ES2902669T3 - Compositions and methods for immunotherapy - Google Patents

Abstract

A T cell comprising: a) a chimeric antigen receptor (CAR) that binds CD19, wherein binding of the CAR to the first antigen is capable of activating the T cell, and b) an inhibitory chimeric antigen receptor (iCAR) comprising an antigen-binding domain that binds PSMA, and at least a portion of a signaling domain of an immunoinhibitory receptor selected from CTLA-4, PD-1, LAG-3, 2B4, and BTLA, wherein binding of iCAR to PSMA reduces T cell cytotoxicity against cells that are positive for both CD19 and PSMA.

Description

DESCRIPTION

Compositions and methods for immunotherapy

INTRODUCTION

Immune-responsive cells are disclosed that include an antigen recognition receptor that binds a first antigen and an inhibitory chimeric antigen receptor (iCAR) that binds a second antigen, where binding of the recognition receptor of antigen to the first antigen activates the immune-sensitive cell and binding of iCAR to the second antigen inhibits the immune-sensitive cell. Methods of producing the immune-responsive cells and methods of treating cancers using the immune-responsive cells are also disclosed.

Background of the invention

T cell-based therapies have curative potential in bone marrow and organ transplantation, immunotherapy against cancer, viral infections, and autoimmune diseases. However, the main treatment-related complications arise from inadvertent T cell reactivity against normal tissues, such as in graft-versus-host disease (GVHD) after infusion of donor lymphocytes or "target reactivity outside the tumor" in autologous T-cell targeted therapy.

The use of donor lymphocyte infusion (DLI) in allogeneic bone marrow transplantation (BMT; 25,000 annually worldwide) has produced significant curative benefits in certain subsets of patients. Recently, DLI has been shown to provide effective therapy for patients with metastatic renal cell carcinoma, breast, and ovarian cancer, with ongoing trials to treat metastatic solid tumors. The key efficacy of DLI, termed graft-versus-leukemia (GVL) in the setting of hematologic malignancies, is limited by the induction of both acute and chronic graft-versus-host disease (GVHD) (rates greater than 40%), to the point that several groups have concluded that the current DLI regimen cannot improve survival unless GVHD induction is decreased.

Similarly, cancer immunotherapy trials have reported "on-target but off-tissue" adverse events of CAR and TCR-modified T cells, including, for example, B-cell aplasia in patients with chronic lymphocytic leukemia (CLL). treated with cells expressing CAR anti-CD19, lethal acute respiratory distress syndrome (ARDS) due to cross-reactivity of T cells with CAR anti-ERBB2 in lung epithelium, and deaths from myonecrosis in patients with melanoma and myeloma treated with a TCR Mage-A3. Chicaybam et al. Human Gene Therapy 2010, 21(10), 1418 discloses Jurkat cells transduced with an activating anti-CD19 CAR and an inhibitory anti-CD20 CAR. Preliminary data indicates that contact with CD19-expressing cells would provide T cell activation, whereas contact with CD19/CD20-expressing cells would result in inhibition.

Nonspecific immunosuppression and T-lymphocyte elimination are currently the only means of controlling undesirable T-lymphocyte responses, at the cost of nullifying therapeutic benefit and causing serious secondary complications. They are based on the onset of symptoms, which can be severe and occasionally unmanageable, thus limiting the use of an otherwise effective cellular treatment.

Strategies to prevent the consequences of cellular side effects are urgently needed. Current approaches do not use the higher order complexity of T-cell therapies to their advantage in managing undesirable side effects. Therefore, novel therapeutic strategies are urgently required.

Summary of the invention

Immune-responsive cells (eg, T cells, natural killer (NK) cells, cytotoxic T cells (CTLs), and regulatory T cells) are disclosed, which express an antigen-binding receptor (eg, CAR or TCR) that has immune cell activation activity and an inhibitory chimeric antigen receptor (iCAR) that selectively reduces or eliminates the immune activity of the immune-sensitive cell. Thus, the off-target effects of the immunoresponsive cell are reduced. The decrease in immune activity may be reversible. Methods of using said immunosensitive cells for the treatment of neoplasms, infectious diseases and other pathologies are disclosed.

An isolated immune-responsive cell is disclosed that includes an antigen-recognition receptor that binds to a first antigen, where binding activates the immune-responsive cell, and an inhibitory chimeric antigen receptor that binds to a second antigen, where binding inhibits immunosensitive cell. In a first aspect, the present invention provides a T lymphocyte comprising: a) a chimeric antigen receptor (CAR) that binds to CD19, wherein the binding of the CAR to the first antigen is capable of activating the T lymphocyte and b) a inhibitory chimeric antigen receptor (iCAR) comprising an antigen-binding domain that binds PSMA, and at least a portion of a signaling domain of an immunoinhibitory receptor selected from CTLA-4, PD-1, LAG-3, 2B4, and BTLA, where binding of iCAR to PSMA reduces T cell cytotoxicity against cells that are both CD19 and PSMA positive.

A method of reducing tumor burden in a subject is disclosed, the method involving administering an effective amount of an immune-responsive cell that includes an antigen recognition receptor that binds to a first antigen, where binding activates the immune-responsive cell, and an inhibitory chimeric antigen receptor that binds to a second antigen, where the binding inhibits the immune-responsive cell, thereby inducing tumor cell death in the subject. The method can selectively target tumor cells compared to non-tumor cells. In some examples, the method reduces the number of tumor cells. In some examples, the method reduces tumor size. In other embodiments, the method eradicates the tumor in the subject.

Further disclosed is a method of increasing the survival of a subject having neoplasia, the method involving the administration of an effective amount of an immunoresponsive cell that includes an antigen recognition receptor that binds to a first antigen, where binding activates the immune-responsive cell, and an inhibitory chimeric antigen receptor that binds to a second antigen, where the binding inhibits the immune-responsive cell, thereby treating or preventing neoplasia in the subject.

The present invention provides the claimed T cell for use in reducing tumor burden and/or tumor size in a subject, and/or increasing survival of a subject having a neoplasm in a subject. In certain embodiments, the neoplasm is selected from the group consisting of hematologic cancer, B-cell leukemia, multiple myeloma, lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma, ovarian cancer, cancer prostate cancer, pancreatic cancer, lung cancer, breast cancer, sarcoma, and acute myeloid leukemia (AML).

A method for producing a cell that is immunoresponsive to a specific antigen that includes an antigen recognition receptor that binds to a first antigen is disclosed, the method involving the introduction into the immune responsive cell of a nucleic acid sequence that encodes an antigen receptor. inhibitory chimeric antigen that binds to a second antigen, where the binding inhibits the immune-responsive cell. The present invention provides a method for producing a T cell of the claims, the method comprising: introducing into a T cell a first nucleic acid sequence encoding CAR and a second nucleic acid encoding iCAR.

Also disclosed herein is an inhibitory chimeric antigen receptor (iCAR) that includes an extracellular domain that binds antigen; a transmembrane domain operably linked to the extracellular domain; and an intracellular domain that activates intracellular signaling to decrease an immune response, the intracellular domain being operably linked to the transmembrane domain. In some embodiments, the intracellular signaling domain is selected from the group consisting of a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, and a BTLA polypeptide. In certain embodiments, the transmembrane domain is selected from the group consisting of a CD4 polypeptide, a CD8 polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, and a BTLA polypeptide. Additionally, the inhibitory chimeric antigen receptor (iCAR) may further comprise a Fab, scFv, ligand, specific ligand, or polyvalent ligand. In some embodiments, binding of an antigen to iCAR activates the intracellular signaling domain that decreases the immune response.

A nucleic acid sequence encoding an inhibitory chimeric antigen receptor that binds to an antigen is disclosed, where binding activates an intracellular signaling domain that decreases an immune response.

A vector is disclosed that includes a nucleic acid sequence encoding an inhibitory chimeric antigen receptor that binds to an antigen, where binding activates an intracellular signaling domain that decreases an immune response.

In a related aspect, the invention provides a pharmaceutical composition (for the treatment of neoplasia or pathogen infection) that includes an effective amount of an immunoresponsive cell of the claims in a pharmaceutically acceptable carrier.

A kit for the treatment of a neoplasm, pathogen infection, an autoimmune disorder or an allogeneic transplant is disclosed, the kit including an immunosensitive cell that includes an antigen recognition receptor that binds to a first antigen and activates the immunosensitive cell and an inhibitory chimeric antigen receptor that binds to a second antigen and inhibits the immune-responsive cell. The kit may further include written instructions for using said cell for treatment of a subject having neoplasia, pathogen infection, autoimmune disorder or allogeneic transplant.

A method for modulating a graft-versus-leukemia or graft-versus-tumor response in a subject is disclosed, the method involving administering an effective amount of an allogeneic immunoresponsive cell to the subject that includes an inhibitory chimeric antigen receptor that binds to a antigen, where binding inhibits the allogeneic immunosensitive cell, thereby modulating a graft-versus-leukemia response or a graft versus tumor in the subject. In one example, the subject has metastatic breast cancer, hematologic malignancy, or a solid tumor, and the human leukocyte antigen (HLA) is HLA-I. In another example, the subject has a tumor that has undergone epithelial-to-mesenchymal transition (EMT), and the antigen is one or more of an epithelial-mesenchymal transition (EMT) antigen, E-cadherin, and cytokeratin. The binding of the inhibitory chimeric antigen receptor and the antigen can decrease cell death in a cell comprising the antigen. The method may reduce graft versus host disease (GVHD) in the subject or a symptom thereof.

In various examples disclosed herein, the first antigen or antigen recognition receptor antigen is a tumor or pathogen antigen. In particular disclosed examples, the antigen recognition receptor antigen is one or more tumor antigens selected from the following group consisting of CD19, CAIX, CEA, CD5, CD7, CD10, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, a cytomegalovirus (CMV) infected cell antigen, EGP-2, EGP-40, EpCAM, erb-B2,3,4, F8P, acetylcholine receptor fetal, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, k-light chain, KDR, LeY, cell adhesion molecule L1, MAGE-A1, mesothelin, Muc-1, Muc -16, ligands of NKG2D, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2 and WT-1.

In various examples disclosed herein, the second antigen or inhibitory chimeric antigen receptor antigen is CD33, CD38, a human leukocyte antigen (HLA), an organ-specific antigen, a blood-brain barrier, an epithelial-mesenchymal transition (EMT) antigen, E-cadherin, cytokeratin, opioid-binding protein/cell adhesion molecule (OPCML), HYLA2, suppressed in colorectal carcinoma (DCC) scaffold/matrix attachment region-binding protein 1 (SMAR1), cell-surface carbohydrate, or mucin-like O-glycan. In various embodiments of the aspects outlined herein, binding of an antigen to iCAR activates the intracellular signaling domain that decreases the immune response.

In various embodiments of the aspects outlined herein, the inhibitory chimeric antigen receptor is recombinantly expressed. In various embodiments of the aspects outlined herein, the inhibitory chimeric antigen receptor is expressed from a vector. In various embodiments of the aspects outlined herein, the inhibitory chimeric antigen receptor (iCAR) includes an intracellular signaling domain selected from the group consisting of a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide , a 2B4 polypeptide and a BTLA polypeptide. In various embodiments of the aspects outlined herein, the inhibitory chimeric antigen receptor (iCAR) includes a transmembrane domain selected from the group consisting of a CD4 polypeptide, a CD8 polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide , a LAG-3 polypeptide, a 2B4 polypeptide, and a BTLA polypeptide. In various embodiments of the claimed T cell, the inhibitory chimeric antigen receptor (iCAR) includes a Fab, scFv, ligand, specific ligand, or polyvalent ligand. In certain embodiments, the T cell expresses an endogenous or recombinant antigen receptor that is selected from the group consisting of 19-28z, P28z, M28z, and 56-28z.

In various embodiments of the aspects outlined herein, the antigen recognition receptor is the chimeric antigen receptor (CAR). In various embodiments of the aspects outlined herein, the antigen recognition receptor is exogenous or endogenous. In various embodiments of the aspects outlined herein, the antigen recognition receptor is recombinantly expressed. In various embodiments of the aspects outlined herein, the antigen recognition receptor is expressed from a vector. In various embodiments of any of the aspects defined herein, an allogeneic cell has an endogenous T cell receptor.

In various aspects delineated herein, the cell is one or more of a T cell, a natural killer (NK) cell, a cytotoxic T cell (CTL), a regulatory T cell, a stem cell human embryonic cell, a cell of the innate immune system, and a pluripotent stem cell from which lymphoid cells can be differentiated. In some embodiments, the immunoresponsive cell is autologous. In other embodiments, the immunoresponsive cell is not autologous.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one skilled in the art to which this invention pertains. The following references provide those of skill in the art with a general definition 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.), SpringerVerlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

By "activates an immunoresponsive cell" is meant the induction of signal transduction or changes in protein expression in the cell that result in the initiation of an immune response. For example, when CD3 chains cluster in response to ligand binding and receptor inhibition tyrosine motifs (ITAM), a signal transduction cascade occurs. In certain embodiments, when an endogenous TCR or exogenous CAR binds antigen, an immune synapse formation occurs that includes the clustering of many molecules near the bound receptor (eg, CD4 or CD8, CD3y/S/£/ Z, etc.) This clustering of membrane-bound signaling molecules allows the ITAM motifs found within the CD3 chains to become phosphorylated. This phosphorylation, in turn, initiates a T cell activation pathway that ultimately activates transcription factors, such as NF-kB and AP-1. These transcription factors induce global T cell gene expression to increase IL-2 production for proliferation and expression of master regulatory T cell proteins to initiate a T cell-mediated immune response. understands a signal that results in a strong and sustained immune response. In various embodiments, this occurs after activation of immune cells (eg, T cells) or is simultaneously mediated through receptors, including, but not limited to, CD28, CD137 (4-1BB), OX40, and ICOS. . Without wishing to be bound by any particular theory, receiving multiple stimulatory signals is important in mounting a strong and long-term T-cell-mediated immune response. Without receiving these stimulating signals, the T lymphocytes are rapidly inhibited and do not respond to the antigen. While the effects of these costimulatory signals vary and remain partially understood, they generally result in increased gene expression in order to generate long-lasting, proliferative, antiapoptotic T cells that strongly respond to antigen for complete eradication and sustained.

The term "antigen recognition receptor" as used herein refers to a receptor that is capable of activating an immune cell (eg, a T cell) in response to antigen binding. Illustrative antigen recognition receptors can be natural or endogenous T cell receptors, exogenous T cell receptors introduced into a cell, and/or chimeric antigen receptors in which a tumor antigen-binding domain is fused with a signaling domain. intracellular capable of activating an immune cell (for example, a T-lymphocyte).

As used herein, the term "antibody" means not only intact antibody molecules, but also fragments of antibody molecules that retain the ability to bind an immunogen. Such fragments are also well known in the art and are regularly used in vitro and in vivo. Accordingly, as used herein, the term "antibody" means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')2, and Fab. F(ab')2 and Fab fragments that lack the Fc fragment of an intact antibody, are cleared more rapidly from the circulation, and may have less nonspecific tissue binding than an intact antibody ((Wahl et al.,, J Nucl Med 24:316-325 (1983) Antibodies of the invention include complete native antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single-chain V region fragments (scFv), fusion polypeptides and non-antibodies. conventional.

By "affinity" is meant a measure of binding strength. Without being bound by theory, affinity depends on the closeness of the stereochemical fit between antibody combining sites and 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 antigen-antibody binding after formation of reversible complexes. Methods for calculating the affinity of an antibody for an antigen are known in the art, including using binding experiments to calculate affinity. The activity of the antibody in functional assays (eg, flow cytometry assay) also reflects the affinity of the antibody. Antibodies and affinities can be phenotypically characterized and compared using functional assays (eg, flow cytometry assay).

The term "chimeric antigen receptor" or "CAR", as used herein, refers to an antigen-binding domain that is fused with an intracellular signaling domain capable of activating or stimulating an immune cell. More typically, the extracellular binding domain of CAR is composed of a single chain variable fragment (scFv) derived from the fusion of the heavy and light variable regions of a murine or humanized monoclonal antibody. Alternatively, scFvs derived from Fab (rather than an antibody, eg, obtained from Fab libraries) can be used. In various embodiments, this scFv is fused to a transmembrane domain and then to an intracellular signaling domain. "First generation" CARs include those that only provide signals to CD3Z upon antigen binding, "second generation" CARs include those that provide both costimulation (eg, CD28 or CD137) and activation (CD3Z). "Third generation" CARs include those that provide multiple costimulation (eg, CD28 and CD137) and activation (CD3Z). In various embodiments, the CAR is selected to have a high affinity or avidity for the antigen.

The term "inhibitory chimeric antigen receptor" or "iCAR", as used herein, refers to an antigen-binding domain that is fused with an intracellular signaling domain capable of inhibiting or suppressing the immune activity of a host. immune cell. iCARs have immune cell inhibitory potential and are distinct and distinguishable from CARs, which are receptors with immune cell activating potential . For example, CARs are activating receptors, since they include CD3Z, iCARs do not contain activating domains. Therefore, iCARs are distinct from CARs and are not a subset of CARs. In certain embodiments, the antigen-binding domain is fused with a transmembrane domain and the one or more intracellular domains of a receptor. immunoinhibitory. The iCAR transmembrane domain can be a CD8 polypeptide, a CD4 polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, an IAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide. The intracellular domain of iCAR can be a TIA-4 c polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLA polypeptide. In various embodiments, the extracellular binding domain of iCAR is composed of a single chain variable fragment (scFv) derived from the fusion of the heavy and light variable regions of a murine or humanized monoclonal antibody. In certain embodiments, the scFV is a prostate-specific membrane antigen (PSMA)-specific scFV. Alternatively, scFvs derived from Fabs (rather than an antibody, eg, derived from Fab libraries) can be used. iCARs have immunosuppressive activity that inhibits T cell function specifically after antigen recognition.

By "inhibits an immune-responsive cell" or "suppresses an immune-responsive cell" is meant the induction of signal transduction or changes in protein expression in the cell that result in suppression of an immune response (eg, decreased cytokine production). In preferred embodiments, the inhibition or suppression of an immunoresponsive cell is selective and/or reversible.

The term "immunosuppressive activity" refers to the induction of signal transduction or changes in protein expression in a cell (eg, an activated immunosensitive cell) that results in a decreased immune response. Polypeptides known to suppress or decrease an immune response include, but are not limited to, CTLA-4 polypeptides, PD-1 polypeptides, LAG-3 polypeptides, 2B4 polypeptides, or BTLA polypeptides (for example, by binding to their corresponding ligands). . At a minimum, iCAR signaling uses the intracellular portions of such molecules and may also include portions of the transmembrane domain and/or extracellular domain of such molecules as necessary to produce a functional iCAR. The term "immunostimulatory activity" refers to the induction of signal transduction or changes in protein expression in a cell (eg, an active immunoresponsive cell) that results in an increased immune response. Immunostimulatory activity may include proinflammatory activity. Polypeptides known to stimulate or enhance an immune response through their binding include CD28, OX-40, 4-1BB, and their corresponding ligands, including B7-1, B7-2, OX-40L, and 4-1BBL. These polypeptides are present in the tumor microenvironment and activate immune responses against neoplastic cells. In various embodiments, promoting, stimulating, or agonizing the proinflammatory polypeptides and/or their ligands enhance the immune response of the immune-responsive cell.

By "CD3Z polypeptide" is meant a protein that has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to NCBI reference number: NP_932170 or a fragment thereof that has activating activity. or stimulating. An example of CD3Z [SEQ ID NO: 1] is provided below.

1 mkwkalftaa ilqaqlpite aqsfglldpk Icylldgílf iygviltalf Irvkfsrsad

61 apayqqgqnq lynelnlgrr eeydvldkrr grdpemggkp qrrknpqegl ynelqkdkraa

121 eayseigrakg errrgkghdg lyqglstatk dtydalhmqa Xppr

By "CD3Z nucleic acid molecule" is meant a polynucleotide that encodes a CD3Z polypeptide.

By "CD28 polypeptide" is meant a protein that has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to NCBI reference number: NP_006130 or a fragment thereof that has stimulatory activity . An example of CD28 [SEQ ID NO: 2] is provided below.

1 mlrlllalnl fpsíqvtgnk ilvkqspmlv aydnavnlsc kysynlfsre fraslhkgld

61 savevcwyg nysqqlqvys ktgfncdgkl gnesvtfylq nlyvnqtdíy fckievmypp

121 pyldneksng tiihvkgkhl cpsplfpgps kpfwvlvwg gvlacysllv tvafiifwvr

181 skrsrllhsd ymntntprrpg ptrkhyqpya pprdfaayrs

By "CD28 nucleic acid molecule" is meant a polynucleotide that encodes a CD28 polypeptide.

By "19-28z" is meant a protein that has at least 85, 90, 95, 96, 97, 98, 99, or 100% identity to the sequence provided below [SEQ ID NO: 3], including a leader sequence at amino acids 1-18 and is capable of binding CD19.

MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNW

VKQRPGQGLEWIGGJYPGDGDTNYNGKFKGQATLiTADKSSSTAYMQLiSGLTSED

SAVYFCARKTISSWDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQS

PKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYR3SISGVPDR

FTGSG SGTDFTLTITNVQSKDLADYFCQQYNRYPYTS GGGTKLEIKRAAAIEVM

YPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLWVGGVLACYSLLVT

VAF11FWVRSKRSRLLHS DYMNMTPER PGPTRKHYQPYAPPRDFAAYRSRVKFS

RSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY

NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRX

An illustrative nucleic acid sequence encoding a 19-28z polypeptide, including a leader sequence [SEQ ID NO: 4], is provided below.

ccatggctctcccagtgactgccctactgcttcccctagcgcttctcctgcatg cagaggtgaagctgcagcagtctggggctgagctggtgaggcctgggtcctcag tgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaact gggtgaagcagaggcctggacagggtcttgagtggattggacagatttatcctg gagatggtgatactaactacaatggaaagttcaagggteaagceacactgactg cagacaaatcctccagcacagcctacatgcagctcagcggcctaacatctgagg actctgcggtctatttctgtgcaagaaagaccattagttcggtagtagatttct actttgactactggggccaagggaccacggtcaccgtctcctcaggtggaggtg gatcaggtggaggtggatctggtggaggtggatctgacattgagctcacccagt ctccaaaattcatgtccacatcagtaggagacagggtcagcgtcacctgcaagg ccagtcagaatgtgggtactaatgtagcctggtatcaacagaaaccaggacaat ctcctaaaccactgatttactcggcaacctaccggaacagtggagtccctgatc gcttcacaggcagtggatctgggacagatttcactctcaccatcactaacgtgc agtctaaagacttggcagactatttctgteaaeaatataacaggtatcegtaca cgtccggaggggggaccaagctggagatcaaacgggcggccgcaattgaagtta tgtatcctcctccttacctagacaatgagaagagcaatggaaccattatccatg tgaaagggaaacacctttgtccaagtcccctatttcccggaccttctaagccct tttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaa cagtggcctt tattattttctgggtgaggagtaagaggagcaggctcctgcaca gtgactacatgaacatgactccccgccgccccgggcccacccgcaagcattace agccctatgceccaccacgcgacttegcagcctatcgctccagagtgaagttca gcaggagcgcagagccccccgcgtaccagcagggccagaaccagctctataacg agcteaatctaggacgaagagaggagtacgatgttttggacaagagacgtggcc gggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgt acaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatga aaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtcteagta cagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcg

Nucleic acid molecules useful in the methods disclosed herein include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally show substantial identity. Polynucleotides that have "substantial identity" with an endogenous sequence are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pairing to form a double-stranded molecule between complementary polynucleotide sequences (eg, a gene described herein), or portions thereof, under various stringency conditions. (See, eg, Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

For example, a stringent salt concentration will typically be 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 organic solvent, eg, formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably, about 50% formamide. Stringent temperature conditions will typically include temperatures of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, concentration of detergent, eg, sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those of skill in the art. Various levels of stringency are achieved by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30°C in 750mM NaCl, 75mM trisodium citrate, and 1% SDS. In one example, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA). In another example, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations of these conditions will be readily apparent to those skilled in the art.

For most applications, the wash steps following hybridization will also vary in their stringency. Washing stringency conditions can be defined by salt concentration and temperature. As before, the stringency of the wash can be increased by reducing the salt concentration or by increasing the temperature. For example, the stringent salt concentration for the washing steps will preferably be 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 washing steps will 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 one example, the wash steps will occur at 25°C in 30mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In another example, the wash steps will occur at 42°C in 15mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. In another embodiment, the washing steps will occur at 68°C in 15mM NaCl, 1.5mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those of skill 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 Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sam Brook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

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

Sequence identity is generally measured using sequence analysis software (eg, Sequence Analysis Software Package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP or PILEUP/PRETTYBOX programs). Said computer program matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and/or other modifications. Conservative substitutions generally 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 approach to determining degree of identity, a BLAST program can be used, with a probability score between e-3 and e-100 indicating a closely related sequence.

The term "analog" refers to a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

The term "ligand" refers, as used herein, to a molecule that binds to a receptor. In particular, the ligand binds to a receptor on another cell, allowing cell-to-cell recognition and/or interaction.

The term "constitutive expression", as used herein, refers to expression under all physiological conditions.

"Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue or organ. Examples of diseases include neoplasia or cellular infection by pathogens.

By "effective amount" is meant an amount sufficient to have a therapeutic effect. In one embodiment, an "effective amount" is an amount sufficient to stop, enhance, or inhibit the continued proliferation, growth, or metastasis (eg, invasion or migration) of a neoplasm.

By "endogenous" is meant a nucleic acid or polypeptide molecule that is normally expressed in a cell or tissue.

By "forcing tolerance" is meant preventing the activity of autoreactive cells or immunosensitive cells that target transplanted organs or tissues.

By "exogenous" is meant a nucleic acid or polypeptide molecule that is not present endogenously in the cell, or is not present at a level sufficient to achieve the functional effects obtained when overexpressed. Thus, the term "exogenous" would encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and overexpressed nucleic acid molecules and polypeptides.

By "heterologous nucleic acid molecule or polypeptide" is meant a nucleic acid molecule (eg, cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in a cell or 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 a cell or sample.

By "immunoresponsive cell" is meant a cell that functions in an immune response or a progenitor or progeny thereof.

By "increase" is meant that it is positively altered by at least 5%. An alteration can be 5%, 10%, 25%, 30%, 50%, 75%, or even 100%.

The term "isolated cell" refers to a cell that is separated from the molecular and/or cellular components that naturally accompany the cell.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components that normally accompany it as found in its natural state. "Isolate" indicates a degree of separation from the original source or surroundings. "Purify" indicates a degree of separation that is greater than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials that the impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques or of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are normally determined by analytical chemistry techniques, eg polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein gives rise to essentially one band on an electrophoretic gel. For a protein that can undergo modifications, eg, phosphorylation or glycosylation, different modifications can give rise to different isolated proteins, which can be purified separately.

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.

The term "obtain" as in "obtain the agent" is intended to include purchasing, synthesizing, or otherwise acquiring the agent (or indicated substance or material).

"Linker" as used herein shall mean a functional group (eg, chemical or polypeptide) that covalently joins two or more polypeptides or nucleic acids such that they are connected to each other. As used herein, a "peptide linker" refers to one or more amino acids used to couple two proteins (eg, to couple h and Vl domains). An illustrative linker sequence used in the invention is GGGGSGg Gg Sg g Gg S [Se Q ID NO:10].

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

By "neoplasia" is meant a disease characterized by the pathological proliferation of a cell or tissue and its subsequent migration to or invasion of other tissues or organs. The growth of the neoplasm is normally uncontrolled and progressive, and occurs under conditions that would not stimulate, or cause the cessation of, the multiplication of normal cells. Neoplasms may affect various types of cells, tissues, or organs, including, but not limited to, an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus and vagina, or a tissue or cell type thereof. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumor of plasma cells). Illustrative neoplasms for which the invention can be used include, but are not limited to, leukemias (eg, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, erythroleukemia acute, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, cancer prostate, squamous cell carcinoma, basilar carcinoma ocellular, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor , cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma and retinoblastoma).

By "operably linked," as used herein, is meant the joining of two or more biomolecules such that the biological functions, activities, and/or structure associated with the biomolecules are at least preserved. In reference to polypeptides, the term means that the joining of two or more polypeptides results in a fusion polypeptide that retains at least some of the corresponding individual activities of each polypeptide component. The two or more polypeptides can be joined directly or through a linker. In reference to nucleic acids, the term means that a first polynucleotide is positioned adjacent to a second polynucleotide which directs transcription of the first polynucleotide when appropriate molecules (eg, transcriptional activator proteins) bind to the second polynucleotide.

By "pathogen" is meant a virus, bacterium, fungus, parasite or protozoan capable of causing disease.

Illustrative viruses include, but are not limited to, Retroviridae (eg, human immunodeficiency virus, such as HIV-1 (also referred to as HDTV-III, LAVE, or HTLV-III/LAV, or H iV-III; and other isolates, such as e1HIV-LP; Picornaviridae (eg, poliovirus, hepatitis A virus; enteroviruses, human Coxsackieviruses, rhinoviruses, echoviruses); Calciviridae (eg, strains causing gastroenteritis); Togaviridae (eg, viruses equine encephalitis virus, rubella virus); Flaviridae (for example, dengue virus, encephalitis virus, yellow fever virus); Coronoviridae (for example, coronavirus); Rhabdoviridae (for example, vesicular stomatitis virus, rabies); Filoviridae (for example, Ebola virus); Paramyxoviridae (for example, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (for example, influenza virus); Bungaviridae ( e.g. Hantaan virus, Bunga virus, phlebovirus and Nairo virus); Arena viridae (hemorrhagic fever virus); Reoviridae (eg, reoviruses, orbiviruses, and rotaviruses); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma virus, polyoma virus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesviruses; Poxviridae (variola virus, vaccinia virus, smallpox virus); and Iridoviridae (for example, African swine fever); and unclassified viruses (for example, hepatitis delta agent (believed to be a defective satellite of hepatitis B virus), non-A, non-B hepatitis agents (class 1 = internally transmitted; class 2 = parenterally transmitted (ie, hepatitis C); Norwalk and related viruses and astroviruses).

Illustrative bacteria include, but are not limited to, Pasteurella, Staphylococcus, 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 sps (eg 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 hovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, species Pathogenic Campylobacter, Enterococcus sp., Haemophilus influenzae, Bacillus antracis, 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 israelli.

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

By "reduce" is meant negatively alter by at least 5%. An alteration can be 5%, 10%, 25%, 30%, 50%, 75%, or even 100%.

By "recognize" is meant that it selectively binds to a target. A T cell that recognizes a virus normally expresses a receptor that binds to an antigen expressed by the virus.

By "reference" or "control" is meant a standard for comparison. For example, the immune response of a cell expressing a CAR and an iCAR can be compared to the immune response of a corresponding cell expressing a CAR only.

As used herein, the term "single-chain variable fragment" or "scFv" is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin covalently linked to form a heterodimer. VH:VL. The heavy (VH) and light (VL) chains are linked either directly or by a peptide-encoding linker (eg, 10, 15, 20, 25 amino acids), which connects the N-terminus of the Vh to the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The linker is typically rich in glycine for flexibility, as well as serine or threonine for solubility. Despite the removal of the constant regions and the introduction of a linker, the scFv proteins retain the specificity of the original immunoglobulin. Single-chain FV polypeptide antibodies can be expressed from nucleic acid that includes the VH and VL coding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See also US Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and United States Patent Publication Nos. 20050196754 and 20050196754.

By "secreted" is meant a polypeptide that is released from a cell via the secretory pathway by the endoplasmic reticulum, Golgi apparatus, and as a vesicle that transiently fuses at the cell's plasma membrane, releasing proteins out of the cell. the cell.

By "signal sequence" or "leader sequence" is meant a peptide sequence (5, 10, 15, 20, 25, 30 amino acids in length) present at the N-terminus of newly synthesized proteins that directs their entry into the secretory pathway.

By "soluble" is meant a polypeptide that diffuses freely in an aqueous environment (eg, not membrane bound).

By "specifically binds" is meant a polypeptide or fragment thereof that recognizes and binds a biological molecule of interest (eg, a polypeptide), but does not recognize or substantially bind other molecules in a sample, eg. , a biological sample, which naturally includes a polypeptide of the invention.

The term "tumor antigen" as used herein refers to an antigen (eg, a polypeptide, glycoprotein, or glycolipid) that is exclusively or differentially expressed on a tumor cell compared to a normal or non-neoplastic cell. . With reference to the invention, a tumor antigen includes any polypeptide expressed by a tumor that is capable of being recognized by an antigen recognition receptor (eg, CD19, Muc-1) or capable of suppressing an immune response through receptor ligand binding (eg, CD47, PD-L1/L2, 87,112).

By "tissue antigen" is meant an antigen (eg, a polypeptide or glycoprotein or glycolipid) that is exclusively or differentially expressed in a normal or non-neoplastic cell or tissue as compared to a tumor cell.

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

The terms "comprise", "comprising", and are intended to have the broad meaning ascribed to them in United States Patent Law and may mean "includes", "including" and the like.

As used herein, "treatment" refers to clinical intervention in an attempt to alter the course of the disease of the individual or cell being treated and may be performed for prophylaxis or during the course of clinical pathology. The therapeutic effects of the treatment include, without limitation, preventing the onset or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequence of the disease, preventing metastases, slowing the rate of disease progression , improve or palliate the pathology and a remission or an improved prognosis. By preventing the progression of a disease or disorder, a treatment may prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment can prevent the onset of the disorder or a symptom of the disorder in a subject at risk of the disorder or suspected of having the disorder.

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

The term "immunocompromised" as used herein refers to a subject having an immunodeficiency. The subject is highly vulnerable to opportunistic infections, infections caused by organisms that do not usually cause disease in a person with a healthy immune system, but can affect people with a malfunctioning or suppressed immune system.

Other aspects of the invention are described in the following disclosure and are within the scope of the invention.

Brief description of the figures

The following detailed description, given by way of example but not intended to limit the invention to the specific embodiments described, may be read in conjunction with the accompanying drawings.

Figures 1A to 1D represent the strategy, design and expression in primary human T cells of iCAR. (A) T cells with specificity for both tumor and non-target tissues can be restricted to tumor only by using an antigen-specific iCAR introduced into T cells to protect non-target tissue. (B) Schematic diagram of the bicistronic vectors used for iCAR and Pdel. iCAR-P: A spacer, transmembrane domain, and intracellular tail of each inhibitory receptor was cloned into a previously described retroviral vector that has a CD8 leader sequence (LS). IRES, internal ribosome entry sites; hrGFP, humanized Renilla green fluorescent protein reporter. A Pdel control vector was designed with a CD8 spacer and transmembrane (TM) domain, and lacking an intracellular tail. (C) Cell surface expression of iCARs was assessed by flow cytometry on transduced primary human T cells. Dot plots are representative of eight different donors. GAM, goat anti-mouse immunoglobulin G F(ab')2 antibody that binds to the murine-derived extracellular domain of CAR. (D represents flow cytometric analysis of cell surface expression of iCARs using goat-anti-mouse (GAM) staining on transduced primary human T cells.

Figures 2A to 2F show that iCARs protected iPS-fib from TCR-mediated allogeneic reactions. Control iCAR- or Pdel-transduced T cells primed with allogeneic moDC (monocyte-derived dendritic cells) were incubated with iPS-derived fibroblasts (iPS-fib) expressing click beetle luciferase (CBL). acronym), isogenic to moDCs, using a range of E/T ratios. (A) T cells transduced with Pdel, PD-1 iCAR-P, or mutCTLA-4 reacting against target iPS-fib (n = 3 per condition). iPS-fib killing was quantified with the Bright-Glo assay system. (B) Cytokine secretion was assessed in cell culture supernatants from (A) at an E/T ratio of 4:1 at 18 hours. GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN-y, interferon-g; TNF-a, tumor necrosis factor-a. (C) Pdel or iCAR positive T cells were incubated for 24 hours with iPS-fib expressing off-target PSMA (iPS-fib-PSMA), and luciferase signal was quantified (left) (right) (n = 3 for each condition). (D to F) Cytokine secretion measured at 24 hours in cell culture supernatants from (C). Error bars represent ±SEM. *P < 0.01, *** P < 0.001 by analysis of variance (ANOVA) comparing iCAR with Pdel and post hoc analysis with Holm-Sidak corrected multiple t-tests. Raw data and P values are provided in Figures 19A to 19E. Figures 3A to 3D show that the iCARs functioned stoichiometrically. (A) PD-1 Pdel- or iCAR-P-transduced alloreactive T cells were sorted for high or low expression of each corresponding receptor, as shown in Figure 13A, and seeded on iPS-fib-PSMA expressing CBL. iPS-fib-PSMA killing relative to untreated cells was assessed with the Bright-Glo assay system (n=3 for each condition). (B) Cytokine secretion, measured at 24 hours in cell culture supernatant from (A) at a 4:1 E/T ratio. (C) PD-1 iCAR-P transduced alloreactive T cells were incubated with sorted iPS-fib-PSMA to determine high or low levels of PSMA expression as shown in Figure 13B. Killing of each population relative to untreated cells was quantified with the Bright-Glo assay system (n=3 per condition). D) Cytokines from (C) were evaluated at 24 hours. Error bars represent ±SEM. *** P < 0.001 by Student's t-test. Error bars represent ±SEM. *P < 0.01, *** P < 0.001 by ANOVA compared to high P del group and post hoc analysis with Holm-Sidak corrected multiple t-tests. Raw data and P values are provided in Figures 20A and 20B.

Figures 4A and 4B show that iCARs limited allogeneic responses in vivo. NOD/SCID/yc mice were injected intraperitoneally with 1 x 106 iPS-derived fibroblasts expressing CBL/PSMA (iPS-fib-PSMA) and, 7 days later, treated intraperitoneally with 5 x 105 sorted, alloreactive T cells. transduced with Pdel or iCAR-P from PD-1. Untreated mice (without T cells) were used as control. (A) iPS-fib-PSMA survival was assessed by BLI before and at selected time points after T cell infusion. Images of four representative mice from each group are shown. (B) Whole body flux (photons per second) was quantified for each mouse and averaged per group (n = 5 per group).

Error bars represent ±SEM. *P < 0.05, ** P < 0.01 by ANOVA compared to Pdel and post hoc analysis with multiple Holm-Sidak corrected t-tests. Raw data and P values are provided in Figures 21A and 21B.

Figures 5A to 5F show that iCARs inhibited 19-28z CAR-driven human T cell target cell clearance, proliferation and cytokine release. (A) Luminex multiplexed cytokine analysis of culture supernatant 24 hours after seeding of double-sorted 19-28z/Pdel or 19-28z/iCAR-transduced human T cells in AAPC with 3T3-CD19 (target) or 3T3- CD19-PSMA (off target). Data is represented as a ratio of off-target/target values and pooled from three independent experiments (n=6 wells per condition). Error bars represent ±SEM. **P < 0.01, *** P < 0.001 by ANOVA comparing iCAR with Pdel and post hoc analysis with multiple Holm-Sidak corrected t-tests. (B) Absolute count of 19-28z/Pdel or 19-28z/iCAR T cells stimulated on days 0 and 7 with off-target AAPC. Exogenous cytokines were not added. Data are representative of six independent experiments. (C) Proliferation of 19-28z/Pdel or 19-28z/iCAR T cells stimulated on days 0 and 7 with non-specific AAPC relative to proliferation in target AAPC. Exogenous cytokines were not added. Data are representative of six independent experiments. (D) T cells seeded in a 1:1 ratio on AAPC mCherry+ on target and off target. Images at 38 hours and 5 days from one of five independent experiments are shown. Scale bars, 0.5 mm. (E and F) Quantification of mCherry signal from (D) against CD19 targets (E) or non-target CD19-PSMA cells (F), as described in Materials and Methods. Error bars represent ±SEM. **P < 0.01, *** P < 0.001 by Student's t-test. Raw data and P values are provided in Figures 22A to 22C.

Figures 6A to 6F show that iCARs restricted the specificity of target cells with CAR 19-28z in vivo. (A) BLI depicting NALM/6 or NALM/6-PSMA tumor progression in NOD/SCID/yc mice treated with selected PD-1 19-28z/iCAR-P T cells. Untreated mice (without T cells) were used as control. (B) Tumor burden was quantified for each mouse and average total flux per group is shown. (C) Spleen weight of mice from (A) sacrificed on day 21. Each point represents a recipient mouse. (D) Flow cytometric analysis of bone marrow from the femur of (C) to determine the presence of tumor cells (CD19+GFP+) and T lymphocytes (CD19-19-28z/GFP+CD4+cD8+). 19-28z expression was assessed by staining for the LNGFR receptor whose complementary DNA (cDNA) is bound to 19-28z and used as a detection marker. (E and F) The absolute number of tumor cells (E) and CD19i9-28z/GFP+CD4+CD8+ T lymphocytes (F) in the spleens of (C) were quantified by flow cytometry with CountBright beads (n = 4 ). Error bars represent ±SEM. **P < 0.01, *** P < 0.001 by Student's t-test.

Figures 7A to 7D show that the iCAR function was temporary and reversible. (A) PD-1 19-28z/Pdel or 19-28z/iCAR-P T cells were incubated with AAPC on target (T) or off-target (O) for the first stimulation. After 3 or 7 days, cells in each group were restimulated with either target AAPC [T^T(1) or O^T(2)] or off-target [T^O(3) or O^O(4). )] in a cross-linked manner to analyze the effects of the first stimulation on subsequent T cell function. (B) The destruction of target (T) or off-target (O) AAPC at 24 hours after incubation with each pool of T cells (second challenge) was analyzed with the Bright-Glo assay system (n = 3 for each condition). (C) Effector cytokine secretion in cell culture supernatant from (B) was analyzed 24 hours after the second stimulation, and interferon-g (IFN-g) is shown as a representative result (n = 3 for each condition). (D) T cell proliferation on day 7 after the second stimulation (n = 3 for each condition). Error bars represent ±SEM. A statistical comparison was made within each condition (ie, T^-T Pdel vs. iCAR-P of PD-1). *** P < 0.001 by Student's t-test.

Figures 8A to 8E show that iCAR and CAR expressing T cells identified targets in vitro and in vivo. (A) PD-1 19-28z/Pdel or 19-28z/iCAR-P T cells were incubated with a 1:1 mixture of AAPC target (CD19+GFP+, green) and off-target (CD19+PSMA+ mCherry+, red) and time-lapse microscopy was used to visualize real-time killing of each population over 38 hours. Representative images are shown and complete movies were made. Scale bars, 0.1 mm. T cells (B) as in (A), PD-1 19-28z/Pdel or 19-28z/iCAR-P were incubated with a 1:1 mixture of AAPC target (CD19+) and off-target (CD19+ PSMA+ ). Killing of each AAPC population was assessed in parallel experiments in which one of each type of AAPC was labeled with CBL (CD19+CBL+/CD19+PSMA+ mix or CD19+CD19+PSMA+CBL+ mix). Killing was quantified with the Bright-Glo assay system at 38 hours (n=3 for each condition). (C to E) NOD/SCID/yc mice were injected with a 1:1 mixture of NALM/6 and nAl M/6-PSMA cells and treated with 19-28z or 19-28z/iCAR-P T cells from PD -one. (C) After sacrifice, the presence of target and off-target NALM/6 cells in bone marrow was analyzed by flow cytometry. (D) The ratio of target/off-target NALM/6 cells in the bone marrow of sacrificed mice was quantified by flow cytometry. (E) The spleen weight of the treated mice was also recorded at the time of sacrifice. Error bars represent ±SEM. *** P < 0.001 by Student's t-test.

Figures 9A–C show that cell surface expression of CTLA-4 iCAR was increased after T cell activation. (A) Cell surface and intracellular expression of CTLA4 iCAR-P in transduced primary human T cells ( B) Western blot analysis using an antibody specific for the intracellular domain of CTLA4 in non-transduced (2) and CTLA4 iCAR-P transduced (1) primary human T cells. Murine EL4 cells (3) served as a negative control. (C) CTLA4 1928z/iCAR T cells were activated using AAPC 3T3-19 and analyzed for cell surface expression of CTLA4 iCAR at 7 hours and 30 hours after activation (n = 3).

Figure 10 shows that iCAR-P bound to cells expressing PSMA. EL4-wt cells were incubated or EL4PSMA, labeled with the lipophilic dye DiD, with T cells expressing iCAR/GFP in a cell conjugation assay. Conjugates are detected by flow cytometry as double positive DiD/GFP events.

Figures 11A to 11D depict the allogeneic reactivity model using iPS-derived fibroblasts and isogenic moDC. (A) Induced pluripotent stem (iPS) cells were generated from PBMC from donor 1 and used to obtain fibroblasts. PBMCs from donor 1 were also used to obtain moDC, which were pulsed with fibroblast lysates and could prime an allogeneic reaction from PBMCs from a second donor. (B) Microscopy image showing the morphology of iPS-teratoma-derived fibroblasts growing in culture. (C) iPS-fib, lacked expression of pluripotency markers, showed fibroblast morphology, and stained positive for several fibroblast surface markers, including CD90, PDGFr-b2, and CD10. (RED= isotype control; BLUE and GREEN are two independent isolated lines) (D) iPS-fib with positive basal staining for HLA class I, but not class II, and the expression of both increased rapidly after treatment with Recombinant gamma INF.

Figures 12A to 12C depict strong reactivity of iCAR-transduced primary human T cells against allogeneic iPS-derived fibroblasts. (A) iCAR-transduced T cells from one donor were primed with pulsed moDCs with iPS-fib lysates from a different donor, and 6 days later stained for CD25 and HLA-DR activation markers. (B) Primed iCAR-transduced T cells were incubated twice for 18 hours with iPS-fib-luc and killing was quantified using the Bright-Glo assay system (n = 3 for each condition). (C) Cytokines were measured at 18 h in cell culture supernatant from (B) at an E:T ratio of 4:1. Error bars represent /- SEM. Statistical comparison was carried out within each condition (ie, T1T). *** p < 0.001 by Student's t-test.

Figures 13A and 13B depict the iCAR or 19-28z/iCAR T cell transduction and sorting strategy. (A) PD1 (GFP) or Pdel (GFP) iCAR-transduced T cells were sorted for transgene expression based on GFP expression level. Each donor represents a separate experiment. Post-sort analysis was performed to confirm purity. (B) T cells transduced with 19-28z (LNGFR)/iCAR (GFP) or 19-28z/Pdel/GFP were sorted for transgene expression based on GFP expression and LNGFR level. Each donor represents a separate experiment. Post-sorting analysis was performed to confirm the purity and expression of iCAR.

Figures 14A and 14B depict the sorting strategy of T cells expressing high/low iCAR and iPS fibroblasts expressing PSMA. (A) PD1/GFP or Pdel/GFP iCAR-transduced T cells were sorted for low or high transgene expression based on GFP expression level. (B) iPS-derived fibroblasts (iPS-fib) were transduced to express PSMA and sorted using an anti-PSMA antibody (iPS-fib-PSMA bulk type+ sorting). These cells were used for experiments in Figures 2 and 4. A second separate type was used to purify iPS-fib expressing low or high PSMA using an anti-PSMA antibody and these cells were used in the experiments in Figure 3.

Figures 15A to 15D show that iCARs inhibited 19-28z-driven human T cell cytokine release and proliferation. (A) Proliferation of human T cells into artificial antigen-presenting cells (Aa PCs) expressing CD19 or both CD19 and PSMA. (B) Representative INFy cytokine analysis of supernatants at 24 h and 48 h after seeding of human T cells transduced with 19-28z/Pdel or iCAR dually sorted into AAPC CD19 (target) or CD19/PSMA (off-target). target) positive. Data is represented as a ratio of off-target/target values and pooled from three independent experiments. (n = 6 wells per condition). Error bars represent /- SEM. (C) Absolute counts of double-positive 19-28z/Pdel or iCAR T cells stimulated on days 0 and 7 with AAPC CD19+ (target). (D) Comparison of therapeutic responses of T lymphocytes with nAl M/6 and NALM/6-PSMA cells in a xenograft model with NOD/Sc ID/Yc- mice.

Figures 16A to 161 show that basal expression of iCAR did not affect the function of primary human T cells. (A, B) Seven days after 1928z/iCAR transduction, T cells were activated with CD3/CD28 beads and IL-2/INFy levels were assessed after 24 hours (C). At eight days after bead activation, absolute T cell expansion was quantified using Count-Bright beads (D) and the change in percentage of GFP-positive cells in each iCAR group was normalized to unstimulated cells. . (E) 1928z/iCAR T cells were co-cultured for five days with irradiated EL4-WT or EL4-PSMA cells and immunophenotyped using flow cytometry. (F) represents a cell conjugation assay of EL4-wt or EL4-PSMA cells, labeled with the lipophilic DiD dye and incubated with T lymphocytes expressing iCAR. (G) depicts graphs plotting IL-2/INFy levels in T cells 24 hours after CD3/CD28 bead activation, seven days after 1928z/iCAR transduction. (H) is a graph depicting the quantification of absolute expansion of T cells using CountBright beads eight days after bead activation. (I) is a graph depicting the change in percentage of GFP positive cells in each iCAR group that was calculated relative to unstimulated cells.

Figures 17A to 17C depict the biochemical and signaling pathway characterization of PD-1 iCAR. PD-1 19-28z/iCAR cells were exposed to AAPC not expressing antigen (TS), Cd 19 (target), or CD19 and PSMA (off-target) at an E:T ratio of 4:1 for 60 min. . (A) Human phosphoimmunoreceptor array incubated with 100 μg of 19-28z/i-CAR T cell lysate of PD-1 and the corresponding AAPCs. All spots were detected using chemiluminescence on the same X-ray film to standardize exposure levels. (B, C) Quantification of arrays in (A) using scanned X-ray film images analyzed using image analysis software. All pixel density is normalized to each matrix using internal pY controls. (B) Phosphorylation states of SHP1 and SHP2 on target, off target, or control AAPC. (C) Quantification of the phosphorylation levels of 59 ITAM/ITIM-associated immunoreceptors.

Figure 18 shows that iCAR acted directly on the stimulating receptor to block its signaling. Figures 19A to 19E represent raw data and statistical significance test for Figures 2A to 2F. (A) Killing of iPS-fibroblasts was quantified using the Bright-Glo assay system for Pdel, PD-1 iCAR-P or mutCTLA-4 transduced T cells. (B) Cytokine secretion was assessed in cell culture supernatants from (A) at an E:T ratio of 4:1 at 18 h. (C) Pdel or iCAR positive T cells were incubated for 24 hours with iPS-fib expressing off-target PMA (iPS-fib-PSMA) and luciferase signal was quantified. (D) Post hoc analysis for (C) was carried out using multiple corrected t-tests with the Holm-Sidak method. (E) Cytokine secretion measured at 24 h in cell culture supernatants from (C). Raw values of GM-CSF are presented. E:T ratio, effector:target ratio.

Figures 20A and 20B represent raw data and statistical significance tests for Figures 3A to 3D. (A) Killing of iPSfib-PSMA relative to untreated cells was assessed using the Bright-Glo assay system for alloreactive T cells transduced with high and low Pdel or iCAR-P from sorted PD1 iCAR-P. Post hoc analysis was performed using multiple t-tests corrected with the Holm-Sidak method. (B) Killing by iCAR-P transduced alloreactive T cells with iPS-fib_PSMA PD1 sorted for high or low levels of PSMA expression was quantified using the Bright-Glo assay system. Post hoc analysis was performed using multiple t-tests corrected with the Holm-Sidak method.

Figures 21A and 21B represent raw data and statistical significance tests for Figures 4A and 4B. (A) Bioluminescence imaging (BLI) of iPS-fib-PSMA before and at selected time points after T cell infusion. (B) Post hoc analysis was performed using multiple t-tests corrected with the Holm-Sidak method.

Figures 22A to 22C represent raw data and statistical significance test for Figures 5A to 5F. (A) Luminex multiplexed cytokine analysis of culture supernatant at 24 h, data is represented as off-target/target ratio and pooled from three independent experiments (n = 6 wells per condition). Post hoc analysis was performed using multiple t-tests corrected with the Holm-Sidak method. (B) Post hoc analysis was performed using multiple Holm-Sidak corrected t-tests for Figure 5B comparing PD1 19-28z/Pdel and 19-28z/iCAR proliferation. (C) Quantification of mCherry signal against target CD19 cells or off-target CD19-PSMA, as described in Methods. Post hoc analysis was performed using multiple t-tests corrected with the Holm-Sidak method.

Figure 23 is a schematic representation of a target antigen screen for iCAR and CAR.

Detailed description of the invention

Cells are disclosed herein, including genetically modified immune responsive cells (eg, T lymphocytes (including all subsets such as CD4, CD8, memory, wild-type, effector, T-reg, etc.), cells of the immune system innate, natural killer (NK) cells, cytotoxic T cells (CTL) that express at least one combination of an antigen recognition receptor (eg, TCR or CAR) and an inhibitory chimeric antigen receptor (iCAR) that reduces or eliminates selectively the immune activity of the immunosensitive cell, and methods of using such cells for the treatment of neoplasias and other pathologies in which it is desired to reduce "off-target" immune responses. The invention defined by the claims is based, at least in part, on the discovery that inhibitory chimeric antigen receptors (iCARs) that bind to a target antigen (eg, PSMA as shown in the prese nte document) are useful for selectively inhibiting and suppressing an immunoreactive cell. In particular, the iCARs used by the invention decrease or prevent the activation of the immune response of an immunoreactive cell. The present approach provides selective immunogenicity for tumor eradication, while excluding non-tumor tissues from the immune response. Thus, T cells expressing an antigen recognition receptor and an iCAR represent a significant advance over conventional adoptive T cell therapy.

The widespread use of donor leukocyte infusion to treat cancer is hampered by the inability to separate the therapeutic efficacy of graft-versus-tumor (GVT) effect from the potentially lethal effects of graft-versus-tumor disease. host (GVHD). Two general approaches have been used to control the side effects of T cell therapy. The first is the use of immunosuppressive drugs, which work nonspecifically by blocking cell division (cytostatics) and largely limit immune responses (glucocorticoids, immunophilins , etc.) or by targeting T cells for removal/death (antibodies). Although powerful, these approaches are nonspecific in terms of separating T cells that function therapeutically and those that cause deleterious side effects. Additionally, all of these drugs cause significant long-term adverse side effects (susceptibility to infections, heart, kidney, and neurological damage).

The second approach engineers T cells with suicide genes/kill switches. These are genetic approaches that cause the engineered cell to die once a suitable signal is provided. Several of them are based on the introduction of selective enzymatic metabolizers of toxic agents, such as the herpes simplex virus thymidine kinase (HSV-tk). Additionally, the use of an inducible caspase-9 protein that is activated using a specific chemical inducer of dimerization has been a Promising approach to actively induced extensive cell death. The main limitations of these approaches are that they induce cell death in all target cells (thus eliminating beneficial cells). Like conventional immunosuppression, they usually require the appearance of symptoms before their implementation (and therefore possible permanent damage to the patient).

In contrast, the iCAR strategy described herein selectively filters the actions of T cells, restricting activity to off-target sites, while preserving therapeutic functionality against the intended target. As shown herein, iCARs were able to inhibit human alloreactive T cells from attacking a "host" tissue in a new in vitro and in vivo model using iPS-derived human fibroblasts. Exclusive surface antigens present on target tissues of GVDH (eg, CD33 for myeloid lineage or organ-specific antigens) but absent on the target malignancy are candidate iCAR targets to differentiate GVHD from GVT. Similarly, the results described herein show that iCAR-mediated inhibition successfully restricts the "on-target, but off-tissue" effects of CAR-engineered T cells, examples of which include aplasia of B lymphocytes in patients with leukemia treated with T lymphocytes that express a specific CAR of c D19 (Kalos et al., Science translational medicine, 2011. 3(95): 95ra73), acute respiratory distress syndrome (ARDS, for its acronym in English) fatal thought to be the result of anti-ERBB2 CART cell cross-reactivity in the lung epithelium (Morgan et al., Mol. Ther., 2010. 18(4): 843-51) and deaths from Cardiac myonecrosis in melanoma and myeloma patients treated with TCR Mage-A3 (June, Update on Immunotherapy Trials for HIV and Cancer, in Recombinant DNA Advisory Committee, 2012). By recognizing a surface antigen that is expressed on heart cells or lung epithelium but is absent on tumor cells, iCARs could potentially be used to protect from TCR Mage-A3 or CAR anti-ERBB2 cross-reactivity, thus resurrecting therapies that would otherwise be promising. Alternatively, since many tumors actively downregulate HLA molecules to escape immune recognition, HLA-targeted iCARs have the potential to provide simultaneous protection to multiple tissues.

An important requirement for the clinical applicability of iCARs is the maintenance of T cell functionality despite prior iCAR signaling. Interestingly, iCAR-transduced T cells were found to still mount a response against a target antigen after exposure to an inhibitory antigen. This reversibility mimics the behavior of NK cells, in which the phosphorylation state of signaling molecules rather than transcriptional states control rapid functional responses such as cytotoxicity. Anti-PD-1 and anti-CTLA4 antibodies are capable of reversing the impaired function of anergized or depleted T cells, again arguing for their ability to temporarily regulate T cell responses. Biochemical analyzes of the effects of PD-1 and CTLA-4 on the TCR complex depend on phosphorylation states, downstream kinases, and motility rather than apoptosis. Both in vitro and in vivo results demonstrated inhibition in response to off-target cells with sustained therapeutic functionality, although there is a possibility that some of the cells are anergized. In addition to functioning on T cells, CTLA4 and PD-1 also function on B cells, macrophages, and dendritic cells. Therefore, iCARs also have the potential to manipulate other immune phenomena.

The iCARs disclosed herein can be used as a buffering tool to limit systemic cytokine storms or immune cell responses, for example, by introducing antigen into the iCAR, such as a recombinant PSMA-Ig infused in a patient. PSMA-Ig can bind to and activate iCAR and thus can temporarily inhibit T cell activation. This can temporarily break the cyclic spiral that causes cytokine storms and allow T cells to become activated with systemic side effects limited. iCARs have this functionality, as shown by cross-linking the iCAR

The present invention uses a physiological mechanism to restrict T cell side effects. This approach mimics the naturally occurring restriction of T cell reactivity and therefore does not require the removal of valuable therapeutically viable cells. . The present approach takes advantage of the multifaceted functionality of cells as drugs, by using synthetic receptors that guide and direct T cells to perform only the desired functions. In conclusion, inhibitory antigen-specific receptors successfully limited T cell proliferation, cytokine secretion, and cytotoxicity following the involvement of cell-surface-specific antigens, thereby conferring protection to normal tissue while preserving critical therapeutic functions mediated by TCR or CAR. Therefore, iCARs provide a novel strategy to establish safer and more effective T-cell therapies in both autologous and allogeneic settings.

Effect of graft versus leukemia (GVL) and graft versus host disease (GVHD)

Since the first use of allogeneic bone marrow transplants, it has been appreciated that the eradication of leukemia depended on the immune response derived from the donor. The effect of graft-versus-leukemia (GVL) was elucidated and further assessed following successful donor lymphocyte infusion following allogeneic bone marrow transplantation (b Mt ). Donor lymphocyte infusion (DLI) is the most established and widespread use of adoptive immunotherapy for malignancies. Unfortunately, the main source of morbidity and mortality after DLI is the occurrence of graft-versus-host disease (GVDH). Reducing its incidence and severity is an important limitation for broader and potentially curative use of DLI in solid and hematologic tumors. The iCAR strategy aims to solve this problem by separating the beneficial effects of GVL from the dangerous consequences of GVHD. GVDh primarily affects the skin, liver, and intestinal tract, sites that possess exclusive antigens such as alia-minor antigens that may be absent from the target malignancy. Such events support a possible role for iCARs in separating the effects of GVL and GVHD.

In adoptive therapy, redirected T cells have been shown to play a potential curative role in various malignancies. Still, this transformative approach is limited due to cross-reactivity and subsequent toxicity against critical normal tissues (heart, lung). Thus, there is a significant need to develop ways to control T-cell reactivity without affecting their therapeutic function, which the iCAR strategy sets out to do. By recognizing a surface antigen that is expressed on cardiac cells or lung epithelium but is absent on tumor cells, icA r could potentially be used to protect against cross-reactivity of TCR Mage-A3 or CAR anti-ERBB2, reviving thus therapies that would otherwise be promising. Alternatively, since many tumors actively downregulate HLA molecules to escape immune recognition, HLA-targeted iCARs could provide simultaneous protection to multiple tissues.

Allogeneic lymphocytes (with a degree of immune mismatch) can be engineered to express an iCAR targeting HLA-I, an antigen universally expressed in different tissues for the treatment of metastatic breast cancer (a type of cancer with negative regulation of extremely active HLA-I). The cells can be used for administration to the patient by infusion. iCAR protects all normal or non-neoplastic tissues expressing HLA-1, while the tumor is killed due to negative or extremely low HLA-I expression.

Treatment in which a patient undergoes an HSCT for the treatment of a hematologic malignancy or as adjuvant treatment for a solid tumor is disclosed. The patient relapses or has residual disease, which is tested to be HLA-I negative or downregulated. Donor lymphocytes are engineered with an HLA-I targeted iCAR. The patient is infused with the cells. iCAR protects all normal or non-neoplastic tissues expressing HLA-I, while the tumor is killed due to negative or extremely low HLA-I expression.

In another example, a patient has a tumor that originates from a site that is not related to the skin, liver cells, or intestine (the major sites of GVDH-related mortality). Donor lymphocyte cells are engineered with iCARs that target antigens expressed on the skin, liver, intestine, or all three. For example, if the tumor has undergone epithelial-to-mesenchymal transition (EMT), as seen with tumor progression and metastatic tumors, E-cadherin and cytokeratin have been shown to be downregulated as part of this process. E-cadherin is highly expressed in normal skin, liver, and intestine (Tsuchiya et al., Arch. Histol. Cytol., 69(2): 135-145(2006)). Therefore, donor lymphocytes expressing an iCAR against E-cadherin react in a GVL manner against the tumor, but are restricted in their ability to attack the skin, liver, or intestine.

Target antigen selection for iCAR and CAR

A novel method and set of reagents for controlling responses of T lymphocytes or other immunomodulatory cells using synthetic receptors utilizing immunoinhibitory receptor (iCAR) signaling domains are disclosed. An appropriate antigen for iCARs will sometimes use a personalized medicine approach due to the natural variation of tumors. At the same time, depending on the use and type of iCAR, several possible "classes" of antigens have the potential to provide protection for several tissues at the same time. These include: (1) universally expressed immunogenic antigens that are negatively regulated by tumors, but not normal tissues, such as human leukocyte antigens (HLA). (2) Antigens downregulated in tumor progression, especially the achievement of a metastatic phenotype, but are maintained in certain normal tissues. Such antigens include: (3) EMT cell surface antigens (such as E-cadherin and cytokeratins); (4) tumor suppressor cell surface antigens, such as OPCML (Cui et al., PLoS On e. 2008; 3(8): e2990); and (6) other similar antigens such as HYAL2, DCC, SMAR1, and the like. OPCML-v1 is widely expressed at variable levels in all normal adult and fetal tissues (except the placenta and peripheral blood mononuclear cells). (7) Carbohydrates, lipids, and cell surface post-translational modifications, such as mucin-type O-glycans (core 3 O-glycans) (Lee and Fukuda, Methods Enzymol. 2010;479:143-54; Suzuki-Anekoji et al., J Bioi Chem 2011 Sep 16;286(37):32824-33;Bao and Fukuda, Methods Enzymol.2010;479:387-96). (8) Additionally, there are many other altered processes in tumors (metabolism, apoptosis, transport, differentiation, and the like) that each result in downregulation of surface antigens, any of these could be used as potential antigen targets. from iCAR.

A personal approach is disclosed that can be applied to each patient. As described herein, the iCAR antigen can be selected by an algorithmic process, after which the clinician can request a specific receptor suitable for the patient's tumor. This receptor is then introduced along with a stimulatory receptor (ie, TCR or CAR or GVL signals) to provide protection against a target tissue. selected.

It is important to note that the iCAR can bind to the same antigen as the activating CAR. For example, there could be a situation where no antigens are found that are binary in their expression between tumor and normal tissue (ie, totally absent in one and present in the other). Still, if an antigen in the tumor is expressed at a much higher level than in normal tissue, both the stimulating CAR and the iCAR receptor could target that same antigen. In the case of the tumor, because there is a large amount of antigen, the stimulating CAR would dominate and cause the elimination of the tumor. In the case of normal tissue, since the expression level of the antigen is low, iCAR could provide adequate inhibition. This is true because the levels of both/or a single target antigen and the levels of the stimulant and iCAR can affect the response of the immune-responsive cell (FIG. 23). Additionally, the change in the affinity of each receptor can be used to modulate and adjust the response.

The disparity of antigens between a target and non-target tissue may be limited by differences primarily in the level of expression rather than the absolute absence of expression. Additionally, variable expression levels can be expected if various tissues are represented with a common antigen for protection using an iCAR. An example is the expression of HLA molecules, which are found widely in most cell types, but often at different levels of expression. Interestingly, downregulation of HLA molecules represents a major mechanism of tumor escape from T cell immune responses. In such a scenario, DLI T cells engineered with an iCAR against an HLA molecule that is downregulated in malignancy could provide broad protection for various tissue types from GVDH while maintaining a GVL effect.

The combinatorial possibilities of such selection of antigens are only limited by the available antibodies, by the tumor surface antigen profile, and by known tissue-specific antigens.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4)

CTLA-4 is an inhibitory receptor expressed by activated T lymphocytes, which, when coupled with its corresponding ligands (CD80 and CD86; B7-1 and B7-2, respectively), mediates the inhibition or anergy of activated T lymphocytes. In both preclinical and clinical studies, blockade of CTLA-4 by systemic infusion of antibodies potentiated the endogenous antitumor response, albeit, in the clinical setting, with significant unforeseen toxicities.

CTLA-4 contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail. Alternative splice variants, encoding different isoforms, have been characterized. The membrane-bound isoform functions as a homodimer interconnected by a disulfide bond, while the soluble isoform functions as a monomer. The intracellular domain is similar to that of CD28, in that it has no intrinsic catalytic activity and contains a YVKM motif capable of binding PI3K, PP2A, and SHP-2 and a proline-rich motif capable of binding SH3-containing proteins. A role of CTLA-4 in inhibiting T cell responses appears to be directly through SHP-2 and PP2A dephosphorylation of TCR-proximal signaling proteins such as CD3 and LAT. CTLA-4 may also affect signaling indirectly by competing with CD28 for binding to CD80/86. CTLA-4 has also been shown to bind and/or interact with PI3K, CD80, AP2M1, and PPP2R5A.

CTLA-4 has an amino acid sequence as set forth in SEQ ID NO: 5.

1 MACLGFQRHK AQLNLATRTW PCTLLFFLLF IPVFCKAMHV AQPAWLASS RGXASPVCEY

⁶¹ ASPGKATEVRVTVXRQADSQ VTEVCAATYM MGNELTFLDD SICTGTSSGN QVNLTIQGLR

121 AMDTGLYICK VELMYPPPYY LGIGNGTQTY VIDPEPCPDS DFLLWILAAV SSGLPFYSFL

183. I.TAVSLSKML KKRSPLTTGV YVKMPPTEPE CEKQFQPYFI PIN

In accordance with the present invention, a CTLA-4 polypeptide may have an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO : 5 (Homology herein can be determined using a standard computer program such as BLAST or FASTA). In non-limiting embodiments, a CTLA-4 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 5 having at least 20, or at least 30, or at least 40, or at least 50, and up to 222 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, the CTLA-4 polypeptide has an amino acid sequence of amino acids 1 to 223, 1 to 50, 50 to 100, 100 to 140, 141 to 161, 162 to 182, 183 to 223, 141 to 223, 162 to 223, or 183 to 223 of SEQ ID NO: 5. In one embodiment, the CTLA-4 polypeptide has an amino acid sequence of amino acids 183 to 223 of SEQ ID NO: 5. In certain embodiments, the intracellular signaling domain of iCAR includes a CTLA-4 polypeptide having an amino acid sequence from amino acids 183 to 223 of SEQ ID NO: 5. In certain embodiments, the transmembrane domain of iCAR includes a CTLA-4 polypeptide having an amino acid sequence of amino acids 162 to 182 of SEQ ID NO: 5.

In accordance with the present disclosure, a "CTLA-4 nucleic acid molecule" refers to a polynucleotide that encodes a CTLA-4 polypeptide.

Programmed cell death protein 1 (PD-1)

PD-1 is a negative immunoregulator of activated T cells after coupling with their corresponding PD-L1 and PD-L2 ligands expressed on dendritic cells and endogenous macrophages. PD-1 is a 268 amino acid type I membrane protein. PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. The structure of the protein includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located at an immune receptor inhibition tyrosine motif and an immune receptor tyrosine switch motif, with PD-1 downregulating TCR signals. SHPI and SHP-2 phosphatases bind to the cytoplasmic tail of PD-1 after ligand binding. Upregulation of PD-L1 is one mechanism by which tumor cells can evade the host's immune system. In preclinical and clinical trials, blockade of PD-1 by antagonist antibodies induced antitumor responses mediated through the host's endogenous immune system.

PD-1 has an amino acid sequence as set forth in SEQ ID NO: 6.

1 mqipqapwpv vwavlqlgwr pgwfldspdr pwnpptfspa llwtegdna tftcsfsnts 61 esfvlnwyrm spsnqtdkla afpedrsqpg qdcrfrvtql pngrdfhmsv vrarrndsgt 121 ylegaislap kaqikeslra elrvterrae vptahpspsp rpagqfqtlv vgwggllgs 181 lvllvwvlav icsraargti garrtgqplk edpsavpvfs vdygeldfqw rektpeppvp 241 cvpeqteyat ivfpsgmgts sparrgsadg prsaqplrpe dghcswpl

In accordance with the present invention, a PD-1 polypeptide may have an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO : 6. In non-limiting embodiments, a PD-1 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 6 that has at least 20, or at least 30, or at least 40, or at least 50, and up to 287 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, a PD-1 polypeptide has an amino acid sequence of from 1 to 288, 1 to 50, 50 to 100, 100 to 144, 145 to 170, 171 to 191, 192 to 288, 145 to 288, 171 to 288, or 192 to 288 of SEQ ID NO: 6. In one embodiment, the PD-1 polypeptide has an amino acid sequence of amino acids 192 to 288 of SEQ ID NO: 6. In certain embodiments, the intracellular signaling domain of iCAR includes a PD-1 polypeptide having an amino acid sequence of amino acids 192 to 288 of SEQ ID NO: 6. In certain embodiments, the transmembrane domain of iCAR includes a PD polypeptide -1 having an amino acid sequence of amino acids 171 to 191 of SEQ ID NO: 6.

In accordance with the present disclosure, a "PD-1 nucleic acid molecule" refers to a polynucleotide that encodes a PD-1 polypeptide.

Lymphocyte Activating Gene 3 (LAG-3)

Lymphocyte activation protein 3 (LAG-3) is a negative immune regulator of immune cells. LAG-3 belongs to the immunoglobulin (Ig) superfamily and contains 4 extracellular Ig-like domains. The LAG3 gene contains 8 exons. Sequence data, exon/intron organization and chromosomal location indicate a close relationship of LAG3 with CD4. LAG3 has also been called CD223 (group of differentiation 223).

LAG-3 has an amino acid sequence as set forth in SEQ ID NO: 7.

1 mweaqflgll fIqplwvapv kplqpgaeyp wwaqegapa qlpcsptípl qdlsllrrag

swgprprryt vlsvgpgglr sgrlplqprv 121 qldergrqrg dfslwlrpar radageyraa vhlrdralsc rlrlrlgqas mtasppgslr 181 asdwvilncs fsrpdrpasv hwfrnrgqgr vpvresphhh laesflflpq vspmdsgpwg 241 ciltyrdgfn vsimynltvl glepptpltv yagagsrvgl pcrlpagvgt rsfltakwtp 301 pgggpdllvt gdngdftlrl edvsqaqagt ytchihlqeq qlnatvtlaí itvtpksfgs 361 pgslgkllce vtpvsgqerf vwssldtpsq rsfsgpwlea qeaqllsqpw qcqlyqgerl61 vtwqhqpdsg ppaaapghpl

swgprprryt vlsvgpgglr sgrlplqprv 121 dfslwlrpar qldergrqrg vhlrdralsc radageyraa mtasppgslr rlrlrlgqas 181 asdwvilncs fsrpdrpasv hwfrnrgqgr vpvresphhh laesflflpq vspmdsgpwg 241 vsimynltvl ciltyrdgfn glepptpltv yagagsrvgl pcrlpagvgt rsfltakwtp 301 pgggpdllvt gdngdftlrl edvsqaqagt ytchihlqeq qlnatvtlaí itvtpksfgs 361 pgslgkllce vtpvsgqerf vwssldtpsq rsfsgpwlea qeaqllsqpw qcqlyqgerl

421 Igaavyftel sspgaqrsgr apgalpaghl llflilgvls llllvtgafg fhlwrrqwrp 481 rrfsaleqgi hppqaqskie eleqepepep epepepepep epeql

In accordance with the present invention, a LAG-3 polypeptide may have an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO : 7. In non-limiting embodiments, a LAG-3 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 7 that has at least 20, or at least 30, or at least 40, or at least 50, and up to 524 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, a LAG-3 polypeptide has an amino acid sequence of from 1 to 525, 1 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 420, 421 to 450, 451 to 471, 472 to 525, 421 to 525, 451 to 525, or 472 to 525 of SEQ ID NO: 7. In one embodiment, the LAG-3 polypeptide has an amino acid sequence of amino acids 472 to 525 of SEQ ID NO: 7. In certain embodiments, the intracellular signaling domain of iCAR includes a LAG-3 polypeptide having an amino acid sequence of amino acids 472 to 525 of SEQ ID NO: 7. In certain embodiments, the iCAR transmembrane domain includes a LAG-3 polypeptide having an amino acid sequence of amino acids 451 to 471 of SEQ ID NO: 7.

In accordance with the present disclosure, a "LAG-3 nucleic acid molecule" refers to a polynucleotide that encodes a LAG-3 polypeptide.

Natural Killer Cell Receptor 2B4 (2B4)

The natural killer cell receptor 2B4 (2B4) mediates non-MHC-restricted cell killing in NK cells and T cell subsets. To date, the function of 2B4 is still under investigation, the 2B4-S isoform is believed to be an activating receptor, and the 2B4-L isoform is believed to be a negative immune regulator of immune cells. 2B4 is activated by binding to its high-affinity ligand, CD48. 2B4 contains a tyrosine switch motif, a molecular switch that allows the protein to associate with various phosphatases. 2B4 has also been called CD244 (group of differentiation 244).

2B4 has an amino acid sequence as set forth in SEQ ID NO: 8.

1 mlgqwtlil llllkvyqgk gcqgsadhw sisgvplqlq pnsíqtkvds íawkkllpsq

61 ngfhhilkwe ngslpsntsn drfsfivknl sllikaaqqq dsglyclevt sísgkvqtat 121 fgvfvfesll pdkvekprlq gqgkíldrgr cqvalsclvs rdgnvsyawy rgsklíqtag 181 nltyldeevd ingthtytcn vsnpvswesh tlnltqdcqn ahqefrfwpf Ivlivllsal 241 fIgtlacfcv wrrkrkekqs etspkeflti yedvkdlktr rnheqeqtfp gggstiysmi 301 qsqssaptsq epaytlysli qpsrksgsrk rnhspsfnst iyevlgksqp kaqnparlsr

361 kelenfdvys

In accordance with the present invention, a 2B4 polypeptide may have an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO: 8 In non-limiting embodiments, a 2B4 polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 8 having at least 20, or at least 30, or at least 40, or at least 50, and up to 369 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, a 2B4 polypeptide has an amino acid sequence of from 1 to 370, 1 to 50, 50 to 100, 100 to 150, 150 to 215, 216 to 229, 230 to 250, 251 to 370, 216 to 370, 230 to 370, or 251 to 370 of SEQ ID NO: 8. In one embodiment, the 2B4 polypeptide has an amino acid sequence of amino acids 251 to 370 of SEQ ID NO: 8. In certain embodiments, the iCAR intracellular signaling domain includes a 2B4 polypeptide having an amino acid sequence of amino acids 251 to 370 of SEQ ID NO: 8. In certain embodiments, the iCAR transmembrane domain includes a 2B4 polypeptide having an amino acid sequence from amino acids 230 to 250 of SEQ ID NO: 8.

In accordance with the present disclosure, a "2B4 nucleic acid molecule" refers to a polynucleotide that encodes a 2B4 polypeptide.

B and T Lymphocyte Attenuator (BTLA)

B- and T-lymphocyte attenuator (BTLA) expression is induced during T-lymphocyte activation, and BTLA remains expressed on Th1 cells, but not on Th2 cells. Like PD1 and CTLA4, BTLA interacts with a B7 homologue, B7H4. However, unlike PD-1 and CTLA-4, BTLA exhibits T cell inhibition through interaction with tumor necrosis family receptors (TNF-Rs), not just the B7 family of cell surface receptors. . BTLA is a ligand for tumor necrosis factor (receptor) superfamily member 14, (TNFRSF14), also known as herpes virus entry mediator (HVEM). BTLA-HVEM complexes negatively regulate the immune response of T cells. Activation of BTLA has been shown to inhibit the function of human CD8+ cancer-specific T cells. BTLA has also designated CD272 (group of differentiation 272).

BTLA has an amino acid sequence as set forth in SEQ ID NO: 9.

1 MKTLPAMLGT GKLFWVFFLIPYLDXWNIHG KESCDVQLYIKRQSEHSILA GDPFELECPV

61 KYCANRPHVT WCKLNGTTCVKLEDRQTSWK EEKNISFFILHFEPVLPNDN GSYRCSANFQ

121 SNLIESHSTT LYVTDVKSASERPSKDEMAS RPWLLYRLLPLGGLPLLITT CFCLFCCLRR

181 HQGKQKELSD TAGREINLVDAHLKSEQTEA STRQNSQVLLSETGIYDNDP DLCFRMQEGS

241 EVYSNPCLEE NKPGIVYASLNHSVIGPNSR LARNVKEAPTEYASICVRS

In accordance with the present invention, a BTLA polypeptide may have an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homologous to SEQ ID NO: 9 In non-limiting embodiments, a BTLA polypeptide can have an amino acid sequence that is a consecutive portion of SEQ ID NO: 9 having at least 20, or at least 30, or at least 40, or at least 50, and up to 288 amino acids in length. Alternatively or additionally, in various non-limiting embodiments, a BTLA polypeptide has an amino acid sequence of from 1 to 289, 1 to 50, 50 to 100, 100 to 134, 135 to 157, 158 to 178, 179 to 289, 135 to 289, 158 to 289, or 179 to 289 of SEQ ID NO: 9. In one embodiment, the BTLA polypeptide has an amino acid sequence of amino acids 179 to 289 of SEQ ID NO: 9. In certain embodiments, the iCAR intracellular signaling domain includes a BTLA polypeptide having an amino acid sequence of amino acids 179 to 289 of SEQ ID NO: 9. In certain embodiments, the iCAR transmembrane domain includes a BTLA polypeptide having an amino acid sequence of amino acids 158 to 178 of SEQ ID NO: 9.

In accordance with the present disclosure, a "BTLA nucleic acid molecule" refers to a polynucleotide that encodes a BTLA polypeptide.

Hematopoietic cell lineages

Mammalian hematopoietic (blood) cells provide a wide range of physiological activities. Hematopoietic cells are divided into lymphoid, myeloid, and erythroid lineages. The lymphoid lineage, which comprises B, T, and natural killer (NK) cells, is responsible for antibody production, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. . The term "T cells" as used herein refers to lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T lymphocytes are involved in the adaptive immune system. The term "natural killer (NK) cells" as used herein refers to lymphocytes that are part of cell-mediated immunity and function during the innate immune response. They do not require prior activation to perform their cytotoxic effect on target cells. Cytotoxic T lymphocytes (cTl or suppressor T lymphocytes) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.

Cells for use in the methods of the invention

Disclosed herein are cells that express a combination of an antigen recognition receptor that activates an immunoresponsive cell (eg, TCR, CAR) and an inhibitory chimeric antigen receptor (iCAR), and methods of using such cells for the treatment of a disease that requires an enhanced immune response. In one approach, tumor antigen-specific T cells (all subsets, including cD4, CD8, memory, wild-type, effector, T-reg, etc.), innate immune system cells, NK cells, CTL cells, or other immunosensitive cells to express an iCAR that binds to an antigen in non-tumor tissue, for the treatment or prevention of neoplasia. For example, a T cell expressing a 19-28z chimeric antigen receptor that recognizes CD19 is co-expressed with a T cell expressing an iCAR that binds CD33. These cells are administered to a human subject in need of them for the treatment or prevention of hematologic cancers (eg, leukemias, lymphomas, and myelomas). In another approach, viral antigen-specific T cells, NK cells, CTL cells can be used for the treatment of viral diseases. The cells may express an endogenous or recombinant antigen receptor, which may be 19-28z specific for CD19, P28z which is specific for PSMA, M28z which is specific for mesothelin, or 56-28z which is specific for CD56.

A patient's own T cells can be genetically modified to target tumors by introducing genes that encode artificial T cell receptors called chimeric antigen receptors (CARs). CARs include CARs that activate an immune response and iCARs that suppress an immune response.

Tumor antigen-specific T lymphocytes ( and NK cells)

Types of tumor antigen-specific human lymphocytes that can be used in the methods disclosed in the herein are, without limitation, peripheral donor lymphocytes genetically modified to express chimeric antigen receptors (CAR) (Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45), peripheral donor lymphocytes genetically modified to express a full-length tumor antigen recognition T-cell receptor complex comprising the ay heterodimer (Morgan, RA, et al. 2006 Science 314:126-129), tumor-infiltrating lymphocyte (TIL)-derived lymphocyte cultures in English) 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 specific peripheral blood leukocytes of antigen using artificial antigen presenting cells (AAPC) or pulsed dendritic cells (Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, GA, et al. 2003 Blood 102:2498-2505). The T cells may be autologous, nonautologous (eg, allogeneic), or derived in vitro from engineered stem or progenitor cells. T cells can be prepared en bloc as is commonly done with peripheral blood lymphocytes (PBLs) or tumor infiltrating lymphocytes (TILs), T cells can be purified using, for example, CD4, CD8, CD62L .

Any suitable tumor antigen (antigenic peptide) is suitable for use in the tumor-related embodiments described herein. Sources of antigens that activate the immune response include, but are not limited to, cancer proteins. The antigen may be expressed as a peptide or as an intact protein or portion thereof. The intact protein or a portion thereof may be native or mutagenized. Suitable immune response activating antigens include prostate specific membrane antigen (PSMA) and prostate stem cell antigen (PCSA).

Viral antigen-specific T lymphocytes ( and NK cells)

Antigens suitable for use in treating pathogenic infections or other infectious diseases, for example, in an immunocompromised subject include, without limitation, viral antigens present in Cytomegalovirus (CMV), Epstein-Barr virus (EBV), in English), human immunodeficiency virus (HIV), and influenza viruses.

The unpurified source of CTL can be any known in the art, such as fetal, neonatal, or adult bone marrow or other hematopoietic cell source, eg, fetal liver, peripheral blood, or umbilical cord blood. Various techniques can be used to separate the cells. For example, negative selection methods may initially remove non-CTLs. The mAbs are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation for both positive and negative selections.

A large proportion of terminally differentiated cells can be initially removed by relatively crude separation. For example, magnetic bead separations can be used initially to remove large numbers of irrelevant cells. Preferably, at least about 80%, generally at least 70%, of the total hematopoietic cells will be removed prior to cell isolation.

Procedures for separation include, but are not limited to, density gradient centrifugation; reinstatement; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents attached to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and selection with antibody fixed to a solid matrix, eg, a plate, microplate, elutriation, or any other convenient technique.

Separation and analysis techniques include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, for example, a plurality of color channels, obtuse and low-angle light scattering detection channels, impedance channels .

Cells can be selected against dead cells, using dyes associated with dead cells such as propidium iodide (PI). Preferably, the cells are harvested in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable isotonic medium, preferably sterile.

Accordingly, the invention generally provides an immune-responsive cell, such as a virus-specific or tumor-specific T cell, comprising a receptor that binds a first antigen and activates the immune-responsive cell and a receptor that binds a second antigen and activates the immunoresponsive cell. inhibits the immune cell.

Vectors

Genetic modification of immune-responsive cells (eg, T cells, CTL cells, NK cells) can be achieved by transducing a substantially homogeneous cell composition with a recombinant DNA or RNA construct. Preferably, a retroviral vector (either gamma retroviral or lentiviral) is used for the introduction of the DNA or RNA construct into the genome of the host cell. For example, a polynucleotide encoding a receptor that binds an antigen (eg, a tumor antigen, or variant, or fragment thereof), can be cloned into a retroviral vector and expression can be driven by its endogenous promoter, from the retroviral long terminal repeat, or from an alternative internal promoter. Non-viral vectors or RNAs can also be used. Random chromosomal integration or targeted integration (for example, using a nuclease, transcriptional activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and/or clusters of regularly spaced short palindromic repeats (CRISPR) can be used. ), or transgenic expression (for example, using a natural or chemically modified RNA).

For the initial genetic modification of cells to provide viral or tumor antigen-specific cells, a retroviral vector is generally employed for transduction, however, any other suitable viral vector or non-viral delivery system may be used. For subsequent genetic modification of cells to provide cells comprising an antigen presenting complex comprising at least two costimulatory ligands, retroviral gene transfer (transduction) is also effective. Combinations of retroviral vector and a suitable packaging line, where the capsid proteins will be functional to infect human cells, are also suitable. Several amphotropic virus-producing cell lines 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, for example, VSVG, RD114 or GALV enveloped pseudotyped particles and any others known in the art.

Possible transduction methods also include direct co-cultivation of cells with producer cells, eg, by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culture with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, eg, by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J.Clin. Invest. 89:1817.

Transducing viral vectors can be used to express a costimulatory ligand in an immunoresponsive cell. Preferably, the chosen vector exhibits high infection efficiency and stable integration and expression (see, eg, 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 272:263267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94: 10319, 1997). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated virus vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr virus (see also, for example, the vectors of 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 , nineteen ninety five). 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., US Patent No. 5,399,346) .

Non-viral approaches to expression of a protein in the cell can also be employed. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Nat!. Acad. Sci. USA 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). Other non-viral means of gene transfer include transfection. in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for the delivery of DNA to a cell. Transplantation of normal genes into affected tissues of a subject can also be achieved by transferring a normal nucleic acid into a cultivable cell type. ex vivo (eg, an autologous or heterologous primary cell or its progeny), after which the cell (or its progeny) is injected into a target tissue or injected systemically. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (eg, zinc finger nucleases, meganucleases, or TALE nucleases). Transient expression can be obtained by electroporation of RNA.

Expression of eDNA for use in methods of polynucleotide therapy can be driven from any suitable promoter (for example, human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any suitable promoter. appropriate mammalian regulatory element or intron (eg, the enhancer/promoter/intron structure of elongation factor 1a). For example, if desired, enhancers known to preferentially drive gene expression in specific cell types can be used to drive expression of a nucleic acid. Enhancers used may include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation may be mediated by cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoter or regulatory elements described above.

The resulting cells can be grown under conditions similar to those of unmodified cells, thus the modified cells can be expanded and used for various purposes.

Polypeptides and analogs

Also included in the invention are aCD19, aPSMA, CD28, CD3Z, CTLA-4, PD-1, and 19-28z polypeptides or fragments thereof that are modified in ways that enhance their antineoplastic activity (eg, a humanized monoclonal antibody). or inhibit their cytotoxic activity (eg, an iCAR) when expressed in an immune-responsive cell. Methods for optimizing an amino acid sequence or a nucleic acid sequence by producing an alteration in the sequence are disclosed. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. Analogs of any naturally occurring polypeptides disclosed herein are further disclosed. Analogs may differ from a naturally occurring polypeptide of the invention by differences in amino acid sequence, by post-translational modifications, or by both. Analogs disclosed herein will generally exhibit at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to the all or part of a naturally occurring amino acid sequence. 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 more than 100 amino acid residues. Again, in an illustrative approach to determining degree of identity, a BLAST program can be used, with a probability score between e-3 and e-100 indicating a closely related sequence. Modifications include chemical derivatization of polypeptides in vivo and in vitro, eg, acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or after treatment with isolated modifying enzymes. The analogs may also differ from the naturally occurring polypeptides of the invention by alterations in the primary sequence. These include genetic variants, both natural and induced (eg, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by specific mutagenesis as described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed.), CSH Press, 1989, or Ausubel et al., cited above). Also included are cyclized peptides, molecules, and analogs containing moieties other than L-amino acids, eg, D-amino acids or synthetic or non-naturally occurring amino acids, eg, beta or gamma amino acids.

In addition to full-length polypeptides, the invention also provides fragments of any one of the polypeptides or peptide domains of the invention. As used herein, the term "a fragment" means at least 5, 10, 13, or 15 amino acids. In other examples, a fragment has 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 to 80, 100, 200, 300 or more contiguous amino acids. The fragments disclosed herein may be generated by methods known to those of skill in the art or may be the result of normal protein processing (eg, removal of amino acids from the nascent polypeptide that are not necessary for biological activity or removal of amino acids). by alternative splicing of mRNA or alternative protein processing events).

Non-protein analogs have a chemical structure designed to mimic the functional activity of a protein disclosed herein. Such analogs are administered according to the methods disclosed herein. Such analogs may exceed the physiological activity of the parent polypeptide. Analog design methods are well known in the art, and analog synthesis can be carried out according to such methods by modifying the chemical structures such that the resulting analogs enhance the antineoplastic activity of the parent polypeptide when expressed in a immunosensitive cell. These chemical modifications include, but are not limited to, substitution of alternative R groups and varying the degree of saturation at specific carbon atoms of a reference polypeptide. Preferably, the protein analogs are relatively resistant to degradation in vivo, resulting in a longer therapeutic effect after administration. Assays for measuring functional activity include, but are not limited to, those described in the examples below.

costimulatory ligands

Interaction with at least one costimulatory ligand provides a non-antigen specific signal important for full activation of an immune cell (eg, T cell). Costimulatory ligands include, without limitation, tumor necrosis factor (TNF) ligands, cytokines (such as IL-2, IL-12, IL-15, or IL21), and ligands of the immunoglobulin (Ig) superfamily. Tumor necrosis factor (TNF) is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its main role is the regulation of immune cells. Tumor necrosis factor (TNF) ligands share several common features. Most ligands are synthesized as type II (extracellular C-terminus) transmembrane proteins that contain a short cytoplasmic segment and a relatively long extracellular region. TNF ligands include, without limitation, nerve growth factor (NGF), CD40L (CD40L)/CD154, CDI137L/4-1BBL, tumor necrosis factor alpha (TNFa), CD134L/OX40L/CD252 , CD27L/CD70, Fas ligand (FasL), CD30L/CD153, tumor necrosis factor beta (TNFp)/lymphotoxin alpha (LTa), lymphotoxin-beta (LTp), CD257/B cell activating factor (BAFF)/Biys /TANK/Tall-1, glucocorticoid-induced TNF receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14).

The immunoglobulin (Ig) superfamily is a large group of soluble and cell-surface proteins that participate in cell recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins and possess an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, without limitation, CD80, CD86, and ICOS, both of which are ligands for CD28.

Management

Compositions comprising genetically modified immunoresponsive cells of the invention (for example, T lymphocytes, cells of the innate immune system, NK cells, CTL cells, or their progenitors) may be for systemic administration or directly to a subject for the treatment of a neoplasm. . In one embodiment, the cells of the invention are for administration injected directly into an organ of interest (eg, an organ affected by neoplasia). Alternatively, compositions comprising genetically modified immune-responsive cells are for indirect administration to the organ of interest, eg, by administration into the circulatory system (eg, tumor vasculature). Differentiating and expanding agents can be provided before, during, or after administration of the cells to increase the production of T cells, innate immune system cells, NK cells, or CTL cells in vitro or in vivo.

The modified cells can be administered in any physiologically acceptable vehicle, usually intravascularly, but can also be introduced into bone or other convenient site where the cells can find an appropriate site for regeneration and differentiation (eg, the thymus). Typically, at least 1x105 cells will be administered, eventually reaching 1x1010 or more. The genetically modified immunoresponsive cells of the invention may comprise a purified population of cells. Those skilled in the art can readily determine the percentage of genetically modified immune-responsive cells in a population using several well-known methods, such as fluorescence-activated cell sorting (FACS). Preferred ranges of purity in populations comprising 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 about 70 to about 75%, about 75 to about 80%, 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%. Dosages can be easily adjusted by those skilled in the art (eg, a decrease in purity may require an increase in dosage). The cells can be introduced by injection, catheter, or the like. If desired, factors may also be included, including, but not limited to, interleukins, eg, IL-2, IL-3, IL-6, IL-11, IL7, IL12, IL15, IL21, as well as the other interleukins, colony stimulating factors, such as G-, M- and GM-CSF, interferons, eg interferon gamma and erythropoietin.

Compositions of the invention include pharmaceutical compositions comprising genetically modified immunoresponsive cells or their progenitors and a pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject and administered to the same or a different compatible subject. The peripheral blood-derived immunoresponsive cells of the invention or their progeny (eg, obtained in vivo, ex vivo, or in vitro ) may be for administration by localized injection, including administration by catheter, systemic injection, localized injection, intravenous injection or parenteral administration. When a therapeutic composition of the present invention (eg, a pharmaceutical composition containing a genetically modified immune-responsive cell) is administered, it will generally be formulated in an injectable unit dose form (solution, suspension, emulsion).

formulations

Compositions of the invention comprising genetically modified immunoresponsive cells may conveniently be provided as sterile liquid preparations, for example isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which may be buffered at a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific fabrics. Liquid or viscous compositions may comprise carriers, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the genetically modified immune responsive cells used in the practice of the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients as desired. Such compositions may be mixed 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 may contain auxiliary substances such as wetting, dispersing, or emulsifying agents (eg, methylcellulose), pH buffering agents, gelling additives or viscosity enhancers, preservatives, flavoring agents, colors, and the like, depending on the route of administration and the desired preparation. Standard texts such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th edition, 1985 can be consulted to prepare suitable preparations, without undue experimentation.

Various additives may be added that enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid and the like. Prolonged absorption of the injectable dosage form can be achieved by the inclusion of absorption delaying agents, for example, alum monostearate and gelatin. According to the present disclosure, however, any carrier, diluent or additive used would have to be compatible with the genetically modified immunoresponsive cells or their progenitors.

The compositions can be isotonic, that is, they can have the same osmotic pressure as blood and tear fluid. 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 particularly preferred for buffers containing sodium ions.

The viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose is preferred because it is readily and inexpensively available and easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The preferred concentration of thickening agent will depend on the agent selected. The important point is to use an amount that achieves the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, eg, liquid dosage form (for example, whether the composition is to be formulated in a solution, a suspension , gel, or other liquid form, such as a timed-release form or a liquid-filled form).

Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the genetically modified immunoresponsive cells as described herein. This will not present any problem to those skilled in chemical and pharmaceutical principles, or the problems can be easily avoided by reference to conventional texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited in the this document.

A consideration related to the therapeutic use of the genetically modified immunoresponsive cells of the invention is the number of cells needed to achieve optimal effect. The amount of cells to be administered will vary for the subject being treated. In one embodiment, between 104 and 1010 between 105 and 109 or between 106 and 108 genetically modified immunoresponsive cells of the invention will be administered to a subject. More efficient cells can be administered in even smaller amounts. In some embodiments, at least about 1 x 108, 2 x 108, 3 x 108, 4 x 108, and 5 x 108 genetically modified immunoresponsive cells of the invention will be administered to a human subject. Precise determination of what would be considered an effective dose may be based on individual factors for each subject, including the size, age, sex, weight, and condition of the particular subject. Dosages can be readily determined by those skilled in the art from this disclosure and knowledge in the art.

Those skilled in the art can readily determine the amount of cells and optional additives, carriers and/or carriers in compositions for administration to a patient. Typically, any additive (in addition to the one or more active cells and/or the one or more agents) is present in an amount from 0.001 to 50% (by weight) 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, most 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 human, and for any particular method of administration, it is therefore preferred to determine: toxicity, such as by determining the lethal dose (LD) and LD50 in an suitable animal model, eg, a rodent such as a mouse; and, the dose of the one or more compositions, concentration of components therein and administration time of the compositions), which elicits an adequate response. Such determinations do not require undue experimentation based on the knowledge of those skilled in the art, this disclosure, and the documents cited herein. And, the time for sequential administrations can be determined without undue experimentation.

Treatment methods disclosed herein

Methods of treating neoplasms in a subject are disclosed herein. Also contemplated herein are methods of treating a pathogen infection or other infectious disease in a subject, such as an immunocompromised human subject. The disclosed methods comprise administering a T cell of the claims, an innate immune system cell, NK cell, or CTL cell disclosed herein in an amount effective to achieve the desired effect, whether palliation of an existing condition or prevention of recurrence. . For treatment, the amount administered is an amount effective to produce the desired effect. An effective amount may be provided in one or a series of administrations. An effective amount can be given as a bolus or by continuous infusion.

An "effective amount" (or "therapeutically effective amount") is an amount sufficient to effect a beneficial or desired clinical result after treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the knowledge of one of ordinary skill in the art. Various factors are normally taken into account when determining a suitable dosage 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 effective form and concentration of the administered antigen-binding fragment.

For adoptive immunotherapy using antigen-specific T cells, cell doses in the range of 106-1010 (eg, 109) are typically infused. After the administration of the genetically modified cells in the host and their subsequent differentiation, T lymphocytes that are specifically directed against the specific antigen are induced. "Induction" of T cells can include inactivation of antigen-specific T cells such as by deletion or anergy. Inactivation is particularly useful in establishing or re-establishing tolerance, such as in autoimmune disorders. The modified cells can be administered by any method known in the art including, but not limited to, intravenous, subcutaneous, intraganglionic, intratumoral, intrathecal, intrapleural, intraperitoneal, and directly to the thymus.

Therapeutic methods disclosed herein

Methods of increasing an immune response in a subject in need thereof are disclosed herein. Methods of treating or preventing a neoplasm in a subject are disclosed. The present invention provides the T cell of the claims for use in reducing tumor burden and/or tumor size in a subject, and/or increasing survival of a subject having a neoplasm in a subject in in which T cells are particularly useful for the treatment of subjects having hematologic cancers (eg, leukemias, lymphomas, and myelomas) or ovarian cancer, which are not amenable to conventional therapeutic interventions. Human subjects suitable for therapy typically comprise two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are those who carry a clinically measurable tumor. A clinically measurable tumor is one that can be detected based on the tumor mass (for example, by palpation, CT scan, ultrasound, mammography, or X-ray; positive biochemical or histopathological markers alone are insufficient to identify this population). A pharmaceutical composition is incorporated into this invention for administration to these subjects to elicit an antitumor response, with the aim of palliating their condition. Ideally, in turn, reduction or mass occurs as a result, but any clinical improvement is a benefit. Clinical improvement includes decreased risk or rate of progression or reduced pathologic consequences of the tumor.

A second group of suitable subjects is known in the art as the "adjuvant group". These are individuals who have had a history of neoplasia, but have responded to another mode of therapy. Prior therapy may have included, but is not restricted to, surgical resection, radiation therapy, and traditional chemotherapy. As a result, these individuals do not have a clinically measurable tumor. However, they are suspected to be at risk of disease progression, either near the site of the original tumor site or by metastasis. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made based on the characteristics observed before or after the initial treatment. These features are known in the clinical art and are appropriately defined for each different neoplasm. Typical features of high-risk subgroups are those in whom the tumor has invaded neighboring tissues or those who show lymph node involvement.

Another group has a genetic predisposition to neoplasia, but has not yet shown clinical signs 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 treatment prophylactically to prevent breast cancer. appearance of neoplasia until it is suitable for preventive surgery.

Human subjects with neoplasms having any of the following neoplasms: glioblastoma, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate and small cell lung cancer) are especially 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 neural ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, ductal adenocarcinoma pancreatic, small cell and non-small cell lung adenocarcinomas, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and liver metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovium, 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 carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, tumor Wilms' r, 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 adenocarcinoma and lobular, squamous and adenocarcinomas of the cervix, uterine and ovarian epithelial carcinomas, prostate adenocarcinomas, transitional squamous cell carcinoma of the bladder, lymphomas of B and T cells (nodular and diffuse), plasmacytoma, acute and chronic leukemias, malignant melanoma, sarcomas of soft tissues and leiomyosarcomas.

Subjects may have an advanced form of disease, in which case the goal of treatment may include mitigation or reversal of disease progression and/or amelioration of side effects. Subjects may have a history of the disease, for which they have already been treated, in which case the therapeutic goal will typically include a decrease or delay in the risk of recurrence.

Accordingly, a method of treating or preventing neoplasia in a subject is disclosed, the method comprising administering an effective amount of an immune-responsive cell comprising a receptor that binds a tumor antigen and activates the immune-responsive cell (eg, TCR, CAR) and a vector encoding an inhibitory chimeric antigen receptor (iCAR) that binds to a target antigen and suppresses the immune-responsive cell. As a consequence of the surface expression of a receptor that binds to a tumor antigen and activates the immunosensitive cell (eg, TCR, CAR) and a vector encoding an inhibitory chimeric antigen receptor (iCAR) that binds to a target antigen and suppresses the immunosensitive cell, adoptively transferred human T lymphocytes or NK cells are endowed with selective cytolytic activity.

In one example, the neoplasm is selected from the group consisting of hematologic cancers (eg, leukemias, lymphomas, and myelomas), ovarian cancer, sarcoma, and acute myeloid leukemia (AML), prostate cancer, breast cancer, bladder cancer , brain cancer, colon cancer, bowel cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, and throat cancer. In another example, the tumor antigen is one or more of carbonic anhydrase IX (CAlX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38 , CD41, CD44, CD49f, CD56, CD74, CD123, CD133, CD138, an antigen from a cytomegalovirus (CMV)-infected cell (for example, a 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), k light chain, receptor domain Kinases Insertion (KDR), Lewis Y (LeY), L1 Cell Adhesion Molecule (L1CAM), Melanoma Antigen Family A 1 (MAGE-AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), mesothelin (MSLN), NKG2D ligands, testicular cancer antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane (PSMA), tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor-R2 (VEGF-R2), or Wilms tumor protein (WT-1).

Other methods of treating subjects with a pathogen infection (eg, viral infection, bacterial infection, fungal infection, parasitic infection, or protozoal infection) are disclosed. The disclosed methods are particularly useful for enhancing an immune response in an immunocompromised subject. Illustrative viral infections amenable to treatment using a method disclosed herein include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), human immunodeficiency virus (VHIV), and influenza virus infections. Accordingly, a method of treating or preventing a pathogen infection in a subject is disclosed, the method comprising administering an effective amount of an immunocompromised cell as described herein.

kits

Kits for the treatment or prevention of a neoplasm, pathogen infection, immune disorder or allogeneic transplant are disclosed. The disclosed kit may include a therapeutic or prophylactic composition containing an effective amount of an immunoresponsive cell comprising an activating antigen receptor and an inhibitory chimeric antigen receptor (iCAR) in unit dosage form. The kit may comprise a sterile container containing a therapeutic or prophylactic vaccine; such containers can be boxes, ampoules, bottles, vials, tubes, pouches, sachets, blister packs, or other suitable form of packaging known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing medicaments.

If desired, the immunoresponsive cell is provided along with instructions for administering the cell to a subject having or at risk of neoplasia, pathogen infection, immune disorder, or allogeneic transplant. The instructions will generally include information on the use of the composition for the treatment or prevention of neoplasia, pathogen infection, immune disorder or allogeneic transplantation. The instructions may also include at least one of the following: description of the therapeutic agent; dosage regimen and administration for the treatment or prevention of 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 (where present), or as a label applied to the container, or as a separate sheet, booklet, card, or sheet supplied in or with the container.

examples

The practice of the present invention employs, unless otherwise indicated, standard techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the purview of the skilled person. Such techniques are explained in detail in references 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, I 996); "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 techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in the preparation and practice of the invention. Techniques particularly useful for particular embodiments will be discussed in the sections below.

The following examples are presented to provide those 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 their invention.

Example 1. Inhibitory Chimeric Antigen Receptor (iCAR) Diverts Off-Target Responses.

SUMMARY

T cell therapies have shown long-term efficacy and curative potential for the treatment of some cancers. However, their use is limited by damage to neighboring tissues, as seen in graft-versus-host disease after infusion of donor lymphocytes or in "on-target, off-tumor" toxicities caused by some therapies. with engineered T cells. Non-specific immunosuppression and irreversible T-lymphocyte depletion are currently the only means of controlling such deleterious responses, but at the cost of nullifying therapeutic benefits or causing secondary complications. Based on the physiological paradigm of inhibitory immune receptors, inhibitory antigen-specific chimeric antigen receptors (iCARs) were designed to preemptively restrict T cell responses. The results presented below demonstrate that iCARs based on CTLA-4- or PD-1 can selectively limit cytokine secretion, cytotoxicity, and proliferation induced through the endogenous T cell receptor or an activating chimeric receptor. The initial effect of iCAR is temporary, thus allowing T cells to function after a subsequent encounter with antigen recognized by their activating receptor. iCARs thus provide a safe, self-regulating, dynamic switch to prevent, rather than treat, the consequences of inadequate T cell specificity.

INTRODUCTION

T cell therapies have demonstrated clinical efficacy in bone marrow and organ transplantation, immunotherapy against cancer, viral infections, and autoimmune diseases (1-6). Unfortunately, T cells are also involved in detrimental side effects. "On-target but off-tumor" adverse events using both T-cell receptor (TCR) and chimeric antigen receptor (CAR)-engineered T cells have been reported in cancer immunotherapy clinical trials. These include B-cell aplasia in patients with chronic lymphocytic leukemia treated with T cells expressing CAR anti-CD 19 (7-9), fatal acute respiratory distress syndrome after infusion with CAR anti-ERBB2 T cells which is thought to be the result of cross-reactivity in the lung epithelium (10) and TCR-induced deaths from cardiac myonecrosis or neurological toxicity in patients treated with TCRs that recognize testicular cancer antigens (11-13). Similarly, the curative benefits of donor lymphocyte infusion (DLI) in allogeneic bone marrow transplantation are hampered by the induction of both acute and chronic graft-versus-host disease (GVDH) and bone marrow aplasia. bone (14). Strategies to separate the beneficial effects of graft versus tumor (GVT) from GVHD have had limited success to date (15).

The current approach to curb T cell-mediated toxicities relies on the use of immunosuppressive regimens such as high-dose corticosteroid therapy, which exert cytostatic or cytotoxic effects on T cells, to restrain immune responses (16). Although effective, this approach fails to discriminate between beneficial and detrimental T cell functions. Additionally, immunosuppressive drugs cause substantial side effects, such as susceptibility to infections and cardiac, renal, and neurological damage (14). Suicide gene engineering strategies, which may use selective enzymatic metabolizers of toxic agents, such as herpes simplex virus thymidine kinase (17) or inducible caspase-9 (18), or antibody-mediated depletion strategies targeting engineered ectopic epitopes on T cells (19, 20), also indiscriminately suppress T cell therapeutic efficacy. Additionally, these approaches are reactive because they are implemented after detrimental side effects are observed. Therefore, strategies that avoid unwanted T cell reactivity are highly desirable.

Physiologic regulation of T cell activation is achieved by several mechanisms including inhibitory immune receptors, which play a critical role in attenuating or terminating T cell responses (21, 22). Inhibitory receptors can be upregulated during T cell priming to reduce immune responses or expressed basally to regulate activation thresholds. Thus, mice deficient for the inhibitory receptor CTLA-4 show massive T cell activation and proliferation and eventually succumb to severe systemic autoimmune disease with infiltration of activated T cells (23). Similarly, loss of PD-1, another inhibitory receptor specifically expressed on activated T cells, causes progressive arthritis and glomerulonephritis in C57BL/6 mice and accelerated insulitis in nonobese diabetic (NOD) mice (24 , 25). Modulation of these receptors and their downstream signaling pathways has a substantial influence on T cell functions. In vitro binding of CTLA-4 or PD-1 during T cell stimulation blocks activation, proliferation, and cytokine release. (26). Notably, anti-CTLA-4 and anti-PD-1 antibodies have shown clinical promise in derepressing responses against T cells in some patients with melanoma, lung, and kidney cancer (22, 27, 28). Blockade of both CTLA-4 and PD-1 is also being actively investigated to reverse immune dysfunction and viral persistence in chronic hepatitis B and HIV infection (29, 30). However, like nonspecific immunosuppression, antibody-mediated inhibitory checkpoint blockade is not antigen-specific and thus does not distinguish between beneficial and detrimental T cell populations.

A genetic engineering strategy was used to take advantage of the natural inhibitory physiology of T cells and regulate T cell responses selectively for antigen. An inhibitory chimeric antigen receptor (CAR (iCAR) that has a surface antigen recognition domain combined with a powerful acute inhibitory signaling domain was designed to limit T cell sensitivity despite simultaneous engagement of an activating receptor ( Figure 1A) As shown below, in human primary T cells, PD-1- and CTLA-4-based iCARs reversibly restrict critical TCR functions or CAR activation and thus allow discrimination between target and non-target cells in vitro and in vivo.

MATERIALS AND METHODS

Study design

The purpose of this study was to create a synthetic receptor that could limit the toxicity of T lymphocytes towards a target cell in a reversible and antigen-dependent manner. Two such receptors using intracellular tails of CTLA-4 or PD-1 and an scFv (against PSMA) targeting domain were designed and tested to see if they could block (i) TCR-driven T cell functionality or (ii) by CAR in vitro and in vivo. In vitro, the focus was on testing (i) cytotoxicity, (ii) cytokine secretion, and (iii) T cell proliferation. In vivo experiments tested the integrated ability of iCAR to protect a cellular target using imaging vivo and endpoint analysis (dictated by the group of untreated mice). Experimental procedures were approved by the Institutional Animal Care and Use Committee of Memorial Sloan-Kettering Cancer Center (MSKCC). The general design of the experiments was to expose T lymphocytes (expressing iCAR or the Pdel control receptor) to target cells (expressing or lacking PSMA) and comparing the groups trying to question iCAR function, always in the presence of controls. internal. T cells lacking iCAR could not contaminate the results by sorting the T cells to be iCAR or iCAR/CAR double positive (using reporter genes). Each experiment was performed multiple times using different donor T cells (T cells were never pooled). In most cases, data were presented using a representative experiment (with sample replicates of more than three) to avoid confounding variables such as differences due to transduction and sorting efficiencies, donor-related variability, and E/R ratios. T.

Inhibitory Chimeric Antigen Receptor ( iCAR) Design

Each inhibitory chimeric antigen receptor (iCAR) was designed with the UniProt sequence annotation using two approaches. First, using commercial gene synthesis or cDNA, the intracellular domain of each receptor was cloned in place of the previously described Pz1 receptor CD28/CD3Z domain (Stephan et al., Nature medicine, 2007. 13(12): 1440-9), thus using the transmembrane and hinge domains of CD8. A CD8 polypeptide may have an amino acid sequence as set forth below:

MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTS

GCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSD

FRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQP

LSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNH

RNRRRVCKCPRPWKSGDKPSLSARYV [SEQ ID NO: 11]

The transmembrane and hinge domains of CD8 may have an amino acid sequence of amino acids 137 to 209 of SEQ ID NO: 11. Alternatively or additionally, the transmembrane and hinge domains of CD4 may also be used and were tested. Nucleic acid sequences and amino acid sequences of the iCAR constructs are provided in Appendix A. Alternatively, transmembrane domains and amino acids up to the first topological extracellular domain noted were included (for PD-1 amino acids 145 to 288: for CTLA4 amino acids 161 to 223), to use the endogenous hinge region of each receptor. These constructs were cloned into the P28z vector after the PSMA scFv. No significant functional differences were observed between the receptors generated by the two approaches. Additionally, versions of each iCAR were created that lacked the targeting domain, but retained the transmembrane and intracellular portions of each receptor. The control receptor Pdel was designed by excising the CD28/CD3Z domain from P-28z (34). iCARs must be clearly distinguished from CARs, all of which trigger T cell activation, in marked contrast to iCARs. The nucleic acid sequence of the PSMA scFv is given below:

atggCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATG CAGAGGTGCAGCTGCAGcagtcaggacctgaactggtgaagcctgggacttc agtgaggatatcctgcaagacttctggatacacattcactgaatataccata cactgggtgaagcagagccatggaaagagccttgagtggattggaaacatca atcctaacaatggtggtaccacctacaatcagaagttcgaggacaaggccac attgactgtagacaagtcctccagtacagcctacatggagctccgcagccta acatctgaggattctgcagtctattattgtgcagctggttggaactttgact actggggccaagggaccacGGTCACCgtctcctcaggtggaggTggAtcagg TggaggtggAtctggTggAggTggatcTGACATTGTGATGACCCAGTCTCAC

AAAT TCAT GTCCACAT CAGTAG GAGACAGGGTCAGCAT CATCT GTAAGGCCA

GTCAAGAT GTGGGTACT GCTGTAGAC TGGTAT CAACAGAAAC CAGGACAAT C

TCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGTCCCTGAT

CGCTT CACAGGCAGTGGATCT GGGACAGACTTCAC TCT CACCAT TACTAAT G

TT CAGTCT GAAGACTTGGCAGATTAT T TCT GTCAGCAATATAACAGC TAT CC

CCTCACGTTCGGTGCTGGGACCATGCTGGACCTGAAACGGgcggccgcA

[SEQ ID NO: 12]

The amino acid sequence of the PSMA scFv is given below:

MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTI

HWVKQSHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSL

TSEDSAVYYCAAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSH

KFMSTSVGDRVSIICKASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPD

RFTGSGSGTDFTLTITNVQSEDLADYFCQQYNSYPLTFGAGTMLDLKRAAA

[SEQ ID NO: 13]

Receptors, eg, PD-1, CTLA-4, 2B4, LAG-3, and BTLA-4, were also tested with a CD19 scFV target. The nucleic acid sequence of the CD19 scFV is provided below:

ATGGCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAG

GTGAAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATT

TCCTGCAAGGCTTCTGGCTATGCATTCAGTAGCTACTGGATGAACTGGGTGAAGCAG

AGGCCTGGACAGGGTCTTGAGTGGATTGGACAGATTTATCCTGGAGATGGTGATACT

AACTACAATGGAAAGTTCAAGGGTCAAGCCACACTGACTGCAGACAAATCCTCCAGC

ACAGCCTACATGCAGCTCAGCGGCCTAACATCTGAGGACTCTGCGGTCTATTTCTGT

GCAAGAAAGACCATTAGTTCGGTAGTAGATTTCTACTTTGACTACTGGGGCCAAGGG

ACCACGGTCACCGTCTCCTCAGGTGGAGGTGGATCAGGTGGAGGTGGATCTGGTGGA

GGTGGATCTGACATTGAGCTCACCCAGTCTCCAAAATTCATGTCCACATCAGTAGGA

GACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGG

TATCAACAGAAACCAGGACAATCTCCTAAACCACTGATTTACTCGGCAACCTACCGG

AACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTC

ACCATCACTAACGTGCAGTCTAAAGACTTGGCAGACTATTTCTGTCAACAATATAAC

AGGTATTCCGTACACGTCCGGAGGGGGGACCAAGCTGGAGATCAAACGGgcggccgcA [SEQ ID NO: 14]

The amino acid sequence of the CD19 scFV is given below:

MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWM

NWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGL

TSEDSAVYFCARKTISSWDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSD

IELTQSPKEMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATY

RNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLE

IKRAAAMALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYA

FSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSS STAY

MQLSGLTSEDSAVYFCARKTISSWDFYFDYWGQGTTVTVSSGGGGSGGGGS

GGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPL

IYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSG

GGTKLEIKRAAA [SEQIDNO:15]

The PSMA target scFV and the CD19 target scFV can be interchanged due to the modular nature of iCARs, provided the appropriate structural considerations are appreciated.

Conjugation assay, Western blots and GAM staining.

The cell surface expression of each iCAR was analyzed using a previously described goat anti-mouse staining protocol (Markley et al., Blood, 2010. 115(17): 3508-19). The cell conjugation assay was performed as previously described (Burshtyn and Davidson, Natural killer cell conjugate assay using two-color flow cytometry. Methods in molecular biology, 2010. 612: 89-96). Briefly, EL4 or EL4-PSMA cells were labeled with lipophilic DiD dye (Invitrogen) and mixed in a 1:1 ratio with T cells in FACS tubes, incubated at 37°C for 5 min, and analyzed in a flow cytometer. Western blot analysis was performed using standard protocols with a Bio-rad Mini-PROTEAN Tetra system. The intracellular tail of CTLA-4 was detected using the polyclonal antibody C-19 that recognizes the end of CTLA-4 (Santa Cruz Biotechnology).

Retroviral Vectors and Virus Production

Plasmids encoding the SFG oncoretroviral vector were prepared using standard molecular biology techniques. Syntheses of 19-28z-IRES-LNGFR, CD19, PSMA, GFP, mCherry, and click beetle luciferase (CBL) vectors have been described (Markley et al., Blood, 2010. 115(17): 3508-19; Stephan et al., Nature medicine, 2007. 13(12): 1440-9; Brentjens et al., Clin. Cancer Res., 2007. 13(18:1): 5426-35). Retroviral producers were prepared from the supernatant of plasmid-transfected H29 cells (Stephan et al., Nature medicine, 2007. 13(12): 1440-9).

cell lines

EL4-CD19, EL4-PSMA, and NIH3T3-CD19 and NIH3T3-PSMA artificial antigen presenting cells (AAPC) have been described (Gade (2005); Stephan et al., Nature medicine, 2007. 13(12): 1440 -9; Maher et al., Nature Biotechnology, 2002. 20(1): 70-75; Markley et al., Blood, 2010. 115(17): 3508-19; Brentjens et al., Clin. Cancer Res., 2007. 13(18:1): 5426-35). NIH3T3-CD19-PSMA, NIH3T3-CD19-mCherry, NIH3 T3-CD 19-GFP and NIH3T3-CD19-CBL, as well as NALM/6-CBL and NALM/6-PSMA-CBL were obtained after transduction with the supernatants corresponding retrovirals from H29-producing cells. All comparative groups of cell lines were sorted for equivalent expression of CD19, GFP, or mCherry using a MoFlo sorter.

Peripheral Blood Leukocyte ( PBL) Collection and Retroviral Transduction

Peripheral blood was obtained from healthy donors after informed consent according to a protocol approved by the Memorial Sloan-Kettering Cancer Center (MSKCC) institutional review board. PBLs were isolated using Ficoll-Paque and activated with phytohemagglutinin (PHA) for 48 hours. Activated T cells were transduced for three consecutive days by centrifugation on retronectin-coated retroviral vector-linked plates (Takara). Cells were fed every 3 days with RPMI medium supplemented with 20 U of IL-2. Ten days after transduction, enhanced GFP (marking iCARs) and LNGFR (marking 19-28z) based FACS selection was used to isolate positive cells on a MoFlo sorter. Post-sorting analysis was performed to ensure equivalent expression of both reporters.

Generation of iPS-derived fibroblasts

Peripheral blood lymphocytes were activated with PHA, transduced with retroviral supernatants (f-citrin-P2A-Myc-E2A-Sox2 and f-vexGFP-P2A-Okt4-T2A-Klf4), and seeded after 24 hours into cells. FEM feeders (Themeli (2013)). The medium was changed to human ES medium with fibroblast growth factor (FGF) (8 ng/mL) on day 5 after transduction and half the medium changed daily thereafter. . T-iPS colonies appeared approximately 22-25 days after transduction. A subcutaneous xenograft teratoma assay was performed using the T-iPS-1.10 cell line. At three months, the teratoma was removed and treated with collagenase 100 U/ml (Invitrogen) and dispase 2 U/ml (Invitrogen) for two hours at 37°C to generate a loose cell suspension. Cells were sorted for HLA-ABC positive cells and after one week in culture in RPMI supplemented with 1% L-glutamine, 1% penicillin, 1% streptomycin, and 10% fetal bovine serum (FBS), generated reproducibly and spontaneously iPS-fib.

Flow cytometry

All flow cytometry analysis was performed on an LSRII cytometer (BD Biosciences) and analyzed with FlowJo software, Ver. 9.6 (TreeStar). Anti-human LNGFR, CD45, CD140b, CD10, HLA-ABC, HLA-DR, CD80, CD86 and CD62L were obtained from BD Biosciences; anti-human CD4, CD8, CD3, CD19, CD90 and 4',6-diamidino-2-phenylindole (DAPI) were obtained from Invitrogen; human anti-PSMA was obtained from Medical & Biological Laboratories; anti-human CCR7 was obtained from R&D; anti-human Foxp3 (236A/E7) and Foxp3 isotype were obtained from eBioscience.

In vitro T cell assays

In general, for proliferation assays, effector cytokine production, and cytotoxicity assays, serial dilutions of purified T lymphocytes were plated on the corresponding AAPC (irradiated with 40-50 Gy and plated 24 hours before at 3 x 104/well) in plates. 96-well flat bottom (with outer wells of the plate containing medium only to minimize the effects of evaporation). iPS fibroblasts were not irradiated when used as targets. Fresh medium was added every 3-4 days or after medium color changes. Cytokine production was quantified by enzyme-linked immunosorbent assay (ELISA) kits (eBioscience) or Luminex assays (Invitrogen) as indicated in the text according to the manufacturer's instructions. T cell counts were calculated using viable cell number (DAPI) and CountBright counts (Invitrogen) on an LSR II (BD) flow cytometer by collecting whole wells. All in vitro culture experiments were performed in RPMI supplemented with 1% L-glutamine, 1% penicillin, 1% streptomycin, and 10% FBS. Exogenous cytokines were not administered at any time unless explicitly indicated.

Luciferase CTL Assay

Cytotoxic T lymphocyte (CTL) assays using bioluminescence as a readout were performed as described previously (Fu et al., PloS one, 2010. 5(7): e11867). Briefly, all in vitro luciferase assays were performed with the Bright-Glo Luciferase Assay System (Promega) and 96-well optical black-bottom microplates (Nunc), and performed according to the manufacturer's protocol with minor adjustments. All target cells were engineered to express CBL with an aGFP reporter to ensure equivalent levels of expression. Culture medium was removed at 50 μl per well, 50 μl of prepared luciferase reagent was added to each well of the 96-well plates, and the plates were incubated for 5 minutes to completely lyse the cells. Measurements were carried out with the IVIS Imaging System 100 Series (Xenogen). Living Image software version 2.6 (Xenogen) was used to quantify photon emission intensities.

CTL fluorescence and time-lapse microscopy

All microscopy images were performed using a Zeiss AxioVert 200M equipped with a live imaging system. Time-lapse videos were acquired and compiled using multidimensional acquisition in MetaMorph software (Molecular Devices). For CTL experiments, the signal from positive mCherry AAPCs was quantified using the built-in morphometric analysis function in MetaMorph.

moDC and priming

Monocyte-derived dendritic cells were generated using the Mo-DC generation toolkit (Miltenyi) from the same donor as the T-iPS cells. moDCs were pulsed for 24 hours at the immature stage (day 5-6) with iPS-Fib lysates, which were generated through six freeze-thaw cycles. DC maturation was confirmed by flow cytometry of CD8O, CD86, and HLA-DR. Priming was performed as described previously (Yuan et al., Journal of immunology, 2005. 174(2): 758-66). Briefly, the first round of priming was performed using a T cell/moDC ratio of 1:30, the second round using a ratio of 1:10 to 1:30. RPMI supplemented with 1% L-glutamine, 1% penicillin, 1% streptomycin, 10% human AB serum (CellGro) and 5 ng/ml human IL-15 (R&D Systems) were used. On day three, 20 U/ml IL-2 was added.

Proteome Profiler Array

T cells were exposed to AAPC at an E/T ratio of 4:1 for 60 min, washed, lysed and incubated (100 µg) in human phosphoimmunoreceptor matrix according to the manufacturer's protocol (R & DSystems). . All spots were detected using chemiluminescence on the same x-ray film to standardize exposure levels. Scanned X-ray film images were analyzed with image analysis software. All pixel density was normalized in each matrix with internal pY controls.

Mouse models and quantitative bioluminescence

For the NALM/6 studies, 6-12 week old male NOD/SCID/yc mice (Jackson Laboratory) were inoculated intravenously with 5 x 10 5 tumor cells (same dose for single or mixed tumor experiments). NALM/6 cells were engineered to express CBL with a GFP reporter. Four days later, 3 x 105 sorted T cells were infused intravenously; cell dose was based on percentage GFP+ 19-28z+ confirmed by post-sort analysis. Mice were sacrificed at 21 days (no control T cells had paralysis of the hind limbs). For iPS-fib studies, 6-12 week old NOD/SCID/Yc (null) mice were inoculated intraperitoneally with 1 x 10 6 cells prepared in a 1:1 mixture of ice-cold RPMI and Matrigel mix (BD Biosciences ). Eight days later, 5 x 10 5 GFP-sorted T cells primed twice with moDC were infused intraperitoneally; cell dose was based on percent GFP+ confirmed by post-sort analysis. Additionally, an in vitro luciferase CTL assay was performed to establish equivalent allogeneic reactivity in all groups using iPS-Fib as the target. In both models, D-luciferin (Xenogen, 150 mg/kg intraperitoneally) was used as a substrate for click beetle luciferase, and bioluminescence images were collected on an IVIS 100 imaging system. Software version 2.6 was used. Living Image to acquire and quantify bioluminescence imaging data sets as described previously (Markley et al., Blood, 2010. 115(17): 3508-19). Mice were cared for according to Memorial Sloan-Kettering Cancer Center (MSKCC) institutional guidelines.

Statistical methods

Data are presented as the mean ± standard deviation/standard error of the mean as indicated in the txt. Results were analyzed by Student's t-test for unpaired (two-tailed) data or by ANOVA as indicated in the text and statistical significance was defined as p < 0.05. Pairwise multiple comparisons were performed using multiple t-tests corrected for multiple comparisons with the Holm-Sidak method. All exact p-values are provided. All statistical analyzes were performed in Prism version 6.0 software (GraphPad).

RESULTS

1. iCARs are Well Expressed on the Cell Surface of Primary Human T Lymphocytes

Without wishing to be bound by any particular theory, it was hypothesized that an antigen-specific single-chain variable fragment (scFv) or Fab fused to the signaling domains of immunoinhibitory receptors (CTLA-4, PD-1, LAG-3 , 2B4 or BTLA) through a transmembrane region would inhibit the function of T lymphocytes specifically after antigen recognition. These receptors are called iCARs as they have immune cell inhibitory potential, chimeric receptors that bind to a specific antigen and are distinct from CARs, a term used to describe receptors with immune cell activating potential.

A scFv specific for human prostate-specific membrane antigen (PSMA) was used as a model surface antigen (31). This scFv has been extensively studied and is being investigated in phase 1 trials for prostate cancer immunotherapy (32). PSMA is overexpressed in metastatic prostate cancer, but is also found in normal kidney, liver, colon, and brain astrocytes (33). Five different PSMA-specific iCARs (termed iCAR-Ps) were generated that had intracellular domains of CTLA-4, PD-1, LAG-3, 2B4, and BTLA, respectively. A control receptor, Pdel, was generated that possessed only the targeting scFv and a transmembrane domain but lacked a cytoplasmic domain (Figure (1B). Each iCAR was cloned into a bicistronic retroviral vector with an IRES-GFP reporter module ( Figure 1B) Following transduction of primary human T cells from peripheral blood mononuclear cells, CTLA4 iCARs were well expressed on the cell surface, and PD-1 and Pdel iCAR-Ps were expressed on the cell surface at levels similar to the P28z receptor (34), a CD28/CD3Z-based, PSMA-specific, dual-signaling receptor currently used in a clinical trial (Figures 1C and 1D).Phosphoarrays were used to investigate whether PD1 iCAR was transducing a signal upon receptor engagement.The state of phosphorylation was analyzed using the RnD human phosphoimmunoreceptor array following exposure to 3t3-D versus 3T3-S targets.1928z-P-PD1 cells are incubated ron with 3T3 TS, 3T3-S or 3T3-D cells. Two hours were allowed for receptor docking, and then cell lysates were analyzed for phospho state. Cells exposed to 3T3-D had significant increases in phosphorylation of SHP-1, SHP-2, as well as 2B4, all downstream targets of PD1 signaling, significantly, in the case of 3t3-S exposure, all three showed a decrease in signal strength (FIG. 18). In the case of CTLA4 iCAR, strong intracellular expression was observed by Western blot and intracellular flow cytometry, but limited cell surface expression (Figures 1C, 1D and 9A-C). Surface expression was restored using a mutant CTLA4 tail at Y165G, a key residue for cell surface trafficking, to construct a mutCTLA4 iCAR (Figures 1C, 1D and 9A-C). This finding is consistent with physiological trafficking of CTLA-4, which is constitutively internalized by resting T cells and degraded by interaction with the AP-2 endocytic adapter complex through its YVKM tyrosin motif (35). Indeed, upregulation of CTLA-4 iCAR-P was observed on the cell surface after T cell activation (Figure 9C), and constitutive surface expression was restored using a Y165G tyrosin motif mutant to construct iCAR-P. of mutCTLA-4, which showed expression on the cell surface in resting cells (figure 1C). Recognition of PSMA by iCAR was demonstrated using a cell conjugation assay in which iCAR-expressing T cells bound to mouse thymoma EL4 cells expressed PSMA (FIG. 10A).

2. iCARs limit TCR responses in an antigen-restricted manner

In addition to their use in autologous targeted T cells, iCARs have potential utility for prevention of GVDH in post-transplant donor infusion. Therefore, the efficacy of iCARs in protecting an untransformed surrogate normal tissue in the context of alloreactivity, a powerful immune response underlying graft rejection, GVDH, and graft-versus-tumor (GVT) therapeutic responses, was evaluated. Furthermore, to study the effect of iCARs on endogenous TCR-driven primary human T cell responses, a model of alloreactivity was established (FIG. 11A) using allogeneic dendritic cells (DC) as cell primers. DC antigen presenting and isogenic fibroblasts as targets (FIG. 11A). In this model, iCAR- or Pdel-engineered T cells were primed with monocyte-derived dendritic cells (moDC), which is an extremely potent stimulator of the endogenous T-cell receptor (TCR), and then tested against fibroblasts expressing the PSMA antigen or not. To obtain isogenic replenishable fibroblasts for DCs without requiring iterative skin biopsies, induced pluripotent stem cells (iPSC) stable fibroblast cell lines, termed iPS-fib, were obtained from iPSCs (Figures 11B-D). iPS-fib showed replicative senescence and contact inhibition, and could be readily transduced, passaged, and implanted into NOD/severe combined immunodeficiency (SCID)/Yc' mice where they persisted for weeks without tumor formation. To acquire potent alloreactive T cells with endogenous TCR specificity against iPS-fib, moDCs were pulsed with isogenic iPS-fib lysates. This priming culture system stimulated strong cytotoxicity and cytokine secretion from various T cell donors, producing both CD4 and CD8 driven responses (FIGS. 12A-C).

To investigate the ability of iCARs to restrict alloreactivity against PSMA+ cells, iCAR- or Pdel-expressing T cells primed with two rounds of pulsed moDC were sorted and then incubated together with iPS-fib or iPS-fib expressing PSMA ( figure 13A) (36). All groups of T cells efficiently killed iPS-fib, demonstrating allogeneic cytotoxicity (Figures 2A and 2B), but iCAR-positive T cells were significantly inhibited in their ability to kill iPS-fib-PSMA+ cells (Figure 2C). Cytotoxicity by T cells expressing PD-1-based iCAR was reduced by up to 95% at low effector-to-target (E/T) ratios. Because cytotoxicity occurs rapidly and has a low activation threshold relative to other T cell responses, cytokine secretion was also tested. PD-1 iCAR caused the strongest inhibition of cytokine secretion (79-88% reduction), while mutCTLA-4 iCAR caused a 55-71% reduction (Figure 2D-F). These results suggested that iCARs might limit reactivity in an antigen-dependent manner.

3. iCARs work stoichiometrically

We investigated whether PD-1 iCAR-P could provide differential levels of inhibition depending on its level of expression or that of the target antigen. Primed T cells were sorted for high or low levels of PD-1 or Pdel iCAR-P expression and exposed to iPS-fib-PSMA (FIG. 14A). A stoichiometric relationship was observed between the destruction of T lymphocytes, the release of cytokines and the level of iCAR expressed. T cells sorted by low levels of PD-1 iCAR-P expression could provide 50% inhibition only up to 1:1 E/T ratios, but high levels of PD-1 iCAR-P expression allowed a 80% inhibition up to 8:1 E/T ratios and even 50% inhibition at 16:1 (Figures 3A and 3B). To examine the impact of iCAR antigen expression level, iPS-fib were sorted for high or low PSMA expression and challenged with sorted PD-1 iCAR-P T cells (FIG. 14B). iPS-fib with high PSMA expression inhibited at least 80% of PD-1 iCAR-P T cell killing and cytokine secretion over a range of E/T ratios (1:1 to 4:1) , while iPS-fib with low PSMA expression did not provide the same level of inhibition (Figures 3C and 3D).

4. iCARs limit allogeneic responses in vivo

To investigate whether an iCAR could protect a tissue from T cell-mediated killing in vivo, iPS-fib-PSMA+ cells (also expressing CBL) were injected intraperitoneally into NOD/SCID/yc mice (FIG. 14B). The cells established nodules that could be monitored by bioluminescence imaging (BLI). Five days after injection of 1 x 106 iPS-fib-PSMA+ cells, mice were treated with 5 x 105 T cells expressing Pdel or PD-1-iCAR-P primed with moDC. The Pdel group eliminated iPS-fib-PSMA+ cells with a significant decrease in BLI signal (from 7 to 22-fold), while the PD-1-iCAR-P group was unable to eliminate nodules with similar BLI. to untreated control mice with T cells (Figures 4A and 4B). These results provide evidence that an iCAR can limit a TCR-driven response in an antigen-specific manner in vivo.

5. iCARs can inhibit the activation of chimeric antigen receptors

To study the effect of iCARs on the modulation of activating CARs, 19-28z, a widely characterized second-generation CAR currently in clinical trials, was used; 19-28z provides activation and costimulation of CD28 in response to CD19 antigen (9, 34). Primary T cells were transduced with 19-28z CAR and iCAR-P receptors, sorted for dual expression, and plated on previously indicated artificial antigen-presenting cells (AAPC) expressing CD19 or both CD19 and PSMA, respectively, which they model target and non-target tissues (FIGS. 13B and 15A). Chimeric antigen receptor (CAR) 19-28z, a widely characterized second-generation CAR currently in clinical trials, was used to provide activation and costimulation signals in response to CD19 antigen (Brentjens et al., Blood, 2011. 118(18 ): 4817-28; Maher et al., Nature Biotechnology, 2002. 20(1): 70-75). Although T cells from control groups (19-28z alone or 19-28z/Pdel) showed similar cytokine secretion in both AAPCs, iCAR-expressing T cells showed a marked decrease in cytokine secretion when exposed to off-target cells relative to on-target cells (FIG. 5A and 15B). PD-1 iCAR-P caused the strongest reduction in cytokine levels (71 to 89%), while mutCTLA-4 iCAR-P caused lesser reduction (48 to 67%), and iCAR -P of LAG-3, BTLA and 2B4 caused 30% inhibition.

19-28z provides a strong proliferation signal, induced by AAPC expressing CD19. Although 19-28z/Pdel T cells were similarly expanded on either AAPC, mutCTLA-4 or PD-1 iCAR-expressing T cells showed reduced accumulation in the presence of off-target AAPCs, causing PD-1 iCAR-P showed a 90% cumulative decrease in T cell accumulation after the second AAPC stimulation (Figures 5B, 5C and 15C). As the ability of iCARs to block cytokine secretion and proliferation by T cells was demonstrated, iCARs were evaluated for their effect on cytotoxicity, which occurs rapidly and has a lower activation threshold than other lymphocyte functions. T. In this co-culture system, the fate of these AAPCs, which were modified to further express mCherry, was examined by quantitative microscopy (FIG. 5D). In less than 38 hours, all groups of 19-28z/iCAR-P and control double-positive T cells lysed the target cells (FIG. 5E). When exposed to non-target CD19+PSMA+ cells, mutCTLA-4 and pD-1 based iCARs caused a 67 and 91% reduction in cytotoxicity, respectively (FIG. 5F). In the case of PD-1, AAPCs persisted for 5 days, while the iCAR effect of mutCTLA-4 was more limited (FIG. 5D). Therefore, the PD-1-based iCAR was selected for further evaluation in vivo .

To assess the role of PD-1 iCAR in vivo, a CD19+ NALM/6 B-cell leukemia cell line was tested with PSMA, and T-cell therapeutic responses were compared to NALM/6 and NALM cells. /6-PSMA in a previously established NOD/SCID/yc mouse xenograft model (Markley et al., Blood, 2010.

115(17): 3508-19; Brentjens et al., Clin. Cancer Res., 2007. 13(18:1): 5426-35) (FIG. 15D). Five days after systemic tumor infusion, mice were treated with a single dose of 3 x 10 5 double-positive sorted 19-28z/PD-1+iCARP+ T cells. Bioluminescent imaging (BLI) of tumor burden showed significant differences (3- to 10-fold reduction) in eradication of NALM/6-PSMA (off-target) compared to NALM/6 (on-target) (Figures 6A and 6B). Although initially confined to the bone marrow, NALM/6 leukemia eventually invades the spleen, the weight of which provides an index of late-stage disease burden. After treatment with 19-28z/PD-1-iCAR-P T cells, NALM/6-PSMA mice did not show significant differences in weight of the spleen relative to the "no T cells" control group, but the spleen weights of the NALM/6 mice were 2.6-fold lower (FIG. 6C). Flow cytometric analyzes confirmed the decreased number of NALM/6 cells in the spleen and bone marrow, unlike the NALM/6-PSMA group (Figures 6D and 6E). In parallel, a greater persistence of T lymphocytes was observed in the NALM/6 group than in the NALM/6-PSMA group (Figures 6D and 6F). These findings established that PD-1-based iCAR selectively prevents clearance of "off-target" NALM/6-PSMA cells in vivo while allowing therapeutic response against "on-target" NALM/6 cells to proceed. ".

6. iCARs work temporarily and reversibly

An attractive aspect for the clinical utility of iCARs is functional reversibility, that is, the resurgence of T-lymphocyte functionality after previous contact with non-target inhibitory tissue. Efficient recognition of PSMA by iCARs was demonstrated by the formation of conjugates of iCAR-expressing T cells with PSMA-expressing EL4 cells, but not with wild-type EL4 cells (FIG. 16FA). Constitutive iCAR expression did not affect T cell proliferative capacity (after activation with CD3/CD28 or DC beads), cytokine secretion, or surface marker expression compared with control T cells ( Figures 16A-E and 16G-I). To assess the temporal characteristics of iCAR-medicated inhibition, sequential stimulation of T cells by target and off-target cells was established to analyze the potential for killing, cytokine secretion, and proliferation in four different sequences. T cells 19-28z/Pdel or 19-28z/PD-1-iCAR-P were exposed to either target (CD19+) or off-target (CD19+PSMA+) AAPC as the first stimulation, followed by exposure to either AAPC at a second stimulation (FIG. 7A). At the second stimulation, both sets of T cells killed target cells equally well regardless of the first stimulation target (Figure 7B), supporting that 19-28z/iCAR-P T cells exposed to non-target cells in the first stimulation killed the target cells and proliferated during the second stimulation, as well as the T lymphocytes that were exposed to the target cells in both stimulations. Control T cells expressing 19-28z/Pdel did not show reduced functionality under the same conditions. Additionally, T cells that were activated in the first stimulation could still be inhibited after exposure to iCAR ligand presented by off-target AAPCs in the second stimulation, suggesting that iCARs might regulate an activated T cell. T cells exposed to non-target cells at both stimulations were also found to have greater inhibition of their killing ability at the second exposure (Figures 7C and 7D).

Corroborating these functional findings, double-positive PD-1, 19-28z/Pdel, and 19-28z/PD-1-iCAR-P iCAR T cells were found to differentially phosphorylate the regulatory phosphatases SHP-1 and SHP-2 (Figs. 17A-C). Exposure to CD19+ target AAPCs showed lower levels of SHP-1 and SHP-2 phosphorylation compared to baseline levels observed after exposure to CD19-lacking AAPCs, consistent with previous studies demonstrating dephosphorylation and subsequent blockade of the suppressive effects of SHP-1/2 on the activation of T lymphocytes (39, 40). In contrast, after exposure to off-target AAPC expressing CD19 and PSMA, SHP-1 and SHP-2 showed higher levels of phosphorylation, supporting that PD-1 iCAR recruits the same biochemical pathways as the PD-1 molecule. 1 endogenous.

7. iCAR and CAR dual-expressing T cells distinguish targets in vitro and in vivo

We evaluated whether T cells expressing the PD-1-based iCAR could distinguish between target cells in vitro and especially in vivo by protecting non-target cells in the presence of target cells within the same organism. This scenario was first addressed in an in vitro co-culture system that mixes GFP+CD19+ target AAPC and non-target mCherry+CD19+PSMA+ AAPC in a 1:1 ratio. Time-lapse microscopy was performed to analyze the effect of 19-28z/Pdel or 19-28z/iCAR-P T cells. Both target and non-target cells were killed at a similar rate by 19-28z/Pdel T cells, but 19-28z/iCAR-P T cells preferentially killed target cells without affecting non-target cells (Figure 8A). Crossover experiments with Click beetle luciferase (CBL)-transduced versions of the AAPCs were used to quantify this selectivity. At 38 hours, 19-28z/iCAR-P T cells removed most (85%) of target AAPCs but few (10%) of non-target cells, corroborating time-lapse microscopy results. time (figure 8B).

To test whether the same selectivity could be achieved in vivo, NOD/SCID/Yc- mice were injected with a mixture of NALM/6 and NALM/6-PSMA tumor cells and these animals were treated with T cells transduced with 19-28z or 19-28z/iCAR-P. After sacrifice, mice treated with 19-28z T cells showed a 3-fold reduction in the number of PSMA+ cells in the spleen and bone marrow compared to mice treated with 19-28z T cells/iCAR-P (Figs. 8C and 8D). Consequently, the iCAR-treated group had a 3.3-fold increase in spleen weight and a higher overall tumor burden (FIG. 8E). These experiments demonstrate that, in the presence of a mixture of target and off-target cells, an iCAR can selectively protect off-target cells without overriding target cell rejection, both in vivo and in vitro.

ANALYSIS

In this example, a genetic approach was taken to constrain the specificity of T cells and it was shown that T cells can be engineered to have an endogenous regulatory targeting mechanism to deliver tumor-specific immunotherapy. An antigen recognition domain was successfully combined with the signaling domains of the immune inhibitory receptors CTLA-4 and PD-1 to achieve antigen-specific suppression of T cell cytotoxicity, cytokine release, and proliferation. This preliminary demonstration study demonstrates the potential of iCARs as a strategy to limit T-cell function at off-target sites and thus divert immune responses away from undesired target tissues.

The crux of the strategy with iCAR is based on three critical properties. The first is that basal iCAR expression does not inhibit T cell function in the absence of antigen. Endogenous CTLA-4 or PD-1 signaling requires the presence of the corresponding ligands to exert its effect. Similarly, expression of the iCARs described herein was not found to affect basal T cell functions. Other inhibitory receptors that are restricted to subsets of T cells may act in concert to fine-tune the regulation of T cell responses (21, 22). Receptors such as LAG-3, 2B4, and BTLA and their combination (eg, as a single second-generation iCAR with multiple combined cytoplasmic domains) warrant further investigation.

The second key property is the maintenance of T cell functionality despite prior iCAR docking. It was found that iCAR-transduced T cells were still able to mount a response against a target antigen after prior exposure to an inhibitory antigen. This reversibility is reminiscent of the behavior of natural killer cells, in which the phosphorylation state of signaling molecules rather than transcriptional changes control rapid functional responses, such as cytotoxicity (41). Anti-PD-1 and anti-CTLA-4 antibodies are able to reverse the impaired function of anergized or depleted T cells, again arguing for their ability to temporarily regulate T cell responses (22). Additionally, biochemical analyzes of the effects of PD-1 and CTLA-4 on the TCR complex depend on phosphorylation states, downstream kinases, and motility rather than apoptosis (40, 42-44). Although both in vitro and in vivo results demonstrate inhibition in response to off-target cells with sustained therapeutic functionality, there is still the possibility that some of the cells may become anergic over time (42). Finally, an infusion of T cells is stochastic, with some T cells quickly finding their target and clearing it, while other T cells will encounter the inhibitory cells first. T cells that repeatedly encounter off-target cells may not expand, a welcome outcome for the iCAR approach, which aims to allow therapeutic responses to proceed while reducing the immune attack on normal tissues . The overall expansion of the infused T cell population will integrate these different pathways occurring at the clonal level, with some T cells expanding while others are suppressed, possibly resulting in the disappearance of all infused T cells over time. Under the experimental conditions, enough T cells persisted for 3 weeks to eliminate the target tumor. In such circumstances, a second or third infusion of T cells may be infused if necessary, which may be clinically advantageous as discussed elsewhere (9). The eventual induction of anergy and clonal clearance as a means of protecting off-target tissues while allowing tumor clearance to proceed should be contrasted with gene suicide strategies where adverse reactivity must manifest before clearance is triggered. of T cells, resulting in termination of therapeutic responses as well.

The iCAR-mediated immune response is useful for the control of graft-versus-host disease after infusion of donor lymphocytes for the treatment of cancer and chronic infections (specifically allowing the beneficial properties of DLI with limited toxicities). Additionally, iCARs are useful for monitoring non-tissue target toxicity of adoptive T cells designed for the treatment of cancer and chronic infections. This raises the possibility of reviving promising therapies, which have unacceptable toxicity profiles, such as involuntary cardiac or pulmonary recognition after adoptive T cell therapy). Therefore, iCARs provide a novel strategy to establish safe and effective T-cell therapies in both autologous and allogeneic settings.

Third, the iCAR approach is antigen-specific and therefore requires the ability to identify tissue-specific target antigens that are absent or downregulated in the tumor but are expressed by non-target tissues. This issue has not been investigated as extensively as the search for tumor antigens, although efforts, such as the Protein Atlas database, are underway to characterize the "surfaceme" of all human tissues (45). One strategy is to use broad classes of surface antigens that are negatively regulated on tumor cells. An example is represented by human leukocyte antigen (HLA) molecules, which are found on virtually all cell types, but are downregulated in tumors as a mechanism of tumor escape from T cell immune responses (46). Thus, allogeneic T cells expressing an iCAR against a host HLA molecule that is downregulated in the tumor can selectively promote the GVT effect. The iCAR approach may be of particular interest in establishing DLI as a means of protecting target tissues from GVHD without altering GVT responses. Another class of antigens compatible with a similar strategy includes cell surface tumor suppressor antigens, such as OPCML, HYAL2, DCC, and SMAR1 (47-49). OPCML-v1, for example, is widely expressed in all normal adult and fetal tissues, but is regulated negatively on flashlights and breast and prostate cancer. Cell surface carbohydrates, lipids, and post-translational modifications, such as mucin-type O-glycans (core 3 O-glycans) have also been found to be negatively regulated by tumors (50). Another candidate target is E-cadherin, which is highly expressed in normal skin, liver and intestine, the main targets of GVDH (51), but is negatively regulated by tumor cells that undergo an epithelial to mesenchymal transition, indicating tumor progression. and metastasis (52).

A major limitation of this study is the lack of availability of a strong clinically relevant human "normal tissue" model, especially one that allows the utilization of human cells, human antigens, and human TCRs, CARs, and iCARs. We attempted to bridge this gap by establishing iPS cells combined with DC from the same donor to derive an alloreactivity reaction using human T cells, human target antigens, and human iCARs. Simply, co-incubation of HLA mismatched allogeneic T cells with iPS or iPS-fib cells did not produce alloreactivity. The use of isogenic DC was essential to generate a powerful alloreactivity. The nature of this alloreactivity was not defined, so it is possible that the responses that were blocked did not influence the mechanisms involved in GVDH.

The expression level of the iCARs was shown to be critical. In settings of high level of activating receptor or antigen expression and/or low expression of iCAR or iCAR-target antigen, sufficient blocking could not be achieved. In most assays, icA r reduced but did not abolish T cell function, rarely exceeding 90% inhibition in any assay. When applying the iCAR strategy in a clinical setting, the functionality of each iCAR should be optimized based on receptor affinity, receptor expression level (i.e., promoter strength), and selection of appropriate target antigens based, in part, on in its level of expression. These will also need to balance with the activating receptor to achieve inhibition at off-target sites. In the case of targeted CAR therapy, an optimized CAR/iCAR ratio could be achieved by careful vector design.

In conclusion, a preliminary demonstration study was provided that inhibitory antigen-specific receptors can successfully redirect T cell proliferation, cytokine secretion, and cytotoxicity following the involvement of cell surface-specific antigens, thus bypassing the toxicity of T cells away from one tissue while maintaining critical effector function against another expressing the same antigen. This was demonstrated in responses mediated by TCR or CAR. This approach prevents, or at least reduces, unwanted target tissue damage and thus obviates the need to irreversibly remove therapeutic T cells after unacceptable toxicity has occurred. It is a paradigm shifting approach that harnesses the multifaceted functionality of cells as drugs by using synthetic receptors that guide and educate T cells to only perform beneficial functions. This dynamic safety switch may find useful applications in various autologous and allogeneic T cell therapies.

REFERENCES

1. J.N. Blattman, P.D. Greenberg, Cancer immunotherapy: A treatment for the masses. Science 305, 200-205 (2004).

2. N. P. Restifo, M. E. Dudley, S. A. Rosenberg, Adoptive immunotherapy for cancer: Harnessing the T cell response. Nat. Rev. Immunol. 12, 269-281 (2012).

3. M. Sadelain, R. Brentjens, I. Riviere, The promise and potential pitfalls of chimeric antigen receptors. curr. opinion Immunol. 21, 215-223 (2009).

4. C. J. Turtle, M. Hudecek, M. C. Jensen, S. R. Riddell, Engineered T cells for anti-cancer therapy. curr. opinion Immunol. 24, 633-639 (2012).

5. B. R. Blazar, W. J. Murphy, M. Abedi, Advances in graft-versus-host disease biology and therapy. Nat. Rev. Immunol. 12, 443-458 (2012).

6. T. M. Brusko, A. L. Putnam, J. A. Bluestone, Human regulatory T cells: Role in autoimmune disease and therapeutic opportunities. Immunol. Rev 223, 371-390 (2008).

7. M. Kalos, B. L. Levine, D. L. Porter, S. Katz, S. A. Grupp, A. Bagg, C. H. June, T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci.Transl. Med. 3, 95ra73 (2011).

8. R. J. Brentjens, I. Riviere, J. H. Park, M. L. Davila, X. Wang, J. Stefanski, C. Taylor, R. Yeh, S. Bartido, O. Borquez-Ojeda, M. Olszewska, Y. Bernal, H Pegram, M. Przybylowski, D. Hollyman, Y. Usachenko, D. Pirraglia, J. Hosey, E. Santos, E. Halton, P. Maslak, D. Scheinberg, J. Jurcic, M. Heaney, G. Heller , M. Frattini, M. Sadelain, Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood 118, 4817-4828 (2011).

9. R. J. Brentjens, M. L. Davila, I. Riviere, J. Park, X. Wang, L. G. Cowell, S. Bartido, J. Stefanski, C. Taylor, M. Olszewska, O. Borquez-Ojeda, J. Qu, T Wasielewska, Q. He, Y. Bernal, I. V. Rijo, C. Hedvat, R. Kobos, K. Curran, P. Steinherz, J. Jurcic, T. Rosenblat, P. Maslak, M. Frattini, M. Sadelain, CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci.Transl. Med. 5, 177ra38 (2013).

10. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA, Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol. Ther. 18, 843-851 (2010).

11. R. A. Morgan, N. Chinnasamy, D. Abate-Daga, A. Gros, P. F. Robbins, Z. Zheng, M. E. Dudley, S. A. Feldman, J. C. Yang, R. M. Sherry, G. Q. Phan, M. S. Hughes, U.S. Kammula, A. D. Miller, C. J. Hessman, A. A. Stewart, N. P. Restifo, M. M. Quezado, M. Alimchandani, A. Z. Rosenberg, A. Nath, T. Wang, B. Bielekova, S. C. Wuest, N. Akula, F. J. McMahon, S. Wilde, B. Mosetter, D. J. Schendel, C. M. Laurencot, S. A. Rosenberg, Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy. J. Immunother. 36, 133-151 (2013).

12. B. J. Cameron, A. B. Gerry, J. Dukes, J. V. Harper, V. Kannan, F. C. Bianchi, F. Grand, J. E. Brewer, M. Gupta, G. Plesa, G. Bossi, A. Vuidepot, A. S. Powlesland, A. Legg, K. J. Adams, A. D. Bennett, N. J. Pumphrey, D. D. Williams, G. Binder-Scholl, I. Kulikovskaya, B. L. Levine, J. L. Riley, A. Varela-Rohena, E. A. Stadtmauer, A. P. Rapoport, G. P. Linette, C. H. June, N. J. Hassan , M. Kalos, B. K. Jakobsen, Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells. Sci.Transl. Med. 5, 197ra103 (2013).

13. G. P. Linette, E. A. Stadtmauer, M. V. Maus, A. P. Rapoport, B. L. Levine, L. Emery, L. Litzky, A. Bagg, B. M. Carreno, P. J. Cimino, G. K. Binder-Scholl, D. P. Smethurst, A. B. Gerry, N. J. Pumphrey, A. D. Bennett, J. E. Brewer, J. Dukes, J. Harper, H. K. Tayton-Martin, B. K. Jakobsen, N. J. Hassan, M. Kalos, C. H. June, Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma. Blood 122, 863-871 (2013).

14. J. L. Ferrara, J. E. Levine, P. Reddy, E. Holler, Graft-versus-host disease. Lancet 373, 1550-1561 (2009).

15. E. Kotsiou, J. K. Davies, New ways to separate graft-versus-host disease and graft-versustumour effects after allogeneic haematopoietic stem cell transplantation. Br. J. Haematol. 160, 133-145 (2013).

16. G. Akpek, S. M. Lee, V. Anders, G. B. Vogelsang, A high-dose pulse steroid regimen for controlling active chronic graft-versus-host disease. Biol. Blood Marrow Transplant. 7, 495-502 (2001).

17. M. T. Lupo-Stanghellini, E. Provasi, A. Bondanza, F. Ciceri, C. Bordignon, C. Bonini, Clinical impact of suicide gene therapy in allogeneic hematopoietic stem cell transplantation. Hmm. Gene Ther. 21, 241-250 (2010).

18. A. Di Stasi, S. K. Tey, G. Dotti, Y. Fujita, A. Kennedy-Nasser, C. Martinez, K. Straathof, E. Liu, A. G. Durett, B. Grilley, H. Liu, C. R. Cruz, B. Savoldo, A. P. Gee, J. Schindler, R. A. Krance, H. E. Heslop, D. M. Spencer, C. M. Rooney, M. K. Brenner, Inducible apoptosis as a safety switch for adoptive cell therapy. N. Engl. J. Med. 365, 1673 1683 (2011).

19. I. Vogler, S. Newrzela, S. Hartmann, N. Schneider, D. von Laer, U. Koehl, M. Grez, An improved bicistronic CD20/tCD34 vector for efficient purification and in vivo depletion of gene-modified T cells for adoptive immunotherapy. Mol. Ther. 18, 1330-1338 (2010).

20. E. Kieback, J. Charo, D. Sommermeyer, T. Blankenstein, W. Uckert, A safeguard eliminates T cell receptor gene-modified autoreactive T cells after adoptive transfer. proc. Natl. Acad. Sci.U.S.A. 105, 623-628 (2008).

21. D.M. Pardoll, The blockade of immune checkpoints in cancer Immunotherapy. Nat Rev Cancer 12, 252-264 (2012).

22. P. Sharma, K. Wagner, J. D. Wolchok, J. P. Allison, Novel cancer immunotherapy agents with survival benefit: Recent successes and next steps. Nat Rev Cancer 11, 805-812 (2011).

23. E. A. Tivol, F. Borriello, A. N. Schweitzer, W. P. Lynch, J. A. Bluestone, A. H. Sharpe, Loss of CTLA-4 leads to massive lymphoproliferation and fatalmultiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4. Immunity 3, 541-547 (1995).

24. Nishimura H, Nose M, Hiai H, Minato N, Honjo T, Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity 11, 141-151 (1999).

25. J. Wang, T. Yoshida, F. Nakaki, H. Hiai, T. Okazaki, T. Honjo, Establishment of NOD-Pdcd1-/-mice as an efficient animal model of type I diabetes. proc. Natl. Acad. Sci.U.S.A. 102, 11823-11828 (2005).

26. R. V. Parry, J. M. Chemnitz, K. A. Frauwirth, A. R. Lanfranco, I. Braunstein, S. V. Kobayashi, P. S. Linsley, C. B. Thompson, J. L. Riley, CTLA-4 and PD-1 receptors inhibit T-cell activation by distinct mechanisms. Mol. Cell. Biol. 25, 9543-9553 (2005).

27. S.L. Topalian, F.S.Hodi, J.R. Brahmer, S.N. Gettinger, D.C. Smith,D. F. McDermott, J. D. Powderly, R. D. Carvajal, J. A. Sosman, M. B. Atkins, P. D. Leming, D. R. Spigel, S. J. Antonia, L. Horn, C. G. Drake, D. M. Pardoll, L. Chen, W. H. Sharfman, R. A. Anders, J. M. Taube, T. L. McMiller , H. Xu, A. J. Korman, M. Jure-Kunkel, S. Agrawal, D. McDonald, G. D. Kollia, A. Gupta, J. M. Wigginton, M. Sznol, Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N. Engl. J.Med. 366, 2443-2454 (2012).

28. F.S. Hodi, S.J. O'Day, D.F. McDermott, R.W. Weber, J.A. Sosman, J.B. Haanen, R. Gonzalez, C. Robert, D. Schadendorf, J.C. Hassel, W. Akerley, A.J. van den Eertwegh, J. Lutzky, P. Lorigan, J. M. Vaubel, G. P. Linette, D. Hogg, C. H. Ottensmeier, C. Lebbe, C. Peschel, I. Quirt, J. I. Clark, J. D. Wolchok, J. S. Weber, J. Tian, M. J. Yellin, G. M. Nichol, A. Hoos, W. J. Urba, Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J.Med. 363, 711-723 (2010).

29. V. A. Pedicord, W. Montalvo, I. M. Leiner, J. P. Allison, Single dose of anti-CTLA-4 enhances CD8+ T-cell memory formation, function, and maintenance. proc. Natl. Acad. Sci.U.S.A. 108, 266-271 (2011).

30. V. Velu, K. Titanji, B. Zhu, S. Husain, A. Pladevega, L. Lai, TH Vanderford, L. Chennareddi, G. Silvestri, GJ Freeman, R. Ahmed, RR Amara, Enhancing SIV- specific immunity in vivo by PD-1 blockade. Nature 458, 206-210 (2009).

31. TP Gade, W. Hassen, E. Santos, G. Gunset, A. Saudemont, MC Gong, R. Brentjens, XS Zhong, M. Stephan, J. Stefanski, C. Lyddane, JR Osborne, IM Buchanan, SJ Hall, WD Heston, I. Riviere, SM Larson, JA Koutcher, M. Sadelain, Targeted elimination of prostate cancer by genetically directed human T lymphocytes. Cancer Res. 65, 9080-9088 (2005).

32. M. Sadelain, R. Brentjens, I. Riviere, The basic principles of chimeric antigen receptor design. Cancer Discov.

3, 388-398 (2013).

33. Y. Kinoshita, K. Kuratsukuri, S. Landas, K. Imaida, P. M. Rovito Jr., C. Y. Wang, G. P. Haas, Expression of prostate-specific membrane antigen in normal and malignant human tissues. World J. Surg. 30, 628-636 (2006).

34. J. Maher, R. J. Brentjens, G. Gunset, I. Riviere, M. Sadelain, Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRz/CD28 receptor. Nat. Biotechnol. 20, 70-75 (2002).

35. W. A. Teft, M. G. Kirchhof, J. Madrenas, A molecular perspective of CTLA-4 function. Annu. Rev Immunol. 24, 65-97 (2006).

36. X. Fu, L. Tao, A. Rivera, S. Williamson, X. T. Song, N. Ahmed, X. Zhang, A simple and sensitive method for measuring tumor-specific T cell cytotoxicity. PLOS One 5, e11867 (2010).

37. R. J. Brentjens, E. Santos, Y. Nikhamin, R. Yeh, M. Matsushita, K. La Perle, A. QuintasCardama, S. M. Larson, M. Sadelain, Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts. Clin. Cancer Res. 13, 5426-5435 (2007).

38. J. C. Markley, M. Sadelain, IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell-mediated rejection of systemic lymphoma in immunodeficient mice. Blood 115, 3508-3519 (2010).

39. J. M. Chemnitz, R. V. Parry, K. E. Nichols, C. H. June, J. L. Riley, s Hp-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J. Immunol. 173, 945-954 (2004).

40. T. Yokosuka, M. Takamatsu, W. Kobayashi-Imanishi, A. Hashimoto-Tane, M. Azuma, T. Saito, Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2. J. Exp. Med. 209, 1201-1217 (2012).

41. Y. T. Bryceson, E. O. Long, Line of attack: NK cell specificity and integration of signals. curr. opinion Immunol.

20, 344-352 (2008).

42. S. Amarnath, C. W. Mangus, J. C. Wang, F. Wei, A. He, V. Kapoor, J. E. Foley, P. R. Massey, T. C. Felizardo, J. L. Riley, B. L. Levine, C. H. June, J. A. Medin, D. H. Fowler, The PDL1 -PD1 axis converts human TH1 cells into regulatory T cells. Sci.Transl. Med. 3, 111ra120 (2011).

43. K.S. Peggs, S.A. Quezada, J.P. Allison, Cell intrinsic mechanisms of T-cell inhibition and application to cancer therapy. Immunol. Rev 224, 141-165 (2008).

44. C.E. Rudd, The reverse stop-signal model for CTLA4 function. Nat. Rev. Immunol. 8, 153-160 (2008).

45. Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, Forsberg M, Zwahlen M, Kampf C, Wester K, Hober S, Wernerus H, Bjorling L, F. Ponten, Towards a knowledge-based Human Protein Atlas. Nat. Biotechnol.

28, 1248-1250 (2010).

46. M. Campoli, S. Ferrone, HLA antigen changes in malignant cells: Epigenetic mechanisms and biological significance. Oncogene 27, 5869-5885 (2008).

47. Y. Cui, Y. Ying, A. van Hasselt, K. M. Ng, J. Yu, Q. Zhang, J. Jin, D. Liu, J. S. Rhim, S. Y. Rha, M. Loyo, A. T. Chan, G. Srivastava , G. S. Tsao, G. C. Sellar, J. J. Sung, D. Sidransky, Q. Tao, OPCML is a broad tumor suppressor for multiple carcinomas and lymphomas with frequently epigenetic inactivation. PLOS One 3, e2990 (2008). 48. K. Singh, D. Mogare, R. O. Giridharagopalan, R. Gogiraju, G. Pande, S. Chattopadhyay, p53 target gene SMAR1 is dysregulated in breast cancer: Its role in cancer cell migration and invasion. PLOS One 2, e660 (2007).

49. L. Meimei, L. Peiling, L. Baoxin, L. Changmin, Z. Rujin, H. Chunjie, Lost expression of DCC gene in ovarian cancer and its inhibition in ovarian cancer cells. Med. Oncol. 28, 282-289 (2011).

50. Tsuboi S, Hatakeyama S, Ohyama C, Fukuda M, Two opposing roles of O-glycans in tumor metastasis. TrendsMol. Med. 18, 224-232 (2012).

51. B. Tsuchiya, Y. Sato, T. Kameya, I. Okayasu, K. Mukai, Differential expression of N-cadherin and E-cadherin in normal human tissues. Arch. Histol. Cytol. 69, 135-145 (2006).

52. C.L. Chaffer, R.A. Weinberg, A perspective on cancer cellmetastasis. Science 331, 1559-1564 (2011).

53. M. T. Stephan, V. Ponomarev, R. J. Brentjens, A. H. Chang, K. V. Dobrenkov, G. Heller, M. Sadelain, T cellencoded CD80 and 4-1BBL induce auto- and transcostimulation, resulting in potent tumor rejection. Nat Med 13, 1440-1449 (2007).

54. D. N. Burshtyn, C. Davidson, Natural killer cell conjugate assay using two-color flow cytometry. MethodsMol. Biol. 612, 89-96 (2010).

55. M. Themeli, C. C. Kloss, G. Ciriello, V. D. Fedorov, F. Perna, M. Gonen, M. Sadelain, Generation of tumortargeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat. Biotechnol. 31, 928 933 (2013).

56. J. Yuan, J. B. Latouche, J. L. Reagan, G. Heller, I. Riviere, M. Sadelain, J. W. Young, Langerhans cells derived from genetically modified human CD34+ hemopoietic progenitors are more potent than peptide-pulsed Langerhans cells for inducing antigen- specific CD8+ cytolytic T lymphocyte responses. J. Immunol. 174, 758-766 (2005).

Appendix A

Nucleic acid sequence of "P-PD1tm-PD1". "P-PD1tm-PD1" is an ¡CAR that includes a PD-1 transmembrane domain, a PD-1 cytoplasmic domain, and a PSMA svFV

atggCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAG GTGCAGCTGCAGcagtcaggacctgaactggtgaagcctgggacttcagtgaggata tcctgcaagacttctggatacacattcactgaatataccatacactgggtgaagcag agecatggaaagagccttgagtggattggaaacatcaatcctaacaatggtggtace acctacaatcagaagttcgaggacaaggccacattgactgtagacaagtcctccagt acagcctacatggagctccgcagcctaacatctgaggattctgcagtctattattgt gcagctggttggaactttgactactggggccaagggaccacGGTCACCgtctcctca ggtggaggTggAtcaggTggaggtggAtctggTggAggTggatcTGACATTGTGATG

AC CCAG TCTCACAAATT CAT GTCCACAT CAGTAGGAGACAGGGT CAGCAT CAT CTGT

AAG GC CAGTCAAGAT GTGG GTACT GCTG TAGAC TGGTAT CAACAGAAAC CAG GACAA

TCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGGTCCCTGATCGC

TTCACAG GCAG TGGATC TGGGACAGACT TCACT CTCACCAT TACTAATGTTCAGTCT

GAAGAC TTGG CAGAT TATTTCTGT CAGCAATATAACAG CTATCCCCT CACGT TCGG T

GCTGGGACCATGCTGGACCTGAAACGGgcggccgcAgagagaagggcagaagtgccc acagcccaccccagcccctcacccaggccagccggccagttccaaaccctggtggtt ggtgtcgtgggcggcctgctgggcagcctggtgctgctagtctgggtcctggccgtc atctgctcccgggccgcacgagggacaataggagccaggcgcaccggccagcccctg aaggaggacccctcagccgtgcctgtgttctctgtggactatggggagctggatttc cagtggcgagagaagaccccggagccccccgtgccctgtgtccctgagcagacggag tatgccaccattgtctttcctagcggaatgggcacctcatcccccgcccgcaggggc tcagccgacggccctcggagtgcccagccactgaggcctgaggatggacactgctct tggcccctctga [SEQ ID NO: 16]

Amino acid sequence of "P-PD1tm-PD1"

MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQ

SHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYC

AAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKEMSTSVGDRVSIIC

KASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQS

EDLADYFCQQYNSYPLTFGAGTMLDLKRAAAERRAEVPTAHPSPSPRPAGQFQTLW

GWGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDF

QWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCS

WPL [SEQ ID NO: 17]

Nucleic acid sequence of "P-CTLA-4tm-CTLA-4wt". "P-CTLA-4tm-CTLA-4wt" is an iCAR that includes a CTLA-4 transmembrane domain, a wild-type CTLA-4 cytoplasmic domain, and a PSMA svFV.

atggCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAG GTGCAGCTGCAGcagtcaggacctgaactggtgaagcctgggacttcagtgaggata tcctgcaagacttctggatacacattcactgaatataccatacactgggtgaagcag agecatggaaagagccttgagtggattggaaacatcaatcctaacaatggtggtace acctacaatcagaagttcgaggacaaggccacattgactgtagacaagtcctccagt acagcctacatggagctccgcagcctaacatctgaggattctgcagtctattattgt gcagctggttggaactttgactactggggccaagggaccacGGTCACCgtctcctca ggtggaggTggAtcaggTggaggtggAtctggTggAggTggatcTGACATTGTGATG

AC CCAG TCTCACAAATT CATGT CCACAT CAGTAGGAGACAGGGT CAGCAT CAT CTGT

AAG GCCAG TCAAGAT G TGGG TACTG CTGTAGAC TG GTAT CAACAGAAAC CAGGACAA

TCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGGTCCCTGATCGC

TT CACAG GCAG TGGATC TGGGACAGACT TCAC TCT CACCAT TACTAATGTTCAGTCT

GAAGAC TTGGCAGAT TATTTCTGT CAG CAATATAACAG CTATCCCCT CAC GTT CGGT

GCTGGGACCATGCTGGACCTGAAACGGgcggccgcACTGGGCATAGGCAACGGAACC

CAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTGGATC

CTTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCT

TTGAGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCTATGTGAAAATG

CC CCCAACAGAG CCAGAATGT GAAAAGCAAT TTCAGCCTTATTTTATTCCCATCAAT

TGA [SEQ ID NO: 18]

Amino acid sequence of "P-CTLA-4tm-CTLA-4wt"

MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQ

SHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYC

AAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKEMSTSVGDRVSIIC

KASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQS

EDLADYFCQQYNSYPLTFGAGTMLDLKRAAALGIGNGTQIYVIDPEPCPDSDFLLWI

LAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

[SEQ ID NO: 19]

Nucleic acid sequence of "P-CTLA-4tm-CTLA-4mut" "P-CTLA-4tm-CTLA-4mut" is an ¡CAR that includes a mutant CTLA-4 transmembrane domain (Y165G mutant), a CTLA cytoplasmic domain -1 and a PSMA target svFV. Two other mutant versions of CTLA-4 were also made: mutant Y182G and mutant Y165G & Y182G.

atggCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAG GTGCAGCTGCAGcagtcaggacctgaactggtgaagcctgggacttcagtgaggata tcctgcaagacttctggatacacattcactgaatataccatacactgggtgaagcag agecatggaaagagccttgagtggattggaaacatcaatcctaacaatggtggtace acctacaatcagaagttcgaggacaaggccacattgactgtagacaagtcctccagt acagcctacatggagctccgcagcctaacatctgaggattctgcagtctattattgt gcagctggttggaactttgactactggggccaagggaccacGGTCACCgtctcctca ggtggaggTggAtcaggTggaggtggAtctggTggAggTggatcTGACATTGTGATG

AC CCAGT CTCACAAAT TCATG TCCACAT CAG TAGGAGACAG GGT CAGCAT CATC TGT

AAG GCCAG TCAAGAT G TGGGTAC TGC TGTAGAC TG GTATCAACAGAAAC CAGGACAA

TCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGGTCCCTGATCGC

TT CACAG GCAG TGGATC TGGGACAGAC TTCAC TCT CACCAT TACTAATGTT CAGTC T

GAAGAC T TGGCAGAT TATTTCTGT CAG CAATATAACAG CTATCCCCT CAC GTTC GGT

GCTGGGACCATGCTGGACCTGAAACGGgcggccgcACTGGGCATAGGCAACGGAACC

CAGATTTATGTAATTGATCCAGAACCGTGCCCAGATTCTGACTTCCTCCTCTGGATC

CTTTGCAGCAGTTAGTTCGGGGTTGTTTTTTTATAGCTTTCTCCTCACAGCTGTTTCT

TTGAGCAAAATGCTAAAGAAAAGAAGCCCTCTTACAACAGGGGTCGGTGTGAAAATG

CC CCCAACAGAG CCAGAAT GT GAAAAG CAAT TTCAGCCTTATTTTATTCCCAT CAAT

TGA [SEQ ID NO:20]

Amino acid sequence of "P-CTLA-4tm-CTLA-4mut"

MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQ

SHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYC

AAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKEMSTSVGDRVSIIC

KASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQS

EDLADYFCQQYNSYPLTFGAGTMLDLKRAAALGIGNGTQIYVIDPEPCPDSDFLLWI

LAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVGVKMPPTEPECEKQFQPYFIPIN

[SEQ ID NO:21]

Nucleic acid sequence of "P-LAG3tm-LAG3". "P-LAG3tm-LAG3" is an ¡CAR that includes a LAG3 transmembrane domain, a LAG3 cytoplasmic domain, and a PSMA svFV

atggCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAG GTGCAGCTGCAGcagtcaggacctgaactggtgaagcctgggacttcagtgaggata tcctgcaagacttctggatacacattcactgaatataccatacactgggtgaagcag agecatggaaagagccttgagtggattggaaacatcaatcctaacaatggtggtace acctacaatcagaagttcgaggacaaggccacattgactgtagacaagtcctccagt acagcctacatggagctccgcagcctaacatctgaggattctgcagtctattattgt gcagctggttggaactttgactactggggccaagggaccacGGTCACCgtctcctca ggtggaggTggAtcaggTggaggtggAtctggTggAggTggatcTGACATTGTGATG

AC CCAG TCTCACAAATT CAT GTCCACAT CAGTAGGAGACAGGGT CAGCAT CAT CTGT

AAG GCCAG TCAAGAT GTGGG TACTG CTGTAGAC T GGTAT CAACAGAAAC CAGGACAA

TCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGGTCCCTGATCGC

T TCACAG GCAG TGGATC TGGGACAGACT T CACTC TCACCAT TACTAATGTTCAGTCT

GAAGAC TTGG CAGAT TATTTCTGTCAG CAATATAACAG CTATCCCCT CAC GT TCGGT

GCTGGGACCATGCTGGACCTGAAACGGgcggccgcACTTGGAGCAGCAGTGTACTTC

ACAGAGCTGTCTAGCCCAGGTGCCCAACGCTCTGGGAGAGCCCCAGGTGCCCTCCCA

GCAGGCCACCTCCTGCTGTTTCTCATCCTTGGTGTCCTTTTCTCTGCTCCTTTTGGTG

ACTGGAGCCTTTGGCTTTCACCTTTGGAGAAGACAGTGGCGACCAAGACGATTTTCT

G CCTTAGAG CAAG GGAT TCAC CCTC CGCAG GCTCAGAG CAAGATAGAG GAGC TGGAG

CAAGAACCGGAGCCGGAGCCGGAGCCGGAACCGGAGCCCGAGCCCGAGCCCGAGCCG

GAGCAGCTCTGA [SEQIDNO:22]

Amino acid sequence of "P-LAG3tm-LAG3"

MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQ

SHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYC

AAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKEMSTSVGDRVSIIC

KASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQS

EDLADYFCQQYNSYPLTFGAGTMLDLKRAAALGAAVYFTELSSPGAQRSGRAPGALP

AGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELE

QEPEPEPEPEPEPEPEPEPEPEQL [SEQ ID NO:23]

Nucleic acid sequence of "P-BTLAtm-BTLA". "P-BTLAtm-BTLA" is an iCAR that includes a BTLA transmembrane domain, a BTLA cytoplasmic domain, and a PSMA svFV

atggCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAG GTGCAGCTGCAGcagtcaggacctgaactggtgaagcctgggacttcagtgaggata tcctgcaagacttctggatacacattcactgaatataccatacactgggtgaagcag agecatggaaagagccttgagtggattggaaacatcaatcctaacaatggtggtace acctacaatcagaagttcgaggacaaggccacattgactgtagacaagtcctccagt acagcctacatggagctccgcagcctaacatctgaggattctgcagtctattattgt gcagctggttggaactttgactactggggccaagggaccacGGTCACCgtctcctca ggtggaggTggAtcaggTggaggtggAtctggTggAggTggatcTGACATTGTGATG

ACCCAG TCT CACAAATT CAT GTCCACAT CAGTAGGAGACAGGGT CAGCAT CAT CTGT

AAGG CCAGT CAAGAT GTG GGTAC TGCTG TAGAC TGGTAT CAACAGAAAC CAG GACAA

TCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGGTCCCTGATCGC

TTCACAG GCAG TG GATCTGGGACAGACT TCAC TCTCACCAT TACTAATGTTCAGTCT

GAAGAC TTG GCAGAT TATTTCTGT CAGCAATATAACAG CTATCCCCT CACG TTCGG T

GCTGGGACCATGCTGGACCTGAAACGGgcggccgcAGATGTAAAAAGTGCCTCAGAA

CGACCCTCCAAGGACGAAATGGCAAGCAGACCCTGGCTCCTGTATAGTTTACTTCCT

TTGGGGGGATTGCCTCTACTCATCACTACCTGTTTCTGCCTGTTCTGCTGCCTGAGA

AGGCAC CAAG GAAAG CAAAAT GAAC TC TCTGACACAG CAGGAAG GGAAAT TAACC TG

GTTGAT GCT CACC TTAAGAG TGAG CAAACAGAAG CAAG CAC CAGGCAAAAT TCCCAA

GTACTGCTATCAGAAACTGGAATTTATGATAATGACCCTGACCTTTGTTTCAGGATG

CAGGAAG GG TCTGAAG TT TATTC TAATC CATG CCTGGAAGAAAACAAAC CAG GCAT T

GTTTATGCTTCCCTGAACCATTCTGTCATTGGACCGAACTCAAGACTGGCAAGAAAT

GTAAAAGAAG CAC CAACAGAATAT GCAT CCATAT GTG TGAG GAGTTAA [SEQ ID

NO:24]

Amino acid sequence of "P-BTLAtm-BTLA"

MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQ

SHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYC

AAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKEMSTSVGDRVSIIC

KASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQS

EDLADYFCQQYNSYPLTFGAGTMLDLKRAAADVKSASERPSKDEMASRPWLLYSLLP

LGGLPLLITTCFCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQ

VLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARN

VKEAPTEYASICVRS [SEQIDNO:25]

Nucleic acid sequence of "P-2B4tm-2B4". "P-2B4tm-2B4" is an ¡CAR that includes an ¡CAR that includes a 2B4 transmembrane domain, a 2B4 cytoplasmic domain, and a PSMA svFV

atggCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAG GTGCAGCTGCAGcagtcaggacctgaactggtgaagcctgggacttcagtgaggata tcctgcaagacttctggatacacattcactgaatataccatacactgggtgaagcag agecatggaaagagccttgagtggattggaaacatcaatcctaacaatggtggtace acctacaatcagaagttcgaggacaaggccacattgactgtagacaagtcctccagt acagcctacatggagctccgcagcctaacatctgaggattctgcagtctattattgt gcagctggttggaactttgactactggggccaagggaccacGGTCACCgtctcctca ggtggaggTggAtcaggTggaggtggAtctggTggAggTggatcTGACATTGTGATG

AC CCAG TCT CACAAAT TCATGTC CACAT CAGTAG GAGACAG G GTCAG CATCAT CTG T

AAG G CCAGT CAAGAT GTGG GTAC TGCTG TAGAC TGGTAT CAACAGAAAC CAG GACAA

TCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGGTCCCTGATCGC

TTCACAG GCAG TGGATC TGGGACAGAC T TCAC TCTCACCAT TACTAATGTT CAGTC T

GAAGAC TTG GCAGAT TATTTCTGT CAGCAATATAACAG CTATCCCCT CACG TTCGG T

GCTGGGACCATGCTGGACCTGAAACGGgcggccgcACAGGACTGTCAGAATGCCCAT

CAGGAATTCAGATTTTGGCCGTTTTTGGTGATCATCGTGATTCTAAGCGCACTGTTC

CTTGGCACCCTTGCCTGCTTCTGTGTGTGGAGGAGAAAGAGGAAGGAGAAGCAGTCA

GAGACCAGT CCCAAGGAATT TTT GACAAT TTACGAAGATGT CAAGGAT CTGAAAACC

AGGAGAAAT CACGAG CAG GAGCAGAC TTTTCCTG GAG GGGG GAGCAC CATC TACTC T

ATGAT CCAGT CCCAGTCTTCTGCTCCCACGT CACAAGAACC TGCATATACAT TATAT

TCAT TAATTCAGCCTTCCAG GAAG TCTG GATC CAGGAAGAG GAACCACAGCCCTTCC

TTCAATAG CAC TAT CTAT GAAGT GATTGGAAAGAGT CAACC TAAAGCCCAGAACCC T

GCTCGATTGAGCCGCAAAGAGCTGGAGAACTTTGATGTTTATTCCTAG [SEQ ID

NO:26]

Amino acid sequence of "P-2B4tm-2B4".

MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQ

SHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYC

AAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKEMSTSVGDRVSIIC

KASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQS

EDLADYFCQQYNSYPLTFGAGTMLDLKRAAAQDCQNAHQEFRFWPFLVIIVILSALF

LGTLACFCVWRRKRKEKQSETSPKEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYS

MIQSQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNHSPSFNSTIYEVIGKSQPKAQNP

ARLSRKELENFDVYS [SEQ ID NO:27]

Nucleic acid sequence of "complete tail of CTLA4, hinge of CD8, and tm", which is an ¡CAR that includes hinge and transmembrane domains of CD8, an intracellular tail of CTLA-4, and an svFV of PSMA

atggCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAG GTGCAGCTGCAGcagtcaggacctgaactggtgaagcctgggacttcagtgaggata tcctgcaagacttctggatacacattcactgaatataccatacactgggtgaagcag agecatggaaagagccttgagtggattggaaacatcaatcctaacaatggtggtace acctacaatcagaagttcgaggacaaggccacattgactgtagacaagtcctccagt acagcctacatggagctccgcagcctaacatctgaggattctgcagtctattattgt gcagctggttggaactttgactactggggccaagggaccacGGTCACCgtctcctca ggtggaggTggAtcaggTggaggtggAtctggTggAggTggatcTGACATTGTGATG

ACC CAGTC TCACAAAT TCATGT CCACAT CAG TAGGAGACAG GGTCAG CAT CATC TGT

AAG GCCAG TCAAGAT GT GGGTAC TGC TGTAGAC TGG TATCAACAGAAAC CAG GACAA

TCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGGTCCCTGATCGC

TTCACAG G CAGT GGATC TGGGACAGACT TCAC TCTCACCAT TACTAATGTTCAGTCT

GAAGAC T TGGCAGAT TATTTCTGT CAG CAATATAACAG CTATCCCCT CAC GTTC GGT

GCTGGGACCATGCTGGACCTGAAACGGgcggccgcACCCACCACGACGCCAGCGCCG CGACCACCAACCCCGGCGCCCACGATCGCGTCGCAGCCCctgtccctgcgcccagag gcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgt gatatctacatctgggcgcccCtggccgggacttgtggggtccttctcctgtcactg gttatcaccctttactgcaaccacagagcaccggcgGTTTCTTTGAGCAAAATGCTA

AAGAAAAGAAGCCCTCTTACAACAGGGGTCGGTGTGAAAATGCCCCCAACAGAGCCA

GAATGTGAAAAGCAATTTCAGCCTTATTTTATTCCCATCAATTGA [SEQ IDNO:28]

Amino acid sequence of "complete tail of CTLA4, hinge of CD8 and tm"

MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQ

SHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYC

AAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKEMSTSVGDRVSIIC

KASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQS

EDLADYFCQQYNSYPLTFGAGTMLDLKRAAAPTTTPAPRPPTPAPTIASQPLSLRPE

ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRAPAVSLSKML

KKRSPLTTGVGVKMPPTEPECEKQFQPYFIPIN [SEQ IDNO:29]

Nucleic acid sequence of "P-CD8tm-PD1". "P-CD8tm-PD1" is an ¡CAR that includes a transmembrane domain of CD8, a cytoplasmic domain of PD-1, and an svFV of PSMA

atggCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAG GTGCAGCTGCAGcagtcaggacctgaactggtgaagcctgggacttcagtgaggata tcctgcaagacttctggatacacattcactgaatataccatacactgggtgaagcag agecatggaaagagccttgagtggattggaaacatcaatcctaacaatggtggtace acctacaatcagaagttcgaggacaaggccacattgactgtagacaagtcctccagt acagcctacatggagctccgcagcctaacatctgaggattctgcagtctattattgt gcagctggttggaactttgactactggggccaagggaccacGGTCACCgtctcctca ggtggaggTggAtcaggTggaggtggAtctggTggAggTggatcTGACATTGTGATG

AC CCAG TCTCACAAATT CATGT CCACAT CAGTAGGAGACAGGGT CAGCAT CAT CTGT

AAG GCCAG TCAAGAT G TGGG TACTG CTGTAGAC TG GTAT CAACAGAAAC CAGGACAA

TCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGGTCCCTGATCGC

TT CACAG GCAG TGGATC TGGGACAGACT TCAC TCT CACCAT TACTAATGTTCAGTCT

GAAGAC TTGGCAGAT TATTTCTGT CAG CAATATAACAG CTATCCCCT CAC GTT CGGT

GCTGGGACCATGCTGGACCTGAAACGGgcggccgcACCCACCACGACGCCAGCGCCG CGACCACCAACCCCGGCGCCCACGATCGCGTCGCAGCCCctgtccctgcgcccagag gcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgt gatatctacatctgggcgcccCtggccgggacttgtggggtccttctcctgtcactg gttatcaccctttactgcaaccacagaatgcattgctcccgggccgcacgagggaca ataggagccaggcgcaccggccagcccctgaaggaggacccctcagccgtgcctgtg ttctctgtggactatggggagctggatttccagtggcgagagaagaccccggagccc cccgtgccctgtgtccctgagcagacggagtatgccaccattgtctttcctagcgga atgggcacctcatcccccgcccgcaggggctcagccgacggccctcggagtgcccag ccactgaggcctgaggatggacactgctcttggcccctctga [SEQ ID NO: 30]

Amino acid sequence of "P-CD8tm-PD1"

MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQ

SHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYC

AAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKEMSTSVGDRVSIIC

KASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQS

EDLADYFCQQYNSYPLTFGAGTMLDLKRAAAPTTTPAPRPPTPAPTIASQPLSLRPE

ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRMHCSRAARGT

IGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSG

MGTSSPARRGSADGPRSAQPLRPEDGHCSWPL [SEQ IDNO:31]

Nucleic acid sequence of "P-CD8tm-CTLA4wt". "P-CD8tm-CTLA4wt" is an iCAR that includes a CD8 transmembrane domain, a wild-type CTLA-4 cytoplasmic domain, and a PSMA svFV

atggCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAG GTGCAGCTGCAGcagtcaggacctgaactggtgaagcctgggacttcagtgaggata tcctgcaagacttctggatacacattcactgaatataccatacactgggtgaagcag agecatggaaagagccttgagtggattggaaacatcaatcctaacaatggtggtace

acctacaatcagaagttcgaggacaaggccacattgactgtagacaagtcctccagt acagcctacatggagctccgcagcctaacatctgaggattctgcagtctattattgt gcagctggttggaactttgactactggggccaagggaccacGGTCACCgtctcctca ggtggaggTggAtcaggTggaggtggAtctggTggAggTggatcTGACATTGTGATG

ACC CAGTC TCACAAATT CATGT CCACAT CAGTAGGAGACAGGGT CAGCAT CATC TGT

AAG GCCAG TCAAGAT GT GGGTAC TGC TGTAGAC TGG TATCAACAGAAAC CAG GACAA

TCTCCTAAACTACTGATTTATTGGGCATCCACTCGGCACACTGGAGGTCCCTGATCGC

TTCACAG G CAGT GGATC TGGGACAGACT TCAC TCTCACCAT TACTAATGTTCAGTCT

GAAGAC T TGGCAGAT TATTTCTGT CAG CAATATAACAG CTATCCCCT CAC GTTC GGT

GCTGGGACCATGCTGGACCTGAAACGGgcggccgcACCCACCACGACGCCAGCGCCG CGACCACCAACCCCGGCGCCCACGATCGCGTCGCAGCCCctgtccctgcgcccagag gcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgt gatatctacatctgggcgcccCtggccgggacttgtggggtccttctcctgtcactg gttatcaccctttactgcaaccacagagcaccggcgATGCTAAAGAAAAGAAGCCCT

CTTACAACAG GGGTCTATGT GAAAAT GCCCC CAACAGAG C CAGAAT GTGAAAAG CAA

TTTCAGCCTTATTTTATTCCCATCAATTGA [SEQ ID NO:32]

Amino acid sequence of "P-CD8tm-CTLA4wt"

MALPVTALLLPLALLLHAEVQLQQSGPELVKPGTSVRISCKTSGYTFTEYTIHWVKQ

SHGKSLEWIGNINPNNGGTTYNQKFEDKATLTVDKSSSTAYMELRSLTSEDSAVYYC

AAGWNFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSHKEMSTSVGDRVSIIC

KASQDVGTAVDWYQQKPGQSPKLLIYWASTRHTGVPDRFTGSGSGTDFTLTITNVQS

EDLADYFCQQYNSYPLTFGAGTMLDLKRAAAPTTTPAPRPPTPAPTIASQPLSLRPE

ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRAPAMLKKRSP

LTTGVYVKMPPTEPECEKQFQPYFIPIN [SEQ ID NO:33]

Claims (10)

1. A T lymphocyte comprising: a) a chimeric antigen receptor (CAR) that binds to CD19, where the binding of the CAR to the first antigen is capable of activating the T lymphocyte, and b) an inhibitory chimeric antigen receptor (iCAR) comprising an antigen-binding domain that binds PSMA, and at least a portion of a signaling domain of an immunoinhibitory receptor selected from CTLA-4, Pd-1, LAG -3, 2B4 and BTLA, where binding of iCAR to PSMA reduces T cell cytotoxicity against cells that are positive for both CD19 and PSMA. 2. The T cell of claim 1, wherein said iCAR is recombinantly expressed. 3. The T lymphocyte of claims 1 or 2, wherein the iCAR is expressed from a vector and/or wherein the CAR is expressed from a vector. 4. The T lymphocyte of any one of claims 1-3, wherein said iCAR further comprises a transmembrane domain, optionally wherein the transmembrane domain comprises a CD4 polypeptide, a CD8 polypeptide, a CTLA-4 polypeptide, a PD- 1, a LAG-3 polypeptide, a 2B4 polypeptide, or a BTLa polypeptide. 5. The T cell of any one of claims 1-4, wherein the iCAR antigen-binding domain comprises a Fab or an scFv. 6. The T cell of any one of claims 1-5, wherein the CAR provides co-stimulation with CD28. 7. A T cell of any one of claims 1-6 for use in a subject in reducing tumor burden and/or tumor size in a subject, and/or in increasing survival of a subject who has a neoplasm. 8. The T lymphocyte for the use of claim 7, wherein the tumor and/or the neoplasm are selected from hematologic cancer, B-cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin's lymphoma and acute myeloid leukemia (AML). 9. A method for producing a T cell of any one of claims 1-6, the method comprising: introducing into a T cell a first nucleic acid sequence encoding CAR and a second nucleic acid encoding iCAR. 10. A composition comprising an effective amount of a T cell of any one of claims 1-6, optionally wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.