DNVSIG3 AND DNVSIG8 RECEPTORS AND METHODS OF USE THEREOF

Abstract

Dominant negative (DN) receptor mutants of the V-Set and Immunoglobulin Domain-containing 3 (VSIG3) receptor and the V-Set and Immunoglobulin Domain-containing 8 (VSIG8) receptor for use in applications directed to modulating the downstream signaling effects of VSIG3 and VSIG8 and their corresponding ligands, and methods for their production are described. The present invention is directed to the use of dnVSIG3 and / or dnVSIG8 receptor mutants to reduce the immunosuppressive effects associated with functional forms of VSIG3 and / or VSIG8 in the presence of their cognate ligands.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 225,840, filed July 26, 2021, the entire contents of which are specifically incorporated by reference.

[0002] Field The present disclosure relates to the field of dominant negative (DN) receptor variants of the V-Set and Immunoglobulin Domain-containing 3 (VSIG3) receptor and the V-Set and Immunoglobulin Domain-containing 8 (VSIG8) receptor for use in applications directed to modulating downstream signaling effects of VSIG3 and VSIG8 and their corresponding ligands, and methods of making the same. The present disclosure is further directed to recombinant cells comprising DNVSIG3 and / or DNVSIG8 receptor variants, and methods of administering the cells to a subject in need thereof. More specifically, the present disclosure relates to recombinant human immune cells comprising DNVSIG3 and / or DNVSIG8 receptor variants, further comprising modified T cell receptors and / or modified chimeric antigen receptors.

[0003] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text form in lieu of a paper copy and is incorporated by reference into the specification. The text file containing the sequence listing is named 125400_1235_Sequence_Listing.txt. The text file is approximately 102kb, was created on June 16, 2021, and has been submitted electronically via EFS-Web. [Background technology]

[0004] background Recent advances in cancer immunotherapy have demonstrated that cancer cells can be eliminated by harnessing the immune system. Most of these treatments rely on eliciting an immune response through the administration of monoclonal antibodies that block various immunosuppressive signals. However, relying on an endogenous response makes it difficult to achieve high therapeutic efficacy while avoiding toxic autoimmune side effects. For more precise treatment, T cells can be engineered with modified antigen receptors, such as chimeric antigen receptors (CARs), to recognize and exert cytotoxic effects on cells expressing specific antigens. A challenge to the success of these therapies is the immunosuppressive microenvironment that redirected T cells encounter as they infiltrate the tumor bed.

[0005] To achieve therapeutic success within solid tumors, T cells with modified antigen receptors must overcome immunosuppressive signals. One approach is to block immunosuppressive pathways such as PD-1 and / or CTLA-4 and enhance genetic responses in combination with recombinant T cell therapy. Many cancers, such as prostate cancer, are known to secrete molecules that create an immunosuppressive environment. Identifying the pathways associated with these immunosuppressive molecules provides an opportunity to mitigate the signaling that ultimately leads to these immunosuppressive effects. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present disclosure is to fill a need for reducing the immunosuppressive effects of immunosuppressive molecules on recombinant T cells used in cancer immunotherapy. This need is filled by the use of recombinant T cells expressing engineered dominant negative forms of V-set and immunoglobulin domain containing 3 (VSIG3) and / or V-set and immunoglobulin domain containing 8 (VSIG8) that reduce or eliminate signaling in VSIG3 and / or VSIG8. [Means for solving the problem]

[0007] Summary of disclosure The present disclosure is generally directed to dominant-negative VSIG3 and VSIG8 receptors, in which the wild-type receptor has been modified such that signaling at the receptor is disrupted as compared to the wild-type receptor. The present disclosure further relates to polynucleotides, vectors, cells, and methods of administration related to the dominant-negative VSIG3 and VSIG8 receptors described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In some embodiments, the disclosure is broadly directed to modified polypeptides comprising an amino acid sequence that shares at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with any one of SEQ ID NOs:20-28. In some embodiments, the amino acid sequence shares at least about 95% sequence identity with SEQ ID NO:20. In some embodiments, the amino acid sequence shares at least about 99% sequence identity with SEQ ID NO:20. In some embodiments, the amino acid sequence is SEQ ID NO:20.

[0009] In some embodiments, the disclosure is broadly directed to modified polypeptides comprising an amino acid sequence that shares at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with any one of SEQ ID NOs:61-70. In some embodiments, the amino acid sequence shares at least about 95% sequence identity with SEQ ID NO:61. In some embodiments, the amino acid sequence shares at least about 99% sequence identity with SEQ ID NO:61. In some embodiments, the amino acid sequence is SEQ ID NO:61.

[0010] In some aspects, a polynucleotide encoding a polypeptide described herein further comprises a signal peptide sequence. In some aspects, the disclosure is broadly directed to a polynucleotide sequence encoding any one of the polypeptides described herein. In some aspects, the disclosure is broadly directed to a polynucleotide sequence comprising a nucleic acid sequence that shares at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with any one of SEQ ID NOs: 50-59. In some aspects, the amino acid sequence shares at least about 95% sequence identity with SEQ ID NO: 50. In some aspects, the amino acid sequence shares at least about 99% sequence identity with SEQ ID NO: 50. In some aspects, the nucleic acid sequence is SEQ ID NO: 50.

[0011] In some embodiments, the disclosure is broadly directed to polynucleotide sequences including a nucleic acid sequence that shares at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity with any one of SEQ ID NOs: 72-81. In some embodiments, the amino acid sequence shares at least about 95% sequence identity with SEQ ID NO: 72. In some embodiments, the amino acid sequence shares at least about 99% sequence identity with SEQ ID NO: 72. In some embodiments, the nucleic acid sequence is SEQ ID NO: 72.

[0012] In some aspects, the disclosure is directed broadly to compositions comprising any one or more of the polypeptides described herein.

[0013] In some aspects, the present disclosure is directed broadly to vectors comprising any one or more of the polynucleotide sequences described herein that encode any one of the recombinant dominant negative receptors described herein. In some aspects, any one or more of the polynucleotide sequences described herein are operably linked to one or more polypeptides.In some aspects, any one or more of the polynucleotide sequences described herein are operably linked to one or more polypeptides.

[0014] In some aspects, the vector is a viral vector. In some aspects, the viral vector is selected from the group consisting of a lentiviral vector, a gamma retroviral vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a poxvirus vector, a herpes virus vector, and an engineered hybrid virus vector. In some aspects, the vector is a lentiviral vector.

[0015] In some aspects, the disclosure is directed broadly to a cell comprising any one or more of the polypeptides described herein. In some aspects, the polypeptide is a dominant-negative receptor that has abolished activity compared to the activity of wild-type VSIG3. In some aspects, the polypeptide is a dominant-negative receptor that has abolished activity compared to the activity of wild-type VSIG8.

[0016] In some aspects, the disclosure is directed broadly to a cell comprising any one or more of the polynucleotides described herein. In some aspects, the disclosure is directed broadly to a cell comprising any one or more of the vectors described herein. In some aspects, the cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell, and a human cell. In some aspects, the cell is a human cell. In some aspects, the human cell is an immune cell. In some aspects, the immune cell is a T cell.

[0017] In some aspects, a cell, such as a T cell, comprises a modified antigen receptor. In some aspects, the modified antigen receptor is a T cell receptor or a chimeric antigen receptor (CAR). In some aspects, the dominant negative receptor is incapable of signal transduction. In some aspects, the extracellular domain of the dominant negative receptor is capable of binding to its corresponding ligand. In some aspects, the corresponding ligand is VISTA. In some aspects, expression of wild-type VSIG3 is downregulated in the cell. In some aspects, expression of wild-type VSIG8 is downregulated in the cell. In some aspects, expression of wild-type VSIG3 and VSIG8 is downregulated in the cell.

[0018] In some embodiments, the modified antigen receptor is a CAR, the CAR comprising a binder selected from the group consisting of prostate specific membrane antigen (PSMA), Tn glycoform of mucin 1 (TnMUC1), mesothelin, glypican 2 (GPC2), fibroblast activation protein (FAP), folate receptor alpha (FRα), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit alpha 2 (IL-13Rα2), and any combination thereof. In some embodiments, the modified antigen receptor is a CAR, the CAR comprising a co-stimulatory domain selected from the group consisting of CD2, 4-1BB, ICOS, and CD27. In some embodiments, the modified antigen receptor is a CAR, the CAR comprises a switch receptor and / or a dominant negative receptor, and the receptor is selected from the group consisting of PD1 / CD28, PDL1 / CD28, CTLA4 / CD28, BTLA / CD28, BTLA / ICOS, TIM3 / CD28, TIGIT / CD226, dnTGFβ, TGFβ / IL-12R, TGFβ / CD28, TGFβ / OX40, IFNγ / CD28, IFNγ / OX40, and IFNγ / IL-12R. In some embodiments, the binder comprises a combination of EGFR and IL-13Rα2. In some embodiments, the modified antigen receptor is a CAR, the CAR comprises a CD3ζ signaling domain.

[0019] In some aspects, the disclosure is broadly directed to a method of administering any one or more recombinant cells described herein comprising polynucleotides and / or polypeptides corresponding to recombinant dominant negative VSIG3 and / or VSIG8 receptors, the method comprising administering to a subject a composition comprising the cells. In some aspects, the cells are autologous to the subject. In some aspects, the cells are allogeneic to the subject. In some aspects, the disclosure is broadly directed to a method of generating modified cells comprising introducing into a cell any one or more of the vectors described herein.

[0020] In some aspects, the cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell, and a human cell. In some aspects, the cell is a human immune cell. In some aspects, the immune cell is a T cell. In some aspects, the T cell comprises a modified antigen receptor.

[0021] In some embodiments, the modified antigen receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the modified antigen receptor is a CAR, and the CAR comprises a binder selected from the group consisting of prostate specific membrane antigen (PSMA), Tn glycoform of mucin 1 (TnMUC1), mesothelin, glypican 2 (GPC2), fibroblast activation protein (FAP), folate receptor alpha (FRα), and a combination of epidermal growth factor receptor (EGFR) and interleukin-13 receptor subunit alpha 2 (IL-13Rα2). In some embodiments, the binder comprises a combination of EGFR and IL-13Rα2.

[0022] In some embodiments, the modified antigen receptor is a CAR, and the CAR comprises a co-stimulatory domain selected from the group consisting of CD2, 4-1BB, ICOS, and CD27. In some embodiments, the modified antigen receptor is a CAR, and the CAR comprises a switch receptor and / or a dominant negative receptor, and the receptor is selected from the group consisting of PD1 / CD28, PDL1 / CD28, CTLA4 / CD28, BTLA / CD28, BTLA / ICOS, TIM3 / CD28, TIGIT / CD226, dnTGFβ, TGFβ / IL-12R, TGFβ / CD28, TGFβ / OX40, IFNγ / CD28, IFNγ / OX40, and IFNγ / IL-12R. In some embodiments, the modified antigen receptor is a CAR, and the CAR comprises a CD3ζ signaling domain.

[0023] The above summary, the following drawing description, and the detailed description are all exemplary and explanatory and are intended to provide further details of the invention, but should not be construed as limiting. Other objects, advantages, and novel features will become readily apparent to those skilled in the art from the following detailed description of the invention. [Brief description of the drawings]

[0024] [Figure 1] Figure 1 shows the characteristics of wild-type VSIG3 and recombinant dominant-negative VSIG3 (dnVSIG3). Both VSIG3 and dnVSIG3 span the lipid bilayer of the cell membrane and both contain extracellular and transmembrane domains. dnVSIG3 lacks most or all of the intracellular domain and is incapable of downstream signaling, whereas VSIG3 contains an intact intracellular domain capable of downstream signaling.

[0025] [Diagram 2] FIG. 2 shows the characteristics of VSIG3 and dnVSIG3, highlighting the relative differences in length of wild-type VSIG3 versus truncated dnVSIG3.

[0026] Detailed Description of the Disclosure I. Dominant negative mutants Dominant negative mutants represent an important class of mutations in which one or more mutations in a receptor interfere with or destroy the function of the wild-type version of the receptor compared to the unmodified receptor.

[0027] In some embodiments, the dominant negative mutant comprises an extracellular domain capable of binding to its corresponding ligand, hi some embodiments, the extracellular domain of the dominant negative mutant does not exhibit a difference in affinity for its corresponding ligand.

[0028] In some embodiments, interference with / disruption of receptor function results in a complete loss of receptor function, in some embodiments, the extracellular domain of the receptor is capable of binding to its corresponding ligand or binding partner, but is unable to generate intracellular signaling due to the absence or alteration of the receptor domain, and thus cells expressing this mutated receptor are unable to respond in the presence of the corresponding receptor ligand compared to the wild type receptor.

[0029] In some embodiments, interference with / disruption of receptor function results in a decrease in the ability of the receptor to transmit a signal from the outside of the receptor to the inside of the receptor. In some embodiments, the decrease in the ability of the receptor to transmit a signal from the outside of the receptor to the inside of the receptor is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. II. VSIG3 and VSIG8

[0030] The dominant negative mutant receptors of the present disclosure are directed to V-set and immunoglobulin domain containing 3 (VSIG3) and V-set and immunoglobulin domain containing 8 (VSIG8).

[0031] VSIG3 is known to have at least one binding partner or ligand, which is V-domain-containing immunoglobulin inhibitor of T cell activation (VISTA). The VSIG3 / VISTA pathway has been reported to be capable of inhibiting the function of human T cells. VSIG3 has been associated with inhibition of human T cell proliferation in the presence of T cell receptor signaling, and also with a significant reduction in cytokine and chemokine production in human T cells, including IFN-γ, IL-2, IL-17, CCL5 / Rantes, CCL3 / MIP-1α, and CXCL11 / I-TAC. See Wang et al., Immunology 156(1):74-85 (2019). Although Wang describes VSIG3 as a ligand for VISTA, VSIG3 is believed to be a transmembrane receptor that contains an extracellular domain, a transmembrane domain, and an intracellular domain.

[0032] When expressed on T cells, the dnVSIG3 receptor engages VISTA, a checkpoint inhibitor expressed primarily on tumor and myeloid cells, and blocks the immunosuppressive signaling triggered by VSIG3-VISTA ligation.

[0033] The wild-type human VSIG3 amino acid sequence is associated with Uniprot Accession Q5DX21. This amino acid sequence consists of 431 amino acids (SEQ ID NO: 1). The corresponding nucleic acid sequence encoding this wild-type human VSIG3, the coding sequence, consists of 1,296 nucleotides (SEQ ID NO: 31). The amino acid sequence of SEQ ID NO: 1 includes the signal peptide (positions 1-22), the extracellular domain (positions 23-241), the transmembrane domain (positions 242-262), and the intracellular domain (positions 263-431). Wild-type human VSIG3 has 18 additional naturally occurring variants, called isoforms or splice variants. The VSIG3 sequence and the dominant negative VSIG3 (DNVSIG3) sequence are described in the table below.

[0034] In some embodiments, the dominant negative VSIG3 comprises the following mutations compared to the wild type:

[0035] (1) at least about 5 residues, at least about 10 residues, at least about 15 residues, at least about 20 residues, at least about 25 residues, at least about 30 residues, at least about 35 residues, at least about 40 residues, at least about 45 residues, at least about 50 residues, at least about 55 residues, at least about 60 residues, at least about 65 residues, at least about 70 residues, at least about 75 residues, at least about 80 residues, at least about 85 residues, at least about 90 residues, or at least about 95 residues from the N-terminus or C-terminus of the intracellular domain , a truncation of at least about 100 residues, at least about 105 residues, at least about 110 residues, at least about 115 residues, at least about 120 residues, at least about 125 residues, at least about 130 residues, at least about 135 residues, at least about 140 residues, at least about 145 residues, at least about 150 residues, at least about 155 residues, at least about 160 residues or at least about 165 residues, or any number of residues falling between any of these values, e.g., about 7 residues, about 52 residues, about 93 residues, etc.

[0036] (2) about 5 residues, about 10 residues, about 15 residues, about 20 residues, about 25 residues, about 30 residues, about 35 residues, about 40 residues, about 45 residues, or about 50 residues from the N-terminus or C-terminus of the intracellular domain, Truncation of about 55 residues, about 60 residues, about 65 residues, about 70 residues, about 75 residues, about 80 residues, about 85 residues, about 90 residues, about 95 residues, about 100 residues, about 105 residues, about 110 residues, about 115 residues, about 120 residues, about 125 residues, about 130 residues, about 135 residues, about 140 residues, about 145 residues, about 150 residues, about 155 residues, about 160 residues or about 165 residues, or any number of residues falling between any of these values, e.g., about 7 residues, about 52 residues, about 93 residues, etc.

[0037] (3) about 5 consecutive residues, about 10 consecutive residues, about 15 consecutive residues, about 20 consecutive residues, about 25 consecutive residues, about 30 consecutive residues, about 35 consecutive residues, about 40 consecutive residues, about 45 consecutive residues, about 50 consecutive residues, about 55 consecutive residues, about 60 consecutive residues, about 65 consecutive residues, about 70 consecutive residues, about 75 consecutive residues, about 80 consecutive residues, about 85 consecutive residues, about 90 consecutive residues, about 95 consecutive residues, about 100 consecutive residues, about 105 ... Contiguous residues, about 110 contiguous residues, about 115 contiguous residues, about 120 contiguous residues, about 125 contiguous residues, about 130 contiguous residues, about 135 contiguous residues, about 140 contiguous residues, about 145 contiguous residues, about 150 contiguous residues, about 155 contiguous residues, about 160 contiguous residues or about 165 contiguous residues, or any number of residues falling between any of these values, e.g., about 7 residues, about 52 residues, about 93 residues, etc.

[0038] (4) Deletion of the entire intracellular domain.

[0039] (5) C-terminal truncation of the intracellular domain such that the resulting intracellular domain consists of about 1 to about 30 residues of the N-terminal portion of the intracellular domain. In some embodiments, the obtained intracellular domain consists of about 5 to about 30 residues, about 10 to about 30 residues, about 15 to about 30 residues, about 20 to about 30 residues, about 25 to about 30 residues, about 5 to about 25 residues, about 10 to about 25 residues, about 15 to about 25 residues, about 20 to about 25 residues, about 5 to about 20 residues, about 10 to about 20 residues, about 15 to about 20 residues, about 5 to about 15 residues, about 10 to about 15 residues, or about 5 to about 10 residues of the N-terminal portion of the intracellular domain.

[0040] (6) A deletion of about 1 to about 10, about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 2 to about 8, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 4 to about 6, about 4 to about 5, about 1 to about 5, about 2 to about 3, or about 1 to about 2 residues from the transmembrane domain.

[0041] In some embodiments, any one of the mutations (1) to (5) is further combined with the mutation (6).

[0042] In some embodiments, any polynucleotide sequence encoding any one of the dnVSIG3 receptors is contemplated. In some embodiments, the polynucleotide sequence is codon-optimized. A codon-optimized polynucleotide sequence encodes the same amino acid sequence as a non-codon-optimized polynucleotide sequence. [Table 1-1] [Table 1-2]

[0043] VSIG8 has been described to be involved in the inhibition of the production of cytokines such as IL-2, IFN-γ, IL-17, IL-6, and IL-19, and chemokines such as MCP-1, MCP-10, IP-10, and other proteins such as IGFBP3 and RBP4 in anti-CD3-activated human CD3 T cells. Furthermore, it has been described to significantly reduce IFN-γ and IL-2 production in both CD4 and CD8 T cells in the presence of T cell receptor signaling and to significantly suppress anti-CD3-induced human T cell proliferation. VSIG8 has also been associated with a significant reduction in the conversion of naive CD4 positive T cells to Th1 cells. See Wang et al., J. Immunol. 200(1 Supplement) 47.4 (2018). VSIG3 is a transmembrane receptor that contains an extracellular domain, a transmembrane domain, and an intracellular domain, and is considered to be a kind of binding partner (ligand) for VISTA. The interaction between VISTA and VSIG8 / dnVSIG8 is believed to work in the same manner as described above for VSIG3.

[0044] The wild-type human VSIG8 amino acid sequence consists of 414 amino acids (SEQ ID NO: 60). The nucleic acid sequence encoding wild-type human VSIG8, the coding sequence, consists of 1245 nucleotides (SEQ ID NO: 71). The amino acid sequence of SEQ ID NO: 60 includes a signal peptide (positions 1-21), an extracellular domain (positions 22-263), a transmembrane domain (positions 264-284), and an intracellular domain (positions 285-414). The wild-type human VSIG8 protein is associated with Uniprot Accession P0DPA2.

[0045] In some embodiments, the dominant negative VSIG8 comprises the following mutations compared to the wild type:

[0046] (1) A truncation of at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65 residues, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 105, at least about 110, at least about 115, at least about 120, at least about 125, or at least about 130 residues, or any number of residues between these values, e.g., about 7 residues, about 52 residues, about 93 residues, etc., from the N-terminus or C-terminus of the intracellular domain.

[0047] (2) A truncation of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125 or about 130 residues from the N-terminus or C-terminus of the intracellular domain, or any number of residues between these values, e.g., about 7 residues, about 52 residues, about 93 residues, etc.

[0048] (3) A deletion of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125 or about 130 contiguous residues within the intracellular domain, or any number of contiguous residues between any of these values, e.g., about 7 residues, about 52 residues, about 93 residues, etc.

[0049] (4) Deletion of the entire intracellular domain.

[0050] (5) C-terminal truncation of the intracellular domain such that the resulting intracellular domain consists of about 1 to about 30 residues of the N-terminal portion of the intracellular domain. In some embodiments, the obtained intracellular domain consists of about 5 to about 30 residues, about 10 to about 30 residues, about 15 to about 30 residues, about 20 to about 30 residues, about 25 to about 30 residues, about 5 to about 25 residues, about 10 to about 25 residues, about 15 to about 25 residues, about 20 to about 25 residues, about 5 to about 20 residues, about 10 to about 20 residues, about 15 to about 20 residues, about 5 to about 15 residues, about 10 to about 15 residues, or about 5 to about 10 residues of the N-terminal portion of the intracellular domain.

[0051] (6) A deletion of about 1 to about 10, about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 2 to about 8, about 2 to about 6, about 2 to about 5, about 2 to about 4, about 4 to about 6, about 4 to about 5, about 1 to about 5, about 2 to about 3, or about 1 to about 2 residues from the transmembrane domain.

[0052] In some embodiments, any one of the mutations (1) to (5) is further combined with the mutation (6).

[0053] The VSIG8 sequence and the dominant negative VSIG8 (DNVSIG8) sequence are set forth in Table 1 above.

[0054] In some embodiments, any polynucleotide sequence encoding any one of the dnVSIG3 receptors is contemplated. In some embodiments, the polynucleotide sequence is codon-optimized. A codon-optimized polynucleotide sequence encodes the same amino acid sequence as a non-codon-optimized polynucleotide sequence. III. Effects of dnVSIG3 and / or dnVSIG8

[0055] In some embodiments, cells expressing dnVSIG3 and / or dnVSIG8 exhibit a decreased amount of one or more cytokines produced by the cells when the cells are in the presence of a cognate ligand that binds to the extracellular domain of dnVSIG3 and / or dnVSIG8, compared to the wild-type form of the corresponding VSIG3 or VSIG8.

[0056] In some embodiments, the one or more cytokines are interleukins. In some embodiments, the interleukins can be selected from IL-1α, IL-1β, IL-1ra (antagonist), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, EL-17C, IL-17D, IL-17E, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A / B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35.

[0057] In some embodiments, the cytokine may be selected from the TNF family, for example one or more cytokines selected from the following list: TNF, in particular TNFα, LTα, LTβ, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L (CD154), FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK ligand. Further examples of preferred cytokines may be selected from the following list: FLT3 ligand, G-CSF, GM-CSF, IFNα / β / ω, IFNγ, LIF, M-CSF, MIF, OSM, stem cell factor, TGFβ1, TGFβ2, TGFβ3, TSLP ligand.

[0058] In some embodiments, cells expressing dnVSIG3 and / or dnVSIG8 exhibit a decreased amount of one or more chemokines produced by the cells when the cells are in the presence of a cognate ligand that binds to the extracellular domain of dnVSIG3 and / or dnVSIG8, compared to the wild-type form of the corresponding VSIG3 or VSIG8.

[0059] In some aspects, the one or more chemokines may be selected from CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, XCL1, and XCL2.

[0060] In some aspects, the reduction in the amount of one or more cytokines and / or chemokines produced by cells expressing dnVSIG3 and / or dnVSIG8 when the cells are in the presence of a cognate ligand that binds to the extracellular domain of dnVSIG3 and / or dnVSIG8 is at least about a reduction.

[0061] In some embodiments, the reduction in the amount of any one or more cytokines and / or any one or more chemokines compared to unmodified VSIG3 or VSIG8 is a reduction of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or at least about 95%.

[0062] In some embodiments, the reduction in the amount of any one or more cytokines and / or any one or more chemokines compared to unmodified VSIG3 or VSIG8 is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% reduction. IV. Vectors and Cells

[0063] The nucleic acids of the disclosure can be present in an expression vector and / or a cloning vector. Expression vectors may include selection markers, origins of replication and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. A large number of suitable vectors and promoters are known to those of skill in the art, and many are commercially available for generating recombinant constructs of interest. The following vectors are provided by way of example and should not be construed as limiting in any way: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540 and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0064] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker functional in the expression host may also be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus, poliovirus, adenovirus-based viral vectors) (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35:2543-2549; Borras et al., Gene Ther. (1999) 6:515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92:7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5:1088-1097; WO 94 / 12649; WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984; and WO 95 / 00655); Adeno-associated viruses (e.g., Ali et al., Hum. Gene Ther. (1998) 9:81-86; Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94:6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38:2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690; Rolling et al., Hum. Gene Ther. (1999) 10:641-648; Ali et al., Hum. Mol. Genet. (1996) 5:591-594; Srivastava, WO 93 / 09239; Samulski et al. al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90:10613-10617. SV40, herpes simplex virus, human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94:10319-23; Takahashi et al., J. Virol. (1999) 73:7812-7816), Retroviral vectors (e.g., vectors derived from retroviruses such as murine leukemia virus, spleen necrosis virus, and Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0065] Additional expression vectors suitable for use include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, recombinant hybrid virus vectors, transposon-mediated vectors, etc. Viral vector technology is well known in the art and described, for example, in Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0066] In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584, WO 01 / 29058, WO 01 / 29058, and U.S. Pat. No. 6,326,193).

[0067] In some embodiments, an expression vector (e.g., lentiviral vector) may be used to introduce a dominant negative receptor into immune cells or their precursors (e.g., T cells). Thus, an expression vector (e.g., lentiviral vector) of the invention may include a nucleic acid encoding a dominant negative receptor. In some embodiments, an expression vector (e.g., lentiviral vector) will include additional elements that aid in the functional expression of the dominant negative receptor encoded therein. In some embodiments, an expression vector that includes a nucleic acid encoding a dominant negative receptor further includes a mammalian promoter. In one embodiment, the vector further includes an elongation factor-1 alpha promoter (EF-1 alpha promoter). Use of the EF-1 alpha promoter may increase the efficiency of expression of a downstream transgene (e.g., a nucleic acid sequence encoding a dominant negative receptor). Physiological promoters (e.g., EF-1 alpha promoter) are less likely to induce integration-mediated genotoxicity and may negate the ability of a retroviral vector to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those of skill in the art and can be incorporated into the vectors of the invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises a non-requisite cis acting sequence that may improve titer and gene expression. One non-limiting example of a non-essential cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which are important for efficient reverse transcription and nuclear import. Other non-essential cis acting sequences are known to those of skill in the art and can be incorporated into the vectors (e.g., lentiviral vectors) of the present invention. In some embodiments, the vector further comprises a posttranscriptional regulatory element.Post-transcriptional regulatory elements may improve RNA translation, improve transgene expression, and stabilize RNA transcripts. One example of a post-transcriptional regulatory element is the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE). Thus, in some embodiments, the vector of the present invention further comprises a WPRE sequence. A variety of post-transcriptional regulatory elements are known to those skilled in the art and can be incorporated into the vector of the present invention (e.g., lentiviral vectors). The vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long terminal repeats (LTRs). The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the end of retroviral DNA that includes the U3, R, and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. In one embodiment, the vector of the present invention (e.g., lentiviral vectors) comprises a 3'U3 deleted LTR. Thus, the vector of the present invention (e.g., lentiviral vector) may contain any combination of elements described herein to increase the efficiency of functional expression of the transgene. For example, the vector of the present invention (e.g., lentiviral vector) may contain a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, a 3'U3 deleted LTR' in addition to a nucleic acid encoding a dominant negative receptor.

[0068] The vector of the present invention may be a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3'LTR enhancer promoter region (U3 region) is modified (e.g., by deletion or substitution). The self-inactivating vector may prevent viral transcription beyond the first round of viral replication. As a result, the self-inactivating vector can only infect and integrate into the host genome (e.g., mammalian genome) once and cannot pass further. Therefore, the self-inactivating vector may greatly reduce the risk of creating a replication-competent virus.

[0069] In some embodiments, the nucleic acid of the present invention may be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled in the art, and any known method can be used to synthesize RNA comprising a sequence encoding a dominant negative receptor of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, for example, Zhao et al. Cancer Res. (2010) 15:9053. Introduction of RNA comprising a nucleotide sequence encoding a dominant negative receptor of the present disclosure into a host cell can be performed in vitro, ex vivo, or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a dominant negative receptor of the present disclosure.

[0070] To assess the expression of a polypeptide or a portion thereof, the expression vector introduced into the cells may contain a selectable marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selectable markers include, but are not limited to, antibiotic resistance genes.

[0071] Reporter genes are used to identify potentially transfected cells and to evaluate the function of regulatory sequences. In general, reporter genes are genes that encode a polypeptide that is not present or expressed in the recipient organism or tissue and whose expression is indicated by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is evaluated at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include, but are not limited to, genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82).

[0072] In some embodiments, the cell comprises dnVSIG3 and / or dnVSIG8. In some embodiments, the cell comprises a polynucleotide encoding dnVSIG3 and / or dnVSIG8. In some embodiments, the cell comprises a vector further comprising a polynucleotide encoding dnVSIG3 and / or dnVSIG8. In some embodiments, the cell expresses dnVSIG3 and / or dnVSIG8.

[0073] In some aspects, the cells of the disclosure include higher eukaryotic cells, such as mammalian cells or insect cells. In some aspects, the cells of the disclosure include lower eukaryotic cells, such as yeast cells. In some aspects, the cells of the disclosure include prokaryotic cells. In some aspects, the cells of the disclosure include bacterial cells, fungal cells, yeast cells, plant cells, animal cells, and human cells.

[0074] In some embodiments, the human cell is an immune cell. In some embodiments, the immune cell is a T cell, e.g., a CD8 positive T cell (e.g., a CD8 positive naive T cell, a central memory T cell, or an effector memory T cell), a CD4 positive T cell, a natural killer T cell (e.g., an NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), or a dendritic cell. In some embodiments, the cell is a monocyte or a granulocyte, e.g., a bone marrow cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil. In some embodiments, the cell is an induced pluripotent stem (iPS) cell, a cell derived from an iPS cell, e.g., an iPS cell that has been generated from a subject and engineered to alter (e.g., induce a mutation) or manipulate the expression of one or more target genes and differentiated into, e.g., a T cell, e.g., a CD8 positive T cell (e.g., a CD8 positive naive T cell, a central memory T cell, or an effector memory T cell), a CD4 positive T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0075] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the total T cell population, CD4 positive cells, CD8 positive cells, and subpopulations thereof defined by function, activation state, maturity, differentiation, expansion, recirculation, localization and / or persistence potential, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation. Among the subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MATT) cells, spontaneous and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, δ / γ T cells. In some embodiments, any number of T cell lines available in the art can be used.

[0076] In some aspects, the methods of the disclosure include isolating immune cells from a subject, preparing, treating, culturing, and / or recombining immune cells. In some aspects, preparing the recombinant cells includes one or more culturing and / or preparation steps. Cells for recombination as described may be isolated from a biological sample, such as a sample obtained or derived from a subject. In some aspects, the subject from which the cells are isolated is a subject with a disease or condition or a subject in need of cell therapy, or a subject to whom cell therapy is to be administered. The subject in some aspects is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, in which cells are isolated, treated, and / or recombined. Thus, the cells in some aspects are primary cells, such as primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject, as well as samples obtained from one or more processing steps, such as separation, centrifugation, genetic modification (e.g., transduction with a viral vector), washing, and / or incubation. The biological sample may be a sample obtained directly from a biological source or a sample that has been processed. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0077] In some embodiments, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Examples of samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsils, or other organs and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources for cell therapy, e.g., adoptive cell therapy. V. Chimeric Antigen Receptors (CARs) and Modified T Cell Receptors

[0078] In some aspects, the cells of the application are modified T cells expressing one or more chimeric antigen receptors (CARs). In some aspects, the cells of the application are modified T cells expressing one or more modified T cell receptors (TCRs).

[0079] In some embodiments, the CAR or TCR comprises a binding domain that binds to one or more tumor antigens. In some embodiments, the tumor antigens include 5T4, 707-AP, 9D7, AFP, AlbZIP, HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4 / m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, beta-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA15-3 / CA27-29, CA19-9, CA72-4, CA125, calreticulin, CAMEL, CAS P-8 / m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, coactosin-like protein, collagen XXIII, COX-2, CT-9 / BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / m, EGFR, ELF2 / m, EMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FAP, FGF-5, FN, FRα, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GA GE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC2, GPC3, GPNMB / m, HAGE, HAST-2, hepsin, Her2 / neu, HERV-K-MEL, HLA-A*0201-R171, HLA-A11 / m, HL A-A2 / m, HNE, homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Rα2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205 / m, KK-LC-1, K-Ras / m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, Mammaglobin A, MART-1 / Melan-A, MART-2, MART-2 / m, Matrix protein 2 2, MC1R, M-CSF, ME1 / m, mesothelin, MG50 / PXDN, MMP11, MN / CAIX antigen, MRP-3, MUC-1, TnMuc-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class I / m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-B, NY-ESO-1, OA1, OFA-iLRP, OGT, OGT / m, OS-9, OS-9 / m, osteocalcin, osteopontin, p15, p190minor, bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-kinase, Pin-1, Pml / PARα, POTE, PRAME, PRDX5 / m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / CD168, RU1, RU2, S-100, SAGE, SART-1, SAR T-2, SART-3, SCC, SIRT2 / m, Sp17, SSX-1, SSX-2 / HOM-MEL-40, SSX-4, STAMP-1, STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFβ, TGFβRll, TGM-4, TPI / m, TRAG-3, TRG, TRP-1, TRP-2 / 6b, TRP / INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2 / FLK-1 and WT1. In some embodiments, the CAR comprises one or more binding domains that bind two or at least two of the above antigens. See at least U.S. Patent Publication No. 2019 / 0275083A1.

[0080] In some embodiments, the tumor antigen is preferably selected from the group consisting of p53, CA125, EGFR, Her2 / neu, hTERT, PAP, MAGE-A1, MAGE-A3, mesothelin, MUC-1, GP100, MART-1, tyrosinase, PSA, PSCA, PSMA, STEAP-1, VEGF, VEGFR1, VEGFR2, Ras, CEA, or WT1, more preferably PAP, MAGE-A3, WT1, and MUC-1.

[0081] In some embodiments, the CAR comprises a costimulatory domain. In some embodiments, the costimulatory domain is selected from CD2, 4-1BB, ICOS, and CD27. In some embodiments, the CAR comprises two costimulatory domains selected from CD2, 4-1BB, ICOS, and CD27. In some embodiments, the CAR comprises one or more signaling domains. In some embodiments, the CAR comprises a CD3 zeta signaling domain.

[0082] In some embodiments, the CAR comprises one or more switch receptors. In some embodiments, the CAR comprises a second or third dominant negative receptor that is not dnVSIG3 or dnVSIG8. In some embodiments, the receptor is selected from the group consisting of PD1 / CD28, PDL1 / CD28, CTLA4 / CD28, BTLA / CD28, BTLA / ICOS, TIM3 / CD28, TIGIT / CD226, dnTGFβ, TGFβ / IL-12R, TGFβ / CD28, TGFβ / OX40, IFNγ / CD28, IFNγ / OX40, and IFNγ / IL-12R. VI. Generation of Modified Immune Cells

[0083] The present disclosure provides methods for producing or generating modified immune cells or precursors thereof (e.g., T cells) for tumor immunotherapy, e.g., adoptive immunotherapy. The cells are generally engineered by introducing one or more nucleic acids encoding one or more dominant negative receptors. In some embodiments, one or more nucleic acids encoding one or more dominant negative receptors are introduced into T cells that have already been modified to express one or more CARs or one or more TCRs.

[0084] In some embodiments, one or more nucleic acids encoding one or more dominant negative receptors of interest are introduced into cells by expression vectors. Expression vectors comprising nucleic acid sequences encoding the dominant negative receptors of interest of the present invention are provided herein. Suitable expression vectors include lentiviral vectors, gamma retroviral vectors, foamy viral vectors, adeno-associated viral (AAV) vectors, adenoviral vectors, recombinant hybrid viruses, naked DNA (including but not limited to transposon mediated vectors, e.g., Sleeping Beauty, Piggybak, and integrase, e.g., Phi31). Other suitable expression vectors are described herein.

[0085] An expression vector comprising a nucleic acid of the present disclosure can be introduced into a host cell by any means known to one of skill in the art. The expression vector may optionally include a viral sequence for transfection. Alternatively, the expression vector may be introduced by fusion, electroporation, biolistics, transfection, lipofection, etc. The host cell may be grown and expanded in culture prior to the introduction of the expression vector, followed by appropriate treatment for the introduction and integration of the vector. The host cell can then be grown and screened by a marker present in the vector. Various markers that may be used are known in the art and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. In some embodiments, the host cell is an immune cell or a precursor thereof, such as a T cell, a NK cell, or a NKT cell.

[0086] The present disclosure also provides genetically modified cells comprising one or more stably expressed dominant negative receptors of the present disclosure. In some embodiments, the genetically modified cells are genetically modified T lymphocytes (T cells), naive T cells (TN), memory T cells (e.g., central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells) and macrophages capable of generating therapeutically relevant progeny. In one embodiment, the genetically modified cells are autologous cells.

[0087] Modified cells (e.g., comprising the subject dominant negative receptors) can be produced by stably transfecting host cells with an expression vector comprising the nucleic acid of the present disclosure. Additional methods for generating modified cells of the present disclosure include, but are not limited to, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, phototransformation, gene electrotransfer and / or hydrodynamic delivery) and / or particle-based methods (e.g., impalefection, use of gene guns and / or magnetofection). Transfected cells expressing the subject dominant negative receptors of the present disclosure can be propagated ex vivo.

[0088] Physical methods for introducing an expression vector into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Chemical methods for introducing an expression vector into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0089] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") is available from Sigma, St. Louis, Missouri; dicetyl phosphate ("DCP") is available from K&K Laboratories, Inc., Plainview, New York; cholesterol ("Choi") is available from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc., Birmingham, Alabama. Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform can be used as the only solvent, as it evaporates more easily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar membrane lipid vesicles formed by the formation of closed lipid bilayers or aggregates. Liposomes are characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). Compositions that have structures in solution that differ from normal vesicular structures are also included. For example, lipids may adopt micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0090] Regardless of the method used to introduce exogenous nucleic acid into a host cell or expose the cell to an inhibitor of the invention, various assays can be performed to confirm the presence of the nucleic acid in the host cell. Such assays include molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, biochemical methods such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot), or by the assays described herein, to identify agents within the scope of the invention.

[0091] In some aspects, the nucleic acid introduced into the host cell is RNA. In another embodiment, the RNA is mRNA, including in vitro transcribed RNA or synthetic RNA. RNA can be produced by in vitro transcription using a template generated by polymerase chain reaction (PCR). DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable DNA source.

[0092] PCR can be used to generate templates for in vitro transcription of mRNA that is then introduced into cells. Methods for performing PCR are well known in the art. Primers used in PCR are designed to have a region that is substantially complementary to a region of DNA used as a template for PCR. "Substantially complementary" as used herein refers to a sequence of nucleotides to which most or all of the bases in the primer sequence are complementary. A substantially complementary sequence is capable of annealing or hybridizing with the intended DNA target under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify a portion of a gene that is normally transcribed in cells (open reading frame), including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of a gene that encodes a particular domain of interest. In one embodiment, primers are designed to amplify the coding region of a human cDNA, including all or part of the 5' and 3' UTRs. Primers useful for PCR are generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides that are substantially complementary to nucleotides on a DNA template that are upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to a position 5' of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a region of nucleotides that are substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to a position 3' of the DNA sequence to be amplified relative to the coding strand.

[0093] Chemical structures capable of promoting RNA stability and / or translation efficiency may also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is 0-3000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be altered by different methods, including but not limited to designing primers for PCR that anneal to different regions of the UTR. Using this approach, one skilled in the art can alter the length of the 5' and 3' UTRs required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0094] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating UTR sequences into the forward and reverse primers or by other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest serves to modify the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce the stability of mRNA. Thus, the 3' UTR can be selected or designed to enhance the stability of the transcribed RNA based on the properties of UTRs that are well known in the art.

[0095] In one embodiment, the 5'UTR can include the Kozak sequence of the endogenous gene. Alternatively, if a non-endogenous 5'UTR has been added to the gene of interest by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5'UTR sequence. The Kozak sequence increases the translation efficiency of some RNA transcripts, but it appears that it is not necessary for all RNAs to allow efficient translation. It is known in the art that the Kozak sequence is necessary for many mRNAs. In other embodiments, the 5'UTR can be derived from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs can be used in the 3' or 5'UTR to inhibit exonuclease degradation of the mRNA.

[0096] To allow synthesis of RNA from a DNA template without the need for gene cloning, a transcription promoter should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter will be incorporated into the PCR product upstream of the open reading frame to be transcribed. In one embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. The consensus nucleotide sequences of T7, T3 and SP6 promoters are known in the art.

[0097] In one embodiment, the mRNA has both a 5'-cap and a 3' poly(A) tail, which determine ribosome binding, initiation of translation, and mRNA stability in cells. On circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatameric products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3'UTR produces normal-sized mRNA that is posttranscriptionally polyadenylated but ineffective for eukaryotic transfection.

[0098] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of a transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0099] The polyA / T segment of the transcribed DNA template can be generated during PCR by using a reverse primer containing a polyT tail, such as a 100T tail (size can be 50-5000T), or after PCR by any other method, including but not limited to DNA ligation or in vitro recombination. The poly(A) tail provides stability to the RNA and reduces its degradation. In general, the length of the poly(A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100-5000 adenosines.

[0100] The poly(A) tail of the RNA can be further extended after in vitro transcription using a poly(A) polymerase such as E. coli poly(A) polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides increases the translation efficiency of the RNA by approximately 2-fold. Furthermore, attachment of various chemical groups to the 3' end improves the stability of the mRNA. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. The ATP analogs can further increase the stability of the RNA.

[0101] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein comprises a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0102] In some embodiments, RNA, such as in vitro transcribed RNA, is electroporated into cells. Any solute suitable for cell electroporation can be included, including factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, detergents, etc.

[0103] In some embodiments, the nucleic acid encoding the subject dominant negative receptor of the present disclosure is RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known in the art, and any known method can be used to synthesize RNA comprising a sequence encoding the subject dominant negative receptor. Methods for introducing RNA into a host cell are known in the art. See, for example, Z Zhao et al. Cancer Res. (2010) 15:9053. Introduction of RNA comprising a nucleotide sequence encoding a dominant negative receptor into a host cell can be performed in vitro, ex vivo, or in vivo. For example, a host cell (e.g., NK cell, cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding one or more of the subject dominant negative receptors.

[0104] The disclosed methods can be applied to modulating T cell activity in basic research and therapy in the areas of cancer, stem cells, acute and chronic infectious diseases, and autoimmune diseases, including assessing the ability of genetically modified T cells to kill targeted cancer cells.

[0105] The methods also provide the ability to control expression levels over a broad range, for example by altering the promoter or the amount of input RNA, allowing expression levels to be individually regulated. Furthermore, PCR-based mRNA production techniques greatly facilitate the design of mRNAs with various structures and combinations of domains.

[0106] One advantage of the RNA transfection method of the present invention is that RNA transfection is essentially transient and does not require vectors. RNA transgenes can be delivered to lymphocytes and expressed there after a short period of in vitro cell activation, as a minimal expression cassette, without the need for additional viral sequences. Under these conditions, the transgene is unlikely to be integrated into the host cell genome. Cell cloning is not required, since RNA transfection is efficient and can uniformly modify the entire lymphocyte population.

[0107] Genetic modification of T cells with in vitro transcribed RNA (IVT-RNA) utilizes two different strategies, both of which are being tested successively in various animal models. Cells are transfected with in vitro transcribed RNA by lipofection or electroporation. To sustain the expression of the introduced IVT-RNA for a long time, it is desirable to stabilize the IVT-RNA using various modifications.

[0108] Several IVT vectors are known in the literature that are genetically modified to be utilized in a standardized manner as templates for in vitro transcription and to generate stabilized RNA transcripts. Currently, protocols used in the art are based on plasmid vectors with the following structure: a 5' RNA polymerase promoter that allows RNA transcription, followed by the gene of interest flanked on the 3' and / or 5' by untranslated regions (UTRs) and a 3' polyadenylation cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenylation cassette by a type II restriction enzyme (the recognition sequence corresponds to the cleavage site). The polyadenylation cassette thus corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization, extending or masking the poly(A) sequence at the 3' end. It is not clear whether this non-physiological overhang affects the amount of protein produced in cells from such constructs.

[0109] In another embodiment, the RNA construct is delivered into the cell by electroporation. See, for example, the formulations and methodologies for electroporation of nucleic acid constructs into mammalian cells taught in US Patent Publication Nos. 2004 / 0014645, 2005 / 0052630A1, 2005 / 0070841A1, 2004 / 0059285A1, and 2004 / 0092907A1. The various parameters, including the electric field strength required for electroporation of any known cell type, are generally known in the relevant research literature, as well as in numerous patents and applications in this field. See, for example, US Patent Nos. 6,678,556, 7,171,264, and 7,173,116. Devices for therapeutic applications of electroporation are commercially available, for example, MedPulser® DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.) and are described in U.S. Patent Nos. 6,567,694, 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482. Electroporation can also be used to transfect cells in vitro, for example, as described in U.S. Patent Publication No. 2007 / 0128708A1. Electroporation can also be utilized to deliver nucleic acids to cells in vitro. Thus, electroporation-mediated administration of nucleic acids, including expression constructs, to cells using any of the many available devices and electroporation systems known to those skilled in the art provides an exciting new means to deliver RNA of interest to target cells.

[0110] In some embodiments, immune cells (e.g., T cells) can be incubated or cultured before, during, and / or after introduction of a nucleic acid molecule encoding a subject dominant negative receptor. In some embodiments, cells (e.g., T cells) can be incubated or cultured before, during, or after introduction of a nucleic acid molecule encoding a subject dominant negative receptor, e.g., before, during, or after transduction of the cells with a viral vector (e.g., a lentiviral vector) encoding a dominant negative receptor. In some embodiments, the method includes activating or stimulating the cells with a stimulatory or activating agent (e.g., an anti-CD3 / anti-CD28 antibody) prior to introducing a nucleic acid molecule encoding a subject dominant negative receptor. VII. Pharmaceutical Compositions

[0111] Also provided are populations of immune cells of the present disclosure that contain one or more dominant negative receptors, compositions enriched for such cells and / or such cells, e.g., compositions in which recombinant receptor expressing cells represent at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more of the total cells in the composition, or specific types of cells, such as T cells or CD8+ or CD4+ cells. Included among the compositions are pharmaceutical compositions and formulations for administration, such as adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to a subject, e.g., a patient.

[0112] Also provided are compositions comprising cells for administration, including pharmaceutical compositions and formulations, such as compositions in unit dose form that contain the number of cells for administration in a given dose or fraction thereof. Pharmaceutical compositions and formulations generally include any one or more pharma- ceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

[0113] The term "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. A "pharmaceutical acceptable carrier" refers to ingredients in a pharmaceutical formulation other than the active ingredient that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some embodiments, the choice of carrier is determined in part by the particular cells and / or the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition can include a preservative. Suitable preservatives include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight compounds such as glycerol, glycerol, tert-butyl esters ... Proteins such as low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, counterions that form salts such as sodium, metal complexes (e.g., Zn-protein complexes) and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0114] In some embodiments, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0115] The formulation may include an aqueous solution. The formulation or composition may also contain multiple active ingredients useful for the particular indication, disease or condition being treated with the cells, preferably active ingredients with complementary activities on the cells, where the respective activities do not adversely affect each other. Suitably, such active ingredients are present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharma- ceutical active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine and / or vincristine. The pharmaceutical composition in some embodiments comprises the cells in an amount effective to treat or prevent the disease or condition, e.g., a therapeutically or prophylactically effective amount. The therapeutic or prophylactic effect in some embodiments is monitored by periodic evaluation of the treated subject. The desired dose may be delivered by a single bolus of cells, multiple boluses of cells, or continuous infusion of cells.

[0116] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. The compositions in some embodiments are provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid formulations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within an appropriate viscosity range to provide longer contact times with specific tissues. The liquid or viscous composition may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0117] Sterile injectable solutions can be prepared by incorporating the cells in a solvent such as a suitable carrier, diluent or excipient, e.g., sterile water, saline, glucose, dextrose, etc. The composition can contain auxiliary substances such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening agents, preservatives, flavorings and / or coloring agents, depending on the desired route of administration and formulation. In some aspects, standard texts can be consulted for preparing appropriate formulations.

[0118] Various additives can be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0119] The formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane. In some embodiments, the formulations used for in vivo administration are generally free of microorganisms. In some embodiments, the formulations used for in vivo administration are generally free of viruses. In some embodiments, the formulations used for in vivo administration are generally free of pathogenic viruses. VIII. Treatment method

[0120] The modified cells (e.g., T cells) described herein may be included in a composition for immunotherapy. The composition may include a pharmaceutical composition and may further include a pharma- ceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified T cells may be administered.

[0121] In one aspect, the invention includes a method for adoptive cell transfer therapy comprising administering the modified T cells of the invention to a subject in need thereof. In another aspect, the invention includes a method of treating a disease or condition in a subject comprising administering a population of modified T cells to a subject in need thereof.

[0122] Also included are methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject a modified cell (e.g., a modified T cell) of the invention. In one embodiment, a method of treating a disease or condition in a subject in need thereof comprises administering to the subject a modified cell (e.g., a modified T cell) comprising a subject CAR and / or a dominant negative receptor. In one embodiment, a method of treating a disease or condition in a subject in need thereof comprises administering to the subject a modified cell (e.g., a modified T cell) comprising a subject CAR (e.g., a CAR having affinity for PSMA on a target cell) and a dominant negative receptor and / or a switch receptor. In one embodiment, a method of treating a disease or condition in a subject in need thereof comprises administering to the subject a modified cell (e.g., a modified T cell) comprising a subject CAR (e.g., a CAR having affinity for PSMA on a target cell), a dominant negative receptor and / or a switch receptor, wherein the modified cell is capable of expressing and secreting a bispecific antibody.

[0123] Methods for administering immune cells for adoptive cell therapy are known and can be used in combination with the provided methods and compositions. For example, methods for adoptive T cell therapy are described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al., U.S. Patent No. 4,690,915 to Rosenberg, and Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85. See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933, Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9, Davila et al. (2013) PLoS ONE 8(4):e61338. In certain embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by autologous transplantation, where cells are isolated and / or otherwise prepared from the subject to be treated, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., a patient, in need of treatment, and the isolated and processed cells are administered to the same subject.

[0124] In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by allogeneic transplantation, in which cells are isolated and / or otherwise prepared from a subject other than the subject who will or will ultimately receive cell therapy, e.g., a first subject. In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0125] The modified immune cells of the present invention can be administered to animals, preferably mammals, and even more preferably humans, to treat cancer. Additionally, the cells of the present invention can be used to treat any condition associated with cancer, particularly cellular immune responses against tumor cells where it is desired to treat or alleviate the disease. Types of cancer that can be treated with the modified cells or pharmaceutical compositions of the present invention include carcinomas, blastomas and sarcomas and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies, such as sarcomas, carcinomas, and melanomas. Other exemplary 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, thyroid cancer, and the like. The cancer can be a non-solid tumor (such as a blood tumor) or a solid tumor. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0126] The cells of the invention can be administered at a dose and by a route and at times determined in appropriate preclinical and clinical experiments and trials. The cell composition can be administered multiple times at doses within these ranges. Administration of the cells of the invention can be combined with other methods useful for treating the desired disease or condition as determined by one of skill in the art.

[0127] Administration of the cells of the invention can be performed by any convenient method known to one of skill in the art. The cells of the invention can be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions herein can be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intravenously (iv) or intraperitoneally. In other examples, the cells of the invention are injected directly into a site of inflammation in a subject, a site of local disease in a subject, a lymph node, an organ, a tumor, etc.

[0128] In certain embodiments, the cells or individual subpopulations of cells are comprised of about 1 million to about 100 billion cells, e.g., in the range of about 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, In some cases, the subject may be administered about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), or in some cases, about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value within these ranges.

[0129] In some embodiments, the dose of total cells and / or the dose of individual subpopulations of cells is greater than or equal to 1×10 5 Cells / kg or approximately 1 x 10 5 cells / kg ~ approx. 1×10 11 cells / kg, 10 4 , and 10 11 Or about 10 11 Cells / kilogram (kg) body weight, e.g., about 10 5 ~about 10 6 Cells / kg body weight, e.g., 1 x 10 5 cells / kg, approximately 1×10 5 cells / kg, approximately 1.5×10 5 cells / kg, approximately 2×10 5 Cells / kg or approximately 1 x 10 6 For example, in some embodiments, the cells are in the range of 10 4 Or about 10 4 ~10 9 Or about 10 9 T cells / kilogram (kg) body weight, e.g., about 10 5~about 10 6 T cells / kg body weight, e.g., 1×10 5 T cells / kg body weight or approximately 1 × 10 5 T cells / kg body weight, approximately 1.5×10 5 T cells / kg, approximately 2×10 5 T cells / kg, or approximately 1 × 10 6 In another exemplary embodiment, a suitable dose range for modified cells for use in the methods of the present disclosure is, but is not limited to, about 1×10 5 cells / kg ~ approx. 1×10 6 cells / kg, approximately 1×10 6 cells / kg ~ approx. 1×10 7 cells / kg, approximately 1×10 7 cells / kg ~ approx. 1×10 8 cells / kg, approximately 1×10 8 cells / kg ~ approx. 1×10 9 cells / kg, approximately 1×10 9 cells / kg ~ approx. 1×10 10 cells / kg, approximately 1×10 10 cells / kg ~ approx. 1×10 11 In an exemplary embodiment, a suitable dose for use in the methods of the present disclosure is about 1×10 cells / kg. 8 In an exemplary embodiment, a suitable dose for use in the methods of the present disclosure is about 1×10 7 In other embodiments, a suitable dose is about 1×10 7 Total cells ~ approx. 5 x 10 7 In some embodiments, a suitable dose is about 1×10 8 Total cells ~ approx. 5 x 10 8 In some embodiments, a suitable dose is about 1.4×10 7 Total cells ~ approx. 1.1 x 10 9 In an exemplary embodiment, a suitable dose for use in the methods of the present disclosure is about 7×10 9 In an exemplary embodiment, a suitable dose is about 1×10 7 Total cells ~ approx. 3 x 10 7 Total cells.

[0130] In some embodiments, the dose of modified cells is administered to a subject in need thereof in a single dose or multiple doses. In some embodiments, the dose of modified cells is administered in multiple doses, for example, once a week or about every 7 days, once every two weeks or about every 14 days, once every three weeks or about every 21 days, once every four weeks or about every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion. IX.Definitions

[0131] Although the following terms are believed to be well understood to those of skill in the art, the following definitions are provided to facilitate description of the presently disclosed subject matter.

[0132] The term "a" or "an" may refer to one or more of that entity, i.e., to multiple referents. Thus, the terms "a" or "an," "one or more," and "at least one" are used interchangeably herein. Furthermore, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements is present, unless the context clearly requires that there is only one element.

[0133] Throughout this specification, references to "one embodiment," "an embodiment," "one aspect," or "an aspect" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0134] As used herein, the term "about" or "approximately" preceding a numerical value indicates a value within plus or minus a range of 10% of that value.

[0135] As will be appreciated by those skilled in the art, for all purposes, particularly in terms of providing a written description, all ranges disclosed herein encompass all possible subranges and combinations of subranges. Any range listed is fully described and can be readily recognized as being divisible into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, upper third, etc. Additionally, as will be appreciated by those skilled in the art, all terms such as "up to," "at least," "greater than," "less than," etc. refer to ranges that are inclusive of the recited numbers and that can subsequently be broken down into subranges as described above. Finally, as will be appreciated by those skilled in the art, ranges include individual members. Thus, for example, a group having 1-3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to a group having 1, 2, 3, 4, 5 cells, etc.

[0136] As used herein, a "control" is a substitute sample used in an experiment for comparison purposes. A control can be "positive" or "negative." As used herein, a "control sample" or "reference sample" refers to a sample or reference that serves as a control for comparison with an experimental sample. For example, an experimental sample contains compounds A, B, and C in a vial, and a control may be the same type of sample treated similarly to the experimental sample, but lacking one or more of compounds A, B, or C.

[0137] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount that results in prevention of one or more outcomes or an increase in one or more outcomes.

[0138] As used herein, the terms "individual," "patient," or "subject" may be an individual organism, a vertebrate, a mammal, or a human. In a preferred embodiment, the individual, patient, or subject is a human.

[0139] As used herein, the terms "drug" or "pharmaceutical active agent" or "bioactive agent" or "active agent" are used interchangeably and refer to any organic or inorganic compound or substance that has biological activity and is adapted or used for therapeutic purposes. Proteins, hormones, anti-cancer drugs, analgesics, anesthetics, small molecule compounds and mimetics, oligonucleotides, DNA, RNA and gene therapy are included in the broader definition of "drug". As used herein, reference to a drug, as well as reference to other chemical compounds herein, is meant to include pharmaceutically acceptable forms of the compound, including isomers such as diastereomers and enantiomers, salts, solvates, and polymorphs, specific crystalline forms, and, where applicable, racemic mixtures and pure isomers of the compounds described herein.

[0140] As used herein, the term "injectable" refers to the ability to inject a composition of the present disclosure through a needle.

[0141] As used herein, the phrase "dominant-negative receptor" refers to a variant of a particular receptor that contains a dominant-negative mutation resulting in a modified polypeptide that disrupts the activity of the wild-type receptor. The disruption of activity may be a complete or partial disruption of the receptor's ability to participate in signal transduction. Dominant-negative receptors further refer to molecules designed to reduce the effect of negative signaling molecules, such as the effect of negative signaling molecules on modified immune cells of the present disclosure. Modified immune cells that contain dominant-negative receptors may bind to negative signaling molecules in the microenvironment of the modified immune cells, reducing the effect of the negative signaling molecules on the modified immune cells.

[0142] As used herein, the term "nucleic acid" refers to any DNA or RNA molecule and is used synonymously with polynucleotide. When referring to a nucleic acid or nucleic acid sequence encoding a specific protein and / or peptide, the nucleic acid or nucleic acid sequence also preferably includes regulatory sequences that allow its expression, i.e., transcription and / or translation of the nucleic acid sequence encoding the specific protein or peptide, respectively, in a suitable host, such as a human.

[0143] As used herein, the term "peptide" refers to a polymer of amino acid monomers. Usually, the monomers are linked by peptide bonds. The term "peptide" does not limit the length of the polymer chain of amino acids. In some embodiments of the invention, a peptide may, for example, contain less than 50 monomer units. Longer peptides, also called polypeptides, usually have 50-600 monomer units, more specifically 50-300 monomer units.

[0144] As used herein, the term "protein" refers to one or more peptides and / or polypeptides folded into a three-dimensional form that facilitates a biological function.

[0145] As used herein, the phrases "protein variants" or "peptide variants" refer to proteins / peptides that may be modified compared to the wild type and have an amino acid sequence that differs from the original sequence in one or more mutations, such as one or more substitutions, insertions and / or deletions of amino acids. In some embodiments, these fragments and / or variants have the same biological function or specific activity, such as its specific antigenicity, compared to the full-length native protein. "Variants" of proteins or peptides as defined in the context of the present invention may contain conservative amino acid substitution(s) compared to their native, i.e. non-mutated, physiological sequences. These amino acid sequences and the nucleotide sequences encoding them fall in particular under the term variant as defined herein. Substitutions in which amino acids from the same class are exchanged for one another are called conservative substitutions. In particular, these are amino acids with aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, side chains with hydroxyl functions, for example, which can enter into hydrogen bridges. This means, for example, that an amino acid with a polar side chain is replaced by another amino acid that also has a polar side chain, or, for example, that an amino acid characterized by a hydrophobic side chain is replaced by another amino acid that also has a hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine). Insertions and substitutions are possible in particular at sequence positions that do not cause modifications in the three-dimensional structure or affect the binding regions. Modifications of the three-dimensional structure due to insertions or deletions can be easily determined, for example, using CD spectroscopy (circular dichroism spectroscopy).

[0146] As used herein, "encoding" refers to the inherent property, and biological properties resulting therefrom, of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, mRNA, etc., to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (i.e., rRNA, tRNA, mRNA) or a defined amino acid sequence. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually listed in the sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0147] As used herein, "identity" refers to the identity of subunit sequences between two polymer molecules, particularly between two amino acid molecules, e.g., between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, e.g., if each position in the two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or the degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions, e.g., if half of the positions in the two sequences (e.g., 5 positions in a polymer 10 amino acids in length) are identical, then the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) are matched or identical, then the two amino acid sequences are 90% identical.

[0148] As used herein, the phrase "operably linked" or "operatively 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 to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0149] As used herein, the term "vector" refers to a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the introduction of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0150] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that, when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to an immune response detected in the absence of the composition of the invention. Immune responses can be readily assessed by a number of art-recognized methods. One of skill in the art will appreciate that the amount of the composition administered herein will vary and can be readily determined based on a number of factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0151] As used herein, the term "autologous" is meant to refer to any material derived from the same individual that is later reintroduced into that individual. "Allogeneic" refers to any material derived from a different animal of the same species. "Xenogeneic" refers to any material derived from an animal of a different species.

[0152] The term "chimeric antigen receptor" or "CAR" as used herein refers to an artificial T cell receptor that is expressed on an immune cell and engineered to specifically bind to an antigen. CARs may be used as a therapy by adoptive cell transfer. T cells are taken from a patient and modified to express a receptor specific for an antigen or a particular form of an antigen. In some embodiments, the CAR has specificity for a selected target, for example, cells expressing prostate specific membrane antigen. CARs may also include an extracellular domain that includes an intracellular activation domain, a transmembrane domain, and a tumor-associated antigen binding region.

[0153] As used herein, the term "downregulation" refers to the reduction or elimination of gene expression of one or more genes.

[0154] The present technology is not limited with respect to the specific aspects described in this application, which are intended as single illustrations of individual aspects of the technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and devices within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be included within the scope of the present technology. It is to be understood that the present technology is not limited to specific methods, reagents, compound compositions, or biological systems, which may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0155] Example 1 The purpose of this example was to illustrate the preparation of dnVSIG3.

[0156] dnVSIG3 (SEQ ID NO:20) was generated by modifying the amino acid sequence of VSIG3 wild-type variant 6 (SEQ ID NO:6) and truncating the C-terminus of the protein by 166 amino acids. This dnVSIG3 is characterized by the almost complete loss of the intracellular domain, with only four amino acids remaining in the intracellular domain.

[0157] This example demonstrates the successful preparation of dnVSIG3.

[0158] The method illustratively described herein can be suitably carried out in the absence of any element or elements, limitations or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising", "including", "containing" and the like are to be interpreted expansively without limitation. Furthermore, the terms and expressions used herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude equivalents of the illustrated and described features or portions thereof. It is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, although the present disclosure is specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the disclosure embodied therein disclosed herein may be incorporated by those skilled in the art, and such modifications and variations are considered to be within the scope of the present disclosure.

[0159] The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings included in the generic disclosure also constitutes part of the method. This includes the general description of the method with a proviso or negative limitation removing any subject matter from the genus, regardless of whether the excised material is specifically described herein. The present technology is not limited in terms of the specific embodiments described in this application, which are intended as examples of individual aspects of the technology. As will be apparent to those skilled in the art, the present technology is susceptible of many modifications and variations without departing from its spirit and scope. In addition to those enumerated herein, methods and apparatuses that are functionally equivalent within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of the present technology. It is to be understood that the present technology is not limited to specific methods, reagents, compounds compositions, or biological systems. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0160] Those skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages set forth above, as well as those inherent therein. Such modifications and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the disclosure and are defined by the following claims, which present non-limiting embodiments of the disclosure.

[0161] Furthermore, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual members or subgroups of members of the Markush group.

[0162] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

[0163] However, reference to any references, articles, publications, patents, patent publications and patent applications cited herein is not, and should not be construed as, an acknowledgment or any form of suggestion that they constitute available prior art or form part of the common general art in any country throughout the world.

Claims

1. A modified polypeptide comprising an amino acid sequence that shares at least about 90% sequence identity with any one of SEQ ID NOs: 20 to 30.

2. (a) The amino acid sequence shares at least about 95% sequence identity with any one of SEQ ID NOs: 20 to 30, and / or (b) The amino acid sequence shares at least about 95% sequence identity with SEQ ID NO: 20, and / or (c) The amino acid sequence shares at least about 99% sequence identity with SEQ ID NO: 20, and / or (d) The amino acid sequence is SEQ ID NO: 20, The polypeptide according to claim 1.

3. A modified polypeptide comprising an amino acid sequence that shares at least about 90% sequence identity with any one of SEQ ID NOs: 61 to 70.

4. (a) The amino acid sequence shares at least about 95% sequence identity with any one of SEQ ID NOs: 61 to 70, and / or (b) The amino acid sequence shares at least about 95% sequence identity with SEQ ID NO: 61, and / or (c) The amino acid sequence shares at least about 99% sequence identity with SEQ ID NO: 61, and / or (d) The amino acid sequence is SEQ ID NO: 61, The polypeptide according to claim 3.

5. The polypeptide according to any one of claims 1 to 4, further comprising a signal peptide sequence.

6. (a) Encoding the polypeptide according to claim 1 or 3, and / or (b) Comprising a nucleic acid sequence that shares at least about 90% sequence identity with any one of SEQ ID NOs: 50 to 59, and / or (c) Comprising a nucleic acid sequence that shares at least about 90% sequence identity with any one of SEQ ID NOs: 72 to 81, A polynucleotide sequence.

7. (a) The nucleic acid sequence of 6(b) shares at least about 95% sequence identity with any one of SEQ ID NOs: 50 to 59, and / or (b) The nucleic acid sequence of 6(b) shares at least about 99% sequence identity with any one of SEQ ID NOs: 50 to 59, and / or (c) The nucleic acid sequence of 6(b) is SEQ ID NO: 50, and / or (d) The nucleic acid sequence of 6(c) shares at least about 95% sequence identity with any one of SEQ ID NOs: 72 to 81, and / or (e) The nucleic acid sequence of 6(c) shares at least about 99% sequence identity with any one of SEQ ID NOs: 72 to 81, and / or (f) The nucleic acid sequence of 6(c) is SEQ ID NO: 72, The polynucleotide sequence according to claim 6.

8. (a) The polypeptide according to claim 1 or 3, or (b) A composition comprising the polynucleotide according to claim 6.

9. (a) Comprising the polynucleotide sequence according to claim 6, optionally, the vector is a viral vector, and / or (b) Comprising the polynucleotide sequence according to claim 6, the polynucleotide sequence is operably linked to one or more polypeptides, optionally, the vector is a viral vector, and / or (c) Comprising the polynucleotide sequence according to claim 6, optionally, the polynucleotide sequence is operably linked to one or more polypeptides, the vector is a viral vector, and further the viral vector is selected from the group consisting of lentiviral vectors, gammaretroviral vectors, foamy viral vectors, adeno-associated viral vectors, adenoviral vectors, poxviral vectors, herpesviral vectors and recombinant hybrid viral vectors, and / or (d) A vector comprising the polynucleotide sequence according to claim 6, optionally, the polynucleotide sequence is operably linked to one or more polypeptides, and the vector is a lentiviral vector.

10. (a) The polypeptide according to claim 1 or 3, wherein the polypeptide is a dominant negative receptor as compared to the activity of the wild-type VSIG3 receptor, and / or (b) The polynucleotide according to claim 6, wherein the polynucleotide is a dominant negative receptor as compared to the activity of the wild-type VSIG8 receptor, and / or (c) The polypeptide according to claim 1 or 3 and the polynucleotide according to claim 6, and / or (d) The polynucleotide according to claim 6, and / or (e) A cell comprising the vector according to claim 9.

11. (a) The cell is selected from the group consisting of bacterial cells, fungal cells, yeast cells, animal cells and human cells, and / or (b) The cell is a human cell, and / or (c) The cell is a human cell that is an immune cell, and / or (d) The cell is a human immune cell that is a T cell, and / or (e) The cell is a human immune cell that is a T cell, and the T cell comprises a modified antigen receptor, and / or (f) The cell is a human immune cell that is a T cell, the T cell comprises a modified antigen receptor, the modified antigen receptor is a T cell receptor or a chimeric antigen receptor (CAR), and / or, (g) The cell is a human immune cell that is a T cell, the T cell comprises a modified antigen receptor, the modified antigen receptor is a chimeric antigen receptor (CAR), the CAR comprises a binder selected from the group consisting of prostate-specific membrane antigen (PSMA), Tn glycoform of mucin 1 (TnMUC1), mesothelin, glypican 2 (GPC2), fibroblast activation protein (FAP), folate receptor α (FRα), epidermal growth factor receptor (EGFR), interleukin-13 receptor subunit α2 (IL-13Rα2), and any combination thereof, and / or, (h) The cell is a human immune cell that is a T cell, the T cell comprises a modified antigen receptor, the modified antigen receptor is a chimeric antigen receptor (CAR), the CAR comprises a binder, and the binder comprises a combination of EGFR and IL-13Rα2, and / or, (i) The cell is a human immune cell that is a T cell, the T cell comprises a modified antigen receptor, the modified antigen receptor is a chimeric antigen receptor (CAR), the CAR comprises a co-stimulatory domain selected from the group consisting of CD2, 4-1BB, ICOS, and CD27, and / or, (j) The cell is a human immune cell that is a T cell, the T cell comprises a modified antigen receptor, the modified antigen receptor is a chimeric antigen receptor (CAR), the CAR comprises a switch receptor and / or a dominant negative receptor, and the receptor is selected from the group consisting of PD1 / CD28, PDL1 / CD28, CTLA4 / CD28, BTLA / CD28, BTLA / ICOS, TIM3 / CD28, TIGIT / CD226, dnTGFβ, TGFβ / IL-12R, TGFβ / CD28, TGFβ / OX40, IFNγ / CD28, IFNγ / OX40, and IFNγ / IL-12R, and / or, (k) The cell is a human immune cell that is a T cell, the T cell comprises a modified antigen receptor, the modified antigen receptor is a chimeric antigen receptor (CAR), the CAR comprises a CD3ζ signaling domain, the cell according to claim 10.

12. (a) The dominant negative receptor is incapable of signal transduction, and / or, (b) The dominant negative receptor is incapable of signal transduction, the extracellular domain of the dominant negative receptor is capable of binding to the corresponding ligand, and / or (c) The dominant negative receptor is incapable of signal transduction, the extracellular domain of the dominant negative receptor is capable of binding to the corresponding ligand which is VISTA, and / or (d) The expression of wild-type VSIG3 is downregulated, and / or (e) The expression of wild-type VSIG8 is downregulated. The cell according to claim 10.

13. A method of administering the cell according to claim 10, the method comprising the step of administering a composition comprising the cell to a subject, optionally (a) the cell is autologous to the subject, or (b) the cell is allogeneic to the subject.

14. A method of generating a modified cell, the method comprising introducing the vector according to claim 9 into the cell.

15. (a) The cell is selected from the group consisting of bacterial cells, fungal cells, yeast cells, animal cells and human cells, and / or (b) The cell is a human immune cell, and / or (c) The cell is a human immune cell, and the human immune cell is a T cell, and / or (d) The cell is a human T cell, and the T cell comprises a modified antigen receptor, and / or (e) The cell is a human T cell comprising a modified antigen receptor, and the modified antigen receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and / or (f) The cell is a human T cell comprising a modified antigen receptor, and the modified antigen receptor is a chimeric antigen receptor (CAR), and the CAR comprises a binder selected from the group consisting of prostate-specific membrane antigen (PSMA), Tn glycoform of mucin 1 (TnMUC1), mesothelin, glypican 2 (GPC2), fibroblast activation protein (FAP), folate receptor α (FRα), and a combination of epidermal growth factor receptor (EGFR) and interleukin-13 receptor subunit α2 (IL-13Rα2), and / or (g) The cell is a human T cell comprising a modified antigen receptor, and the modified antigen receptor is a chimeric antigen receptor (CAR), and the CAR comprises a binder, and the binder comprises a combination of EGFR and IL-13Rα2, and / or The (h) cell is a human T cell comprising a modified antigen receptor, the modified antigen receptor is a chimeric antigen receptor (CAR), the CAR comprises a co-stimulatory domain selected from the group consisting of CD2, 4-1BB, ICOS and CD27, and / or, The (i) cell is a human T cell comprising a modified antigen receptor, the modified antigen receptor is a chimeric antigen receptor (CAR), the CAR comprises a switch receptor and / or a dominant negative receptor, the receptor is selected from the group consisting of PD1 / CD28, PDL1 / CD28, CTLA4 / CD28, BTLA / CD28, BTLA / ICOS, TIM3 / CD28, TIGIT / CD226, dnTGFβ, TGFβ / IL-12R, TGFβ / CD28, TGFβ / OX40, IFNγ / CD28, IFNγ / OX40 and IFNγ / IL-12R, and / or, The (j) cell is a human T cell comprising a modified antigen receptor, the modified antigen receptor is a chimeric antigen receptor (CAR), the CAR comprises a CD3ζ signaling domain. The method according to claim 14.

16. A composition for use in treating a subject in need thereof, the composition comprising the cells according to claim 10, optionally, (a) the cells are autologous to the subject. Or, (b) the cells are allogeneic to the subject, composition.

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