Switch costimulatory receptors

Abstract

To provide a fusion protein that when displayed on a cell can convert a negative signal into a positive signal in the cell.SOLUTION: The fusion protein is a chimeric protein comprising at least two domains, where the first domain is a polypeptide that is associated with a negative signal and the second domain is a polypeptide that is associated with a positive signal. Also provided are cells engineered to express fusion proteins being switch receptors that are able to switch negative signals to positive signals for enhancement of an immune response.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 61 / 513,259, filed July 29, 2011, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Background of the Invention A general principle of the immune system is that T cells sense the microenvironment and are then activated or inhibited depending on the sensed signal. The CD28 gene family is composed of two genes, CD28 and ICOS, that transmit positive signals, and three genes, CTLA4, PD-1, and BTLA, that deliver negative signals (Riley et al., 2005, Blood 105:13-21). The ligands for PD-1 are PDL1 and PDL2. It is well known that PD-1 ligands are often expressed in the tumor microenvironment, and contact of PDL1 or PDL2 with PD-1 on T cells can result in T cell inactivation.

[0003] Currently, the only approach to prevent the negative signals delivered by PD-1 or BTLA ligands is to administer antagonistic antibodies or fusion proteins that bind to PD-1 or BTLA, an approach currently being tested in early-phase clinical trials (Cheever et al., 2008, Immunol Rev 222:357-68). Another approach would be to administer small molecules that can inhibit PD-1 or BTLA signaling. The current approach to prevent T cell inactivation by PD-1 is to administer systemic treatment to patients with PD-1 antagonistic antibodies.

[0004] All of the above approaches have the limitation that systemic treatment prevents inactivation of T cells present in both the tumor microenvironment and the immune system as a whole, which is predicted to lead to autoimmune or systemic inflammatory syndromes in some patients (Beck et al., 2006, J Clin Oncol 24:2283-9 (Non-Patent Document 3); Blansfield et al., 2005, J Immunother 28:593-8 (Non-Patent Document 4); Dougan et al., 2009, Annual Review of Immunology 27:83-117 (Non-Patent Document 5)).

[0005] Thus, there is an urgent need in the art for compositions and methods for effective forms of adoptive therapy, which the present invention addresses. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Riley et al.,2005,Blood 105:13-21 [Non-patent document 2] Cheever et al.,2008,Immunol Rev 222:357-68 [Non-patent document 3] Beck et al.,2006,J Clin Oncol 24:2283-9 [Non-patent document 4] Blansfield et al.,2005,J Immunother 28:593-8 [Non-Patent Document 5] Dougan et al.,2009,Annual Review of Immunology 27:83-117 Summary of the Invention

[0007] The present invention provides a fusion protein comprising a first domain that is a polypeptide associated with a negative signal and a second domain that is a polypeptide associated with a positive signal.

[0008] In one embodiment, the first domain is at least a portion of an extracellular domain of a polypeptide associated with negative signaling, and the second domain is at least a portion of an intracellular domain of a polypeptide associated with positive signaling.

[0009] In one embodiment, the fusion protein further comprises a transmembrane domain, hi another embodiment, the transmembrane domain is the transmembrane domain of a polypeptide associated with a negative signal or the transmembrane domain of a polypeptide associated with a positive signal.

[0010] In one embodiment, the polypeptide associated with negative signaling is selected from the group consisting of CTLA4, PD-1, and BTLA.

[0011] In one embodiment, the polypeptide associated with a positive signal is selected from the group consisting of CD28 and ICOS.

[0012] The present invention also provides cells engineered to express a fusion protein comprising a first domain that is a polypeptide associated with a negative signal and a second domain that is a polypeptide associated with a positive signal.

[0013] In one embodiment, the cell further comprises a chimeric antigen receptor (CAR) comprising the antigen recognition domain of a specific antibody and the intracellular domain of the CD3 zeta chain.

[0014] The present invention also provides a vector comprising a first domain that is a polypeptide associated with a negative signal and a second domain that is a polypeptide associated with a positive signal.

[0015] The present invention provides methods for treating a cancer patient, hi one embodiment, the method comprises administering to the patient T cells that have been genetically engineered to express a fusion protein comprising a first domain that is a polypeptide associated with negative signaling and a second domain that is a polypeptide associated with positive signaling.

[0016] In one embodiment, the T cells are further genetically engineered to express a CAR comprising the antigen recognition domain of a specific antibody and the intracellular domain of the CD3 zeta chain.

[0017] In one embodiment, the T cells are autologous T cells. [The present invention 1001] A fusion protein comprising a first domain and a second domain, wherein the first domain is a polypeptide associated with a negative signal and the second domain is a polypeptide associated with a positive signal. [The present invention 1002] The fusion protein of the present invention 1001, wherein the first domain is at least a portion of the extracellular domain of a polypeptide associated with a negative signal, and the second domain is at least a portion of the intracellular domain of a polypeptide associated with a positive signal. [The present invention 1003] The fusion protein of the present invention 1001 further comprising a transmembrane domain. [The present invention 1004] The fusion protein of the present invention 1003, wherein the transmembrane domain is a transmembrane domain of a polypeptide associated with a negative signal or a transmembrane domain of a polypeptide associated with a positive signal. [The present invention 1005] 1001. The fusion protein of the present invention, wherein the polypeptide involved in negative signaling is selected from the group consisting of CTLA4, PD-1, and BTLA. [The present invention 1006] 1001. The fusion protein of the present invention, wherein the polypeptide associated with a positive signal is selected from the group consisting of CD28 and ICOS. [The present invention 1007] A cell engineered to express a fusion protein comprising a first domain and a second domain, wherein the first domain is a polypeptide associated with a negative signal and the second domain is a polypeptide associated with a positive signal. [The present invention 1008] 1007. The cell of claim 1007, further comprising a chimeric antigen receptor (CAR) comprising an antigen recognition domain of a specific antibody and an intracellular domain of the CD3 zeta chain. [The present invention 1009] A vector comprising a first domain and a second domain, wherein the first domain is a polypeptide associated with a negative signal and the second domain is a polypeptide associated with a positive signal. [The present invention 1010] 1. A method of treating a patient with cancer, comprising administering to the patient T cells that have been genetically engineered to express a fusion protein comprising a first domain and a second domain, wherein the first domain is a polypeptide associated with negative signaling and the second domain is a polypeptide associated with positive signaling. [The present invention 1011] The method of claim 1010, wherein the T cells are further genetically engineered to express a chimeric antigen receptor (CAR) comprising the antigen recognition domain of a specific antibody and the intracellular domain of the CD3 zeta chain. [The present invention 1012] The method of claim 1011, wherein the T cells are autologous T cells.

[0018] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0019] [Figure 1A]Figure 1, comprising Figures 1A-1C, is a series of images demonstrating that BTLA signals can be transduced into other signals in the form of chimeric costimulatory receptors (CCRs), also called switch receptors. Figure 1A is an image depicting a schematic of a chimeric switch receptor. [Figure 1B] FIG. 1B is an image demonstrating that surface expression of BTLA was detected by HVEM-Fc fusion protein at different time points as indicated. [Figure 1C] Figure 1C shows images of IL-2 produced by electroporated T cells stimulated with either a BTLA ligand-negative cell line (KTPloCD86A2) or a BTLA ligand-HVEM-positive cell line (KTPloCD86A2 HVEM). IL-2 production was assayed by ELISA 24 hours after stimulation. The results showed that by fusing the BTLA extracellular domain with the intracellular domains of both ICOS and CD3ζ, T cells could be activated by stimulation with the BTLA ligand-HVEM-expressing cell line. This indicates that BTLA signals can be transduced into other signals in the form of chimeric costimulatory receptors. [Figure 2A] Figure 2, comprising Figures 2A-2C, is a series of images demonstrating that BTLA signals can be transduced into CD28 signals through the BTLA-CD28 CCR. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 3A] Figure 3, including Figures 3A and 3B, is a series of images demonstrating that BTLA signaling can be converted to ICOS signaling through BTLA-ICOS CCR. The results showed that ICOS signaling converted from BTLA-ICOS enhanced Th17 cell production. [Figure 3B] See legend to Figure 3A. [Figure 4A] Figure 4, comprising Figures 4A-4D, is a series of images demonstrating that PD1 signals can be transduced into CD28 signals. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 5A] Figure 5, comprising Figures 5A and 5C, is a series of images demonstrating reversal of PD1 inhibition by PD1-CD28 CCR co-transduction. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6A] Figure 6, comprising Figures 6A-6C, is a series of images demonstrating the conversion of PD1 signals to ICOS signals. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7] FIG. 7 is an image demonstrating that the inhibitory effect of PD1wt on cytokine production is rescued by a PD1 chimeric construct. [Figure 8] FIG. 8 is an image demonstrating that the PD-1 chimeric receptor does not affect granzyme B production. [Figure 9] FIG. 9 is an image demonstrating that minimal differences in the killing activity of CD8 T cells were observed in the presence or absence of PD1. [Figure 10] FIG. 10 is an image showing the effect of PD-1 chimeric receptors on T cell proliferation. [Figure 11] FIG. 11 is an image showing that PD1-CD28 chimeric receptors increase the number of CD8 T cells. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description The present invention generally relates to fusion protein receptors that, when displayed on a cell, can convert a negative signal into a positive signal for the cell. Fusion proteins are chimeric proteins because they contain at least two domains: a first domain is a polypeptide associated with a negative signal and a second domain is a polypeptide associated with a positive signal. In one embodiment, the first domain binds to an inhibitor and activates the fusion protein, resulting in a positive signal transmitted through the second domain and transduced into the cell. In this way, the fusion protein can convert an otherwise negative signal into a positive intracellular signal. Therefore, the present invention can be considered to encompass switch receptors that can switch a negative signal into a positive signal for enhanced immune response. Enhanced immune response can treat diseases associated with inappropriate immune responses.

[0021] The present invention is based on the discovery that T cells can be engineered to express switch receptors to take advantage of the fact that T cells sense the microenvironment and are activated or inhibited depending on the signal they sense. For example, the present invention takes advantage of the fact that ligands that inhibit T cell activity are present in the tumor microenvironment. T cells are engineered to express switch receptors whose first domain is activated by an inhibitory ligand in the tumor microenvironment and can, by signaling through the second domain of the switch receptor, switch an otherwise inhibitory signal into a positive signal for the T cell. Thus, the present invention provides therapies that offer an improved therapeutic index with less toxicity, and also provides the ability to provide a single treatment that is effective and avoids the need for continuous administration of antibodies.

[0022] In some cases, the cells are genetically modified before being administered to a patient in need thereof. Preferably, the cells can be genetically modified to stably express the desired switch receptor of the present invention. In other cases, the cells can be further modified to express an antibody binding domain (e.g., a chimeric antigen receptor (CAR)) on the surface that confers novel antigen specificity that is MHC-independent. A CAR combines the antigen recognition domain of a specific antibody with the intracellular domain of the CD3ζ chain or the FcγRI protein into a single chimeric protein. In this regard, the cells are engineered to express both the switch receptor and the CAR.

[0023] The modified cells of the present invention are capable of replicating in vivo, resulting in long-term persistence that may result in sustained tumor control.

[0024] The present invention further provides methods of making the switch receptors of the invention and methods of using these switch receptors in the research and treatment of cancer.

[0025] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0026] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0027] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0028] As used herein, "about" when referring to measurable values ​​such as amounts, time periods, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for practicing the disclosed methods.

[0029] The term "antibody," as used herein, refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody may be a complete immunoglobulin derived from natural or recombinant sources, or an immunoreactive portion of a complete immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. Antibodies in the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0030] The term "antigen" or "Ag," as used herein, is defined as a molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that virtually any macromolecule, including proteins or peptides, can function as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response encodes an "antigen," as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen may be synthetically produced or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0031] The term "anti-tumor effect," as used herein, refers to a biological effect that can be manifested as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an amelioration of various physiological symptoms associated with a cancerous condition. An "anti-tumor result" can also be primarily manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the development of tumors.

[0032] As used herein, the term "autologous" may be used to refer to material derived from the same individual that is later reintroduced into the individual.

[0033] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0034] "Xenogeneic" refers to a graft derived from an animal of a different species.

[0035] As used herein, "having biological or immunological activity" refers to a fusion protein of the present invention having a similar structural function (not necessarily to the same extent) and / or a similar regulatory function (not necessarily to the same extent) and / or a similar biochemical function (not necessarily to the same extent) and / or immunological activity (not necessarily to the same extent) as the individual wild-type proteins that are the building blocks of the fusion protein of the present invention.

[0036] The term "cancer," as used herein, is defined as a disease characterized by the rapid, unregulated growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0037] "Chimeric protein" means a single polypeptide unit that contains two distinct polypeptide domains that do not occur naturally within the same polypeptide unit. Typically, such chimeric proteins are made by expression of a cDNA construct, but may also be made by protein synthesis methods known in the art.

[0038] The term "derivative" as used herein in reference to an amino acid sequence refers to a chemical modification of the fusion protein of the present invention.

[0039] "Encoding" refers to the inherent property of a specific nucleotide sequence of a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a distinct sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a distinct sequence of amino acids, and the biological properties attributed thereto. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces that protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is generally provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0040] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may contain introns.

[0041] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein effective to achieve a particular biological result. Such result may include, but is not limited to, inhibition of viral infection as determined by any suitable means in the art.

[0042] As used herein, "endogenous" refers to material that originates from or is produced within an organism, cell, tissue, or system.

[0043] As used herein, "exogenous" refers to material introduced from or produced outside an organism, cell, tissue, or system.

[0044] The term "expression," as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0045] "Expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) into which a recombinant polynucleotide may be incorporated.

[0046] As used herein, the term "fusion protein" refers to a chimeric protein containing amino acid sequences from two or more different proteins. Typically, fusion proteins result from in vitro recombinant techniques well known in the art.

[0047] As used herein, "homologous" refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both molecules is occupied by the same monomer subunit; for example, if a position in each of two DNA molecules is occupied by adenine, they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in two sequences (e.g., 5 positions in a polymer 10 subunits long) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0048] The term "immune response," as used herein, means the detectable result of the stimulation and / or activation of immune cells.

[0049] "Immune response," as that term is used herein, refers to a process that results in the activation and / or elicitation of effector functions in either T cells, B cells, natural killer (NK) cells, and / or antigen-presenting cells. Thus, an immune response, as understood by those skilled in the art, includes, but is not limited to, detectable antigen-specific or allogeneic activation of helper T cell responses or cytotoxic T cell responses, production of antibodies, T cell-mediated activation of allergic reactions, and the like.

[0050] "Immune cell," as that term is used herein, refers to any cell involved in the initiation of an immune response. Such cells include, but are not limited to, T cells, B cells, NK cells, antigen-presenting cells, etc.

[0051] As used herein, "instructional material" includes publications, records, graphics, or other media of expression that can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kits of the invention may, for example, be affixed to a container containing the nucleic acids, peptides, and / or compositions of the invention, or may be shipped together with the container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the compounds be used cooperatively by the recipient.

[0052] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0053] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0054] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, in that a nucleotide sequence that encodes a protein may, in some versions, contain introns.

[0055] As used herein, "lentivirus" refers to a genus of the Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; it can deliver significant amounts of genetic information to the DNA of host cells, and is therefore one of the most efficient gene delivery vectors.HIV, SIV, and FIV are all examples of lentiviruses.Vector derived from lentiviruses offer a means to achieve significant levels of gene transfer in vivo.

[0056] The term "modulating" an immune response, as used herein, means mediating a detectable increase or decrease in the level of immune response in a mammal compared to the level of immune response in the mammal in the absence of treatment or compound, and / or compared to the level of immune response in an otherwise identical but untreated mammal. The term encompasses interfering with and / or affecting native signals or responses in a mammal, preferably a human, thereby mediating a beneficial therapeutic response.

[0057] "Negative signal," as used herein, means a signal that induces a typical cascade of intracellular events associated with, inter alia, decreased proliferation, decreased activation, decreased cell processing, etc.

[0058] "Positive signal," as used herein, means a signal that induces a typical cascade of intracellular events associated with, inter alia, increased proliferation, increased activation, increased cell processing, etc.

[0059] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed into a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0060] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0061] The term "polynucleotide," as used herein, is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide, as used herein, are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cellular genomes using conventional cloning techniques and PCR™, etc., and synthetic means.

[0062] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise a protein or peptide sequence. Polypeptides include peptides or proteins containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, e.g., commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, e.g., commonly referred to in the art as proteins (of which there are many types). "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0063] The term "promoter," as used herein, is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0064] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be a core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0065] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the production of the gene product in a cell under most or all physiological conditions of the cell.

[0066] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, substantially causes production of the gene product in a cell only when an inducer corresponding to the promoter is present in the cell.

[0067] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene, causes production of a gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0068] The term "subject" is intended to include living organisms (eg, mammals) in which an immune response can be elicited.

[0069] As used herein, a "substantially purified" cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in its naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous population of cells. In other cases, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0070] The term "therapeutic" as used herein means treatment and / or prophylaxis. A therapeutic effect is obtained by suppressing, alleviating, or eradicating a disease state.

[0071] The terms "transfected" or "transformed" or "transduced," as used herein, refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary host cell and its progeny.

[0072] The phrases "under transcriptional control" or "operably linked," as used herein, mean that the promoter is in the correct location and orientation relative to the polynucleotide to control the initiation of transcription and expression by RNA polymerase of the polynucleotide.

[0073] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0074] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand, which mediates a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGFβ and / or rearrangement of cytoskeletal structures.

[0075] "Activated," as used herein, refers to a state of T cells that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells undergoing cell division. Activation can also be associated with the generation of an immune response (e.g., mitogens such as ConA or PHA), detectably upregulating surface markers such as CD25, the IL2 receptor, and initiating a phosphorylation cascade involving p56lck, resulting in the release of cytokines and interleukins, and, inter alia, binding to nascent DNA strands. 3 It increases DNA synthesis, which can be assessed by assessing the level of 3H-thymidine incorporation, causing cell proliferation.

[0076] The term "specifically binds," as used herein, refers to an antibody or ligand that recognizes and binds to a cognate binding partner protein present in a sample (e.g., a stimulatory molecule and / or costimulatory molecule present on a T cell), but does not substantially recognize or bind to other molecules in the sample.

[0077] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is true regardless of the breadth of the range.

[0078] explanation The present invention relates to the discovery that chimeric switch receptors can be designed to switch negative signaling signals to positive signals. In one embodiment, the switch receptor is a chimeric protein comprising a first protein or a fragment thereof associated with negative signaling and a second protein or a fragment thereof associated with positive signaling. Examples of proteins associated with negative signaling include, but are not limited to, CTLA-4, PD-1, BTLA, etc. Examples of proteins associated with positive signaling include, but are not limited to, CD28, ICOS, etc.

[0079] The present invention relates to chimeric switch receptors and related fusion proteins, and methods of treating cancer with these proteins.

[0080] In one embodiment, the present invention provides cells (e.g., T cells or natural killer cells) engineered to express a chimeric switch receptor that exhibits anti-tumor properties. In some cases, the engineered cells are also engineered to express a chimeric antigen receptor (CAR). In some cases, the engineered cells of the present invention exhibit enhanced IL-2 and IFN-γ production. In some cases, the engineered cells of the present invention are biased to secrete IL-17. Thus, when infused into a patient, the engineered cells of the present invention can eliminate tumor cells in vivo in the patient.

[0081] composition In one aspect, the present invention provides a switch receptor that, when expressed in a cell, converts a negative signal into a positive intracellular signal. For example, the switch receptor has a first domain comprising a polypeptide that delivers a negative signal and a second domain comprising a polypeptide that delivers a positive signal.

[0082] In one embodiment, polypeptides capable of delivering a negative signal include, but are not limited to, CTLA4, PD-1, BTLA, and the like.

[0083] In one embodiment, polypeptides capable of delivering a positive signal include, but are not limited to, ICOS, CD28, and the like.

[0084] Suitable first domains for polypeptides that deliver negative signals include variants or derivatives of wild-type CTLA4. Preferably, the first domain of the switch receptor of this embodiment is at least a portion of the extracellular domain of the CTLA protein, specifically the portion of the extracellular domain required for binding to the natural ligand of CTLA. Variants of the wild-type extracellular domain or the portion of the extracellular domain responsible for binding to the natural ligand of CTLA are also included in the present invention, as long as they provide a level of biological activity similar to that of the wild-type protein.

[0085] Suitable first domains for polypeptides that deliver negative signals include variants or derivatives of wild-type PD-1. Preferably, the first domain of the switch receptor of this embodiment is at least a portion of the extracellular domain of the PD-1 protein, specifically the portion of the extracellular domain required for binding to PD-1's natural ligand. Variants of the wild-type extracellular domain or the portion of the extracellular domain responsible for binding to PD-1's natural ligand are also included in the present invention, so long as they provide a level of biological activity similar to that of the wild-type protein.

[0086] Suitable first domains for polypeptides that deliver negative signals include variants or derivatives of wild-type BTLA. Preferably, the first domain of the switch receptor of this embodiment is at least a portion of the extracellular domain of the BTLA protein, specifically the portion of the extracellular domain required for binding to BTLA's natural ligand. Variants of the wild-type extracellular domain or the portion of the extracellular domain responsible for binding to BTLA's natural ligand are also included in the present invention, so long as they provide a level of biological activity similar to that of the wild-type protein.

[0087] Suitable second domains for polypeptides that deliver positive signals include variants or derivatives of the ICOS protein. Preferably, the second domain of the switch receptor in this embodiment is at least a portion of the intracellular domain (also called the endodomain) of the ICOS protein, specifically the portion necessary for inducing a signal to an intracellular component of a cell. Variants of the intracellular domain of the wild-type ICOS protein or the portion of the intracellular domain responsible for signaling are also included in the present invention, as long as they provide a level of biological activity similar to that of the wild-type protein.

[0088] Suitable second domains for polypeptides that deliver positive signals include variants or derivatives of the CD28 protein. Preferably, the second domain of the switch receptor in this embodiment is at least a portion of the intracellular domain (also called the endodomain or cytoplasmic domain) of the CD28 protein, specifically the portion necessary for inducing a signal to an intracellular component of a cell. Variants of the intracellular domain of the wild-type CD28 protein or the portion of the intracellular domain responsible for signaling are also included in the present invention, as long as they provide a level of biological activity similar to that of the wild-type protein.

[0089] Switch receptors of the invention include polypeptides corresponding to the cytoplasmic, transmembrane, and extracellular domains, as well as polypeptides corresponding to smaller portions of the cytoplasmic, transmembrane, and extracellular domains. In one embodiment, the switch receptor includes the transmembrane domain of a first polypeptide that delivers a negative signal. In another embodiment, the switch receptor includes the transmembrane domain of a second polypeptide that delivers a positive signal.

[0090] In yet additional aspects of the invention, the first polypeptide delivering the negative signaling component of any of the switch receptors described herein may be replaced with another inhibitory protein, i.e., a protein that prevents activation of an immune response and / or induces apoptosis in other cell types, such as T cells or B cells, natural killer (NK) cells, NKT cells, lymphocyte precursor cells, dendritic cells, monocytes / macrophages, tissue-based macrophage lineage cells with antigen-presenting capacity, and any of a number of non-professional antigen-presenting cells, e.g., endothelial cells. Examples of inhibitory proteins include, but are not limited to, PD-1, CTLA-4, BTLA, CD160, CD161, and CD94; ligands for LAG-3 and CD244 (see 2011, Wherry, Nat Immunol. 131:492-9).

[0091] Any suitable first polypeptide that delivers a negative signal can be used according to the present invention, provided that it binds to its corresponding ligand and, through this binding event, results in activation of the switch receptor. According to one embodiment of the present invention, contact of a first polypeptide that delivers a negative signal of a switch receptor with its corresponding ligand results in activation of a second polypeptide that delivers a positive signal of the switch receptor. In this way, a negative signal can be converted into a positive signal. That is, the first polypeptide of the switch receptor of the present invention can induce an intracellular signaling pathway such that activation of the second polypeptide of the present invention results in conversion of the negative signal into a positive signal. Thus, a unique property of the first polypeptide of the switch receptor of the present invention is that it converts a natural trans-signal that would naturally result in a negative signal in a cell into a positive signal that induces the cell to exhibit anti-tumor properties.

[0092] Similarly, any suitable second polypeptide can be used so long as it is capable of sending a positive signal to the cell, i.e., a signal that is distinct from the trans-signal associated with the first polypeptide component of the switch receptor. The second polypeptide can be a protein that sends a positive or activating signal. Preferred examples of second polypeptides of the present invention include, but are not limited to, CD28, CD27, ICOS, CD137 (4-1BB), and TCRζ.

[0093] In one embodiment, the present invention takes advantage of microenvironments that are teeming with ligands or proteins that inhibit the immune system and lead to undesirable disease states. That is, switch receptors can be engineered to contain a first domain that binds to immunoinhibitory factors within the microenvironment and converts the signal normally associated with the immunoinhibitory factor into a positive signal that activates cells to mount an enhanced immune response.

[0094] A preferred chimeric protein of the present invention is BTLA:ICOS. Genetic chimerization and recombinant expression of a BTLA sequence with an ICOS sequence results in a chimeric BTLA:ICOS "switch receptor" that exhibits structural and functional characteristics attributable to both BTLA and ICOS. Cells engineered to express BTLA:ICOS can divert inhibitory signaling to stimulatory signals, thereby enhancing T cell function. In some cases, cells are engineered to express the BTLA:ICOS switch receptor in combination with a CAR.

[0095] Another preferred chimeric protein of the present invention is PD1:CD28. Genetic chimerization and recombinant expression of the PD1 sequence with the CD28 sequence results in a chimeric PD1:CD28 "switch receptor" that exhibits structural and functional characteristics attributable to both PD1 and CD28. Cells engineered to express PD1:CD28 can redirect inhibitory signaling into stimulatory signals, thereby enhancing T cell function. In some cases, cells are engineered to express the PD1:CD28 switch receptor in combination with a CAR.

[0096] Another preferred chimeric protein of the present invention is CTLA4:CD28. Genetic chimerization and recombinant expression of the CTLA4 sequence with the CD28 sequence results in a chimeric CTLA4:CD28 "switch receptor" that exhibits structural and functional characteristics attributable to both CTLA4 and CD28. Cells engineered to express CTLA4:CD28 can redirect inhibitory signaling into stimulatory signals, thereby enhancing T cell function. In some cases, cells are engineered to express the CTLA4:CD28 switch receptor in combination with a CAR.

[0097] The protein of the present invention can exist in multiple forms.For example, the protein of the present invention can be in the form of a linear or branched polypeptide.Linear chimeric proteins can be produced by recombinant DNA technology.For example, chimeric transcription cassettes can be assembled using restriction endonuclease site overlap or polymerase chain reaction (PCR)-based splice-by-overlap extension.

[0098] Branched polypeptide chimeric proteins can be easily generated by template-assembled synthetic peptide (TASP) technology (Mutter, Trends Biochem. Sci. 13:260-265 (1988)). According to this method, peptide units are synthesized separately and covalently coupled to a multifunctional support, such as a core peptide, using chemical coupling reagents. For example, a cyclic decapeptide analog of gramicidin S, in which two antiparallel β-sheet segments (lys-ala-lys) are connected by two β-turns, can be used as the core peptide. A segment condensation strategy can be used to attach the first and second proteins to the ε-amino groups of the four lysine side chains.

[0099] The proteins of the present invention may exist as two or more separate proteins linked together by a bridge, such as a chemical bond. For example, two or more protein components can be directly and covalently linked to each other in a branched structure using a chemical cross-linking reagent such as dithio-bis(succinimidyl propionate) (DSP). This methodology can, for example, directly link a first protein and a second protein.

[0100] The specific first and second polypeptides of the chimeric switch receptors of the present invention can vary depending on the disease being treated. Typically, for example, when treating cancer or viral infections, a second polypeptide that stimulates an immune cell response is used. When treating immune system disorders in which a pathogenic immune response is present, an inhibitory second polypeptide is used. Thus, in the case of cancer and viral diseases, a switch receptor that converts an inhibitory signal into an activating immune activation signal is desired. In contrast, in the case of autoimmune diseases, a chimeric switch receptor that converts an activating signal into an inhibitory immune signal is desired. In this situation, the immunoinhibitory second protein component can be directed to various pathogenic immune effectors, including T cells, B cells, natural killer cells, and antigen-presenting cells.

[0101] Thus, the present invention provides a switch receptor that, when expressed in a cell, converts a positive signal into a negative intracellular signal, e.g., the switch receptor contains a first domain comprising a polypeptide that delivers a positive signal; and a second domain comprising a polypeptide that delivers a negative intracellular signal.

[0102] genetic modification The present invention encompasses cells (e.g., T cells) transduced with a lentiviral vector (LV). In one embodiment, the LV encodes a switch receptor of the invention comprising a first domain comprising a polypeptide that delivers a negative signal and a second domain comprising a polypeptide that delivers a positive signal.

[0103] In one embodiment, the cells may be further transduced with LVs encoding a chimeric antigen receptor (CAR), which combines the antigen recognition domain of a specific antibody with the intracellular domain of the CD3ζ chain or the FcγRI protein into a single chimeric protein.

[0104] Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenes and their transmission to daughter cells. Lentiviral vectors have the added advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0105] Briefly summarized, expression of the natural or synthetic nucleic acids of the present invention is typically achieved by operably linking a nucleic acid encoding the desired polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for controlling the expression of the desired nucleic acid sequence.

[0106] Nucleic acids can be cloned into many types of vectors, including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0107] Additionally, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-3 (3 rdViruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0108] Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription.

[0109] An example of a promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of a polynucleotide sequence operably linked to it. However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0110] To assess the expression of CAR polypeptide or a portion thereof, the expression vector introduced into cells may also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a group of cells that are intended to be transfected or infected by a viral vector.In other aspects, the selection marker may be carried on a separate piece of DNA and used in co-transfection techniques.To enable expression in host cells, both the selection marker and the reporter gene may be flanked by appropriate regulatory sequences.Useful selection markers include, for example, antibiotic resistance genes, such as neo.

[0111] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that encode polypeptides that are not present or expressed in recipient organisms or tissues and whose expression is manifested by some easily detectable property, such as enzymatic activity. After the DNA is introduced into the recipient cells, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared using known techniques or commercially available. Generally, the construct with the minimal 5'-flanking region that exhibits the highest expression level of the reporter gene is identified as the promoter. Such a promoter region can be linked to a reporter gene and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0112] Methods for introducing and expressing genes into cells are known in the art. With regard to expression vectors, the vector can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0113] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle gun, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL volumes 1-3 (3rd ed., Cold Spring Harbor Press, NY 2001).

[0114] Biological methods for introducing a polynucleotide of interest into host cells include the use of DNA vectors and RNA vectors.Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus type I, adenovirus, adeno-associated virus, etc.See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0115] Chemical means for introducing polynucleotides into host cells include macromolecular complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems such as lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle).

[0116] When a non-viral delivery system is used, an exemplary delivery vehicle is liposomes. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, nucleic acids can be associated with lipids. The lipid-associated nucleic acid can be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension within lipids, contained in or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to a specific structure in solution. For example, they may exist in a bilayer structure as micelles, or may have a "collapsed" structure. They may simply be dispersed in solution or form aggregates of heterogeneous size or shape. Lipids are fatty substances that may be naturally occurring or synthetic. For example, lipids include the lipid droplets naturally occurring in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0117] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma (St. Louis, MO); dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure containing a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, lipids may be in a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0118] Therapeutic applications The present invention involves a type of cell therapy in which T cells are genetically modified to express a switch receptor and the engineered T cells are infused into a recipient in need thereof. The infused cells are capable of killing tumor cells in the recipient. Unlike antibody therapy, the engineered T cells of the present invention are capable of replicating in vivo, resulting in long-term persistence that can result in sustained tumor control.

[0119] The present invention also relates to methods for treating a patient for a disease, comprising administering to the patient an effective amount of an engineered switch receptor of the present invention. A variety of diseases can be treated by the methods of the present invention, including, but not limited to, cancers such as ovarian cancer, breast cancer, colon cancer, glioblastoma multiforme, prostate cancer, and leukemia; viral infections such as chronic viral infections with HBV, HCV, HTLV-1, HTLV-II, EBV, HSV-I, HSV-II, and KSHV; and autoimmune diseases such as arthritis, asthma, graft-versus-host disease, organ rejection, psoriasis, systemic lupus erythematosus, atopic allergy, inflammatory bowel disease, multiple sclerosis, allergic dermatitis, Sjogren's syndrome, progressive systemic sclerosis, autoimmune thyroiditis, autoimmune diabetes, autoimmune liver disease, and myelodysplastic syndrome.

[0120] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include kidney or renal cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, squamous cell cancer (e.g., epithelial squamous cell cancer), cervical cancer, ovarian cancer, prostate cancer, liver cancer, bladder cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer or stomach cancer. Hematological malignancies including gastrointestinal stromal tumor (GIST), pancreatic cancer, head and neck cancer, glioblastoma, retinoblastoma, astrocytoma, theca cell tumor, virilizing tumor, hepatocellular carcinoma, non-Hodgkin's lymphoma (NHL), multiple myeloma, and acute hematological malignancies, endometrial or uterine cancer, endometriosis, fibrosarcoma, choriocarcinoma, salivary gland cancer, vulvar cancer, thyroid cancer, esophageal cancer, liver cancer, anal cancer, penile cancer, nasopharyngeal cancer, laryngeal cancer, Kaposi's sarcoma, melanoma, skin cancer, schwannoma, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract cancer, thyroid cancer carcinomas), Wilms' tumor, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky mass disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia, chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (such as that associated with brain tumors), and Meigs' syndrome. "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0121] In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from cancers, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.

[0122] In one embodiment, the tumor antigens of the present invention comprise one or more antigenic cancer epitopes that are immunologically recognized by tumor-infiltrating lymphocytes (TILs) derived from mammalian cancer tumors.

[0123] Malignant tumors express numerous proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-associated molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidates for target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0124] For the treatment of cancer, the switch receptors of the present invention may optionally be administered to a patient in combination with other chemotherapeutic agents. Suitable chemotherapeutic agents include, for example, alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonic acids such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine. Ethyleneimines and methylamelamines, including methylamelamine; chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, nitrogen mustards such as uracil mustard; carmustine, chlorozotocin Nitrosoureas such as chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6- Antibiotics such as diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, Pyrimidine analogs such as floxuridine and 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folinic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; and Bestrabsil. bucil); bisantrene; edatrexate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; Podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (trademark); razoxane; sizophyllan; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;Taxanes, such as paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE™, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronic acid; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin, capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0125] Also included are antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene; and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0126] Also included are chemotherapeutic agents that can sensitize tumor cells to TRAIL and overcome TRAIL resistance, such as proteasome inhibitors and histone deacetylase (HDAC) inhibitors, cycloheximide, imatinib mesylate and other protein tyrosine kinase inhibitors, 17-allylamino-17-demethoxygeldanamycin, arsenic trioxide, and X-linked inhibitor of apoptosis protein small molecule antagonists; and pharmaceutically acceptable salts, acids, or derivatives of any of these.

[0127] Additional information regarding methods of cancer treatment is provided in US Pat. No. 7,285,522, which is incorporated by reference in its entirety.

[0128] Thus, in a preferred embodiment, the switch receptor of the present invention can be used to treat breast cancer. In another preferred embodiment, the switch receptor of the present invention can be used to treat colon cancer. In another embodiment, the switch receptor of the present invention can be used to treat liver cancer. In another preferred embodiment, the switch receptor of the present invention can be used to treat ovarian cancer. In another embodiment, the switch receptor of the present invention can be used to treat leukemia. In another embodiment, the switch receptor of the present invention can be used to treat melanoma. In a further embodiment, the switch receptor of the present invention can be used to treat alloimmune diseases, such as transplant rejection or graft-versus-host disease or host-versus-graft disease.

[0129] Typically, for each disease indication, a small "library" of candidate switch receptors can be generated and comparatively evaluated in appropriate well-established ex vivo and in vivo models to determine relative efficacy and toxicity.

[0130] The specific first and second proteins used in the method will vary depending on the disease being treated. Generally, in the case of cancer, a switch receptor that converts an inhibitory signal into an activating immune transactivation signal is desired. Without wishing to be bound by a particular theory, the anti-tumor immune response elicited by the engineered cells of the present invention can be an active or passive immune response. The response can be part of an adoptive immunotherapy approach.

[0131] For ex vivo immunization, at least one of the following is performed in vitro prior to administering the cells to a mammal: (i) expanding the cells, (ii) introducing a nucleic acid encoding a switch receptor of the invention into the cells, or (iii) cryopreserving the cells.

[0132] Ex vivo techniques are well known in the art and are described more fully below. Briefly, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a switch receptor of the invention. The engineered cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be human, and the engineered cells can be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0133] Techniques for ex vivo expansion of hematopoietic stem and progenitor cells are described in U.S. Patent No. 5,199,942, incorporated herein by reference, and may be applied to the cells of the present invention. Other suitable methods are known in the art, and therefore the present invention is not limited to a particular method of ex vivo expansion of cells. Briefly, ex vivo culture and expansion of T cells involves the following steps: (1) collecting mammalian-derived CD34+ hematopoietic stem and progenitor cells from peripheral blood collection or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cell growth factors described in U.S. Patent No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand can be used for cell culture and expansion.

[0134] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response to an antigen in a patient.

[0135] In general, cells activated and expanded as described herein can be utilized in the treatment and prevention of diseases occurring in immunocompromised individuals. In particular, the engineered cells of the present invention are used in the treatment of cancer. In certain embodiments, the cells of the present invention are used in the treatment of patients at risk of developing cancer. Thus, the present invention provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of the engineered T cells of the present invention to a subject in need thereof.

[0136] The engineered T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with other components, such as a diluent and / or IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present invention may comprise the target cell populations described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0137] The pharmaceutical compositions of the present invention can be administered in a manner appropriate for the disease to be treated (or prevented). The appropriate dosage can be determined by clinical trials, but the amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease.

[0138] When an "immunologically effective amount," "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is referred to, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). Generally, pharmaceutical compositions comprising the T cells described herein are administered in doses of 10 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6It can be said that the T cell composition can be administered at a dosage of 1000 cells / kg body weight (including all integer values ​​within these ranges). The T cell composition can also be administered multiple times at these dosages. The cells can be administered by using injection techniques commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0139] In certain embodiments, it may be desirable to administer activated T cells to a subject, then draw blood again (or perform apheresis), activate the T cells according to the present invention, and reinfuse the activated and expanded T cells back into the patient. This process can be performed multiple times, every few weeks. In certain embodiments, T cells can be activated from a blood draw of 10 cc to 400 cc. In certain embodiments, T cells are activated from a blood draw of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Without being bound by theory, the use of this multiple blood draw / multiple reinfusion protocol may select for certain populations of T cells.

[0140] Administration of the compositions of the present invention can be carried out in any convenient manner, including aerosol inhalation, injection, oral ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intralymph node, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by iv injection. The T cell compositions may also be injected directly into a tumor, lymph node, or site of infection.

[0141] In certain embodiments of the invention, using the methods described herein or other methods known in the art to expand T cells to therapeutic levels, the activated and expanded cells are administered to the patient along with (e.g., before, simultaneously with, or after) a number of appropriate treatment modalities, including, but not limited to, antiviral therapy, treatment with agents such as cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efalizumab treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody treatments, cytoxin, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993; Isoniemi, supra). In a further embodiment, the cell compositions of the invention are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) T cell-depleting therapy using either bone marrow transplantation, chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the invention are administered after B cell-depleting therapy, such as an agent reactive with CD20, e.g., Rituxan.For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after the transplant, the subject receives an infusion of the expanded immune cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0142] The dosages of the above treatments administered to patients will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Estimation of dosages for human administration can be performed according to art-recognized practices. Doses for CAMPATH, for example, will generally range from 1 to about 100 mg for an adult patient and will generally be administered daily for 1 to 30 days. A preferred daily dose is 1 to 10 mg per day, although in some cases larger doses of up to 40 mg per day may be used (as described in U.S. Pat. No. 6,120,766). [Example]

[0143] Experimental Example The present invention will be described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and, unless otherwise specified, are not intended to be limiting. Therefore, the present invention should in no way be construed as being limited to the following examples, but should instead be construed to encompass any and all variations that become evident as a result of the teachings provided herein.

[0144] Without further description, one of ordinary skill in the art can, using the foregoing description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0145] Example 1: Switch Receptor The results presented herein demonstrate that it is possible to engineer chimeric receptors to convert negative signals into positive signals in T cells. Experiments were designed to develop a method to circumvent systemic, and therefore immune system-wide, inhibition of tumor inhibitors. Briefly, T cells were engineered to express chimeric receptors encoding the PD-1 extracellular domain (without the inhibitory PD-1 domain) and the stimulatory CD28 or ICOS signaling domain. The orientation of the chimeric receptors placed the PD-1 extracellular domain extracellularly and the CD28 or ICOS stimulatory domain intracellularly. Thus, because the intracellular signal is delivered by the CD28 or ICOS signaling endodomain rather than the native inhibitory PD-1 endodomain, T cell interaction with tumor antigens in the tumor microenvironment is positively influenced by PD-1 ligand contact.

[0146] The materials and methods utilized in these experiments are described below.

[0147] material and method Switch receptor generation Constructs were designed for testing BTLA switch receptors. Below are the sequences of each construct cloned into the pGEM.64A-based IVT vector: TIFF2025142038000002.tif61140TIFF2025142038000003.tif231139TIFF2025142038000004.tif231138TIFF2025142038000005.tif120138

[0148] T cell transduction A method for T cell preparation using paramagnetic polystyrene beads coated with anti-CD3 and anti-CD28 monoclonal antibodies has been described (Laport et al., 2003, Blood 102:2004-2013). Lentiviral transduction was performed as described (Levine et al., 2006, Proc Natl Acad Sci USA 103:17372-17377). Electroporation of T cells with RNA has been described as a method for expressing these receptors (Zhao et al., 2010, Cancer Res 70:9062). The use of adenoviral vectors has been described (Schroers et al., 2004, Exp Hematol 32:536). Numerous other approaches for expressing proteins in T cells have been described (June et al., 2009, Nat Rev Immunol 9:704).

[0149] Cytokine analysis Quantification of soluble cytokine factors was performed using Luminex bead array technology and kits purchased from Life technologies (Invitrogen). The assay was performed according to the manufacturer's protocol with an eight-point standard curve generated using a three-fold dilution series.

[0150] The results of the experiment are described below.

[0151] The results presented herein demonstrate that chimeric receptors can be engineered and expressed on T cells to transduce negative signals into positive signals in T cells. Thus, the present invention provides adoptive therapy with T cells or NK cells using cells genetically modified to express a T cell receptor (TCR) or chimeric antigen receptor (CAR).

[0152] Experiments were designed to develop target-specific methods to circumvent systemic, and thus immune system-wide, inhibition of PD-1. Target-specific methods include, for example, administering T cells expressing chimeric receptors encoding the PD-1 extracellular domain and encoding the stimulatory CD28 or ICOS signaling domain, rather than the inhibitory PD-1 domain, in the intracellular compartment of the T cells. Thus, because the intracellular signal is delivered by the CD28 or ICOS signaling endodomain, rather than the native PD-1 endodomain, T cell interactions with tumor antigens in the tumor microenvironment will be positively influenced by PD-1 ligand contact.

[0153] BTLA signal conversion In a similar approach, we constructed a chimeric receptor encoding BTLA. BTLA, along with PD-1, is a member of the CD28 family. BTLA has several ligands, including HVEM, which are often expressed on tumor cells and other cells within the tumor microenvironment. BTLA interaction with its natural ligands on cells is known to negatively regulate T cell immune responses (Paulos et al., 2010, J Clin Invest 120:76-80; Derre et al., 2010, J Clin Invest 120:157-67; Chemnitz et al., 2006, J Immunol 176:6603-14). To circumvent this, we constructed a chimeric receptor composed of the BTLA extracellular domain and an intercellular signaling endodomain containing the CD28 or ICOS domain.

[0154] Briefly, molecular cloning was used to construct the exemplary chimeric receptor depicted in Figure 1. To test whether ligation of the BTLA CCR expressed on the T cell surface could properly express and transmit signals to CD3ζ through the ICOS intracellular domain, IVT mRNA encoding the indicated BTLA CCR was electroporated into stimulated T cells, and surface expression of BTLA was detected by HVEM-Fc fusion protein at different time points as indicated (Figure 1B). Electroporated T cells were stimulated with either a BTLA ligand-negative cell line (KTPolCD86A2) or a BTLA ligand-HVEM-positive cell line (KTCD86FluHVEM). 24 hours after stimulation, IL-2 produced by the T cells was assayed by ELISA (Figure 1C). The results demonstrated that fusing the BTLA extracellular domain to both the ICOS and CD3ζ intracellular domains enabled T cells to be activated by stimulation with a BTLA ligand-HVEM-expressing cell line. This indicates that BTLA signals can be transduced into other signals in the form of chimeric costimulatory receptors (CCRs).

[0155] The next set of experiments was designed to assess whether the inhibitory signal of BTLA could be converted into a costimulatory signal of CD28 through the BTLA-CD28 CCR. To find an appropriate window in which CD28 signaling could be detected, different doses (µg / 0.1 ml T cells) of RNA were electroporated into T cells, and CAR (CD19z, CD19-28Z) expression was detected by FACS. The upper panel shows the histogram and percentage of transgene expression, and the lower panel shows the MFI of transgene expression (Figure 2A). IL-2 production of T cells electroporated with RNA as described in Figure 2A was stimulated by the CD19-positive cell line K562-CD19, and IL-2 production was assayed by ELISA, as shown in Figure 2B. The upper panel shows IFNγ, and the lower panel shows IL-2 production. The results show that there was no detectable IL-2 production for CD19z RNA electroporated T cells, unlike CD19-28z RNA electroporated T cells, which showed IL-2 production of over 300 pg / ml at a 1.5 μg RNA dose. IFNγ production was detectable at similar levels for both CD19z and CD19-28Z electroporated T cells at a 1.5 μg RNA dose. Therefore, 1.5 μg RNA was used as the RNA dose for testing BTLA-CD28 signal transduction polypeptides.

[0156] T cells were co-electroporated with 1.5 μg CD19z and BTLA CCR as indicated and stimulated with K562 expressing CD19 (K562CD19) or both CD19 and HVEM (K562CD19 / HVEM). A K562 line expressing mesothelin (with or without HVEM) was used as a control (Figure 2C). The results show that full-length (wild-type) BTLA suppressed the production of both IL-2 and IFNγ. When stimulated with the CD19 / HVEM double-positive cell line, there was no detectable IL-2 production for T cells electroporated with CD19z alone, whereas T cells electroporated with CD19-28z and stimulated with the CD19 / HVEM double-positive cell line produced over 400 pg / ml of IL-2. However, when T cells were co-electroporated with both CD19z RNA and BTLA-CD28 CCR RNA, IL-2 production when stimulated with a CD19 / HVEM double-positive cell line was four-fold higher than that of CD19-28z-electroporated T cells. T cells co-electroporated with both CD19z RNA and BTLA-CD28 CCR RNA produced higher IFNγ than CD19z- or CD19-28z-electroporated T cells when stimulated with a CD19 / HVEM double-positive cell line or CD19-positive K562 cells expressing low levels of HVEM (Figure 2C). The results presented here demonstrate that the inhibitory signal of BTLA can be transduced into a CD28 signal through the BTLA-CD28 CCR.

[0157] The next set of experiments was designed to test whether inhibitory BTLA signals could be converted into costimulatory ICOS signals through the BTLA-ICOS CCR. Briefly, the conversion of BTLA signals into ICOS signals was examined in a Th17-biased system as shown in Figure 3A. Resting CD4 T cells were co-electroporated with CD19z and BTLA CCR (treatment) as indicated and stimulated with a CD19 / HVEM double-positive cell line (groups 1 and 2, duplicates), or T cells were electroporated with BTLA-ICOS alone and stimulated with plate-bound HVEM-Fc and OKT3 (group 3). As described (Paulos et al., 2010, Science Translational Medicine), CD3 / ICOS beads or CD3 / CD28 beads were used as positive and negative controls, respectively. All cultures were performed in the presence of a Th17 cytokine cocktail. On different days (as indicated), T cells were stimulated with PMA / ionomycin, and IL-17A and IFNγ were detected by intracellular cytokine staining (Figure 3B). The results showed that ICOS signaling converted from BTLA-ICOS enhanced Th17 cell production in the presence of HVEM inhibitory signals.

[0158] Conversion of PD-1 signaling to CD28 signaling To test whether ligation of PD1 CCR expressed on the surface of T cells could be functionally expressed to signal through the intracellular domains of CD28, CD27, or ICOS, IVT RNA encoding PD1 CCR was used (sequence shown below). TIFF2025142038000006.tif230139

[0159] PD1 CCR RNA and CD19z RNA as indicated were co-electroporated into stimulated T cells, and the transgenes were detected with anti-PD1 and anti-CAR Abs at the indicated time points (Figure 4A). Co-electroporated T cells as described in Figure 4A were co-cultured with Nalm6 (a human B cell leukemia line) expressing PD-L1 or GFP as a control, or K562-CD19 expressing PD-L1 or ICOSL as a control. IFNγ production was assayed 24 hours after co-culture. When co-cultured with CD19 / PD-L1 double-positive cell lines, T cells co-transfected with CD19z and PD1-CD28 (PD1-28) showed significantly higher IFNγ production than T cells electroporated with CD19z alone or with cytoplasmic-truncated PD1 or PD1-CD27 (PD1-27). When T cells were co-electroporated with CD19z and full-length (wild-type) PD1 and stimulated with a CD19 / PD-L1 double-positive cell line, potent T cell inhibition was observed (Figure 4B).

[0160] Co-electroporated T cells as described in Figure 4A were co-cultured with Nalm6 (a human B cell leukemia line) expressing PD-L1 or GFP as a control, or K562-CD19 expressing PD-L1 or ICOSL as a control. IL-2 production was assayed 24 hours after co-culture. When co-cultured with a CD19 / PD-L1 double-positive cell line, T cells co-transduced with CD19z and PD1-CD28 (PD1-28) showed significantly higher IL-2 production than T cells electroporated with CD19z alone or with cytoplasmic domain-truncated PD1 or PD1-CD27 (PD1-27). When T cells were co-electroporated with CD19z and full-length PD1 and stimulated with a CD19 / PD-L1 double-positive cell line, potent T cell inhibition was observed (Figure 4C).

[0161] Co-electroporated T cells as described in Figure 4A were tested in a flow cytometry-based CTL assay. T cells co-transduced with CD19z and PD1 showed significantly reduced killing ability against Nalm6-PD-L1 targets, whereas T cells co-electroporated with PD1 CCR showed no significant difference when Nalm6-PD1 was used as the target compared to T cells electroporated with CD19Z alone. When PD1 ligand-negative Nalm6 was used as the target, no significant difference was found for all T cell groups, including T cells co-electroporated with CD19z and PD1.

[0162] The next set of experiments was designed to reverse PD1 inhibition by PD1-CD28 CCR co-transduction. To mimic a tumor microenvironment or chronic infection in which T cells are PD1-positive, T cells were stimulated with CD3 / CD28 beads or OKT3 / PBMC / IL2 and co-electroporated with CD19z (10 μg) and PD1 (5 μg), with additional PD1 CCR (10 μg) as indicated. One day after electroporation, expression of CAR and PD1 and / or PD1 CCR was detected by FACS (Figure 5A).

[0163] Electroporated T cells as shown in Figure 5A were cocultured with Nalm6 or K562-CD19, which express PD-L1. After overnight coculture, IL-2 production was assayed. Results showed that the amount of IL-2 produced by T cells electroporated with CD19z alone was reduced compared to the same T cells cocultured with a CD19-positive cell line without PD-L1. However, in the presence of PD1, IL-2 production was completely blocked when cocultured with a PD-L1-positive cell line, except for T cells co-electroporated with PD1-CD28 (PD1-28), which showed much higher IL-2 production than CD19Z-only T cells. PD1 expression on T cells had minimal effect on T cells when cocultured with a PD-L1-negative cell line (Figure 5B).

[0164] Electroporated T cells as shown in Figure 5A were co-cultured with Nalm6 or K562-CD19 expressing PD-L1. After overnight co-culture, IFNγ production was assayed (Figure 5C). A cytokine production profile similar to that of IL-2 production shown in Figure 5B was observed. The results presented herein demonstrate that the PD1-CD28 CCR can reverse the inhibitory effects of PD1 and promote T cell effector function.

[0165] Conversion of PD1 signaling to ICOS signaling CD4 T cells were electroporated with PD1 (or PD1 variant) mRNA and CD19-z CAR mRNA (10 μg each). Four hours after electroporation (day 0), T cells were mixed with K562 cells expressing PD-L1 and CD19 in R10 culture medium (0.5:1 = K562:T cells) in the presence of IL1 (10 ng / ml), IL6 (10 ng / ml), IL23 (20 ng / ml), and neutralizing antibodies against IL4 and IFNγ (10 μg / ml). On the indicated days (days 5, 9, and 12 after electroporation), cells were incubated with PMA (3 μg / ml), ionomycin (1 μg / ml), and GolgiStop for 4 hours for intracellular cytokine staining. Surface staining for CD4 was performed, followed by intracellular staining for IFNγ, IL17, and IL2. CD4 T cells stimulated with anti-CD28 / CD3 beads and anti-ICOS / CD3 beads were used as controls (Figure 6A). Cytokine production was enhanced in cells expressing PD1-ICOS compared with PD1 wild-type or tailless PD1, particularly on day 9. Cell proliferation of stimulated T cells is shown in Figure 6C.

[0166] PD-1 chimeric receptor PD-1 is upregulated on the surface of exhausted CD8 T cells in patients with chronic viral infections. Blockade of PD-1:PD-L1 signaling restores the function of exhausted CD8 T cells that express PD-1. Many tumors express PD-L1, providing an immunosuppressive microenvironment.

[0167] The goal of the following experiments is to direct adoptively transferred T cells to overcome the inhibitory tumor microenvironment by introducing PD-1 chimeric receptors into the tumor site.

[0168] We observed that the inhibitory effect of PD1wt on cytokine production was rescued by the PD1 chimeric construct (Figure 7). However, the PD1 chimeric receptor did not affect granzyme B production (Figure 8). Similarly, minimal differences were observed in the killing activity of CD8 T cells in the presence or absence of PD1 (Figure 9).

[0169] The next set of experiments was designed to evaluate the effect of PD-1 chimeric receptors on T cell proliferation (Figure 10). PD1-CD28 chimeric receptors were observed to increase the number of CD8 T cells (Figure 11).

[0170] In summary, the results presented herein demonstrate that PD-1 chimeric constructs do not exhibit the inhibitory effect exhibited by PD-1wt. PD1-CD28 appears to increase TNFα, IL2, and IFNγ production in CD4 T cells. PD-1 chimeric receptors did not exhibit increased cytotoxicity over that of T cells expressing CD19CARz alone. PD1-CD28 increased the number of CD8 T cells over that of T cells expressing CD19CARz alone.

[0171] Conversion of co-inhibitory signaling to positive costimulation The results presented herein demonstrate that switch receptors were expressed in T cells by electroporation or by lentiviral vectors. It was observed that CAR and switch receptors can be expressed in the same T cells. When cells expressing CAR or TCR and switch receptors are exposed to tumor cells bearing BTLA or PD-1 ligands, the T cells are shown to have a positive immune response, rather than the usual inhibitory response.

[0172] When these chimeric receptors were expressed in T cells, it was observed that, in the case of the BTLA switch receptor, interaction with the natural ligand HVEM on tumor cells stimulated the T cells to express functions associated with positive antitumor effects, including the secretion of IFNγ.

[0173] Another important finding from these studies was that interaction of HVEM:BTLA with the chimeric receptor resulted in enhanced IL-17 secretion, a marker of TH17 cells, cells known to be useful for tumor immunotherapy (Martin-Orozco et al., 2009, Immunity 31:787-98; Paulos et al., 2010, Science Translational Medicine 2:55-78; Garaude et al., 2010, Sci Transl Med 2(55):55ps2). For example, the results presented herein demonstrate that T cells expressing a CAR and a BTLA switch receptor with an ICOS signaling domain were biased to secrete large amounts of IL-17.

[0174] Furthermore, the results presented herein show that T cells expressing a CAR and a PD1 switch receptor with a deleted domain containing CD28 are protected from inhibition, kill tumor cells, and secrete cytokines (IL-2 and IFNγ) when they express the PD-1 switch receptor.

[0175] Without wishing to be bound by any particular theory, it is believed that by expressing a chimeric antigen receptor (CAR) on T cells together with a switch receptor for PD-1 or BTLA and then introducing it into the tumor microenvironment, the T cells have enhanced antitumor effects and exhibit a TH17 phenotype. The adoptive T cell transfer method for tumor immunotherapy of the present invention is also applicable to the field of vaccine therapy, such as for chronic viral infections, including HIV or other viruses such as EBV, HCV, or CMV. This technology can also be easily incorporated into other clinical trials currently using genetically modified T cells with TCRs. For example, the switch receptor of the present invention can be used in conjunction with T cells with TCRs specific for cancer antigens such as MAGE-A3 and NY-ESO-1, and it is believed that including the switch receptor with these T cells will increase the efficacy of the T cells.

[0176] The disclosures of all individual patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. While the present invention has been disclosed with respect to specific embodiments, it will be apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

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gctcacctta agagtgagca aacagaagca agcaccaggc aaaattccca agtactgcta 660 tcagaaactg gatttatga taatgaccct gaccttgtt tcaggatgca ggaagggtct 720 gaagtttatt ctaatccatg cctggaagaa aaaaccag gcattgttta tgcttccctg 780 aaccattctg tcattggacc gaactcaaga ctggcagaa atgtaaaga agcaccaca 840 gatatgcat ccatatgtgt gaggagttaa 870 <210> 2 <211> 685 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 2 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaatt atttgggtc 60 ttctttta tcccatatct ggacatctgg aacatccatg ggaaagaatc atgtgatgta 120 cagctttata taagagaca atctgacac tccatcttag caggagatcc ctttgaacta 180 gatgccctg tgaaatactg tgctaacagg cctcatgtga cttggtgcaa gctcaatgga 240 acacatgtg taaacttga agatagacaa acagttga aggagaga gaacttca 300 tttttcattc tacattttga accagtgctt cctaatgaca atgggtcata ccgctgttct 360 gcaaattttc agtctaatct cattgaaagc cactcaacaa ctctttatgt gacagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaaatggcaa gcagaccctg gctcctgtat 480 agtttacttc ctttgggggg attgcctcta ctcatcacta cctgtttctg cctgttctgc 540 tgcctggagg agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg 600 ccgccccggg cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc 660 ctatcgctcc tgataagcgg ccgca 685 <210> 3 <211> 701 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 3 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaaatt attttgggtc 60 ttcttcttaa tcccatatct ggacatctgg aacatccatg ggaaagaatc atgtgatgta 120 cagctttata taaagagaca atctgaacac tccatcttag caggagatcc ctttgaacta 180 gatgccctg tgaaatactg tgctaacagg cctcatgtga cttggtgcaa gctcaatgga 240 acacatgtg taaacttga agatagacaa acagttga aggagaga gaacttca 300 ttttcattc tacattttga accagtgctt cctaatgaca atggtcata ccgctgttct 360 gcaaattttc agtctaatct cattgaaagc cactcaacaa ctctttatgtlogagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaatggca gcagaccctg gctcctgtat 480 agtttactc ctttgggggg attgcctcta ctcatcacta cctgttctc cctgttctgc 540 tgcctggaag gaaataga tcaacaag gagaagtcc tgtggagcct gcagagcctt 600 gtcgttacag ctgccccagg gaggaggagg gcagcaccat cccatccag gaggattacc 660 gaaaaccgga gcctgcctgc tcccctgat aagcggccgc a 701 <210> 4 <211> 685 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 4 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaatt atttgggtc 60 ttctttta tcccatatct ggacatctgg aacatccatg ggaaagaatc atgtgatgta 120 cagctttata taagagaca atctgacac tccatcttag caggagatcc ctttgaacta 180 gatgccctg tgaaatactg tgctaacagg cctcatgtga cttggtgcaa gctcaatgga 240 acacatgtg taaacttga agatagacaa acagttga aggagaga gaacttca 300 ttttcattc tacattttga accagtgctt cctaatgaca atggtcata ccgctgttct 360 gcaaattttc agtctaatct cattgaaagc cactcaacaa ctctttatgtlogagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaatggca gcagaccctg gctcctgtat 480 agtttctggt tacccatagg atgtgcagcc ttgttgtag tctgcatttt gggatgcata 540 cttattgagg agtagagga gcaggctcct gcacagtgac tacatgaca tgactccccg 600 ccgccccggg cccaccgca agcattacca gcctatgcc cccaccgcg acttcgcagc 660 ctatcgctcc tgatataagcgg ccgca 685 <210> 5 <211> 701 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 5 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaatt atttgggtc 60 ttcttttta tcccatatct ggacatctgg aacatccatg ggaaagaatc atgtgatgta 120 cagctttata taagagaca atctgacac tccatcttag caggagatcc ctttgaacta 180 gatgccctg tgaaatactg tgctaacagg cctcatgtga cttggtgcaa gctcaatgga 240 acacatgtg taaacttga agatagacaa acagttga aggagaga gaacttca 300 ttttcattc tacattttga accagtgctt cctaatgaca atggtcata ccgctgttct 360 gcaaattttc agtctaatct cattgaaagc cactcaacaa ctctttatgtlogagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaatggca gcagaccctg gctcctgtat 480 agtttctggt tacccatagg atgtgcagcc ttgttgtag tctgcatttt gggatgcata 540 cttattgaag gaaataga tcaacaag gagaagtcc tgtggagcct gcagagcctt 600 gtcgttacag ctgccccagg gaggaggagg gcagcaccat cccatccag gaggattacc 660 gaaaaccgga gcctgcctgc tccccctgat aagcggccgc a 701 <210> 6 <211> 672 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 6 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaaatt atttgggtc 60 ttcttcttaa tcccatatct ggacatctgg aacatccatg ggaaagaatc atgtgatgta 120 cagctttata taaagagaca atctgaacac tccatcttag caggagatcc ctttgaacta 180 gaatgccctg tgaaatactg tgctaacagg cctcatgtga cttggtgcaa gctcaatgga 240 acaacatgtg taaaacttga agatagacaa acaagttgga aggaagagaa gaacatttca 300 ttttcattc tacatttga accagtgctt cctaatgaca atgggtcata ccgctgttct 360 gcaaatttc agtctaatct cattgaaagc cactcaacaa ctctttatgt gacagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaaatggcaa gcagaccctg gctcctgtat 480 agttacttc ctttgggggg attgcctcta ctcatcacta cctgtttctg cctgttctgc 540 tgcctgtgtt ggcttacaa aaagaagtat tcatccagtg tgcacgaccc taacggtgaa 600 tacatgttca tgagagcagt gaacacagcc aaaaaatcta gactcacaga tgtgacccta 660 taggcggccg about 672 <210> 7 <211> 1011 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 7 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaatt atttgggtc 60 ttcttttta tcccatatct ggacatctgg aacatccatg ggaaagaatc atgtgatgta 120 cagctttata taagagaca atctgacac tccatcttag caggagatcc ctttgaacta 180 gatgccctg tgaaatactg tgctaacagg cctcatgtga cttggtgcaa gctcaatgga 240 acacatgtg taaacttga agatagacaa acagttga aggagaga gaacttca 300 ttttcattc tacattttga accagtgctt cctaatgaca atggtcata ccgctgttct 360 gcaaattttc agtctaatct cattgaaagc cactcaacaa ctctttatgtlogagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaaatggcaa gcagaccctg gctcctgtat 480 agtttacttc ctttgggggg attgcctcta ctcatcacta cctgtttctg cctgttctgc 540 tgcctgtgtt ggcttacaaa aaaagtt tcatccagtg tgcacgaccc taacggtgaa 600 tacatgttca tgagagcagt gaacacagcc aaaaaatcta gactcacaga tgtgacccta 660 720 ctctataacg agctcaatct agcacgaaga gagagtacg atgttttgga caagagacgt 780 ggccgggacc ctgagatggg gggaaagccg your area accctcagga aggcctgtac 840 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaggcgag 900 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 960 acctacgacg cccttcacat gcaggccctg ccccctcgct aagcggccgc a 1011 <210> 8 <211> 672 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 8 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaatt atttgggtc 60 ttctttta tcccatatct ggacatctgg aacatccatg ggaaagaatc atgtgatgta 120 cagctttata taagagaca atctgacac tccatcttag caggagatcc ctttgaacta 180 gatgccctg tgaaatactg tgctaacagg cctcatgtga cttggtgcaa gctcaatgga 240 acacatgtg taaacttga agatagacaa acagttga aggagaga gaacttca 300 ttttcattc tacattttga accagtgctt cctaatgaca atggtcata ccgctgttct 360 gcaaattttc agtctaatct cattgaaagc cactcaacaa ctctttatgtlogagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaatggca gcagaccctg gctcctgtat 480 agtttctggt tacccatagg atgtgcagcc ttgttgtag tctgcatttt gggatgcata 540 cttattgtt ggcttacaa aaagaagtat tcatccagtg tgcacgaccc taacggtgaa 600 tacatgttca tgagagcagt gaacacagcc aaaaaatcta gactcacaga tgtgacccta 660 taggcggccg about 672 <210> 9 <211> 1011 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 9 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaatt atttgggtc 60 ttctttta tcccatatct ggacatctgg aacatccatg ggaaagaatc atgtgatgta 120 cagctttata taagagaca atctgacac tccatcttag caggagatcc ctttgaacta 180 gatgccctg tgaaatactg tgctaacagg cctcatgtga cttggtgcaa gctcaatgga 240 acacatgtg taaacttga agatagacaa acagttga aggagaga gaacttca 300 ttttcattc tacattttga accagtgctt cctaatgaca atggtcata ccgctgttct 360 gcaaattttc agtctaatct cattgaaagc cactcaacaa ctctttatgtlogagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaatggca gcagaccctg gctcctgtat 480 agtttactc ctttgggggg attgcctcta ctcatcacta cctgttctc cctgttctgc 540 tgcctgtgtt ggcttacaa aaagaagtat tcatccagtg tgcacgaccc taacggtgaa 600 tacatgttca tgagagcagt gaacacagcc aaaaaatcta gactcacaga tgtgacccta 660 720 ctctataacg agctcaatct agcacgaaga gagagtacg atgttttgga caagagacgt 780 ggccgggacc ctgagatggg gggaaagccg your area accctcagga aggcctgtac 840 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaggcgag 900 cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 960 acctacgacg cccttcacat gcaggccctg ccccctcgct aagcggccgc a 1011 <210> 10 <211> 690 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 10 atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60 ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120 ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180 gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240 gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300 cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360 tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420 gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480 aggccagccg gccagttcca aaccctggtg ttctggttac ccataggatg tgcagccttt 540 gttgtagtct gcattttggg atgcatactt atttgttggc ttacaaaaaa gaagtattca 600 tccagtgtgc acgaccctaa cggtgaatac atgttcatga gagcagtgaa cacagccaaa 660 aaatctagac tcacagatgt gaccctataa 690 <210> 11 <211> 717 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 11 atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60 ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120 ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180 gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240 gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300 cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360 tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420 gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480 aggccagccg gccagttcca aaccctggtg ttttgggtgc tggtggtggt tggtggagtc 540 ctggcttgct atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag 600 aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 660 cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctcctaa 717 <210> 12 <211> 720 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 12 atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60 ccaggatggt tcttagactc cccagacagg ccctggacc cccccacctt ctccccagcc 120 ctgctcgtgg tgaccgagg ggacaacgcc accttcacct gcagctctc cacacatcg 180 gagagcttcg tgctaactg gtaccgcatg agccccagca accagacgga caagctggcc 240 gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300 cccaacgggc gtgacttcca catgagcgtg gtcaggggccc ggcgcaatga cagcggcacc 360 tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaagag cctgcgggca 420 gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480 aggccagccg gccagttcca aaccctgtg atccttgtga tctctctgg aatgttcctt 540 gttttcaccc tggccggggc cctgttccctc catcaacgaa ggaatatag atcaacaaa 600 ggagaaagtc ctgtggagcc tgcagagcct tgtcgttaca gctgccccag ggaggaggag 660 ggcagcacca tccccatcca ggaggattac cgaaaaccgg agcctgcctg ctccccctaa 720

Claims

1. A fusion protein comprising a first domain and a second domain, wherein the first domain is a polypeptide associated with a negative signal and the second domain is a polypeptide associated with a positive signal.

2. The fusion protein of claim 1, wherein the first domain is at least a portion of the extracellular domain of a polypeptide associated with a negative signal, and the second domain is at least a portion of the intracellular domain of a polypeptide associated with a positive signal.

3. The fusion protein of claim 1, further comprising a transmembrane domain.

4. 4. The fusion protein of claim 3, wherein the transmembrane domain is a transmembrane domain of a polypeptide associated with a negative signal or a transmembrane domain of a polypeptide associated with a positive signal.

5. The fusion protein of claim 1, wherein the polypeptide associated with a negative signal is selected from the group consisting of CTLA4, PD-1, and BTLA.

6. The fusion protein of claim 1, wherein the polypeptide associated with a positive signal is selected from the group consisting of CD28 and ICOS.

7. A cell engineered to express a fusion protein comprising a first domain and a second domain, wherein the first domain is a polypeptide associated with a negative signal and the second domain is a polypeptide associated with a positive signal.

8. The cell of claim 7, further comprising a chimeric antigen receptor (CAR) comprising an antigen recognition domain of a specific antibody and an intracellular domain of the CD3 zeta chain.

9. A vector comprising a first domain and a second domain, wherein the first domain is a polypeptide associated with a negative signal and the second domain is a polypeptide associated with a positive signal.

10. 1. A method of treating a patient with cancer, comprising administering to the patient T cells that have been genetically engineered to express a fusion protein comprising a first domain and a second domain, wherein the first domain is a polypeptide associated with negative signaling and the second domain is a polypeptide associated with positive signaling.

11. The method of claim 10, wherein the T cells are further genetically engineered to express a chimeric antigen receptor (CAR) comprising the antigen recognition domain of a specific antibody and the intracellular domain of the CD3 zeta chain.

12. 12. The method of claim 11, wherein the T cells are autologous T cells.

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