# Patent

## Title
Constitutive expression of costimulatory ligands on adoptively transferred T lymphocytes

## Abstract

The present invention provides immunoresponsive cells, including T cells, cytotoxic T cells, regulatory T cells, and Natural Killer (NK) cells, expressing at least one of an antigen-recognizing receptor and a co-stimulatory ligand and methods of use therefore for the treatment of neoplasia and other pathologies where an increase in an antigen-specific immune response is desired.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is the U.S. national phase, pursuant to 35 U.S.C. §371, of PCT international application Ser. No. PCT/US2008/004251, filed Mar. 31, 2008, designating the United States and published in English on Oct. 9, 2008 as publication WO 2008/121420 A1, which claims priority to U.S. provisional application Ser. No. 60/921,144, filed on Mar. 30, 2007. The entire contents of the aforementioned patent applications are incorporated herein by this reference.
Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; “application cited documents”), and each of the PCT and foreign applications or patents corresponding to and/or claiming priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference, and may be employed in the practice of the invention. More generally, documents or references are cited in this text, either in a Reference List before the claims, or in the text itself; and, each of these documents or references (“herein cited references”), as well as each document or reference cited in each of the herein cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.


STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
The United States government has certain rights in this invention by virtue of grant numbers PO1 CA59350.


BACKGROUND OF THE INVENTION
Prostate cancer is the most frequent cancer in males in the United States and the cause of nearly 31,000 deaths per year. When diagnosed early, cancer can be effectively treated by surgery or radiation. Postsurgical residual disease requires radiation and/or hormonal therapy, which may prevent tumor progression and metastasis. At present, there is no curative treatment for hormone refractory, metastatic prostate cancer. Immunotherapy is a targeted therapy that in principle provides for the treatment of such cancers. Obstacles remain to induce tumor immunity, which requires the expansion of cytotoxic T lymphocytes to numbers sufficient to mediate tumor rejection. Among the mechanisms limiting efficient T cell priming and tumor rejection is the inherent absence of costimulatory ligands on many malignancies.
SUMMARY OF THE INVENTION
The present invention generally provides immunoresponsive cells, including T cells and Natural Killer (NK) cells, expressing at least one of an antigen-recognizing receptor and a co-stimulatory ligand and methods of use therefore for the treatment of neoplasia, infectious disease, and other pathologies.
In one aspect, the invention generally provides an immunoresponsive cell comprising a receptor that binds an antigen and an exogenous co-stimulatory ligand.
In another aspect, the invention provides a virus specific T cell expressing a vector (e.g., an expression vector) encoding a polypeptide selected from any one or more of CD80, 4-1BBL, OX40L, CD70 and CD30L. In one embodiment, the virus specific T cell recognizes a virus selected from any one or more of Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), and influenza virus antigens.
In yet another aspect, the invention provides a tumor antigen-specific T cell expressing a vector encoding a polypeptide selected from the group consisting of CD80, 4-1BBL, OX40L, CD70 and CD30L. In one embodiment, the cell expresses CD80 and 4-1BBL. In another embodiment, the vector is a retroviral vector (e.g., gamma-retroviral or lentiviral); it may also be non-viral.
In yet another aspect, the invention provides a method of modulating an immune response in a subject, the method comprising administering an effective amount of an immunoresponsive cell of any previous aspect. In one embodiment, the method increases or reduces an immune response. In another embodiment, the method increases self-tolerance or increases tolerance to an organ transplant.
In yet another aspect, the invention provides a method of treating or preventing a neoplasia in a subject, the method comprising administering an effective amount of an immunoresponsive cell comprising a receptor that binds a tumor antigen and a vector encoding a co-stimulatory ligand. In one embodiment, the neoplasia is selected from the any one or more of prostate cancer, colon cancer, breast cancer, and glioblastoma. In another embodiment, the tumor antigen is prostate-specific membrane antigen, CD19, NY-ESO-1, WT-1, hTERT, or mesothelin.
In another aspect, the invention provides a method of enforcing tolerance in a subject, the method comprising administering an effective amount of an immunoresponsive cell comprising a receptor that binds an antigen and a vector encoding a co-stimulatory ligand. In one embodiment, the method prevents or reduces an autoimmune disease or a disease associated with allogeneic transplantation.
In yet another aspect, the invention provides a method of treating or preventing a pathogen infection in a subject, the method comprising administering an effective amount of an immunoresponsive cell comprising a receptor that binds a viral antigen and a vector encoding a co-stimulatory ligand. In one embodiment, the pathogen is a virus, bacteria, fungus, protozoa or parasite. In another embodiment, the virus is selected from any one or more of Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), and influenza virus. In yet another embodiment, the cell is a T cell, a Natural Killer (NK) cell, or a cytotoxic T lymphocyte (CTL).
In still another aspect, the invention provides method for producing an antigen-specific immunoresponsive cell, the method comprising introducing into the immunoresponsive cell a nucleic acid sequence that encodes a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an antigen-binding domain coupled to an intracellular signaling domain that activates an immunoresponsive cell. In one embodiment, the immunoresponsive cell is a T cell, CTL, or NK cell. In another embodiment, the antigen-binding domain is a tumor antigen-binding domain. In yet another embodiment, the tumor antigen is prostate specific membrane antigen (PSMA). In yet another embodiment, the intracellular signaling domain activates a T cell, CTL cell, or NK cell. In yet another embodiment, the intracellular signaling domain is the ζ-chain signaling domain.
In another aspect, the invention provides a method of treating a neoplasia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell comprising a tumor antigen and an antigen presenting complex comprising at least two co-stimulatory ligands, wherein at least one of the two co-stimulatory ligands is selected from any one or more of a tumor necrosis factor (TNF) ligand and an immunoglobulin (Ig) superfamily ligand and combinations thereof, thereby treating cancer in the subject.
In another aspect, the invention provides a method of treating a neoplasia in a subject, the method comprising administering to the subject a therapeutically effective amount of a Natural Killer (NK) cell comprising a tumor antigen and an antigen presenting complex comprising at least two co-stimulatory ligands, wherein at least one of the two co-stimulatory ligands is selected from any one or more of a tumor necrosis factor (TNF) ligand and an immunoglobulin (Ig) superfamily ligand and combinations thereof, thereby treating cancer in the subject. In one embodiment, the TNF ligand is selected from any one or more of 4-1BBL, OX40L, CD70, CD30L, and LIGHT. In another embodiment, the Ig superfamily ligand is selected from CD80 and CD86. In yet another embodiment, the cell expresses at least two co-stimulatory ligands, where one is a TNF ligand (e.g., 4-1BBL) and the other is an Ig superfamily ligand (e.g., CD80).
In another aspect, the invention provides a method of treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a T cell comprising a receptor specific for a viral antigen and an antigen presenting complex comprising at least two co-stimulatory ligands, wherein at least one of the two co-stimulatory ligands is selected from any one or more of a tumor necrosis factor (TNF) ligand and an immunoglobulin (Ig) superfamily ligand and combinations thereof, thereby treating the infectious disease in the subject.
In yet another aspect, the invention provides method of treating an infectious disease in a subject, comprising administering to the subject a therapeutically effective amount of a Natural Killer (NK) cell comprising a receptor specific for a viral antigen and an antigen presenting complex comprising at least two co-stimulatory ligands, wherein at least one of the two co-stimulatory ligands is selected from any one or more of a tumor necrosis factor (TNF) ligand and an immunoglobulin (Ig) superfamily ligand and combinations thereof, thereby treating the infectious disease in the subject. In one embodiment, the subject is an immunocompromised subject. In another embodiment, the TNF ligand is selected from any one or more of 4-1BBL, OX40L, CD70, LIGHT and CD30L. In yet another embodiment, the Ig superfamily ligand is selected from any one or more of CD80 and CD86. In yet another embodiment, the at least two co-stimulatory ligands are a TNF ligand and an Ig superfamily ligand. In another embodiment, the TNF ligand is 4-1BBL and the Ig superfamily ligand is CD80. In another embodiment, the antigen recognition complex is constitutively expressed on the surface of the cell. In another embodiment, the viral antigen is an antigen specific for Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), or influenza virus. In another embodiment, the at least two co-stimulatory ligands are constitutively expressed on the surface of the cell.
In another aspect, the invention provides a pharmaceutical composition comprising an effective amount of an immunoresponsive cell of any previous aspect in a pharmaceutically acceptable excipient.
In another aspect, the invention provides a pharmaceutical composition for the treatment of a neoplasia comprising an effective amount of a tumor antigen-specific T cell of any previous aspect in a pharmaceutically acceptable excipient.
In another aspect, the invention provides a pharmaceutical composition for the treatment of a pathogen infection comprising an effective amount of a viral-specific T cell of the previous aspect in a pharmaceutically acceptable excipient. In one embodiment, the composition further comprising a cytokine selected from any one or more of IL-2, IL-3, IL-6, IL-11, IL7, IL12, IL15, IL21, granulocyte macrophage colony stimulating factor, alpha, beta or gamma interferon and erythropoietin.
In another aspect, the invention provides a kit comprising an immunoresponsive cell comprising a receptor that binds an antigen and an exogenous co-stimulatory ligand. In one embodiment, the kit further comprises written directions for using said cell for the treatment of a subject having neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant.
In various embodiments of any previous aspect, the method further comprises the step of obtaining the immunoresponsive cell or co-stimulatory ligand. In still other embodiments of the previous aspects, the co-stimulatory ligand is constitutively or inducibly expressed. In yet other embodiments of the previous aspects, at least two co-stimulatory ligands are constitutively expressed. In various embodiments of any previous aspect, the cell is selected from any one or more of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell. In still other embodiments of the previous aspects, the antigen is a tumor or pathogen antigen, e.g., any one or more of prostate-specific membrane antigen (PSMA), Carcinoembryonic Antigen (CEA), IL13Ralpha, her-2, CD19, NY-ESO-1, HIV-1 Gag, Lewis Y, Mart-1, gp100, tyrosinase, WT-1, hTERT, mesothelin. In still other embodiments of the previous aspects, the cell expresses a recombinant and/or an endogenous antigen receptor. In still other embodiments of the previous aspects, the co-stimulatory ligand is a tumor necrosis factor (TNF) ligand (e.g., 4-1BBL, OX40L, CD70, LIGHT, and CD30L) or an immunoglobulin (Ig) superfamily ligand (e.g., CD80 and CD86). In still other embodiments of the previous aspects, the immunoresponsive cell expresses at least one TNF ligand and at least one Ig superfamily ligand. In still other embodiments of the previous aspects, the cell expresses 4-1BBL and CD80. In still other embodiments of the previous aspects, an antigen presenting complex and/or co-stimulatory ligands are constitutively or inducibly expressed on the surface of the T cell. In still other embodiments of the previous aspects, the co-stimulatory ligand is expressed in a retroviral vector. In another embodiment, the tumor antigen is prostate-specific membrane antigen, CD19, NY-ESO-1, WT-1 or hTERT. In still other embodiments, the cell expresses a recombinant or an endogenous receptor for the antigen. In still other embodiments, the co-stimulatory ligand is a tumor necrosis factor (TNF) ligand or an immunoglobulin (Ig) superfamily ligand. In still other embodiments, the TNF ligand is selected from any one or more of 4-1BBL, OX40L, CD70, LIGHT, and CD30L. In various embodiments, the intracellular signaling domain is a ζ-chain signaling domain. In other embodiments, in the one costimulatory signal is provided through an engineered antigen receptor and the other by overexpressing a costimulatory ligand. In still other embodiments, the Ig superfamily ligand is selected from the group consisting of CD80 and CD86.
DEFINITIONS
By “CD80 polypeptide” is meant a protein having at least 85% identity to NCBI Reference No: NP—005182 or a fragment thereof that acts as an Ig superfamily ligand.
By “CD80 nucleic acid molecule” is meant any polynucleotide encoding a CD80 polypeptide. An exemplary CD80 nucleic acid molecule is NM—005191.
By “4-1BBL polypeptide” is meant a protein having at least 85% identity to NCBI Reference No: P41273 or NP—001552 or a fragment thereof that that acts as a tumor necrosis factor (TNF) ligand.
By “4-1BBL nucleic acid molecule” is meant a polynucleotide encoding a 4-1BBL polypeptide.
By “OX40L polypeptide” is meant a protein having at least 85% identity to NCBI Reference No: BAB18304 or NP—003317 or a fragment thereof that is a tumor necrosis factor (TNF) ligand.
By “OX40L nucleic acid molecule” is meant a polynucleotide encoding a OX40L polypeptide.
By “CD70 polypeptide” is meant a protein having at least 85% identity to NCBI Reference No: NP—001243 or a fragment thereof that acts as a tumor necrosis factor (TNF) ligand.
By “CD70 nucleic acid molecule” is meant a polynucleotide encoding a CD70-polypeptide.
By “Light polypeptide” is meant a protein having at least 85% identity to NCBI Reference No: NP—742011 or a fragment thereof that acts as a tumor necrosis factor (TNF) ligand.
By “Light nucleic acid molecule” is meant a polynucleotide encoding a Light polypeptide.
By “CD30L polypeptide” is meant a protein having at least 85% identity to NCBI Reference No: AAB97877 or a fragment thereof that acts as a tumor necrosis factor (TNF) ligand.
By “CD30L nucleic acid molecule” is meant a polynucleotide encoding a CD30L polypeptide.
By “CD86 polypeptide” is meant a protein having at least 85% identity to NCBI Reference No: P42081 or a fragment thereof that acts as an Ig superfamily ligand.
By “CD86 nucleic acid molecule” is meant a polynucleotide encoding a CD86 polypeptide.
By “Pz1 polypeptide” is meant a protein having at least 85% identity to the protein described by Gong et al., Neoplasia 1:123-7, 1999 or a fragment thereof.
By “PZ1 polypeptide” is meant a protein having at least 85% identity to the protein described by Maher et al. Nature Biotechnology, Vol 20, January 2002, 70-75 or a fragment thereof. The ζchain signaling domain is also described by Maher et al. Nature Biotechnology, Vol 20, January 2002, 70-75.
Nucleic acid molecules useful in the methods of the invention 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 with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily 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, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash 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 wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42.degree. C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example,
Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence.
By “analog” is meant a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.
The term “ligand” as used herein refers to a molecule that binds to a receptor. In particular, the ligand binds a receptor on another cell, allowing for cell-to-cell recognition.
The term “constitutive expression” as used herein refers to expression under all physiological conditions.
The term “chimeric antigen receptor” (CAR) as used herein refers to a tumor antigen-binding domain that is fused to an intracellular signaling domain capable of activating T cells. Most commonly, the CAR's extracellular binding domain is derived from a murine or humanized monoclonal antibody.
By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasia or pathogen infection of cell.
By “effective amount” is meant an amount sufficient to arrest, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion, or migration) of a neoplasia.
By “enforcing tolerance” is meant preventing the activity of self-reactive cells or immunoresponsive cells that target transplanted organs or tissues.
By “exogenous” is meant a nucleic acid molecule or polypeptide that is not endogenously present in the cell, or not present at a level sufficient to achieve the functional effects obtained when over-expressed. The term “exogenous” would therefore encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and over-expressed nucleic acid molecules and polypeptides.
By a “heterologous nucleic acid molecule or polypeptide” is meant a nucleic acid molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptide that is not normally present in a cell or 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 “isolated cell” is meant a cell that is separated from the molecular and/or cellular components that naturally accompany the cell.
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 “obtaining” as in “obtaining the agent” is intended to include purchasing, synthesizing or otherwise acquiring the agent (or indicated substance or material).
By “modulate” is meant positively or negatively alter. Exemplary modulations include a 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. Neoplasia growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells. Neoplasias can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, 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. Neoplasias include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumor of the plasma cells). Illustrative neoplasms for which the invention can be used include, but are not limited to leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., 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, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile 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). In one embodiment, screening methods of the invention identify compositions that are useful for treating breast or lung cancer.
By “receptor” is meant a polypeptide, or portion thereof, present on a cell membrane that selectively binds one or more ligand.
By “recognize” is meant selectively binds a target. A T cell that recognizes a virus typically expresses a receptor that binds an antigen expressed by the virus.
By “pathogen” is meant a virus, bacteria, fungi, parasite or protozoa capable of causing disease.
Exemplary viruses include, but are not limited to, Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astroviruses).
Exemplary bacteria include, but are not limited to, Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., 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 “specifically binds” is meant a polypeptide or fragment thereof that recognizes and binds a polypeptide of interest, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
The term “tumor antigen” as used herein refers to any polypeptide expressed by a tumor that is capable of inducing an immune response.
By “virus antigen” is meant a polypeptide expressed by a virus that is capable of inducing an immune response.
The terms “comprises”, “comprising”, and are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean “includes”, “including” and the like.
As used herein, “treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for 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 who has an immunodeficiency. The subject is very vulnerable to opportunistic infections, infections caused by organisms that usually do not cause disease in a person with a healthy immune system, but can affect people with a poorly functioning or suppressed immune system.
Other aspects of the invention are described in the following disclosure and are within the ambit 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 specific embodiments described, may be understood in conjunction with the accompanying drawings.
FIGS. 1a-1f show that CD80 and 4-1BBL co-expression in human T-cells elicits robust proliferative responses after cyclic stimulations through their endogenous T cell receptor or through a chimeric antigen receptor without antigen presenting cell (APC)-provided costimulation. 0.4×106 purified human T lymphocytes was activated with antibody to CD3 (OKT3) and quantified the expansion of T lymphocytes retrovirally transduced with CD80, 4-1BBL, the combination of the two, or a control vector (FIG. 1a,b). Transduction efficiencies were assessed by FACS analysis 48 hr after gene transfer (FIG. 1a). The absolute count of CD8+ T cells transduced with the indicated costimulatory ligand and stimulated weekly with 10 μg/ml plate-bound OKT3 is graphed in FIG. 1b. The investigation was extended to Cytomegalovirus (CMV)-specific memory donor T cells (FIG. 1c,d) and genetically redirected autologous T cells (FIG. 1e,f). CMV-pp65-specific T lymphocytes, briefly expanded on HLA-A*0201+pp65+ artificial APCs and transduced with CD80, 4-1BBL, or a combination of the two (FIG. 1c). Absolute counts of CD8+pp65+ T cells enriched and transduced as described in c after weekly restimulation with HLA-A*0201+ pp65-presenting Caco-2 tumor cells are shown in FIG. 1d. To rapidly generate prostate cancer-reactive human T lymphocytes (FIG. 1e,f), peripheral blood T cells were retrovirally transduced with the chimeric antigen receptor Pz1 (Gade et al., Cancer Res. 65:9080-9088, 2005), a non-HLA restricted antigen receptor specific for the tumor antigen PSMA. The Pz1 receptor comprises a PSMA-binding single-chain antibody fragment fused to the human CD3 ζ signaling domain. In FIG. 1e, primary human CD3+ T lymphocytes was retrovirally transduced with Pz1 alone or in combination with CD80 and 4-1BBL. Transduction efficiencies were assessed by FACS analysis. The T cell expansion of Pz1-transduced human T lymphocytes cocultured weekly with LNCaP tumor monolayers is shown in FIG. 1f. Costimulatory ligands were expressed on the T cell (top) or the tumor cell (bottom). Respective activation condition are depicted in cartoons shown in the right panels of b,d and f. Data are representative of three independent experiments. Each point in b, d and f represents the mean±s.e.m. of three randomly picked wells. * indicates<104 cells.
FIGS. 2a-2c show the eradication of established—prostate-specific membrane antigen (PSMA) prostate carcinoma tumors in Scid/beige mice by Pz1+ T-cells transduced with CD80 and 4-1BBL. FIG. 2a shows in vivo bioluminescent imaging and corresponding coronal MRI scans of firefly luciferase+PC3-PSMA tumors in Scid/beige mice four weeks after systemic inoculation with 4×106 tumor cells (day 0 of T-cell treatment), and eighteen days after adoptive transfer of 4×106 CD8+Pz1+ or Pz1+CD80+4-1BBL+ T lymphocytes. An equal number of T-cells bearing the human CD19-targeting chimeric antigen receptor 19z were injected in the control group. Pseudocolor images superimposed on conventional photographs are shown. The same animals imaged before and after treatment using bioluminescent imaging and MRI are shown. The two mice represent a total of n=10. FIG. 2b shows three graphs. Bioluminescent tumor signal quantified per animal every two days over a time period of 28 days. Acquisitions with saturated pixels—although included in the figure to allow direct visual comparison between groups—were not used for photon quantification but repeated at a shorter acquisition time. The graph shows photons/second/cm2/surface radius (sr) versus days after T cell injection. Every line corresponds to one animal with each dot representing the average photon count of the ventral and dorsal acquisition per animal at any given time point. Survival is illustrated in the Kaplan-Meier curve in FIG. 2c. 
FIGS. 3a-3d show robust, yet tumor antigen dependent, in vivo proliferation of CD80+4-1BBL+ T lymphocytes. FIG. 3a shows comparative in vivo bioluminescent imaging of adoptively transferred T cells in PC3-PSMA tumor bearing Scid/beige mice on days 0, 8 and 18 after the injection of 4×106CD8+ clickbeetle luciferase (click-luc)-expressing Pz1-transduced or Pz1+CD80+4-1BBL+ transduced T-lymphocytes. As an antigen specificity control, an equal number of 19z+CD80+4-1BBL+ T cells were infused. T-cell treatment started as in FIG. 2 four weeks after the systemic injection of 4×106 PC3-PSMA tumor cells. The five mice per group shown in each panel represent a total of n=8/group. Acquisitions with saturated pixels—although shown in the figure to allow direct visual comparison were not used for photon quantification but repeated at a shorter acquisition time. FIG. 3b shows three graphs. Clickbeetle luciferase signal intensities from sequential bioluminescence imaging were collected every 2 days after T-cell transfer over a sixteen day time period. Every line represents one animal with each dot showing the average photon count of the ventral and dorsal acquisition per animal at any given time point. FIG. 3c shows a multicolor FACS analysis of a lung single-cell suspension prepared from a representative animal infused either with Pz1+ or Pz1+CD80+4-1BBL+ T lymphocytes, 6 d after T cell transfer. Cells were stained with idiotypic antiserum specific for the chimeric antigen receptor Pz1. In FIG. 3d absolute pulmonary Pz1+ T cell numbers (total cell counts of viable Trypan blue-negative cells×percentages of Pz1+ T cells). Shown bar graphs represent the mean±s.e.m. of three mice. FIGS. 4a-4f show costimulation of T cells in cis as a results of the colocalization of CD80, 4-1BBL and their receptors CD28 and 4-1BB into the immunological synapse after induced T-cell/tumor cell cluster formation. In FIG. 4a-d, peripheral blood human T lymphocytes were transduced with Pz1 and restimulated with LNCaP-CD80 cells before a second gene transfer with the bicistronic vector encoding the dsRed-monomer-4-1BBL fusion protein and CD80. After negative magnetic CD8+ isolation, T cells were labeled with FITC-Choleratoxin B (CTB), and incubated with unmodified LNCaP tumor cells or alone. Fixed conjugates were permeabilized, stained with the indicated antisera and visualized by confocal microscopy. T cell-LNCaP cell clusters from three independent experiments were randomly chosen. Scale bars, 10 μm. The numbers of clusters with clear concentrations of the indicated costimulatory ligand or receptor at the T cell-APC junction over the total number of analyzed clusters is shown in the lower right panels of FIGS. 4a and b. FIG. 4a is a set of ten confocal micrographs exemplifying the polarization of 4-1BBL—expressed as a ds-Red fusion protein in combination with CD80 on CD8+ Pz1 transduced T cells—into the immunological synapse. T-cell/LnCaP tumor clusters were incubated for 50 minutes before fixation, permeabilization and incubation with anti 4-1BB antiserum. In FIG. 4b, fixed T cell-tumor cell clusters were incubated with anti-CD80 and anti-CD28 antisera. Again, colocalization of CD80 with its receptor CD28 in the immunological synapse after tumor antigen encounter is visualized. FIG. 4c illustrates the augmented accumulation of granzyme-B at the immunological synapse. This accumulation is dependent on a functional engagement of 4-1BB by its ligand 4-1BBL, which is expressed on the same T cell surface. Primary human T lymphocytes were genetically modified as in FIG. 4a,b. As indicated, retroviral vectors encoding Pz1 also express control shRNA or 4-1BB targeting shRNA under the control of the U6 promoter in their 3′LTR as described herein below. Cell conjugates in the top (Pz1+ control shRNA) and middle (and Pz1+CD80+dsred 4-1BBL+ control shRNA) row represent CD80+dsred 4-1BBL-untransduced and transduced T-lymphocytes, respectively, cultured in the same well and conjugated to LnCaP on the same glass slide. FIG. 4d shows the relative recruitment index (RRI) and the relative intensity, calculated as described herein below, of Granzyme-B-Alexa 647 at the T cell-antigen presenting cell interphase. Data points in each group show the calculated value of 35 analyzed conjugates (symbols) and their mean (−) of three independent experiments. *P=0.0001; **P<0.0001. In FIG. 4e,f, show NF-κB-luciferase assays in isolated single T cell clones. These results corroborate autocostimulation as an operant mechanism of delivering costimulatory signals. A CD3+CD28+4-1BB-Jurkat T cell clone stably transfected with an NF-κB-luciferase reporter was retrovirally transduced with a tricistronic vector coexpressing 4-1BB, 4-1BBL and CD80 or a control vector. To preclude bystander costimulation, transduced T lymphocytes were subcloned into OKT-3-coated 96-well plates before the first detectable surface expression of encoded proteins. Twelve hours after activation, the presence of single T cells in the wells was microscopically confirmed and the bioluminescent signal was measured on a single-cell level. FIG. 4e shows 36 bioluminescence acquisitions of transduced single T cells assembled with Adobe Illustrator software. Respective bioluminescent single-cell signals were quantified, normalized to background bioluminescence, and plotted in FIG. 4f. 
FIGS. 5a-5e show that CD80 and 4-1BBL-displaying T lymphocytes trans-costimulate unmodified, antigen specific bystander T-cells through physical contact. FIG. 5a shows a set of 4 confocal micrographs of a Pz1+ T cell engaging an LNCaP tumor cell while in physical T cell-Tcell contact with a bystander Pz1+ T lymphocyte transduced with CD80 and dsRed-4-1BBL. Cell clusters were induced and analyzed as described in FIG. 4a,b. Scale bars, 10 μm. In FIG. 5b-d, peripheral T lymphocytes of a cytomegalovirus (CMV)-seropositive HLA A2.1+ donor were transduced with Pz1 or co-transduced with Pz1CD80 and 4-1BBL. In parallel, CMV-specific, genetically unmodified cytotoxic T lymphocytes (CTLs) from the same donor were enriched by artificial antigen presenting cell (AAPC) co-culture as described herein below. These cells were labeled with carboxyfluoroscein succinimidyl ester (CFSE). Bead-sorted Pz1+ (FIG. 5b) or Pz1+CD80+4-1BBL+ (FIG. 5c,) T lymphocytes were admixed to expanded CMV-reactive pp65+ CTLs at a 1:1 ratio and exposed to irradiated Caco-2 tumor cells retrovirally transduced to present surface pp65 in an HLA A2.1-dependent, as well as PSMA in an HLA-independent manner. Alternatively, a transwell membrane separated Pz1+CD80+4-1BBL+ T-cells from pp65+ T-cells, both engaging Caco-2 tumor (FIG. 5d). Respective co-culture conditions are depicted in cartoons shown on the left. Two days after tumor antigen contact intracellular Granzyme B levels gated on CD8+ T-cells were quantified by FACS analysis (middle column). Granzyme B-antigen presenting cell (APC) mean fluorescent intensities (MFI) of the CFSE− (Pz1+) and CFSE+ (pp65+) T cell sub-populations are summarized on top of each profile. On day 7 CFSE dilutions and Pz1+ compared to pp65+ T-cell fractions were analyzed by flow cytometry after pp65 tetramer and CD8 staining (right column)). CFSE-MFIs indicated on top of each profile are based on pp65+-populations after CD8+-gating. Total cell counts of CD8+pp65+ T-cells after the 7 day cultures are graphed in (FIG. 5e). Each bar graph represents the mean±s.e.m of three randomly chosen wells. Data are representative of two independent experiments. No exogenous cytokines were added at any point of the co-culture.
FIGS. 6a and 6b show that the accumulation of adoptively transferred PSMA-redirected T lymphocytes at tumor sites is augmented by Pz1+CD80+4-1BBL+, but not 19z+CD80+4-1BBL+ T-cells in Scid/beige mice. FIG. 6a shows dual in vivo bioluminescence imaging of external Gaussia luciferase (x-gaus-luc) in RM1.PGLS and Raji tumors in addition to Clickbeetle luciferase (Click-luc) in tumor targeting T-cells. Two weeks after the systemic injection of 1×106CD19+x-gaus-luc+Raji tumors and two days after the subsequent infusion of 5×105 PSMA+x-gaus-luc+RM1.PGLS tumor cells into the same animals, established tumors in the bone marrow (Raji) and lung (RM1) were treated with a combination of three cell populations of T cells transduced as indicated. Each animal received a total of 12×106 CD8+ chimeric antigen receptor+ T-cells (4×106 T-cells/transduction condition). Notably, the T-cell population listed third (Pz1+, Pz1+CD80+4-1BBL+, 19z+CD80+4-1BBL+, left, middle, right row, respectively) were injected twelve hours after the combined injection of T-cells listed first and second to avoid T-cell/T-cell interaction in the lung due to crowding and not as a result of selective tumor antigen binding. At the indicated time points x-gaus-luc+ tumor cells or click-luc+ T-cells were monitored by bioluminescent imaging. On day 0 and day 4 a time period of at least 4 hours between tumor and T-cell imaging ensured the bioluminescent signal to return to background levels. A total number of n=5 Scid/beige mice were imaged per treatment group. FIG. 5b shows a series of 6 graphs quantitating clickbeetle luciferase signal intensities from sequential bioluminescence imaging every day after T-cell transfer for a four day time period. Every line represents one animal with each dot showing the average photon count measured over the pulmonary area (top) or both femurs (bottom), respectively at any given time point.
FIG. 7 shows that dual T cell-expressed CD80 and 4-1BBL elicits superior T cell expansion compared to CD28 and/or 4-1BB signaling elements fused in series with the Pz1 chimeric antigen receptor ζ signaling domain. Primary T cells were transduced with Pz1, P28z (Maher et al. Nature Biotechnology, Vol 20, January 2002, 70-75), which contains the CD28 signaling domain in series with the ζ chain, or P284-1BBz which includes both, CD28 and 4-1BB signaling regions. Alternatively, T lymphocytes were co-transduced with P28z and 4-1BBL (noted P28Z+4-1BBL). Pz1+CD80+4-1BBL refers to Pz1+T cells that co-express both ligands, CD80 and 4-1BBL. Transduced T cells were stimulated weekly (indicated by arrows) on LNCaP tumor monolayers under conditions outlined in detail under FIG. 1. The fold expansion of the CD8+ Pz1-transduced human T lymphocytes population is graphed.
FIG. 8 provides amino acid sequences for CD80, 4-1BBL, OX40L, CD70, LIGHT, and CD30L.



DETAILED DESCRIPTION OF THE INVENTION
The present invention generally provides cells, including genetically modified immunoresponsive cells (e.g., T cells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL) cells) expressing at least one of an antigen-recognizing receptor and a co-stimulatory ligand and methods of use therefore for the treatment of neoplasia and other pathologies where an increase in an antigen-specific immune response is desired. The invention is based, at least in part, on the discovery that the constitutive retroviral expression of CD80 and 4-1BBL in co-transduced human T cells targeting prostate specific membrane antigen mounted a robust tumor-antigen-dependent T-cell proliferation coupled with a profound in vivo rejection of disseminated well-established prostate carcinoma tumors. Furthermore, CD80 and 4-1BBL expressing T cells provided costimulation of bystander T cells in trans in a contact dependent and antigen-specific manner at the tumor site. Taken together, the concept of genetically modified T cells as a constitutive pool of costimulatory ligands to optimally costimulate themselves in addition to enhancing the immunogenicity within the tumor microenvironment represents a significant advance over conventional adoptive T cell therapy. Furthermore, as demonstrated ex vivo using enriched CMV-specific T lymphocytes, this approach is not limited to the treatment of neoplasias, but is amenable to a wide range of applications where an increase in an antigen-specific immune response is desired, such applications include not only the treatment of neoplasias, but also for the enhancement of an immune response against a pathogen infection or an infectious disease and to reinforce immune tolerance in regulatory T cells in the context of autoimmunity or allogeneic transplantation.
Hematopoietic Cell Lineages
Mammalian hematopoietic (blood) cells provide a diverse range of physiologic activities. Hematopoietic cells are divided into lymphoid, myeloid and erythroid lineages. The lymphoid lineage, comprising B, T and natural killer (NK) cells, provides for the production of antibodies, 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 chiefly responsible for cell-mediated immunity. T cells 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 act during the innate immune response. They do not require prior activation in order to perform their cytotoxic effect on target cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.
Cells for use in the Methods of the Invention
The present invention provides cells expressing at least one of an antigen-recognizing receptor and a co-stimulatory ligand 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, NK cells, CTL cells or other immunoresponsive cells are used as shuttles for the selective enrichment of one or more co-stimulatory ligands for the treatment or prevention of neoplasia. For example, a T cell expressing a co-stimulatory ligands 4-1BBL and CD80 are constitutively co-expressed in a T cell that expresses a chimeric antigen receptor PZ1 that recognizes and binds Prostate Specific Membrane Antigen (PSMA). Such cells are administered to a human subject in need thereof for the treatment or prevention of prostate cancer. In another approach, viral antigen-specific T cells, NK cells, CTL cells can be used for the treatment of viral diseases. For example, CD80 and 4-1BBL are expressed in cytomegalovirus (CMV)-specific cytotoxic T lymphocytes for the treatment of CMV.
Tumor Antigen-Specific T Lymphocytes (and NK Cells)
Types of tumor antigen-specific human lymphocytes that can be used in the methods of the invention include, without limitation, peripheral donor lymphocytes genetically modified to express chimeric antigen receptors (CARs) (Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45), peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex comprising the α and β heterodimer (Morgan, R. A., et al. 2006 Science 314:126-129), lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies (Panelli, M. C., et al. 2000 J Immunol 164:495-504; Panelli, M. C., et al. 2000 J Immunol 164:4382-4392), and selectively in vitro-expanded antigen-specific peripheral blood leukocytes employing artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells (Dupont, J., et al. 2005 Cancer Res 65:5417-5427; Papanicolaou, G. A., et al. 2003 Blood 102:2498-2505). The T cells may be autologous, allogeneic, or derived in vitro from engineered progenitor or stem cells.
Any suitable tumor antigen (antigenic peptide) is suitable for use in the tumor-related embodiments described herein. Sources of antigen include, but are not limited to cancer proteins. The antigen can be expressed as a peptide or as an intact protein or portion thereof. The intact protein or a portion thereof can be native or mutagenized. One suitable antigen is prostate specific membrane antigen (PSMA).
Viral Antigen-Specific T Lymphocytes (and NK Cells)
Suitable antigens for use in the treatment of pathogen infection or other infectious disease, for example, in an immunocompromised subject include, without limitation, viral antigens present in Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), and influenza virus.
The unpurified source of CTLs may be any known in the art, such as the bone marrow, fetal, neonate or adult or other hematopoietic cell source, e.g., fetal liver, peripheral blood or umbilical cord blood. Various techniques can be employed to separate the cells. For instance, negative selection methods can remove non-CTLs initially. 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 a relatively crude separation. For example, magnetic bead separations can be used initially to remove large numbers of irrelevant cells. Preferably, at least about 80%, usually 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; resetting; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix, e.g. plate, chip, elutriation or any other convenient technique.
Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels.
The cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI). Preferably, the cells are collected in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, preferably sterile, isotonic medium.
Accordingly, the invention generally provides an immunoresponsive cell, such as a virus or tumor specific T cell comprising a receptor that binds an antigen and an exogenous co-stimulatory ligand (e.g., CD80, 4-1BBL, OX40L, CD70 and CD30L).
Vectors
Genetic modification of immunoresponsive cells (e.g., T cells, CTL cells, NK cells) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA construct. Preferably, a retroviral vector (either gamma-retroviral or lentiviral) is employed for the introduction of the DNA construct into the cell. For example, a polynucleotide encoding a co-stimulatory ligand protein (e.g., tumor necrosis factor (TNF) ligand, such as 4-1BBL, OX40L, CD70, LIGHT, and CD30L, or an Ig superfamily ligand, such as CD80 and CD86), or a receptor that binds an antigen, or a variant, or a fragment thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Non-viral vectors may be used as well.
Co-Stimulatory Ligands
The interaction with at least one co-stimulatory ligand provides a non-antigen-specific signal required for full activation of a T cell. Co-stimulatory ligands include, without limitation, tumor necrosis factor (TNF) ligands, cytokines (such as IL-2, IL-12, IL-15 or IL21), and immunoglobulin (Ig) superfamily ligands.
TNF Ligands
Tumor necrosis factor (TNF) is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its primary role is in the regulation of immune cells. Tumor necrosis factor (TNF) ligands share a number of common features. The majority of the ligands are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. TNF ligands include, without limitation, nerve growth factor (NGF), CD40L (CD40L)/CD154, CD137L/4-1BBL, tumor necrosis factor alpha (TNFα), CD134L/OX40L/CD252, CD27L/CD70, Fas ligand (FasL), CD30L/CD153, tumor necrosis factor beta (TNFβ/lymphotoxin-alpha (LTα), lymphotoxin-beta (LTβ), CD257/B cell-activating factor (BAFF)/Blys/THANK/Tall-1, glucocorticoid-induced TNF Receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF14).
Ig Superfamily Ligands
The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. These proteins share structural features with immunoglobulins—they possess an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, without limitation, CD80 and CD86, both ligands for CD28.
For initial genetic modification of the cells to provide tumor or viral antigen-specific cells, a retroviral vector is generally employed for transduction, however any other suitable viral vector or delivery system can be used. For subsequent genetic modification of the cells to provide cells comprising an antigen presenting complex comprising at least two co-stimulatory ligands, retroviral gene transfer (transduction) likewise proves effective. Combinations of retroviruses and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various 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 suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art.
Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.
Other transducing viral vectors can be used to express a co-stimulatory ligand of the invention in an immunoresponsive cell. Preferably, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, 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, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).
Non-viral approaches can also be employed for the expression of a protein in cell. 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. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically.
cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can 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 can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
The resulting cells can then be grown under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.
Polypeptides and Analogs
Also included in the invention are PZ1, P28z, 4-1BBL, OX40L, CD70, LIGHT, and CD30L polypeptides or fragments thereof that are modified in ways that enhance their anti-neoplastic activity when expressed in an immunoresponsive cell. The invention provides methods for optimizing an amino acid sequence or nucleic acid sequence by producing an alteration in the sequence. Such alterations may include certain mutations, deletions, insertions, or post-translational modifications. The invention further includes analogs of any naturally-occurring polypeptide of the invention. Analogs can differ from a naturally-occurring polypeptide of the invention by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring amino, acid sequence of the invention. The length of 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 exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the invention by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., .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 embodiments a fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments at least 60 to 80, 100, 200, 300 or more contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).
Non-protein analogs have a chemical structure designed to mimic the functional activity of a protein of the invention. Such analogs are administered according to methods of the invention. Such analogs may exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs increase the anti-neoplastic activity of the original polypeptide when expressed in an immunoresponsive cell. These chemical modifications include, but are not limited to, substituting 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 in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below.
Administration
Compositions comprising genetically modified immunoresponsive cells of the invention (e.g., T cells, NK cells, CTL cells, or their progenitors) can be provided systemically or directly to a subject for the treatment of a neoplasia, pathogen infection, or infectious disease. In one embodiment, cells of the invention are directly injected into an organ of interest (e.g., an organ affected by a neoplasia). Alternatively, compositions comprising genetically modified immunoresponsive cells are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature). Expansion and differentiation agents can be provided prior to, during or after administration of the cells to increase production of T cells, NK cells, or CTL cells in vitro or in vivo.
The modified cells can be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). Usually, at least 1×105 cells will be administered, eventually reaching 1×1010, or more. Genetically modified immunoresponsive cells of the invention can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of genetically modified immunoresponsive cells in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Preferable ranges of purity in populations comprising genetically modified immunoresponsive cells are about 50 to about 55%, about 55 to about 60%, and 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 still more preferably the purity is about 85 to about 90%, about 90 to about 95%, and about 95 to about 100%. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage).
The cells can be introduced by injection, catheter, or the like. If desired, factors can also be included, including, but not limited to, interleukins, e.g. IL-2, IL-3, IL-6, and IL-11, as well as the other interleukins, the colony stimulating factors, such as G-, M- and GM-CSF, interferons, e.g. .gamma.-interferon 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 subject or a different, compatible subject.
Peripheral blood derived immunoresponsive cells of the invention or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present invention (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

Formulations

Compositions of the invention comprising genetically modified immunoresponsive cells can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally 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 tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.
Sterile injectable solutions can be prepared by incorporating the genetically modified immunoresponsive cells utilized in practicing the present invention in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.
Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. 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 pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the present invention, however, any vehicle, diluent, or additive used would have to be compatible with the genetically modified immunoresponsive cells or their progenitors.
The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of this invention may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
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 economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount that will achieve 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, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or 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 in the present invention. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.
One consideration concerning the therapeutic use of genetically modified immunoresponsive cells of the invention is the quantity of cells necessary to achieve an optimal effect. The quantity of cells to be administered will vary for the subject being treated. In a one embodiment, between 104 to 1010, between 105 to 109, or between 106 and 108 genetically modified immunoresponsive cells of the invention are administered to a human subject. In preferred embodiments, at least about 1×1082×108, 3×108, 4×108, and 5×108 genetically modified immunoresponsive cells of the invention are administered to a human subject. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.
The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and/or carrier in compositions and to be administered in methods of the invention. Typically, any additives (in addition to the active stem cell(s) and/or agent(s)) are present in an amount of 0.001 to 50% (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, preferably about 0.0001 to about 1 wt %, still more preferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %, and still more preferably about 0.05 to about 5 wt %. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it is preferred to determine therefore: toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.
Methods of Treatment
Provided herein are methods for treating neoplasia in a subject. Also contemplated herein are methods for treating a pathogen infection or other infectious disease in a subject, such as an immunocompromised human subject. The methods comprise administering a T cell, NK cell, or CTL cell of the invention in an amount effective to achieve the desired effect, be it palliation of an existing condition or prevention of recurrence. For treatment, the amount administered is an amount effective in producing the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion.
An “effective amount” (or, “therapeutically effective amount”) is an amount sufficient to effect a beneficial or desired clinical result upon 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 skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the antigen-binding fragment administered.
For adoptive immunotherapy using antigen-specific T cells, cell doses in the range of 109 are typically infused. Upon administration of the genetically modified cells into the host and subsequent differentiation, T cells are induced that are specifically directed against the specific antigen. “Induction” of T cells can include inactivation of antigen-specific T cells such as by deletion or anergy. Inactivation is particularly useful to establish or reestablish 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, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal and directly to the thymus.
Therapeutic Methods
The invention provides methods for increasing an immune response in a subject in need thereof. In one embodiment, the invention provides methods for treating or preventing a neoplasia in a subject. The invention provides therapies that are particularly useful for the treatment of subjects having prostate cancer, or metastatic prostate cancer that is not amenable to conventional therapeutic interventions. Suitable human subjects for therapy typically comprise two treatment groups that can be distinguished by clinical criteria. Subjects with “advanced disease” or “high tumor burden” are those who bear a clinically measurable tumor. A clinically measurable tumor is one that can be detected on the basis of tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram or X-ray; positive biochemical or histopathologic markers on their own are insufficient to identify this population). A pharmaceutical composition embodied in this invention is administered to these subjects to elicit an anti-tumor response, with the objective of palliating their condition. Ideally, reduction in tumor mass occurs as a result, but any clinical improvement constitutes a benefit. Clinical improvement includes decreased risk or rate of progression or reduction in pathological 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 been responsive to another mode of therapy. The prior therapy can have included (but is not restricted to, surgical resection, radiotherapy, and traditional chemotherapy. As a result, these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts, and are suitably defined for each different neoplasia. Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.
Another group have a genetic predisposition to neoplasia but have not yet evidenced clinical signs of neoplasia. For instance, women testing positive for a genetic mutation associated with breast cancer, but still of childbearing age, can wish to receive one or more of the antigen-binding fragments described herein in treatment prophylactically to prevent the occurrence of neoplasia until it is suitable to perform preventive surgery.
Human neoplasia subjects having any of the following neoplasias: glioblastoma, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate and small cell lung cancer) are especially appropriate 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, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinomas, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma, and liver metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the uterine cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinomas, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and diffuse) plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcomas and leiomyosarcomas.
The subjects can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and/or amelioration of side effects. The subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of recurrence.
Accordingly, the invention provides a method of treating or preventing a neoplasia in a subject, the method comprising administering an effective amount of an immunoresponsive cell comprising a receptor that binds a tumor antigen and a vector encoding a co-stimulatory ligand. In one embodiment, the neoplasia is selected from the group consisting of prostate cancer, colon cancer, breast cancer, and glioblastoma. In another embodiment, the tumor antigen is prostate-specific membrane antigen, CD19, NY-ESO-1, WT-1 or hTERT.
In another approach, the invention provides a method of enforcing tolerance in a subject, the method comprising administering an effective amount of an immunoresponsive cell comprising a receptor that binds an antigen and a vector encoding a co-stimulatory ligand. In one embodiment, the method prevents or reduces an autoimmune disease or a disease associated with allogeneic transplantation.
As a consequence of constitutive surface expression of co-stimulatory ligands, adoptively transferred human T or NK cells are endowed with augmented proliferative, cytolytic, and survival capacities in an intrinsically poorly immunogenic tumor or immunodeficient environment devoid of co-stimulatory ligands. Furthermore, subsequent to their localization to tumor or viral infection and their proliferation, co-stimulatory ligand-expressing T cells turn the tumor or viral infection site into a highly conductive environment for a wide range of immune cells involved in the physiological anti-tumor or antiviral response (tumor infiltrating lymphocytes, NK-, NKT-cells, dendritic cells, and macrophages).
In other embodiments, the invention provides methods for treating subjects with a pathogen infection (e.g., viral infection, bacterial infection, fungal infection, parasite infection, or protozoal infection. The invention is particularly useful for enhancing an immune response in an immunocompromised subject. Exemplary viral infections susceptible to treatment using a method of the invention include, but are not limited to, Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), and influenza virus infections.
Accordingly, the invention provides a method of treating or preventing a pathogen infection in a subject, the method comprising administering an effective amount of an immunoresponsive cell as described herein.
Kits
The invention provides kits for the treatment or prevention of a neoplasia, pathogen infection, immune disorder or allogeneic transplant. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of an immunoresponsive cell comprising one or more co-stimulatory ligands in unit dosage form. In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic vaccine; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired the immunoresponsive cell is provided together with instructions for administering the cell to a subject having or at risk of developing a neoplasia, pathogen infection, immune disorder or allogeneic transplant. The instructions will generally include information about the use of the composition for the treatment or prevention of neoplasia, pathogen infection, immune disorder or allogeneic transplant. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a neoplasia, pathogen infection, immune disorder or allogeneic transplant or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
Recombinant methods are well known in the art. The practice of the invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (Gait, ed., 1984); “Animal Cell Culture” (Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (Wei & Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (Miller & Calos, eds., 1987); “Current Protocols in Molecular Biology” (Ausubel et al., eds., 1987); “PCR: The Polymerase Chain Reaction”, (Mullis et al., eds., 1994); and “Current Protocols in Immunology” (Coligan et al., eds., 1991). These techniques are applicable to the production of the polynucleotides and polypeptides, and, as such, can be considered in making and practicing the invention. Particularly useful techniques for are discussed in the sections that follow.
EXAMPLES
The following examples are provided as a further description of the invention, and to illustrate but not limit the invention.
Example 1
T Cells Co-expressing CD80 and 4-1BBL Elicit Robust Proliferative Responses after Cyclic Stimulations through their Endogenous T Cell Receptor or through a Chimeric Antigen Receptor without Antigen Presenting Cell (APC)-provided Costimulation
To assess whether constitutive expression of costimulatory ligands in T cells could substitute for APC-mediated costimulation, the T cell responses of human primary T cells were first investigated in three experimental systems. Using anti-CD3 (OKT3)-mediated T cell activation, the expansion of peripheral blood T lymphocytes transduced with CD80 and 4-1BBL was quantified (FIG. 1a), which were compared to T cells transduced with either ligand alone or none. Recurrent T cell receptor (TCR)-stimulation alone in the absence of costimulatory ligands failed to expand T cells and rapidly induced a decline in T cell number following the first restimulation (FIG. 1b). In sharp contrast, OKT3-stimulated CD80+4-1BBL+ T cells triggered a mean 237-fold greater proliferation over 21 days. In comparison, T cells transduced with either ligand alone exhibited a mean 8.1-fold reduced proliferation (p<0.0001). Based on these observations, the concept of T-cell mediated costimulation was extended to two clinically relevant applications of adoptive T cell therapy, using cytomegalovirus (CMV)-specific memory donor T cells and differentiation antigen-specific, genetically redirected autologous T cells. CMV pp65-specific T lymphocytes briefly expanded on HLA-A*0201+pp65+ artificial APCs24 were readily transduced with CD80 and 4-1BBL (FIG. 1c). Following exposure to the HLA-A*0201+ pp65-transduced colonic tumor cell line Caco-2, T cells equipped with the costimulatory ligand pair CD80 and 4-1BBL exhibited a significantly greater (209-fold, p<0.0001) expansion, compared to a continuously declining T cell number in the control groups (FIG. 1d).
To rapidly generate tumor-reactive human T lymphocytes, peripheral blood T cells were retrovirally transduced with the chimeric antigen receptor Pz1 (Gade et al., Cancer Res. 65:9080-9088, 2005), a non-HLA-restricted antigen receptor specific for the tumor antigen PSMA. The Pz1 receptor comprises a PSMA-binding single chain antibody fragment fused to the human CD3 ζ signaling domain and is analogous in structure to other chimeric antigen receptors currently in use in clinical trials. Pz1+ T cells coexpressing CD80 and 4-1BBL (FIG. 1e) mounted a robust proliferative response following three weekly stimulations with PSMA+, CD80−, CD86−, 4-1BBL−LNCaP cells (mean 1042-fold enrichment, FIG. 1f, upper panel). This expansion was 9-fold, greater (p<0.0001) than that obtained when expressing CD80 and 4-1BBL in the tumor cells rather than in the T cells (FIG. 1f, lower panel). Further analyses documented the greater induction of IL-2 and IFN-γ by exposure to PSMA in T cells coexpressing CD80 and 4-1BBL, as well as their greater antigen-specific cytolytic potential and decreased susceptibility to apoptosis, when compared to conventionally stimulated T lymphocytes (data not shown). In parallel studies, other members of TNF ligand family5, including OX40L, CD27L (CD70) or CD30L, with or without CD80, were investigated and the combination of CD80 and 4-1BBL was found to be the most potent (data not shown).
In aggregate, these in vitro studies demonstrate the ability of T lymphocytes coexpressing CD80 and 4-1BBL cells to strongly potentiate suboptimal TCR-activation and, furthermore, to substitute for the lack of costimulation provided by the APC.
Example 2
T Cells Co-expressing CD80 and 4-1BBL Eradicate Established, Systemic Tumors
To investigate the potency of our CD80+4-1BBL+ T cells in vivo, a model of multifocal, established prostate cancer utilizing PSMA−PC-3 tumor cells (Gong et al., Neoplasia 1:123-7, 1999, which is hereby incorporated by reference in its entirety) was developed. Using dual-modality bioluminescence and magnetic resonance imaging, tumors were visualized four weeks after intravenous inoculation, prior to initiating adoptive T cell therapy. The lungs, cervical lymph nodes, bone marrow, and liver were identified as the main sites of disease (FIG. 2a). In this model, animals were treated four weeks after tumor inoculation with a single intravenous infusion of 8×106 PSMA-targeted T cells, expressing either CD80, 4-1BBL, both, or neither.
In control mice treated with 8×106 CD19-targeted T cells, which, like untransduced T cells, fail to lyse PSMA+ tumor targets in vitro tumor burden steadily progressed until mice had to be sacrificed (FIG. 2b,c). Treatment with Pz1+ T cells resulted in a short-term reduction of tumor burden, followed by terminal tumor progression (FIG. 2b), yielding a modest 12-day survival advantage (p=0.0001, FIG. 2c). Constitutive expression of either CD80 or 4-1BBL alone in PSMA-targeted T cells only marginally augmented this therapeutic response, extending the median survival to 63 and 66 days, respectively (p=0.077, p=0.056, respectively). T cells coexpressing CD80 and 4-1BBL induced major responses and reduced the tumor burden 3.3-fold (p=0.0028) four days after adoptive T cell transfer. A 1375-fold reduction was obtained after sixteen days, relative to the Pz1+ T cell treatment group (p=0.0002). Seven of ten treated animals remained tumor-free 200 days after infusion of Pz1+CD80+4-1BBL+ T lymphocytes, and none in all other treatment or control groups (FIG. 2c). The three mice that failed therapy initially showed marked tumor regression before relapsing (FIG. 2b) and survived for 100 days (FIG. 2c).
Example 3
In Vivo T Cell Expansion is Robust and Antigen-Specific
To track and quantify in vivo T-cell migration and accumulation in relation to tumor localization and tumor burden, adoptively transferred T cells were additionally marked with Click Beetle Red-luciferase (Ponomarev et al., Eur J Nucl Med Mol Imaging. 2004 May; 31(5):740-51) (CBR-luc, FIG. 3a). Serial imaging of mice treated with Pz1+ T cells showed a progressive increase in signal that reached a peak four days after T cell injection (FIG. 3b). A low-level signal remained detectable up to day 18. In the case of Pz1+CD80+4-1BBL+ T cells, peak signal was detected on day 8 (41-fold higher nadir photon count than Pz1+ T cells, p=0.0009), which was followed by a gradual signal decline, although bioluminescence could still be detected until day 100 in some animals (FIG. 3b). Importantly, the effect of CD80 and 4-1BBL co-expression was abrogated in 19z1+ T lymphocytes (FIG. 3b), consistent with the need for antigen stimulation for expansion to occur. Flow cytometric analyses and T cell counts of lung single cell suspensions in three treated mice per group determined the actual T cell number to be highly concordant with acquired bioluminescent signal intensities (FIG. 3c,d). These studies, therefore, demonstrate that T cells coexpressing CD80 and 4-1BBL expand in an antigen-dependent manner before eventually entering a contraction phase resulting in substantial, if not complete, T cell clearance.
Example 4
Coalescence of the Costimulatory Ligands CD80 and 4-1BBL with Their Respective Receptors CD28 and 4-1BB in the Immunological Synapse Precedes Functional T Cell Auto-costimulation
To address whether constitutively expressed costimulatory ligands activate T cells in cis, the question of whether these ligands colocalize with their cognate receptors during T-cell activation was first examined. Both CD28 and 4-1BB amplify T cell receptor (TCR) signaling after recruitment into central membrane compartments of the immunological synapse31-33. Therefore, colocalization of CD80 and 4-1BBL to the T-cell tumor cell contact area is likely a prerequisite for auto-costimulation. To visualize 4-1BBL distribution by confocal microscopy, the cytoplasmic domain was fused to monomeric dsRed and this protein was coexpressed with CD80 in Pz1-transduced CD8+ T lymphocytes. Immediately prior to admixing with unmodified CD28−, 4-1BB−LNCaP cells, T cells were labeled with fluorescein isothiocyanate-conjugated cholera toxin β subunit (FITC-CTB) to visualize lipid raft clusters after synapse formation. Following LNCaP tumor engagement, 4-1BBL and CD80, as well as 4-1BB and CD28, mobilized into choleratoxin-FITC-positive T-cell-tumor cell contact areas (FIG. 4a,b). To investigate the functional consequence of this interaction, the colocalization of Granzyme B (GRB. Defined, albeit sparse, GRB condensation was localized near the contact zone in clusters of Pz1-expressing CD8+ T cells and their cognate tumor targets (FIG. 4c,d)) was examined and quantified. The median GRB recruitment to T-cell tumor cell junctions was amplified 2.2-fold in CD80+4-1BBL+ T cells (p=0.0001). To confirm that these observed differences were indeed a result of 4-1BB engagement with 4-1BBL within the same cell, 4-1BB expression was knocked down by stable coexpression of 4-1BBL and 4-1BB-specific shRNA (data not shown). Knock-down of 4-1BB significantly diminished GRB density near the synapse (2.02-fold reduction, p<0.0001), despite the presence of 4-1BBL at the T-cell junction in all imaged cell clusters. These findings strongly suggest that the costimulatory ligand functionally engages its receptor on the same T-cell surface after antigen-induced coalescence in the immunological synapse.
To further demonstrate auto-costimulation, a single cell assay was devised in which the endogenous 4-1BB/4-1BBL interaction could be analyzed following the earliest expression of 4-1BBL in recently transduced T cells. To achieve this, firefly luciferase was first expressed under the transcriptional control of NF-κB, a key downstream effector of 4-1BB signaling, in a clone of CD3+CD28+4-1BB−Jurkat T cells (JNL). To preclude NF-κB induction by sporadic bystander costimulation, JNL cells were exposed to retroviral vectors encoding 4-1BB, 4-1BBL and CD80, and subcloned by limiting dilution 4 hours thereafter, well before the first detectable surface gene expression (data not shown), and immediately stimulated by plate-bound OKT3. As illustrated in FIG. 4e, isolated CD80+CD28+4-1BBL+4-1BB+ T cells markedly up-regulated NF-κB, in contrast to JNL cells transduced with control vector. The luminescence signal was acquired in 36 wells containing a single, multiply transduced cell as described in Methods. Coexpression of the two ligand pairs increased NF-κB-dependent signal 3.8-fold (relative to control JNL; p<0.0001; FIG. 4f). Since at any point of this assay the sole source of costimulatory ligands was the isolated T cell's own surface, this NF-κB upregulation could only reflect the impact of auto-costimulation.
Example 5
In Vitro Trans-Costimulation of Antigen-Specific Bystander T Cells
The constitutive expression of costimulatory ligands may also enable genetically modified T cells to costimulate T cells in trans. Notably, three-cell clusters comprised of CD80+4-1BBL+ and CD80−4-1BBL− T cells were occasionally noted in the confocal studies shown in FIG. 4 (FIG. 5a). To provide functional evidence for trans-costimulation, a coculture system was devised in which PSMA-specific T cells expressing CD80 and 4-1BBL were admixed with carboxyfluoroscein succinimidyl ester (CFSE)-labeled T cells that were not transduced with CD80 or 4-1BBL. It was found that these CFSE-labeled, CMV pp65-specific T cells were effectively costimulated by autologous, bystander Pz1+CD80+4-1BBL+ T lymphocytes (FIG. 5b,c). Physical contact between the two T cell populations was a prerequisite for the CMV-reactive cytotoxic T lymphocytes to expand, as their separation by a transwell membrane greatly reduced the strong induction of GRB and the robust T-cell expansion mounted in pp65 responder cells, consistent with a cell contact-dependent mechanism (FIG. 5d).
Example 6
In Vivo Trans-Costimulation of Tumor-Infiltrating T Cells
These observations prompted us to investigate whether T cell-mediated trans-costimulation is operative in vivo. To this end, two previously described animal models were combined: the RM1-PSMA tumor model in which tumors are confined to the lungs (Gade et al., Cancer Res. 65:9080-9088, 2005), and the Raji tumor model, in which tumor cells selectively colonize bone marrow (Brentjens et al., Nat Med. 2003 March; 9(3):279-86). All mice were treated with Pz1-transduced T cells, which also expressed CBR-luc (FIG. 3). Luciferase-negative Pz1+ T cells, either expressing or lacking CD80 and 4-1BBL, were subsequently infused into the animals. The expression of Gaussia-luciferase (Gau-Luc) in the tumor cells, and of CBR-luc in Pz1+T cells, allowed the use of dual bioluminescence imaging to simultaneously monitor tumor progression and the spatial and temporal accumulation of T cells. Comparable tissue distributions and bioluminescent signals of CBR-luc+ lymphocytes were observed six hours (day 0) after adoptive transfer in all treatment groups (FIG. 6a). On day 2, the effect of Pz1+CD80+4-1BBL+ T cells on CBR-luc+Pz1+ T cells was still modest (median 1.2-fold, relative to Pz1+ T cells as bystander, p=0.0947). The CBR-luc signal subsequently decayed in mice given control Pz1+ T cells (FIG. 6a). In contrast, this photon count recorded over the thoracic area augmented 6.5-fold on average in mice given Pz1+CD80+4-1BBL+ T lymphocytes (p=0.0122, FIG. 6a,b). Importantly, this effect was selective for the PSMA-targeted T cells, since co-injected CBR-luc+19z1-transduced T cells, which infiltrated the established Raji tumors in both femurs, did not significantly enhance thoracic bioluminescent signal at any time point (median 1.5-fold increase on day 4, p=0.0947, FIG. 6a,b). Collectively, these in vitro and in vivo data indicate that CD80+4-1BBL+ T cells locally enhance T-cell responses by providing costimulation in trans.
The constitutive display of CD80 on T lymphocytes serves as a costimulatory ligand for CD28 but could also engage negative regulators of T cell expansion, such as CTLA-4 at high affinities (Hodi et al. Clin Cancer Res. 2007 September 15:13:5238-42). To bypass therapeutically undesirable T cell auto-inhibition mediated by T cell-expressed CD80, T lymphocytes were co-transduced with the chimeric antigen receptor P28z (Maher et al. Nature Biotechnology, Vol 20, January 2002, P 70-75) and expressed 4-1BBL on the T cell surface (FIG. 7). The dual fusion receptor P28z contains both TCR and CD28 signaling moieties. Whereas signaling through P28z failed to invoke a sustained T cell expansion, the co-expression of 4-1BBL markedly enhanced the proliferative response more than 10-fold by day 14. The synergistic CD80-4-1BBL costimulatory signal, however, was still strongest when both full length ligands were expressed on the surface of Pz1-transduced T lymphocytes (approximately two-fold higher than P28Z+4-1BBL, FIG. 7). CD28 signals relayed by a CD28 signaling element fused into the chimeric antigen receptor construct can therefore endow Pz1+4-1BBL+ T lymphocytes with enhanced proliferative properties, although it is not as robust a T cell proliferation than observed in Pz1+4-1BBL+CD80+ T cells.
A major goal of cancer immunotherapy is to provide safe and effective costimulation to tumor-reactive T lymphocytes. Using a genetic approach, it was demonstrated that T cells themselves provide potent costimulation. Constitutive expression of costimulatory ligands in human primary CMV-specific cells and PSMA-targeted T cells not only compensated for the absence of these ligands on APCs, but also induced a proliferative response surpassing that elicited by conventional APCs (FIG. 1). The survey of a panel of costimulatory ligands indicated that CD80 and 4-1BBL provided the strongest T-cell activation under our experimental conditions, enabling robust T cell expansion following repeated weekly antigenic stimulation. CD80+4-1BBL+ T cells exhibited superior proliferation, cytokine secretion, in vitro survival and in vivo expansion and persistence, resulting in a 40- to 50-fold greater T cell biomass one week after infusion in tumor-bearing mice, when compared to PSMA-specific T cells that were not transduced with CD80 and 4-1BBL (FIG. 3). Whereas animals treated with non-CD80/4-1BBL-transduced PSMA-specific T cells uniformly succumbed to disease, infusion of the same T cells rendered CD80+4-1BBL+ effectively rejected established, systemic PC-3 tumors in the majority of treated mice (FIG. 2). These findings, obtained in a very challenging tumor model, underscore the biological activity and remarkable potency of constitutive, high-level expression of costimulatory ligands in T cells.
To investigate the mechanism underlying this potentiated response, the spatial distribution of CD80 and 4-1BBL and their receptors on T cells before and after tumor engagement were first examined. CD28 and 4-1BB have been shown to accumulate in close proximity to the TCR within the central supramolecular activation cluster of immunological synapses. Costimulatory ligands expressed by APCs, including CD80 CD40L and CD70, polarize to the synapse, supporting the notion that the immunological synapse provides an ordered contact area to facilitate and propagate costimulatory ligand-receptor interactions. Consistent with this model, T-cell-encoded CD80 and 4-1BBL polarized towards the contact zone between T cells and LNCaP tumor cells with, together with CD28 and 4-1BB (FIG. 4). The functional consequence of this colocalization is illustrated by the accumulation of GRB, which is ferried towards the T cell-APC interface along microtubules following antigen contact in synapses containing 4-1BB+4-1BBL+ foci but not in T cells lacking 4-1BB (FIG. 4). The immunological synapse is thus apparently well suited to orchestrate auto-costimulation.
Auto-costimulation was demonstrated in a single cell assay in which de novo expression of 4-1BB and 4-1BBL in recently transduced T cells activated NF-κB without any possible contribution from a neighboring T cell. NF-κB expression was visualized in real-time, after physical separation of single clones shortly after transduction, several hours before costimulatory ligand expression could be detected (FIG. 4). Auto-costimulation may thus allow T lymphocytes to override the poor costimulatory capacity of suboptimal APCs, including tumor cells. Furthermore, constitutive cell surface expression of 4-1BBL in T cells ensures costimulation ‘on demand’ by ensuring that the ligand remains ready to engage transiently upregulated receptors such as 4-1BB. One might speculate that the physical interaction of 4-1BBL with its receptor may occur during early raft reorganization or between opposing plasma membrane folds at the center of the synapse. This effect appears to be self-regulated as CD80+4-1BBL+ T-cell expansion was gradually attenuated in response to repeated in vitro antigen exposure (FIG. 1). In vivo T-cell expansion was also self-limited (FIG. 3), which may be due to tumor elimination that deprives the T cells of antigenic stimulation and attenuated costimulatory signaling.
Without wishing to be bound by theory, our studies also support trans-costimulation as a mechanism for enhancing immunity through high-level expression of CD80 and 4-1BBL in T lymphocytes. The proliferative and effector functions of T cells engaging APC-presented antigen in the absence of CD80 or 4-1BBL were thus amplified by in vitro coculture or in vivo coadministration of T cells displaying both ligands. In vitro cell mixing and transwell studies indicated that trans-costimulation required cell-cell contact (FIG. 5), in agreement with trans effects reported for other cell types such as fibroblasts or bystander tumor cells. Trans-costimulation was also induced in vivo, in organ-specific fashion, as illustrated in mice bearing two different tumors segregating to different sites (FIG. 6).
Trans-costimulation mediated by T cells paves the way for exciting new therapeutic approaches since costimulation may be delivered to neighboring T cells within the tumor microenvironment. This feature is especially valuable as dendritic cells frequently fail to fully upregulate costimulatory ligands within the tumor microenvironment. Although soluble factors such as interleukin-2 may contribute to T cell aid, the dependence on cell-cell contact effectively restricts trans-costimulation to other tumor-infiltrating T cells. Without wishing to be bound by theory, the effect of T cell competition on the in vivo frequency of trans-costimulation. in trans may ultimately broaden the anti-tumor immune response via the recruitment of a diverse population of endogenous tumor-infiltrating lymphocytes and thus help to prevent tumor antigen escape.
In summary, it was shown that CD80 and 4-1BBL expression in human T lymphocytes is a biologically efficacious means to circumvent the lack of conventional APC-mediated costimulation in the tumor microenvironment. This approach is applicable to T cells activated through a transduced antigen receptor as well as their endogenous TCR. T cell-mediated costimulation, whether in auto, trans or both, may thus be useful in a wide range of malignancies and infectious diseases that are being treated by adoptive T cell therapy.
EMBODIMENTS OF THE INVENTION
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

## Claims

1. An immunoresponsive cell comprising a receptor that binds an antigen and an exogenous co-stimulatory ligand, wherein the exogenous co-stimulatory ligand is 4-1BBL and/or CD80 or CD86.

2. The immunoresponsive cell of claim 1, wherein the co-stimulatory ligand is constitutively or inducibly expressed.

3. The immunoresponsive cell of claim 1, wherein at least two co-stimulatory ligands are constitutively expressed.

4. The immunoresponsive cell of claim 1, wherein the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

5. The immunoresponsive cell of claim 1, wherein the antigen is a tumor or pathogen antigen.

6. The immunoresponsive cell of claim 3, wherein the antigen is selected from the group consisting of prostate-specific membrane antigen (PSMA), Carcinoembryonic Antigen (CEA), IL13Ralpha, her-2, CD19, NY-ESO-1, HIV-1 Gag, Lewis Y, Mart-1, gp100, tyrosinase, WT-1, hTERT, and mesothelin.

7. The immunoresponsive cell of claim 1, wherein the cell expresses a recombinant or an endogenous antigen receptor that is Pz1 or P28z.

8. The immunoresponsive cell of claim 1, wherein the co-stimulatory ligand is a tumor necrosis factor (TNF) family ligand or an immunoglobulin (Ig) superfamily ligand.

9. The immunoresponsive cell of claim 8, wherein the TNF family ligand is 4-1BBL.

10. The immunoresponsive cell of claim 8, wherein the Ig superfamily ligand is selected from the group consisting of CD80 and CD86.

11. The immunoresponsive cell of claim 1, wherein the immunoresponsive cell expresses 4-1BBL.

12. The immunoresponsive cell of claim 1, wherein the immunoresponsive cell expresses 4-1BBL and CD80.

13. The immunoresponsive cell of claim 1, wherein an endogenous or recombinant antigen receptor or antigen presenting complex is constitutively expressed on the surface of the T cell.

14. The immunoresponsive cell of claim 1, wherein the co-stimulatory ligand is constitutively expressed on the surface of the T cell.

15. An immunoresponsive cell comprising a receptor that binds an antigen and an exogenous co-stimulatory ligand, wherein the co-stimulatory ligand is expressed in a retroviral vector.

16. A virus specific T cell expressing a vector encoding a polypeptide selected from the group consisting of CD80 and 4-1BBL.

17. The virus specific T cell of claim 16, wherein the T cell recognizes a virus selected from the group consisting of Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), and influenza virus antigens.

18. A tumor antigen-specific T cell expressing a vector encoding a polypeptide selected from the group consisting of CD80 and 4-1BBL.

19. The tumor antigen-specific T cell of claim 18, wherein the cell expresses CD80 and 4-1BBL.

20. The tumor antigen-specific T cell of claim 18, wherein the vector is a retroviral vector.

21. The immunoresponsive cell of claim 15, wherein the immunoresponsive cell expresses 4-1BBL and/or CD80 or CD86.

22. The immunoresponsive cell of claim 15, wherein the co-stimulatory ligand is constitutively or inducibly expressed.

23. The immunoresponsive cell of claim 15, wherein at least two co-stimulatory ligands are constitutively expressed.

24. The immunoresponsive cell of claim 1, wherein the cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), and a regulatory T cell.

25. The immunoresponsive cell of claim 15, wherein the antigen is a tumor or pathogen antigen.

26. The immunoresponsive cell of claim 25, wherein the antigen is selected from the group consisting of prostate-specific membrane antigen (PSMA), Carcinoembryonic Antigen (CEA), IL13Ralpha, her-2, CD19, NY-ESO-1, HIV-1 Gag, Lewis Y, Mart-1, gp100, tyrosinase, WT-1, hTERT, and mesothelin.

27. The immunoresponsive cell of claim 15, wherein the cell expresses a recombinant or an endogenous antigen receptor that is Pz1 or P28z.

28. The immunoresponsive cell of claim 15, wherein the co-stimulatory ligand is a tumor necrosis factor (TNF) family ligand or an immunoglobulin (Ig) superfamily ligand.

29. The immunoresponsive cell of claim 28, wherein the TNF family ligand is 4-1BBL.

30. The immunoresponsive cell of claim 21, wherein the immunoresponsive cell expresses CD80.

31. The immunoresponsive cell of claim 21, wherein the immunoresponsive cell expresses CD86.

32. The immunoresponsive cell of claim 21, wherein the immunoresponsive cell expresses 4-1BBL.

33. The immunoresponsive cell of claim 21, wherein the immunoresponsive cell expresses 4-1BBL and CD80.

34. The immunoresponsive cell of claim 15, wherein an endogenous or recombinant antigen receptor or antigen presenting complex is constitutively expressed on the surface of the T cell.

35. The immunoresponsive cell of claim 15, wherein the co-stimulatory ligand is constitutively expressed on the surface of the T cell.

36. An immunoresponsive cell comprising a receptor that binds an antigen and an exogenous co-stimulatory ligand, wherein the immunoresponsive cell is a T-cell or NK cell and expresses 4-1BBL and/or CD80 or CD86.