WO2021244654A1

WO2021244654A1 - Activation induced cytokine production in immune cells

Info

Publication number: WO2021244654A1
Application number: PCT/CN2021/098502
Authority: WO
Inventors: Zhongyuan TU; Yafeng Zhang; Shu Wu; Fengyuan TANG; Wang ZHANG; Baowei LIU
Original assignee: Nanjing Legend Biotech Co., Ltd.
Priority date: 2020-06-05
Filing date: 2021-06-05
Publication date: 2021-12-09

Abstract

Therapeutic immunoresponsive cells: (a) express an antigen receptor (e.g., CAR or TCR) directed toward a target antigen of interest, and (b) express and secrete one or more cytokines or costimulatory proteins at high levels when the immunoresponsive cells are activated and at low levels or not at all when the immunoresponsive cells are not activated.

Description

ACTIVATION INDUCED CYTOKINE PRODUCTION IN IMMUNE CELLS

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to international application PCT/CN2020/094741, filed on June 5, 2020.

The foregoing applications, and all documents cited therein or during their prosecution ( “appln cited documents” ) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ( “herein cited documents” ) , and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

SEQUENCE STATEMENT

The instant application contains a Sequence Listing, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said ASCII copy, was created June 4, 2021, is named 55570-99001_ST25. txt and is 116, 709 bytes in size.

FIELD OF THE INVENTION

The invention relates to therapeutic immunoresponsive cells which (a) express an antigen receptor (e.g., CAR or TCR) directed toward a target antigen of interest, and (b) express and secrete one or more cytokines or costimulatory proteins at high levels when the immunoresponsive cell is activated and at low levels of not at all when the immunoresponsive cell is not activated.

BACKGROUND OF THE INVENTION

Immunotherapy with chimeric antigen receptor (CAR) T cells offers a promising method to improve cure rates and decrease morbidities for patients with cancer. In this regard, CD19-specific CAR T cell therapies have achieved dramatic objective responses for a high percent of patients with CD19-positive leukemia or lymphoma (1-2) . Most patients with other hematologic tumor or solid tumors however, have experienced transient or no benefit from CAR T cell therapies (3-5) . Novel strategies are therefore needed to improve CAR T cell function for patients with these tumors. One obstacle for the field is limited CAR T cell persistence after infusion into patients. The other obstacle is the hostile of tumor microenvironment suppresses CAR T cell function.

Besides CAR-T therapy, people also use other immune cells for cell therapy, especially for allogenic cell therapy, such as engineered NK cells, NKT cells and γδ T cells (6) . Persistence and potency are also two main factors for clinical outcome.

Improvements in the quality and fitness of chimeric antigen receptor (CAR) -engineered immune cells, through CAR design or manufacturing optimizations, could enhance the therapeutic potential of CAR-T cells (7-8) .

Recently, people have demonstrated that CAR-T cells expanded in IL-15 (CAR-T/IL-15) preserve a less-differentiated stem cell memory (Tscm) phenotype, defined by expression of CD62L ⁺CD45RA ⁺CCR7 ⁺, as compared with cells cultured in IL-2 (CAR-T/IL-2) (9) . CAR-T with IL-15 armor cells exhibited reduced expression of exhaustion markers, higher antiapoptotic properties, and increased proliferative capacity upon antigen challenge. In this regard, people have tested CAR T cells that constitutively secrete different cytokines to enhance CAR T cell survival and anti-tumor activity (10) .

Although the opportunities and benefits of armored CAR T-cells are apparent, there are also unintended and detrimental off-target side effects as systemic administration of cytokines to causes significant toxicity. In the case of IL-12, the toxicities of elevated systemic levels have been well described (11) . Specifically, preclinical toxicity assessments of recombinant IL-12 in mice, guinea pigs, and squirrel monkeys indicated that IL-12 caused adverse toxicity in hematopoietic, intestinal, hepatic and pulmonary tissues. In case of interleukin-15 (IL-15) , concern of T-cell leukemogenicity has tempered the clinical application (12) . It is also reported that CAR-T cells with NFAT-induced IL-12 demonstrate enhanced the CAR-T potency (13) . However, while NFAT may be dephosphorylated and transferred to the nucleus in activated T cells, it also promotes T cell “tolerance” and “anergy. ” Ablation of NFAT1 from T cells results in improved rather than impaired tumor control. In addition, NFAT signaling pathway rely on sustained Ca ²⁺ entry upon TCR engagement. It is already known that CAR-T cells form non-classical immune synapses and do not activate Ca ²⁺ pathway (14) . In this regard, NFAT responsive promoters may not work well in CAR modified immune cells. Therefore, a strategy to safely deliver cytokine signals to engineered immune cells remains elusive. It is urgent to find one universal activation controlled promoter for all CAR modified immune cells.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention provides a novel platform to provide a stimulatory signal to T-cells, NK cells, and the like upon engagement with a defined disease-or tumor-associated antigen. The platform provides means for more efficient and effective expression of cytokines and costimulatory molecules from effector cells by harnessing the activation state of the effector cells to limit their production in cells where there has not been engagement of an antigen receptor (e.g., CAR or TCR) , and promote their expression once the antigen receptor is engaged and the immune cell is activated. The platform provides improved immune cell potency and persistence for therapeutic applications and reduces toxicity related to uncontrolled expression of cytokines. The invention provides inducible promoters that are activated by immune cell engagement but display low basal activity.

In certain embodiments, systems, cells, and therapies of the invention comprise a chimeric antigen receptor (CAR) or other target binding molecule operatively linked to a stimulatory element such as, but not limited to CD3ζ, that promotes toxicity of the cell to the target, and in addition expression of a cytokine or other agent that promotes potency and/or persistence when expressed in the cell, operatively linked to an expression element that is induced to high levels of expression when the cell is activated and expressed at low levels or not at all when the cell is not activated. In certain embodiments, expression of a cytokine is driven by expression elements such as promoters and transcription factor binding sites that are active in an immune cell and subject to the activation state of the immune cell. A non-limiting example of a promoter active in an immune cell is the IFNβ promoter. Non-limiting examples of transcription factor binding sites that are subject to the activation state of an immune cell include NF-κB, NFAT, and AP-1 binding sites. When the immune cell is activated by antigen engagement there is activation and nuclear translocation of certain transcription factors, including but not limited to activator protein-1 (AP-1) , nuclear factor of activated T-cells (NFAT) , and nuclear factor-κ-light chain enhancer of activated B cells (NF-κB) transcriptional factors, which bind to their respective sites at the promoter to stimulate transcription. Thus, a cytokine encoding sequence or other sequence operatively linked to a promoter and transcription factor binding sites for AP-1, NF-κB, NFAT, or other transcription factor that operates at the binding site when the cell is activate is expressed at high levels when the cell is activated and at low or undetectable levels when the cell is not activated.

In an aspect, the invention provides a nucleic acid which comprises (i) a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , or a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes a T cell receptor (TCR) , or a first nucleic acid sequence comprising a first regulatory region operatively linked to an antigen binding domain fused to a component of T cell receptor (TCR) complex, and (ii) a second nucleic acid sequence comprising a second regulatory region, operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the second regulatory region comprises a promoter and one or more binding sites, wherein the binding sites bind to transcription factors that are active in activated immune cells. A CAR comprises an antigen binding domain that is selective for a target, a transmembrane domain llinked to the extracellular domain, and an intracellular signaling domain. Components of a T cell receptor complex include a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain, a CD3 epsilon chain, a CD3 zeta chain, and functional fragments thereof. The first nucleic acid sequence is expressed in an effector cell to express the CAR, TCR or antigen binding domain fused to a component of a T cell receptor (TCR) complex, and the second nucleotide sequence is expressed at a high level when the effector cell is activated and a low level when the effector cell is not activated. In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are transcribed in the same direction when the nucleic acid is in an activated effector cell. In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are transcribed in opposite directions in an activated effector cell. In certain embodiments, there is provided a vector which comprises the nucleic acid. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a lentiviral vector. In certain embodiments, there is provided a cell which comprises the nucleic acid. In certain embodiments, the nucleic acid is introduced into a cell, for example by transduction or transfection. In certain embodiments, the cell is an autologous cell. In other embodiments, the cell is an allogeneic cell.

In an aspect, the invention provides a system which comprises (i) a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , or a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes a T cell receptor (TCR) , or a first nucleic acid sequence comprising a first regulatory region operatively linked to an antigen binding domain fused to a component of a T cell receptor (TCR) complex, and (ii) a second nucleic acid sequence comprising a second regulatory region, operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the second regulatory region comprises a promoter and one or more binding sites, wherein the binding sites bind to transcription factors that are active in activated immune cells. In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are located on the same DNA. In certain embodiments, the first nucleic acid sequence and the second nucleic acid sequence are located on different DNAs. The first nucleic acid sequence is expressed in an effector cell to express a CAR, TCR or antigen binding domain fused to CD3 chain of T cell receptor (TCR) complex, and the second nucleotide sequence is expressed at a high level when the effector cell is activated and a low level when the effector cell is not activated. In certain embodiments, there is provided a vector which comprises the nucleic acid sequences. In certain embodiments, there is provided two or more vectors which comprise the nucleic acid sequences. For example, there can be a first vector which comprises the first nucleic acid sequence and a second vector which provides the second nucleic acid sequence. Also, in certain embodiments, there can be more than one CAR, TCR or antigen binding domain fused to CD3 chain of T cell receptor (TCR) complex expressed, and in certain embodiments, there can be expressed in activated cells a cytokine and a costimulatory molecule or more than one cytokine or more than one costimulatory molecule. In certain embodiments, the vector is a viral vector. In certain embodiments, the vector is a lentiviral vector. In certain embodiments, there is provided a cell which comprises the first nucleic acid sequence and the second nucleic acid sequence. In certain embodiments, the nucleic acid sequence is introduced into a cell, for example by transduction or transfection. In certain embodiments, the cell is an autologous cell. In other embodiments, the cell is an allogeneic cell.

In an aspect, there is provided a system comprising (i) a chimeric antigen receptor (CAR) , or a T cell receptor (TCR) , or antigen binding domain fused to a component of T cell receptor (TCR) complex; and (ii) a nucleic acid sequence comprising a regulatory region operatively linked to a nucleic acid sequence that encodes a cytokine and/or a costimulatory protein, wherein the regulatory region comprises a promoter and one or more binding sites that bind to one or more transcription factors that are active in activated immune cells. The nucleic acid sequence encoding the cytokine and/or the costimulatory protein is expressed at a high level when the effector cell is activated and a low level when the effector cell is not activated. In certain embodiments, there is provided a vector which comprises the nucleic acid sequence encoding the cytokine and/or the costimulatory protein. In certain embodiments, there is expressed in activated cells a cytokine and a costimulatory molecule or more than one cytokine or more than one costimulatory molecule. In certain embodiments, the cytokine and the costimulatory molecule or the more than one cytokine or the more than one costimulatory molecule are encoded by more than one nucleic acid sequence and in certain embodiments, the nucleic acid sequences are provided in more than one vector.

In an aspect, there is provided an immune cell which comprises (i) a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , and (ii) a second nucleic acid sequence comprising a second regulatory region, operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the second regulatory region comprises a promoter and one or more binding sites that bind to one or more transcription factors that are active in activated immune cells. In certain embodiments, the first and second nucleic acid sequences are encoded on one DNA. In certain embodiments, the first and second nucleic acid sequences are encoded on different DNAs. In certain embodiments, the first and second nucleic acid sequences are transcribed in the same direction. In certain embodiments, the first and second nucleic acid sequences are transcribed in different directions.

In an aspect, there is provided an immune cell which comprises (i) a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes a T cell receptor (TCR) , or a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes an antigen binding domain fused to a component of a T cell receptor (TCR) complex, and (ii) a second nucleic acid sequence comprising a second regulatory region, operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the second regulatory region comprises a promoter and one or more binding sites that bind to one or more transcription factors that are active in activated immune cells. In certain embodiments, the first and second nucleic acid sequences are encoded on one DNA. In certain embodiments, the first and second nucleic acid sequences are encoded on different DNAs. In certain embodiments, the first and second nucleic acid sequences are transcribed in the same direction. In certain embodiments, the first and second nucleic acid sequences are transcribed in different directions.

In an aspect, there is provided a composition comprising nucleic acids, systems, vectors, or cells of the invention. In certain embodiments, the composition is a pharmaceutical composition. In certain embodiments, the composition is a therapeutic composition.

In an aspect, there is provided a method of making or modifying an immune cell, which comprises introducing into the cell a nucleic acid that comprises a regulatory region operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the regulatory region comprises a promoter and one or more transcription factor binding sites, wherein the transcription factor binding sites bind to transcription factors that are active in activated immune effector cells.

In an aspect, there is provided a method of making an immune cell, which comprises introducing into a cell (i) a first nucleic acid comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , or a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes a T cell receptor (TCR) , or a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes an antigen binding domain fused to a component of a T cell receptor (TCR) complex, and (ii) a second nucleic acid comprising a second regulatory region operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the second regulatory region comprises a promoter and one or more transcription factor binding sites, wherein the transcription factor binding sites bind to transcription factors that are active in activated immune effector cells.

In an aspect, there is provided a method for making or modifying an immune cell to include a binding site against a cancer antigen, the method comprising: introducing into the cell a first nucleic acid encoding a chimeric antigen receptor (CAR) , TCR or antigen binding domain fused to a component of a TCR complex comprising a sequence encoding a single-chain variable fragment (scFv) or a single domain antibody (sdAb) , specific for the cancer antigen operatively linked to a transmembrane domain and a T-cell signaling domain, and introducing into the cell a second nucleic acid encoding a cytokine operatively linked to a promoter and one or more binding sites that bind to one or more transcription factors that are active in activated effector cells, whereby the immune cells express the receptor, and express and secrete the cytokine when activated.

In an aspect, there is provided a method for making or modifying an immune cell to include a binding site against a disease antigen, the method comprising: introducing into the cell a first nucleic acid encoding a chimeric antigen receptor (CAR) , TCR or antigen binding domain fused to CD3 chain of TCR complex comprising a sequence encoding a single-chain variable fragment (scFv) or a single domain antibody (sdAb) specific for the disease antigen operatively linked to a transmembrane domain and a T-cell signaling domain, and introducing into the cell a second nucleic acid encoding a cytokine operatively linked to a promoter and one or more binding sites that bind to one or more transcription factors that are active in activated effector cells, whereby the immune cells express the receptor, and express and secrete the cytokine when activated.

In an aspect, there is provided a pharmaceutical composition comprising an anti-tumor effective amount of a population of effector cells, wherein the effector cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , wherein the CAR comprises at least one extracellular antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, and a nucleic acid sequence that encodes a cytokine or a costimulatory protein operatively linked to a regulatory region which comprises a promoter and one or more binding sites, wherein the binding sites bind to transcription factors that are active in activated immune cells.

In an aspect, there is provided a pharmaceutical composition comprising an anti-tumor effective amount of a population of effector cells, wherein the effector cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , wherein the CAR comprises an extracellular antigen recognition domain specific for one or more antigens (such as tumor antigens) or epitopes, a transmembrane domain, and an intracellular signaling domain.

In an aspect, the CAR comprising an extracellular antigen recognition domain that is selective for a target, a transmembrane domain operatively linked to the extracellular domain, and an intracellular signaling domain comprising a primary intracellular signaling domain of an immune effector cell. In certain embodiments, the primary intracellular signaling domain comprises or is derived from an intracellular signaling domain of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In certain embodiments, the intracellular signaling domain further comprises an intracellular co- stimulatory domain. In certain embodiments, the intracellular signaling domain does not comprise an intracellular co-stimulatory domain. In an aspect, the intracellular co-stimulatory domain comprises or is derived from an intracellular signaling domain of a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof. In certain embodiments, the transmembrane domain comprises CD8α transmembrane region or CD28 transmembrane region. In certain embodiments, the CAR further comprising a CD8α hinge or CD28 hinge between the antigen recognition domain that is selective for a target and the transmembrane domain.

In certain embodiments, the TCR or TCR complex comprises (a) TCR chain selected from an alpha chain, a beta chain, a gamma chain and a delta chain of a T cell receptor, (b) an epsilon chain, a delta chain, and/or a gamma chain of cluster of differentiation 3 (CD3) , or (c) a CD3 zeta chain.

In the various aspects of the invention, in non-limiting embodiments, the cells comprise an αβ T cell, γδ T cell, Vδ1 cell, Vδ2 cell, Vδ3 cell, Vδ5 cell, NKT cell, iNKT cell, or NKT like cell. The cells are also referred to herein as immunoresponsive cells, T cells, Tumor Infiltrating Lymphocytes, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTLs) , Natural Killer T (NK-T) cells or regulatory T cells, and cell which express an antigen recognizing receptor (e.g., CAR or TCR) directed toward a target antigen of interest.

In the various aspect of the invention, in non-limiting embodiments, the cytokine or costimulatory protein comprises IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL21, IL-23, CCL19, leptin or one or more of the cytokines or costimulatory proteins or one or more of the cytokines or costimulatory proteins expressed with a cytokine or costimulatory protein other than IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL21, IL-23, CCL19, or leptin. Functional cytokines and costimulatory molecules can be full-length proteins or truncated proteins with function maintained. The cytokines can be expressed in soluble form, or membrane bound form.

In certain embodiments of the invention, in non-limiting embodiments, the promoter for expression of a cytokine or stimulatory molecule comprises an IFN-β promoter, an IL-2 promoter, a BCL-2 promoter, an IL-6 promoter, an IFN-γ promoter, an IL-12 promoter, an IL-4 promoter, an IL-15 promoter, or an IL-21 promoter. In certain embodiments, the promoter comprises a viral promoter, such as, without limitation, a Herpes simplex virus (HSV) thymidine kinase (TK) promoter or a mini-TK promoter. In certain embodiments, the promoter comprises an artificial promoter, including but not limited to a YB-TATA promoter. The promoter can be a “minimal promoter” from the above. Minimal promoters are described in the art and may be selected to minimize the basal level of transcription in cells that are not activated.

In the various aspects of the invention, in non-limiting embodiments, the transcription factor binding sites comprise one or more copies of one more binding sites of NF-κB, AP-1, NFAT1, NFAT2, NFAT3, NFAT4, Myc, NR4A, TOX1, TOX2, TOX3, TOX4, STAT 1, STAT2, STAT3, STAT4, STAT5, or STAT6. In certain embodiments, there are multiple copies of one or more of the transcription factor binding sites. In certain embodiments, the transcription factor binding sites comprise T cell intrinsic regulatory elements (TIREs) which refers to NF-κB, AP-1, and NFAT regulatory elements suitable singly, in multiples, and/or in combinations to control expression in T cells. In certain embodiments, the transcription factor binding sites comprise immune cell regulatory elements, including but not limited to binding sites for AHR, ATM, BATF, Bcl-6, Blimp-1 (Prdm1) , C/EBP β, CREB, E4BP4 (NFIL3) , EOMES, ETS1, FOXO1, FOXP3, GATA3, HIF-1α, ID2, IKZF1 (Ikaros) , IKZF2 (Helios) , IKZF3 (Aiolos) , IRF1, IRF2, IRF4, IRF5, IRF7, IRF8, GFI1, c-Jun, Ki-67, c-MAF, NR4A1 (Nur77) , NR4A2, NR4A3 (NOR-1) , p53, PCNA, PPARγ, RelB, RORα (RORA; NR1F1) , RORγ2 (RORγt) , SMAD3/4, SPI1, SSRP1, T-bet (Tbx21) , TCF1, TCF4 (E2-2) , TCL1, Th-POK, TOX, TOX2, XBP-1, ZAP-70, or ZEB2.

When there are different transcription factor binding sites, they can be in different proportions and in any arrangement or order. Non-limiting examples include any combination of A and B, wherein there are from 2-10 or more binding sites, wherein A and B represent different transcription factor binding sites. Further non-limiting examples include any combination of A, B and C, wherein there are from 3-15 or more binding sites, wherein A, B, and C represent different transcription factor binding sites. For two or more transcription factor binding sites, non-limiting embodiments include A and B in proportions of about 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2, 1: 1, 2: 8, 2: 7, 2: 6, 2: 5, 2: 4, 2: 3, 2: 2, 2: 1, 3: 7, 3: 6, 3: 5, 3: 4, 3: 3, 3: 2, 3: 1, 4: 6, 4: 5, 4: 4, 4: 3, 4: 2, 4: 1, 5: 5, 5: 4, 5: 3, 5: 2, 5: 1, 6: 4, 6: 3, 6: 2, 6: 2, 7: 3, 7: 2, 7: 1, 8: 2, 8: 1, and 9: 1, wherein A is a transcription factor binding site for one transcription factor and B is a binding site for a different transcription factor. For three or more transcription factor binding sites, non-limiting embodiments include A and B in proportions as above, and A and C in proportions of about 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2, 1: 1, 2: 8, 2: 7, 2: 6, 2: 5, 2: 4, 2: 3, 2: 2, 2: 1, 3: 7, 3: 6, 3: 5, 3: 4, 3: 3, 3: 2, 3: 1, 4: 6, 4: 5, 4: 4, 4: 3, 4: 2, 4: 1, 5: 5, 5: 4, 5: 3, 5: 2, 5: 1, 6: 4, 6: 3, 6: 2, 6: 2, 7: 3, 7: 2, 7: 1, 8: 2, 8: 1, or 9: 1, wherein A is a transcription factor binding site for one transcription factor and C is a binding site for a different transcription factor. In particular embodiments, transcription factor binding sites comprise A, AA, AAA, AAAA, AAAAA, B, BB, BBB, BBBB, BBBBB, AB, BA, AABB, BBAA, AAABBB, BBBAAA, AAAABBBB, BBBBAAAA, AAAAABBBBB, or BBBBBAAAAA wherein A represents an NF-κB binding site and B represents an AP-1 binding site. In certain embodiments, the second regulatory region comprises from three to ten NF-κB binding sites, from three to ten AP-1 binding sites, or from three to ten binding sites, each selected from NF-κB and AP-1 binding sites. In ceratin embodiments transcription factor binding sites are arranged and spaced to take advantage of cooperative binding of transcription factors at adjacent or “composite” sites, e.g., AB, ABAB, ABABAB, ABABABAB, or ABABABABABAB.

In certain embodiments, transcription factor binding sites comprise A _n and B _n and C _n sites, wherein each n is 1, 2, 3, 4, or 5 and the A, B, , and C binding sites are in any order. In certain embodiments, the transcription factor binding sites comprise A _nB _nC _n, wherein each n is independently 1, 2, 3, 4, or 5, wherein A represents one of an NF-κB binding site, an AP-1 binding site, or an NFAT binding site, B represents a second of an NF-κB binding site, an AP-1 binding site, or an NFAT binding site, and C represents a third of an NF-κB binding site, an AP-1 binding site, or an NFAT binding site.

In certain embodiments, transcription factor binding sites comprise ABC, AABC, ABBC, ABCC, AABBC, AABCC, ABBCC, AABBCC, AAABBCC, AABBBCC, AABBCCC, AAABBBCC, AAABBCCC, AAABBBCCC, AAAABBBBCCCC, AAAAABBBCCC, AAABBBBBCCC, AAABBBCCCCC, AAAAABBBBBCCC, AAAAABBBCCCCC, or AAAAABBBBBCCCCC. In certain embodiments, the second regulatory region comprises from three to ten NF-κB binding sites, from three to ten AP-1 binding sites, from three to ten NFAT binding sites or from three to fifteen binding sites, each of which comprises a NF-κB, AP-1, or NFAT binding site.

In the various aspect of the invention, in non-limiting embodiments, as exemplified herein, the first nucleic acid sequence and the second nucleic acid sequence each comprises a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20 or the complement thereof.

In an aspect, the invention provides for use of the compositions of the invention to make CAR T and CAR NK cells as well as methods of making the cells.

In an aspect, the invention provides for use of compositions of the invention to treat cancers and diseases as well as methods of administering the compositions to treat cancers, and other diseases including but not limited to infectious diseases. In certain embodiments, they are prophylactic, including to delay or prevent metastasis or recurrence of a cancer. Kits for all aspects of the invention, including kits for making and testing CAR-αβT cells, CAR-γδT or NK cells and kits for administering the CAR-αβT cells, CAR-γδT cells and NK cells.

In certain embodiments, the invention provides a method for treating a subject with cancer, the method comprising administering a therapeutic population of immune cells which comprise (i) a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , and (ii) a second nucleic acid sequence comprising a second regulatory region, operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the second regulatory region comprises a promoter and one or more binding sites that bind to one or more transcription factors that are active in activated immune cells. In certain embodiments, the first and second nucleic acid sequences are encoded on one DNA. In certain embodiments, the first and second nucleic acid sequences are encoded on different DNAs. In certain embodiments, the first and second nucleic acid sequences are transcribed in the same direction. In certain embodiments, the first and second nucleic acid sequences are transcribed in different directions.

In certain embodiments, the invention provides a method for treating a subject with cancer, the method comprising administering a therapeutic population of immune cells which comprise (i) a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes a T cell receptor (TCR) , or a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes an antigen binding domain fused to a component of a T cell receptor (TCR) complex, and (ii) a second nucleic acid sequence comprising a second regulatory region, operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the second regulatory region comprises a promoter and one or more binding sites that bind to one or more transcription factors that are active in activated immune cells. In certain embodiments, the first and second nucleic acid sequences are encoded on one DNA. In certain embodiments, the first and second nucleic acid sequences are encoded on different DNAs. In certain embodiments, the first and second nucleic acid sequences are transcribed in the same direction. In certain embodiments, the first and second nucleic acid sequences are transcribed in different directions.

Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC) , such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53 (c) EPC and Rule 28 (b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent (s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises, ” “comprised, ” “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes, ” “included, ” “including” and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG. 1. CAR armored with constitutive cytokine expression. The CAR construct and cytokine are expressed on the same transcript. P2A indicates a short, virus-derived peptide sequence that mediates a ribosome-skipping event and enables generation of separate peptide products from one mRNA.

FIG. 2. CAR armored with inducible cytokine expression under an NF-κB (A) or AP-1 combined with NF-κB (B) inducible elements. The CAR construct and cytokine are expressed in opposite directions from their respective promoters.

FIG. 3. Schematic illustration of CAR constructs used in following studies. CAR: conventional unarmored CAR; CAR-7X19: CAR armored with constitutive expression of IL7 and CCL19; CAR-i7X19: CAR armored with inducible expression of IL7 and CCL19. The induced expression is driven by NFAT-AP-1-NF-κB motif derived elements

FIG. 4A-F. CAR surface expression of unarmored (A) or armored CAR-7X19 (B) , CAR-i7X19 (C) on αβ T cells. CAR surface expression of unarmored (D) or armored CAR-15 (E) , CAR-i15 (F) on γδ T cells.

FIG. 5. IL-7 (A) , CCL19 (B) and IL-15 (C) cytokine production by CAR-T cells outlined in FIG. 1-3 with or without TAA stimulation

FIG. 6. Anti-tumor cytotoxicity of CAR-T cells against DLL3-positive (A) or BCMA-positive (B) tumor cells with designs outlined in FIG. 1-3

FIG. 7. IFN-γ production of CAR-T cells against DLL3-positive (A) or BCMA-positive (B) tumor cells with designs outlined in FIG. 1-3

FIG. 8. Persistence and expansion of CAR-T cells against DLL3-positive (A and B) or BCMA-positive (C and D) tumor cells via repeated stimulations with designs outlined in FIG. 1-3.

FIG. 9. Analysis of exhaustion markers at round 3 in different CAR-T cells during repeptitive assay described in FIG. 8.

FIG. 10. IFN-γ production of CAR-T cells against DLL3-positive (A) or BCMA-positive (B) tumor cells via repeated stimulations with designs outlined in FIG. 1-3

FIG. 11. In vivo anti-tumor effect of CAR-T cells described in FIG. 3. Tumor growth was monitored by bioluminescence (A) or caliper (B) over the exepriemtnal time course. Tumor cell intrinsic bioluminescence analysis indicates that inducible IL7 and CLL19 can substantially promote comparable tumor eradicative activity of CAR-T cells as constitutive expressed IL7 and CCL19. The striking difference between FIG. 11 and FIG. 12 indicates that cytokine armored CAR-T cells could induce pseudoprogression as reflected by inflammated tumor microenviroment.

FIG. 12. In vivo pharmacokinetics of CAR-T cells, described in FIG. 3, in peripheral blood suggested that both inducible and constitutive expression of IL7 and CCL19 can substantially enhance CAR-T cell expansion in vivo.

FIG. 13. In vivo pharmacokinetics of CAR-T cells, described in FIG. 3, in peripheral blood suggested that both inducible and constitutive expression of IL7 and CCL19 can substantially enhance CAR-T cell expansion in vivo. IL7 (A) and CCL19 (B) expression in vivo in murine peripheral blood at different time points. While constitutive expression of IL7 and CCL19 can be detected in serum, inducible expressed cytokine was much lower, suggesting its local release within tumor vicinity.

DETAILED DESCRIPTION OF THE INVENTION

The invention generally relates to improved compositions and methods for treating tumors, neoplastic diseases, autoimmune diseases, infectious diseases, and other disorders. In particular embodiments, the invention relates to improved adoptive cell therapy using genetically modified immune effector cells. Genetic approaches offer a potential means to enhance immune recognition and eliminate disease cells. One promising strategy is to genetically engineer immune effector cells to express chimeric antigen receptors (CAR) that redirect cytotoxicity toward cancer cells.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

Provided herein are nucleic acids, vectors, cells, and therapies comprising recombinant nucleic acids and proteins. The invention provides nucleic acids and methods for making effector cells that express chimeric antigen receptors (CARs) comprising an antigen binding domain selective for a disease-or tumor-associated target antigen, a transmembrane domain operatively linked to the extracellular domain, and an intracellular signaling domain, and advantageously also express ‘armoring” components in an activation dependent manner. That is, the effectors cells express the armoring components in high levels when the effector cells is engaged with a target and activated, but express the armoring components at a low level or not at all when the effector cell is not activated. In certain embodiments, the armoring agent is a cytokine. For example, cytokine production by an effector cells is advantageous for process of target cells killing, but excessive cytokine production is often detrimental to a treatment subject. According to the invention, controlled expression of a cytokine or other molecule is dependent on a promoter that is active in the effector cell, and transcription factor binding sites incorporated near or adjacent to the promoter that bind to transcription factors which are active when the effector cell is activated, and inactive when the effector cell is not activated. The invention is generally applicable to any CAR cell or construct, as described in further detail below.

Cytokines

Cytokines useful for enhancing immune cell fitness and/or enhancing immune cell cytotoxicity include, without limitation, IL-7, IL-12, IL-15, and IL-21.

The following have been constructed and characterized in vitro and in vivo and are non-limiting: anti-CD19-BBζ/IL-7 CAR, anti-CD19-BBζ/IL-15 CAR, anti-CD19–BBζ/IL-21 CAR, anti-BCMA-BBζ/IL-7 CAR, anti-BCMA-BBζ/IL-15 CAR, anti-BCMA-BBζ/IL-21 CAR, anti-CD19-BBζ/NFkB-IL-7 CAR, anti-CD19-BBζ/NFkB-IL-15 CAR, anti-CD19–BBζ/NFkB-IL-21 CAR, anti-BCMA-BBζ/NFkB-IL-7 CAR, anti-BCMA-BBζ/NFkB-IL-15 CAR, anti-BCMA-BBζ/NFkB-IL-21 CAR, anti-CD19-BBζ/NFkBAP-1-IL-7 CAR, anti-CD19-BBζ/NFkBAP-1-IL-15 CAR, anti-CD19-BBζ/NFkBAP-1-IL-21 CAR, anti-BCMA-BBζ/NFkBAP-1-IL-7 CAR, anti-BCMA-BBζ/NFkBAP-1-IL-15 CAR, and anti-BCMA-BBζ/NFkBCAP-1-IL-21 CAR.

Transcription Factor Binding Sites

In an aspect, the invention provides regulated expression dependent on the host cell type and activation state of the host cell.

This aspect of the invention is exemplified herein by regulated expression in immune cells using promoters and transcription factor binding sites that are active and can be modulated in cells of the immune system.

NF-κB and AP-1 are transcriptional factors that play an important role in gene transcription in activated immune cells. Both TCR and CAR based signaling pathways activate NF-κB and AP-1 transcriptional factors. T cell-NF-κB plays an important role in tumor control. It is also investigated that that stimulation of NK cells or γδ T cells with specific cell targets results in an increased binding activity of NF-κB and AP-1 transcription factors.

The NF-κB transcription factor family in mammals consists of five proteins, p65 (RelA) , RelB, c-Rel, p105/p50 (NF-κB1) , and p100/52 (NF-κB2) that associate with each other to form distinct transcriptionally active homo-and heterodimeric complexes. They all share a conserved 300 amino acid long amino-terminal Rel homology domain (RHD) , and sequences within the RHD are required for dimerization, DNA binding, interaction with IκBs, as well as nuclear translocation. (Oeckinghaus et al., 2009, The NF-κB Family of Transcription Factors and Its Regulation, Cold Spring Harb Perspect Biol. 2009 Oct; 1 (4) : a000034) .

NF-κB exerts its fundamental role as transcription factor by binding to variations of the consensus DNA sequence of 5′-GGGRNYYYCC-3′ (in which R is a purine, (i.e., A or G) , Y is a pyrimidine (i.e., C or T) , and N is any nucleotide) known as κB sites. How NF-κB selectively recognizes a small subset of relevant κB sites from the large excess of potential binding sites (about 1.4×10 ⁴ estimated in human genome) is a critical step for stimulus-specific gene transcription. At a molecular level, DNA-binding differences of individual NF-κB dimers have been linked to dimer-specific roles in gene regulation (Hoffmann et al., 2006, Transcriptional regulation via the NF-kappaB signaling module. Oncogene 25: 6706; Natoli G., 2006, Tuning up inflammation: how DNA sequence and chromatin organization control the induction of inflammatory genes by NF-kappaB. FEBS Lett. 580: 2843) . Much work has been carried out to identify structural features of NF-κB: DNA complexes and how distinctive features of NF-κB proteins and DNA sequences contribute to specific complex formation (Siggers et al., 2012, Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-κB family DNA binding. Nat Immunol. 13 (1) : 95; Mulero et al., 2019, Genome reading by the NF-κB transcription factors. Nucleic Acids Res. 47 (19) : 9967) . The presence of NF-κB sites is observed to be a minimal requirement for NF-κB regulation but not sufficient for gene induction (Wan et al., 2009, Specification of DNA Binding Activity of NF-κB Proteins, Cold Spring Harb Perspect Biol. 1 (4) : a000067. ) .

The dimeric transcription factor complex Activator Protein-1 (AP-1) is a group of proteins involved in a wide array of cell processes and a critical regulator of nuclear gene expression during T-cell activation. AP-1 transcription factors are homo-or hetero-dimmer forming proteins that belong to a group of DNA binding proteins called Basic -Leucine Zipper domain (bZIP) proteins. Dimerization between members of the AP-1 family occurs through a structure which is known as leucine zipper, comprised of a heptad of repeats of leucine residues along a α-helix, which can dimerize with another α-helix via formation of a coiled–coil structure with contacts between hydrophobic leucine zipper domain. Adjacent to the leucine zipper lies a basic DNA binding domain which is rich in basic amino acids and is responsible for DNA-binding in either 12-O-tetradecanoylphorbol-13-acetate (TPA) response elements (5′-TGAG/CTCA-3′) or cAMP response elements (CRE, 5′-TGACGTCA-3′) (Shaulian et al. AP-1 as a regulator of cell life and death. Nat. Cell Biol. 4: E131; Atsaves, 2019, AP-1 Transcription Factors as Regulators of Immune Responses in Cancer. Cancers 11 (7) : 1037) .

Nuclear factor of activated T cells (NFAT) is a family of transcription factors identified in activated T cells. All NFAT proteins share a conserved DNA-binding domain (DBD) that specifies binding to the DNA core sequence (A/T) GGAAA. NFAT1, 2 and 4 are expressed in cells of the immune system. NFAT1 is constitutively expressed in normal human T cells, whereas NFAT2 is induced by activation. NFAT proteins are also expressed in various non-lymphoid tissues, where they are involved in the regulation of diverse cellular functions in organs other than the immune system. NFAT proteins interact with other transcription factors such as AP1, FOXP3, and BATF when translocated to the nucleus. AP-1 forms a complex with NFATs and induces various cytokines such as IL-2, IL-4, and IFN-γ and other T cell activation-induced proteins and composite NFAT: AP-1 binding sites have been described in numerous genes involved in immune responses.

The Myc proteins (c-Myc, L-Myc, S-Myc, and N-Myc) are a family of transcription factors that regulate growth and cell cycle entry by their ability to induce expression of genes required for these processes. In normal cells, mitogen stimulation leads to a burst of Myc expression in G1 phase, facilitating entry into the cell cycle. MYC plays a role in regulating a range of innate and adaptive immune cells, and is a key transcription factor that regulates immune cell maturation, development, proliferation and activation, including macrophages, T cells, dendritic cells, and natural killer (NK) cells.

Another useful transcriptional control mechanism of the invention involves the NR4A1 family of transcription factors (e.g., NR4A1, NR4A2, and NR4A3) . When NR4A1 is overexpressed in naive T cells, there is upregulation of genes related to anergy and exhaustion, downregulation of genes related to effector programs, reduced TH1 and TH17 differentiation in CD4+ T cells, and reduced IFNγ production by CD8+ T cells. Ablation of NR4A1 enhances effector functions of CD4+ and CD8+ T cells, increases expansion, and blocks the formation of tolerance. (Liu X. et al., 2019, Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction. Nature. 2019 Feb 27) . According to the invention, NR4A is a useful transcription factor to maintain expression of cytokines. Incorporation of NR4A binding elements in constructs of the invention boosts cytokine expression and prolongs cytokine release by the CAR T cells.

Similarly, TOX transcription factors act as mediators of T cell exhaustion. TOX and TOX2 as well as NR4A family members have been shown to be highly induced in CD8 ⁺CAR ⁺ PD-1 ^high TIM3 ^high ( “exhausted” ) TILs. (Seo, H. et al., 2019, TOX and TOX2 transcription factors cooperate with NR4A transcription factors to impose CD8 ⁺ T cell exhaustion, PNAS June 18, 2019 116 (25) : 12410) . Other TOX family members include TOX3 and TOX4. TOX transcription factors normally activate transcription through cAMP response element (CRE) sites and protect against cell death by inducing antiapoptotic and repressing pro-apoptotic transcripts. According to the invention, TOX family binding elements are used to increase and/or prolong cytokine expression. An example of a cAMP response element (CRE) is the response element for CREB which contains the highly conserved nucleotide sequence, 5'-TGACGTCA-3’.

Another group of useful transcription factors involved in transcription activation in immune cells are members of signal transducer and activator of transcription (STAT) family proteins, including STAT3, STAT4, STAT5A, STAT5B, and, STAT6, which mediate response to cytokines and growth factors. STAT proteins dimerize through reciprocal pTyr-SH2 domain interactions, and translocate to the nucleus where they bind to specific STAT-response elements in the target gene promoters and regulate transcription. There are 10 or so STAT-response elements, in general consisting of a palindromic sequence, TT N _i AA, where i is 4, 5, or 6. Recognition of this sequence by a particular STAT depends on the value of i as well as on the specific sequence for N _i. For example, binding of STAT3 is better if N is 4, STAT1 if N is 5, and STAT6 if N is 6. (Schindler, U. et al., 1995, Components of a Stat recognition code: evidence for two layers of molecular selectivity. Immunity 2: 689.; Seidel, H.M. et al., 1995, Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity. Proc. Natl. Acad. Sci. USA 92: 3041) .

The transcription factor binding sites can used singly or in multiples, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more transcription factor binding sites. The transcription factors can be the same or different, and can be mixed in varying ratios and in any order. Example constructs shown herein comprise 3 sequential NF‐κB binding sites, 5 sequential NF‐κB binding sites, three sequential NF‐κB three sequential AP-1 binding sites, or 5 sequential NF‐κB and 5 sequential AP-1 sites.

Promoters

In certain embodiments, cytokine expression is driven by an IFN-β promoter or functional promoter fragment thereof. The IFN-β promoter is well known and characterized (see, e.g, Vodjdani G. et al., 1988. Structure and characterization of a murine chromosomal fragment containing the interferon beta gene. J Mol Biol. 204 (2) : 221-31) and an IFN-β promoter fragment (SEQ ID NO: 54) sufficient to drive cytokine expression is exemplified herein.

In certain embodiments, cytokine expression is driven by an ELAM promoter or functional promoter fragment thereof (e.g., SEQ ID NO: 55) . The ELAM promoter is well known and characterized (see, e.g, Schindler U., Baichwal VR., 1994. Three NF-kappa B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression. Mol Cell Biol. 14 (9) : 5820-31. It contains three NF-κB binding site, thus making the promoter fragment sufficient to drive cytokine expression is exemplified herein.

In certain embodiments, cytokine expression is driven by an IL-2 promoter or functional promoter fragment thereof. The T cell growth factor, IL-2, is the major cytokine that is produced during the primary response of T cells. IL-2 expression is controlled tightly at the transcriptional level, and extensive analysis of the IL-2 gene established a minimal promoter region, which extends about 300 bp relative to the transcription start site, that is known to be sufficient for IL-2 induction upon T cell activation in vitro. (Jain, J. et al., 1995, Transcriptional regulation of the IL-2 gene. Curr. Opin. Immunol. 7: 333–342; Serfling, E. et al., 1995, The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation. Biochim. Biophys. Acta. 1263: 181–200) .

In certain embodiments, cytokine or costimulatory protein expression is driven by a BCL-2 promoter or functional promoter fragment thereof. In certain embodiments, the promoter fragment is a minimal BCL-2 promoter.

In certain embodiments, cytokine or costimulatory protein expression is driven by an IL-6 promoter or functional promoter fragment thereof. In certain embodiments, the promoter fragment is a minimal IL-6 promoter.

In certain embodiments, cytokine or costimulatory protein expression is driven by an IFN-γ promoter or functional promoter fragment thereof. In certain embodiments, the promoter fragment is a minimal IFN-γ promoter.

In certain embodiments, cytokine or costimulatory protein expression is driven by an IL-12 promoter or functional promoter fragment thereof. In certain embodiments, the promoter fragment is a minimal IL-12 promoter.

In certain embodiments, cytokine or costimulatory protein expression is driven by an IL-4 promoter or functional promoter fragment thereof. In certain embodiments, the promoter fragment is a minimal IL-4 promoter.

In certain embodiments, cytokine or costimulatory protein expression is driven by an IL-21 promoter or functional promoter fragment thereof. In certain embodiments, the promoter fragment is a minimal IL-21 promoter.

In certain embodiments, cytokine or costimulatory protein expression is driven by a tk promoter or functional promoter fragment thereof. In certain embodiments, the promoter fragment is a mini tk promoter.

In certain embodiments, cytokine or costimulatory protein expression is driven by a TATA promoter or functional promoter fragment thereof. In certain embodiments, the promoter fragment is an YB TATA promoter.

Minimal promoters

Minimal promoters are described in the art and may be selected to minimize the basal level of transcription in cell that are not activated. For example, Parvin et al. describes a eukaryotic minimal promoter of IgH transcription that can be reconstituted in vitro in a minimal reaction that contains only TATA-binding protein (TPB) , TFIIB and RNA polymerase II (pol II) when the template is negatively coiled. (Parvin et al., 1993, DNA topology and a minimal set of basal factors for transcription by RNA polymerase II. Cell 73: 522) . Butler (Butler et al, 2002, The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes & Dev. 16: 2583) refers to the core promoter as the minimal stretch of contiguous DNA sequence that is sufficient to direct accurate initiation of transcription by the RNA polymerase II machinery. According to Butler, a core promoter typically encompasses the site of transcription initiation and extends either upstream or down stream for an additional ～35 nucleotides and in many instances will comprise only about 40 nt, include the TATA box, initiator (Inr) , TFIIB recognition element (BRE) , and downstream core promoter element (DPE) that are commonly found in core promoters but also notes that each of these core promoter elements is found in some but not all core promoters. These are distinct from other cis-acting DNA sequences that regulate RNA polymerase II transcription such as the proximal promoter, enhancers, silencers, and boundary/insulator elements which contain recognition sites for a variety of sequence-specific DNA-binding factors that are involved in transcriptional regulation. The proximal promoter is the region in the immediate vicinity of the transcription start site (roughly from -250 to +250 nt) . Enhancers and silencers can be located many kbp from the transcription start site and act either to activate or to repress transcription.

Minimal or core promoters can be engineered or selected for low basal transcription, for high level expression when induced, to optimize fold induction, or a combination thereof and the expression characteristics can be modified by varying the copy number and spacing of regulatory elements. Promoters useful in the invention include, without limitation, viral promoters and promoter elements and minimal or core viral promoters and promoters synthesized therefrom, for example the miniTK promoter obtained from Herpes simplex virus (HSV) thymidine kinase, the core promoter from cytomegalovirus (CMV) , and the core promoter from simian virus 40 (SV40) . A non-limiting example of a minimal promoter engineered for low expression in the uninduced state and high inducibility is the YB promoter (SEQ ID NO: 48) described by Hansen st al., 2014, Transplantation of prokaryotic two-component signaling pathways into mammalian cells. Proc. Natl. Acad. Sci. USA 111 (44) : 15705-15710. Elements of the HSV promoter are characterized, for example by McKnight, S. L. et al., 1981, Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell 25: 385–398. An exemplary minimal or core TK promoter comprises SEQ ID NO: 45. In certain embodiments, a nucleic acid sequence for expression of a cytokine or costimulatory protein comprises a functional fragment of a tk promoter operably linked to binding sites that bind to one or more transcription factors that are active in activated immune cells. In certain embodiments, the binding sites for transcription factors that are active in activated immune cells comprise binding sites for transcription factors that are activated in T cells. Such binding sites may be referred to herein as T cell intrinsic regulatory elements (TIREs) . An exemplary non-limiting example of a tk promoter operably linked to T cell intrinsic regulatory elements comprises SEQ ID NO: 46. SEQ ID NO: 46 comprises 5 NFAT binding sites, five (5) AP-1 binding sites and five (5) NF-κB binding sites. The sequences of transcription factor binding sites, being active in T cells, constitutes an example of a TIRE. SEQ ID NO: 46 comprises optional tk promoter elements which need not be present in all embodiments of the invention that comprise a core TK promoter, for example two Sp1 elements, and CAAT box. Also, tk promoter elements such as the octamer sequence may be included in embodiments of the invention.

Chimeric antigen receptors (CARs)

The invention, which provides engagement dependent regulation of expression, is used with any CAR, including but not limited to what are referred to as first-generation, second-generation, third-generation, and “armored” CARs.

The term “chimeric antigen receptor” or “CAR” as used herein refers to an artificially constructed hybrid protein or polypeptide containing a binding moiety (e.g. an antibody) linked to immune cell (e.g. T cell) signaling or activation domains. In some embodiments, CARs are synthetic receptors that retarget T cells to tumor surface antigens (Sadelain et al., Nat. Rev. Cancer 3 (l) : 35-45 (2003) ; Sadelain et al., Cancer Discovery 3 (4) : 388-398 (2013) ) . CARs can provide both antigen binding and immune cell activation functions onto an immune cell such as a T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition can give T-cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a mechanism of tumor escape.

In certain embodiments, the chimeric receptor comprises an extracellular antigen recognition domain specific for one or more antigens (such as tumor antigens) or epitopes, a transmembrane domain, and an intracellular signaling domain of a T cell, γδ T cell, NK cell or NKT cell and/or co-stimulatory receptors. “CAR-T” refers to a T cell, γδ T cell, NK cell or NKT cell that expresses a CAR. “Anti-CD19 CAR” refers to a CAR having an extracellular binding domain specific for CD19, “anti-BCMA CAR” refers to a CAR having an extracellular binding domain specific for BCMA.

Several “generations” of CARs have been developed. First-generation CAR T-cells utilize an intracellular domain from the CD3ζ-chain of the TCR, which provides so called ‘signal 1, ’ and induces cytotoxicity against targeted cells. Engagement and signaling via the CD3ζ chain is required for T-cell stimulation and proliferation but is not often sufficient for sustained proliferation and activity in the absence of a second signal or ‘signal 2. ’ Second-generation CARs were developed to enhance efficacy and persistence in vivo after reinfusion into a subject and contain an second costimulatory signaling domain (CD28 or 4- 1BB) intracellular domain that functions to provide ‘signal 2’ to mitigate anergy and activation-induced cell death seen with first generation CAR T-cells. Third-generation CARs are further optimized by use of two distinct costimulatory domains in tandem, e.g., CD28/4-1BB/CD3ζ or CD28/OX-40/CD3ζ. (see, e.g., Yeku et al., 2016, Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy. Biochem Soc Trans. 44 (2) : 412) . CARs have been further optimized or “armored” to secrete active cytokines or express costimulatory ligands that further improve efficacy and persistence. There are several variants of armored CAR T-cells, designed for example to express IL-12, CD40L, or 4-1BBL.

Intracellular signaling domain

The intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as T cell) . In certain embodiments, the primary intracellular signaling domain is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In certain embodiments, the primary intracellular signaling domain is derived from CD3ζ (i.e., “a CD3ζ intracellular signaling domain” ) . In certain embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence. In certain embodiments, the intracellular signaling domain comprises both a primary intracellular signaling domain (e.g., a CD3ζ intracellular signaling domain) and an intracellular co-stimulatory domain. In certain embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain but does not comprise an intracellular co-stimulatory domain. In certain embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence but does not comprise a primary intracellular signaling domain.

Co-stimulatory Domains

"Co-stimulatory domain" (CSD) as used herein refers to the portion of the CAR which enhances the proliferation, survival and/or development of memory cells. The CARs of the invention may comprise one or more co-stimulatory domains. Each costimulatory domain comprises a costimulatory domain of any one or more of, for example, members of the TNFR superfamily, CD28, CD137 (4-lBB) , CD134 (OX40) , DaplO, CD27, CD2, CD5, ICAM-1, LFA-1 (CD1 la/CD18) , Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof. Further costimulatory domains used with the invention comprise one or more of: 2B4/CD244/SLAMF4, 4-1BB/TNFSF9/CD137, B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF-R/TNFRSF13C, BAFF/BLyS/TNFSF13B, BLAME/SLAMF8, BTLA/CD272, CD100 (SEMA4D) , CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55) , CD18, CD19, CD2, CD200, CD229/SLAMF3, CD27 Ligand/TNFSF7, CD27/TNFRSF7, CD28, CD29, CD2F-10/SLAMF9, CD30 Ligand/TNFSF8, CD30/TNFRSF8, CD300a/LMIR1, CD4, CD40 Ligand/TNFSF5, CD40/TNFRSF5, CD48/SLAMF2, CD49a, CD49D, CD49f, CD53, CD58/LFA-3, CD69, CD7, CD8 α, CD8 β, CD82/Kai-1, CD84/SLAMF5, CD90/Thy1, CD96, CDS, CEACAM1, CRACC/SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1/CLEC7A, DNAM1 (CD226) , DPPIV/CD26, DR3/TNFRSF25, EphB6, GADS, Gi24/VISTA/B7-H5, GITR Ligand/TNFSF18, GITR/TNFRSF18, HLA Class I, HLA-DR, HVEM/TNFRSF14, IA4, ICAM-1, ICOS/CD278, Ikaros, IL2R β, IL2R γ, IL7R α, Integrin α4/CD49d, Integrin α4β1, Integrin α4β7/LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT/TNFSF14, LTBR, Ly108, Ly9 (CD229) , lymphocyte function associated antigen-1 (LFA-1) , Lymphotoxin-α/TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1) , NTB-A/SLAMF6, OX40 Ligand/TNFSF4, OX40/TNFRSF4, PAG/Cbp, PD-1, PDCD6, PD-L2/B7-DC, PSGL1, RELT/TNFRSF19L, SELPLG (CD162) , SLAM (SLAMF1) , SLAM/CD150, SLAMF4 (CD244) , SLAMF6 (NTB-A) , SLAMF7, SLP-76, TACI/TNFRSF13B, TCL1A, TCL1B, TIM-1/KIM-1/HAVCR, TIM-4, TL1A/TNFSF15, TNF RII/TNFRSF1B, TNF-α, TRANCE/RANKL, TSLP, TSLP R, VLA1, and VLA-6.

CAR ligand-binding domains

CARs typically employ scFv domains of antibodies to target cell surface antigens of target cells. These binding domains consist of a variable heavy and variable light chains fused together with a flexible linker. The variable domains are derived within an antibody, determining antigen specificity. TCR-like antibody based CARs are a class of CARs which express scFvs from antibodies that specifically recognize MHC class molecules and its loaded peptide (Dahan et al., 2012, T-cell-receptor-like antibodies -generation, function and applications. Expert Reviews in Molecular Medicine. 14: e6) . This specificity can be utilized to target cancers based on recognition of mutated intracellular proteins. If mutated peptide sequences are loaded onto the MHC, they could effectively generate neo-epitopes, which can be used to distinguish a cancerous cell from a normal cell by a CAR that only recognizes the specific MHC/peptide combination.

Antigen binding domains take many forms. Non-limiting examples include bispecific receptors (Zakaria Grada, et al. TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy. Molecular Therapy, 2013, 2, e105) , single domain VHH based CARs (De Meyer T, et a., VHH-based products as research and diagnostic tools. Trends Biotechnol. 2014 May; 32 (5) : 263-70) , and “universal” CARs comprising avidin that binds to any antigen receptor that incorporates biotin (Huan Shi, et al. Chimeric antigen receptor for adoptive immunotherapy of cancer: latest research and future prospects. Molecular Cancer, 2014, 13: 219) .

The term “antigen binding domain” as used herein refers to an antibody fragment including, but not limited to, a diabody, a Fab, a Fab’, a F (ab’) 2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv) , a (dsFv) 2, a bispecific dsFv (dsFv-dsFv') , a disulfide stabilized diabody (ds diabody) , a single domain antibody (sdAb) , a single chain variable fragment (scFv) an scFv dimer (bivalent diabody) , a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a bivalent domain antibody, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. An antigen binding domain is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds. In some embodiments, an antigen-binding fragment may comprise one or more complementarity detrmining regions (CDRs) from a particular human antibody grafted to frameworks (FRs) from one or more different human antibodies.

The antigen binding domain can be made specific for any disease-associated antigen, including but not limited to tumor-associated antigens (TAAs) and infectious disease-associated antigens. In certain embodiments, the ligand binding domain is bispecific. Antigens have been identified in most of the human cancers, including Burkitt lymphoma, neuroblastoma, melanoma, osteosarcoma, renal cell carcinoma, breast cancer, prostate cancer, lung carcinoma, and colon cancer. TAAs include, without limitation, CD19, CD20, CD22, CD24, CD33, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα) , mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP and HER-2. TAAs further include neoantigens, peptide/MHC complexes, and HSP/peptide complexes.

In certain embodiments, the antigen binding domain comprises a T-cell receptor or binding fragment thereof that binds to a defined tumour specific peptide-MHC complex. The term “T cell receptor, ” or “TCR, ” refers to a heterodimeric receptor composed of αβ or γδ chains that pair on the surface of a T cell. Each α, β, γ, and δ chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition through the complementarity determining regions (CDR) , followed by a constant domain (C) that is anchored to cell membrane by a connecting peptide and a transmembrane (TM) region. The TM region associates with the invariant subunits of the CD3 signaling apparatus. Each of the V domains has three CDRs. These CDRs interact with a complex between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544; Murphy (2012) , xix, 868 p. ) .

In certain embodiments, the antigen binding domain comprises a natural ligand of a tumor expressed protein or tumor-binding fragment thereof. For example, the transferrin receptor 1 (TfR1) , also known as CD71, is a homodimeric protein that is a key regulator of cellular iron homeostasis and proliferation. Although TfR1 is expressed at a low level in a broad variety of cells, it is expressed at higher levels in rapidly proliferating cells, including malignant cells in which overexpression has been associated with poor prognosis. In an embodiment of the invention, the antigen binding domain comprises transferrin or a transferrin receptor-binding fragment thereof.

In certain embodiments, the antigen binding domain is specific to a defined tumor associated antigen, such as but not limited to FRα, CEA, 5T4, CA125, SM5-1 or CD71. In certain embodiments, the tumor associated antigen can be a tumor-specific peptide-MHC complex. In certain such embodiments, the peptide is a neoantigen. In other embodiments, the tumor associated antigen it a peptide-heat shock protein complex.

Another class of CARs is based on NK cells, γδ cell and NK cell receptors. NK cells have robust antitumor activity and many of their receptors can recognize various stress induced or overexpressed ligands on different tumors, thereby activating NK cell cytotoxicity. (Spear P. et al., 2013, NKG2D ligands as therapeutic targets. Cancer Immunity 13: 8) . NK receptor based CARs are engineered with intact extracellular binding domains of NK cell receptors fused to cytoplasmic activation and costimulatory domains. The NKG2D CAR has been shown to recognize myelomas, lymphomas, and ovarian cancers. (Gacerez et al., 2016, How chimeric antigen receptor design affects adoptive T cell therapy. J. Cell Physiol. 231 (12) : 2590) .

In certain embodiments, targeting domains of CARs of the invention target tumor-associated antigens. In certain embodiments, the tumor-associated antigen is selected from: 707-AP, a biotinylated molecule, a-Actinin-4, abl-bcr alb-b3 (b2a2) , abl-bcr alb-b4 (b3a2) , adipophilin, AFP, AIM-2, Annexin II, ART-4, BAGE, BCMA, b-Catenin, bcr-abl, bcr-abl p190 (e1a2) , bcr-abl p210 (b2a2) , bcr-abl p210 (b3a2) , BING-4, CA-125, CAG-3, CAIX, CAMEL, Caspase-8, CD171, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v7/8, CD70, CD123, CD133, CDC27, CDK-4, CEA, CLCA2, CLL-1, CTAG1B, Cyp-B, DAM-10, DAM-6, DEK-CAN, DLL3, EGFR, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6/AML, FAP, FBP, fetal acetylcholine receptor, FGF-5, FN, FR-α, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, GPC3, GPC-2, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2 (FGF6) , HST-2/neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR/FUT, Lewis Y, L1-CAM, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, Malic enzyme, Mammaglobin-A, MART-1/Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, Myosin, NA88-A, Neo-PAP, NKG2D, NPM/ALK, N-RAS, NY-ESO-1, OA1, OGT, oncofetal antigen (h5T4) , OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, Survivin, Survivin-2B, SYT/SSX, TAG-72, TEL/AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2/INT2, TRP-2-6b, Tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ-light chain.

Spacer

Spacer domain or extracellular spacer domain (ESD) refers to a spacer region between the antigen binding domain and the costimulatory receptor. The spacer provides flexibility to access the targeted antigen and receptor ligand. In certain embodiments long spacers are employed, for example to target membrane-proximal epitopes or glycosylated antigens (see Guest R.D. et al. The role of extracellular spacer regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens. J. Immunother. 2005; 28: 203–211; Wilkie S. et al., Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor. J. Immunol. 2008; 180: 4901–4909) . In other embodiments, CARs bear short spacers, for example to target membrane distal epitopes (see Hudecek M. et al., Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clin. Cancer Res. 2013; 19: 3153–3164; Hudecek M. et al., The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol. Res. 2015; 3: 125–135) . In certain embodiments, the spacer comprises all or part of or is derived from an IgG hinge, including but not limited to IgG1, IgG2, or IgG4. By “derived from an Ig hinge” is meant a spacer comprising insertions, deletions, or mutations in an IgG hinge. In certain embodiments, a spacer can comprise all or part of one or more antibody constant domains, such as but not limited to CH2 and/or CH3 domains. In certain embodiments, in a spacer comprising all or part of a CH2 domain, the CH2 domain is modified so as not to bind to an Fc receptor. For example, Fc receptor binding in myeloid cells has been found to impair CAR T cell functionality. In certain embodiments, the spacer comprises all or part of an Ig-like hinge from CD28, CD8, or other protein comprising a hinge region. In certain embodiments of the invention that comprise a spacer, the spacer is from 1 and 50 amino acids in length. Other extracellular spacer domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

Transmembrane Domain

"Transmembrane domain" (TMD) as used herein refers to the region of the CAR which crosses the plasma membrane. The transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins) , an artificial hydrophobic sequence or a combination thereof. Although the main function of the transmembrane is to anchor the CAR in the T cell membrane, in certain embodiments, the transmembrane domain influences CAR function. In certain embodiments, the transmembrane domain is from CD4, CD8α, CD28, or ICOS. Gueden et al. associated use of the ICOS transmembrane domain with increased CAR T cell persistence and overall anti-tumor efficacy (Guedan S. et al., Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight. 2018; 3: 96976) . In an embodiment, the transmembrane domain comprises a hydrophobic α helix that spans the cell membrane. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In certain embodiments, the transmembrane domain is a human transmembrane domain. In certain embodiments, the transmembrane domain comprises human CD8α transmembrane domain. In certain embodiments, the transmembrane domain comprises human CD28 transmembrane domain.

Hinge region

The chimeric receptors of the present application may comprise a hinge domain that is located between the extracellular domain and the transmembrane domain. A hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular domain relative to the transmembrane domain of the effector molecule can be used. The hinge domain may contain about 10-100 amino acids, e.g., about any one of 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. In some embodiments, the hinge domain may be at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

In certain embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. In certain embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In certain embodiments, the hinge domain is derived from CD8, such as CD8α. In certain embodiments, the hinge domain is a portion of the hinge domain of CD8α, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. In certain embodiments, the hinge domain is derived from CD28.

Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the chimeric receptor systems described herein. In certain embodiments, the hinge domain is the hinge domain that joins the constant domains CH1 and CH2 of an antibody. In certain embodiments, the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In certain embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In certain embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In certain embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In certain embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In certain embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In certain embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.

Non-naturally occurring peptides may also be used as hinge domains for the chimeric receptors described herein. In certain embodiments, the hinge domain between the C-terminus of the extracellular ligand-binding domain of an Fc receptor and the N-terminus of the transmembrane domain is a peptide linker, such as a (GxS) n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

The term “T-cell receptor” or “TCR” refers to a molecule on the surface of a T cell or T lymphocyte that is responsible for recognizing an antigen. TCR is a heterodimer which is composed of two different protein chains. In some embodiments, the TCR of the present disclosure consists of an alpha (α) chain and a beta (β) chain and is referred as αβ TCR. αβ TCR recognizes antigenic peptides degraded from protein bound to major histocompatibility complex molecules (MHC) at the cell surface. In some embodiments, the TCR of the present disclosure consists of a gamma (γ) and a delta (δ) chain and is referred as γδ TCR. γδ TCR recognizes peptide and non-peptide antigens in a MHC-independent manner. γδ T cells have shown to play a prominent role in recognizing lipid antigens. In particular, the γ chain of TCR includes but is not limited to Vγ2, Vγ3, Vγ4, Vγ5, Vγ8, Vγ9, Vγ10, a functional variant thereof, and a combination thereof; and the δ chain of TCR includes but is not limited to δ1, δ2, δ3, a functional variant thereof, and a combination thereof. In some embodiments, the γδ TCR may be Vγ2/Vδ1TCR, Vγ2/Vδ2 TCR, Vγ2/Vδ3 TCR, Vγ3/Vδ1 TCR, Vγ3/Vδ2 TCR, Vγ3/Vδ3 TCR, Vγ4/Vδ1 TCR, Vγ4/Vδ2 TCR, Vγ4/Vδ3 TCR, Vγ5/Vδ1 TCR, Vγ5/Vδ2 TCR, Vγ5/Vδ3 TCR, Vγ8/Vδ1 TCR, Vγ8/Vδ2 TCR, Vγ8/Vδ3 TCR, Vγ9/Vδ1 TCR, Vγ9/Vδ2 TCR, Vγ9/Vδ3 TCR, Vγ10/Vδ1 TCR, Vγ10/Vδ2 TCR, and/or Vγ10/Vδ3 TCR. In some examples, the γδ TCR may be Vγ9/Vδ2 TCR, Vγ10/Vδ2 TCR, and/or Vγ2/Vδ2 TCR.

Variants

In some embodiments, amino acid sequence variants of the antibody moieties or other moieties provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody moiety. Amino acid sequence variants of an antibody moiety may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody moiety, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody moiety. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

In some embodiments, antibody binding domain moieties comprising one or more amino acid substitutions, deletions, or insertions are provided. Sites of interest for mutational changes include the antibody binding domain heavy and light chain variable regions (VRs) and frameworks (FRs) . Amino acid substitutions may be introduced into a binding domain of interest and the products screened for a desired activity, e.g., retained/improved antigen binding or decreased immunogenicity. In certain embodiments, amino acid substitutions may be introduced into one or more of the primary co-stimulatory receptor domain (extracellular or intracellular) , secondary costimulatory receptor domain, or extracellular co-receptor domain. Accordingly, the invention encompasses CoStAR proteins and component parts particularly disclosed herein as well as CoStAR proteins and component parts having at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to the amino acid sequences particulary disclosed herein. The terms “percent similarity, ” “percent identity, ” and “percent homology” when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program BestFit. Other algorithms may be used, e.g. BLAST, psiBLAST or TBLASTN (which use the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410) , FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448) ,

Particular amino acid sequence variants may differ from a reference sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids. In some embodiments, a variant sequence may comprise the reference sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues inserted, deleted or substituted. For example, 5, 10, 15, up to 20, up to 30 or up to 40 residues may be inserted, deleted or substituted.

In some preferred embodiments, a variant may differ from a reference sequence by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative substitutions. Conservative substitutions involve the replacement of an amino acid with a different amino acid having similar properties. For example, an aliphatic residue may be replaced by another aliphatic residue, a non-polar residue may be replaced by another non-polar residue, an acidic residue may be replaced by another acidic residue, a basic residue may be replaced by another basic residue, a polar residue may be replaced by another polar residue or an aromatic residue may be replaced by another aromatic residue. Conservative substitutions may, for example, be between amino acids within the following groups:

Conservative substitutions are shown in the Table below.

Amino acids may be grouped into different classes according to common side-chain properties: a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that influence chain orientation: Gly, Pro; aomatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

Cells

The cells used in the present invention may be any lymphocyte that is useful in adoptive cell therapy, such as a T-cell or a natural killer (NK) cell, an NKT cell, a γδ T cell or T regulatory cell. The cells may be allogeneic or autologous.

T cells or T lymphocytes are a type of lymphocyte that have a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells) , by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarised below. Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumour cells, and are also implicated in transplant rejection. CTLs express the CD8 molecule at their surface.

These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells) . Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO. Regulatory T cells (Treg cells) , formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.

Two major classes of CD4+ Treg cells have been described -naturally occurring Treg cells and adaptive Treg cells. Naturally occurring Treg cells (also known as CD4 ⁺CD25 ⁺FoxP3 ⁺ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD11c ⁺) and plasmacytoid (CD123 ⁺) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response.

Natural Killer Cells (or NK cells) are a type of cytolytic cell which form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner. NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes.

In certain embodiments, therapeutic cells of the invention comprise autologous cells engineered to express a construct or system of the invention. In certain embodiments, therapeutic cells of the invention comprise allogeneic cells engineered to express a construct or system of the invention.. Autologous cells may be advantageous in avoiding graft-versus-host disease (GVHD) due to CAR-or TCR-mediated recognition of recipient alloantigens. Also, the immune system of a recipient could attack the infused CAR-or TCR-bearing cells, causing rejection. In certain embdoiments, to prevent GVHD, and to reduce rejection, endogenous TCR is removed from allogeneic cells by genome editing.

Nucleic Acids

An aspect of the invention provides a nucleic acid sequence of the invention, encoding any of the CARs, TCRs, cytokines, polypeptides, or proteins described herein (including functional portions and functional variants thereof) . As used herein, the terms “polynucleotide” , “nucleotide” , and “nucleic acid” are intended to be synonymous with each other. It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed, e.g. codon optimization. Nucleic acids according to the invention may comprise DNA or RNA. They may be single stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

The terms “variant” , “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.

The nucleic acid sequences may be joined by a sequence allowing co-expression of the two or more nucleic acid sequences. For example, the construct may rearranged and comprise an internal promoter. There can be expression of multiple cytokines, using for example, an additional promoter, an internal ribosome entry sequence (IRES) sequence or a sequence encoding a cleavage site. The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into the discrete proteins without the need for any external cleavage activity. Various self-cleaving sites are known, including the Foot-and Mouth disease virus (FMDV) and the 2A self-cleaving peptide. The co-expressing sequence may be an internal ribosome entry sequence (IRES) . The co-expressing sequence may be an internal promoter.

Vectors

In an aspect, the present invention provides a vector which comprises a nucleic acid sequence or nucleic acid construct of the invention.

Such a vector may be used to introduce the nucleic acid sequence (s) or nucleic acid construct (s) into a host cell so that it expresses one or more CAR, TCR or antigen binding domain fused to CD3 chain of TCR complex, and inducible cytokine according to the first aspect of the invention and, optionally, one or more other proteins of interest (POI) . The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector or synthetic mRNA.

The nucleic acids of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.

Vectors derived from retroviruses, such as the lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene or transgenes and its propagation in daughter cells. The vector may be capable of transfecting or transducing a lymphocyte including a T cell or an NK cell. The present invention also provides vectors in which a nucleic acid of the present invention is inserted. The expression of natural or synthetic nucleic acids encoding a TCR, CAR or antigen binding domain fused to CD3 chain of TCR complex and inducible cytokine is typically achieved by operably linking a nucleic acid encoding the CAR, TCR or antigen binding domain fused to CD3 chain of TCR complex polypeptide or portions thereof to one promoters and the cytokine expressing portion to another promoter, and incorporating the construct into an expression vector.

Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

One example of a suitable CAR, TCR or antigen binding domain fused to CD3 chain of TCR complex promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-1α (EF-1α) . However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumour virus (MMTV) , human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.

The vectors can be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals, see also, WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193) . In some embodiments, the nucleic acid construct of the invention is a multi-cistronic construct comprising two promoters; one promoter driving the expression of the TCR or CAR. In some embodiments, the dual promoter constructs of the invention are uni-directional. In other embodiments, the dual promoter constructs of the invention are bi-directional. In order to assess the expression of the CAR or TCR polypeptide and cytokine polypeptides, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or transduced through viral vectors.

Sources of Cells

Prior to expansion and genetic modification, a source of cells (e.g., immune effector cells, e.g., T cells or NK cells) is cells obtained from a subject. The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals) . Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumours.

In one aspect, immune cells such as T cells or NK cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.

A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, T cells are isolated by incubation with anti-CD3/anti-CD28-conjugated beads, such as

M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one aspect, the time period is about 30 minutes. In a further aspect, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumour infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein) , subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In certain aspects, it may be desirable to perform the selection procedure and use the "unselected" cells in the activation and expansion process. "Unselected" cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD16, HLA-DR, and CD8. In certain aspects, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, CD137, PD1, TIM3, LAG-3, CD150 and FoxP3+. Alternatively, in certain aspects, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar method of selection.

The methods described herein can include, e.g., selection of a specific subpopulation of immune effector cells, e.g., T cells, that are a T regulatory cell-depleted population, CD25+ depleted cells, using, e.g., a negative selection technique, e.g., described herein. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%of CD25+ cells.

A specific subpopulation of effector cells that specifically bind to a target antigen can be enriched for by positive selection techniques. For example, in some embodiments, effector cells are enriched for by incubation with target antigen-conjugated beads for a time period sufficient for positive selection of the desired abTCR effector cells. In some embodiments, the time period is about 30 minutes. In some embodiments, the time period ranges from 30 minutes to 36 hours or longer (including all ranges between these values) . In some embodiments, the time period is at least one, 2, 3, 4, 5, or 6 hours. In some embodiments, the time period is 10 to 24 hours. In some embodiments, the incubation time period is 24 hours. For isolation of effector cells present at low levels in the heterogeneous cell population, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate effector cells in any situation where there are few effector cells as compared to other cell types. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention.

T cells or NK cells for stimulation can also be frozen after a washing step. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20%DMSO and 8%human serum albumin, or culture media containing 10

%Dextran

40 and 5%Dextrose, 20%Human Serum Albumin and 7.5%DMSO, or 31.25%Plasmalyte-A, 31.25%Dextrose 5%, 0.45%NaCl, 10

%Dextran

40 and 5%Dextrose, 20%Human Serum Albumin, and 7.5%DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80℃ at a rate of 1℃ per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C or in liquid nitrogen.

Allogeneic CAR and TCR effector cells

In embodiments described herein, the immune effector cell can be an allogeneic immune effector cell, e.g., T cell, γδ Tcell or NK cell. For example, the cell can be an allogeneic γδ T cell, e.g., an allogeneic γδ T cell with endogenous T cell receptor (TCR) or allogeneic αβ T cell lacking expression of endogenous T cell receptor (TCR) and/or human leukocyte antigen (HLA) , e.g., HLA class I and/or HLA class II.

A T cell lacking a functional endogenous TCR can be, e.g., engineered such that it does not express any functional TCR on its surface, engineered such that it does not express one or more subunits that comprise a functional TCR (e.g., engineered such that it does not express (or exhibits reduced expression) of TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon, and/or TCR zeta) or engineered such that it produces very little functional TCR on its surface. Alternatively, the T cell can express a substantially impaired TCR, e.g., by expression of mutated or truncated forms of one or more of the subunits of the TCR. The term "substantially impaired TCR" means that this TCR will not elicit an adverse immune reaction in a host.

A T cell or NK cell described herein can be, e.g., engineered such that it does not express a functional HLA on its surface. For example, a cell described herein can be engineered such that cell surface expression HLA, e.g., HLA class I and/or HLA class II, is downregulated. In some aspects, downregulation of HLA may be accomplished by reducing or eliminating expression of beta-2 microglobulin (B2M) .

In some embodiments, the cell can lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II. Modified cells that lack expression of a functional TCR and/or HLA can be obtained by any suitable means, including a knock out or knock down of one or more subunit of TCR or HLA. For example, the T cell or NK cell can include a knock down of TCR and/or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription-activator like effector nuclease (TALEN) , or zinc finger endonuclease (ZFN) .

In some embodiments, the allogeneic cell can be a cell which does not expresses or expresses at low levels an inhibitory molecule, e.g. a cell engineered by any method described herein. For example, the cell can be a cell that does not express or expresses at low levels an inhibitory molecule, e.g., that can decrease the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5) , LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276) , B7-H4 (VTCN1) , HVEM (TNFRSF14 or CD270) , KIR, A2aR, MHC class I, MHC class II, Gal9, adenosine, and TGFR beta. Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA, RNA or protein level, can optimize a CAR-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR) , a transcription-activator like effector nuclease (TALEN) , or a zinc finger endonuclease (ZFN) , e.g., as described herein, can be used.

siRNA and shRNA to inhibit endogenous TCR or HLA

In some embodiments, endogenous TCR expression and/or HLA expression can be inhibited using siRNA or shRNA that targets a nucleic acid encoding a TCR and/or HLA, and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5) , LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276) , B7-H4 (VTCN1) , HVEM (TNFRSF14 or CD270) , KIR, A2aR, MHC class I, MHC class II, Gal9, adenosine, and TGFR beta) , in a T cell.

Expression of siRNA and shRNAs in immune cells can be achieved using any conventional expression system, e.g., such as a lentiviral expression system. Exemplary shRNAs that downregulate expression of components of the TCR are described, e.g., in US Publication No.: 2012/0321667. Exemplary siRNA and shRNA that downregulate expression of HLA class I and/or HLA class II genes are described, e.g., in U.S. publication No.: US 2007/0036773.

CRISPR to inhibit endogenous TCR or HLA

"CRISPR" or "CRISPR to inhibit TCR and/or HLA” as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. "Cas" , as used herein, refers to a CRISPR-associated protein. A "CRISPR/Cas" system refers to a system derived from CRISPR and Cas which can be used to silence or mutate a TCR and/or HLA gene, and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5) , LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276) , B7-H4 (VTCNl) , HVEM (TNFRSF14 or CD270) , KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta) .

Naturally-occurring CRISPR/Cas systems are found in approximately 40%of sequenced eubacteria genomes and 90%of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845.

Activation and Expansion of Immune Cells

T cells may be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

Generally, the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30 (8) : 3975-3977, 1998; Haanen et al., J. Exp. Med. 190 (9) : 13191328, 1999; Garland et al., J. Immunol Meth. 227 (l-2) : 53-63, 1999) .

In some embodiments, expansion can be performed using flasks or containers, or gas-permeable containers known by those of skill in the art and can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, about 7 days to about 14 days, about 8 days to about 14 days, about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, or about 13 days to about 14 days. In some embodiments, the second TIL expansion can proceed for about 14 days.

In certain embodiments, the expansion can be performed using non-specific T-cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15) . The non-specific T-cell receptor stimulus can include, for example, an anti-CD3 antibody, such as about 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, N. J. or Miltenyi Biotech, Auburn, Calif. ) or UHCT-1 (commercially available from BioLegend, San Diego, Calif., USA) . CAR-or TCR-expressing cells can be expanded in vitro by including one or more antigens, including antigenic portions thereof, such as epitope (s) , of a cancer, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1: 26-35 (27 L) or gpl 00: 209-217 (210M) , optionally in the presence of a T-cell growth factor, such as 300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. CAR or TCR cells may also be rapidly expanded by re-stimulation with the same antigen (s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the cells can be further stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. In some embodiments, the stimulation occurs as part of the expansion. In some embodiments, the expansion occurs in the presence of irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

In certain embodiments, the cell culture medium comprises IL-2. In some embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL, or between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In certain embodiments, the cell culture medium comprises OKT3 antibody. In some embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, about 1 μg/mL or between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, or between 50 ng/mL and 100 ng/mL of OKT3 antibody.

In certain embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 are employed as a combination during the expansion. In some embodiments, IL-2, IL-7, IL-15, and/or IL-21 as well as any combinations thereof can be included during the expansion. In some embodiments, a combination of IL-2, IL-15, and IL-21 are employed as a combination during the expansion. In some embodiments, IL-2, IL-7, and IL-21 as well as any combinations thereof can be included.

In certain embodiments, the expansion can be conducted in a supplemented cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells.

In certain embodiments, the expansion culture media comprises about 500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15, about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100 IU/mL of IL-15, or about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15, or about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 300 IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 200 IU/mL of IL-15, or about 180 IU/mL of IL-15.

In some embodiments, the expansion culture media comprises about 20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about 10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, or about 0.5 IU/mL of IL-21, or about 20 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 12 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 10 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21, or about 2 IU/mL of IL-21. In some embodiments, the cell culture medium comprises about 1 IU/mL of IL-21, or about 0.5 IU/mL of IL-21.

In some embodiments the antigen-presenting feeder cells (APCs) are PBMCs. In an embodiment, the ratio of CAR-or TCR-expressing cells to PBMCs and/or antigen-presenting cells in the expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500, or between 1 to 50 and 1 to 300, or between 1 to 100 and 1 to 200.

In certain aspects, the primary stimulatory signal and the costimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation) . Alternatively, one agent may be coupled to a surface and the other agent in solution. In one aspect, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain aspects, both agents can be in solution. In one aspect, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

In one aspect, the two agents are immobilized on beads, either on the same bead, i.e., “cis, ” or to separate beads, i.e., “trans. ” By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co -immobilized to the same bead in equivalent molecular amounts. In one aspect, a 1: 1 ratio of each antibody bound to the beads for CD4+T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti CD3: CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1: 1. In one particular aspect an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1: 1. In one aspect, the ratio of CD3: CD28 antibody bound to the beads ranges from 100: 1 to 1: 100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3: CD28 is less than one. In certain aspects of the invention, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2: 1. In one particular aspect, a 1: 100 CD3: CD28 ratio of antibody bound to beads is used. In one aspect, a 1: 75 CD3: CD28 ratio of antibody bound to beads is used. In a further aspect, a 1: 50 CD3: CD28 ratio of antibody bound to beads is used. In one aspect, a 1: 30 CD3: CD28 ratio of antibody bound to beads is used. In one preferred aspect, a 1: 10 CD3: CD28 ratio of antibody bound to beads is used. In one aspect, a 1: 3 CD3: CD28 ratio of antibody bound to the beads is used. In yet one aspect, a 3: 1 CD3: CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1: 500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain aspects the ratio of cells to particles ranges from 1: 100 to 100: 1 and any integer values in-between and in further aspects the ratio comprises 1: 9 to 9: 1 and any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3-and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1: 100, 1: 50, 1: 40, 1: 30, 1: 20, 1: 10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, and 15: 1 with one preferred ratio being at least 1: 1 particles per T cell. In one aspect, a ratio of particles to cells of 1: 1 or less is used. In one particular aspect, a preferred particle: cell ratio is 1: 5. In further aspects, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one aspect, the ratio of particles to cells is from 1: 1 to 10: 1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1: 1 to 1: 10 (based on cell counts on the day of addition) . In one particular aspect, the ratio of particles to cells is 1: 1 on the first day of stimulation and adjusted to 1: 5 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1: 1 on the first day, and 1: 5 on the third and fifth days of stimulation. In one aspect, the ratio of particles to cells is 2: 1 on the first day of stimulation and adjusted to 1: 10 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1: 1 on the first day, and 1: 10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, ratios will vary depending on particle size and on cell size and type. In one aspect, the most typical ratios for use are in the neighborhood of 1: 1, 2: 1 and 3: 1 on the first day.

In further aspects of the present invention, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative aspect, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further aspect, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

Preparation of CAR-and TCR-expressing cells of the invention

Viral-and non-viral-based genetic engineering tools can be used to generate CAR-T cells, resulting in permanent or transient expression of therapeutic genes. Retrovirus-based gene delivery is a mature, well-characterized technology, which has been used to permanently integrate CARs into the host cell genome (Scholler J., e.g. Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci. Transl. Med. 2012; 4: 132ra53; Rosenberg S.A. et al., Gene transfer into humans-immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction. N. Engl. J. Med. 1990; 323: 570–578) .

Non-viral DNA transfection methods can also be used. For example, Singh et al describes use of the Sleeping Beauty (SB) transposon system developed to engineer CAR T cells (Singh H., et al., Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system. Cancer Res. 2008; 68: 2961–2971) and is being used in clinical trials (see e.g., ClinicalTrials. gov: NCT00968760 and NCT01653717) . The same technology is applicable to engineer T-cells, NK cells and the like according to the invention.

Multiple SB enzymes have been used to deliver transgenes. Mátés describes a hyperactive transposase (SB100X) with approximately 100-fold enhancement in efficiency when compared to the first-generation transposase. SB100X supported 35-50%stable gene transfer in human CD34 (+) cells enriched in hematopoietic stem or progenitor cells. (Mátés L. et al., Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat. Genet. 2009; 41: 753–761) and multiple transgenes can be delivered from multicistronic single plasmids (e.g., Thokala R. et al., Redirecting specificity of T cells using the Sleeping Beauty system to express chimeric antigen receptors by mix-and-matching of VL and VH domains targeting CD123+ tumors. PLoS ONE. 2016; 11: e0159477) or multiple plasmids (e.g., Hurton L. V. et al., Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Proc. Natl. Acad. Sci. USA. 2016; 113: E7788–E7797) . Such systems are used with CoStARs of the invention.

Morita et al, describes the piggyBac transposon system to integrate larger transgenes (Morita D. et al., Enhanced expression of anti-CD19 chimeric antigen receptor in piggyBac transposon-engineered T cells. Mol. Ther. Methods Clin. Dev. 2017; 8: 131–140) Nakazawa et al. describes use of the system to generate EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor (Nakazawa Y et al, PiggyBac-mediated cancer immunotherapy using EBV-specific cytotoxic T-cells expressing HER2-specific chimeric antigen receptor. Mol. Ther. 2011; 19: 2133–2143) . Manuri et al used the system to generate CD-19 specific T cells (Manuri P.V.R. et al., piggyBac transposon/transposase system to generate CD19-specific T cells for the treatment of B-lineage malignancies. Hum. Gene Ther. 2010; 21: 427–437) .

Transposon technology is easy and economical. One potential drawback is the longer expansion protocols currently employed may result in T cell differentiation, impaired activity and poor persistence of the infused cells. Monjezi et al describe development minicircle vectors that minimize these difficulties through higher efficiency integrations (Monjezi R. et al., Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors. Leukemia. 2017; 31: 186–194) . These transposon technologies can be used for CoStARs of the invention.

Pharmaceutical Compositions

The present invention also relates to a pharmaceutical composition containing a vector or cell of the invention expressing an activatable cytokine and CAR or TCR together with a pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active polypeptides and/or compounds.

In some embodiments, a pharmaceutical composition is provided comprising a vector or cell described above and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is provided comprising a nucleic acid encoding a CAR/cytokine construct according to any of the embodiments described above and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition is provided comprising an effector cell expressing a CAR/cytokine construct described above and a pharmaceutically acceptable carrier. Such a formulation may, for example, be in a form suitable for intravenous infusion.

As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

An aspect of the invention provides a population of modified effector cells expressing a recombinant CAR, TCR or antigen binding domain fused to CD3 chain of TCR complex and activatable cytokine. A suitable population may be produced by a method described above.

The population of modified effector cells may be for use as a medicament. For example, a population of modified T cells as described herein may be used in cancer immunotherapy therapy, for example adoptive T cell therapy.

Other aspects of the invention provide the use of a population of modified effector cells as described herein for the manufacture of a medicament for the treatment of cancer, a population of modified effector cells as described herein for the treatment of cancer, and a method of treatment of cancer may comprise administering a population of modified T cells as described herein to an individual in need thereof.

The population of modified effector cells may be autologous i.e. the modified effector cells were originally obtained from the same individual to whom they are subsequently administered (i.e. the donor and recipient individual are the same) . A suitable population of modified T cells for administration to the individual may be produced by a method comprising providing an initial population of T cells obtained from the individual, modifying the T cells to express a cAMP PDE or fragment thereof and an antigen receptor which binds specifically to cancer cells in the individual, and culturing the modified T cells.

The population of modified effector cells may be allogeneic i.e. the modified effector cells were originally obtained from a different individual to the individual to whom they are subsequently administered (i.e. the donor and recipient individual are different) . The donor and recipient individuals may be HLA matched to avoid GVHD and other undesirable immune effects.

Following administration of the modified effector cells, the recipient individual may exhibit a cell mediated immune response against cancer cells in the recipient individual. This may have a beneficial effect on the cancer condition in the individual.

Cancer conditions may be characterised by the abnormal proliferation of malignant cancer cells and may include leukaemias, such as AML, CML, ALL and CLL, lymphomas, such as Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma, and solid cancers such as sarcomas, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterus cancer, ovary cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, oesophageal cancer, pancreas cancer, renal cancer, adrenal cancer, stomach cancer, testicular cancer, cancer of the gall bladder and biliary tracts, thyroid cancer, thymus cancer, cancer of bone, and cerebral cancer, as well as cancer of unknown primary (CUP) .

Cancer cells within an individual may be immunologically distinct from normal somatic cells in the individual (i.e. the cancerous tumor may be immunogenic) . For example, the cancer cells may be capable of eliciting a systemic immune response in the individual against one or more antigens expressed by the cancer cells. The tumor antigens that elicit the immune response may be specific to cancer cells or may be shared by one or more normal cells in the individual.

An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse) , murine (e.g. a mouse) , canine (e.g. a dog) , feline (e.g. a cat) , equine (e.g. a horse) , a primate, simian (e.g. a monkey or ape) , a monkey (e.g. marmoset, baboon) , an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon) , or a human.

In preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals) may be employed.

Method of Treatment

The term “therapeutically effective amount” refers to an amount of a CAR-or TCR-and activatable cytokine or composition thereof as disclosed herein, effective to "treat" a disease or disorder in an individual. In the case of cancer, the therapeutically effective amount as disclosed herein can reduce the number of cancer cells; reduce the tumor size or weight; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent a composition for expressing a CAR or TCR and activatable cytokine herein can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. In some embodiments, the therapeutically effective amount is a growth inhibitory amount. In some embodiments, the therapeutically effective amount is an amount that improves progression free survival of a patient. In the case of infectious disease, such as viral infection, the therapeutically effective amount of a cell or composition as disclosed herein can reduce the number of cells infected by the pathogen; reduce the production or release of pathogen-derived antigens; inhibit (i.e., slow to some extent and preferably stop) spread of the pathogen to uninfected cells; and/or relieve to some extent one or more symptoms associated with the infection. In some embodiments, the therapeutically effective amount is an amount that extends the survival of a patient.

Cells, including αβ T cells, γδ T cells, NK cells, and the like, expressing CARs or TCRs and activatable cytokines for use in the methods of the present may either be created ex vivo either from a patient's own peripheral blood (autologous) , or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (allogenic) , or peripheral blood from an unconnected donor (allogenic) . Alternatively, the cells may be derived from ex-vivo differentiation of inducible progenitor cells or embryonic progenitor cells to αβ T cells, γδ T cells, or NK cells. In these instances, T-cells expressing an activatable cytokine with CAR, TCR or antigen binding domain fused to CD3 chain of TCR complex, are generated by introducing DNA or RNA coding for the activatable cytokine with CAR, TCR or antigen binding domain fused to CD3 chain of TCR complex, by one of many means including transduction with a viral vector, transfection with DNA or RNA.

αβ T cells, γδ T cells, or NK cells expressing an activatable cytokine with CAR or TCR of the present invention may be used for the treatment of haematological cancers or solid tumors.

A method for the treatment of disease relates to the therapeutic use of a vector or cell, including a αβ T cell, γδ T cell, or NK cell, of the invention. In this respect, the vector, or αβ T cell, or γδ T cell, or NK cell may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease. The method of the invention may cause or promote T-cell mediated killing of cancer cells. The vector, the vector, or αβ T cell, or γδ T cell, or NK cell according to the present invention may be administered to a patient with one or more additional therapeutic agents. The one or more additional therapeutic agents can be co-administered to the patient. By “co-administering” is meant administering one or more additional therapeutic agents and the vector, or T or NK cell of the present invention sufficiently close in time such that the vector, or T or NK cell can enhance the effect of one or more additional therapeutic agents, or vice versa. In this regard, the vectors or cells can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the vectors or cells and the one or more additional therapeutic agents can be administered simultaneously. One co-administered therapeutic agent that may be useful is IL-2, as this is currently used in existing cell therapies to boost the activity of administered cells. However, IL-2 treatment is associated with toxicity and tolerability issues.

As mentioned, for administration to a patient, the effector cells of the invention can be allogeneic or autologous to the patient. In certain embodiments, allogeneic cells are further genetically modified, for example by gene editing, so as to minimize or prevent GVHD and/or a patient’s immune response against the effector cells.

The effector cells are used to treat cancers and neoplastic diseases associated with a target antigen. Cancers and neoplastic diseases that may be treated using any of the methods described herein include tumours that are not vascularized, or not yet substantially vascularized, as well as vascularized tumours. The cancers may comprise non-solid tumours (such as hematological tumours, for example, leukemias and lymphomas) or may comprise solid tumours. Types of cancers to be treated with the effector cells of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumours, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumours/cancers and pediatric tumours/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia) , chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia) , polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms) , multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumours are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumours can be benign or malignant. Different types of solid tumours are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas) . Examples of solid tumours, such as sarcomas and carcinomas, include adrenocortical carcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, stomach cancer, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid cancer (e.g., medullary thyroid carcinoma and papillary thyroid carcinoma) , pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumour, cervical cancer (e.g., cervical carcinoma and pre-invasive cervical dysplasia) , colorectal cancer, cancer of the anus, anal canal, or anorectum, vaginal cancer, cancer of the vulva (e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, and fibrosarcoma) , penile cancer, oropharyngeal cancer, esophageal cancer, head cancers (e.g., squamous cell carcinoma) , neck cancers (e.g., squamous cell carcinoma) , testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumour, fibroma, fibroadenoma, adenomatoid tumours, and lipoma) , bladder carcinoma, kidney cancer, melanoma, cancer of the uterus (e.g., endometrial carcinoma) , urothelial cancers (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureter cancer, and urinary bladder cancer) , and CNS tumours (such as a glioma (such as brainstem glioma and mixed gliomas) , glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases) .

When “an immunologically effective amount, ” “an anti-tumour effective amount, ” “a tumour-inhibiting effective amount, ” or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumour size, extent of infection or metastasis, and condition of the patient (subject) . It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10 ⁴ to 10 ⁹ cells/kg body weight, in some instances 10 ⁵ to 10 ⁶ cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988) .

Combination Therapies

An effector cell of the invention described herein may be used in combination with other known agents and therapies. Administered "in combination" , as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery" . In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

An effector cell expressing an activatable cytokine and CAR or TCR described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the CAR-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

The cellular therapy of the invention and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The cellular therapy can be administered before the other treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.

When administered in combination, the therapy and the additional agent (e.g., second or third agent) , or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of the effector cell therapy, the additional agent (e.g., second or third agent) , or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy. In other embodiments, the amount or dosage of the cellular therapy, the additional agent (e.g., second or third agent) , or all, that results in a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%lower) than the amount or dosage of each agent used individually, e.g., as a monotherapy, required to achieve the same therapeutic effect.

In further aspects, an activatable cytokine with CAR or TCR-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation, peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108: 963-971.

In certain instances, compounds of the present invention are combined with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, anti-nausea agents (or anti-emetics) , pain relievers, cytoprotective agents, and combinations thereof.

In one embodiment, a CAR or TCR with activatable cytokine-expressing cell described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin) ) , a vinca alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine) , an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide) , an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab) , an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine) ) , an mTOR inhibitor, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib) , an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide) .

General Chemotherapeutic agents considered for use in combination therapies include busulfan

busulfan injection

cladribine

cyclophosphamide

cytarabine, cytosine arabinoside

cytarabine liposome injection

daunorubicin hydrochloride

daunorubicin citrate liposome injection

dexamethasone, , doxorubicin hydrochloride

etoposide

fludarabine phosphate

hydroxyurea

Idarubicin

mitoxantrone

Gemtuzumab Ozogamicin

In embodiments, general chemotherapeutic agents considered for use in combination therapies include anastrozole

bicalutamide

bleomycin sulfate

busulfan

busulfan injection

capecitabine

N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin

carmustine

chlorambucil

cisplatin

cladribine

cyclophosphamide

cytarabine, cytosine arabinoside

cytarabine liposome injection

dacarbazine

dactinomycin (Actinomycin D, Cosmegan) , daunorubicin hydrochloride

daunorubicin citrate liposome injection

dexamethasone, docetaxel

doxorubicin hydrochloride

etoposide

fludarabine phosphate

5-fluorouracil

flutamide

tezacitibine, Gemcitabine (difluorodeoxycitidine) , hydroxyurea

Idarubicin

ifosfamide

irinotecan

L-asparaginase

leucovorin calcium, melphalan

6-mercaptopurine

methotrexate

mitoxantrone

mylotarg, paclitaxel

phoenix (Yttrium90/MX-DTPA) , pentostatin, polifeprosan 20 with carmustine implant

tamoxifen citrate

teniposide

6-thioguanine, thiotepa, tirapazamine

topotecan hydrochloride for injection

vinblastine

vincristine

and vinorelbine

Treatments can be evaluated, for example, by tumour regression, tumour weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, quality of life, protein expression and/or activity. Approaches to determining efficacy of the therapy can be employed, including for example, measurement of response through radiological imaging.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

Examples

Example 1: Plasmid construction, virus preparation, titer evaluation

Chimeric antigen receptors armored with different cytokines, were designed as shown in FIG. 1-3 and SEQ ID NO: 1 to SEQ ID NO: 22 and SEQ ID NO: 31 to SEQ ID NO: 47. To generate viral particles comprising polynucleic acids encoding any of the systems disclosed herein, lentivirus packaging plasmid mixture including pMDLg/pRRE (Addgene#11251) , pRSV-Rev (Addgene#11253) , and pMD2. G (Addgene#11259) were pre-mixed with a PLVX-EF1A (including target system) vector at a pre-optimized ratio with polyetherimide (PEI) , mixed properly, and incubated at room temperature for 5 minutes. The transfection mix is added dropwise to 293-T cells and mixed gently. Transfected 293-T cells were incubated overnight at 37℃ and 5%CO ₂. Twenty-four hours post-transfection, supernatants were collected and centrifuged at 4℃, 500 g for 10 min to remove any cellular debris. Centrifuged supernatants were filtered through a 0.45 μm PES filter to concentrate the viral supernatants post-ultracentrifugation. After centrifugation, the supernatants were carefully discarded and the virus pellets were rinsed with pre-chilled DPBS. The concentration of virus were measured. Virus was aliquoted and stored at -80℃. Viral titers were determined by functional transduction on a T cell line. Specifically, SEQ ID NO: 22, 3 and 7 were chosen for demonstration with gamma/delta T cells in the following examples and named CAR, CAR-15 and CAR-i15 hereafter.

Briefly, the lentiviral vector was modified using pLVX-Puro (Clontech#632164) by replacing the original promoter with human elongation factor 1α promoter (hEF1α) and by removing the puromycin resistance gene with EcoRI and BamHI by GenScript. PLVX-EF1A was further subjected to the lentivirus packaging procedure as described above.

Example 2: T cell transduction and FACS analysis of transduced T cells

Cell expansion

Leukocytes were collected in R10 medium, then mixed with 0.9%NaCl solution at a 1: 1 (v/v) ratio. Lymphoprep medium of 3 mL in volume was added to a 15 mL centrifuge tube and slowly layered to form 6 mL of diluted lymphocyte mix. The lymphocyte mix was centrifuged at 800 g for 30 minutes without brakes at 20 ℃. Lymphocyte buffy coat was then collected with a 200 μL pipette. The harvested fraction was diluted at least 6 fold of 0.9%NaCl or R10 to reduce the density of the solution before further centrifugation at 250g for 10 minutes at 20℃. The supernatant was aspirated completely, and 10 mL of R10 was added to the cell pellet. The mixture was further centrifuged at 250 g for 10 minutes at 20℃. The supernatant was then aspirated. Two milliliters R10 pre-warmed at 37℃ with 100 IU/mL IL-2 was added to the cell pellet, and the cell pellet was gently re-suspended. Cells were quantified and the PBMC sample was ready for experimentation.

Gamma/delta T cells were activated by addition of 5 μM Zoledronate and 1000 IU/mL IL-2 to PBMCs and cultured for 14 days with periodical change of media supplemented with 1000 IU/mL IL-2.

Alternatively, gamma/delta T cells can be isolated from PBMC or umbilical cord blood (UCB) and then stimulated by anti-gamma/delta TCR antibody and anti-CD3 (OKT3) followed by co-incubation of K562-based artificial antigen-presenting cells (aAPCs) at an 1: 2 ratio for at least 10 days.

αβ T cells transduction

Pre-activated alpha/beta T cells were collected and resuspended in 1640 medium containing 300 IU/mL IL-2. A lentiviral vector encoding the system of Example 1 was diluted to MOI = 5 with the same medium and infected with 1 × 10 ⁶ activated T cells. The pre-activated T cells were transduced with lentivirus stock in the presence of 8 μg/ml polybrene with centrifugation at 1000 g, 32 ℃ for 1h. The transduced cells were then transferred to the cell culture incubator for transgene expression under suitable conditions. The following day, the transduced cells were centrifuged and replaced with fresh media. Cell density was measured every other day, and fresh media were added to continue the expansion.

γδ T cells transduction

Forty-eight hours post-activation, γδ T cells were transduced with lentiviral vectors encoding the system of Example 1 at an MOI of 5 with 5 pg/ml polybrene. Cells were cultured in AIM-V supplemented with IL-2 (1000 IU/ml) in a humidified chamber with periodical change of media as determined by the pH of the culture media for further expansion. Cells were harvested 10 days post-transduction and the total number, purity and transduction efficiency were determined. Cells could be further enriched with a negative TCRγ/δ+ T cell isolation kit (Miltenyi Biotec) before future applications or cryopreserved.

Example 3: Quantification of transgene expression

On day 3 and onwards post transduction, cells were evaluated for expression of the system of Example 1 by flow cytometry. An aliquot of cells was collected from the culture before washed, pelleted, and resuspended in diluted antibodies (eBioscience Anti-human TCR beta PE, anti-CAR Ab) at a dilution factor of 100 in PBS+0.5%FBS –50-100 μl per sample. Cells were resuspended in about 50 to 100 μl of solution. Cells were incubated at 4℃ for 30 minutes. Viability dye eFluor780 or SYTOX Blue viability stain was also added according to manufacturer’s instructions. Post-incubation, cells were washed twice in PBS and resuspended in 100 to 200 μl PBS for analysis. The mean fluorescence of the system was quantified by flow cytometry.

For anti-CD19 CAR-T (SEQ ID NO: 21) staining, cells were stained with Alexa Fluor 488-labeled human CD19 protein (Genscript) . Flow cytometry analysis for all experiments was performed by using FlowJo (Tree Star, Inc. ) .

For anti-BCMA CAR-T (SEQ ID NO: 22) staining, cells were stained with Alexa Fluor 488-labeled mouse-anti-camel sdAb antibodies (Genscript) . Flow cytometry analysis for all experiments was performed by using FlowJo (Tree Star, Inc. ) .

As illustrated in FIG. 3, pro-inflammatory polypeptides interleukin 7 (IL7) and chemokine (C-C motif) ligand 19 (CCL19) were selected. Conventional CAR-T cells (CAR) as well as CAR-T cells with constitutive expression of IL7 and CCL19 (CAR-7X19) were used as internal references to demonstrate the potency of T cell intrinsic regulatory elements (TIRE) -mediated inducible expression (CAR-i7X19) .

For CAR-αβ-T cells, CAR expression was determined at 4 days post infection by a rabbit Anti-VHH antibody (GenScript) via flow cytometry (BD FACsCelesta) . CAR positive rate and geometric mean expression (mean fluorescent intensity, MFI) was further analyzed by Flowjo 7.6. Notably, TIRE did not have any significant effect on CAR expression (FIG. 4A-4C) .

For CAR-γδ-T cells, as shown in FIG. 4D-4F, CAR expression were determined to be 42.4%, 35.5%and 24.4%for CAR-, CAR-15-and CAR-i15-transduced γδ T cells, respectively. Analysis of CAR expression demonstrated that TIRE did not drastically impact CAR expression on γδ-T cells.

Example 4. AP-1, NF-κB-induced expression of CAR-T armors

To investigate the potency of a TIRE-induced expression of armors, IL7 and CCL19 secretion from CAR-αβ T cells were evaluated in the absence and presence of TAA stimulation.

In brief, 1 × 10 ⁵ DLL3-targeting CAR+ αβ T cells were cultured alone or together with 5 × 10 ⁵ DLL3-expressing SHP-77 cells (E: T=1: 5) . Cell supernatants were harvested after two days and analyzed for IL7 and CCL19 production using Human IL-7 ELISA kit (R&D, #DY207) and Human CCL19 ELISA kit (R&D, #DY361) by PHERAstar Fsx (manufactured by BMG LABTECH) .

As shown in FIG. 5, while CAR-T cells with constitutive expression of IL7 (FIG. 5A) and CCL19 (FIG. 5B) show high expression of IL9 and CCL19, CAR-i7X19 secreted about 10%of amount of IL7 and CCL19 at the basel level without TAA stimulation. Strikingly, upon TAA engagement, CAR-i7X19 significantly up-regulates the release of IL7 and CCL19 by about 6 fold and 2 fold, respectively, suggesting a robust of activation of the TIRE induced protein expression.

To investigate the potency of an AP-1, NF-κB-induced expression of armors, IL-15 secretion from CAR-T cells were evaluated in the absence and presence of TAA stimulation.

In brief, 0.5 × 10 ⁵ BCMA-targeting CAR+ T cells were cultured alone or together with 2 x 10 ⁵ BCMA-expressing H929 cells (E: T=1: 4) . Cell supernatants were harvested 48 hours after initial incubation and analyzed for IL-15 production using Human IL-15 ELISA kit (R&D, #D1500) by PHERAstar Fsx (manufactured by BMG LABTECH) .

As shown in FIG. 5C, while CAR-T cells with constitutive expression of IL-15 displayed high expression of IL-15, CAR-i15 secreted about 5%of amount of IL-15 at the basel level without TAA stimulation. In comparison, upon TAA engagement, CAR-i7X19 significantly up-regulated the release of IL-15 by about 10 folds while the level remained unchanged with CAR-15. This suggests the robust of activation of the AP-1, NF-κB induced protein expression in γδ T cells.

Example 5: In vitro Cytotoxicity assay of CAR-T cells

Cytotoxicity of designed CARs, as well as their control γδ T cells was determined in a 20 h co-culture assay. In the experiments, the effector cells were centrifugally collected, then diluted to the desired concentrations with 1640 phenol-red free medium (Invitrogen) with 2%heat inactivated FBS (Invitrogen) . The target cell, H929, exhibited decent expression of target antigens BCMA. The target cell, Raji, exhibited decent expression of target antigens CD19. The effector cells were co-cultured at different effector to target ratios (E: T = 4: 1, 2: 1, 1: 1.0.5: 1 and 0.25: 1) at 37℃ for 20 h in 96 well plates. Additional wells contain assay buffer only (1640 phenol red-free medium plus 2%hiFBS) , target cell only (T) , effector cell only (E) and max release of target cell (1%solution of triton-X 100) . Each condition was performed in triplicate, and the cytotoxicity of effector cells was detected by LDH assay kit (Roche) . After completion of the 20 hr co-culture, the assay plate was centrifuged, and supernatants were collected in a new 96-well plate. The supernatant plate was diluted with an equal volume of the LDH assay reagent according to the manufacture’s manual. The assay plate was incubated for about 30 min at 15℃-25℃. The absorbance of the plate was measured at 492 nm and 650 nm using Flexstation reader (Molecular Devices) . The specific cytotoxicity was calculated by the formula: %Target cell lysis = 100* [ (OD _CAR-T cell _{+Target cell}) - (OD _CAR-T cell) - (OD _Target cell) + (OD _{Buffer background}) ] / (OD _{Target Maximum release} –OD _{Target Minimum release}) .

For CAR-αβ T cells, as shown in FIG. 6A, in contrast to little cytotoxicity of untransduced cells against tumor cells, specific cytotoxicity was observed in CAR-T cell groups. Although no significant difference was found between three groups at the ratio with E:T = 1: 1, a slight increase of efficacy was observed in CAR-T cells armored with either constitutive or a TIRE-induced expression of IL7 and CCL19 at low E: T ratio of 0.3: 1.

For CAR-γδ T cells, we demonstrated that TAA-inducible, AP-1, NF-κB-responsive design, CAR-i15, displayed similar level of short-term cytotoxicity towards BCMA-positive target cells, RPMI 8226 and H929 (FIG. 6B) .

Example 6. Cytokine release of CAR-T cells

To further analyze the effect of CAR-T cells against tumor cells, interferon-γrelease was analyzed in the culture supernatant using the HTRF human IFN gamma kit (Cisbio, #62HIFNGPEH) .

For CAR-αβ T cells, as shown in FIG. 7A, in contrast to little secretion of IFN-γby untransduced T cells, CAR-T cells secreted functional amount of IFN-γ. Of note, while the cytotoxicity was comparable between different CAR-T cells, or even slighter higher by CAR-T cells with a TIRE-inducible armors, the IFN-γ release was visibly lower comparing to conventional CAR or CAR with a constitute armor. This set of results indicate that TIRE CAR-T cells might be equipped with as potent cytotoxicity as conventional CAR T cells but be kept under relative less active state.

For CAR-γδ T cells , while CAR-i15 displayed similar level of anti-tumor cytotoxicity demonstrated in the previous example, we found that it consistently secreted significantly less IFN-γ than CAR-15 and even unarmored CAR-γδ T cells (FIG. 7B) . This might suggest that inducible expression of IL-15, via AP-1 and NF-κB in this particular case, can lead to a lower cytokine armor expression, thus displaying a safer profile than the constitutive armor expression designs.

Example 7: Persistent cytotoxicity assayed by repetitive TAA stimulations

Persistence of CAR-αβ T cells was evaluated in a repetitive tumor challenge assay. In brief, 0.75 × 10 ⁴ CAR+ T cells were co-cultured with 3 × 10 ⁵ SHP-77 cells in a 24 well. Three days later, cells were harvested to determine the relative ratio of viable T cell and tumor cell. CAR+ T was quantified and re-plated with fresh SHP-77 cells at a ratio of 1: 4 for the next round. IFN-γ release in the supernatant was determined at the end of each round. The exhaustion markers of CAR-T cells were evaluated by the end of the third round.

As shown in FIG. 8A and 8B, although a slight higher cytotoxicity was observed in CAR-7X19 and CAR-i7X19, no obvious difference was observed in these two groups in comparison to conventional CAR-T cells in T cell persistence (FIG. 8A) and expansion (FIG. 8B) . However, the T cell exhaustion markers (PD-1, TIM-3 and LAG-3) were strikingly less expressed in the group of CAR-i7X19 CAR-T cells (FIG. 9) . In line with comparable persistence and expansion, no difference was observed in IFN-γ released during repetitive tumor cell stimulations (FIG. 10A) .

Persistence of CAR-γδ T cells were evaluated with a repetitive tumor challenge assay. In brief, 1 × 10 ⁵ CAR+ γδ T cells were co-cultured with 3 × 10 ⁵ H929 cells in a 24 well. Two days later, cells were harvested to determine the relative ratio of viable T cell and tumor cell. CAR+ T was quantified and re-plated with fresh H929 cells at a ratio of 1: 3 for the next round. IFN-γ release in the supernatant was determined at the end of each round.

As shown in FIG. 8C and 8D, both CAR-15 and CAR-i15 displayed better persistence in anti-tumor cytotoxicity (FIG. 8C) and expansion (FIG. 8D) compared to unarmored CAR-T cells. Similar to the observation made with short-term anti-tumor cytotoxicity assays, IFN-γ released during repetitive tumor cell stimulations was consistently lower in CAR-i15 group than CAR-15 (FIG. 10B) . Taken together, TAA-induced, AP-1-, NF-κB responsive-IL-15 armored CAR-γδ T cells displayed more persistent anti-tumor cytotoxicity while maintaining a safer profile characterized by lower IFN-γ production.

Example 8. Therapeutic Effect in Tumor Model

To further demonstrate the anti-tumor efficacy of a TIRE-mediated inducible IL7 and CCL19 derived armors in CAR-αβ T cells, a SHP77 derived xenotransplantations was used in vivo. To this end, NCG mice (NOD-Prkdc Cd5 I12rg Cd /NjuCrl) were subcutaneously injected with SHP-77/FF-Luc cells. A single dose of untransduced T cells (2.43 × 10 ⁶) or CAR+ T cells (2.5 × 10 ⁵) was administered intravenously to tumor engrafted mice 14 days after tumor inoculation. Tumor length (L) and width (W) was measured by caliper every 3-4 days after CAR-T cells treatment. Tumor volume was estimated using formula: V = (W2× L) /2. Moreover, fluorescence intensity derived from tumor cells was measured once a week (total flux (photons/sec) ) using in vivo imaging system (IVIS) . CAR-T proliferation in peripheral blood were monitored once a week for 5 weeks. Plasma were harvested were analyzed IL7 and CCL19 production at 14 and 21 days post injection.

As shown in FIG. 11, thirty-three days after treatment, the NCG mice treated with CAR-T cells all showed reduced tumor burden comparing with the untransduced T cell-treated group (FIG. 11A and 11B) . In comparison to conventional CAR-T cells, the anti-tumor efficacy is more pronounced in CAR-T cell armored constitutive IL7 × CCL19 or inducible IL7 × CCL19 (FIG. 11A) . In striking contrast to tumor regression determined by bioluminescence imaging, tumor size determined by caliper show significant relapse in the group of CAR-T cell armored constitutive IL7 × CCL19 (FIG. 11B) , suggesting that pseudoprogression judged by the tumor size was most likely induced by IL7 and CLL19-mediated local inflammatory response. T cell expansion in peripheral blood was further determined in FIG. 12. A notable increase of CAR+ T cells (FIG. 12) were observed at day 33 post infusion, confirming a beneficial effect of constitutive IL7 × CCL19 and inducible IL7 × CCL19 in promoting CAR-T cell proliferation. Expression level of IL7 and CLL19 was further evaluated in FIG. 13. In comparison to constitutive IL7 × CCL19 CAR, a significantly reduction of peripheral levels of IL7 (FIG. 13A) and CCL19 (FIG. 13B) was observed in the group of CAR-T armored with a TIRE-mediated inducible IL7 × CCL19 CAR.

Collectively, these results strikingly suggested that the superiority of CAR-T cell armored with TIRE-derived induction of IL7 × CCL19 in tumor regression and CAR-T expansion, and further protecting mice from disease progression. This set of data further indicated that a TIRE-mediated expression armors have negelectable side-effect in predisposing of systemic inflammation.

Anti-tumor activity of an exemplary anti-BCMA CAR-T is assessed in vivo in an RPMI-8226 xenograft model. Briefly, one million (1×10 ⁶) RPMI-8226 cells stably expressing the firefly luciferase reporter are implanted subcutaneously/intravenously on day 0 in NOD/SCID IL-2RγCnull (NSG) mice. Fourteen days after tumor inoculation, mice are treated with intravenous injection of 1 × 10 ⁶ armored CAR-alpha/beta T or gamma/delta T or mock T cells or phosphate-buffered saline (PBS) . Tumor progression was monitored by bioluminescent imaging (BLI) once a week. In addition, T cell proliferation is monitored via FACS analysis from plasma drawn from blood.

Anti-tumor activity of an exemplary anti-CD19 CAR-T is assessed in vivo in an Raji xenograft model. Briefly, one million (1×10 ⁶) Raji cells stably expressing the firefly luciferase reporter are implanted subcutaneously/intravenously on day 0 in NOD/SCID IL-2RγCnull (NSG) mice. Seven days after tumor inoculation, mice are treated with intravenous injection of 1 × 10 ⁶ armored CAR-αβ T or γδ T or mock T cells or phosphate-buffered saline (PBS) . Tumor progression was monitored by bioluminescent imaging (BLI) once a week. In addition, T cell proliferation is monitored via FACS analysis from plasma drawn from blood.

For toxicity evaluations, clinical symptoms were observed every day, while the animals’ body weights and the fluorescence intensities triggered by tumor-Luc cells were measured every week. Blood (0.2 mL) was taken every week for detecting the humanized cytokine profiles (IL-15, IFN-γ and TNF) in mice.

Example 9: Exemplary constructs

The cytokines IL-7, IL-12, IL-15, IL-21 have clear function and increase immune cell fitness and/or increase immune cell cytotoxicity. The following constructs, reflected in Table 1 were constructed and characterized in vitro and in vivo: anti-CD19–BBζ/IL-7 CAR, anti-CD19–BBζ/IL-15 CAR, anti-CD19–BBζ/IL-21 CAR, anti-BCMA–BBζ/IL-7 CAR, anti-BCMA–BBζ/IL-15 CAR, anti-BCMA–BBζ/IL-21 CAR, anti-CD19–BBζ/NFkB-IL-7 CAR, anti-CD19–BBζ/NFkB-IL-15 CAR, anti-CD19–BBζ/NFkB-IL-21 CAR, anti-BCMA–BBζ/NFkB-IL-7 CAR, anti-BCMA–BBζ/NFkB-IL-15 CAR, anti-BCMA–BBζ/NFkB-IL-21 CAR, anti-CD19–BBζ/NFkB-AP-1-IL-7 CAR, anti-CD19–BBζ/NFkB-AP-1-IL-15 CAR, anti-CD19–BBζ/NFkB-AP-1-IL-21 CAR, anti-BCMA–BBζ/NFkB-AP-1-IL-7 CAR, anti-BCMA–BBζ/NFkB-AP-1-IL-15 CAR, anti-BCMA–BBζ/NFkB-CAP-1-IL-21 CAR. SEQ ID NOs: 1-4 are amino acid sequences corresponding to the structure depicted in FIG. 1. SEQ ID NOs: 5-20 are nucleotide sequences corresponding to the structure depicted in FIG. 2, beginning at the 5’ end with the start codon of the CAR and terminating at the 3’ end at the NF-κB and AP-1 binding sites which regulate transcription of the cytokine in the opposite direction. Constructs corresponding to FIG. 2 comprise a minimal IFNβ promoter and NF-κB and/or AP-1 binding motifs in the following numbers and combinations: 3x NF-κB, 5x NF-κB, 3x NF-κB and 3xAP-1, 5x NF-κB and 5x AP-1.

Nucleotide and Amino Acid Sequences

SEQ ID NO: 1 (Human IL-15-armored anti-CD19 CAR amino acid sequence)

SEQ ID NO: 2 (Human IL-7-armored anti-CD19 CAR amino acid sequence)

SEQ ID NO: 3 (Human IL-15-armored anti-BCMA CAR amino acid sequence)

SEQ ID NO: 4 (Human IL-7-armored anti-BCMA CAR amino acid sequence)

SEQ ID NO: 5 (3NF-κB inducible human IL-15-armored anti-CD19 CAR nucleic acid sequence)

SEQ ID NO: 6 (3NF-κB inducible human IL-7-armored anti-CD19 CAR nucleic acid sequence)

SEQ ID NO: 7 (3NF-κB inducible human IL-15-armored anti-BCMA CAR nucleic acid sequence)

SEQ ID NO: 8 (3NF-κB inducible human IL-7-armored anti-BCMA CAR nucleic acid sequence)

SEQ ID NO: 9 (3NF-κB/3AP-1 inducible human IL-15-armored anti-CD19 CAR nucleic acid sequence)

SEQ ID NO: 10 (3NF-κB/3AP-1 inducible human IL-7-armored anti-CD19 CAR nucleic acid sequence)

SEQ ID NO: 11 (3NF-κB/3AP-1 inducible human IL-15-armored anti-BCMA CAR nucleic acid sequence)

SEQ ID NO: 12 (3NF-κB/3AP-1 inducible human IL-7-armored anti-BCMA CAR nucleic acid sequence)

SEQ ID NO: 13 (5NF-κB inducible human IL-15-armored anti-CD19 CAR nucleic acid sequence)

SEQ ID NO: 14 (5NF-κB inducible human IL-7-armored anti-CD19 CAR nucleic acid sequence)

SEQ ID NO: 15 (5NF-κB inducible human IL-15-armored anti-BCMA CAR nucleic acid sequence)

SEQ ID NO: 16 (5NF-κB inducible human IL-7-armored anti-BCMA CAR nucleic acid sequence)

SEQ ID NO: 17 (5NF-κB/5AP-1 inducible human IL-15-armored anti-CD19 CAR nucleic acid sequence)

SEQ ID NO: 18 (5NF-κB/5AP-1 inducible human IL-7-armored anti-CD19 CAR nucleic acid sequence)

SEQ ID NO: 19 (5NF-κB/5AP-1 inducible human IL-15-armored anti-BCMA CAR nucleic acid sequence)

SEQ ID NO: 20 (5NF-κB/5AP-1 inducible human IL-7-armored anti-BCMA CAR nucleic acid sequence)

SEQ ID NO: 21 (Anti-CD19 CAR amino acid sequence)

SEQ ID NO: 22 (Anti-BCMA CAR amino acid sequence)

SEQ ID NO: 23 (Human IL-15 amino acid sequence)

SEQ ID NO: 24 (Human IL-7 amino acid sequence)

SEQ ID NO: 25 (1NF-κB nucleic acid sequence)

SEQ ID NO: 26 (3NF-κB nucleic acid sequence)

SEQ ID NO: 27 (5NF-κB nucleic acid sequence)

SEQ ID NO: 28 (1NF-κB/1AP-1 nucleic acid sequence)

SEQ ID NO: 29 (3NF-κB/3AP-1 nucleic acid sequence)

SEQ ID NO: 30 (5NF-κB/5AP-1 nucleic acid sequence)

SEQ ID NO: 31 (the polypeptide sequence of human CD8a signal peptide)

SEQ ID NO: 32 (the polypeptide sequence of human CD8a hinge)

SEQ ID NO: 33 (the polypeptide sequence of human CD8a Transmembrane)

SEQ ID NO: 34 (the polypeptide sequence of human 4-1BB Topological domain)

SEQ ID NO: 35 (the polypeptide sequence of human CD3 zeta chain Topological domain)

SEQ ID NO: 36 (the polypeptide sequence of human CCL19)

SEQ ID NO: 37 (the polypeptide sequence of P2A)

SEQ ID NO: 38 (the polypeptide sequence of DLL3 binder)

SEQ ID NO: 39 (the nucleotide sequence of NFAT response element)

SEQ ID NO: 40 (the nucleotide sequence of AP-1 response element)

SEQ ID NO: 41 (the nucleotide sequence of NF-κB response element)

SEQ ID NO: 42 (the nucleotide sequence of 5NFAT response element)

SEQ ID NO: 43 (the nucleotide sequence of 5AP-1 response element)

SEQ ID NO: 44 (the nucleotide sequence of 5NF-κB response element)

SEQ ID NO: 45 (the nucleotide sequence of 5NFAT-5AP-1 -5NF-κB response element)

SEQ ID NO: 46 (the nucleotide sequence of mini-TK)

SEQ ID NO: 47 (the nucleotide sequence of TIRE with mini-TK promoter)

SEQ ID NO: 48 (the nucleotide sequence of the YB minimal promoter)

SEQ ID NO: 49 (the nucleotide sequence of TIRE with YB-minimal promoter)

SEQ ID NO: 50 (human IL-7/CCL19-armored anti-DLL3 CAR nucleic acid sequence)

SEQ ID NO: 51 (TIRE-miniTK promoter inducible human IL-7/CCL19-armored anti-DLL3 CAR nucleic acid sequence)

SEQ ID NO: 52 (human IL-15-armored anti-DLL3 CAR nucleic acid sequence)

SEQ ID NO: 53 (TIRE-miniTK promoter inducible human IL-15-armored anti-DLL3 CAR nucleic acid sequence)

SEQ ID NO: 54 (IFN-βpromoter)

SEQ ID NO: 55 (ELAM promoter)

REFERENCES

1. Hay KA, Turtle CJ. Chimeric Antigen Receptor (CAR) T Cells: Lessons Learned from Targeting of CD19 in B-Cell Malignancies. Drugs. 2017; 77 (3) : 237–245.

2. Boyiadzis, M.M., Dhodapkar, M.V., Brentjens, R.J. et al. Chimeric antigen receptor (CAR) T therapies for the treatment of hematologic malignancies: clinical perspective and significance. j. immunotherapy cancer 2018; 6, 137

3. Ma S, Li X, Wang X, et al. Current Progress in CAR-T Cell Therapy for Solid Tumors. Int J Biol Sci. 2019; 15 (12) : 2548–2560.

4. Kheng N, Shaun OB, et al. CAR T Cell Therapy for Solid Tumors. Annu Rev Med. e 2017 68: 1, 139-152

5. Gill S. How close are we to CAR T-cell therapy for AML? Best Pract Res Clin Haematol. 2019 Dec; 32 (4) : 101104

6. Rotolo R, Leuci V, Donini C, et al. CAR-Based Strategies beyond T Lymphocytes: Integrative Opportunities for Cancer Adoptive Immunotherapy. Int J Mol Sci. 2019; 20 (11) : 2839.

7. Roddie C, O'Reilly M, et al. Manufacturing chimeric antigen receptor T cells: issues and challenges. Cytotherapy. 2019 Mar; 21 (3) : 327-340.

8. Stock S, Schmitt M, Sellner L, et al. Optimizing Manufacturing Protocols of Chimeric Antigen Receptor T Cells for Improved Anticancer Immunotherapy. Int J Mol Sci. 2019; 20 (24) . E6223.

9. Darya A, Robyn A, et al. IL15 Enhances CAR-T Cell Antitumor Activity by Reducing mTORC1 Activity and Preserving Their Stem Cell Memory Phenotype. Cancer Immunol Res. 2019; 7 (5) .

10. Chen Y, Sun C, et al. Eradication of Neuroblastoma by T Cells Redirected with an Optimized GD2-Specific Chimeric Antigen Receptor and Interleukin-15. Clin Cancer Res. 2019; 1; 25 (9) : 2915-2924

11. Car D, Eng M, et al. The toxicology of interleukin-12: a review. Toxicol Pathol. 1999; 27 (1) : 58-63.

12. Steel JC, Waldmann TA, Morris JC. Interleukin-15 biology and its therapeutic implications in cancer. Trends Pharmacol Sci. 2012; 33 (1) : 35–41.

13. Liu Y, Shengmeng D, et al. Armored Inducible Expression of IL-12 Enhances Antitumor Activity of Glypican-3–Targeted Chimeric Antigen Receptor–Engineered T Cells in Hepatocellular Carcinoma. J Immunol. 2019, 203 (1) : 198-207.

14. Davenport AJ, Cross RS, Watson KA, et al. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity [published correction appears in Proc Natl Acad Sci U S A. 2019 May 28; 116 (22) : 11075-11076.

* * *

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

A nucleic acid which comprises

(i) a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , or

a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes a T cell receptor (TCR) , or

a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes an antigen binding domain fused to a component of T cell receptor (TCR) complex; and

(ii) a second nucleic acid sequence comprising a second regulatory region, operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the second regulatory region comprises a promoter and one or more regulatory elements, wherein the regulatory elements bind to transcription factors that are active in activated immune cells.
The nucleic acid of claim 1, wherein the first nucleic acid and the second nucleic acid are transcribed in opposite directions.
The nucleic acid of claim 1, wherein the one or more regulatory elements bind to transcription factors that are active in T cells.
The nucleic acid of claim 1, wherein the one or more regulatory elements comprise T cell intrinsic regulatory elements (TIREs) .
A vector which comprises the nucleic acid of claim 1.
A system for expressing an antigen receptor in an immune cell which comprises

(i) a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , or

a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes a TCR, or

a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes an antigen binding domain fused to a component of a TCR complex; and

(ii) a second nucleic acid sequence comprising a second regulatory region operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the second regulatory region comprises a promoter and one or more binding sites that bind to one or more transcription factors that are active in activated immune cells;

or

(i) a chimeric antigen receptor (CAR) , or TCR, or antigen binding domain fused to a component of a TCR complex, ; and

(ii) a nucleic acid sequence comprising a regulatory region operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the regulatory region comprises a promoter and one or more regulatory elements that bind to one or more transcription factors that are active in activated immune cells.
The system of claim 6, wherein the one or more regulatory elements bind to transcription factors that are active in T cells.
An immune cell which comprises the nucleic acid of any one of claims 1 to 4 or the vector of claim 5 or the system of any one of claims 6 to 7.
An immune cell which comprises

(i) a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , or

a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes a TCR, or

a first nucleic acid sequence comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes an antigen binding domain fused to a component of a TCR complex; and

(ii) a second nucleic acid sequence comprising a second regulatory region, operatively linked to a nucleic acid sequence that encodes one or more cytokine and/or costimulatory protein, wherein the second regulatory region comprises a promoter and one or more regulatory elements that bind to one or more transcription factors that are active in activated immune cells.
The immune cell of claim 8, where the first nucleic acid is linked to the second nucleic acid.
The immune cell of claim 9, wherein the one or more regulatory elements bind to transcription factors that are active in T cells.
The immune cell of any one of claims 8 to 11, wherein the cell comprises an αβ T cell, γδ T cell, Vδ1 cell, Vδ2 cell, Vδ3 cell, Vδ5 cell, NK cell, NKT cell, iNKT cell, or NKT like cell.
The nucleic acid of claim 1 to 4, the vector of claim 5, the system of claim 6 to 7, or the cell of claims 8 to 11, wherein the one or more cytokine or costimulatory protein comprises one or more of IL-7, IL-9, IL-10, IL-12, IL-15, IL-18, IL21, IL-23, CCL19 or leptin.
The nucleic acid, vector, system, or cell of any preceding claim, wherein the promoter comprises an IFN-β promoter, an IL-2 promoter, an BCL-2 promoter, an IL-6 promoter, an IFN-γ promoter, an IL-12 promoter, an IL-4 promoter, an IL-15 promoter, an IL-21 promoter, a viral promoter, a herpes simplex virus (HSV) thymidine kinase (TK) promoter, or a YB-TATA promoter.
The nucleic acid, vector, system, or cell of claim 14, wherein the promoter is a minimal promoter.
The nucleic acid, vector, system, or cell of any preceding claim, wherein the second regulatory region comprises from one to ten transcription factor binding sites.
The nucleic acid, vector, system, or cell of any preceding claim, wherein the transcription factor binding sites comprise one or more copies of one more binding sites of NF-κB, AP-1, Myc, NR4A, TOX1, TOX2, TOX3, TOX4, STAT1, STAT2, STAT3, STAT4, STAT5, or STAT6.
The nucleic acid, vector, system, or cell of any preceding claim, wherein the second regulatory region comprises from three to ten NF-κB binding motifs, from three to ten AP-1 binding motifs, from three to ten binding motifs, each selected from NF-κB and AP-1 binding motifs or from three to ten binding motifs, each selected from NFAT, NF-κB and AP-1 binding motifs.
The nucleic acid, vector, system, or cell of any preceding claim, where the CAR comprises an extracellular antigen recognition domain that is selective for a target, a transmembrane domain operatively linked to the extracellular domain, and an intracellular signaling domain comprising a primary intracellular signaling domain of an immune effector cell.
The nucleic acid, vector, system, or cell of claim 19, wherein the primary intracellular signaling domain of the CAR is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d.
The nucleic acid, vector, system, or cell of claim 19 or 20, wherein the intracellular signaling domain of the CAR further comprises an intracellular co-stimulatory domain.
The nucleic acid, vector, system, or cell of claim 19 or 20, wherein the intracellular signaling domain of the CAR does not comprise an intracellular co-stimulatory domain.
The nucleic acid, vector, system, or cell of any one of claims 1 to 22, wherein the CAR comprises an extracellular antigen recognition domain that is selective for a target, a transmembrane domain operatively linked to the extracellular domain, and an intracellular signaling domain comprising an intracellular co-stimulatory domain.
The nucleic acid, vector, system, or cell of claim 21 or 23, wherein the intracellular co-stimulatory domain is derived from a co-stimulatory molecule selected from the group consisting of CD27, CD28, 4-1BB, OX40, CD40, PD-1, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, DAP10, DAP12, CD83, ligands of CD83 and combinations thereof.
The nucleic acid, vector, system, or cell of claims 19 to 24, wherein the transmembrane domain comprises CD8α transmembrane region or CD28 transmembrane region.
The CAR of any one of claims 19 to 25, further comprising a CD8α hinge or CD28 hinge between the antigen recognition domain that is selective for a target and the transmembrane domain.
The nucleic acid, vector, system, or cell of any one of claims 1 to 13, where the TCR or TCR complex comprising (a) TCR chain selected from an alpha chain, a beta chain, a gamma chain and a delta chain of a T cell receptor, (b) an epsilon chain, a delta chain, and/or a gamma chain of cluster of differentiation 3 (CD3) , or (c) a CD3 zeta chain.
The nucleic, vector, system, or cell of any preceding claim, wherein the first nucleic acid sequence and the second nucleic acid sequence each comprises a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%identical to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20 or the complement thereof.
A composition comprising the immune cell of any one of claims 8 to 28.
A composition comprising the immune cell of any one of claims 8 to 28 and a physiologically acceptable excipient.
A method of modifying an immune cell, which comprises introducing into the cell a nucleic acid that comprises a regulatory region operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the regulatory region comprises a promoter and one or more transcription factor binding sites, wherein the transcription factor binding sites bind to transcription factors that are active in activated immune effector cells.
A method of making an immune cell, which comprises introducing into a cell

(i) a first nucleic acid comprising a first regulatory region operatively linked to a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , or

a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes a T cell receptor (TCR) , or

a first nucleic acid sequence comprising a first regulatory region, operatively linked to a nucleic acid sequence that encodes an antigen binding domain fused to a component of a T cell receptor (TCR) complex; and

(ii) a second nucleic acid comprising a second regulatory region operatively linked to a nucleic acid sequence that encodes a cytokine or a costimulatory protein, wherein the second regulatory region comprises a promoter and one or more transcription factor binding sites, wherein the transcription factor binding sites bind to transcription factors that are active in activated immune effector cells.
A method for modifying an immune cell to have a binding site against a cancer antigen, the method comprising: introducing into the cell a first nucleic acid encoding a chimeric antigen receptor (CAR) , TCR or antigen binding domain fused to a component of a TCR complex comprising a sequence encoding a single-chain variable fragment (scFv) or a single domain antibody (sdAb) specific for the cancer antigen linked to a transmembrane domain and a T-cell signaling domain; and introducing into the cell a second nucleic acid encoding a cytokine operatively linked to a promoter and one or more binding sites that bind to one or more transcription factors that are active in activated effector cells, whereby the immune cells express the CAR, TCR, or antigen binding domain fused to a component of a TCR complex, and express and secrete the cytokine when activated.
The method of any one of claims 31 to 33, wherein the immune cell is an αβ T cells, γδ T cell, Vδ1 cell, Vδ2 cell, Vδ3 cell, Vδ5 cell, NK cell, NKT cell, iNKT cell, or NKT like cell.
A pharmaceutical composition comprising an anti-tumor effective amount of a population of effector cells, wherein the effector cells comprise a nucleic acid sequence that encodes a chimeric antigen receptor (CAR) , and a nucleic acid sequence that encodes a cytokine or a costimulatory protein operatively linked to a regulatory region which comprises a promoter and one or more binding sites, wherein the binding sites bind to transcription factors that are active in activated immune cells.
A pharmaceutical composition comprising an anti-tumor effective amount of a population of effector cells, wherein the effector cells comprise a nucleic acid sequence that encodes a TCR or antigen binding domain fused to a component of a TCR complex, and a nucleic acid sequence that encodes a cytokine or a costimulatory protein operatively linked to a regulatory region which comprises a promoter and one or more binding sites, wherein the binding sites bind to transcription factors that are active in activated immune cells.
The pharmaceutical composition of claim 35 or 36, wherein the effector cells comprise T cells.
The pharmaceutical composition of claim 35 or 36, wherein the effector cells comprise NK cells.
The pharmaceutical composition of claim 35 or 36, wherein the effector cells comprise γδ T cells and/or α/β T cells.
The pharmaceutical composition of claim 35 or 36, wherein the effector cells are modified cells of a human having a hematological cancer.
The pharmaceutical composition of claim 40, wherein the hematological cancer is leukemia or lymphoma.
The pharmaceutical composition of claim 40, wherein the hematological cancer is chronic lymphocytic leukemia (CLL) , acute lymphocytic leukemia (ALL) , chronic myelogenous leukemia (CML) , mantle cell lymphoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma or multiple myeloma.
The pharmaceutical composition of claim 35 or 36, wherein the tumor comprises an adult carcinoma comprising oral or pharynx cancer (tongue, mouth, pharynx, head and neck) , digestive system cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, interhepatic bile duct, gallbladder, pancreas) , respiratory system cancer (larynx, lung and bronchus) , bones and joint cancer, soft tissue cancer, skin cancer (melanoma, basal and squamous cell carcinoma) , pediatric tumor (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma) , tumor of the central nervous system (brain, astrocytoma, glioblastoma, glioma) , or cancer of the breast, the genital system (uterine cervix, uterine corpus, ovary, vulva, vagina, prostate, testis, penis, endometrium) , the urinary system (urinary bladder, kidney and renal pelvis, ureter) , the eye and orbit, the endocrine system (thyroid) , and the brain and other nervous system, or any combination thereof.
A method of providing an anti-tumor immunity in a subject comprising administering to the subject an effective amount of immune cells of any one of claims 8 to 28 or immune cells modified by the method of any one of claims 31 to 34.
A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of immune cells of any one of claims 8 to 28 or immune cells that are modified by the method of any one of claims 31 to 34, wherein the modified cells treat the cancer.
A prophylactic method of delaying or preventing metastasis or recurrence of a cancer in a subject, the method comprising administering to the subject an effective amount of immune cells of any one of claims 8 to 28 or immune cells modified by the method of any one of claims 29 to 32.
A kit for making a chimeric antigen receptor T-cell or γδ T cell or NK cell or for treating a tumor in a subject, comprising a container comprising the nucleic acid, system, vector, host cell, or composition of any of claims 1 to 43, and instructions for using the kit.
Use of the nucleic acid, system or vector of any of claims 1 to 28 to make a chimeric antigen receptor effector cell.
Use of the nucleic acid, system, vector, host cell, or composition of any of claims 1 to 42, to treat a tumor in a subject.