RU2832516C2 - Compositions and methods for immunotherapy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is an expression vector containing a first polynucleotide coding a chimeric antigen receptor and a second polynucleotide coding a chimeric costimulatory receptor. Also disclosed is a cytotoxic T-cell containing said expression vector.

EFFECT: invention provides enhanced and selective cytolytic activity of T-cells.

18 cl, 21 dwg, 1 tbl, 1 ex

Description

Cross-reference to related applications

[0001] This application is a non-provisional application that claims priority from U.S. Provisional Application Serial No. 61/709,072, filed October 2, 2012, the entire contents of which are hereby incorporated by reference.

List of sequences

[0002] This application contains a sequence listing created on September 30, 2013; the file, in ASCII format, is designated 3314040AWO_Sequence Listing_ST25.txt and is 27.5 kilobytes in size. The full contents of the sequence listing file are hereby incorporated by reference into the application.

Background of the invention

[0003] Prostate cancer is the most common malignancy in men in the United States, causing approximately 31,000 deaths per year. When diagnosed early, the cancer can be effectively treated with surgery or radiation. Postoperative residual disease requires radiation and/or hormonal therapy, which can prevent tumor progression and metastasis. There is currently no curative treatment for hormone-refractory, metastatic prostate cancer. Immunotherapy is a targeted therapy that has the potential to cure these malignancies.

[0004] Targeted T-cell therapies using genetically modified autologous T cells have begun to show evidence of therapeutic efficacy in melanoma and slow-growing B-cell malignancies. Current T-cell engineering techniques redirect a patient's T cells to tumor antigens via a transduced T-cell receptor (TCR) or chimeric antigen receptor (CAR). The newly discovered ability to induce potent immune responses, however, necessitates restricting immune attacks to the tumor and avoiding reactions against normal tissues that may express the target antigen. Unfortunately, the limited availability of truly tumor-restricted antigens often prevents highly specific targeting. Among the limitations that prevent highly specific targeting is the limited availability of truly tumor-restricted antigens. Accordingly, new treatments for neoplasia are urgently needed.

Brief summary of the invention

[0005] The present invention generally relates to immunoresponsive cells, including T cells and natural killer (NK) cells, expressing an antigen-binding receptor (e.g., CAR or TCR) having immunocyte activation activity and a chimeric costimulatory receptor (CCR), and to methods of using them in this manner to treat neoplasia, infectious disease, and other pathologies.

[0006] In one aspect, the invention relates to an isolated immunoresponsive cell having an antigen-recognizing receptor that binds a first antigen with low affinity, wherein the binding activates the immunoresponsive cell, and a chimeric costimulatory receptor (CCR) that binds a second antigen and stimulates the immunoresponsive cell.

[0007] In another aspect, the invention relates to a method for inducing tumor cell death in a subject, wherein the method comprises administering an effective amount of immunoresponsive cells comprising an antigen-recognizing receptor that binds a first antigen with low affinity, wherein the binding activates the immunoresponsive cell, and a chimeric costimulatory receptor (CCR) that binds a second antigen and stimulates the immunoresponsive cell, thereby inducing tumor cell death in the subject.

[0008] In another aspect, the invention relates to a method of treating or preventing neoplasia in a subject, wherein the method comprises administering an effective amount of immunoresponsive cells comprising an antigen recognition receptor that binds a first antigen with low affinity, wherein the binding activates the immunoresponsive cell, and a chimeric costimulatory receptor (CCR) that binds a second antigen and stimulates the immunoresponsive cell, thereby treating or preventing neoplasia in the subject.

[0009] In another aspect, the invention relates to a method of treating prostate cancer in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of T cells comprising an antigen recognition receptor that binds PSCA or CD19 with low affinity, wherein the binding activates an immunoresponsive cell, and a chimeric costimulatory receptor (CCR) that binds PSMA and stimulates the immunoresponsive cell, thereby curing prostate cancer in the subject.

[0010] In another aspect, the invention relates to a method for producing an antigen-specific immunoresponsive cell, wherein the method comprises introducing into the immunoresponsive cell a nucleic acid sequence encoding a chimeric costimulatory receptor (CCR), wherein the chimeric costimulatory receptor has an antigen-binding domain linked to an intracellular signaling domain that stimulates the immunoresponsive cell, wherein the immunoresponsive cell has an antigen-recognizing receptor that binds a first antigen with low affinity, wherein the binding activates the immunoresponsive cell.

[0011] In a related aspect, the invention relates to a pharmaceutical composition comprising an effective amount of immunoreactive cells of the invention (e.g., tumor antigen-specific T cells in a pharmaceutical composition for treating neoplasia) in a pharmaceutically acceptable excipient.

[0012] In a further aspect, the invention relates to a kit for treating a neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant, wherein the kit comprises an immunoresponsive cell having an antigen recognition receptor that binds a first antigen and activates the immunoresponsive cell, and a chimeric costimulatory receptor (CCR) that binds a second viral antigen and stimulates the immunoresponsive cell. The kit may further comprise written instructions for using the immunoresponsive cells to treat a subject having a neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant.

[0013] In various embodiments of any of the aspects described herein, the immunoresponsive cell is selected as having an antigen-recognizing receptor with low affinity. This may include selecting the immunoresponsive cell as having an antigen-recognizing receptor that binds a first antigen with low affinity. In various embodiments of any of the aspects described herein, the antigen-recognizing receptor is selected as having low affinity for expression in the cell. This may include introducing a second nucleic acid sequence encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a second antigen-binding domain coupled to a second intracellular signaling domain that activates the immunoresponsive cell. In various embodiments of any of the aspects described herein, the antigen-recognizing receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In various embodiments, the intracellular signaling domain of said antigen-recognizing receptor is the signaling domain of the CD3 chain. In various embodiments, the intracellular signaling domain of the chimeric costimulatory receptor (CCR) is the signaling domain of CD97, CD11a-CD18, CD2, ICOS, CD27, CD154, CD5, OX40, 4-1BB, or CD28.

[0014] In various embodiments of any of the aspects described herein, the antigen recognition receptor is exogenous or endogenous. In various embodiments of any of the aspects described herein, the antigen recognition receptor is expressed recombinantly. In various embodiments, the antigen recognition receptor is expressed from a vector. In various embodiments, the chimeric costimulatory receptor (CCR) is expressed from a vector. In particular embodiments, the immunoresponsive cell expresses a recombinant or endogenous antigen receptor that is 19z1 or Pz1.

[0015] In various embodiments of any of the aspects described herein, the immunoresponsive cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, or a pluripotent stem cell that can differentiate into lymphoid cells. Various embodiments of any of the aspects described herein relate to an immunoresponsive cell of any of claims 1-9, wherein said immunoresponsive cell is autologous.

[0016] In various embodiments of any of the aspects described herein, the antigen is a tumor or pathogen antigen. In various embodiments of any of the aspects described herein, one or more antigen-binding domains are tumor antigen-binding domains. In various embodiments of any of the aspects described herein, the antigens or tumor antigens are selected from CAIX, CEA, CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL13R-a2, x-light chain, KDR, LeY, cell adhesion molecule LI, MAGE-AI, MUC1, mesothelin, NKG2D ligands, NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2 and WT-1. In various embodiments, the first and second antigens are selected from CD133, cytomegalovirus (CMV) infected cell antigen, erbB2, KDR mesothelin, NKG2D ligands, NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, CD19, VEGF-R2 and WT-1. In particular embodiments, the first and second antigens are selected from HER2, MUC1, CD44, CD49f, EpCAM, CEA, CD133, cytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, KDR, mesothelin, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, VEGF-R2 or WT-1. In particular embodiments, the first and second antigens are selected from CD10 and CD19. In other embodiments, the first and second antigens are selected from CD56 and CD138. In particular embodiments, the first and second antigens are selected from mesothelin, folate receptor-a, CD44 and CD133.

[0017] In various embodiments of any of the aspects described herein, the neoplasia is selected from the group consisting of prostate cancer, breast cancer, B-cell leukemia, multiple myeloma, and ovarian cancer. In various embodiments of any of the aspects described herein, the method reduces the number of tumor cells, reduces the size of the tumor, and/or kills the tumor in the subject.

[0018] In various embodiments, the neoplasia is prostate cancer, and the first and second tumor antigens are different antigens selected from PSCA, PSMA, CD19, CD133, cytomegalovirus (CMV)-infected cell antigen, erb-B2, KDR mesothelin, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), VEGF-R2, and WT-I. In various embodiments, the neoplasia is breast cancer, and the first and second tumor antigens are different antigens selected from HER2, MUC1, CD44, CD49f, EpCAM, CEA, CD133, cytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, KDR, mesothelin, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, VEGF-R2 or WT-1. In particular embodiments, the neoplasia is B-cell leukemia, and the first and second tumor antigens are selected from CDIO and CD19. In particular embodiments, the neoplasia is multiple myeloma, and the first and second tumor antigens are selected from CD56 and CD138. In various embodiments, the neoplasia is ovarian cancer and the first and second tumor antigens are different antigens selected from mesothelin, folate receptor-a, CD44, and CD133.

[0019] The invention relates to compositions and methods for targeting T cells to tumor cells. The compositions and articles defined by the invention were isolated or otherwise manufactured in accordance with the examples provided below. Other features and advantages of the invention are apparent from the detailed description and from the claims.

Brief description of the figures

[0020] Fig. 1A-C are graphs depicting vector design and expression by transduction of primary human T cells for a chimeric antigen receptor (CAR) and a chimeric costimulatory receptor (CCR). ( A ) depicts the production of CARs by fusing the heavy and light chains of the immunoglobulin variable domains to the transmembrane domain of CD8, which is fused to the cytosolic signaling domains of CD3. By utilizing an internal ribosome entry site (IRES) to provide bicistronic expression, CAR expression can be readily detected by correlation with dsRED fluorescence (data not shown). CCRs were generated by fusing scFvs to the transmembrane and signaling domain of CD28 ¹⁵ fused to the cytosolic signaling domain of 4-1BB (also known as CD137) ²¹ . CCR expression can be correlated with bicistronic hrGFP expression (data not shown). Abbreviations: LTR, Long terminal repeat; SD, Splice donor site; SA, Splice acceptor site; VH or VL, Heavy or light chain variable domains, respectively; EC, Extracellular domain; TM, Transmembrane domain; C, Cytosolic domain; IRES, Internal ribosome binding site; dsRED, Discosoma sp. red fluorescent protein, hrGFP, Human recombinant green fluorescent protein. ( B ) Representative transduction efficiencies of primary human T cells using these retroviral vectors are depicted. ( C ) Transduction of CTLs by different and multiple CARs for the present studies is depicted.

[0021] Figures 2A-D show that dual receptor, CAR/CCR, mediated activation of human T cells provides active CTL function, long-term proliferation, and enhanced tumor killing upon binding of two antigens. ( A ) shows that T cells expressing the chimeric receptors lysed antigen-positive cells when a CD19-specific CAR was expressed by the T cells in CTL assays, compared to untransduced or P28BB-transduced T cells. Graphs are representative of n>4 experiments, where error bars represent the standard deviation from the mean of 3 replicates. ( B ) Long-term T-cell proliferation as measured by absolute T-cell counts after 31 days is shown for T cells not expressing, expressing one or both chimeric receptors, cocultured with human tumor cell lines expressing both antigens or either antigen alone. Arrows indicate restimulation of T cells using freshly irradiated tumor cells. Only when dual receptor-expressing T cells encounter both antigens is robust long-term proliferation observed. Graphs are representative of n>4 experiments, with error bars representing the standard deviation from the mean of 3 replicates. ( C ) shows the efficiency of systemic tumor killing by tumor-sensitive T cells (TTS) assessed by intravenous (IV) infusion of 1.0 x ¹⁰⁶ T cells into NSG mice bearing luciferase-expressing CD19 ⁺ PSMA ⁺ human PC3 prostate tumors. Tumor burden was quantified weekly using BLI. Images of two representative mice from each group are shown, with tumor luminescence pixel intensity represented by color. Mean tumor burden is plotted with error bars representing the standard deviation from the mean of 6 mice per group. ( D ) shows selective killing of DP tumors using a triple-tumor mouse model by subcutaneous injection of 1 x ¹⁰⁶ cells of each PC3 tumor, CD19-only positive cells into the left flanks, PSMA-only positive cells into the right flanks, and both CD19 and PSMA-positive cells into the backs of the mice. T cells expressing either 19z1, P28BB, or both 19z1 + P28BB of the chimeric receptors were infused intravenously 7 days after tumor infusion. Representative images of 2 mice per group bearing these tumors are shown, with tumor fluorescence represented by color. Tumors were quantified using calipers, and tumor volumes were plotted against time for each tumor. Error bars represent standard deviations from the mean of 6 mice. Statistical significance was determined using two-tailed independent samples t-tests to compare values obtained for 19z1 T cells and 19z1+P28BB T cells, and p- values are presented as * for <0.05 or ** for <0.01.

[0022] Figures 3A-E show that tumor-sensitive T cells (TTS) selectively killed human prostate tumors when targeting two prostate tumor antigens. ( A ) shows the evaluation of three different PSCA-specific scFvs for their assembly into bispecific antibodies that also have specificity for CD3. T cells were co-cultured at a 20:1 ratio with PSCA ⁺ PC3 tumor cells, and antibodies were added at different amounts and specific lysis was measured. ( B ) shows the preparation of CARs using anti-PSCA scFvs that exhibited different potencies in cytotoxicity assays. CAR-mediated specific lysis of PSCA-expressing target cells confirmed the reduced efficacy of the Lz1 scFv by requiring a 50-fold higher effector:target ratio to achieve the same level of lysis for either Hz1 or Mz1. ( C ) and ( D ) show how selective killing of systemic prostate tumors expressing PSCA and PSMA was examined using these ineffective scFvs. Tumors ( Fig. 5 ) engrafted and were treated as described in Fig. 2. After 14 days, 1.0× ¹⁰⁶ chimeric receptor T cells positive for Mz1+P28BB ( Fig. 3C ) or Lz1+P28BB ( Fig. 3D ) were administered by intravenous infusion. Shown are images of two representative mice from each group, with tumor luminescence indicated by color (blue = 5 × 10 ⁵ to red = 2 × 10 ⁷ photons). Mean tumor burden was quantified by luminescence and plotted with error bars representing the standard deviation from the mean of 5 mice per group. Two PSMA tumor-injected mice (green line) died after day 49, and thus the mean luminescence value is an average of 3 values for days 56 and 63. Fig. 3E Selective antitumor responses to PSCA ⁺ PSMA ⁺ tumors alone were achieved by Lz1+ P28BB T cells in mice also bearing ^PSCA− PSMA ⁺ and PSCA ^{+ PSMA−} tumors, similar to Fig. 2D . Statistical significance was determined using two-tailed independent samples t-tests to compare values obtained for Lz1 T cells and Lz1+P28BB T cells, and p values are presented as * for <0.05 or ** for <0.01.

[0023] Figures 4A-D show that enhanced cytokine secretion and BclxL expression was detected for TTS cells when co-cultured on DP tumors. ( A ) shows a multiplex cytokine assay for untransduced T cells or T cells transduced with 19z1, P28BB, or both 48 hours after stimulation with the primary antigen using either untransduced PC3 cells (Blank) or CD19 ⁺ PSMA ⁺ PC3 cells. Error bars represent the standard deviation from the mean of 2 biological replicates. ( BD ) shows a multiplex cytokine assay for untransduced T cells or T cells transduced with Hz] (B), Mz1 (C), and Lz1 (D) anti-PSCA CAR, P28BB CCR, or both CAR + CCRs shown 48 hours after stimulation with a second antigen using either empty cells or PSCA + PSMA + PC3 cells. ( E ) shows a Western blot assay for Bc1xL performed using cell lysates from untransduced T cells or T cells transduced with 19z1, P28BB, or both 24 hours after initial antigen stimulation. Total Akt was used as a loading control.

[0024] Figure 5 shows the preparation of tumor cells for expression of GFP-firefly luciferase (GFP/Luc) fusion protein and tumor antigens. Untransduced PC3 cells (empty) were transduced with GFP/Luc and either CD19, PSMA, PSCA, or a combination of the two antigens using retroviral expression constructs. Cells were purified by double FACS purification for GFP/Luc, CD19, PSMA, and/or PSCA.

[0025] Fig. 6A-C illustrate the concept of tumor-responsive T cells. In ( A ) it is shown that TTS cells expressing an effective CAR become strongly stimulated by A ⁺ 113 ⁺ cells to promote an immune response against A ⁺ cells. CAR ⁺ CCR ⁺ cells can bind tumor antigen-bearing A ⁺ cells with CAR providing CD3 activation signals. This can lead to transient cell lysis. CAR ⁺ CCR ⁺ cells can bind tumor antigen-bearing B ⁺ cells with CCR providing CD28 and CD137 signals. This signal alone is not sufficient to induce lysis or proliferation. Only when CAR ⁺ CCR ⁺ cells bind tumor antigen-bearing A ⁺ B ⁺ cells with CAR and CCR can both activation and stimulation be achieved. This results in potent T cell lysis, proliferation, enhanced cytokine secretion, upregulation of BcIxL, and the ability to selectively kill tumors in vivo . However, depending on CAR efficiency, these CAR ⁺ CCR ⁺ cells can potentially be recycled to kill cells positive for only one CAR-specific antigen. Figure 6B shows that by reducing CAR efficiency, TTs can be reactivated by binding CCR upon encountering A ⁺ B ⁺ cells to selectively respond to and kill A ⁺ 13 ⁺ cells while avoiding a response to A+ cells. Figure ( C ) shows that by co-expressing one CAR providing a TCR activation signal upon binding to a tumor antigen and a second CAR providing stimulation signals upon binding to a different tumor antigen, T cells can only kill tumors expressing both antigens, but not tumors expressing either antigen alone.

Detailed description of the invention

[0026] The contents of all patents, published applications and other references mentioned in this application are herein incorporated by reference in this specification.

[0027] 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 pertains. The following references provide one of ordinary skill in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below unless otherwise defined.

[0028] By "activates an immunoresponsive cell" is meant the induction of signaling or changes in protein expression in a cell that lead to the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and immunoreceptor tyrosine-binding inhibitory motifs (ITAMs), a signaling cascade is formed. In particular embodiments, when an endogenous TCR or exogenous CAR binds an antigen, an immunological synapse is formed that induces the clustering of multiple molecules near the bound receptor (e.g., CD4 or CD8, CD3///, etc.). This clustering of membrane-bound signaling molecules allows the ITAM motifs contained within the CD3 chains to become phosphorylated. This phosphorylation, in turn, initiates the T cell activation pathway, ultimately activating transcription factors such as NF-KB and AP-1. These transcription factors induce global expression of T cell genes to increase IL-2 production for proliferation and expression of T cell master regulatory proteins to initiate a T cell-mediated immune response. By "stimulates an immunoresponsive cell" is meant a signal that results in a strong and persistent immune response. In various embodiments, this occurs following activation of the immune cell (e.g., T cell) or in parallel mediated through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, and ICOS. Without being bound by a particular theory, receiving multiple stimulatory signals is important for establishing a strong and persistent T cell-mediated immune response. Without receiving these stimulatory signals, T cells quickly become inhibited and unresponsive to the antigen. While the effects of these costimulatory signals are variable and remain partially understood, they typically result in increased gene expression to produce long-lived, proliferative, and anti-apoptotic T cells that actively respond to antigen for complete and permanent destruction.

[0029] The term "antigen recognition receptor," as used herein, refers to a receptor that is capable of activating an immune cell (e.g., a T cell) in response to binding of an antigen. Exemplary antigen recognition receptors can be native or endogenous T cell receptors or chimeric antigen receptors in which a tumor antigen binding domain is fused to an intracellular signaling domain capable of activating an immune cell (e.g., a T cell). In various embodiments, an antigen recognition receptor is selected that has low or minimal affinity or avidity for the antigen.

[0030] By "affinity" is meant a measure of the strength of binding between an antibody and a simple hapten or antigenic determinant. Without being bound by theory, affinity depends on the closeness of the stereochemical match between the binding sites of the antibody and the antigenic determinants, on the size of the area of contact between them, and on the distribution of charged and hydrophobic groups. Affinity also includes the term "avidity", which refers to the strength of the antigen-antibody bond after formation of reversible complexes. Methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of binding experiments to calculate affinity. In the case of binding of an antibody (Ab) to an antigen (Ag), the affinity constant (expressed as the inverse of the dissociation constant) is used.

The chemical equilibrium of antibody binding is also the ratio of the binding rate constants (k forward) and the dissociation rate constants (k reverse). Two antibodies may have the same affinity, but one may have both high binding and dissociation rate constants, while the other may have both low binding and dissociation rate constants.

Antibody activity in functional assays (e.g., cell lysis assay) also reflects the affinity of the antibody. In various embodiments, the receptor that recognizes the antigen has low affinity. Low affinity includes micromolar and nanomolar affinities (e.g., 10 ^-5 , 50 ^-6 , 10 ^-6 , 5×10 ^-7 , 10 ^-7 , 5×10 ^-8 , 10 ^-8 , 5×10 ^-9 , 10 ^-9 M). The antibody and affinities can be phenotypically characterized and compared using a functional assay (e.g., cell lysis assay).

[0031] By "affinity" is meant a measure of the strength of binding between an antibody and a sample. The term "chimeric costimulatory receptor" (CCR), as used herein, refers to a specific type of chimeric antigen receptor (CAR) that mediates costimulation independent of activation. When expressed on immunoresponsive cells in combination with an antigen-recognizing receptor (e.g., a CAR or TCR that activates the cell), the CCR targets a second antigen. In particular embodiments, the CCR has a medium or high affinity for its target antigen.

[0032] The term "chimeric antigen receptor" (CAR), as used herein, refers to a tumor antigen binding domain fused to an intracellular signaling domain capable of activating or stimulating T cells. Most commonly, the extracellular binding domain of a CAR consists of a single-chain variable fragment (scFv) derived from a fusion of the variable heavy and light regions of a murine or humanized monoclonal antibody. Alternatively, Fab-derived scFvs (instead of antibody-derived, such as those derived from Fab libraries) can be used. In various embodiments, these scFvs are fused to a transmembrane domain and then to an intracellular signaling domain. "First generation" CARs include CARs that exclusively provide CD3 signals upon antigen binding, "second generation" CARs include CARs that provide both costimulation (e.g., CD28 or CD137) and activation (CD3). "Third generation" CARs include CARs that provide multiple costimulation (e.g., CD28 and CD137) and activation (CD3). In CAR applications to date, CARs having high affinity or avidity for an antigen are selected, which is separate and distinct from the invention described herein.

[0033] By "CD3 polypeptide" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to NCBI Reference No. NP_932170, or a fragment thereof having activating or stimulatory activity. An exemplary CD3 is provided in Table 1 below. By "CD3 nucleic acid molecule" is meant a polynucleotide encoding a CD3 polypeptide.

[0034] By "CD8 polypeptide" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to NCBI Reference No. NP_001759 or a fragment thereof having stimulatory activity. An exemplary CD8 is provided in Table 1 below. By "CD8 nucleic acid molecule" is meant a polynucleotide encoding a CD8 polypeptide.

[0035] By "CD28 polypeptide" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to NCBI Reference No. NP_006130, or a fragment thereof having stimulatory activity. An exemplary CD28 is provided in Table 1 below. By "CD28 nucleic acid molecule" is meant a polynucleotide encoding a CD28 polypeptide.

[0036] By "4-1BB polypeptide" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to NCBI Reference No. P41273 or NP_001552, or a fragment thereof, that acts as a tumor necrosis factor (TNF) ligand. An exemplary 4-1BB is provided in Table 1 below. By "4-1BBL nucleic acid molecule" is meant a polynucleotide encoding a 4-1 BBL polypeptide.

[0037] By "CD80 polypeptide" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to NCBI Reference No: NP_005182, or a fragment thereof, that functions as an Ig superfamily ligand. An exemplary CD80 polypeptide is provided in Table 1 below.

[0038] By "CD80 nucleic acid molecule" is meant any polynucleotide encoding a CD80 polypeptide. An exemplary CD80 nucleic acid molecule is NM_005191.

[0039] By "OX4OL polypeptide" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI Reference No. BAB18304 or NP_003317 or a fragment thereof, which is a ligand for tumor necrosis factor (TNF). By "OX40L nucleic acid molecule" is meant a polynucleotide encoding an OX4OL polypeptide.

[0040] By "19z1 polypeptide" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the sequence shown below and having activating activity when binding to CD19.

[0041] By "P28z polypeptide" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the sequence shown below.

[0042] By "CD19" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the sequence shown below and capable of binding CD19.

[0043] By "PSMA" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the sequence shown below and capable of binding PSMA.

[0044] By "P28BB" is meant a protein having at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the sequence shown below and having activating activity when bound to PSMA.

Table 1 SEQ ID NO. Name 1 CD3ζ mkwkalftaa ilqaqlpite aqsfglldpk lcylldgilf iygviltalf lrvkfsrsad apayqqgqnq lynelnlgrr eeydvldkrr grdpemggkp qrrknpqegl ynelqkdkma eayseigmkg errrgkghdg lyqglsta tkdtydalhm
qalppr 2 CD8 malpvtalll plalllhaar psqfrvspld rtwnlgetve lkcqvllsnp tsgcswlfqp rgaaasptfl lylsqnkpka aegldtqrfs gkrlgdtfvl tlsdfrrene gyyfcsalsn simyfshfvp vflpakpttt paprpptpap tiasqplslr peacrpaagg avhtrgldfa cdiyiwapla gtcgvlllsl vitlycnhrn rrrvckcprp vvksgdkpsl saryv 3 CD28 mlrllllalnl fpsiqvtgnk ilvkqspmlv aydnavnlsc kysynlfsre fraslhkgld savevcvvyg nysqqlqvys ktgfncdgkl gnesvtfylq nlyvnqtdiy fckievmypp pyldneksng tiihvkgkhl cpsplfpgps kpfwvlvvvg gvlacysllv tvafiifwvr skrsrllhsd ymnmtprrpg ptrkhyqpya pprdfaayrs 4 4-1BB mgnscyniva tlllvlnfer trslqdpcsn cpagtfcdnn rnqicspcpp nsfssaggqr tcdicrqckg vfrtrkecss tsnaecdctp gfhclgagcs mceqdckqgq eltkkgckdc cfgtfndqkr gicrpwtncs ldgksvlvng tkerdvvcgp spadlspgas svtppapare pghspqiisf flaltstall fllffltlrf svvkrgrkkl lyifkqpfmr pvqttqeedg cscrfpeeee ggcel 5 CD80 mghtrrqgts pskcpylnff qllvlaglsh fcsgvihvtk evkevatlsc ghnvsveela qtriywqkek kmvltmmsgd mniwpeyknr tifditnnls ivilalrpsd egtyecvvlk yekdafkreh laevtlsvka dfptpsisdf eiptsnirri icstsggfpe phlswlenge elnainttvs qdpetelyav sskldfnmtt nhsfmcliky ghlrvnqtfn wnttkqehfp dnllpswait lisvngifvi ccltycfapr crerrrnerl rresvrpv 6 OX40L mervqpleen vgnaarprfe rnklllvasv iqglglllcf tyiclhfsal qvshrypriq sikvqfteyk kekgfiltsq kedeimkvqn nsviincdgf ylislkgyfs qevnislhyq kdeeplfqlk kvrsvnslmv asltykdkvy lnvttdntsl ddfhvnggel ilihqnpgef cvl 7 19z1 MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKAS GYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQA TLTADKSSSTAYMQLSGLTSEDSAVYECARKTISSWDFYFDY WGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGD RVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPD RFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTK LEIKRAAAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRVKFSR SAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR 8 P28z MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTF TEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSS STAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTVTVSSGGGGSGGG GSGGGGSDIVMTQSHKFMSTSVGDRVSIIКASQDVGTAVDWYQQКP GQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYF CQQYNSYPLTFGAGTMLDLKRAAAAIEVMYPPPYLDNEKSNGTIIHVK GKHLСPSPLFPPGPSКPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEW 9 CD19 IGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVY FCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIEL TQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSA TYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTS GGGTKLEIKR 10 PSMA MMALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYT FTEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKS SSTAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTVTVSSGGGGSGG GGSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQK PGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADY FCQQYNSYPLTFGAGTMLDLKR 11 P28BB MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTF TEYTIHWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSS STAYMELRSLTSEDSAVYYCAAGWNFDYWGQGTTVTVSSGGGGSGGG GSGGGGSDIVMTQSHKFMSTSVGDRVSIICKASQDVGTAVDWYQQKP GQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQSEDLADYF CQQYNSYPLTFGAGTMLDLKRAAAAIEVMYPPPYLDNEKSNGTIIHVK GKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKR SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRFSVVKRGRK KLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE

[0045] Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule encoding a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to the endogenous nucleic acid sequence, but typically have substantial identity. Polynucleotides having "substantial identity" to the endogenous sequence are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant to pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene as described herein), or portions thereof, under conditions of varying stringency. (See, for example, Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0046] For example, the stringent salt concentration is typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent, such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions typically include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Variation of additional parameters such as hybridization time, concentration of detergent, such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA are well known to those skilled in the art. Different levels of stringency are achieved by combining these different conditions as needed. In a preferred embodiment, hybridization occurs at 30°C in 750 mM NaCl, 75 mM trisodium citrate and 1% SDS. In a more preferred embodiment, hybridization occurs at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide and 1001.1 g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization occurs at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Suitable variations of these conditions will be readily apparent to those skilled in the art.

[0047] For most applications, the wash steps following hybridization can also be varied in stringency. The stringency conditions of the wash can be determined by salt concentration and by temperature. As above, the stringency of the wash can be increased by decreasing the salt concentration or by increasing the temperature. For example, the stringent salt concentration for the wash steps is preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps typically include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the wash steps occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash steps occur at 42 degrees C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In another embodiment, the wash steps occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be readily apparent to those skilled in the art.

[0048] Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmei (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0049] By "substantially identical" is meant a polypeptide or nucleic acid molecule that has at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0050] Sequence identity is typically measured using sequence analysis software (e.g., the sequence analysis software package from the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software aligns identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an illustrative method, the BLAST program can be used to determine the degree of identity, where a probability between e-3 and e-100 indicates a closely related sequence.

[0051] By "analog" is meant a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0052] The term "ligand" as used herein refers to a molecule that binds to a receptor. In particular, a ligand binds a receptor on another cell, allowing cell-to-cell recognition.

[0053] The term "constitutive expression" as used herein refers to expression under all physiological conditions.

[0054] A "disease" is any condition or disorder that damages a cell, tissue, or organ or interferes with its normal functioning. Examples of diseases include neoplasia or infection of a cell by a pathogen.

[0055] By "effective amount" is meant an amount sufficient to arrest, alleviate, or inhibit ongoing proliferation, growth, or metastasis (e.g., invasion or migration) of the neoplasia.

[0056] By "forced resistance" is meant the prevention of the activity of autoreactive cells or immunoreactive cells targeting transplanted organs or tissues.

[0057] By "exogenous" is meant a nucleic acid molecule or polypeptide that is not present endogenously in a cell, or is not present at a level sufficient to achieve the functional effects obtained by overexpression. The term "exogenous" thus encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and overexpressed nucleic acid and polypeptides.

[0058] By "heterologous nucleic acid molecule or polypeptide" is meant a nucleic acid molecule (e.g., a cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or a sample obtained from a cell. This nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in the cell or sample.

[0059] By "immunoreactive cell" is meant a cell that functions in the immune response, or its precursor or progeny.

[0060] By "isolated cell" is meant a cell that has been separated from the molecular and/or cellular components that naturally accompany the cell.

[0061] The terms "isolated," "purified," or "biologically pure" refer to a material that is free to varying degrees from components that normally accompany it as found in its natural state. "Isolation" means a degree of separation from the original source or environment. "Purification" means a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not significantly affect the biological properties of the protein or cause other adverse effects. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA methods, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" may mean that the nucleic acid or protein forms essentially a single band in an electrophoretic gel. For a protein that can be modified, such as by phosphorylation or glycosylation, different modifications may form different isolated proteins that can be purified separately.

[0062] The term "tumor antigen binding domain" as used herein refers to a domain capable of specifically binding a particular antigenic determinant or set of antigenic determinants present on a tumor.

[0063] By "modulate" is meant to change positively or negatively. Illustrative modulations include changes of 1%, 2%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0064] "Neoplasia" is a disease characterized by abnormal proliferation of cells or tissues and their subsequent migration to or invasion of other tissues or organs. The growth of neoplasia is usually uncontrolled and progressive, and occurs under conditions that do not cause proliferation or cause cessation of proliferation of normal cells. Neoplasias can involve a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph node, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, genitourinary tract, ureter, urethra, uterus, and vagina, or their tissue or cell type. Neoplasias include malignancies such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). Illustrative neoplasias for which the invention can be used include, but are not limited to, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's lymphoma), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, endometrial cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In one embodiment, the screening methods of the invention identify compositions useful for treating breast or lung cancer.

[0065] By "receptor" is meant a polypeptide or portion thereof present on a cell membrane that selectively binds one or more ligands.

[0066] By "recognizes" is meant to selectively bind to the target. A T cell that recognizes a virus typically expresses a receptor that binds the antigen expressed by the virus.

[0067] A "pathogen" is a virus, bacterium, fungus, parasite, or protozoan that can cause disease. Illustrative viruses include, but are not limited to, Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 (also referred to as HDTV-III, LAVE, or HTLV-III/LAV, or HIV-III; and other isolates such as HIV-LP); Picornaviridae (e.g., polioviruses, hepatitis A virus; enteroviruses, human coxsackieviruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue fever viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., Ebola viruses); Paramyxoviridae (eg, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (eg, influenza viruses); Bungaviridae (eg, Hantaan viruses, bungaviruses, phleboviruses, and nairoviruses); Arena Viridae (hemorrhagic fever viruses); Reoviridae (eg, reoviruses, orbiviruses, and rotaviruses); Birnaviridae ; Hepadnaviridae (hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papillomaviruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (smallpox viruses, vaccinia viruses, poxviruses); and Iridoviridae (eg, African swine fever virus); and unclassified viruses (eg, hepatitis delta agent (presumed to be a defective satellite of hepatitis B virus), non-A, non-B hepatitis agents (class 1 = internally transmitted; class 2 = parenterally transmitted (i.e., hepatitis C); Norwalk virus and related viruses, and astroviruses).

[0068] Illustrative bacteria include, but are not limited to, Pasteurella , Staphylococci, Streptococcus , Escherichia coli , Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include, but are not limited to, Helicobacter pylori , Borelia burgdoiferi , Legionella pneumophilia , Mycobacteria species (e.g., M. tuberculosis , M. avium , M. intracellulare , M. kansaii , M. gordonae ), Staphylococcus aureus , Neisseria gonorrhoeae , Neisseria meningitidis , Listeria monocytogenes , Streptococcus pyogenes (group A Streptococcus ), Streptococcus agalactiae (group B Streptococcus ), Streptococcus (viridans group), Streptococcus faecalis , Streptococcus bovis , Streptococcus (anaerobic species), Streptococcus pneumoniae , pathogenic Campylobacter sp. , Enterococcus sp , Haemophilus influenzae , Bacillus anthracis , corynebacterium diphtheriae , corynebacterium sp. , Erysipelothrix rhusiopathiae , Clostridium perfringers , Clostridium tetani , Enterobacter aerogenes , Klebsiella pneumoniae , Pasturella multocida , Bacteroides sp. , Fusobacterium nucleatum , Streptobacillus moniliformis , Treponema pallidium , Treponema pertenue , Leptospira , Rickettsia and Actinomyces israelii .

[0069] By "specifically binds" is meant a polypeptide or fragment thereof that recognizes and binds the polypeptide of interest, but that does not substantially recognize and bind other molecules in a sample, such as a biological sample, that naturally includes the polypeptide of the invention.

[0070] The term "tumor antigen" as used herein refers to any polypeptide expressed by a tumor that is capable of inducing an immune response.

[0071] By "viral antigen" is meant a polypeptide expressed by a virus that is capable of inducing an immune response.

[0072] The terms "comprises," "comprising," are intended to have the broad meaning ascribed to them by the U.S. Patent Law and may mean "includes," "including," etc.

[0073] As used herein, "treatment" refers to a clinical intervention in an attempt to alter the course of a disease in an individual or cell being treated, and may be performed either prophylactically or during the course of a clinical pathology. Therapeutic effects of treatment include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, reducing or alleviating the condition of the disease, and causing remission or improvement in prognosis. By preventing the progression of a disease or disorder, treatment may prevent deterioration due to the disorder in an affected or diagnosed subject or in a subject suspected of having the disorder, but treatment may also prevent the onset of a disorder or a symptom of a disorder in a subject at risk for the disorder or suspected of having the disorder.

[0074] The term "subject" as used herein refers to a vertebrate, preferably a mammal, more preferably a human.

[0075] The term "immunocompromised" as used herein refers to a subject suffering from an immunodeficiency. The subject is highly susceptible to opportunistic infections, infections caused by organisms that do not normally cause disease in a person with a healthy immune system, but can affect people with a poorly functioning or suppressed immune system.

[0076] Other aspects of the invention are described in the following description and are within the scope of the invention.

[0077] The present invention generally relates to cells, including genetically modified immunoresponsive cells (e.g., T cells, natural killer (NK) cells, cytotoxic T lymphocyte (CTL) cells) expressing at least a combination of an antigen-recognizing receptor (e.g., a TCR or CAR) and a chimeric costimulatory receptor (CCR), and to methods of using them thereby to treat neoplasia and other pathologies where an increase in an antigen-specific immune response is desired. The invention is based, at least in part, on the discovery that the simultaneous engagement of two antigens co-expressed by a tumor cell via an antigen-recognizing receptor and a chimeric costimulatory receptor is useful for activating and stimulating immunoresponsive cells without systemic effects. In particular, reactivity against tissues expressing either antigen alone is preferably minimal, including T cell activation in the presence of both antigens but not either antigen alone. T cell activation is mediated by a TCR or CAR targeting an antigen (e.g., CD19 or prostate stem cell antigen, PSCA). Costimulation is independently mediated by a “chimeric costimulatory receptor” (CCR) ^12,13 targeting a second antigen (e.g., prostate-specific membrane antigen, PSMA). This approach results in enhanced reactivity against dual antigen (DP)-positive tumors but fails to prevent enhanced reactivity against single antigen (SP)-positive tumors. It has been found that tumor-sensitive T cells can be induced to differentiate DP tumors from SP tumors by reducing T cell activation to a level where T cell activation is ineffective on its own, but is functionally restored at the tumor site by CCR recruited via an independent, co-expressed antigen. This approach provides immunogenicity within the tumor microenvironment for tumor killing while sparing SP cells that are normal or non-neoplastic, and represents a significant advance over conventional adoptive T cell therapies.

[0078] Moreover, this method is not limited to the treatment of neoplasias, but is suitable for a wide range of applications in which an increase in an antigen-specific immune response is desired, where such applications include not only the treatment of neoplasias, but also the enhancement of the immune response against infection with a pathogen or an infectious disease and the enhancement of immune resistance of regulatory T cells in the context of autoimmunity or allogeneic transplantation.

Hematopoietic cell lines

[0079] Hematopoietic (blood) cells provide a wide variety of physiological activities. Hematopoietic cells are divided into lymphoid, myeloid, and erythroid lineages. The lymphoid lineage, which contains B, T, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign antigens in the blood, detection of cells foreign to the host, etc. The term "T cells" as used herein refers to lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the acquired immune system. The term "natural killer (NK) cells" as used herein refers to lymphocytes that are part of cell-mediated immunity and act during the innate immune response. They do not require prior activation to exert their cytotoxic effect on target cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic cells or tumor cells.

Cells for use in the methods of the invention

[0080] The present invention relates to cells expressing a combination of an antigen-recognizing receptor that activates an immunoresponsive cell (e.g., a TCR, CAR) and a chimeric costimulatory receptor (CCR), and to methods of using such cells to treat a disease requiring an enhanced immune response. In one method, tumor antigen-specific T cells, NK cells, CTL cells, or other immunoresponsive cells are used as shuttles to selectively enrich for one or more costimulatory ligands to treat or prevent neoplasia. For example, a T cell expressing a chimeric antigen receptor 19z1 that recognizes CD19 co-expressed in a T cell expressing a chimeric costimulatory receptor P28BB that recognizes and binds prostate-specific membrane antigen (PSMA). Such cells are administered to a human subject in need thereof to treat or prevent prostate cancer. In another way, viral antigen-specific T cells, NK cells, CTL cells can be used to treat viral diseases. For example, a chimeric co-stimulatory antigen receptor that recognizes the first CMV antigen and a chimeric antigen receptor that recognizes and binds the second CMV antigen are co-expressed on cytotoxic T lymphocytes to treat CMV.

Tumor antigen-specific T lymphocytes (and NK cells)

[0081] Types of tumor antigen-specific human lymphocytes that can be used in the methods of the invention include, but are not limited to, peripheral donor lymphocytes genetically modified to express chimeric antigen receptors (CARs) (Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45), peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex containing a heterodimer of a and p (Morgan, RA, et al. 2006 Science 314:126-129), lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies (Panelli, MC, et al. 2000 J Immunol 164:495-504; Panelli, MC, et al. 2000 J Immunol 164:4382-4392), and selectively expanded in vitro antigen-specific peripheral blood leukocytes using artificial antigen-presenting cells (AAPCs) or sensitized dendritic cells (Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, GA, et al. 2003 Blood 102:2498-2505). T cells can be autologous, allogeneic, or derived in vitro from engineered progenitors or stem cells.

[0082] Any suitable tumor antigen (antigenic peptide) is suitable for use in the tumor-related embodiments described herein. Antigen sources include, but are not limited to, cancer proteins. The antigen can be expressed in the form of a peptide or in the form of an intact protein or portion thereof. The intact protein or portion thereof can be natural or mutagenized. Suitable antigens include prostate-specific membrane antigen (PSMA) and prostate stem cell antigen (PCSA).

Viral antigen-specific T lymphocytes (and NK cells)

[0083] Suitable antigens for use in treating an infection with a pathogen or other infectious disease, such as in an immunocompromised subject, include, but are not limited to, viral antigens present in cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus.

[0084] The crude CTL source may be any source known in the art, such as bone marrow, a fetal, neonatal or adult source, or other hematopoietic cells, such as fetal liver, peripheral blood or cord blood. Various methods can be used to separate the cells. For example, negative selection methods can first remove non-CTL cells. mAbs are particularly useful for identifying markers associated with specific cell lines and/or differentiation stages for both positive and negative selections.

[0085] A large percentage of terminally differentiated cells can be initially removed by relatively coarse separation. For example, magnetic bead separations can be used initially to remove large amounts of irrelevant cells. Preferably, at least about 80%, typically at least 70%, of the total hematopoietic cells are removed before cell isolation.

[0086] Separation methods include, but are not limited to, density gradient centrifugation; rosetting; attachment to cell density modifying particles; magnetic separation using antibody-coated magnetic beads; affinity chromatography; cytotoxic agents attached to or used in combination with mAbs, including, but not limited to, complement and cytotoxins; and panning using antibody attached to a solid matrix, such as a plate, chip, settling, or any other convenient method.

[0087] Separation and analysis methods include, but are not limited to, flow cytometry, which may have varying degrees of sophistication, such as multiple color channels, small angle channels, and subdued light scatter, resistance detection.

[0088] Cells can be selected from dead cells using dyes that bind to dead cells, such as propidium iodide (PI). Preferably, the cells are collected in a medium containing 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, preferably sterile, isotonic medium.

[0089] Accordingly, the invention generally relates to an immunoresponsive cell, such as a virus-specific or tumor-specific T cell, comprising a receptor that binds a first antigen and activates the immunoresponsive cell and a receptor that binds a second antigen and stimulates the immunoresponsive cell.

Vectors

[0090] Genetic modification of immunoresponsive cells (e.g., T cells, CTL cells, NK cells) can be accomplished by transducing a substantially homogeneous composition of cells with a recombinant DNA construct. Preferably, a retroviral vector (either gamma-retroviral or lentiviral) is used to introduce the DNA construct into the cell. For example, a polynucleotide encoding a receptor that binds an antigen (e.g., a tumor antigen, or a variant or fragment thereof) can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from a retroviral long terminal repeat, or from a promoter specific for the target cell type of interest. Non-viral vectors can also be used.

[0091] For the initial genetic modification of cells to produce tumor- or viral antigen-specific cells, a retroviral vector is typically used for transduction, but any other suitable viral vector or non-viral delivery system can be used. For subsequent genetic modification of cells to produce cells containing an antigen-presenting complex containing at least two costimulatory ligands, retroviral gene transfer (transduction) has similarly proven to be effective. Combinations of a retroviral vector and a suitable packaging line are also useful, wherein the capsid proteins are rendered functional for infection of human cells. Various cell lines that produce amphotropic viruses are known, including, but not limited to, PA12 (Miller, et al . (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are also suitable, such as particles pseudotyped with the VSVG, RD114, or GALV envelope, and any others known in the art.

[0092] Possible transduction methods also include direct co-cultivation of cells with producer cells, such as by Bregni, et al. (1992) Blood 80:1418-1422, or culturing with viral supernatant alone or concentrated vector preparations from storage in the presence or absence of appropriate growth factors and polycations, such as by Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0093] Other transducing viral vectors can be used to express the costimulatory ligand of the invention in an immunoresponsive cell. Preferably, the selected vector exhibits high infection efficiency and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated viral vectors, vaccinia virus, bovine papillomavirus, or a herpes virus such as Epstein-Barr virus (see also, for example, the vectors from Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409,1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0094] Non-viral methods can also be used to express protein in a cell. For example, a nucleic acid molecule can be introduced into a cell by nucleic acid injection in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465,1990).

[0095] Other non-viral methods of gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also potentially provide advantages for delivering DNA into a cell. Transplantation of normal genes into diseased tissues of a subject can also be accomplished by transferring normal nucleic acid into a culturable cell type ex vivo (e.g., autologous or heterologous primary cells or their progeny), after which the cell (or its progeny) is injected into the target tissue or injected systemically. Recombinant receptors can also be engineered or produced using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases). Transient expression can be achieved by electroporation of RNA. Expression of cDNA for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters) and can be regulated through any suitable mammalian regulatory element or intron (e.g., elongation factor 1c enhancer/promoter/intron structures). For example, if desired, enhancers known to preferentially direct gene expression in particular cell types can be used to direct nucleic acid expression. Enhancers can include, but are not limited to, enhancers characterized as tissue-specific or cell-specific enhancers. Alternatively, if the genomic clone is used as a therapeutic construct, regulation can be mediated by related regulatory sequences or, if desired, regulatory sequences obtained from a heterologous source, including any of the promoters or regulatory elements described above.

[0096] The resulting cells can then be grown under conditions similar to those of unmodified cells, whereby the modified cells can be propagated and used for a variety of purposes.

[0097] The invention also relates to polypeptides 19z1, CD19, CD8, CD3, dsRed, P28BB, PSMA, CD28, 4-1BB, GFP or fragments thereof modified by methods that enhance their antineoplastic activity when expressed in an immunoresponsive cell. The invention relates to methods for optimizing an amino acid sequence or a nucleic acid sequence by producing changes in the sequence. Such changes may include specific mutations, deletions, insertions, or post-translational modifications. The invention further relates to analogs of any naturally occurring polypeptide of the invention. Analogs may differ from a naturally occurring polypeptide of the invention by differences in amino acid sequence, by post-translational modifications, or by both. Analogues of the invention typically have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to all or part of a naturally occurring amino acid sequence of the invention. The length of the sequence comparison is at least 5, 10, 15 or 20 amino acid residues, preferably at least 25, 50 or 75 amino acid residues, and more preferably greater than 100 amino acid residues. Again, as an illustrative method for determining the degree of identity, the BLAST program can be used, with a probability value between e ³ and e ^-100 indicating a closely related sequence. Modifications include chemical derivatization of polypeptides in vivo and in vitro , such as acetylation, carboxylation, phosphorylation or glycosylation; such modifications may occur during the synthesis or processing of the polypeptides or subsequent treatment with isolated modifying enzymes. Analogs may also differ from the naturally occurring polypeptides of the invention by changes in the primary sequence. They include genetic variants, both natural and induced (e.g., arising by random mutagenesis by irradiation or exposure to ethane methyl sulfate, or by site-directed mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules and analogs containing residues other than L-amino acids, e.g., D-amino acids, or unnatural or synthetic amino acids, e.g., - or - amino acids.

[0098] In addition to full-length polypeptides, the invention also provides fragments of any of the domains of the polypeptides or peptides of the invention. As used herein, the term "fragment" means at least 5, 10, 13, or 15 amino acids. In other embodiments, the fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments at least 60-80, 100, 200, 300, or more contiguous amino acids. Fragments of the invention can be produced by methods known to those skilled in the art, or can be produced by normal protein processing (e.g., removal of amino acids not required for biological activity from a nascent polypeptide, or removal of amino acids through alternative mRNA splicing events or alternative protein processing).

[0099] Non-protein analogs have a chemical structure designed to mimic the functional activity of a protein of the invention. Such analogs are administered by the methods of the invention. Such analogs can exceed the physiological activity of the parent polypeptide. Methods for designing analogs are well known in the art, and the synthesis of analogs can be carried out in accordance with such methods by modifying the chemical structures such that the resulting analogs increase the anti-neoplastic activity of the parent polypeptide when expressed in an immunoresponsive cell. These chemical modifications include, but are not limited to, substitution of alternative R groups and alteration of the degree of saturation of carbon atoms of the reference polypeptide. Preferably, the protein analogs are relatively resistant to degradation in vivo , resulting in a more prolonged therapeutic effect after administration. Assays for measuring functional activity include, but are not limited to, the assays described in the examples below.

Costimulatory ligands

[00100] Interaction with at least one costimulatory ligand provides a non-antigen-specific signal that is important for the full activation of immune cells (e.g., T cells). Costimulatory ligands include, but are not limited to, tumor necrosis factor (TNF) ligands, cytokines (such as IL-2, IL-12, IL-15, or IL21), and immunoglobulin (Ig) superfamily ligands.

[00101] Tumor necrosis factor (TNF) is a cytokine involved in systemic inflammation and stimulates the acute phase response. Its primary role is the regulation of immunocytes. Tumor necrosis factor (TNF) ligands share a number of common features. Most ligands are synthesized as type II transmembrane proteins (with an extracellular C-terminus) containing a short cytoplasmic fragment and a relatively long extracellular region. TNF ligands include, but are not limited to, nerve growth factor (NGF), CD4OL (CD4OL)/CD154, CD137L/4-1BBL, tumor necrosis factor alpha (TNFa), CD134L/OX4OL/CD252, CD27L/CD70, Fas ligand (FasL), CD3OL/CD153, tumor necrosis factor beta (TNF(3)/lymphotoxin-alpha (LTa), lymphotoxin-beta (ur(3), CD257/B-cell activating factor (BAFF)/Blys/THANK/Ta11-1, glucocorticoid-inducible TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface proteins and soluble proteins that are involved in cell recognition, binding, or adhesion processes. These proteins share structural properties with immunoglobulins - they possess an immunoglobulin domain (folded). Ligands of the immunoglobulin superfamily include, but are not limited to, CD80 and CD86, both ligands for CD28.

[00102] Compositions comprising genetically modified immunoresponsive cells of the invention (e.g., T cells, NK cells, CTL cells, or precursors thereof) can be administered systemically or directly to a subject to treat a neoplasia, infection with a pathogen, or an infectious disease. In one embodiment, the cells of the invention are directly administered to an organ of interest (e.g., an organ affected by a neoplasia). Alternatively, compositions comprising genetically modified immunoresponsive cells are administered indirectly to an organ of interest, such as by administration into the circulatory system (e.g., tumor vasculature). Expansion and differentiation agents can be provided before, during, or after administration of the cells to enhance the production of T cells, NK cells, or CTL cells in vitro or in vivo .

[00103] The modified cells can be administered in any physiologically acceptable carrier, typically intravascularly, although they can also be administered into bone or other convenient site where the cells can find a suitable site for regeneration and differentiation (e.g., the thymus). Typically, at least 1 x 10 ⁵ cells are administered, eventually reaching 1 x 10 ¹⁰ or more. The genetically modified immunoresponsive cells of the invention can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of genetically modified immunoresponsive cells in a population using various well-known methods, such as fluorescence-activated cell sorting (FACS). Preferred ranges of purity in populations containing genetically modified immunoresponsive cells are from about 50 to about 55%, from about 55 to about 60%, and from about 65 to about 70%. More preferably, the purity is from about 70 to about 75%, from about 75 to about 80%, from about 80 to about 85%; and even more preferably, the purity is from about 85 to about 90%, from about 90 to about 95%, and from about 95 to about 100%. Those skilled in the art can easily adjust the doses (e.g., a decrease in purity may require an increase in the dose). The cells can be administered by injection, catheter, or the like. If desired, factors can also be included, including, but not limited to, interleukins, such as IL-2, IL-3, IL-6, and IL-11, as well as other interleukins, colony stimulating factors such as G-, M-, and GM-CSF, interferons such as gamma interferon, and erythropoietin.

[00104] Compositions of the invention include pharmaceutical compositions comprising genetically modified immunoresponsive cells or progenitors thereof and a pharmaceutically acceptable carrier. Administration may be autologous or heterologous. For example, immunoresponsive cells or progenitors may be obtained from one subject and administered to the same subject or to another, compatible subject. The peripheral blood-derived immunoresponsive cells of the invention or their progeny (e.g., obtained in vivo , ex vivo , or in vitro ) may be administered by localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present invention (e.g., a pharmaceutical composition comprising genetically modified immunoresponsive cells), it is typically formulated in a unit dose form (solution, suspension, emulsion) suitable for injection.

Compositions

[00105] Compositions of the invention containing genetically modified immunoresponsive cells can be conveniently provided in the form of sterile liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions that can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat more convenient for administration, especially by injection. Viscous compositions, on the other hand, can be formulated within a suitable viscosity range to provide longer periods of contact with particular tissues. Liquid or viscous compositions can contain carriers, which can be a solvent or dispersion medium, for example, water, saline, phosphate buffered saline, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

[00106] Sterile injectable solutions can be prepared by incorporating the genetically modified immunoresponsive cells used in the practice of the present invention in the required amount in a suitable solvent with various amounts of other ingredients as desired. Such compositions can be present in admixture with a suitable carrier, diluent or excipient such as sterile water, physiological saline, glucose, dextrose or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-increasing additives, preservatives, flavoring agents, coloring agents and the like, depending on the desired route of administration and preparation. Standard texts such as REMINGTON'S PHARMACEUTICAL SCIENCE, 17th edition, 1985, the contents of which are incorporated herein by reference, may be consulted to obtain suitable preparations without undue experimentation.

[00107] Various additives can be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Extended absorption of the injectable pharmaceutical form can be achieved by means of adsorption delaying agents, for example, aluminum monostearate and gelatin. According to the present invention, however, any carrier, diluent or additive used must be compatible with the genetically modified immunoreactive cells or their progeny.

[00108] The compositions may be isotonic, i.e., they may have the same osmotic pressure as blood and tears. The desired isotonicity of the compositions of this invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is preferred, especially for buffers containing sodium ions.

[00109] The viscosity of the compositions, if desired, can be maintained at a selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and economically available and is easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The preferred concentration of the thickening agent depends on the agent selected. The important point is to use an amount that can achieve the selected viscosity. It is understood that the choice of suitable carriers and other additives may depend on the precise route of administration and the nature of the particular dosage form, for example, a liquid dosage form (e.g., whether the composition is intended to be formulated in a solution, suspension, gel, or other liquid form such as a sustained release form or a liquid-filled form).

[00110] Those skilled in the art will appreciate that the components of the compositions should be selected to be chemically inert and not to interfere with the viability or efficacy of the genetically modified immunoresponsive cells as described herein. This is not a problem for those skilled in the art of chemical and pharmaceutical principles, or the problems can be readily avoided by reference to standard texts or by simple experimentation (not involving undue experimentation) from this disclosure and the documents cited herein. One consideration regarding the therapeutic use of the genetically modified immunoresponsive cells of the invention is the number of cells required to achieve an optimal effect. The number of cells to be administered may vary for the subject to be treated. In one embodiment, between 10 ⁴ and 10 ¹⁰ , between 10 ⁵ and 10 ⁹ , or between 10 ⁶ and 10 ⁸ genetically modified immunoresponsive cells of the invention are administered to a human subject. More effective cells can be administered in even smaller amounts. In some embodiments, at least about 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ and 5×10 ⁸ of the genetically modified immunoresponsive cells of the invention are administered to a human subject. The precise determination of what can be considered an effective dose can be based on factors individual to each subject, including the height, age, sex, weight and condition of the particular subject. Those skilled in the art can readily determine doses from this disclosure and knowledge in the art.

[00111] One skilled in the art can readily determine the amount of cells and optional additives, supports and/or carriers in the compositions and for administration by the methods of the invention. Typically, any additives (in addition to the active cell(s) and/or agent(s)) are present in an amount of 0.001-50% (w/w) of a solution in phosphate-buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as from about 0.0001 to about 5% by weight, preferably from about 0.0001 to about 1% by weight, even more preferably from about 0.0001 to about 0.05% by weight or from about 0.001 to about 20% by weight, preferably from about 0.01 to about 10% by weight, and even more preferably from about 0.05 to about 5% by weight. Of course, for any composition to be administered to an animal or a human, and for any particular route of administration, it is preferable to determine in this way: toxicity, for example by determining the lethal dose (LD) and LD50 in a suitable animal model, for example rodents such as mice; and the dose of the composition(s), the concentration of the components therein, and the timing of administration of the composition(s) that elicit a suitable response. Such determinations do not require undue experimentation, due to the knowledge of the person skilled in the art, this disclosure, and the documents cited herein. And the timing of subsequent administrations can be determined without undue experimentation.

Treatment methods

[00112] Provided herein are methods for treating a neoplasia in a subject. Also provided herein are methods for treating an infection with a pathogen or another infectious disease in a subject, such as a human subject with an immune deficiency. The methods include administering T cells, NK cells, or CTL cells of the invention in an amount effective to achieve a desired effect, whether alleviating an existing condition or preventing a relapse. For treatment, the amount administered is an amount effective to achieve the desired effect. The effective amount can be provided in a single administration or in a series of administrations. The effective amount can be provided in a bolus or by continuous perfusion.

[00113] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to produce the effect of an advantageous or desired clinical result in a treatment. An effective amount may be administered to a subject in one or more doses. With respect to treatment, an effective amount is an amount sufficient to attenuate, alleviate, stabilize, reverse, or slow the progression of a disease, or otherwise reduce the pathological consequences of a disease. An effective amount is typically determined by a physician on a case-by-case basis and is within the skill of the art. Several factors are typically taken into account in determining an appropriate dose to achieve an effective amount. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the form and effective concentration of the antigen-binding fragment being administered.

[00114] For adoptive immunotherapy using antigen-specific T cells, doses of cells in the range of 10 ⁶ -10 ¹⁰ (e.g., 10 ⁹ ) are typically infused. By introducing genetically modified cells into the host and allowing them to differentiate, T cells are induced that are specifically directed against the specific antigen. "Induction" of T cells may include inactivation of antigen-specific T cells, such as by deletion or anergy. Inactivation is particularly useful for the development or restoration of resistance, such as in autoimmune disorders. The modified cells may be administered by any route known in the art, including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal, and directly into the thymus.

[00115] The invention relates to methods for increasing the immune response in a subject in need thereof. In one embodiment, the invention relates to methods for treating or preventing neoplasia in a subject. The invention relates to therapies that are particularly useful for treating subjects suffering from prostate cancer or metastatic prostate cancer that are refractory to conventional therapeutic interventions. Suitable human subjects for therapy typically comprise two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are subjects bearing a clinically measurable tumor. A clinically measurable tumor is a tumor that can be detected based on tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram, or X-ray; positive biochemical or histopathological markers alone are insufficient to identify this population). The pharmaceutical composition encompassed by this invention is administered to these subjects to induce an antitumor response, with the aim of alleviating their condition. Ideally, the result is a reduction in tumor mass, but any clinical improvement is an advantage. Clinical improvement includes a reduced risk or rate of progression or a reduction in the pathological consequences of the tumor.

[00116] The second group of suitable subjects is known in the art as the "adjuvant group." These are individuals who have a history of neoplasia but who have responded to another form of therapy. Previous therapy may include, but is not limited to, surgical resection, radiotherapy, and conventional chemotherapy. As a result, these individuals do not have clinically measurable tumor. However, they are suspected of being at risk for disease progression, either near the original tumor site or due to metastasis. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial therapy. These features are known in the clinical art and are appropriately defined for each different neoplasia. Features typical of the high-risk subgroups are features that the tumor has invaded adjacent tissues or that show lymph node involvement.

[00117] Another group has a genetic predisposition to neoplasia, but does not yet have proven clinical evidence of neoplasia. For example, women who test positive for a genetic mutation associated with breast cancer, but are still of childbearing age, may wish to receive one or more of the antigen-binding fragments described herein in prophylactic treatment to prevent the occurrence of neoplasia until preventive surgery is appropriate.

[00118] Human subjects with neoplasia, suffering from any of the following neoplasias: glioblastoma, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate cancer and small cell lung cancer), are particularly suitable subjects. Suitable carcinomas further include any known in the field of oncology, including, but not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small and large cell adenocarcinomas of the lung, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its metastases to the liver, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bile duct carcinomas, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous cell and adenocarcinomas of the cervix, epithelial carcinomas of the uterus and ovary, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B- and T-cell lymphomas (nodular and diffuse), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcomas and leiomyosarcomas.

[00119] Subjects may have a late stage form of the disease, in which case the goal of treatment may include reducing or reversing the progression of the disease and/or alleviating side effects. Subjects may have a history of a condition for which they have already been treated, in which case the goal of therapy may typically include reducing or delaying the risk of relapse.

[00120] Accordingly, the invention relates to a method of treating or preventing a neoplasia in a subject, wherein the method comprises administering an effective amount of immunoresponsive cells comprising a receptor that binds a tumor antigen and activates the immunoresponsive cell (e.g., TCR, CAR), and a vector encoding a receptor that binds another tumor antigen and stimulates the immunoresponsive cell. In one embodiment, the neoplasia is selected from the group consisting of prostate cancer, breast cancer, malignant blood cell tumors (e.g., leukemias, lymphomas, and myelomas), ovarian cancer, malignant bladder tumor, malignant brain tumor, colon cancer, malignant intestinal tumor, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, glioblastoma, and malignant throat tumor. In another embodiment, the tumor antigen is one or more of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen (e.g., cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine protein kinase erb-B2,3,4, folate binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human epidermal growth factor receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), interleukin-13 receptor alpha-2 subunit (IL-13Ra2), ic-light chain, kinase insertion domain-containing receptor (KDR), Lewis antigen Y (LeY), LI cell adhesion molecule (L1CAM), melanoma family A antigen 1 (MAGE-A1), mucin 1 (MUC1), mesothelin (MSLN), NKG2D ligands, testicular malignancy antigen NY-ES0-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), endothelial growth factor vascular endothelial growth factor R2 (VEGF-R2) or Wilms tumor protein (WT-1).

[00121] As a consequence of expressing on the surface a receptor that binds a tumor antigen and activates an immunoreactive cell (e.g., TCR, CAR) and a vector encoding a receptor that binds another tumor antigen and stimulates an immunoreactive cell (e.g., CCR), adoptively transferred human T or NK cells acquire enhanced and selective cytolytic activity at the tumor site. Moreover, following their localization to the tumor or viral infection and their proliferation, costimulatory ligand-expressing T cells convert the tumor or viral infection site into a highly conductive environment for a wide range of immune cells involved in the physiological antitumor or antiviral response (tumor-infiltrating lymphocytes, NK, NKT cells, dendritic cells, and macrophages).

[00122] In other embodiments, the invention relates to methods of treating subjects with an infection with a pathogen (e.g., a viral infection, a bacterial infection, a fungal infection, a parasitic infection, or a protozoan infection). The invention is particularly useful for enhancing an immune response in a subject with an immune deficiency. Exemplary viral infections susceptible to treatment using the method of the invention include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (HIV), and influenza virus infections.

[00123] Accordingly, the invention relates to a method of treating or preventing infection with a pathogen in a subject, wherein the method comprises administering an effective amount of immunoresponsive cells as described herein.

Sets

[00124] The invention relates to kits for treating or preventing a neoplasia, infection with a pathogen, an immune disorder, or an allogeneic transplant. In one embodiment, the kit includes a therapeutic or prophylactic composition comprising an effective amount of immunoresponsive cells comprising an activating antigen receptor and a costimulatory antigen receptor, in a unit dosage form. In particular embodiments, the cells further comprise a costimulatory ligand. In some embodiments, the kit comprises a sterile container containing a therapeutic or prophylactic vaccine; such containers may be boxes, ampoules, bottles, vials, tubes, bags, packets, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing drugs.

[00125] If desired, the immunoresponsive cells are provided along with instructions for administering the cells to a subject having or at risk of developing a neoplasia, pathogen infection, immune disorder, or allogeneic transplant. The instructions typically include information on using the composition to treat or prevent a neoplasia, pathogen infection, immune disorder, or allogeneic transplant. In other embodiments, the instructions include at least one of the following: a description of the drug; a dosing and administration schedule for treating or preventing a neoplasia, pathogen infection, immune disorder, or allogeneic transplant or symptoms thereof; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or in the form of a label attached to the container, or in the form of a separate sheet, brochure, card, or folder supplied in or with the container.

EXAMPLES

[00126] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are well within the skill of the art. Such techniques are fully explained in literature such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, 1989); Oligonucleotide Synthesis (Gait, 1984); Animal Cell Culture (Freshney, 1987); Methods in Enzymology Handbook of Experimental Immunology (Weir, 1996); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, 1987); Current Protocols in Molecular Biology (Ausubel, 1987); "PCR: The Polymerase Chain Reaction," (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These methods are useful for producing the polynucleotides and polypeptides of the invention, and as such, may be provided for the making and practicing of the invention. Particularly suitable methods for particular embodiments are discussed in the following sections.

[00127] The following examples are provided to provide one skilled in the art with a complete disclosure and description of how to perform and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors consider to be their invention.

Example 1: T cells co-expressing a chimeric antigen receptor (CAR) and a chimeric costimulatory receptor (CCR) killed established tumors

[00128] The invention relates to "tumor-responsive T cells" that simultaneously engage two antigens co-expressed by a tumor cell. Importantly, it was found that reactivity against tissues expressing either antigen alone should be negligible, triggering only T cell activation in the presence of both antigens, but not either antigen alone. The invention is based, at least in part, on the discovery that the combination provides selective T cell immunoreactivity, and thus makes the method clinically relevant. First, T cell activation should be directed to a single antigen (e.g., CD19 or prostate stem cell antigen, PSCA), which may be mediated by a T cell receptor (TCR) or a chimeric antigen receptor (CAR). Costimulation is independently mediated by a “chimeric costimulatory receptor” (CCR) ^12,13 targeting a second antigen (e.g., prostate-specific membrane antigen, PSMA). This pathway results in enhanced immunoreactivity against dual-antigen-positive (DP) tumors but fails to prevent enhanced immunoreactivity against single-antigen-positive (SP) tumors. A second principle important for tumor-sensing T cells to differentiate DP tumors from SP tumors is to reduce T cell activation to the point where it is ineffective on its own but is functionally restored at the tumor site by CCRs engaged by an independent, co-expressed antigen. Because CAR and CCR recognize cell surface antigens rather than HLA-peptide complexes, T cells engineered in this manner target tumors directly and cannot be costimulated by interactions with cells that cross-present target antigens. As shown here, this approach results in selective tumor killing in mice bearing multiple tumors.

[00129] To demonstrate that both T cell activation and costimulation signals can be provided in vivo using two separate antigen-specific receptors, a combination of a CAR providing a CD3 activation signal upon recognition of the B cell marker CD19 ¹⁴ and a CCR specific for PSMA ^12,15 was evaluated. Because of the synergy between CD28 and 4-1BB ^16,17 , including tandem cytoplasmic domains ^18,21 , the 4-1BB cytoplasmic domain was added to the PSMA P28 CCR ¹⁵ as described ² (Fig. 1A). Primary human peripheral blood T cells were transduced with the 19z1 and/or P28BB receptors and showed readily detectable expression of both receptors, with transduction efficiencies ranging from 45-70% (Fig. 1B). Four T cell groups were analyzed in all subsequent studies, including anti-CD19 CAR (19z1), anti-PSMA CCR (P28BB), both anti-CD19 CAR and anti-PSMA CCR in combination (19z1+P28BB), and a mock-transduced control group (Mock) (Fig. 1C). In vitro cytotoxic and proliferative responses to CD19 and/or PSMA showed that anti-CD19 cytotoxicity was, as expected, conferred by 19z1 and unchanged in the presence of PSMA.

[00130] Quantitative comparison of T cell groups normalized by the fraction of 19z1-transduced T cells for the 19z1 and 19z1+P28BB groups, and by the P28BB-transduced fraction in the P28BB group, showed that 19z1 and 19z1+P28BB T cells specifically lysed 40-47% of CD19 ⁺ targets at a 50:1 E:T ratio, while P28BB-transduced T cells were unable to lyse PSMA ⁺ targets (Fig. 2A). However, when re-exposed to these antigens in the absence of exogenous cytokine, only 19z1+P28BB T cells showed active proliferation with a 58-fold increase over 31 days when co-cultured on artificial antigen-presenting cells (AAPCs) expressing both antigens. In comparison, 19z1 or P28BB T cells showed only a modest increase over the first 14 days, as did 19z1+P28BB T cells on CD19 ⁺ PSMA ⁻ APC (Fig. 2B). Further evidence of a stronger T cell activation in the presence of both antigens was provided by quantifying cytokine production and induction of the anti-apoptotic molecule BclxL in 19z1+P28BB T cells, which were significantly higher in the presence of CD19 ⁺ PSMA ⁺ APC than in the presence of either antigen alone (Figs. 4A, 4E).

[00131] The ability of these dual receptor-expressing T cells to kill established systemic human prostate tumors in vivo was first tested in immunodeficient NOD/SCID-yC KO (NSG) mice bearing double positive (CD19+PSMA+) tumor cells. NSG mice were systemically inoculated with 2.0 x 10 ⁶ firefly luciferase-expressing PC3 tumor cells expressing both CD19 and PSMA (Fig. 5) and treated 19 days later with a single intravenous infusion of 1.0 x 10 ⁶ 19z1, 19z1+P28BB, P28BB, or control T cells. Thirty-five days later, mice injected with P28BB T cells or control T cells were sacrificed due to tumor burden. In contrast, mice treated with 19z1 T cells had a marked reduction in tumor burden. Surprisingly, mice treated with 19z1+P28BB T cells had undetectable tumor burden (Fig. 2C). During 70 days of post-infusion monitoring, CD19+ tumors eventually relapsed in mice treated with 19z1 T cells, whereas complete remission continued in all mice treated with 19z1+P28BB T cells (Fig. 2C). This result clearly indicated that tumor eradication had been achieved.

[00132] These findings, however, raised concerns due to potential background killing of CD19 ^{+ PSMA−} tumors by 19z1+P28BB T cells. It was possible that T cell immunoreactivity might be undesirably enhanced in recipients bearing double positive CD19 ⁺ PSMA ⁺ tumors due to T cell recirculation. To test this hypothesis, mice were infused subcutaneously with CD19 ^{+ PSMA−} tumors in the left flanks, ^CD19− PSMA ⁺ tumors in the right flanks, and CD19 ⁺ PSMA ⁺ tumors in the backs. One week later, mice were injected intravenously with one of 19z1, P28BB, or 19z1+P28BB T cells (1.0 x 10 ⁶ cells). Mice treated with P28BB T cells had progression of all three tumors and had to be sacrificed within 35 days (Fig. 2D). In mice treated with 19z1 T cells, CD19 ^{+ PSMA−} and CD19 ⁺ PSMA ⁺ tumors underwent significant reduction in size compared to their progression in P28BB T cell recipients, before eventually progressing. Consistent with preliminary results, mice treated with 19z1+P28BB T cells showed complete eradication of CD19 ⁺ PSMA ⁺ tumors. However, as hypothesized, rejection of CD19 ^{+ PSMA−} tumors was also significantly enhanced and exceeded that observed in 19z1 T cell recipients (Fig. 2D, lower panels). Thus, the method of splitting a signal targeting two antigens failed to restrict T cell reactivity and protect single-antigen tumors.

[00133] To address the single antigen reactivity issue, it was hypothesized that T cell activation could be minimized, almost to the point of inhibition, to be restored only at the site of dual antigen expression by engaging the correct CCR. Thus, a CAR with reduced activity was envisioned. For these experiments, a clinically relevant combination of antigens targeting PSCA and PSMA was used. Three PSCA-specific scFvs with different binding affinities for PSCA were evaluated (Figure 3A). While scFv HzI efficiently lysed tumor cells down to the picogram range, scFv Lz1 required 1,000-10,000-fold more antibody to achieve similar specific lysis efficiency. These scFvs were used to generate three CD3-based CARs with different activities in cytotoxicity assays (Figure 3B). Two of these CARs, HzI and MzI, directed moderate lytic activity against PSCA ⁺ targets (20% specific lysis at an E:T ratio of 50:1). In contrast, the third CAR, Lz1, achieved only 10%, qualifying it as an ineffective antigen receptor. This hierarchy was further confirmed in cytokine release assays, which showed enhanced cytokine secretion by 19z1+P28BB T cells (Fig. 4A) and Hz1+P28BB T cells (Fig. 4B) compared to cells with each receptor alone. This enhancement was less in Mz1+P28BB T cells (Fig. 4C) and even further reduced in Lz1+P28BB T cells (Fig. 4D).

[00134] To evaluate the therapeutic efficacy and targeting profile of PSCA ⁺ PSMA ^- reactive T cells, the antitumor activity of these T cells was tested in animals bearing PSCA ⁺ PSMA ^- and/or PSCA ⁺ PSMA ⁺ tumors. First, to test the ability of Mz1 + P28BB and Lz1 + P28BB T cells to selectively kill PSCA ⁺ PSMA ⁺ cells, mice were inoculated intravenously with 2 × 10 ⁶ FFLuc-expressing PC3 cells positive for PSMA, PSCA, or both (Figure 5). Fourteen days later, one group of mice received 1 × 10 ⁶ Mz1 + P28BB CAR + T cells by intravenous infusion, and another group received 1 × 10 ⁶ Lz1 + P28BB CARP T cells. For mice bearing PSCA+PSMA+ tumors treated with the more potent MzI+P28BB T cells, tumors showed greater tumor regression than mice treated with Lz1+PBB T cells (Fig. 3C). Similar to the CD19 experiment (Fig. 2C), these tumors eventually relapsed and progressed. However, in mice bearing PSCA+PSMA+ tumors, Mz1+P28BB T cells induced active and durable tumor killing. Consistent with the lower activity of LzI+P28BB T cells, tumor killing in mice bearing PSCA+PSMA+ tumors treated with Lz1+P28BB T cells was slower but nonetheless equally successful, resulting in robust tumor killing and long-term survival of all treated mice (Fig. 3C). Tumor killing was not enhanced in control mice bearing either PSCAVSMA- or PSCA-PSMA+ tumors (Fig. 3C). To more rigorously assess background activity against PSCAVSMA- tumors, we tested them in the context of mice also bearing PSCA+PSMA+ and PSCAPSMA+ tumors. Lz1+P28BB T cells mediated the killing of PSCA+PSMA+ tumors without enhancing the killing of PSCA+PSMA tumors (Fig. 3E), which was not different from the killing induced by Lz1 T cells.

[00135] Thus, these results demonstrate the feasibility of reducing T cell activation to the point of preventing immunoreactivity against tissues expressing a single target antigen and preserving T cell activation at a tumor site where two antigens are co-expressed without risking reactivity against tissues expressing a single antigen. In doing so, the results demonstrate the validity of principles for achieving two complementary outcomes that determine specificity and safety: 1) the ability to generate the intended specificity in the absence of a unique target antigen through recognition of a combined antigen; and 2) protection of cells expressing only one of the antigens by titrating activation and costimulation signals so as to substantially limit activation to sites of co-expression of the target antigens.

[00136] Targeted T cell therapies have the potential to provide curative treatment, but their utility is limited by the small number of validated tumor-specific targets. Expression outside tumors does result in “precise tumor knockout” effects that ^2-4 may sometimes be tolerable but are ultimately lethal ¹¹ . The method described herein provides improved targeting by providing titrated activation and costimulation signals through recognition of a combined antigen (Figures 6A-6C).

[00137] During physiological presentation of antigen ^22, T cells are primed in lymph nodes by receiving activating and costimulatory signals and migrate to peripheral sites where T cell effector functions are not as dependent on costimulation. Similarly, T cells recruited via the antigen receptor and CCR can recirculate to other peripheral sites and exhibit enhanced cytolytic activity against tissues expressing only one of the target antigens (Figure 6A). Thus, the present method is designed to overcome this problem of potential systemic effect to bypass cells positive for a single antigen, including non-tumor cells (Figure 6B). In a triple tumor mouse model (PSCA ⁺ , PSMA ^+, and PSCA ⁺ PSMA ⁺ ), we achieved the eradication of PSCAVSMA ⁺ while bypassing PSCA ^{+ PSMA−} and PSCA ⁻ PSMA ⁺ tumors (Fig. 3E).

[00138] As demonstrated by the present studies, this selectivity for DP tumors can be achieved by reducing the potency of the CAR, which creates cells that are less cytotoxic (Figure 3B) and that have reduced levels of cytokine secretion (Figures 4A-4D). While both THI and TH2 cytokine levels are relatively high for both the 19z1 and HzI CARs, use of the less efficient MzI and LzI CARs resulted in decreased levels. The increase in cytokine levels in Lz1+P28BB T cells compared to LzI T cells was minimal, with the exception of IL-2 and IL-13. While IL-2 induces proliferation and can stimulate either TH1 or TH2 responses ²³ , IL-13 is associated with a TH2 response specific to 4-1BB/CD137 signaling ^24,25 .

[00139] PSCA and PSMA are promising targets for the treatment of metastatic prostate cancer ^26,27 although neither is absolutely specific for the prostate. In human subjects, PSCA expression has been detected in prostate cancer and in the renal pelvis, ureter, bladder, and urethra ²⁸ . PSMA expression is highly correlated with primary prostate cancer, metastasis, as well as with type II astrocytes, renal proximal tubules, and intestinal brush border ²⁹ . Thus, dual targeting of PSCA/PSMA is expected to increase targeting of prostate cancer and decrease reactivity against these normal tissues. It is to be appreciated that this principle may be extended to other tumor types expressing the pair of antigens, particularly those that confer true tumor specificity. For example, HER2, MUC1, CD44, CD49f and/or EpCAM can be used in this method to treat breast cancer ³¹ . Similarly, mesothelin, folate receptor-a, CD44 and/or CD133 can be used to treat ovarian cancer ^32,33 . Targeting tumor-initiating cells or tumor stem cells for which unique target antigens/structures have not yet been clearly identified ^34,35 may be a particularly attractive use of this method.

[00140] An important aspect of this method is the limitation and near-abrogation of T cell activation in response to a single antigen. Low affinity or low avidity CARs, which provide only a weak activation signal, have been found to be useful in achieving this effect. Alternatively, an endogenous low affinity or low avidity TCR can be used in combination with a CCR to provide antigen-specific costimulation. Together, the results indicate the benefits of limiting the activity of engineered T cells, combining activity with safety through recognition of the combined antigen by tumor-sensitive T cells.

Construction of gamma-retroviral vector and virus production

[00141] The gamma retroviral vector SFG-19z1 is described in detail ¹⁴ . This backbone construct was used to replace scFvs to generate SFG-HzI, SFG-MzI, and SFG-LzI by direct cloning using the NcoI site located at the 5' scFv and the Nod site located at the 3' scFv. To generate SFG-P28BB, the CD28 and CD137 fusion domains were amplified by PCR with SFG-P28BBz1 and ligated into the 3' PSMA scFv using the 5' NcoI site and the 3' BamHI site to include a stop codon at the 3' BB domain, while the CD3 domain was removed ²¹ . Bicistronic gene expression for CAR to co-express with dsRED and for CCR to co-express with hrGFP was achieved using an internal ribosome binding site as described previously. Vectors were used to transiently transfect cell lines to generate stably virus-producing lines as described previously.

Production of anti-PSCA scFv

[00142] Three novel PSCA-specific scFvs, named HzI, MzI, and LzI, were generated by amplifying the variable heavy (VH) and variable light (VL) domains conferring specificity to the PSCA antigen for non-overlapping epitopes using degenerate primers with a hybrid as described previously ³⁶ . These VH and VL domains were fused together using a linker and used to replace the CD19 scFv in the SFG-19z1 backbone using the 5'-SphI to 3'-NotI sites.

Isolation, retroviral transduction and culture of primary human T cells

[00143] Peripheral blood leukocytes were isolated using Ficoll gradients and transduced as described previously. Briefly, after 48 h activation with 2 μg/ml phytohemagglutinin, cells were transduced twice by centrifugation inoculation for 1 h on retronectin-coated plates over a further 48 h and 20 U/ml IL-2 were added. After vector expression was allowed for 3 days, transduction efficiency was determined by flow cytometry and total unsorted cells were used for various assays or adoptive transfers.

Generation of antigen-expressing tumor cell lines

[00144] The human prostate tumor line PC3 was obtained from ATCC and transduced using ¹⁶ retroviruses to produce PC3-GFP/Luc, which were then used to produce 10 PC3-CD19, PC3-PSMA, PC3-CD19-PSMA, PC3-PSCA, and PC3-PSCA-PSMA by retroviral transduction.

Chromium Release CTL Kill Assays

[00145] Target cells expressing the desired antigen were labeled with ^51Cr and co-cultured with T cells in decreasing effector:target ratios. After 4 hours of culture, the supernatant was removed and used to measure the radioactivity released from the chromium. Specific lysis was determined by subtracting the background radioactivity of target cells not co-cultured with T cells and dividing by the radioactivity measured for target cells completely lysed using 0.2% Triton X-100.

Long-term T cell proliferation assays

[00146] Tumor cells expressing the desired antigen were irradiated with 30 Gy prior to co-culture with 1.0 x 10 ⁶ T cells at an effector:target ratio of 5:1. T cells were counted weekly using an Invitrogen Countess cell counter and then restimulated with irradiated tumor cells. No exogenous cytokines were added to these co-cultures.

Obtaining tumor models in mice

[00147] PC3 tumor cells were infused into NOD/SCID-IL2Ry mice obtained either from Jackson Laboratories or bred in-house according to protocol 04-10-024, which was approved by the MSKCC Institutional Animal Care and Use Committee. For systemic tumor experiments, 2.0 x 10 ⁶ tumor cells were infused into mice with 1.0 x 10 ⁶ chimeric receptor-positive T cells infused 14 days later. For subcutaneous tumor experiments, 1.0 x 10 ⁶ tumor cells were injected into the tumor site, allowed to engraft for 7 days, after which 1.0 x 10 ⁶ chimeric positive T cells were infused IV.

Quantification of tumor load

[00148] For experiments with systemic tumors, bioluminescence imaging (BLI) was used to quantify tumor burden by correlating the amount of tumor burden with luminescence using the MS 100 system (Caliper Life Sciences) as described previously. For subcutaneous tumors, calipers were used to measure tumor size. Tumor volume was calculated by multiplying the length, width, and height of each tumor.

Bispecific antibody-mediated tumor lysis

[00149] Bispecific antibodies containing PSCA-specific scFv fused to CD3-specific scFv were added at varying amounts to untransduced T cells co-cultured with PSCA ⁺ PC3 at a ratio of 20:1, respectively, in standard 4-hour chromium release assays.

Flow cytometry

[00150] Cells were analyzed using an LSRII flow cytometer or sorted using a FACSAria cell sorter (BD Biosciences) as previously described ¹⁶ . Detection of the chimeric receptor on the cell surface can be achieved directly using AF647-conjugated goat anti-mouse antibody (Invitrogen). Antibodies for CD4-PE-Cy7, CD8-Pacific Blue, and CD19-APC were obtained from Invitrogen, while anti-PSCA antibodies were purified from hybridoma supernatants and anti-PSMA antibodies were obtained from MBL International.

Cytokine analysis

[00151] Supernatants were collected 48 hours after the second tumor stimulation from long-term T cell proliferation experiments and used for cytokine analysis using a custom-made HCYTMAG-60K multiplex system (Miilipore) and analyzed using a Luminex 100 device (Luminex) as described previously ²¹ .

Western blot analysis

[00152] Cells were harvested 24 hours after initial tumor stimulation from long-term T cell proliferation experiments for use in Western blot analysis of BclxL expression. Western blotting was performed as previously described ²¹ using primary antibodies against Bcl _x L and Akt (Ceil Signaling Technology).

[00153] It is evident from the above description that changes and modifications can be made to the invention described herein to adapt it to various uses and conditions. Such embodiments are also within the scope of the following claims.

[00154] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. A reference to an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[00155] This application may be related to U.S. Patent Application No. 12/593,751, a U.S. national phase application under 35 U.S.C. §371, International Patent Application No. PCT/US2008/004251, filed March 8, 2010, which claims priority from U.S. Provisional Application Ser. No. 60/921,144, filed March 30, 2007, the entire contents of which are hereby incorporated by reference.

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

165 170 175

Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met

180 185 190

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

195 200 205

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

210 215 220

<210> 4

<211> 255

<212> PRT

<213> Homo sapiens

<400> 4

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

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

35 40 45

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

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

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

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

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

145 150 155 160

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

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

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

245 250 255

<210> 5

<211> 288

<212> PRT

<213> Homo sapiens

<400> 5

Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr

1 5 10 15

Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys

20 25 30

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

35 40 45

Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile

50 55 60

Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp

65 70 75 80

Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr

85 90 95

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

100 105 110

Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg

115 120 125

Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr

130 135 140

Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile

145 150 155 160

Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu

165 170 175

Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp

180 185 190

Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met

195 200 205

Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg

210 215 220

Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro

225 230 235 240

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

245 250 255

Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg

260 265 270

Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val

275 280 285

<210> 6

<211> 182

<212> PRT

<213> Homo sapiens

<400> 6

Met Glu Arg Val Gln Pro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg

1 5 10 15

Pro Arg Phe Glu Arg Asn Lys Leu Leu Leu Val Ala Ser Val Ile Gly

20 25 30

Gly Leu Gly Leu Leu Leu Cys Phe Thr Tyr Ile Cys Leu His Phe Ser

35 40 45

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

50 55 60

Gln Phe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln

65 70 75 80

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

85 90 95

Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu

100 105 110

Val Asn Ile Ser Leu His Tyr Gln Asp Glu Glu Pro Leu Phe Gln Leu

115 120 125

Lys Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr Tyr

130 135 140

Lys Asp Lys Val Tyr Leu Asn Tyr Thr Thr Asp Asn Thr Ser Leu Asp

145 150 155 160

Asp Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn Pro

165 170 175

Gly Glu Phe Cys Val Leu

180

<210> 7

<211> 451

<212> PRT

<213> Homo sapiens

<400> 7

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

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

20 25 30

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

35 40 45

Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu

50 55 60

Trp Ile Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly

65 70 75 80

Lys Phe Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr

85 90 95

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

100 105 110

Glu Cys Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp

115 120 125

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

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr

145 150 155 160

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

165 170 175

Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Asn Phe Ile Arg Val Lys Glu Ser Arg Ser Ala Glu Pro Pro Ala Tyr

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Pro Pro Arg

450

<210> 8

<211> 478

<212> PRT

<213> Homo sapiens

<400> 8

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Trp Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Val Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

180 185 190

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

195 200 205

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

210 215 220

Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn

225 230 235 240

Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys Arg

245 250 255

Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

260 265 270

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

275 280 285

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

290 295 300

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

305 310 315 320

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

325 330 335

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

340 345 350

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

355 360 365

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475

<210> 9

<211> 245

<212> PRT

<213> Homo sapiens

<400> 9

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Glu Ser Ser Tyr

20 25 30

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

35 40 45

Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe

50 55 60

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

65 70 75 80

Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys

225 230 235 240

Leu Glu Ile Lys Arg

245

<210> 10

<211> 257

<212> PRT

<213> Homo sapiens

<400> 10

Met Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu

1 5 10 15

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

20 25 30

Pro Gly Thr Ser Val Arg Ile Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

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

50 55 60

Glu Trp Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Asp Val Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser

180 185 190

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

195 200 205

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

210 215 220

Thr Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr

225 230 235 240

Asn Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys

245 250 255

Arg

<210> 11

<211> 412

<212> PRT

<213> Homo sapiens

<400> 11

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Trp Ile Gly Asn Ile Asn Pro Asn Asn Gly Gly Thr Thr Tyr Asn Gln

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Cys Ala Ala Gly Trp Asn Phe Asp Tyr Trp Gly Gln Gly Thr Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Val Gly Thr Ala Val Asp Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

180 185 190

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

195 200 205

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

210 215 220

Asn Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn

225 230 235 240

Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Met Leu Asp Leu Lys Arg

245 250 255

Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu

260 265 270

Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro

275 280 285

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

290 295 300

Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe

305 310 315 320

Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

405 410

<---

Claims (22)

1. An expression vector containing: a. a first polynucleotide encoding a chimeric antigen receptor (CAR) that binds to a first antigen with a K _d of about 1 x 10 ^-8 M to about 1 x 10 ^-5 M, wherein binding of the CAR to the first antigen provides for delivery of an activation signal to a T cell containing said vector, and b. a second polynucleotide encoding a chimeric costimulatory receptor (CCR) comprising (i) an antigen-binding domain that binds to a second antigen and (ii) an intracellular signaling domain, wherein binding of the CCR to the second antigen provides for delivery of a costimulatory signal to the T cell, but does not by itself provide for delivery of an activation signal to the T cell, wherein the T cell containing said vector exhibits a higher degree of cytolytic activity against cells that are positive for both the first antigen and the second antigen, compared to activity against cells that are positive for the first antigen alone, and wherein the T cell containing said vector exhibits substantially absent or insignificant long-term or persistent cytolytic activity against cells that are positive for the first antigen alone. 2. The vector of claim 1, wherein the CAR binds to the first antigen with a K _d of about 5 x 10 ^-8 M or more. 3. The vector of claim 1, wherein the CAR binds to the first antigen with a K _d of about 1 x 10 ^-7 M or more. 4. The vector of claim 1, wherein the CAR binds to the first antigen with a K _d of about 1 x 10 ^-6 M or more. 5. The vector of claim 1, wherein the CAR binds to the first antigen with a binding affinity that is lower than the binding affinity with which the CCR binds to the second antigen. 6. The vector according to any one of claims 1-5, wherein the antigen is a tumor or pathogen antigen. 7. Vector according to any of the items. 1-5, wherein said first and second antigens are selected from the group consisting of CAIX, CEA, CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, cytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, Erb-B2, Erb-B3, Erb-B4, FBP, fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, x-light chain, KDR, LeY, cell adhesion molecule LI, MAGE-AI, MUC1, mesothelin, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2 and WT-1. 8. The vector of any one of claims 1-5, wherein said first and second antigens are different antigens selected from the group consisting of CD133, cytomegalovirus (CMV) infected cell antigen, Erb-B2 KDR, mesothelin, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, CD19, VEGF-R2 and WT-1. 9. The vector of any one of claims 1 to 5, wherein said first and second antigens are selected from the group consisting of HER2, MUC1, CD44, CD49f, EpCAM, CEA, CD133, cytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, Erb-B2, Erb-B3, Erb-B4, FBP, KDR, mesothelin, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, VEGF-R2 and WT-1. 10. The vector of claim 9, wherein said first and second antigens are CD10 and CD19. 11. The vector of claim 9, wherein said first and second antigens are CD56 and CD138. 12. The vector according to claim 9, wherein said first and second antigens are PSCA and PSMA. 13. The vector of claim 9, wherein said first and second antigens are different antigens selected from the group consisting of mesothelin, folate receptor-a, CD44 and CD133. 14. The vector of any one of claims 1-5, wherein the CAR comprises an intracellular signaling domain that comprises the signaling domain of the CD3ζ chain. 15. The vector of any one of claims 1-5, wherein the intracellular CCR signaling domain is the CD97 signaling domain, the CD11a-CD18 signaling domain, the CD2 signaling domain, the ICOS signaling domain, the CD27 signaling domain, the CD154 signaling domain, the CD5 signaling domain, the OX40 signaling domain, the 4-1BB signaling domain, or the CD28 signaling domain. 16. A vector according to any one of paragraphs 1-5, wherein the vector is a viral vector. 17. A vector according to any one of claims 1-5, wherein the vector is a retroviral vector. 18. A cytotoxic T cell comprising an expression vector according to any one of claims 1-5.