Switch co-stimulatory receptor

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 based on U.S. Patent Application No. 61 / 513,259, filed on July 29, 2011, the content of which is hereby incorporated by reference in its entirety into this specification.

Background Art

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

[0003] At present, the only approach to prevent the negative signals delivered by the ligands of PD - 1 or BTLA is to administer antagonistic antibodies or fusion proteins that bind to PD - 1 or BTLA, and this approach is currently being tested in early - phase clinical trials (Cheever et al., 2008, Immunol Rev 222:357 - 68 (Non - Patent Document 2)). Another approach would be to administer small - molecule compounds that can inhibit PD - 1 signaling or BTLA signaling. The current approach to prevent T cell inactivation by PD - 1 is to administer systemic treatment with PD - 1 antagonistic antibodies to patients.

[0004] Each of the above approaches has the limitation that systemic treatment prevents T cells present in both the tumor microenvironment and the entire immune system from becoming inactivated, which is expected to result in autoimmunity or systemic inflammatory syndrome 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] Accordingly, there is an urgent need in the art for compositions and methods for an effective form of adoptive therapy. The present invention addresses this need.

Prior Art Documents

Non - Patent Documents

[0006]

Non - Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

Non - Patent Document 4

Non - Patent Document 5

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 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.

[0009] In one embodiment, the fusion protein further comprises a transmembrane domain. In 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 a negative signal 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 a cell 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 an antigen recognition domain of a specific antibody and an intracellular domain of the CD3ζ 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 a method for treating cancer patients. In one aspect, the method comprises administering to a patient genetically engineered T cells that 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.

[0016] In one aspect, the T cells are further genetically engineered to express a chimeric antigen receptor (CAR) comprising an antigen recognition domain of a specific antibody and an intracellular domain of the CD3ζ chain.

[0017] In one aspect, the T cells are autologous T cells. [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. [Invention 1002] The fusion protein of Invention 1001, wherein the first domain is at least a portion of an extracellular domain of a polypeptide associated with a negative signal and the second domain is at least a portion of an intracellular domain of a polypeptide associated with a positive signal. [Invention 1003] The fusion protein of Invention 1001, further comprising a transmembrane domain. [Invention 1004] The fusion protein of 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. [Invention 1005] The fusion protein of Invention 1001, wherein the polypeptide associated with a negative signal is selected from the group consisting of CTLA4, PD-1, and BTLA. [Invention 1006] The fusion protein of Invention 1001, wherein the polypeptide associated with a positive signal is selected from the group consisting of CD28 and ICOS. [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. [Invention 1008] The cell of Invention 1007, further comprising a chimeric antigen receptor (CAR) comprising an antigen recognition domain of a specific antibody and an intracellular domain of the CD3ζ chain. [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. [Invention 1010] A method for treating a cancer patient, comprising administering to the patient a genetically engineered T cell that expresses 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. [Invention 1011] The method of Invention 1010, wherein the T cell is further genetically engineered to express a chimeric antigen receptor (CAR) comprising an antigen recognition domain of a specific antibody and an intracellular domain of the CD3ζ chain. [Invention 1012] The method of Invention 1011, wherein the T cell is an autologous T cell.

[0018] The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For purposes of illustration of the present invention, presently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact arrangements and instrumentalities of the embodiments shown in the drawings.

Brief Description of the Drawings

[0019]

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Mode for Carrying Out the Invention

[0020] Detailed Description The present invention generally relates to a fusion protein receptor that can convert a negative signal into a positive signal for a cell when displayed on the cell. Since the fusion protein contains at least two domains, it is a chimeric protein, where the first domain is a polypeptide associated with a negative signal and the 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, whereupon a signal is sent through the second domain, resulting in a positive signal that is transmitted to the cell. Thus, the fusion protein can convert what would otherwise be a negative signal into a positive intracellular signal. Accordingly, the present invention can be considered to encompass a switch receptor that can switch a negative signal to a positive signal for enhancing the immune response. Enhancing the 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 a switch receptor in order to take advantage of the fact that T cells sense their microenvironment and are activated or inhibited depending on the signals sensed. For example, the present invention takes advantage of the fact that a ligand that inhibits T cell activity is present within the tumor microenvironment. T cells are engineered to express a switch receptor where the first domain is activated by an inhibitory ligand within the tumor microenvironment and signaling through the second domain of the switch receptor can convert what would otherwise be an inhibitory signal into a positive signal for the T cell. Accordingly, the present invention provides a treatment that offers an improved therapeutic index with less toxicity, and also provides the ability to provide an effective single treatment that avoids the need for continuous administration of an antibody.

[0022] In some cases, the cells are genetically modified before administration to the 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 may be further modified to express on the surface an antibody binding domain (e.g., a chimeric antigen receptor (CAR)) that confers a novel antigen specificity that is MHC-independent. A CAR is a combination of the antigen recognition domain of a specific antibody with the intracellular domain of a CD3ζ chain or an FcγRI protein into a single chimeric protein. In this regard, the cells are engineered to express both the switch receptor and the CAR.

[0023] Since the modified cells of the present invention can replicate in vivo, a long-term persistence is provided that can result in sustained tumor control.

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

[0025] Definitions 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. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but the preferred materials and methods are described herein. The following terminology will be used in the description of the present invention and in the claims.

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

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

[0028] As used herein, "about" means, when referring to a measurable value such as an amount, a time period, etc., that variations of ±20%, ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value are appropriate to practice the disclosed method, and thus includes such variations.

[0029] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. The antibody may be a complete immunoglobulin of natural or recombinant origin, or may be an immunoreactive portion of a complete immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies in the present invention can exist in various 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] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response can include either or both antibody production or activation of specific immunocompetent cells. One of ordinary skill in the art will understand that macromolecules, including virtually all proteins or peptides, can function as antigens. Additionally, an antigen may be derived from recombinant DNA or genomic DNA. Thus, as the term is used herein, one of ordinary skill 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." Further, one of ordinary skill in the art will understand that an antigen need not be encoded by the full-length nucleotide sequence of a gene. The present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and it is readily apparent that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Additionally, one of ordinary skill 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 produced synthetically or may be derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0031] As used herein, the term "anti-tumor effect" refers to a biological effect that can manifest as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in mean survival, or remission of various physiological symptoms associated with the cancerous state. "Anti-tumor results" can also manifest, first and foremost, by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the occurrence of tumors.

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

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

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

[0035] As used herein, "having biological or immunological activity" refers to a fusion protein according to the present invention having a structural function (not necessarily to the same extent) and / or a regulatory function (not necessarily to the same extent) and / or a biochemical function (not necessarily to the same extent) and / or an immunological activity (not necessarily to the same extent) similar to that of the individual wild-type proteins that are 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 through the bloodstream and lymphatic system to other parts of the body. 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 containing two distinct polypeptide domains that do not naturally occur within the same polypeptide unit. Typically, such chimeric proteins are created by the expression of cDNA constructs, but may also be created by protein synthesis methods known in the art.

[0038] With respect to amino acid sequences, the term "derivative" as used herein means a chemical modification of the fusion protein of the present invention.

[0039] "Encoding" refers to the specific nucleotide sequence of a polynucleotide, such as a gene, cDNA, or mRNA, which has a distinct sequence of nucleotides (i.e., rRNA, tRNA, and mRNA), or a distinct sequence of amino acids, and which functions as a template for the synthesis of other polymers and macromolecules in biological processes, along with the biological properties resulting therefrom. Thus, when transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, that gene encodes that protein. A nucleotide sequence that is identical to the mRNA sequence, including both the coding strand, which is generally provided in a sequence listing, and the non-coding strand that 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, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are mutually degenerate versions and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNAs may contain introns.

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

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

[0043] As used herein, "exogenous" refers to a material that is introduced into, or produced outside of, an organism, cell, tissue, or system.

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

[0045] An "expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression regulatory sequence operably linked to a nucleotide sequence to be expressed. The 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 the recombinant polynucleotide is incorporated.

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

[0047] "Homologous," as used herein, refers to 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 of the two molecules is occupied by the same monomeric subunit; e.g., when 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; e.g., if half of the positions in two sequences (e.g., 5 positions in a 10-subunit polymer) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) match or are homologous, the two sequences are 90% homologous.

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

[0049] As used herein, the term "immune response" means a process that results in the activation and / or recruitment of effector functions in any of T cells, B cells, natural killer (NK) cells, and / or antigen-presenting cells. Thus, immune responses include, but are not limited to, detectable antigen-specific or allogeneic activation of helper T cell responses or cytotoxic T cell responses, antibody production, T cell-mediated activation of allergic reactions, etc., as understood by those skilled in the art.

[0050] As used herein, the term "immune cell" means 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, "explanatory materials" include publications, records, graphics, or other media of expression that can be used to inform about the usefulness of the compositions and methods of the present invention. The explanatory materials of the kits of the present invention may, for example, be attached to a container containing the nucleic acids, peptides, and / or compositions of the present invention, or may be shipped together with a container containing the nucleic acids, peptides, and / or compositions. Alternatively, the explanatory materials may be shipped separately from the container, with the intention that the explanatory materials and the compounds be used cooperatively by the recipient.

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

[0053] For the purposes 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, "nucleotide sequences encoding amino acid sequences" 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" may also include introns in that, in some versions, the nucleotide sequence encoding the protein may contain introns.

[0055] "Lentivirus", as used herein, refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of a host cell and are thus one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses present a means of achieving 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] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, nucleic acids and polynucleotides are interchangeable as used herein. One of ordinary skill in the art has 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, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR (trademark), etc., as well as synthetic means.

[0062] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound 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 can constitute the sequence of a protein or peptide. 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, which are also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, which are commonly referred to in the art as proteins (many types exist). "Polypeptide" includes, inter alia, fragments having biological activity, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

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

[0064] As used herein, the term "promoter / control sequence" means a nucleic acid sequence required for the expression of a gene product operably linked to the promoter / control sequence. In some instances, this sequence can be a core promoter sequence, and in other instances, this sequence can also include enhancer sequences and other control elements required for the expression of the gene product. A promoter / control sequence can, for example, be one that expresses a 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, causes the 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 the production of the gene product in a cell only when the cell is of the tissue type corresponding to the promoter.

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

[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 natural state. In some cases, a substantially purified population of cells refers to a homogeneous 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 prevention. A therapeutic effect is obtained by suppression, alleviation, or eradication of a disease state.

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

[0072] The phrases "under transcriptional control" or "functionally linked," as used herein, mean that a promoter is in the correct position and orientation with respect to a polynucleotide to regulate the initiation of transcription and expression of the polynucleotide by RNA polymerase.

[0073] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. A number of 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 be interpreted to also include non-plasmid compounds and non-viral compounds such as, for example, polylysine compounds, liposomes, etc., that facilitate the transfer of nucleic acids into cells. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

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

[0075] "Activation", as used herein, refers to the state of a T cell that has 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 cell" refers, inter alia, to a T cell that is undergoing cell division. Activation may be associated with the generation of an immune response (e.g., mitogens such as ConA or PHA), detectably upregulating surface markers such as CD25, i.e., the IL2 receptor, initiating a phosphorylation cascade involving p56lck, causing the release of cytokines and interleukins, and, inter alia, increasing DNA synthesis, which can be assayed by assaying the level of 3 H-thymidine incorporation into nascent DNA strands, causing cell proliferation.

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

[0077] Range: 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 merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, a recitation of a range should be considered to specifically disclose all the possible subranges and individual numerical values within that range. For example, a range recitation such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0078] Description The present invention relates to the discovery that it is possible to design chimeric switch receptors for switching negative signaling signals to positive signals. In one aspect, the switch receptor is a chimeric protein comprising a first protein or a fragment thereof associated with a negative signal and a second protein or a fragment thereof associated with a positive signal. Examples of proteins associated with negative signals include, but are not limited to, CTLA-4, PD-1, BTLA, etc. Examples of proteins associated with positive signals 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 aspect, 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 production of IL-2 and IFN-γ. In some cases, the engineered cells of the present invention are biased to secrete IL-17. Thus, the engineered cells of the present invention can eliminate in vivo tumor cells in a patient when injected into 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 that includes a polypeptide that delivers a negative signal and a second domain that includes a polypeptide that delivers a positive signal.

[0082] In one aspect, polypeptides having the ability to deliver a negative signal include, but are not limited to, CTLA4, PD-1, BTLA, and the like.

[0083] In one aspect, polypeptides having the ability to deliver a positive signal include, but are not limited to, ICOS, CD28, and the like.

[0084] Suitable first domains for a polypeptide that delivers a negative signal include variants or derivatives of wild-type CTLA4. Preferably, the first domain of the switch receptor in this aspect is at least a portion of the extracellular domain of the CTLA protein, specifically, the portion of the extracellular domain necessary 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 a positive signal 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 referred to as the endodomain or cytoplasmic domain) of the CD28 protein, specifically, the portion necessary to induce a signal to the intracellular components of the cell. Variants of the intracellular domain of the wild-type CD28 protein or portions of the intracellular domain that are 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] The switch receptors of the present invention include polypeptides corresponding to the cytoplasmic domain, transmembrane domain, and extracellular domain, as well as polypeptides corresponding to smaller portions of the cytoplasmic domain, transmembrane domain, and extracellular domain. 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 a further additional aspect of the present invention, the first polypeptide that delivers any negative signal component of the switch receptors described herein may be replaced with another inhibitory protein, i.e., a protein that prevents activation of the immune response and / or induces apoptosis in any of T cells or B cells, natural killer (NK) cells, NKT cells, lymphocyte progenitor cells, dendritic cells, monocytes / macrophages, macrophage lineage cells based on antigen-presenting tissues, and a number of non-professional antigen-presenting cells, such as other cell types like endothelial cells. Examples of inhibitory proteins include, but are not limited to, PD-1, CTLA-4, BTLA, CD160, CD161, and CD94; ligands of 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 in the present invention, provided that it binds to the corresponding ligand and through this binding event brings about activation of the switch receptor. According to one aspect of the present invention, contact of the first polypeptide that delivers a negative signal of the 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 a negative signal into a positive signal. Thus, the unique property of the first polypeptide of the switch receptor of the present invention is to convert a natural trans-signal that would naturally bring about a negative signal to cells into a positive signal that induces cells to exhibit anti-tumor characteristics.

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

[0093] In one aspect, the present invention utilizes a microenvironment in which there are a number of ligands or proteins that inhibit the immune system and bring about an undesirable disease state. That is, the switch receptor can be engineered to include a first domain that binds to an immunosuppressive factor within the microenvironment and converts the signal normally associated with the immunosuppressive factor into a positive signal that activates cells to exhibit an enhanced immune response.

[0094] A preferred chimeric protein of the present invention is BTLA:ICOS. Genetic chimerization and recombinant expression of the BTLA sequence with the 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 redirect 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 redirect inhibitory signaling to 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 redirect inhibitory signaling to 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 proteins of the present invention can exist in a number of forms. For example, the proteins of the present invention can be in the form of linear or branched polypeptides. Linear chimeric proteins can be produced by recombinant DNA techniques. For example, chimeric transcription cassettes can be assembled using restriction endonuclease site overlaps or polymerase chain reaction (PCR)-based splice-by-overlap extension.

[0098] Branched polypeptide chimeric proteins can be readily produced 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 carrier such as a core peptide using a chemical coupling reagent. For example, a cyclic decapeptide analog of gramicidin S in which two antiparallel β-sheet segments (lys-ala-lys) are linked 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, chemical cross-linking reagents such as dithio-bis(succinimidyl propionate) (DSP) can be used to directly covalently link two or more protein components to each other in a branched structure. By this methodology, for example, the first and second proteins can be directly linked.

[0100] The specific first and second polypeptides of the chimeric switch receptor of the present invention can vary depending on the disease to be treated. Typically, for example, when treating cancer or viral infection, a second polypeptide that stimulates an immune cell response is used. When treating an immune system disorder with a pathogenic immune response, 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 activation signal into an inhibitory immune signal is desired. In this context, the immunosuppressive second protein component can be directed to various pathogenic immune effectors, including T cells, B cells, natural killer cells, and antigen-presenting cells.

[0101] Accordingly, the present invention provides a switch receptor that, when expressed in a cell, converts a positive signal into a negative intracellular signal. For example, this switch receptor contains a first domain that includes a polypeptide that delivers a positive signal and a second domain that includes a polypeptide that delivers a negative intracellular signal.

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

[0103] In one embodiment, the cell may be further transduced by an LV that encodes a chimeric antigen receptor (CAR) that 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 enable long-term stable integration of the transgene and its transmission to daughter cells. Lentiviral vectors have additional advantages compared to vectors derived from oncoretroviruses such as murine leukemia virus because they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0105] Briefly summarized, the expression of natural or synthetic nucleic acids of the present invention is typically achieved by operably linking a nucleic acid encoding a 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. A typical cloning vector contains 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 a number of types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particularly important vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0107] Furthermore, the expression vector may be provided to cells in the form of a viral vector. Viral vector technology is well known in the art, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-3 (3 rd(ed., Cold Spring Harbor Press, NY 2001) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain at least one functional origin of replication, promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers in at least one organism (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0108] Additional promoter elements, such as enhancers, control the frequency of transcriptional initiation. Typically, these are located in the region 30 - 110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. Since the spacing between promoter elements is often flexible, promoter function is conserved when elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function either cooperatively or independently to activate transcription.

[0109] Examples of promoters are the immediate early cytomegalovirus (CMV) promoter sequence, which is a strong constitutive promoter sequence capable of driving high-level expression of polynucleotide sequences operably linked thereto. However, other constitutive promoter sequences may 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 leukemia 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 the expression of a polynucleotide sequence operably linked thereto when such expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0110] To assess the expression of the CAR polypeptide or a portion thereof, the expression vector introduced into the cells may contain either or both a selectable marker gene and a reporter gene to facilitate the identification and selection of expressing cells from the population of cells to be transfected or infected through a viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a cotransfection technique. Appropriate control sequences may flank both the selectable marker and the reporter gene to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0111] Reporter genes are used to identify cells that may have been transfected and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that does not exist or is not expressed in the recipient organism or tissue and encodes a polypeptide whose expression is manifested by some readily detectable property, such as enzyme activity. After the DNA has been introduced into the recipient cells, at an appropriate time point, the expression of the reporter gene is assayed. Suitable reporter genes can 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 may be obtained commercially. Generally, a construct having a 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 ligated to the reporter gene and used to evaluate agents for their ability to modulate transcription driven by the promoter.

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

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

[0114] Biological methods for introducing a polynucleotide of interest into a host cell 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 cells, such as human cells. Other viral vectors can be derived from, for example, lentivirus, poxvirus, herpes simplex virus type I, adenovirus, and adeno-associated virus. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

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

[0116] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated within the aqueous interior of a liposome, may be interspersed within the lipid bilayer of a liposome, may be attached to a liposome via a linking molecule associated with both the liposome and the oligonucleotide, may be entrapped within a liposome, may be complexed with a liposome, may be dispersed in a solution containing a lipid, may be mixed with a lipid, may be combined with a lipid, may be contained as a suspension within a lipid, may be contained within or complexed with a micelle, or may be associated with a lipid in some other manner. Compositions related to lipids, lipid / DNA, or lipid / expression vectors are not limited to a particular structure in solution. For example, they may exist as bilayer structures as micelles, or may have a "collapsed" structure. They may simply be dispersed in solution, or may form aggregates of non-uniform size or shape. A lipid is a fatty substance that may be a naturally occurring lipid or a synthetic lipid. For example, lipids include lipid droplets that naturally occur in the cytoplasm, as well as classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0117] Suitable lipids for use can be obtained from commercial suppliers. For example, dimyristoyl 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; dimyristoyl 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 about -20°C. Since chloroform evaporates more readily than methanol, it is used as the sole solvent. "Liposome" is a generic term encompassing various monolayer and multilayer lipid media formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure that includes a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an 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 having structures different from normal vesicular structures in solution are also included. For example, the lipids may assume a micellar structure or may simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0118] Therapeutic application The present invention includes a type of cell therapy in which T cells are genetically modified to express a switch receptor and the engineered T cells are injected into a recipient in need thereof. The injected cells can kill tumor cells in the recipient. Unlike antibody therapy, the engineered T cells of the present invention can replicate in vivo, resulting in long-term persistence that can provide sustained tumor control.

[0119] The present invention also relates to a method of treating a patient for a disease, comprising the step of administering to the patient, in an effective amount, an engineered switch receptor of the present invention. The method of the present invention can treat a variety of diseases 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 by 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 cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include kidney cancer or renal carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma including lung cancer, 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 or stomach cancer including gastrointestinal cancer, gastrointestinal stromal tumor (GIST), pancreatic cancer, head and neck cancer, glioblastoma, retinoblastoma, astrocytoma, meningioma, virilizing tumor, hepatoma, non-Hodgkin lymphoma (NHL), multiple myeloma, and hematological malignancies including acute hematological malignancies, endometrial cancer 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, anaplastic glioma, neuroblastoma, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcoma, urinary tract cancer, thyroid carcinomas, Wilms' tumor, and B cell lymphoma (including low grade / follicular non-Hodgkin lymphoma (NHL); small lymphocyte (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; large tumor lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenström macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia, chronic myelogenous leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with nevus flammeus, edema (such as that associated with a brain tumor), and Meigs syndrome. "Tumor" as used herein refers to the growth and proliferation of all neoplastic cells, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0121] With respect to the present invention, the term "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to an antigen common to a specific 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 aspect, 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 a number of proteins that can function 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 prostate acid phosphatase (PAP) and prostate specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are the oncofetal antigens such as carcinoembryonic antigen (CEA). In B cell lymphoma, the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen unique to each individual tumor. B cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in B cell lymphoma. 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] Regarding treatment for cancer, the switch receptor 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, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptonigrin, tubercidin, ubenimex, dinostatin, and 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; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxyridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, drostanolone propionate, epitioestanol, mepitiostane, testolactone; anti-adrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demeclocycline; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (trademark); razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (「Ara-C」); cyclophosphamide; thiotepa;Taxanes, for example, paclitaxel (TAXOL™, Bristol-Myers Squibb Oncology, Princeton, N.J.) 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; topoisomerase inhibitor RFS2'000; difluoromethylornithine (DMFO); retinoic acid; esperamicin, capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing are included.;

[0125] Antiestrogens, for example, tamoxifen, raloxifene, aromatase inhibitory 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and antihormonal agents that act to control or inhibit the hormonal action on tumors, such as pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing are also included.;

[0126] 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 small molecule antagonists of X-linked apoptosis inhibitor protein; and pharmaceutically acceptable salts, acids, or derivatives of any of these are also included.

[0127] Additional information regarding methods of treating cancer is provided in U.S. Patent No. 7,285,522, which is incorporated herein by reference in its entirety.

[0128] Accordingly, 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 allogeneic immune diseases, such as graft rejection or graft-versus-host disease or host-versus-graft disease.

[0129] Typically, for each disease application, 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 any particular theory, the anti-tumor immune response induced by the engineered cells of the 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) amplification of the cells, (ii) introduction of the nucleic acid encoding the switch receptor of the invention into the cells, or (iii) cryopreservation of 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 a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing the switch receptor of the invention. The engineered cells can then 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] Methods for the ex vivo expansion of hematopoietic stem and progenitor cells are described in U.S. Patent No. 5,199,942, which is incorporated herein by reference and can be applied to the cells of the present invention. Other suitable methods are known in the art, and thus 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 includes the following steps: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammalian source from a peripheral blood collection or a bone marrow explant; 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 the culture and expansion of cells.

[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 for inducing an immune response against an antigen in a patient.

[0135] Generally, the cells activated and expanded as described herein can be utilized in the treatment and prevention of diseases that occur 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. Accordingly, the present invention provides a method for treating or preventing cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the engineered T cells of the present invention.

[0136] The engineered T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or IL-2 or other cytokines or other components such as cell populations. Briefly, the pharmaceutical compositions of the present invention may include 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 being treated (or prevented). Appropriate dosages can be determined by clinical trials, but the amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease.

[0138] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount", or "therapeutic amount" is indicated, 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, degree of infection or metastasis, and the condition of the patient (subject). Generally, the pharmaceutical compositions containing the T cells described herein are in the range of 10 4 ~10 9 cells / kg body weight, preferably in the range of 10 5 ~10 6It can be said that it can be administered at a dosage of cells / kg body weight (including all integer values within these ranges). The T cell composition may be administered multiple times at these dosages. The cells can be administered by using infusion techniques generally known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment plan for a particular patient can be readily determined by those skilled in the medical art by monitoring the patient for signs of the 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 amplified T cells into the patient. This process can be carried out multiple times every few weeks. In certain embodiments, the T cells can be activated from a blood draw of 10 cc to 400 cc. In certain embodiments, the 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 composition 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, intranodally, intramedullarily, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to a patient by intradermal injection or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by i.v. injection. The composition of T cells may be directly injected into a tumor, lymph node, or site of infection.

[0141] In certain aspects of the invention, using the methods described herein or other methods known in the art by which T cells are amplified to therapeutic levels, the activated and amplified cells are administered to a patient (e.g., before, simultaneously, or after) with a number of suitable treatment modalities including, but not limited to, antiviral treatment, 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 aspects, the T cells of the invention may be used in combination with chemotherapies, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs either inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506), or inhibit the p70S6 kinase important for signaling induced by growth factors (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 further aspects, the cell compositions of the invention are administered to a patient (e.g., before, simultaneously, or after) with T cell depletion therapy using any of chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another aspect, the cell compositions of the invention are administered after B cell depletion therapy with an agent that reacts with CD20, e.g., rituxan.For example, in one aspect, a subject can undergo peripheral blood stem cell transplantation after receiving standard treatment with high-dose chemotherapy. In certain aspects, after transplantation, the subject receives an infusion of the amplified immune cells of the present invention. In additional aspects, the amplified cells are administered before or after surgery.

[0142] The dosage of the above-described treatment administered to a patient will vary depending on the condition being treated and the exact nature of the recipient of the treatment. An approximation of the dosage for administration to humans can be carried out by practices recognized in the art. The dosage for CAMPATH, for example, generally ranges from 1 to about 100 mg for adult patients and will generally be administered daily over a period of 1 to 30 days. A preferred daily dosage is 1 to 10 mg per day, although in some cases larger dosages up to 40 mg per day may be used (described in U.S. Patent No. 6,120,766).

Examples

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

[0144] Without further elaboration, one skilled in the art can, using the above description and the following illustrative examples, make and utilize the compounds of the present invention and practice the methods described in the claims. Accordingly, the following working examples are specifically pointed out as preferred embodiments of the present invention and should in no way be construed as limiting the remainder of the disclosure.

[0145] Example 1: Switch Receptor The results presented in this specification 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 avoid inhibition of tumor inhibitors systemically and thus throughout the immune system. Briefly, T cells were engineered to express a chimeric receptor encoding a PD-1 extracellular domain (without the inhibitory PD-1 domain) and a signaling domain of stimulatory CD28 or ICOS. The orientation of the chimeric receptor placed the PD-1 extracellular domain extracellularly and the stimulatory domain of CD28 or ICOS intracellularly in the T cell. Thus, since the intracellular signal is delivered by the signaling end domain of CD28 or ICOS rather than the native inhibitory PD-1 end domain, the interaction of the T cell with tumor antigens in the tumor microenvironment is positively influenced by contact with the PD-1 ligand.

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

[0147] Materials and Methods Switch Receptor Generation Constructs were designed for testing of the BTLA switch receptor. The following are the sequences of each construct cloned into an IVT vector based on pGEM.64A. TIFF0007714619000001.tif61140TIFF0007714619000002.tif231139TIFF0007714619000003.tif231138TIFF0007714619000004.tif120138

[0148] Transduction of T Cells A method for preparing T cells using paramagnetic polystyrene beads coated with anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody 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 U S A 103:17372 - 17377). Electroporation of T cells with RNA has been described as a method for expressing these receptors (2010, Zhao et al., Cancer Res 70:9062). The use of adenoviral vectors has been described (2004, Schroers et al., Exp Hematol 32:536). Numerous other approaches for expressing proteins in T cells have been described (2009, June et al., 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 using an 8 - point standard curve generated using a 3 - fold dilution series.

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

[0151] The results presented herein demonstrate that it is possible to engineer and express chimeric receptors in T cells to convert negative signals to positive signals in T cells. Thus, the present invention provides adoptive therapy with T cells or NK cells of the type that uses genetically modified cells that express a T - cell receptor (TCR) or chimeric antigen receptor (CAR).

[0152] Experiments were designed to develop a target - specific method to avoid systemic and thus inhibition of PD - 1 in the entire immune system. The target - specific method includes, for example, administering T cells that express a chimeric receptor encoding the extracellular domain of PD - 1 and encoding, in the intracellular portion of the T cell, a signaling domain of stimulatory CD28 or ICOS instead of the inhibitory PD - 1 domain. Thus, since the intracellular signal is delivered by the signaling end - domain of CD28 or ICOS rather than the native PD - 1 end - domain, the interaction of T cells with tumor antigens within the tumor microenvironment will be positively affected by contact with the PD - 1 ligand.

[0153] Conversion of BTLA signal In a similar approach, a chimeric receptor encoding BTLA was constructed. BTLA is also a member of the CD28 family along with PD - 1. BTLA often has several ligands, including HVEM, which are expressed on tumor cells and other cells within the tumor microenvironment. The interaction of BTLA with its natural ligand on the cell surface is known to negatively regulate the T - cell immune response (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 bypass this, a chimeric receptor composed of the extracellular domain of BTLA and an inter - cellular signaling end - domain containing the domain of CD28 or ICOS was constructed.

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

[0155] The following experimental set was designed to assess whether the inhibitory signal of BTLA can be converted into a co-stimulatory signal of CD28 through the BTLA-CD28 CCR. To find an appropriate window that can show CD28 signaling, different doses (ug / 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). The IL2 production of T cells electroporated with RNA as described in Figure 2A was stimulated by the CD19-positive cell line K562-CD19, and the 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 indicate that, unlike CD19-28z RNA electroporated T cells, which showed IL-2 production exceeding 300 pg / ml at an RNA dose of 1.5 ug, no detectable IL-2 production was found for CD19z RNA electroporated T cells. IFNγ production could be detected at similar levels for both CD19z electroporated T cells and CD19-28Z electroporated T cells at an RNA dose of 1.5 ug. Therefore, 1.5 ug RNA was used as the RNA dose for testing the BTLA-CD28 signal conversion polypeptide.

[0156] T cells were co-electroporated with 1.5 μg of CD19z and BTLA CCR as indicated and stimulated with K562 expressing CD19 (K562CD19) or both CD19 and HVEM (K562CD19 / HVEM). K562 lines expressing mesothelin (with or without HVEM) were used as controls (Figure 2C). The results show that full-length (wild-type) BTLA suppressed the production of both IL2 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 only, but T cells electroporated with CD19-28z and stimulated with the CD19 / HVEM double-positive cell line produced more than 400 pg / ml of IL-2. However, when T cells were co-electroporated with both CD19z RNA and BTLA-CD28 CCR RNA, the IL-2 production when stimulated with the CD19 / HVEM double-positive cell line was 4-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 electroporated T cells or CD19-28z electroporated T cells when stimulated with the CD19 / HVEM double-positive cell line or CD19-positive K562 expressing low levels of HVEM (Figure 2C). The results presented herein demonstrate that the inhibitory signal of BTLA can be converted to a CD28 signal through BTLA-CD28 CCR.

[0157] The following experimental set was designed to test whether inhibitory BTLA signals can be converted into co-stimulatory ICOS signals through the BTLA-ICOS CCR. Briefly, the conversion of BTLA signals into ICOS signals was tested in a Th17 polarization system as shown in Figure 3A. Resting CD4 T cells were co-electroporated (treated) with CD19z and BTLA CCR as shown, 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 (2010, Paulos et al., Science Translational Medicine), CD3 / ICOS beads and CD3 / CD28 beads were used as positive and negative controls, respectively. All cultures were performed in the presence of a Th17 cytokine cocktail. At different days after stimulation (as shown), T cells were stimulated with PMA / ionomycin, and IL-17A and IFNγ were detected by intracellular cytokine staining (Figure 3B). The results showed that the ICOS signal converted from BTLA-ICOS enhanced Th17 cell production in the presence of the HVEM inhibitory signal.

[0158] Conversion of PD-1 Signals into CD28 Signals IVT RNA encoding PD1 CCR was used (sequence shown below) to test whether ligation of PD1 CCR expressed on the T cell surface can functionally express to transmit signals through the intracellular domain of CD28 or CD27 or ICOS. TIFF0007714619000005.tif230139

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

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

[0161] The 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 the Nalm6-PD-L1 target. However, for T cells co-electroporated with PD1 CCR, when using Nalm6-PD1 as the target, no significant difference was observed compared to T cells electroporated with CD19Z alone. When using PD1 ligand-negative Nalm6 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 co-introducing PD1-CD28 CCR. To mimic the tumor microenvironment or chronic infection where 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) along with the additional PD1 CCR (10 μg) as shown. One day after electroporation, the expression of CAR and PD1 and / or PD1 CCR was detected by FACS (Figure 5A).

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

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

[0165] Conversion of the PD1 signal to the ICOS signal 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), 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), the T cells were mixed with K562 cells expressing PD-L1 and CD19 (0.5:1 = K562:T cells) in R10 culture medium. On the indicated days (5 days, 9 days, and 12 days after electroporation), the cells were incubated for 4 hours with PMA (3 μg / ml), ionomycin (1 μg / ml), and GolgiStop for intracellular cytokine staining. After surface staining for CD4, intracellular staining for IFNγ, IL17, and IL2 was performed. CD4 T cells stimulated with anti-CD28 / CD3 beads and anti-ICOS / CD3 beads were used as controls (Fig. 6A). Cytokine production was enhanced particularly on day 9 in cells expressing PD1-ICOS compared to PD1 wild type or tailless PD1. Cell proliferation of the stimulated T cells is shown in Fig. 6C.

[0166] PD-1 chimeric receptor PD-1 is upregulated on the surface of exhausted CD8 T cells in patients with chronic viral infection. Blocking of the PD-1:PD-L1 signal restores the function of exhausted CD8 T cells expressing PD-1. Many tumors express PD-L1 and provide an immunosuppressive microenvironment.

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

[0168] It was 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, a minimal difference was 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 the PD-1 chimeric receptor on T cell proliferation (Figure 10). It was observed that the PD1-CD28 chimeric receptor increased the number of CD8 T cells (Figure 11).

[0170] In summary, the results presented herein demonstrate that the PD-1 chimeric construct does not exhibit the inhibitory effect shown by PD-1wt. PD1-CD28 appears to increase the production of TNFα, IL2, and IFNγ in CD4 T cells. The PD-1 chimeric receptor did not show increased cytotoxicity compared to that of T cells expressing only CD19CARz. PD1-CD28 increased the number of CD8 T cells compared to that of T cells expressing only CD19CARz.

[0171] Redirecting co-inhibitory signaling to positive co-stimulation The results presented herein demonstrate that the switch receptor was expressed in T cells by electroporation or by a lentiviral vector. It was observed that it was possible to express the CAR and the switch receptor in the same T cell. When cells expressing the CAR or TCR and the switch receptor were exposed to tumor cells having the ligand for BTLA or PD-1, the T cells were shown to have a positive immune response rather than the normal inhibitory response.

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

[0173] Another important result from these studies is that interaction with the chimeric receptor of HVEM:BTLA led to enhanced IL-17 secretion. This is a marker of cells known to be useful for tumor immunotherapy, TH17 cells (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 domain deleted containing CD28 were protected from inhibition, killed tumor cells, and secreted cytokines (IL-2 and IFNγ) when the PD-1 switch receptor was expressed.

[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.

[0177] Sequence information SEQUENCE LISTING <110> THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA <120> SWITCH COSTIMULATORY RECEPTORS <150> US 61 / 513,259 <151> 2011-07-29 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 870 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 1 atgaagacat tgcctgccat gcttggaact gggaaattat tttgggtctt cttcttaatc 60 ccatatctgg acatctggaa catccatggg aaagaatcat gtgatgtaca gctttatata 120 aagagacaat ctgaacactc catcttagca ggagatccct ttgaactaga atgccctgtg 180 aaatactgtg ctaacaggcc tcatgtgact tggtgcaagc tcaatggaac aacatgtgta 240 aaacttgaag atagacaaac aagttggaag gaagagaaga acatttcatt tttcattcta 300 cattttgaac cagtgcttcc taatgacaat gggtcatacc gctgttctgc aaattttcag 360 tctaatctca ttgaaagcca ctcaacaact ctttatgtga cagatgtaaa aagtgcctca 420 gaacgaccct ccaaggacga aatggcaagc agaccctggc tcctgtatag tttacttcct 480 ttggggggat tgcctctact catcactacc tgtttctgcc tgttctgctg cctgagaagg 540 caccaaggaa agcaaaatga actctctgac acagcaggaa gggaaattaa cctggttgat 600 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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 gaatgccctg tgaaatactg tgctaacagg cctcatgtga cttggtgcaa gctcaatgga 240 acaacatgtg taaaacttga agatagacaa acaagttgga aggaagagaa gaacatttca 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 tgcctggaag gaaatataga tcaaacaaag gagaaagtcc tgtggagcct gcagagcctt 600 gtcgttacag ctgccccagg gaggaggagg gcagcaccat ccccatccag gaggattacc 660 gaaaaccgga gcctgcctgc tccccctgat aagcggccgc a 701 <210> 4 <211> 685 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 4 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaaatt attttgggtc 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 tttttcattc tacattttga accagtgctt cctaatgaca atgggtcata ccgctgttct 360 gcaaattttc agtctaatct cattgaaagc cactcaacaa ctctttatgt gacagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaaatggcaa gcagaccctg gctcctgtat 480 agtttctggt tacccatagg atgtgcagcc tttgttgtag tctgcatttt gggatgcata 540 cttattgagg agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg 600 ccgccccggg cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc 660 ctatcgctcc tgataagcgg ccgca 685 <210> 5 <211> 701 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 5 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaaatt attttgggtc 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 tttttcattc tacattttga accagtgctt cctaatgaca atgggtcata ccgctgttct 360 gcaaattttc agtctaatct cattgaaagc cactcaacaa ctctttatgt gacagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaaatggcaa gcagaccctg gctcctgtat 480 agtttctggt tacccatagg atgtgcagcc tttgttgtag tctgcatttt gggatgcata 540 cttattgaag gaaatataga tcaaacaaag gagaaagtcc tgtggagcct gcagagcctt 600 gtcgttacag ctgccccagg gaggaggagg gcagcaccat ccccatccag 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 attttgggtc 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 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 tgcctgtgtt ggcttacaaa aaagaagtat tcatccagtg tgcacgaccc taacggtgaa 600 tacatgttca tgagagcagt gaacacagcc aaaaaatcta gactcacaga tgtgacccta 660 taagcggccg ca 672 <210> 7 <211> 1011 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 7 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaaatt attttgggtc 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 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 tgcctgtgtt ggcttacaaa aaagaagtat tcatccagtg tgcacgaccc taacggtgaa 600 tacatgttca tgagagcagt gaacacagcc aaaaaatcta gactcacaga tgtgacccta 660 tgcagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 720 ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 780 ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 840 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 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 ctgggaaatt attttgggtc 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 tttttcattc tacattttga accagtgctt cctaatgaca atgggtcata ccgctgttct 360 gcaaattttc agtctaatct cattgaaagc cactcaacaa ctctttatgt gacagatgta 420 aaaagtgcct cagaacgacc ctccaaggac gaaatggcaa gcagaccctg gctcctgtat 480 agtttctggt tacccatagg atgtgcagcc tttgttgtag tctgcatttt gggatgcata 540 cttatttgtt ggcttacaaa aaagaagtat tcatccagtg tgcacgaccc taacggtgaa 600 tacatgttca tgagagcagt gaacacagcc aaaaaatcta gactcacaga tgtgacccta 660 taagcggccg ca 672 <210> 9 <211> 1011 <212> DNA <213> Artificial <220> <223> Chemically synthesized <400> 9 aagcttgccg ccatgaagac attgcctgcc atgcttggaa ctgggaaatt attttgggtc 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 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 tgcctgtgtt ggcttacaaa aaagaagtat tcatccagtg tgcacgaccc taacggtgaa 600 tacatgttca tgagagcagt gaacacagcc aaaaaatcta gactcacaga tgtgacccta 660 tgcagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 720 ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 780 ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 840 aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 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 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 atccttgtga tcttctctgg aatgttcctt 540 gttttcaccc tggccggggc cctgttcctc catcaacgaa ggaaatatag atcaaacaaa 600 ggagaaagtc ctgtggagcc tgcagagcct tgtcgttaca gctgccccag ggaggaggag 660 ggcagcacca tccccatcca ggaggattac cgaaaaccgg agcctgcctg ctccccctaa 720

Claims

1. A chimeric switch receptor comprising an extracellular domain associated with a negative signal, a transmembrane domain, and an intracellular domain associated with a positive signal, (a) the chimeric switch receptor is BTLA-ITM-CD28, BTLA-BTM-CD27, BTLA-ITM-CD27, BTLA-BTM-ICOS-CD3ζ, or BTLA-ITM-ICOS-CD3ζ; and (b) when expressed in an immune cell, the chimeric switch receptor converts a negative signal delivered by BTLA into a positive signal delivered by CD28, CD27, CD3ζ, or ICOS in the immune cell, thereby enhancing the anti-tumor properties of the immune cell; Chimeric switch receptors.

2. A chimeric switch receptor comprising an extracellular domain associated with a negative signal, a transmembrane domain, and an intracellular domain associated with a positive signal, the chimeric switch receptor is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8; and When expressed in an immune cell, the chimeric switch receptor converts a negative signal delivered by BTLA into a positive signal delivered by CD28, CD27, CD3ζ, or ICOS in the immune cell, thereby enhancing the anti-tumor properties of the immune cell. Chimeric switch receptors.

3. 3. An isolated cell engineered to express the chimeric switch receptor of claim 1 or 2.

4. The isolated cell of claim 3, 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.

5. 5. The isolated cell of claim 3 or 4, which is a T cell.

6. 5. The isolated cell of claim 3 or 4, which is an autologous T cell.

7. The isolated cell of any one of claims 3 to 6, which secretes IL-17.

8. 8. The isolated cell of any one of claims 3 to 7, which exhibits enhanced production of IL-2 and IFN-γ.

9. A vector comprising a nucleic acid encoding the chimeric switch receptor of claim 1 or 2.

10. 10. The vector of claim 9, which is a lentiviral vector.

11. 11. An isolated cell engineered to express the vector of claim 9 or 10.

12. (a) is a T cell; (b) are autologous T cells, or (c) secrete IL-17; 12. The isolated cell of claim 11.

13. The isolated cell of claim 11 or 12, further comprising a chimeric antigen receptor (CAR) comprising an antigen recognition domain of a specific antibody that binds to a tumor antigen and the intracellular domain of the CD3 zeta chain.

14. A pharmaceutical composition for treating cancer, comprising the isolated cells of any one of claims 3 to 8 and 11 to 13.

15. 15. The pharmaceutical composition of claim 14, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, melanoma, leukemia, and lung cancer.

16. A pharmaceutical composition comprising the chimeric switch receptor of claim 1 or 2, the isolated cell of any one of claims 3 to 8 and 11 to 13, or the vector of claim 9 or 10.

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