser Thr Glu Arg Leu Asp Ile Leu Asp Asp Glu Arg His Val Ile 115 120 125 Ser Phe Ser Val Val Gly Gly Asp His Arg Leu Ser Asn Tyr Arg Ser 130 135 140 Val Thr Thr Leu His Pro Ser Pro Ile Ser Gly Thr Val Val Val Glu 145 150 155 160 Ser Tyr Val Val Asp Val Pro Pro Gly Asn Thr Lys Glu Glu Thr Cys 165 170 175 Asp Phe Val Asp Val Ile Val Arg Cys Asn Leu Gln Ser Leu Ala Lys 180 185 190 Ile Ala Glu Asn Thr Ala Ala Glu Ser Lys Lys Lys Met Ser Leu 195 200 205 <210> 84 <211> 203 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 84 Met Arg Ser Pro Val Gln Leu Gln His Gly Ser Asp Ala Thr Asn Gly 1 5 10 15 Phe His Thr Leu Gln Pro His Asp Gln Thr Asp Gly Pro Ile Lys Arg 20 25 30 Val Cys Leu Thr Arg Gly Met His Val Pro Glu His Val Ala Met His 35 40 45 His Thr His Asp Val Gly Pro Asp Gln Cys Cys Ser Ser Val Val Gln 50 55 60 Met Ile His Ala Pro Pro Glu Ser Val Trp Ala Leu Val Arg Arg Phe 65 70 75 80 Asp Asn Pro Lys Val Tyr Lys Asn Phe Ile Arg Gln Cys Arg Ile Val 85 90 95 Gln Gly Asp Gly Leu His Val Gly Asp Leu Arg Glu Val Met Val Val 100 105 110 Ser Gly Leu Pro Ala Val Ser Ser Thr Glu Arg Leu Glu Ile Leu Asp 115 120 125 Glu Glu Arg His Val Ile Ser Phe Ser Val Val Gly Gly Asp His Arg 130 135 140 Leu Lys Asn Tyr Arg Ser Val Thr Thr Leu His Ala Ser Asp Asp Glu 145 150 155 160 Gly Thr Val Val Val Glu Ser Tyr Ile Val Asp Val Pro Pro Gly Asn 165 170 175 Thr Glu Glu Glu Thr Leu Ser Phe Val Asp Thr Ile Val Arg Cys Asn 180 185 190 Leu Gln Ser Leu Ala Arg Ser Thr Asn Arg Gln 195 200 <210> 85 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 85 Met Pro Thr Ser Ile Gln Phe Gln Arg Ser Ser Thr Ala Ala Glu Ala 1 5 10 15 Ala Asn Ala Thr Val Arg Asn Tyr Pro His His His Gln Lys Gln Val 20 25 30 Gln Lys Val Ser Leu Thr Arg Gly Met Ala Asp Val Pro Glu His Val 35 40 45 Glu Leu Ser His Thr His Val Val Gly Pro Ser Gln Cys Phe Ser Val 50 55 60 Val Val Gln Asp Val Glu Ala Pro Val Ser Thr Val Trp Ser Ile Leu 65 70 75 80 Ser Arg Phe Glu His Pro Gln Ala Tyr Lys His Phe Val Lys Ser Cys 85 90 95 His Val Val Ile Gly Asp Gly Arg Glu Val Gly Ser Val Arg Glu Val 100 105 110 Arg Val Val Ser Gly Leu Pro Ala Ala Phe Ser Leu Glu Arg Leu Glu 115 120 125 Ile Met Asp Asp Asp Arg His Val Ile Ser Phe Ser Val Val Gly Gly 130 135 140 Asp His Arg Leu Met Asn Tyr Lys Ser Val Thr Thr Val His Glu Ser 145 150 155 160 Glu Glu Asp Ser Asp Gly Lys Lys Arg Thr Arg Val Val Glu Ser Tyr 165 170 175 Val Val Asp Val Pro Ala Gly Asn Asp Lys Glu Glu Thr Cys Ser Phe 180 185 190 Ala Asp Thr Ile Val Arg Cys Asn Leu Gln Ser Leu Ala Lys Leu Ala 195 200 205 Glu Asn Thr Ser Lys Phe Ser 210 215 <210> 86 <211> 211 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 86 Met Glu Met Ile Gly Gly Asp Asp Thr Asp Thr Glu Met Tyr Gly Ala 1 5 10 15 Leu Val Thr Ala Gln Ser Leu Arg Leu Arg His Leu His His Cys Arg 20 25 30 Glu Asn Gln Cys Thr Ser Val Leu Val Lys Tyr Ile Gln Ala Pro Val 35 40 45 His Leu Val Trp Ser Leu Val Arg Arg Phe Asp Gln Pro Gln Lys Tyr 50 55 60 Lys Pro Phe Ile Ser Arg Cys Thr Val Asn Gly Asp Pro Glu Ile Gly 65 70 75 80 Cys Leu Arg Glu Val Asn Val Lys Ser Gly Leu Pro Ala Thr Thr Ser 85 90 95 Thr Glu Arg Leu Glu Gln Leu Asp Asp Glu Glu His Ile Leu Gly Ile 100 105 110 Asn Ile Ile Gly Gly Asp His Arg Leu Lys Asn Tyr Ser Ser Ile Leu 115 120 125 Thr Val His Pro Glu Met Ile Asp Gly Arg Ser Gly Thr Met Val Met 130 135 140 Glu Ser Phe Val Val Asp Val Pro Gln Gly Asn Thr Lys Asp Asp Thr 145 150 155 160 Cys Tyr Phe Val Glu Ser Leu Ile Lys Cys Asn Leu Lys Ser Leu Ala 165 170 175 Cys Val Ser Glu Arg Leu Ala Ala Gln Asp Ile Thr Asn Ser Ile Ala 180 185 190 Thr Phe Cys Asn Ala Ser Asn Gly Tyr Arg Glu Lys Asn His Thr Glu 195 200 205 Thr Asn Leu 210 <210> 87 <211> 188 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 87 Met Glu Ala Asn Gly Ile Glu Asn Leu Thr Asn Pro Asn Gln Glu Arg 1 5 10 15 Glu Phe Ile Arg Arg His His Lys His Glu Leu Val Asp Asn Gln Cys 20 25 30 Ser Ser Thr Leu Val Lys His Ile Asn Ala Pro Val His Ile Val Trp 35 40 45 Ser Leu Val Arg Arg Phe Asp Gln Pro Gln Lys Tyr Lys Pro Phe Ile 50 55 60 Ser Arg Cys Val Val Lys Gly Asn Met Glu Ile Gly Thr Val Arg Glu 65 70 75 80 Val Asp Val Lys Ser Gly Leu Pro Ala Thr Arg Ser Thr Glu Arg Leu 85 90 95 Glu Leu Leu Asp Asp Asn Glu His Ile Leu Ser Ile Arg Ile Val Gly 100 105 110 Gly Asp His Arg Leu Lys Asn Tyr Ser Ser Ile Ile Ser Leu His Pro 115 120 125 Glu Thr Ile Glu Gly Arg Ile Gly Thr Leu Val Ile Glu Ser Phe Val 130 135 140 Val Asp Val Pro Glu Gly Asn Thr Lys Asp Glu Thr Cys Tyr Phe Val 145 150 155 160 Glu Ala Leu Ile Lys Cys Asn Leu Lys Ser Leu Ala Asp Ile Ser Glu 165 170 175 Arg Leu Ala Val Gln Asp Thr Thr Glu Ser Arg Val 180 185 <210> 88 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 88 Met Met Asp Gly Val Glu Gly Gly Thr Ala Met Tyr Gly Gly Leu Glu 1 5 10 15 Thr Val Gln Tyr Val Arg Thr His His Gln His Leu Cys Arg Glu Asn 20 25 30 Gln Cys Thr Ser Ala Leu Val Lys His Ile Lys Ala Pro Leu His Leu 35 40 45 Val Trp Ser Leu Val Arg Arg Phe Asp Gln Pro Gln Lys Tyr Lys Pro 50 55 60 Phe Val Ser Arg Cys Thr Val Ile Gly Asp Pro Glu Ile Gly Ser Leu 65 70 75 80 Arg Glu Val Asn Val Lys Ser Gly Leu Pro Ala Thr Thr Ser Thr Glu 85 90 95 Arg Leu Glu Leu Leu Asp Asp Glu Glu His Ile Leu Gly Ile Lys Ile 100 105 110 Ile Gly Gly Asp His Arg Leu Lys Asn Tyr Ser Ser Ile Leu Thr Val 115 120 125 His Pro Glu Ile Ile Glu Gly Arg Ala Gly Thr Met Val Ile Glu Ser 130 135 140 Phe Val Val Asp Val Pro Gln Gly Asn Thr Lys Asp Glu Thr Cys Tyr 145 150 155 160 Phe Val Glu Ala Leu Ile Arg Cys Asn Leu Lys Ser Leu Ala Asp Val 165 170 175 Ser Glu Arg Leu Ala Ser Gln Asp Ile Thr Gln 180 185 <210> 89 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 89 Met Pro Ser Glu Leu Thr Pro Glu Glu Arg Ser Glu Leu Lys Asn Ser 1 5 10 15 Ile Ala Glu Phe His Thr Tyr Gln Leu Asp Pro Gly Ser Cys Ser Ser 20 25 30 Leu His Ala Gln Arg Ile His Ala Pro Pro Glu Leu Val Trp Ser Ile 35 40 45 Val Arg Arg Phe Asp Lys Pro Gln Thr Tyr Lys His Phe Ile Lys Ser 50 55 60 Cys Ser Val Glu Gln Asn Phe Glu Met Arg Val Gly Cys Thr Arg Asp 65 70 75 80 Val Ile Val Ile Ser Gly Leu Pro Ala Asn Thr Ser Thr Glu Arg Leu 85 90 95 Asp Ile Leu Asp Asp Glu Arg Arg Val Thr Gly Phe Ser Ile Ile Gly 100 105 110 Gly Glu His Arg Leu Thr Asn Tyr Lys Ser Val Thr Thr Val His Arg 115 120 125 Phe Glu Lys Glu Asn Arg Ile Trp Thr Val Val Leu Glu Ser Tyr Val 130 135 140 Val Asp Met Pro Glu Gly Asn Ser Glu Asp Asp Thr Arg Met Phe Ala 145 150 155 160 Asp Thr Val Val Lys Leu Asn Leu Gln Lys Leu Ala Thr Val Ala Glu 165 170 175 Ala Met Ala Arg Asn Ser Gly Asp Gly Ser Gly Ser Gln Val Thr 180 185 190 <210> 90 <211> 434 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 90 Met Glu Glu Val Ser Pro Ala Ile Ala Gly Pro Phe Arg Pro Phe Ser 1 5 10 15 Glu Thr Gln Met Asp Phe Thr Gly Ile Arg Leu Gly Lys Gly Tyr Cys 20 25 30 Asn Asn Gln Tyr Ser Asn Gln Asp Ser Glu Asn Gly Asp Leu Met Val 35 40 45 Ser Leu Pro Glu Thr Ser Ser Cys Ser Val Ser Gly Ser His Gly Ser 50 55 60 Glu Ser Arg Lys Val Leu Ile Ser Arg Ile Asn Ser Pro Asn Leu Asn 65 70 75 80 Met Lys Glu Ser Ala Ala Ala Asp Ile Val Val Val Asp Ile Ser Ala 85 90 95 Gly Asp Glu Ile Asn Gly Ser Asp Ile Thr Ser Glu Lys Lys Met Ile 100 105 110 Ser Arg Thr Glu Ser Arg Ser Leu Phe Glu Phe Lys Ser Val Pro Leu 115 120 125 Tyr Gly Phe Thr Ser Ile Cys Gly Arg Arg Pro Glu Met Glu Asp Ala 130 135 140 Val Ser Thr Ile Pro Arg Phe Leu Gln Ser Ser Ser Gly Ser Met Leu 145 150 155 160 Asp Gly Arg Phe Asp Pro Gln Ser Ala Ala His Phe Phe Gly Val Tyr 165 170 175 Asp Gly His Gly Gly Ser Gln Val Ala Asn Tyr Cys Arg Glu Arg Met 180 185 190 His Leu Ala Leu Ala Glu Glu Ile Ala Lys Glu Lys Pro Met Leu Cys 195 200 205 Asp Gly Asp Thr Trp Leu Glu Lys Trp Lys Lys Ala Leu Phe Asn Ser 210 215 220 Phe Leu Arg Val Asp Ser Glu Ile Glu Ser Val Ala Pro Glu Thr Val 225 230 235 240 Gly Ser Thr Ser Val Val Ala Val Val Phe Pro Ser His Ile Phe Val 245 250 255 Ala Asn Cys Gly Asp Ser Arg Ala Val Leu Cys Arg Gly Lys Thr Ala 260 265 270 Leu Pro Leu Ser Val Asp His Lys Pro Asp Arg Glu Asp Glu Ala Ala 275 280 285 Arg Ile Glu Ala Ala Gly Gly Lys Val Ile Gln Trp Asn Gly Ala Arg 290 295 300 Val Phe Gly Val Leu Ala Met Ser Arg Ser Ile Gly Asp Arg Tyr Leu 305 310 315 320 Lys Pro Ser Ile Ile Pro Asp Pro Glu Val Thr Ala Val Lys Arg Val 325 330 335 Lys Glu Asp Asp Cys Leu Ile Leu Ala Ser Asp Gly Val Trp Asp Val 340 345 350 Met Thr Asp Glu Glu Ala Cys Glu Met Ala Arg Lys Arg Ile Leu Leu 355 360 365 Trp His Lys Lys Asn Ala Val Ala Gly Asp Ala Ser Leu Leu Ala Asp 370 375 380 Glu Arg Arg Lys Glu Gly Lys Asp Pro Ala Ala Met Ser Ala Ala Glu 385 390 395 400 Tyr Leu Ser Lys Leu Ala Ile Gln Arg Gly Ser Lys Asp Asn Ile Ser 405 410 415 Val Val Val Val Asp Leu Lys Pro Arg Arg Lys Leu Lys Ser Lys Pro 420 425 430 Leu Asn <210> 91 <211> 423 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 91 Met Asp Glu Val Ser Pro Ala Val Ala Val Pro Phe Arg Pro Phe Thr 1 5 10 15 Asp Pro His Ala Gly Leu Arg Gly Tyr Cys Asn Gly Glu Ser Arg Val 20 25 30 Thr Leu Pro Glu Ser Ser Cys Ser Gly Asp Gly Ala Met Lys Asp Ser 35 40 45 Ser Phe Glu Ile Asn Thr Arg Gln Asp Ser Leu Thr Ser Ser Ser Ser 50 55 60 Ala Met Ala Gly Val Asp Ile Ser Ala Gly Asp Glu Ile Asn Gly Ser 65 70 75 80 Asp Glu Phe Asp Pro Arg Ser Met Asn Gln Ser Glu Lys Lys Val Leu 85 90 95 Ser Arg Thr Glu Ser Arg Ser Leu Phe Glu Phe Lys Cys Val Pro Leu 100 105 110 Tyr Gly Val Thr Ser Ile Cys Gly Arg Arg Pro Glu Met Glu Asp Ser 115 120 125 Val Ser Thr Ile Pro Arg Phe Leu Gln Val Ser Ser Ser Ser Leu Leu 130 135 140 Asp Gly Arg Val Thr Asn Gly Phe Asn Pro His Leu Ser Ala His Phe 145 150 155 160 Phe Gly Val Tyr Asp Gly His Gly Gly Ser Gln Val Ala Asn Tyr Cys 165 170 175 Arg Glu Arg Met His Leu Ala Leu Thr Glu Glu Ile Val Lys Glu Lys 180 185 190 Pro Glu Phe Cys Asp Gly Asp Thr Trp Gln Glu Lys Trp Lys Lys Ala 195 200 205 Leu Phe Asn Ser Phe Met Arg Val Asp Ser Glu Ile Glu Thr Val Ala 210 215 220 His Ala Pro Glu Thr Val Gly Ser Thr Ser Val Val Ala Val Val Phe 225 230 235 240 Pro Thr His Ile Phe Val Ala Asn Cys Gly Asp Ser Arg Ala Val Leu 245 250 255 Cys Arg Gly Lys Thr Pro Leu Ala Leu Ser Val Asp His Lys Pro Asp 260 265 270 Arg Asp Asp Glu Ala Ala Arg Ile Glu Ala Ala Gly Gly Lys Val Ile 275 280 285 Arg Trp Asn Gly Ala Arg Val Phe Gly Val Leu Ala Met Ser Arg Ser 290 295 300 Ile Gly Asp Arg Tyr Leu Lys Pro Ser Val Ile Pro Asp Pro Glu Val 305 310 315 320 Thr Ser Val Arg Arg Val Lys Glu Asp Asp Cys Leu Ile Leu Ala Ser 325 330 335 Asp Gly Leu Trp Asp Val Met Thr Asn Glu Glu Val Cys Asp Leu Ala 340 345 350 Arg Lys Arg Ile Leu Leu Trp His Lys Lys Asn Ala Met Ala Gly Glu 355 360 365 Ala Leu Leu Pro Ala Glu Lys Arg Gly Glu Gly Lys Asp Pro Ala Ala 370 375 380 Met Ser Ala Ala Glu Tyr Leu Ser Lys Met Ala Leu Gln Lys Gly Ser 385 390 395 400 Lys Asp Asn Ile Ser Val Val Val Val Asp Leu Lys Gly Ile Arg Lys 405 410 415 Phe Lys Ser Lys Ser Leu Asn 420 <210> 92 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 92 Met Lys Met Asp Lys Lys Thr Ile Val Trp Phe Arg Arg Asp Leu Arg 1 5 10 15 Ile Glu Asp Asn Pro Ala Leu Ala Ala Ala Ala His Glu Gly Ser Val 20 25 30 Phe Pro Val Phe Ile Trp Cys Pro Glu Glu Glu Gly Gln Phe Tyr Pro 35 40 45 Gly Arg Ala Ser Arg Trp Trp Met Lys Gln Ser Leu Ala His Leu Ser 50 55 60 Gln Ser Leu Lys Ala Leu Gly Ser Asp Leu Thr Leu Ile Lys Thr His 65 70 75 80 Asn Thr Ile Ser Ala Ile Leu Asp Cys Ile Arg Val Thr Gly Ala Thr 85 90 95 Lys Val Val Phe Asn His Leu Tyr Asp Pro Val Ser Leu Val Arg Asp 100 105 110 His Thr Val Lys Glu Lys Leu Val Glu Arg Gly Ile Ser Val Gln Ser 115 120 125 Tyr Asn Gly Asp Leu Leu Tyr Glu Pro Trp Glu Ile Tyr Cys Glu Lys 130 135 140 Gly Lys Pro Phe Thr Ser Phe Asn Ser Tyr Trp Lys Lys Cys Leu Asp 145 150 155 160 Met Ser Ile Glu Ser Val Met Leu Pro Pro Pro Trp Arg Leu Met Pro 165 170 175 Ile Thr Ala Ala Ala Glu Ala Ile Trp Ala Cys Ser Ile Glu Glu Leu 180 185 190 Gly Leu Glu Asn Glu Ala Glu Lys Pro Ser Asn Ala Leu Leu Thr Arg 195 200 205 Ala Trp Ser Pro Gly Trp Ser Asn Ala Asp Lys Leu Leu Asn Glu Phe 210 215 220 Ile Glu Lys Gln Leu Ile Asp Tyr Ala Lys Asn Ser Lys Lys Val Val 225 230 235 240 Gly Asn Ser Thr Ser Leu Leu Ser Pro Tyr Leu His Phe Gly Glu Ile 245 250 255 Ser Val Arg His Val Phe Gln Cys Ala Arg Met Lys Gln Ile Ile Trp 260 265 270 Ala Arg Asp Lys Asn Ser Glu Gly Glu Glu Ser Ala Asp Leu Phe Leu 275 280 285 Arg Gly Ile Gly Leu Arg Glu Tyr Ser Arg Tyr Ile Cys Phe Asn Phe 290 295 300 Pro Phe Thr His Glu Gln Ser Leu Leu Ser His Leu Arg Phe Phe Pro 305 310 315 320 Trp Asp Ala Asp Val Asp Lys Phe Lys Ala Trp Arg Gln Gly Arg Thr 325 330 335 Gly Tyr Pro Leu Val Asp Ala Gly Met Arg Glu Leu Trp Ala Thr Gly 340 345 350 Trp Met His Asn Arg Ile Arg Val Ile Val Ser Ser Phe Ala Val Lys 355 360 365 Phe Leu Leu Leu Pro Trp Lys Trp Gly Met Lys Tyr Phe Trp Asp Thr 370 375 380 Leu Leu Asp Ala Asp Leu Glu Cys Asp Ile Leu Gly Trp Gln Tyr Ile 385 390 395 400 Ser Gly Ser Ile Pro Asp Gly His Glu Leu Asp Arg Leu Asp Asn Pro 405 410 415 Ala Leu Gln Gly Ala Lys Tyr Asp Pro Glu Gly Glu Tyr Ile Arg Gln 420 425 430 Trp Leu Pro Glu Leu Ala Arg Leu Pro Thr Glu Trp Ile His His Pro 435 440 445 Trp Asp Ala Pro Leu Thr Val Leu Lys Ala Ser Gly Val Glu Leu Gly 450 455 460 Thr Asn Tyr Ala Lys Pro Ile Val Asp Ile Asp Thr Ala Arg Glu Leu 465 470 475 480 Leu Ala Lys Ala Ile Ser Arg Thr Arg Glu Ala Gln Ile Met Ile Gly 485 490 495 Ala Ala Pro Asp Glu Ile Val Ala Asp Ser Phe Glu Ala Leu Gly Ala 500 505 510 Asn Thr Ile Lys Glu Pro Gly Leu Cys Pro Ser Val Ser Ser Asn Asp 515 520 525 Gln Gln Val Pro Ser Ala Val Arg Tyr Asn Gly Ser Lys Arg Val Lys 530 535 540 Pro Glu Glu Glu Glu Glu Arg Asp Met Lys Lys Ser Arg Gly Phe Asp 545 550 555 560 Glu Arg Glu Leu Phe Ser Thr Ala Glu Ser Ser Ser Ser Ser Ser Val 565 570 575 Phe Phe Val Ser Gln Ser Cys Ser Leu Ala Ser Glu Gly Lys Asn Leu 580 585 590 Glu Gly Ile Gln Asp Ser Ser Asp Gln Ile Thr Thr Ser Leu Gly Lys 595 600 605 Asn Gly Cys Lys 610 <210> 93 <211> 335 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 93 Met Asn Gly Ala Ile Gly Gly Asp Leu Leu Leu Asn Phe Pro Asp Met 1 5 10 15 Ser Val Leu Glu Arg Gln Arg Ala His Leu Lys Tyr Leu Asn Pro Thr 20 25 30 Phe Asp Ser Pro Leu Ala Gly Phe Phe Ala Asp Ser Ser Met Ile Thr 35 40 45 Gly Gly Glu Met Asp Ser Tyr Leu Ser Thr Ala Gly Leu Asn Leu Pro 50 55 60 Met Met Tyr Gly Glu Thr Thr Val Glu Gly Asp Ser Arg Leu Ser Ile 65 70 75 80 Ser Pro Glu Thr Thr Leu Gly Thr Gly Asn Phe Lys Lys Arg Lys Phe 85 90 95 Asp Thr Glu Thr Lys Asp Cys Asn Glu Lys Lys Lys Lys Met Thr Met 100 105 110 Asn Arg Asp Asp Leu Val Glu Glu Gly Glu Glu Glu Lys Ser Lys Ile 115 120 125 Thr Glu Gln Asn Asn Gly Ser Thr Lys Ser Ile Lys Lys Met Lys His 130 135 140 Lys Ala Lys Lys Glu Glu Asn Asn Phe Ser Asn Asp Ser Ser Lys Val 145 150 155 160 Thr Lys Glu Leu Glu Lys Thr Asp Tyr Ile His Val Arg Ala Arg Arg 165 170 175 Gly Gln Ala Thr Asp Ser His Ser Ile Ala Glu Arg Val Arg Arg Glu 180 185 190 Lys Ile Ser Glu Arg Met Lys Phe Leu Gln Asp Leu Val Pro Gly Cys 195 200 205 Asp Lys Ile Thr Gly Lys Ala Gly Met Leu Asp Glu Ile Ile Asn Tyr 210 215 220 Val Gln Ser Leu Gln Arg Gln Ile Glu Phe Leu Ser Met Lys Leu Ala 225 230 235 240 Ile Val Asn Pro Arg Pro Asp Phe Asp Met Asp Asp Ile Phe Ala Lys 245 250 255 Glu Val Ala Ser Thr Pro Met Thr Val Val Pro Ser Pro Glu Met Val 260 265 270 Leu Ser Gly Tyr Ser His Glu Met Val His Ser Gly Tyr Ser Ser Glu 275 280 285 Met Val Asn Ser Gly Tyr Leu His Val Asn Pro Met Gln Gln Val Asn 290 295 300 Thr Ser Ser Asp Pro Leu Ser Cys Phe Asn Asn Gly Glu Ala Pro Ser 305 310 315 320 Met Trp Asp Ser His Val Gln Asn Leu Tyr Gly Asn Leu Gly Val 325 330 335 <210> 94 <211> 533 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 94 Met Lys Arg Asp His His His His His His Gln Asp Lys Lys Thr Met 1 5 10 15 Met Met Asn Glu Glu Asp Asp Gly Asn Gly Met Asp Glu Leu Leu Ala 20 25 30 Val Leu Gly Tyr Lys Val Arg Ser Ser Glu Met Ala Asp Val Ala Gln 35 40 45 Lys Leu Glu Gln Leu Glu Val Met Met Ser Asn Val Gln Glu Asp Asp 50 55 60 Leu Ser Gln Leu Ala Thr Glu Thr Val His Tyr Asn Pro Ala Glu Leu 65 70 75 80 Tyr Thr Trp Leu Asp Ser Met Leu Thr Asp Leu Asn Pro Pro Ser Ser 85 90 95 Asn Ala Glu Tyr Asp Leu Lys Ala Ile Pro Gly Asp Ala Ile Leu Asn 100 105 110 Gln Phe Ala Ile Asp Ser Ala Ser Ser Ser Asn Gln Gly Gly Gly Gly 115 120 125 Asp Thr Tyr Thr Thr Asn Lys Arg Leu Lys Cys Ser Asn Gly Val Val 130 135 140 Glu Thr Thr Thr Ala Thr Ala Glu Ser Thr Arg His Val Val Leu Val 145 150 155 160 Asp Ser Gln Glu Asn Gly Val Arg Leu Val His Ala Leu Leu Ala Cys 165 170 175 Ala Glu Ala Val Gln Lys Glu Asn Leu Thr Val Ala Glu Ala Leu Val 180 185 190 Lys Gln Ile Gly Phe Leu Ala Val Ser Gln Ile Gly Ala Met Arg Lys 195 200 205 Val Ala Thr Tyr Phe Ala Glu Ala Leu Ala Arg Arg Ile Tyr Arg Leu 210 215 220 Ser Pro Ser Gln Ser Pro Ile Asp His Ser Leu Ser Asp Thr Leu Gln 225 230 235 240 Met His Phe Tyr Glu Thr Cys Pro Tyr Leu Lys Phe Ala His Phe Thr 245 250 255 Ala Asn Gln Ala Ile Leu Glu Ala Phe Gln Gly Lys Lys Arg Val His 260 265 270 Val Ile Asp Phe Ser Met Ser Gln Gly Leu Gln Trp Pro Ala Leu Met 275 280 285 Gln Ala Leu Ala Leu Arg Pro Gly Gly Pro Pro Val Phe Arg Leu Thr 290 295 300 Gly Ile Gly Pro Pro Ala Pro Asp Asn Phe Asp Tyr Leu His Glu Val 305 310 315 320 Gly Cys Lys Leu Ala His Leu Ala Glu Ala Ile His Val Glu Phe Glu 325 330 335 Tyr Arg Gly Phe Val Ala Asn Thr Leu Ala Asp Leu Asp Ala Ser Met 340 345 350 Leu Glu Leu Arg Pro Ser Glu Ile Glu Ser Val Ala Val Asn Ser Val 355 360 365 Phe Glu Leu His Lys Leu Leu Gly Arg Pro Gly Ala Ile Asp Lys Val 370 375 380 Leu Gly Val Val Asn Gln Ile Lys Pro Glu Ile Phe Thr Val Val Glu 385 390 395 400 Gln Glu Ser Asn His Asn Ser Pro Ile Phe Leu Asp Arg Phe Thr Glu 405 410 415 Ser Leu His Tyr Tyr Ser Thr Leu Phe Asp Ser Leu Glu Gly Val Pro 420 425 430 Ser Gly Gln Asp Lys Val Met Ser Glu Val Tyr Leu Gly Lys Gln Ile 435 440 445 Cys Asn Val Val Ala Cys Asp Gly Pro Asp Arg Val Glu Arg His Glu 450 455 460 Thr Leu Ser Gln Trp Arg Asn Arg Phe Gly Ser Ala Gly Phe Ala Ala 465 470 475 480 Ala His Ile Gly Ser Asn Ala Phe Lys Gln Ala Ser Met Leu Leu Ala 485 490 495 Leu Phe Asn Gly Gly Glu Gly Tyr Arg Val Glu Glu Ser Asp Gly Cys 500 505 510 Leu Met Leu Gly Trp His Thr Arg Pro Leu Ile Ala Thr Ser Ala Trp 515 520 525 Lys Leu Ser Thr Asn 530 <210> 95 <211> 345 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 95 Met Ala Ala Ser Asp Glu Val Asn Leu Ile Glu Ser Arg Thr Val Val 1 5 10 15 Pro Leu Asn Thr Trp Val Leu Ile Ser Asn Phe Lys Val Ala Tyr Asn 20 25 30 Ile Leu Arg Arg Pro Asp Gly Thr Phe Asn Arg His Leu Ala Glu Tyr 35 40 45 Leu Asp Arg Lys Val Thr Ala Asn Ala Asn Pro Val Asp Gly Val Phe 50 55 60 Ser Phe Asp Val Leu Ile Asp Arg Arg Ile Asn Leu Leu Ser Arg Val 65 70 75 80 Tyr Arg Pro Ala Tyr Ala Asp Gln Glu Gln Pro Pro Ser Ile Leu Asp 85 90 95 Leu Glu Lys Pro Val Asp Gly Asp Ile Val Pro Val Ile Leu Phe Phe 100 105 110 His Gly Gly Ser Phe Ala His Ser Ser Ala Asn Ser Ala Ile Tyr Asp 115 120 125 Thr Leu Cys Arg Arg Leu Val Gly Leu Cys Lys Cys Val Val Val Ser 130 135 140 Val Asn Tyr Arg Arg Ala Pro Glu Asn Pro Tyr Pro Cys Ala Tyr Asp 145 150 155 160 Asp Gly Trp Ile Ala Leu Asn Trp Val Asn Ser Arg Ser Trp Leu Lys 165 170 175 Ser Lys Lys Asp Ser Lys Val His Ile Phe Leu Ala Gly Asp Ser Ser 180 185 190 Gly Gly Asn Ile Ala His Asn Val Ala Leu Arg Ala Gly Glu Ser Gly 195 200 205 Ile Asp Val Leu Gly Asn Ile Leu Leu Asn Pro Met Phe Gly Gly Asn 210 215 220 Glu Arg Thr Glu Ser Glu Lys Ser Leu Asp Gly Lys Tyr Phe Val Thr 225 230 235 240 Val Arg Asp Arg Asp Trp Tyr Trp Lys Ala Phe Leu Pro Glu Gly Glu 245 250 255 Asp Arg Glu His Pro Ala Cys Asn Pro Phe Ser Pro Arg Gly Lys Ser 260 265 270 Leu Glu Gly Val Ser Phe Pro Lys Ser Leu Val Val Val Ala Gly Leu 275 280 285 Asp Leu Ile Arg Asp Trp Gln Leu Ala Tyr Ala Glu Gly Leu Lys Lys 290 295 300 Ala Gly Gln Glu Val Lys Leu Met His Leu Glu Lys Ala Thr Val Gly 305 310 315 320 Phe Tyr Leu Leu Pro Asn Asn Asn His Phe His Asn Val Met Asp Glu 325 330 335 Ile Ser Ala Phe Val Asn Ala Glu Cys 340 345 <210> 96 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 96 Met Ala Gly Gly Asn Glu Val Asn Leu Asn Glu Cys Lys Arg Ile Val 1 5 10 15 Pro Leu Asn Thr Trp Val Leu Ile Ser Asn Phe Lys Leu Ala Tyr Lys 20 25 30 Val Leu Arg Arg Pro Asp Gly Ser Phe Asn Arg Asp Leu Ala Glu Phe 35 40 45 Leu Asp Arg Lys Val Pro Ala Asn Ser Phe Pro Leu Asp Gly Val Phe 50 55 60 Ser Phe Asp His Val Asp Ser Thr Thr Asn Leu Leu Thr Arg Ile Tyr 65 70 75 80 Gln Pro Ala Ser Leu Leu His Gln Thr Arg His Gly Thr Leu Glu Leu 85 90 95 Thr Lys Pro Leu Ser Thr Thr Glu Ile Val Pro Val Leu Ile Phe Phe 100 105 110 His Gly Gly Ser Phe Thr His Ser Ser Ala Asn Ser Ala Ile Tyr Asp 115 120 125 Thr Phe Cys Arg Arg Leu Val Thr Ile Cys Gly Val Val Val Val Ser 130 135 140 Val Asp Tyr Arg Arg Ser Pro Glu His Arg Tyr Pro Cys Ala Tyr Asp 145 150 155 160 Asp Gly Trp Asn Ala Leu Asn Trp Val Lys Ser Arg Val Trp Leu Gln 165 170 175 Ser Gly Lys Asp Ser Asn Val Tyr Val Tyr Leu Ala Gly Asp Ser Ser 180 185 190 Gly Gly Asn Ile Ala His Asn Val Ala Val Arg Ala Thr Asn Glu Gly 195 200 205 Val Lys Val Leu Gly Asn Ile Leu Leu His Pro Met Phe Gly Gly Gln 210 215 220 Glu Arg Thr Gln Ser Glu Lys Thr Leu Asp Gly Lys Tyr Phe Val Thr 225 230 235 240 Ile Gln Asp Arg Asp Trp Tyr Trp Arg Ala Tyr Leu Pro Glu Gly Glu 245 250 255 Asp Arg Asp His Pro Ala Cys Asn Pro Phe Gly Pro Arg Gly Gln Ser 260 265 270 Leu Lys Gly Val Asn Phe Pro Lys Ser Leu Val Val Val Ala Gly Leu 275 280 285 Asp Leu Val Gln Asp Trp Gln Leu Ala Tyr Val Asp Gly Leu Lys Lys 290 295 300 Thr Gly Leu Glu Val Asn Leu Leu Tyr Leu Lys Gln Ala Thr Ile Gly 305 310 315 320 Phe Tyr Phe Leu Pro Asn Asn Asp His Phe His Cys Leu Met Glu Glu 325 330 335 Leu Asn Lys Phe Val His Ser Ile Glu Asp Ser Gln Ser Lys Ser Ser 340 345 350 Pro Val Leu Leu Thr Pro 355 <210> 97 <211> 344 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 97 Met Ala Gly Ser Glu Glu Val Asn Leu Ile Glu Ser Lys Thr Val Val 1 5 10 15 Pro Leu Asn Thr Trp Val Leu Ile Ser Asn Phe Lys Leu Ala Tyr Asn 20 25 30 Leu Leu Arg Arg Pro Asp Gly Thr Phe Asn Arg His Leu Ala Glu Phe 35 40 45 Leu Asp Arg Lys Val Pro Ala Asn Ala Asn Pro Val Asn Gly Val Phe 50 55 60 Ser Phe Asp Val Ile Ile Asp Arg Gln Thr Asn Leu Leu Ser Arg Val 65 70 75 80 Tyr Arg Pro Ala Asp Ala Gly Thr Ser Pro Ser Ile Thr Asp Leu Gln 85 90 95 Asn Pro Val Asp Gly Glu Ile Val Pro Val Ile Val Phe Phe His Gly 100 105 110 Gly Ser Phe Ala His Ser Ser Ala Asn Ser Ala Ile Tyr Asp Thr Leu 115 120 125 Cys Arg Arg Leu Val Gly Leu Cys Gly Ala Val Val Val Ser Val Asn 130 135 140 Tyr Arg Arg Ala Pro Glu Asn Arg Tyr Pro Cys Ala Tyr Asp Asp Gly 145 150 155 160 Trp Ala Val Leu Lys Trp Val Asn Ser Ser Ser Trp Leu Arg Ser Lys 165 170 175 Lys Asp Ser Lys Val Arg Ile Phe Leu Ala Gly Asp Ser Ser Gly Gly 180 185 190 Asn Ile Val His Asn Val Ala Val Arg Ala Val Glu Ser Arg Ile Asp 195 200 205 Val Leu Gly Asn Ile Leu Leu Asn Pro Met Phe Gly Gly Thr Glu Arg 210 215 220 Thr Glu Ser Glu Lys Arg Leu Asp Gly Lys Tyr Phe Val Thr Val Arg 225 230 235 240 Asp Arg Asp Trp Tyr Trp Arg Ala Phe Leu Pro Glu Gly Glu Asp Arg 245 250 255 Glu His Pro Ala Cys Ser Pro Phe Gly Pro Arg Ser Lys Ser Leu Glu 260 265 270 Gly Leu Ser Phe Pro Lys Ser Leu Val Val Val Ala Gly Leu Asp Leu 275 280 285 Ile Gln Asp Trp Gln Leu Lys Tyr Ala Glu Gly Leu Lys Lys Ala Gly 290 295 300 Gln Glu Val Lys Leu Leu Tyr Leu Glu Gln Ala Thr Ile Gly Phe Tyr 305 310 315 320 Leu Leu Pro Asn Asn Asn His Phe His Thr Val Met Asp Glu Ile Ala 325 330 335 Ala Phe Val Asn Ala Glu Cys Gln 340 <210> 98 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 98 Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu Leu 1 5 10 15 Leu Ala Val Ser Gly Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp 20 25 30 Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met 35 40 45 Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu 50 55 60 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Ala Thr Arg 65 70 75 80 Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly 85 90 95 Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr 100 105 110 Lys <210> 99 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 99 Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Gly Leu Arg Pro Val Gln Ala Gln Ala Gln Ser Asp 20 25 30 Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu Ala Gly Ile Val Met 35 40 45 Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu Ala Val Tyr Phe Leu 50 55 60 Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala Glu Ala Thr Arg Lys 65 70 75 80 Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln 85 90 95 Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln Arg Pro Tyr Tyr Lys 100 105 110 <210> 100 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 100 Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Asp Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu 20 25 30 Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu 35 40 45 Ala Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala 50 55 60 Glu Ala Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr 65 70 75 80 Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr 85 90 95 Gln Arg Pro Tyr Tyr Lys 100 <210> 101 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 101 Met Gly Gly Leu Glu Pro Cys Ser Arg Leu Leu Leu Leu Pro Leu Leu 1 5 10 15 Leu Ala Val Ser Asp Cys Ser Cys Ser Thr Val Ser Pro Gly Val Leu 20 25 30 Ala Gly Ile Val Met Gly Asp Leu Val Leu Thr Val Leu Ile Ala Leu 35 40 45 Ala Val Tyr Phe Leu Gly Arg Leu Val Pro Arg Gly Arg Gly Ala Ala 50 55 60 Glu Ala Thr Arg Lys Gln Arg Ile Thr Glu Thr Glu Ser Pro Tyr Gln 65 70 75 80 Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser Asp Leu Asn Thr Gln 85 90 95 Arg Pro Tyr Tyr Lys 100 <210> 102 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 102 Glu Ser Pro Tyr Gln Glu Leu Gln Gly Gln Arg Ser Asp Val Tyr Ser 1 5 10 15 Asp Leu Asn Thr Gln 20 <210> 103 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 103 Met Ile Pro Ala Val Val Leu Leu Leu Leu Leu Leu Val Glu Gln Ala 1 5 10 15 Ala Ala Leu Gly Glu Pro Gln Leu Cys Tyr Ile Leu Asp Ala Ile Leu 20 25 30 Phe Leu Tyr Gly Ile Val Leu Thr Leu Leu Tyr Cys Arg Leu Lys Ile 35 40 45 Gln Val Arg Lys Ala Ala Ile Thr Ser Tyr Glu Lys Ser Asp Gly Val 50 55 60 Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu Thr Leu Lys 65 70 75 80 His Glu Lys Pro Pro Gln 85 <210> 104 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 104 Asp Gly Val Tyr Thr Gly Leu Ser Thr Arg Asn Gln Glu Thr Tyr Glu 1 5 10 15 Thr Leu Lys His Glu 20 <210> 105 <211> 171 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 105 Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu 1 5 10 15 Ser Gln Val Ser Pro Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg 20 25 30 Val Phe Val Asn Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val 35 40 45 Gly Thr Leu Leu Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile 50 55 60 Leu Asp Pro Arg Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys 65 70 75 80 Asp Lys Glu Ser Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys 85 90 95 Val Glu Leu Asp Pro Ala Thr Val Ala Gly Ile Ile Val Thr Asp Val 100 105 110 Ile Ala Thr Leu Leu Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His 115 120 125 Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu Leu Arg 130 135 140 Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala Gln Tyr 145 150 155 160 Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys 165 170 <210> 106 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 106 Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu 1 5 10 15 Ser Gln Val Ser Pro Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg 20 25 30 Val Phe Val Asn Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val 35 40 45 Gly Thr Leu Leu Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile 50 55 60 Leu Asp Pro Arg Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys 65 70 75 80 Asp Lys Glu Ser Thr Val Gln Val His Tyr Arg Thr Ala Asp Thr Gln 85 90 95 Ala Leu Leu Arg Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp 100 105 110 Asp Ala Gln Tyr Ser His Leu Gly Gly Asn Trp Ala Arg Asn Lys 115 120 125 <210> 107 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 107 Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala Gln Tyr Ser 1 5 10 15 His Leu Gly Gly Asn 20 <210> 108 <211> 207 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 108 Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser 1 5 10 15 Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 20 25 30 Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 35 40 45 Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60 Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp 65 70 75 80 His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95 Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110 Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met 115 120 125 Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu 130 135 140 Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys 145 150 155 160 Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn 165 170 175 Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg 180 185 190 Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 195 200 205 <210> 109 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 109 Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp Leu Tyr Ser 1 5 10 15 Gly Leu Asn Gln Arg 20 <210> 110 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 110 Met Glu Gln Gly Lys Gly Leu Ala Val Leu Ile Leu Ala Ile Ile Leu 1 5 10 15 Leu Gln Gly Thr Leu Ala Gln Ser Ile Lys Gly Asn His Leu Val Lys 20 25 30 Val Tyr Asp Tyr Gln Glu Asp Gly Ser Val Leu Leu Thr Cys Asp Ala 35 40 45 Glu Ala Lys Asn Ile Thr Trp Phe Lys Asp Gly Lys Met Ile Gly Phe 50 55 60 Leu Thr Glu Asp Lys Lys Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp 65 70 75 80 Pro Arg Gly Met Tyr Gln Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro 85 90 95 Leu Gln Val Tyr Tyr Arg Met Cys Gln Asn Cys Ile Glu Leu Asn Ala 100 105 110 Ala Thr Ile Ser Gly Phe Leu Phe Ala Glu Ile Val Ser Ile Phe Val 115 120 125 Leu Ala Val Gly Val Tyr Phe Ile Ala Gly Gln Asp Gly Val Arg Gln 130 135 140 Ser Arg Ala Ser Asp Lys Gln Thr Leu Leu Pro Asn Asp Gln Leu Tyr 145 150 155 160 Gln Pro Leu Lys Asp Arg Glu Asp Asp Gln Tyr Ser His Leu Gln Gly 165 170 175 Asn Gln Leu Arg Arg Asn 180 <210> 111 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 111 Asp Gln Leu Tyr Gln Pro Leu Lys Asp Arg Glu Asp Asp Gln Tyr Ser 1 5 10 15 His Leu Gln Gly Asn 20 <210> 112 <211> 163 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 112 Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu 1 5 10 15 Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys 20 25 30 Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala 35 40 45 Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 50 55 60 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 65 70 75 80 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 85 90 95 Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 100 105 110 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 115 120 125 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 130 135 140 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 145 150 155 160 Pro Pro Arg <210> 113 <211> 164 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 113 Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu 1 5 10 15 Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys 20 25 30 Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala 35 40 45 Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 50 55 60 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 65 70 75 80 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 85 90 95 Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 100 105 110 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 115 120 125 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 130 135 140 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 145 150 155 160 Leu Pro Pro Arg <210> 114 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 114 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 1 5 10 15 Val Leu Asp Lys Arg 20 <210> 115 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 115 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 1 5 10 15 Ser Glu Ile Gly Met Lys 20 <210> 116 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 116 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 1 5 10 15 Ala Leu His Met Gln 20 <210> 117 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 117 Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe 1 5 10 15 Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30 Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45 Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60 Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe 65 70 75 80 Leu Gly Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile Ile Gln Asn Val 85 90 95 Asn Lys Ser His Gly Gly Ile Tyr Val Cys Arg Val Gln Glu Gly Asn 100 105 110 Glu Ser Tyr Gln Gln Ser Cys Gly Thr Tyr Leu Arg Val Arg Gln Pro 115 120 125 Pro Pro Arg Pro Phe Leu Asp Met Gly Glu Gly Thr Lys Asn Arg Ile 130 135 140 Ile Thr Ala Glu Gly Ile Ile Leu Leu Phe Cys Ala Val Val Pro Gly 145 150 155 160 Thr Leu Leu Leu Phe Arg Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu 165 170 175 Asp Ala Gly Asp Glu Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn 180 185 190 Leu Asp Asp Cys Ser Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly 195 200 205 Thr Tyr Gln Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu 210 215 220 Lys Pro 225 <210> 118 <211> 188 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 118 Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu Pro Ala Thr Ile Phe 1 5 10 15 Leu Leu Phe Leu Leu Ser Ala Val Tyr Leu Gly Pro Gly Cys Gln Ala 20 25 30 Leu Trp Met His Lys Val Pro Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45 Asp Ala His Phe Gln Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60 Thr Trp Trp Arg Val Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe 65 70 75 80 Leu Gly Pro Gly Glu Asp Pro Asn Glu Pro Pro Pro Arg Pro Phe Leu 85 90 95 Asp Met Gly Glu Gly Thr Lys Asn Arg Ile Ile Thr Ala Glu Gly Ile 100 105 110 Ile Leu Leu Phe Cys Ala Val Val Pro Gly Thr Leu Leu Leu Phe Arg 115 120 125 Lys Arg Trp Gln Asn Glu Lys Leu Gly Leu Asp Ala Gly Asp Glu Tyr 130 135 140 Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser Met 145 150 155 160 Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly Thr Tyr Gln Asp Val Gly 165 170 175 Ser Leu Asn Ile Gly Asp Val Gln Leu Glu Lys Pro 180 185 <210> 119 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 119 Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp Cys Ser Met Tyr Glu 1 5 10 15 Asp Ile Ser Arg Gly 20 <210> 120 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 120 Arg Pro Arg Arg Ser Pro Ala Gln Asp Gly Lys Val Tyr Ile Asn Met 1 5 10 15 Pro Gly Arg Gly 20 <210> 121 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 121 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser 20 25 30 Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly 35 40 45 Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala 50 55 60 Ala Tyr Arg Ser 65 <210> 122 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 122 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 123 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 123 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210> 124 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 124 His His His His His 1 5 <210> 125 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 125 His His His His His His 1 5 <210> 126 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 126 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 127 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 127 Arg Tyr Ile Arg Ser 1 5 <210> 128 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 128 Phe His His Thr 1 <210> 129 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 129 Trp Glu Ala Ala Ala Arg Glu Ala Cys Cys Arg Glu Cys Cys Ala Arg 1 5 10 15 Ala <210> 130 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <220> <221> MISC_FEATURE <222> (2)..(3) <223> the amino acids in these positions can be any amino acid <220> <221> MISC_FEATURE <222> (4)..(4) <223> the amino acids in this position can be either Leu or Ile <400> 130 Tyr Xaa Xaa Xaa 1 <210> 131 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <220> <221> MISC_FEATURE <222> (2)..(3) <223> the amino acids in these positions can be any amino acid <220> <221> MISC_FEATURE <222> (4)..(4) <223> the amino acids in this positions can be either Leu or Ile <220> <221> MISC_FEATURE <222> (5)..(10) <223> the amino acids in these positions can be any amino acid <220> <221> MISC_FEATURE <222> (11)..(12) <223> the amino acids in these positions may be present or absent such that either one or two amino acids are present. the amino acids in these positions can be any amino acid <220> <221> misc_feature <222> (14)..(15) <223> Xaa can be any naturally occurring amino acid <220> <221> MISC_FEATURE <222> (16)..(16) <223> the amino acids in this positions can be either Leu or Ile <400> 131 Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa 1 5 10 15 <210> 132 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 132 gattacaagg atgacgatga caag 24 <210> 133 <211> 732 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 133 Gly Gly Ala Thr Cys Cys Cys Ala Gly Gly Thr Ala Cys Ala Ala Cys 1 5 10 15 Thr Gly Cys Ala Gly Cys Ala Gly Thr Cys Thr Gly Gly Gly Cys Cys 20 25 30 Thr Gly Ala Gly Cys Thr Gly Gly Ala Gly Ala Ala Gly Cys Cys Thr 35 40 45 Gly Gly Cys Gly Cys Thr Thr Cys Ala Gly Thr Gly Ala Ala Gly Ala 50 55 60 Thr Ala Thr Cys Cys Thr Gly Cys Ala Ala Gly Gly Cys Thr Thr Cys 65 70 75 80 Thr Gly Gly Thr Thr Ala Cys Thr Cys Ala Thr Thr Cys Ala Cys Thr 85 90 95 Gly Gly Cys Thr Ala Cys Ala Cys Cys Ala Thr Gly Ala Ala Cys Thr 100 105 110 Gly Gly Gly Thr Gly Ala Ala Gly Cys Ala Gly Ala Gly Cys Cys Ala 115 120 125 Thr Gly Gly Ala Ala Ala Gly Ala Gly Cys Cys Thr Thr Gly Ala Gly 130 135 140 Thr Gly Gly Ala Thr Thr Gly Gly Ala Cys Thr Thr Ala Thr Thr Ala 145 150 155 160 Cys Thr Cys Cys Thr Thr Ala Cys Ala Ala Thr Gly Gly Thr Gly Cys 165 170 175 Thr Thr Cys Thr Ala Gly Cys Thr Ala Cys Ala Ala Cys Cys Ala Gly 180 185 190 Ala Ala Gly Thr Thr Cys Ala Gly Gly Gly Gly Cys Ala Ala Gly Gly 195 200 205 Cys Cys Ala Cys Ala Thr Thr Ala Ala Cys Thr Gly Thr Ala Gly Ala 210 215 220 Cys Ala Ala Gly Thr Cys Ala Thr Cys Cys Ala Gly Cys Ala Cys Ala 225 230 235 240 Gly Cys Cys Thr Ala Cys Ala Thr Gly Gly Ala Cys Cys Thr Cys Cys 245 250 255 Thr Cys Ala Gly Thr Cys Thr Gly Ala Cys Ala Thr Cys Thr Gly Ala 260 265 270 Ala Gly Ala Cys Thr Cys Thr Gly Cys Ala Gly Thr Cys Thr Ala Thr 275 280 285 Thr Thr Cys Thr Gly Thr Gly Cys Ala Ala Gly Gly Gly Gly Gly Gly 290 295 300 Gly Thr Thr Ala Cys Gly Ala Cys Gly Gly Gly Ala Gly Gly Gly Gly 305 310 315 320 Thr Thr Thr Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly Cys 325 330 335 Cys Ala Ala Gly Gly Gly Ala Cys Cys Ala Cys Gly Gly Thr Cys Ala 340 345 350 Cys Cys Gly Thr Cys Thr Cys Cys Thr Cys Ala Gly Gly Thr Gly Gly 355 360 365 Ala Gly Gly Cys Gly Gly Thr Thr Cys Ala Gly Gly Cys Gly Gly Cys 370 375 380 Gly Gly Thr Gly Gly Cys Thr Cys Thr Ala Gly Cys Gly Gly Thr Gly 385 390 395 400 Gly Cys Gly Gly Ala Thr Cys Gly Gly Ala Cys Ala Thr Cys Gly Ala 405 410 415 Gly Cys Thr Cys Ala Cys Thr Cys Ala Gly Thr Cys Thr Cys Cys Ala 420 425 430 Gly Cys Ala Ala Thr Cys Ala Thr Gly Thr Cys Thr Gly Cys Ala Thr 435 440 445 Cys Thr Cys Cys Ala Gly Gly Gly Gly Ala Gly Ala Ala Gly Gly Thr 450 455 460 Cys Ala Cys Cys Ala Thr Gly Ala Cys Cys Thr Gly Cys Ala Gly Thr 465 470 475 480 Gly Cys Cys Ala Gly Cys Thr Cys Ala Ala Gly Thr Gly Thr Ala Ala 485 490 495 Gly Thr Thr Ala Cys Ala Thr Gly Cys Ala Cys Thr Gly Gly Thr Ala 500 505 510 Cys Cys Ala Gly Cys Ala Gly Ala Ala Gly Thr Cys Ala Gly Gly Cys 515 520 525 Ala Cys Cys Thr Cys Cys Cys Cys Cys Ala Ala Ala Ala Gly Ala Thr 530 535 540 Gly Gly Ala Thr Thr Thr Ala Thr Gly Ala Cys Ala Cys Ala Thr Cys 545 550 555 560 Cys Ala Ala Ala Cys Thr Gly Gly Cys Thr Thr Cys Thr Gly Gly Ala 565 570 575 Gly Thr Cys Cys Cys Ala Gly Gly Thr Cys Gly Cys Thr Thr Cys Ala 580 585 590 Gly Thr Gly Gly Cys Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly 595 600 605 Ala Ala Ala Cys Thr Cys Thr Thr Ala Cys Thr Cys Thr Cys Thr Cys 610 615 620 Ala Cys Ala Ala Thr Cys Ala Gly Cys Ala Gly Cys Gly Thr Gly Gly 625 630 635 640 Ala Gly Gly Cys Thr Gly Ala Ala Gly Ala Thr Gly Ala Thr Gly Cys 645 650 655 Ala Ala Cys Thr Thr Ala Thr Thr Ala Cys Thr Gly Cys Cys Ala Gly 660 665 670 Cys Ala Gly Thr Gly Gly Ala Gly Thr Ala Ala Gly Cys Ala Cys Cys 675 680 685 Cys Thr Cys Thr Cys Ala Cys Gly Thr Ala Cys Gly Gly Thr Gly Cys 690 695 700 Thr Gly Gly Gly Ala Cys Ala Ala Ala Gly Thr Thr Gly Gly Ala Ala 705 710 715 720 Ala Thr Cys Ala Ala Ala Gly Cys Thr Ala Gly Cys 725 730 <210> 134 <211> 244 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 134 Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro 1 5 10 15 Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr 20 25 30 Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu 35 40 45 Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln 50 55 60 Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr 65 70 75 80 Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 85 90 95 Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro 130 135 140 Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser 145 150 155 160 Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly 165 170 175 Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly 180 185 190 Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu 195 200 205 Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln 210 215 220 Gln Trp Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu 225 230 235 240 Ile Lys Ala Ser <210> 135 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 135 ggatcccagg tgcagctgca ggaatctggc cctggcctcg tgaagcccag cgagacactg 60 agcctgacct gtaccgtgtc tggcggctct gtgtccagcg gcagctacta ctggtcctgg 120 atcagacagc cccctggcaa gggcctggaa tggatcggct acatctacta cagcggctcc 180 accaactaca accccagcct gaagtccaga gtgaccatca gcgtggacac cagcaagaac 240 cagttctccc tgaagctgag cagcgtgaca gccgccgata ccgccgtgta ctactgtgcc 300 agagagggca agaacggcgc cttcgacatc tggggccagg gcacaatggt caccgtgtca 360 tctggtggag gaggatctgg gggaggcgga agcggaggcg gcggatctga tattcagatg 420 acccagagcc ccagcagcct gagcgcctct gtgggcgaca gagtgacaat tacctgccgg 480 gccagccaga gcatcagcag ctacctgaac tggtatcagc agaagcccgg caaggccccc 540 aaactgctga tctacgccgc cagctctctg cagtctggcg tgcccagcag attttccggc 600 tctggcagcg gcaccgactt caccctgacc atctctagcc tgcagcccga ggactcgcc 660 acctactact gccagcagag ctacagcacc cccctgacct tggcggagg caccaaggtg 720 gaatcaag 729 <210> 136 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 136 Gly Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 1 5 10 15 Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser 20 25 30 Ser Gly Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly 35 40 45 Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn 50 55 60 Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Lys Leu Ser Val Thr Ala Ala Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Glu Gly Lys Asn Gly Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 130 135 140 Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg 145 150 155 160 Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser 180 185 190 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 210 215 220 Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val 225 230 235 240 Glu Ile Lys <210> 137 <211> 1044 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 137 atggctgga gcgatgaagt taatcttatt gagagcagaa cagtggttcc tctcaataca 60 tgggttttaa tatccaactt caaagtagcc tacaatatcc ttcgtcgccc tgatggaacc 120 tttaaccgac acttagctga gtatctagac cgtaaagtca ctgcaaacgc caatccggtt 180 gatggggttt tctcgttcga tgtcttgatt gatcgcagga tcaatcttct aagcagagtc 240 tatagaccag cttatgcaga tcaagagcaa cctcctagta tttagatct cgagaagcct 300 gttgatggcg acattgtccc tgttatattg ttcttccatg gaggtagctt tgctcattct 360 tctgcaaaca gtgccatcta cgatactctt tgtcgcaggc ttgttggttt gtgcaagtgt 420 gttgttgtct ctgtgaatta tcggcgtgca ccagagaatc catacccttg tgcttatgat 480 gatggttgga ttgctcttaa ttgggttaac tcgagatctt ggcttaaatc caagaaagac 540 tcaaaggtcc atattttctt ggctggtgat agctctggag gtaacatcgc gcataatgtg 600 gctttaagag cgggtgaatc gggaatcgat gttttgggga acattctgct gaatcctatg 660 tttggtggga atgagagaac ggagtctgag aaaagttttgg atggggaaata cttttgtgacg 720 gttagagacc gcgattggta ctggaaagcg ttttacccg agggagaaga tagagagcat 780 ccagcgtgta atccgtttag cccgagaggg aaaagcttag aaggagtgag tttccccaag 840 agtcttgtgg ttgtcgcggg tttggatttg attagagatt ggcagttggc atacgcggaa 900 gggctcaaga aagcgggtca agaggttaag cttatgcatt tagagaaagc aactgttggg 960 ttttacctct tgcctaataa caatcatttc cataatgtta tggatgagat ttcggcgttt 1020 gtaaacgcgg aatgtatgcg tgac 1044 <210> 138 <211> 348 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 138 Put Ala Ala Ser Asp Glu Val Asn Leu Ile Glu Ser Arg Thr Val Val 1 5 10 15 Pro Leu Asn Thr Trp Val Leu Ile Ser Asn Phe Lys Val Ala Tyr Asn 20 25 30 Ile Leu Arg Arg Pro Asp Gly Thr Phe Asn Arg His Leu Ala Glu Tyr 35 40 45 Leu Asp Arg Lys Val Thr Ala Asn Ala Asn Pro Val Asp Gly Val Phe 50 55 60 Ser Phe Asp Val Leu Ile Asp Arg Arg Ile Asn Leu Leu Ser Arg Val 65 70 75 80 Tyr Arg Pro Ala Tyr Ala Asp Gln Glu Gln Pro Pro Ser Ile Leu Asp 85 90 95 Leu Glu Lys Pro Val Asp Gly Asp Ile Val Pro Val Ile Leu Phe Phe 100 105 110 His Gly Gly Ser Phe Ala His Ser Ser Ala Asn Ser Ala Ile Tyr Asp 115 120 125 Thr Leu Cys Arg Arg Leu Val Gly Leu Cys Lys Cys Val Val Val Ser 130 135 140 Val Asn Tyr Arg Arg Ala Pro Glu Asn Pro Tyr Pro Cys Ala Tyr Asp 145 150 155 160 Asp Gly Trp Ile Ala Leu Asn Trp Val Asn Ser Arg Ser Trp Leu Lys 165 170 175 Ser Lys Lys Asp Ser Lys Val His Ile Phe Leu Ala Gly Asp Ser Ser 180 185 190 Gly Gly Asn Ile Ala His Asn Val Ala Leu Arg Ala Gly Glu Ser Gly 195 200 205 Ile Asp Val Leu Gly Asn Ile Leu Leu Asn Pro Met Phe Gly Gly Asn 210 215 220 Glu Arg Thr Glu Ser Glu Lys Ser Leu Asp Gly Lys Tyr Phe Val Thr 225 230 235 240 Val Arg Asp Arg Asp Trp Tyr Trp Lys Ala Phe Leu Pro Glu Gly Glu 245 250 255 Asp Arg Glu His Pro Ala Cys Asn Pro Phe Ser Pro Arg Gly Lys Ser 260 265 270 Leu Glu Gly Val Ser Phe Pro Lys Ser Leu Val Val Val Ala Gly Leu 275 280 285 Asp Leu Ile Arg Asp Trp Gln Leu Ala Tyr Ala Glu Gly Leu Lys Lys 290 295 300 Ala Gly Gln Glu Val Lys Leu Met His Leu Glu Lys Ala Thr Val Gly 305 310 315 320 Phe Tyr Leu Leu Pro Asn Asn Asn His Phe His Asn Val Met Asp Glu 325 330 335 Ile Ser Ala Phe Val Asn Ala Glu Cys Met Arg Asp 340 345 <210> 139 <211> 276 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 139 atgaagagag atcatcatca tcatcatcat caagataaga agactatgat gatgaatgaa 60 gaagacgacg gtaacggcat ggatgagctt ctagctgttc ttggttacaa ggttaggtca 120 tccgaaatgg ctgatgttgc tcagaaactc gagcagcttg aagttatgat gtctaatgtt 180 caagaagacg atctttctca actcgctact gagactgttc actataatcc ggcggagctt 240 tacacgtggc ttgattctat gctcaccgac cttaat 276 <210> 140 <211> 92 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 140 Met Lys Arg Asp His His His His His His Gln Asp Lys Lys Thr Met 1 5 10 15 Met Met Asn Glu Glu Asp Asp Gly Asn Gly Met Asp Glu Leu Leu Ala 20 25 30 Val Leu Gly Tyr Lys Val Arg Ser Ser Glu Met Ala Asp Val Ala Gln 35 40 45 Lys Leu Glu Gln Leu Glu Val Met Met Ser Asn Val Glu Glu Asp Asp 50 55 60 Leu Ser Gln Leu Ala Thr Glu Thr Val His Tyr Asn Pro Ala Glu Leu 65 70 75 80 Tyr Thr Trp Leu Asp Ser Met Leu Thr Asp Lear Asn 85 90 <210> 141 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 141 ggatcccagg tgcagctggt gcagtctggc gccgaagtga aaagaccagg cgccagcgtg 60 caggtctcct gtagagccag cggctacagc atcacacct actacatgca gtgggtgcgc 120 caggccccag gcgctggact ggatgtg ggcgtca accccaggcgg cgtgacaagc 180 tacgcccaga aattccagggg cagagtgacc ctgaccaacg acaccac ccacacagtg 240 tacatgcagc tgaacagcct gaccagcgcc gandaccgccg tgtactactg tgccagatgg 300 gccctgtggg gcgacttcgg catggatgtg tggggcaagg gcaccctcgt gaccgtgtct 360 agcggaggcg gaggatctgg cggaggggga tctggaggcg gcggaagcga catccagatg 420 acccagagcc ctagcaccct gagcgccagc atcggcgata gagtgaccat cacctgtcgg 480 gccagcgagg gcatctatca ctggctggcc tggtatcagc agaagcccgg caaggccccc 540 aagctgctga tctacaaggc cagctctctg gcctctggcg ccctagcag atttctggc 600 agcggctccg gcaccgactt caccctgaca atcagcagcc tgcagcccga cgacttcgcc 660 acctact gccagcagta cagcaactac cccctgacct tcggcggagg caccaagctg 720 hole 729 <210> 142 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> synthetic polypeptide <400> 142 Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro 1 5 10 15 Gly Ala Ser Val Gln Val Ser Cys Arg Ala Ser Gly Tyr Ser Ile Asn 20 25 30 Thr Tyr Tyr Met Gln Trp Val Arg Gln Ala Pro Gly Ala Gly Leu Glu 35 40 45 Trp Met Gly Val Ile Asn Pro Ser Gly Val Thr Ser Tyr Ala Gln Lys 50 55 60 Phe Gln Gly Arg Val Thr Leu Thr Asn Asp Thr Ser Thr Asn Thr Val 65 70 75 80 Tyr Met Gln Leu Asn Ser Leu Thr Ser Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Trp Ala Leu Trp Gly Asp Phe Gly Met Asp Val Trp Gly 100 105 110 Lys Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 130 135 140 Ser Thr Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile Thr Cys Arg 145 150 155 160 Ala Ser Glu Gly Ile Tyr His Trp Leu Ala Trp Tyr Gln Gln Lys Pro 165 170 175 Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser Leu Ala Ser 180 185 190 Gly Ala Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205 Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys 210 215 220 Gln Gln Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu 225 230 235 240 Glu Ile Lys <210> 143 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 143 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 144 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 144 ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60 gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120 aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180 cgcgacttcg cagcctatcg ctcc 204 <210> 145 <211> 111 <212> DNA <213> Artificial Sequence <220> <223> synthetic polynucleotide <400> 145 cggagggacc agaggctgcc ccccgatgcc cacaagcccc ctgggggagg cagtttccgg 60 acccccatcc aagaggagca ggccgacgcc cactccaccc tggccaagat c 111

Claims

1. A heterodimeric conditionally active chimeric antigen receptor (CAR), comprising: a) a first polypeptide, i) a first member of a specific binding pair; and ii) a first regulatory domain; and iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between said first member of a specific binding pair and said first regulatory domain; a first polypeptide comprising: b) a second polypeptide, i) a transmembrane domain; and ii) a second regulatory domain; and iii) a second member of the dimerization pair; and iv) an intracellular signaling domain; The second polypeptide comprises Contains or a) a first polypeptide, i) a first member of a specific binding pair; and ii) a regulatory domain; and iii) a first member of a dimerization pair; and iv) a transmembrane domain interposed between said first member of a specific binding pair and said regulatory domain; a first polypeptide comprising: b) a second polypeptide, i) a second member of said dimerization pair; and ii) an intracellular signaling domain; A second polypeptide comprising: The heterodimeric conditionally active chimeric antigen receptor (CAR).

2. The heterodimeric conditionally active CAR described in claim 1, wherein the first polypeptide comprises a hinge region interposed between the first member of the specific binding pair and the transmembrane domain, the hinge region being an immunoglobulin IgG hinge region or a CD8-derived hinge.

3. The heterodimeric, conditionally active CAR of claim 1, wherein the first member of the specific binding pair is an antibody or antibody fragment, a ligand, or a receptor.

4. The heterodimeric conditionally active CAR of claim 1, wherein the first and second regulatory domains are selected from 4-1BB (CD137), CD28, ICOS, BTLA, OX-40, CD27, CD30, GITR, HVEM, DAP10, DAP12, and CD28.

5. The heterodimeric, conditionally active CAR of claim 1, wherein the intracellular signaling domain is selected from ZAP70 and CD3-zeta. a) the first and second members of the dimerization pair form a homodimer in the presence of a small molecule dimerizer; or b) the first and second members of the dimerization pair form a heterodimer in the presence of a small molecule dimerizer; The heterodimeric conditionally active CAR of claim 1.

7. The method of claim 1, wherein the first and second members of the dimerization pair are a) FK506 binding protein (FKBP) and FKBP; b) FKBP and calcineurin catalytic subunit A (CnA); c) FKBP and cyclophilin; d) FKBP and FKBP-rapamycin-related protein (FRB); e) gyrase B (GyrB) and GryB; f) dihydrofolate reductase (DHFR) and DHFR; g) DmrB and DmrB, h) PYL and ABI; i) Cry2 and CIP; j) GAI and GID1 The heterodimeric conditionally active CAR of claim 1, selected from:

8. i) the first and second regulatory domains are derived from 4-1BB; ii) the first and second members of the dimerization pair are FKBP and FRB; and iii) the signaling domain comprises an ITAM; The heterodimeric conditionally active CAR of claim 1.

9. The heterodimeric, conditionally active CAR of claim 1, wherein the first member of the specific binding pair is a single-chain Fv.

10. The heterodimeric, conditionally active CAR of claim 1, wherein the first member of the specific binding pair binds to an epitope present on a cell, on a solid surface, or in a lipid bilayer.

11. A mammalian cell genetically modified to produce the heterodimeric conditionally active CAR described in claim 1.

12. The cell described in claim 11, which is a T lymphocyte or a NK cell.

13. A nucleic acid comprising a nucleotide sequence encoding the heterodimeric conditionally active CAR described in claim 1.

14. The nucleic acid of claim 13, wherein the nucleotide sequence is functionally linked to a T lymphocyte-specific promoter or a NK cell-specific promoter.

15. A recombinant expression vector comprising the nucleic acid described in claim 13.

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