Compositions and methods for treating cancer using self-driven chimeric antigen receptors

Abstract

Self-driven surface antigen-regulated promoter-therapeutic payload constructs containing an antigen-binding domain are disclosed. Also disclosed are nucleic acids, recombinant expression vectors, host cells, antigen-binding fragments, and pharmaceutical compositions related to the surface antigen-regulated promoter-therapeutic payload constructs. Also disclosed are methods for treating or preventing cancer in a subject, and methods for producing the self-driven surface antigen-regulated promoter-therapeutic payload constructs in T cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT patent application claims priority to U.S. Provisional Patent Application No. 62 / 954,161, filed December 27, 2019, and U.S. Provisional Patent Application No. 62 / 814,759, filed March 6, 2019, the entire contents of each of which are incorporated herein by reference.

[0002] Field of Disclosure This application relates to the field of cancer, and in particular to inducible promoters linked to therapeutic payloads and methods of use thereof. [Background technology]

[0003] background Cancer is one of the most deadly threats to human health. With nearly 1.3 million new cases each year in the United States alone, it is the second leading cause of death after cardiovascular disease, accounting for one in four deaths. Most of these deaths are caused by solid tumors. While significant advances have been made in the medical treatment of some specific cancers, the five-year survival rate for all cancers combined has improved by only about 10% over the past 20 years. Cancer, or malignant tumors, metastasize and grow rapidly and uncontrollably, making them extremely difficult to treat.

[0004] Multiple myeloma ("MM") is a debilitating, often incurable disease, with over 30,000 new cases diagnosed each year in the United States (Source: MM research foundation). MM is the second most common blood cancer in the United States after non-Hodgkin's lymphoma ("NHL") (Smith L, McCourt O, Henrich M, et al. Multiple myeloma and physical activity: a scoping review. BMJ Open. 2015;5:e009576). MM adversely affects plasma cells in the bone marrow, which can lead to bone marrow failure and patient death (National Cancer Institute. A snapshot of myeloma. November 5, 2014. See cancer.gov on the World Wide Web). Complications of myeloma include bone pain, bone loss, anemia, immunosuppression, renal dysfunction, and neuropathy (Mayo Clinic staff. Diseases and conditions: Multiple myeloma: Treatments and drugs. December 4, 2015).

[0005] First-line therapies for MM include proteasome inhibitors, immunomodulatory agents, steroids, histone deacetylase ("HDAC") inhibitors, and chemotherapy. Although these approaches aim to kill MM cells, many of them are also associated with widespread immunosuppression and systemic toxicity.

[0006] Chimeric antigen receptor T cell (CAR T) technology has provided significant advances in the treatment of hematological malignancies such as MM and shows great promise for the treatment of solid tumors. However, this technology currently suffers from two unresolved drawbacks: insufficient CAR T efficacy leading to suboptimal responses, and adverse side effects such as cytokine release syndrome resulting from excessive CAR T activation. The objective of the present invention is to address these two problems by creating CAR T moieties and other therapeutic payloads that can be naturally fine-tuned based on the tumor burden and inflammatory environment in the patient, at any given time during treatment, and in any local microenvironment.

[0007] Chimeric antigen receptors (CARs) are hybrid molecules consisting of three essential units: (1) an extracellular antigen-binding motif, (2) a binding / transmembrane motif, and (3) an intracellular T cell signaling motif (Long AH, Haso WM, Orentas RJ. Lessons learned from a highly active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013;2(4):e23621). The antigen-binding motif of CARs is typically modeled after a single-chain fragment variable fragment (ScFv), the smallest binding domain of an immunoglobulin (Ig) molecule. Other antigen-binding motifs, including receptor ligands (e.g., IL-13, engineered to bind to the IL-13 receptor expressed in tumors), complete immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D), have also been engineered. Other cellular targets (e.g., NK or gamma-delta T cells) are being engineered to express CARs (Brown CE et al. Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012;7(2):e31210). Substantial further effort remains to be expended in identifying the most active T cell populations to transduce with CAR vectors, in identifying optimal culture and expansion techniques, and in elucidating the molecular details of the CAR protein structure itself.

[0008] The binding motif of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or can be designed as a long, flexible linker. Structural motifs, such as those derived from the IgG constant domain, can be used to extend the ScFv binding domain farther from the T cell membrane surface. This may be important for some tumor targets (e.g., disialoganglioside GD2; Orentas et al., this observation unpublished) whose binding domains are particularly close to the tumor cell surface membrane. All signaling motifs used in CARs to date include the CD3-zeta chain, as this core motif is an important signal for T cell activation. The first reported second-generation CARs featured the CD28 signaling domain and CD28 transmembrane sequence. This motif was used in third-generation CARs, which also contained the CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009;183(9):5563-74). With the advent of new technologies, activation of T cells by beads bound to anti-CD3 and anti-CD28 antibodies and the presence of the classical "signal 2" from CD28 no longer needs to be encoded by the CAR itself. Third-generation vectors using bead activation have been shown to perform no better than second-generation vectors in in vitro assays and, furthermore, offer no clear advantage over second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013;121(7):1165-74; Kochenderfer JN et al. Blood. 2012;119(12):2709-20).This is evidenced by the clinical success of second-generation CD28 / CD3-zeta (Lee DW et al. American Society of Hematology Annual Meeting. New Orleans, LA; December 7-10, 2013) and CD19-specific CARs in the CD137 / CD3-zeta signaling mode (Porter DL et al. N Engl J Med. 2011;365(8):725-33). In addition to CD137, other members of the tumor necrosis factor receptor superfamily, such as OX40, can also provide important sustained signals in CAR-transduced T cells (Yvon E et al. Clin Cancer Res. 2009;15(18):5852-60). Equally important are the culture conditions under which CAR T cell populations are cultured.

[0009] T cell-based immunotherapy has become a new frontier in synthetic biology. Multiple promoters and gene products have been designed to target these highly potent cells to the tumor microenvironment, where they can circumvent negative regulatory signals and mediate effective tumor killing. Eliminating unwanted T cells through drug-induced dimerization of an inducible caspase-9 construct using AP1903 offers one way to pharmacologically trigger a powerful switch that can control T cell populations (Di Stasi A et al. N Engl J Med. 2011;365(18):1673-83). Furthermore, generating an effector T cell population that is resistant to the negative regulatory effects of transforming growth factor-β by expressing a dominant-negative receptor demonstrates the extent to which effector T cells can be engineered for optimal antitumor activity (Foster AE et al. J Immunother. 2008;31(5):500-5). Thus, although CARs appear to be able to trigger T cell activation in a manner similar to endogenous T cell receptors, currently, there is no evidence that CARs in vivo + The limited proliferation of T cells, their rapid disappearance after infusion, and poor clinical activity have been major obstacles hindering the clinical application of this technology.

[0010] To date, prior art CAR therapeutic approaches have used CAR T constructs under the control of constitutive promoters, such as human elongation factor 1 alpha (EF1α), phosphoglycerate kinase (PGK), murine leukemia virus (MuLV), murine stem cell virus (MSCV), or other constitutive promoters known in the art, which are often expressed at high levels or artificially induced (via small molecules or soluble components). These approaches do not adequately match the dynamics of antigen expression, which can result in excessive or insufficient effector cell responses at the cellular level and can lead to undertreatment or overtreatment and toxicity for patients at the biological level. Summary of the Invention [Problem to be solved by the invention]

[0011] Thus, there is an urgent and long-felt need in the art to discover new compositions and methods for treating MM and CLL using approaches that can provide specific and effective anti-tumor effects without exhibiting the problems described above (e.g., high toxicity, poor efficacy, etc.). [Means for solving the problem]

[0012] The present invention addresses the above-mentioned needs by providing compositions and therapeutic methods that can be used to treat cancer and other diseases and / or conditions. In particular, the invention disclosed and described herein provides inducible promoter-therapeutic payload constructs that can be used to treat diseases, disorders, or conditions associated with dysregulated expression of various antigens, including, but not limited to, mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38, or combinations thereof, which contain antigen-specific binding domains that result in high surface expression on transduced T cells, high degrees of cytolysis, and in vivo proliferation and persistence of the transduced T cells. Furthermore, the invention disclosed and described herein provides self-driven control of such therapeutic payloads by utilizing surface antigen-regulated inducible promoters that modulate the expression level of one or more therapeutic payloads depending on the expression level of the surface antigen on target cells.

[0013] overview The invention disclosed and described herein is based on the unexpected discovery that regulation or modulation of expression of a therapeutic payload construct by a surface antigen-regulated promoter can be directly correlated with the activity of the therapeutic payload and, therefore, with the expression level of the surface antigen in the environment of the target cell.

[0014] Provided herein are novel self-driven inducible promoter-payload therapeutic constructs comprising a therapeutic payload operably linked to a surface antigen-regulated inducible promoter that regulates the expression level of one or more therapeutic payloads depending on the expression level of a surface antigen on a target cell.

[0015] Without being limited to any particular mechanism of action, as used herein, a "self-driving" surface antigen-regulated inducible promoter refers to utilizing a surface antigen-regulated inducible promoter to drive a therapeutic payload, providing a low basal level of surface expression of the therapeutic payload in the absence of tumor target antigen expression. In the presence of an activating target antigen on the surface of the target cell, the therapeutic payload is activated, which triggers activation of the appropriate signaling pathway, which in turn activates the signal mediator of the surface antigen-regulated inducible promoter, thereby increasing expression of the therapeutic payload beyond the basal level of expression.

[0016] In this way, a positive feedback loop is created in which the higher the expression of a given target antigen, the higher the expression of the therapeutic payload, and vice versa, allowing for highly efficient control of the expression of the therapeutic payload to achieve a precisely tailored T cell response depending on the level of the target present at a particular site and time point. The increased expression of the therapeutic payload leads to optimal antitumor activity and rapid elimination of the target tumor cells. As the tumor cells are quantitatively reduced / eliminated, the level of therapeutic payload expression returns to its basal level of expression.

[0017] In one aspect, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter, wherein the surface antigen-regulated inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells, thereby achieving a precisely controlled T cell response depending on the level of target antigen present in the tumor environment.

[0018] In one embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising a nucleotide sequence comprising SEQ ID NOs: 137 and 138, or a combination thereof, wherein the surface antigen-regulated inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells, thereby achieving a precisely controlled T cell response depending on the level of target antigen present in the tumor environment.

[0019]

[0013] In one aspect, provided herein are self-driven surface antigen-regulated inducible promoter-therapeutic payload constructs comprising at least one therapeutic payload comprising a chimeric antigen receptor (CAR), a cytokine, a chemokine, a transport receptor, a bispecific antibody, a neutralizing / blocking antibody, a T cell stimulating receptor, a truncated inhibitory receptor, a hybrid inhibitory / activating receptor, an anti-apoptotic protein, an shRNA, or a protease, or a combination thereof, operably linked to a surface antigen-regulated inducible promoter comprising a STAT5 response element, an AP-1 response element, or an NF-kappa B response element, or a combination thereof, wherein the surface antigen-regulated inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of a surface antigen on a target cell; also provided are host cells (e.g., T cells) expressing the surface antigen-regulated inducible promoter-therapeutic payload constructs, and nucleic acid molecules encoding the surface antigen-regulated inducible promoter-therapeutic payload constructs.

[0020] In one embodiment, one or more therapeutic payloads (e.g., based on, but not limited to, chimeric antigen receptors (CARs), cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, or proteases) are expressed under the control of a surface antigen-regulated inducible promoter, and the one or more therapeutic payloads are separated by a 2A ribosomal skip sequence or an internal ribosome entry sequence (IRES), or a combination thereof.

[0021] In one embodiment, the surface antigen-regulated inducible promoter-therapeutic payload constructs disclosed herein may include, for example, but are not limited to, a CAR, which may comprise a single molecule expressed on the surface of an effector cell, or may comprise a signaling module expressed by the effector cell and a soluble targeting module, with the soluble targeting module binding to the cell-expressed signaling module to form a fully functional CAR. The CAR exhibits high surface expression on transduced T cells, a high degree of cytolysis, and in vivo proliferation and persistence of the transduced T cells. Also provided are methods of using the disclosed CARs, host cells, and nucleic acid molecules, for example, to treat cancer in a subject. Also provided are methods of using the disclosed surface antigen-regulated inducible promoter-therapeutic payload constructs, host cells, and nucleic acid molecules, for example, to control expression of the therapeutic payload.

[0022] In one embodiment, an isolated polynucleotide encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload is provided, wherein the therapeutic payload is operably linked to a surface antigen-regulated inducible promoter, and the therapeutic payload comprises a CAR comprising a fragment selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, and a single-chain Fv (ScFv).

[0023] In one embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload is provided, wherein the encoded extracellular antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123, or CD38 antigen-binding domain, or a combination thereof.

[0024] In another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload is provided, wherein the encoded extracellular antigen-binding domain comprises at least one heavy chain variable region of an antibody that binds to mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123, or CD38 antigen-binding domain, or a combination thereof.

[0025] In one embodiment, an isolated polynucleotide is provided encoding a surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the therapeutic payload is operably linked to a surface antigen-regulated inducible promoter, and the therapeutic payload comprises a CAR comprising, from N- to C-terminus, at least one extracellular binding domain comprising a mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen binding domain, or a combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain.

[0026] Thus, in one embodiment, an isolated polynucleotide is provided encoding a CAR-based therapeutic payload operably linked to a surface antigen-regulated inducible promoter, wherein at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38 antigen binding domain of the CAR-based therapeutic payload comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 15, 17, 19, 21, 23, 25, 87, 89, 91, 93, 95, 97, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135.

[0027] In one embodiment, an isolated polynucleotide is provided encoding a CAR-based therapeutic payload operably linked to at least one surface antigen-regulated inducible promoter, wherein at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38 antigen binding domain of the CAR-based therapeutic payload comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 16, 18, 20, 22, 24, 26, 88, 90, 92, 94, 98, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136.

[0028] In one embodiment, an isolated polynucleotide is provided encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the therapeutic payload is operably linked to a surface antigen-regulated inducible promoter, and the therapeutic payload comprises a CAR comprising, from N- to C-terminus, at least one extracellular binding domain comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 15, 17, 19, 21, 23, 25, 87, 89, 91, 93, 95, 97, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, or a combination thereof; at least one transmembrane domain; and at least one intracellular signaling domain.

[0029] In one embodiment, an isolated polynucleotide is provided encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the therapeutic payload is operably linked to a surface antigen-regulated inducible promoter, and the therapeutic payload comprises a CAR comprising, from N- to C-terminus, at least one extracellular binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 16, 18, 20, 22, 24, 26, 88, 90, 92, 94, 98, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136, or a combination thereof; at least one transmembrane domain; and at least one intracellular signaling domain.

[0030] In one embodiment, the targeting domain of the CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is a monoclonal antibody, ScFv and 135, wherein the effector cell-expressed component of the CAR contains a binding domain specifically directed to bind to the tag or epitope expressed on the soluble CAR module, thereby specifically binding the soluble component of the CAR to the cell-binding component of the CAR to form a fully functional CAR structure.

[0031] In another embodiment, the targeting domain of the CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is a monoclonal antibody, ScFv and an additional ScFv, wherein the effector cell-expressed component of the CAR contains a tag or epitope that specifically responds to the additional ScFv expressed on the soluble CAR module, thereby specifically binding the soluble component of the CAR to the cell-binding component of the CAR to form a fully functional CAR structure.

[0032] In yet another embodiment, an isolated nucleic acid molecule construct encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload is provided, wherein the extracellular antigen-binding domain of the encoded CAR further comprises at least one lipocalin-based antigen-binding antigen (anticalin) that binds to mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen-binding domain, or a combination thereof.

[0033] In one embodiment, an isolated nucleic acid molecule is provided encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the encoded extracellular mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38, antigen-binding domain is connected to the transmembrane domain by a linker domain.

[0034] In another embodiment, an isolated nucleic acid molecule is provided encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the encoded extracellular mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen binding domain is positioned after a sequence encoding a leader or signal peptide.

[0035] In yet another embodiment, a surface antigen-regulated inducible promoter-therapeutic payload based on a CAR comprises at least one antigen binding domain encoded by a nucleotide sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 15, 17, 19, 21, 23, 25, 87, 89, 91, 93, 95, 97, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135. Isolated nucleic acid molecules encoding constructs are provided, wherein the CAR further encodes an extracellular antigen-binding domain that targets an antigen including (but not limited to) CD19, CD20, CD22, CD33, CD123, CD5, CD7, CD138, BCMA (CD269), ROR1, TSLPR, TEM-1, TEM-7, TEM-8, TEM-9, CD371, CD276, CD99, GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, PRAME TCR, KRAS TCR, or any combination thereof.

[0036] In certain embodiments, an isolated nucleic acid molecule is provided encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the further encoded extracellular antigen binding domain is an anti-CD19 ScFv antigen binding domain, an anti-CD20 ScFv antigen binding domain, an anti-CD22 ScFv antigen binding domain, an anti-ROR1 ScFv antigen binding domain, an anti-mesothelin ScFv antigen binding domain, an anti-CD33 ScFv antigen binding domain, an anti-CD38 ScFv antigen binding domain, an anti-CD123 (IL3RA) ScFv antigen binding domain, an anti-CD138 ScFv antigen binding domain, an anti-BCMA (CD269) ScFv antigen binding domain, an anti-GPC2 ScFv antigen binding domain, an anti-GPC3 ScFv antigen binding domain, an anti-FGFR4 ScFv antigen binding domain, an anti-c-Met ScFv antigen binding domain, an anti-PMSA ScFv antigen binding domain, or an anti-glycolipid F77 The antigen-binding domain of the ScFv includes an anti-EGFRvIII ScFv antigen-binding domain, an anti-GD-2 ScFv antigen-binding domain, an anti-NY-ESO-1 TCR ScFv antigen-binding domain, an anti-MAGE A3 TCR ScFv antigen-binding domain, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or any combination thereof.

[0037] In one embodiment, the CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct provided herein further comprises a linker or spacer domain.

[0038] In one embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein an extracellular antigen-binding domain, an intracellular signaling domain, or both, comprising mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38, or a combination thereof, is linked to a transmembrane domain by a linker or spacer domain.

[0039] In one embodiment, an isolated nucleic acid molecule is provided encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the encoded linker domain is derived from the extracellular domain of CD8, TNFRSF19, or CD28 and is linked to a transmembrane domain.

[0040] In another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded CAR further comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, TNFRSF19, and CD154, or a combination thereof.

[0041] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded intracellular signaling domain further comprises a CD3 zeta intracellular domain.

[0042] In one embodiment, an isolated nucleic acid molecule is provided encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the encoded intracellular signaling domain is located C-terminal to the CD3 zeta intracellular domain.

[0043] In another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or a combination thereof.

[0044] In a further embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0045] In one embodiment, an isolated nucleic acid molecule is provided encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, which further contains a leader sequence or signal peptide, wherein the nucleotide sequence of the leader or signal peptide comprises the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 41, or SEQ ID NO: 43.

[0046] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded leader sequence comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 40, SEQ ID NO: 42, or SEQ ID NO: 44.

[0047] In one embodiment, provided herein is a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct comprising, from N- to C-terminus, at least one antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0048] In one embodiment, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the extracellular antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to the antigen, or at least one heavy chain variable region of an antibody that binds to the antigen, or a combination thereof.

[0049] In another embodiment, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0050] In some embodiments, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the CAR further encodes an extracellular antigen-binding domain comprising CD19, CD20, CD22, CD19 / 22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0051] In one embodiment, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the extracellular antigen-binding domain is an anti-CD19 ScFv antigen-binding domain, an anti-CD20 ScFv antigen-binding domain, an anti-CD22 ScFv antigen-binding domain, an anti-ROR1 ScFv antigen-binding domain, an anti-mesothelin ScFv antigen-binding domain, an anti-CD33 ScFv antigen-binding domain, an anti-CD38 ScFv antigen-binding domain, an anti-CD123 (IL3RA) ScFv antigen-binding domain, an anti-CD138 ScFv antigen-binding domain, an anti-BCMA (CD269) ScFv antigen-binding domain, an anti-GPC2 ScFv antigen-binding domain, an anti-GPC3 ScFv antigen-binding domain, an anti-FGFR4 ScFv antigen-binding domain, an anti-c-Met ScFv antigen-binding domain, an anti-PMSA ScFv antigen-binding domain, an anti-glycolipid F77 ScFv antigen-binding domain, or an anti-EGFRvIII ScFv antigen-binding domain. The antigen-binding domain of the ScFv includes an anti-GD-2 ScFv, an anti-NY-ESO-1 TCR ScFv, an anti-MAGE A3 TCR ScFv, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or any combination thereof.

[0052] In another embodiment, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the extracellular antigen-binding domain is an immunoglobulin variable heavy chain alone (VH) anti-CD19 antigen-binding domain, anti-CD20 VH antigen-binding domain, anti-CD22 VH antigen-binding domain, anti-ROR1 VH antigen-binding domain, anti-mesothelin VH antigen-binding domain, anti-CD33 VH antigen-binding domain, anti-CD38 VH antigen-binding domain, anti-CD123 (IL3RA) VH antigen-binding domain, anti-CD138 VH antigen-binding domain, anti-BCMA (CD269) VH antigen-binding domain, anti-GPC2 VH antigen-binding domain, anti-GPC3 VH antigen-binding domain, anti-FGFR4 VH antigen-binding domain, anti-c-Met VH antigen-binding domain, anti-PMSA VH antigen-binding domain, anti-glycolipid F77 VH antigen-binding domain, anti-EGFRvIII VH antigen-binding domain, anti-GD-2 VH antigen-binding domain, anti-NY-ESO-1 The antibody may comprise a TCR VH antigen-binding domain, an anti-MAGE A3 TCR VH antigen-binding domain, or an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, or any combination thereof.

[0053] In another embodiment, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the extracellular antigen-binding domain comprises a protein or peptide (P) sequence capable of specifically binding to a target antigen, such as an anti-CD19 P antigen-binding domain, an anti-CD20 P antigen-binding domain, an anti-CD22 P antigen-binding domain, an anti-ROR1 P antigen-binding domain, an anti-mesothelin P antigen-binding domain, an anti-CD33 P antigen-binding domain, an anti-CD38 P antigen-binding domain, an anti-CD123 (IL3RA) P antigen-binding domain, an anti-CD138 P antigen-binding domain, an anti-BCMA (CD269) P antigen-binding domain, an anti-GPC2 P antigen-binding domain, an anti-GPC3 P antigen-binding domain, an anti-FGFR4 P antigen-binding domain, an anti-c-Met P antigen-binding domain, an anti-PMSA P antigen-binding domain, an anti-glycolipid F77 P antigen-binding domain, an anti-EGFRvIII P antigen-binding domain, an anti-GD-2 P antigen-binding domain, an anti-NY-ESO-1 TCR P antigen-binding domain, an anti-MAGE The CAR may be derived from a natural or synthetic sequence comprising the A3 TCR P antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof. In another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0054] In yet another embodiment, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein at least one intracellular signaling domain comprises a costimulatory domain comprising a functional signaling domain of a protein selected from the group consisting of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or a combination thereof.

[0055] In another embodiment, the nucleic acid sequence encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct comprises the nucleic acid sequence of SEQ ID NO: 139. In one embodiment, the nucleic acid sequence encodes a CAR-based promoter-therapeutic payload construct comprising the amino acid sequence of SEQ ID NO: 78.

[0056] In another embodiment, the nucleic acid sequence encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct comprises the nucleic acid sequence of SEQ ID NO: 140. In one embodiment, the nucleic acid sequence encodes a CAR-based promoter-therapeutic payload construct comprising the amino acid sequence of SEQ ID NO: 78.

[0057] In another embodiment, the nucleic acid sequence encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct comprises the nucleic acid sequence of SEQ ID NO: 77. In one embodiment, the nucleic acid sequence encodes a CAR-based promoter-therapeutic payload construct comprising the amino acid sequence of SEQ ID NO: 78.

[0058] In one embodiment, the CAR-based surface antigen-regulated inducible promoter-therapeutic payload constructs disclosed herein are modified to express or contain a detectable marker for use in diagnosing, monitoring, and / or predicting treatment outcomes such as progression-free survival in cancer patients, or for monitoring the progress of such treatment.

[0059] In one embodiment, the nucleic acid molecule encoding the CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct may be contained in a vector, such as a viral vector, which may be a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, or a retrovirus vector, or a combination thereof.

[0060] In yet another embodiment, vectors expressing CAR-based surface antigen-regulated inducible promoter-therapeutic payload constructs may be further modified to include one or more operable elements to control the expression of CAR T cells or to eliminate CAR-T cells by a suicide switch. This suicide switch may include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In a preferred embodiment, the CAR-expressing vector may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0061] In another aspect, a host cell is further provided that comprises a nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct. In some embodiments, the host cell is a T cell, such as a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8 + T cells.

[0062] In one embodiment, provided herein is a transduced T cell comprising an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter, wherein the surface antigen-regulated inducible promoter therapeutic payload construct confers on the transduced CAR T cell the ability to achieve a precisely controlled T cell response depending on the level of target antigen present in the tumor environment by mounting an anti-tumor response dependent on the expression level of the surface antigen on the target cell.

[0063] In another aspect, provided herein is a transduced CAR T cell comprising an isolated nucleic acid molecule encoding a CAR operably linked to a surface antigen-regulated inducible promoter, wherein the surface antigen-regulated inducible promoter CAR construct confers on the transduced CAR T cell the ability to achieve a precisely controlled CAR T cell response depending on the level of target surface antigen present in the tumor environment by mounting an anti-tumor response depending on the expression level of the corresponding surface antigen on the target cell, wherein i) in the absence of expression of the tumor target surface antigen, the surface antigen-regulated inducible promoter confers a low basal level of CAR expression; ii) in the presence of an activating target surface antigen on the surface of the target cell, the CAR is activated, which triggers activation of the appropriate signaling pathway, which in turn activates the signal mediator of the surface antigen-regulated inducible promoter, thereby increasing CAR expression above the basal level of expression; iii) higher expression of a given target surface antigen results in higher expression of the CAR, and vice versa, thereby controlling CAR expression with high efficiency, resulting in a CAR that is precisely tailored to the level of the target present at a particular site and time point. A T cell response is achieved, iv) increased CAR expression leads to optimal anti-tumor activity and rapid elimination of targeted tumor cells, and v) as tumor cells are quantitatively reduced / eliminated, the level of CAR expression returns to its basal level of expression.

[0064] In one embodiment, transduced T cells with increased CAR expression lead to optimal anti-tumor activity and rapid elimination of targeted tumor cells, with levels of therapeutic payload expression returning to basal levels of expression (reference: expression levels prior to the anti-tumor response) as tumor cells are quantitatively reduced / eliminated.

[0065] In one embodiment, the transduced T cells are autologous. In another embodiment, the transduced T cells are allogeneic.

[0066] In yet another embodiment, a pharmaceutical composition is provided comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a nucleic acid sequence encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the CAR comprises at least one extracellular antigen-binding domain comprising an antigen-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 16, 18, 20, 22, 24, 26, 88, 90, 92, 94, 98, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136, at least one linker domain, at least one transmembrane domain, and at least one intracellular signaling domain, and wherein the T cells are T cells of a human with cancer. Cancer includes, inter alia, blood cancers such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myeloid leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma), or multiple myeloma (MM), or a combination thereof.

[0067] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct contains a transmembrane domain of a protein selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0068] In another embodiment, a pharmaceutical composition is provided, wherein the human cancer is selected from the group consisting of oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), gastrointestinal cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct), and rectal cancer. duct, gallbladder, pancreas), respiratory tract (larynx, lung, and bronchus), bone and joint cancer, soft tissue cancer, adult cancers including skin cancer (melanoma, basal cell carcinoma, and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), as well as cancers of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testicles, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), brain and other nervous system, or any combination thereof.

[0069] In yet another embodiment, a pharmaceutical composition is provided comprising an antitumor effective amount of a population of human T cells from a human with cancer, wherein the cancer is a refractory cancer that is unresponsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), adult B-cell malignancies including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-lineage ALL (acute lymphocytic leukemia)), multiple myeloma (MM), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other hematological cancers and solid tumors, or any combination thereof.

[0070] In yet another embodiment, a pharmaceutical composition is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct modulates the activity of the therapeutic payload by utilizing an innate effector cell pathway based on innate cell activation, mitogenic state, or a combination thereof.

[0071] In another embodiment, a method of producing CAR-containing T cells (hereinafter "CAR-T cells") is provided, the method comprising transducing T cells with a vector or nucleic acid molecule comprising a surface antigen-regulated inducible promoter-therapeutic payload encoding a CAR (as disclosed) that specifically binds to mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38, or a combination thereof, thereby producing the CAR-T cells.

[0072] In yet another embodiment, a method for generating a population of RNA engineered cells is provided, comprising introducing in vitro transcribed or synthetic RNA of a nucleic acid molecule comprising a surface antigen-regulated inducible promoter-therapeutic payload encoding a disclosed CAR into cells of a subject, thereby generating CAR T cells.

[0073] In yet another aspect, a method of diagnosing a disease, disorder, or condition associated with expression in a cell of a surface antigen-regulated inducible promoter-therapeutic payload construct comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 139, 140, and 141, or a combination thereof, is provided, comprising contacting the cell with a human anti-mesothelin, anti-CD33, anti-CD19, anti-CD19 / CD20, anti-CD22, anti-ROR1, anti-CD123, or anti-CD38 antibody, or fragment thereof, or a combination thereof, wherein the antibody or fragment thereof is selected from the group consisting of SEQ ID NOs: 8, 10, 12, 16, 18, 20, 22, 24, 26, 88, 90, 92, 94, 98, 116, 118, 120, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 , 126, 128, 130, 132, 134, and 136, or a combination thereof; and b) detecting the presence of the antigen mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123, or CD38, or a combination thereof, wherein the presence of mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123, or CD38, or a combination thereof, diagnoses a disease, disorder, or condition associated with expression of mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123, or CD38, or a combination thereof.

[0074] In one embodiment, the disease, disorder, or condition associated with expression of mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38, or a combination thereof, is cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, adult B-cell malignancies including acute lymphoblastic leukemia (ALL), minimal residual disease (MRD) in acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myelogenous leukemia), non-Hodgkin's lymphoma (NHL), pediatric B-cell malignancies (including B-lineage ALL (acute lymphocytic leukemia)), multiple myeloma (MM), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other hematological cancers and solid tumors, or any combination thereof.

[0075] In another embodiment, a method of diagnosing or prognosing or risk assessment for a disease associated with mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 (or a combination thereof) in a mammal is provided, comprising detecting expression of mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38, or a combination thereof, in a sample from the mammal, the step comprising: a) detecting expression of mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38, or a combination thereof, in a sample from the mammal, the step comprising: a) treating the sample with a human anti-mesothelin, anti-CD33, anti-CD19, anti-CD19 / CD20, anti-CD22, anti-CD19 / 22, anti-ROR1, anti-CD123, or anti-CD38 antibody (or an antibody against ...CD38 antibody). or a combination thereof) or a fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 16, 18, 20, 22, 24, 26, 88, 90, 92, 94, 98, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136; and b) detecting the presence of one or more antigens, wherein the presence of the antigen(s) diagnoses a mesothelin-, CD33-, CD19-, CD19 / CD20-, CD22-, CD19 / 22-, ROR1-, CD123-, or CD38-associated disease in the mammal.

[0076] In another embodiment, a method of redirecting a CAR antigen target is provided, comprising contacting a cell with a surface antigen-regulated inducible promoter-therapeutic payload construct comprising a human anti-mesothelin, anti-CD33, anti-CD19, anti-CD19 / CD20, anti-CD22, anti-CD19 / 22, anti-ROR1, anti-CD123, or anti-CD38 antibody, or fragment thereof, wherein the antibody or fragment thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 16, 18, 20, 22, 24, 26, 88, 90, 92, 94, 98, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, and 136. In one embodiment, the cells are selected from the group consisting of mesothelin-, CD33-, CD19-, CD19 / CD20-, CD22-, CD19 / 22-, ROR1-, CD123-, or CD38-expressing tumor cells, tumor-associated macrophages, and any combination thereof.

[0077] In another aspect, a method for eliciting anti-tumor immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with a surface antigen-regulated inducible promoter-therapeutic payload construct comprising a vector or nucleic acid molecule encoding a disclosed therapeutic payload, for example, but not limited to, a CAR, a cytokine, a chemokine, a transport receptor, a bispecific antibody, a neutralizing / blocking antibody, a T cell stimulatory receptor, a truncated inhibitory receptor, a hybrid inhibitory / activating receptor, an anti-apoptotic protein, an shRNA, or a protease, or a combination thereof.

[0078] In another embodiment, a method of treating or preventing cancer in a mammal is provided, comprising administering to the mammal one or more of the disclosed surface antigen-regulated promoter-therapeutic payload constructs in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of host cells expressing a CAR (as disclosed) that specifically binds to mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38, and / or one or more of the aforementioned antigens, under conditions sufficient to form in the subject an immune complex consisting of the antigen-binding domain of the CAR and the extracellular domain of mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38, and / or one or more of the aforementioned antigens.

[0079] In yet another embodiment, a method is provided for treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells comprising one or more of the disclosed surface antigen-regulated inducible promoter-therapeutic payload constructs, wherein the T cells comprise a nucleic acid sequence encoding a CAR, the CAR being selected from the group consisting of SEQ ID NOs: 8, 10, 12, 16, 18, 20, 22, 24, 26, 88, 90, 92, 94, 98, 116, 118, 120, 122, 124, 126, 128, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, , 120, 122, 124, 126, 128, 130, 132, 134, and 136, or any combination thereof, at least one extracellular mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the T cell is a T cell of a subject with cancer.

[0080] In yet another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising an anti-tumor effective amount of a population of T cells comprising one or more of the disclosed surface antigen-regulated inducible promoter-therapeutic payload constructs, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR being selected from the group consisting of SEQ ID NOs: 8, 10, 12, 16, 18, 20, 22, 24, 26, 88, 90, 92, 94, 98, 116, 118, 119, 120, 121, 122, 123, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, The T cell comprises at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen-binding domain comprising the amino acid sequence of 20, 122, 124, 126, 128, 130, 132, 134, and 136, or any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the T cell is a T cell of a subject with cancer. In some embodiments of the methods described above, at least one transmembrane domain comprises a transmembrane domain of the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or a combination thereof.

[0081] In yet another embodiment, a method is provided for generating a persistent population of genetically engineered T cells in a human diagnosed with cancer. In one embodiment, the method comprises administering to the human T cells genetically engineered to express a surface antigen-regulated promoter-therapeutic payload construct (e.g., based on a CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulatory receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptotic protein, shRNA, protease), wherein the self-driven surface antigen-regulated promoter-therapeutic payload construct is selected from the group consisting of SEQ ID NOs: 8, 10, 12, 16, 18, 20, 22, 24, 26, 88, 90, 92, 94, 98, 116, 118, 120, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 2 and at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen binding domain comprising the amino acid sequence of 6, 128, 130, 132, 134, and 136, or any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the persistent population of engineered T cells, or a population of progeny of the T cells, persists in humans for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years after administration.

[0082] In one embodiment, the progeny T cells in the human include memory T cells. In another embodiment, the T cells are autologous T cells.

[0083] In all aspects and embodiments of the methods described herein, any of the cancers, diseases, disorders, or conditions associated with elevated expression of tumor antigens, as described above, can be treated or prevented or ameliorated using one or more of the surface antigen-regulated promoter-therapeutic payload constructs disclosed herein.

[0084] For the avoidance of doubt, the specification and claims disclosed herein specifically and conditionally exclude the Syn-Notch constructs described in U.S. Patent No. 9,670,281, issued June 6, 2017, entitled "Binding-triggered transcriptional switches and methods of use thereof," and U.S. Patent No. 9,834,608, issued December 5, 2017, to Wendell A. Lim et al., respectively, and the NFAT-regulated IL-12 expression described in U.S. Patent No. 8,556,882, issued October 15, 2013, to Richard A. Morgan et al.

[0085] In yet another aspect, a kit is provided for generating a surface antigen-regulated promoter-therapeutic payload construct in a T cell as described above, or for preventing, treating, or ameliorating any of the cancers, diseases, disorders, or conditions associated with elevated expression of a tumor antigen in a subject as described above, comprising a container containing any one of the nucleic acid molecules, vectors, host cells, or compositions disclosed above, or any combination thereof, or instructions for use of the kit.

[0086] It is understood that the surface antigen-regulated promoter-therapeutic payload constructs, host cells, nucleic acids, and methods described above are useful beyond the scope of the specific aspects and embodiments detailed herein. The features and advantages of the present disclosure described above will become more apparent from the following detailed description, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0087]

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[0088] Detailed Description definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly contradicts. For example, the term "an antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." Thus, "comprising an antigen" means "including an antigen" without excluding other elements. The term "and / or" means "and" or "or." Furthermore, unless otherwise specified, it is understood that any and all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for illustrative purposes. Although many methods and materials similar or equivalent to those described herein can be used, particularly preferred methods and materials are described below. In the event of any conflict, the present specification (including explanations of terms) will control. Additionally, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. To facilitate identification of various embodiments, explanations of terms are provided below.

[0089] The term "about," when referring to measurable values ​​such as amounts and durations, is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the stated value, as such variations are appropriate for the practice of the disclosed methods.

[0090] Unless otherwise specified, scientific terms used herein are used in conventional manner.The definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (ed.), The Encyclopedia of Molecular Biology, published by Blackwell Science, 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, 1995; and other similar references.

[0091] The invention disclosed and described herein is based on the unexpected discovery that regulation or modulation of expression of a therapeutic payload construct by a surface antigen-regulated promoter can be directly correlated with the activity of the therapeutic payload and, therefore, with the expression level of the surface antigen in the environment of the target cell.

[0092] The present disclosure provides novel self-driven inducible promoter-therapeutic payload constructs comprising one or more therapeutic payloads operably linked to a surface antigen-regulated inducible promoter, where the surface antigen-regulated inducible promoter regulates the expression level of the one or more therapeutic payloads depending on the expression level of a surface antigen in the environment of a target cell, as well as host cells (e.g., T cells) that express the surface antigen-regulated inducible promoter-therapeutic payload constructs and nucleic acid molecules encoding the surface antigen-regulated inducible promoter-therapeutic payload constructs.

[0093] Without being limited to any particular mechanism of action, as used herein, a "self-driven" surface antigen-regulated inducible promoter refers to the use of a surface antigen-regulated inducible promoter to drive a therapeutic payload, providing a low basal level of therapeutic payload surface expression in the absence of tumor target antigen expression. In the presence of an activating target antigen on the surface of a target cell, the therapeutic payload is activated, triggering the activation of appropriate signaling pathways, which in turn activate the signal mediators of the surface antigen-regulated inducible promoter, thereby increasing therapeutic payload expression beyond the basal level. In this way, a positive feedback loop is created in which increased expression of a given target antigen also increases therapeutic payload expression, and vice versa, resulting in highly efficient control of therapeutic payload expression and achieving a T cell response precisely tailored to the level of target present at a particular site and time point. Increased therapeutic payload expression leads to optimal antitumor activity and rapid elimination of target tumor cells. As tumor cells are quantitatively reduced / eliminated, the level of therapeutic payload expression returns to its basal level.

[0094] This self-driven activation can then be repeated upon renewed exposure to the antigen (e.g., but not limited to, as may occur due to tumor recurrence, metastatic events, tumor migration / spread, etc.). Consequently, this ability to control the timing and magnitude of the T cell response to a self-driven therapeutic payload can improve treatment efficacy, reduce the risk of tumor escape, and reduce treatment-associated toxicity. This is analogous to biologically or endogenously adjusting the dose of treatment depending on the disease state at the cellular level.

[0095] The self-driven surface antigen-regulated promoter-therapeutic payload constructs are then described in detail, including a description of the surface antigen-regulated inducible promoter, the therapeutic payload, and further detailed descriptions of the self-driven CAR-based surface antigen-regulated promoter-therapeutic payload constructs, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression vectors, and host cells, as well as treatment methods, compositions, and kits that use the disclosed self-driven CAR-based surface antigen-regulated promoter-therapeutic payload constructs.

[0096] A. Surface antigen-regulated inducible promoter In one aspect, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter, wherein the surface antigen-regulated inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells, thereby achieving a precisely controlled T cell response depending on the level of target antigen present in the tumor environment.

[0097] In one embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising a nucleotide sequence comprising SEQ ID NOs: 137 and 138, or a combination thereof, wherein the surface antigen-regulated inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells, thereby achieving a precisely controlled T cell response depending on the level of target antigen present in the tumor environment.

[0098] In one embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising a nucleotide sequence comprising SEQ ID NOs: 137 and 138, or a combination thereof, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, wherein the surface antigen-regulated inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells, thereby achieving a precisely controlled T cell response depending on the level of target antigen present in the tumor environment.

[0099] In another embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising a nucleotide sequence comprising SEQ ID NOs: 137 and 138, or a combination thereof, wherein the surface antigen-regulated inducible promoter upregulates the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells, thereby achieving a precisely controlled T cell response depending on the level of target antigen present in the tumor environment.

[0100] In another embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising a nucleotide sequence comprising SEQ ID NOs: 137 and 138, or a combination thereof, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, wherein the surface antigen-regulated inducible promoter upregulates the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells, thereby achieving a precisely controlled T cell response depending on the level of target antigen present in the tumor environment.

[0101] In yet another embodiment, the expression level of a surface antigen-regulated inducible promoter therapeutic payload construct described herein may be upregulated by, for example, but not limited to, about 10-100%, 200%, 300%, 400%, and 500%. Ranges recited herein expressly include all integer values ​​subsumed therein, as if they were expressly recited.

[0102] In one embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising a nucleotide sequence comprising SEQ ID NOs: 137 and 138, or a combination thereof, wherein the surface antigen-regulated inducible promoter down-regulates the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells, thereby achieving a precisely controlled T cell response depending on the level of target antigen present in the tumor environment.

[0103] In another embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising a nucleotide sequence comprising SEQ ID NOs: 137 and 138, or a combination thereof, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, wherein the surface antigen-regulated inducible promoter down-regulates the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells, thereby achieving a precisely controlled T cell response depending on the level of target antigen present in the tumor environment.

[0104] In yet another embodiment, the expression levels of the surface antigen-regulated inducible promoter therapeutic payload constructs described herein may be downregulated by, for example, but not limited to, about 10-100%, 200%, 300%, 400%, and 500%. Ranges recited herein expressly include all integer values ​​subsumed therein, as if they were expressly recited.

[0105] B. Therapeutic Payloads In its broadest aspect, provided herein are self-driven surface antigen-regulated inducible promoter-therapeutic payload constructs comprising at least one therapeutic payload comprising a chimeric antigen receptor (CAR), cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptotic protein, shRNA, or protease, or combinations thereof, operably linked to a surface antigen-regulated inducible promoter comprising a STAT5 response element, an AP-1 response element, or an NF-kappa B response element, or combinations thereof, wherein the surface antigen-regulated inducible promoter regulates the expression level of the therapeutic payload depending on the expression level of a surface antigen on a target cell; also provided are host cells (e.g., T cells) expressing the surface antigen-regulated inducible promoter-therapeutic payload constructs, and nucleic acid molecules encoding the surface antigen-regulated inducible promoter-therapeutic payload constructs.

[0106] In one aspect, provided herein is a self-driven surface antigen-regulated inducible promoter-therapeutic payload construct comprising a therapeutic CAR and at least one therapeutic payload, comprising a CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptotic protein, shRNA, or protease, or combinations thereof, operably linked to a surface antigen-regulated inducible promoter comprising a STAT5 response element, an AP-1 response element, or an NFkappaB response element, or combinations thereof, wherein the surface antigen-regulated inducible promoter regulates the expression level of the therapeutic CAR and therapeutic payload depending on the expression level of a surface antigen on a target cell; also provided are host cells (e.g., T cells) that express the surface antigen-regulated inducible promoter-therapeutic payload construct, and nucleic acid molecules encoding the surface antigen-regulated inducible promoter-therapeutic payload construct.

[0107] In one embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a cytokine comprising IL-2, IL-15, IL-7, TNFa, IFN gamma, IFN beta, IFN alpha, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGF beta, IL-17, IL-18, or any combination thereof.

[0108] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a chemokine, including but not limited to, for example, CCL2, CCL3, CCL4, CCL5, CCL19, CCL21, CCL25, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, or any combination thereof.

[0109] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a trafficking receptor, such as a cytokine receptor, including but not limited to, CCR2, CCR3, CCR4, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR6, SIP1, or any combination thereof, which trafficking receptor serves to assist in the trafficking of the CAR T cell to the tumor site.

[0110] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a bispecific antibody, including but not limited to, a bispecific T cell engaging antibody (BiTE), for example, but not limited to, anti-CD3 and anti-CD19 targeting, or anti-CD3 and anti-CD22 targeting, or anti-CD3 and anti-CD20 targeting, or anti-CD3 and anti-CD33 targeting, or anti-CD3 and anti-CD123 targeting, or anti-CD3 and anti-CD38 targeting, other multi-targeting antibodies, etc.

[0111] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a neutralizing / blocking antibody against, for example, but not limited to, PD-L1, PD-L2, CD95L, TRAIL receptor, IL-6R, IL-1R, TGF beta receptor, PD-1, LAG-3, Tim-3, TGF beta, IL-10, CTLA-4, VISTA, TIGIT, IL-1, IL-1R, etc., expressed as an scFv, or as an IgG, or scFvFc, or VHH, or F(ab), or F(ab)2, or native ligand binding domain, or in other configurations, which neutralizing / blocking antibody enhances T cell lytic function, cytokine release, persistence, proliferative capacity, and prevents T cell checkpoint blockade, exhaustion, apoptosis, and activation-induced cell death.

[0112] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a T cell stimulatory receptor, such as, but not limited to, IL-2Ralpha, IL-15Ralpha, IL-7Ralpha, CXCR5, etc., which serves to enhance the anti-tumor function of T cells.

[0113] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a truncated inhibitory receptor (dominant negative; "dn"), such as, but not limited to, dn-TGF beta receptor II, dn-PD-1, dn-CTLA-4, dn-IL-10 receptor, dn-KLRG1, dn-CD160, dn-TIM3, dn-LAG3, dn-BTLA, dn-VISTA, etc., which truncated inhibitory receptor acts to prevent inhibition of T cell function.

[0114] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a hybrid inhibitory / activating receptor, such as, but not limited to, the ectodomain of PD-1 fused to the endodomain of CD28, the ectodomain of TGF beta receptor II fused to the endodomain of gp130, the ectodomain of IL-10 receptor fused to the endodomain of 4-1BB, or the IL-4 receptor fused to the endodomain of IL-7 receptor, which hybrid inhibitory / activating receptor serves to transduce a T cell inhibitory signal into a T cell activating signal.

[0115] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises an anti-apoptotic protein, such as, but not limited to, BCL-2, MCL-1, CED9, Bfl-1, Brag-1, A-1, or BCL-XL, which serves to prolong T cell persistence and prevent apoptosis.

[0116] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises an shRNA for, for example, but not limited to, PD-1, CTLA-4, KLRG-1, CD160, TGF beta receptor II, IL-10R, etc., which serves to downregulate T cell suppressor factors to enhance the anti-tumor function of T cells.

[0117] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a protease, such as MMP2 or MMP4, which serves to digest tumor stroma and increase T cell entry into the tumor.

[0118] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a peptide, such as an iRGD peptide, which serves to increase the entry of anti-cancer drugs into tumors.

[0119] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises a second CAR T construct, which serves to target a second tumor antigen or an antigen expressed on suppressor cells present in the tumor microenvironment (for example, but not limited to, PD-L1, PD-L2, TRAIL receptors CD33, CD138, etc. present on MDSCs and suppressor B cells).

[0120] In yet another embodiment, one or more therapeutic payloads (such as, but not limited to, based on chimeric antigen receptors (CARs), cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, or proteases) are expressed under the control of a surface antigen-regulated inducible promoter, and the one or more therapeutic payloads are separated by a 2A ribosomal skip sequence or an internal ribosome entry sequence (IRES), or a combination thereof.

[0121] In yet another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct is selected from the group consisting of cytokines including IL-2, IL-15, IL-7, TNFα, IFN gamma, IFN beta, IFN alpha, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGF beta, IL-17, IL-18, transport receptors such as cytokine receptors including, but not limited to, CCR4, CCR6, CXCR5, e.g., CCR4, CCR7, CCR2, and the like, e.g., anti-CD3 and anti-CD19 targeting or other targeting receptors. further comprising bispecific antibodies, including bispecific T cell engaging antibodies (BiTEs), including but not limited to, multitargeting antibodies; neutralizing / blocking antibodies against, for example, but not limited to, PD-L1, IL-6R, IL-1R, expressed as scFv or as IgG or in other configurations; T cell stimulatory receptors; hybrid inhibitory / activating receptors, including but not limited to, truncated inhibitory receptors, such as the ectodomain of PD-1 fused to the endodomain of CD28; anti-apoptotic proteins, including but not limited to, BCL-2 or BCL-XL; shRNA; proteases; second CAR T constructs; or any combination thereof, each of which has biological properties as described above.

[0122] C. Chimeric Antigen Receptors (CARs) In its narrowest aspect, the invention disclosed and described herein is based on the unexpected discovery that regulation or modulation of expression of a therapeutic payload construct by a surface antigen-regulated promoter can be directly correlated with the activity of the therapeutic payload and, therefore, with the expression level of the surface antigen in the environment of the target cell.

[0123] Unlike previously existing CAR T constructs, whose expression is controlled by a constitutive promoter, the CAR-based surface antigen-regulated inducible promoter-therapeutic payload constructs described herein have several advantages over the prior art, including, but not limited to: i) the ability to optimally perform anti-tumor CAR function by tailoring or modulating the timing and magnitude of the anti-tumor response depending on the specific amount of antigen expressed by the tumor at that time; ii) the ability to prevent deleterious T cell hyperactivation (exhaustion, activation-induced cell death, reduced metabolic capacity, rapid terminal differentiation); iii) the ability to reduce or eliminate the risk of toxicity associated with inappropriate or excessive CAR activation; iv) the ability to reduce or eliminate CAR-associated cytokine release syndrome (CRS); or v) the ability to reduce or eliminate CAR-associated neurotoxicity, or any combination thereof.

[0124] Thus, in one embodiment of the invention disclosed and described herein, the at least one CAR-based therapeutic payload construct self-driven by a surface antigen-regulated inducible promoter provided herein comprises at least one chimeric antigen receptor operably linked to a surface antigen-regulated inducible promoter, which regulates the expression level of one or more therapeutic payloads depending on the expression level of a surface antigen on a target cell.

[0125] In one embodiment, the self-driven CAR-based surface antigen-regulated promoter-therapeutic payload constructs provided herein comprise one or more CAR-based therapeutic payloads operably linked to, for example, but not limited to, a surface antigen-regulated inducible promoter, wherein the CAR-based therapeutic payload comprises a CAR comprising, from N- to C-terminus, at least one extracellular binding domain comprising a mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen binding domain, or a combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain.

[0126] CARs are artificially constructed hybrid proteins or polypeptides containing an antibody antigen-binding domain (e.g., a single-chain variable fragment (ScFv)) linked to a T cell signaling domain via a transmembrane domain. Characteristics of CARs include the ability to redirect T cell specificity and reactivity to a selected target in an MHC-independent manner, leveraging the antigen-binding properties of monoclonal antibodies. Because of their ability to recognize antigens without MHC restriction, CAR-expressing T cells have the ability to recognize antigens independent of antigen processing, thereby circumventing a major mechanism of tumor escape. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

[0127] The unique ability to combine functional moieties from different protein domains is an innovative feature of CARs. The choice of these protein domains, as well as the specific binding mode, are important design features. Individual design domains are essential components that can be used in any heterogeneous CAR platform to manipulate lymphocyte function. For example, the selection of extracellular binding domains can enable otherwise ineffective CARs.

[0128] The physicochemical properties of the immunoglobulin-derived protein sequences used to generate the extracellular antigen-binding domain of the self-driven CAR-based surface antigen-regulated promoter-therapeutic payload constructs can be completely neutral or can be such that they significantly reduce the efficacy of therapeutic T cells expressing the self-driven CAR-based surface antigen-regulated promoter-therapeutic payload constructs by self-binding and metabolically exhausting the T cells, independent of the antigen-binding function of the CAR domain. Furthermore, the choice of intracellular signaling domain can also govern the activity and durability of therapeutic lymphocyte populations used in immunotherapy. While the ability to bind target antigens and deliver activation signals to T cells via the extracellular and intracellular domains described above are important CAR design aspects, it has become clear that the choice of source of the extracellular antigen-binding fragment can have a significant effect on the potency of self-driven CAR-based surface antigen-regulated promoter-therapeutic payload constructs and therefore may play a crucial role in the function and clinical utility of self-driven CAR-based surface antigen-regulated promoter-therapeutic payload constructs.

[0129] As disclosed herein, the intracellular T cell signaling domain of a self-driven CAR-based surface antigen-regulated promoter-therapeutic payload construct can include, for example, a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. A T cell receptor signaling domain refers to a portion of a surface antigen-regulated promoter-therapeutic payload construct that includes the intracellular domain of a T cell receptor, such as, but not limited to, the intracellular portion of the CD3 zeta protein. A costimulatory signaling domain refers to a portion of a surface antigen-regulated promoter-therapeutic payload construct that includes the intracellular domain of a costimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand that is necessary for lymphocytes to efficiently respond to antigens.

[0130] The self-driving CAR-based surface antigen-regulated promoter-therapeutic payload constructs of the present invention are now described in detail, including a description of their extracellular antigen-binding domains, transmembrane domains, and intracellular domains, as well as further description of the self-driving CAR-based surface antigen-regulated promoter-therapeutic payload constructs, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression vectors, and host cells, as well as treatment methods, compositions, and kits that use the disclosed self-driving CAR-based surface antigen-regulated promoter-therapeutic payload constructs.

[0131] 1. Extracellular domain In one embodiment, the CAR-based surface antigen-regulated promoter-therapeutic payload construct comprises a target-specific binding element, also referred to as an antigen-binding domain or site. The choice of domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that can serve as ligands for the antigen-binding domain of the therapeutic surface antigen target-specific promoter-payload construct include those associated with viral infection, bacterial infection, and parasitic infection, autoimmune disease, and cancer cells.

[0132] In one embodiment, the surface antigen-regulated promoter-therapeutic payload construct can be designed to target a tumor antigen of interest by engineering a desired antigen-binding domain that specifically binds to an antigen on tumor cells. Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. The choice of antigen-binding domain can depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin. The tumor antigens disclosed herein are included by way of example only, and the list is not intended to be limiting, as other examples will be readily apparent to those skilled in the art.

[0133] In one embodiment, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express multiple proteins that can serve as target antigens for immune attack. Such molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules include those belonging to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins represent truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are also potential target antigens in B-cell lymphomas. Several of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy using monoclonal antibodies, but without much success.

[0134] A type of tumor antigen may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not occur on other cells in the body. TAAs are not unique to tumor cells; instead, they are also expressed on normal cells under conditions that do not induce immune tolerance to the antigen. Expression of the antigen in a tumor may occur under conditions that allow the immune system to respond to the antigen. A TAA may be an antigen that is expressed on normal cells during fetal development, when the immune system is not mature and cannot respond to the antigen, or a TAA may be an antigen that is normally present at very low levels on normal cells but is expressed at significantly higher levels on tumor cells.

[0135] Examples of TSAs or TAAs include, but are not limited to, differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and the like; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, and CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0136] Additionally, in certain embodiments, human extracellular antigen binding domains are used in place of murine-derived binding domains, resulting in self-driven CAR-based surface antigen-regulated promoter-therapeutic payload constructs that function better in vivo, while avoiding the induction of anti-CAR immunity in the host immune response and the killing of CAR-T cell populations associated with murine-based antigen binding domains.

[0137] Self-driving CAR-based surface antigen-regulated promoter-therapeutic payload constructs expressing fully human extracellular ScFv antigen-binding domains exhibit superior activity, including: i) prevention of poor CAR-T persistence and function as observed with murine-derived binding sequences; ii) elimination of the need for localized targeting of the self-driving CAR-based surface antigen-regulated promoter-therapeutic payload construct for efficacy; and iii) the ability to engineer CAR-T cells based on both high- and low-affinity binders to individual antigens. This latter attribute allows researchers to better tailor the efficacy versus toxicity and / or tissue specificity of CAR-T products, since lower-affinity binders may have higher specificity for tumors than normal tissues due to the higher expression of certain antigens in tumors compared to normal tissues, thereby preventing on-target off-tumor toxicity and bystander cell killing.

[0138] In a preferred embodiment, the antigen binding domain portion of the CAR-based surface antigen-regulated promoter-therapeutic payload construct targets antigens including, but not limited to, CD19, CD20, CD22, ROR1, mesothelin, CD33, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, and MAGE A3 TCR.

[0139] In one embodiment, the extracellular antigen-binding domain in a CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise an scFV binder, for example, as disclosed in commonly-assigned issued U.S. Patent No. 10,183,993 (entitled "Compositions and Methods for Treating Cancer with Anti-Mesothelin Immunotherapy," first filed January 9, 2017 as Provisional Patent Application No. 62 / 444,201, issued January 22, 2019, and assigned Docket No. LEN_017 to Lentigen Technology, Inc.).

[0140] In a preferred embodiment, an isolated nucleic acid molecule encoding the extracellular mesothelin antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 87, or a sequence 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular mesothelin antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 88.

[0141] In another embodiment, the nucleic acid sequence encoding the CAR comprises the nucleic acid sequence of SEQ ID NO: 89, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 90, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In addition to scFv sequences that can be used as extracellular antigen-binding domains in CAR-based surface antigen-regulated promoter-therapeutic payload constructs, single-chain antigen binders (as opposed to scFvs) can be incorporated into functional CARs.

[0142] For example, CD33-specific heavy chain-only binders as disclosed in commonly-owned, co-pending non-provisional patent application Ser. No. 15 / 934,770 (Provisional Patent No. 62 / 476,438) (entitled "Compositions and Methods For Treating Cancer With Anti-CD33 Immunotherapy," filed Mar. 24, 2018, and assigned Docket No. LEN_018 to Lentigen Technology, Inc.).

[0143] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD33 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO:91, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD33 antigen-binding domain comprises the amino acid sequence of SEQ ID NO:92, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0144] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG1906 that targets CD33-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 93 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the amino acid sequence of SEQ ID NO: 94 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0145] In one embodiment, the extracellular antigen-binding domain in a CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise an scFV binder, for example, as disclosed in commonly-assigned, co-pending provisional patent application Ser. No. 62 / 773,940, entitled "Compositions and Methods for Treating Cancer with Anti-CD38 Immunotherapy," filed Nov. 30, 2018, and assigned Docket No. LEN_026 to Lentigen Technology, Inc.

[0146] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD38 antigen-binding domain M3803 comprises the nucleotide sequence of SEQ ID NO: 144, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 145, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0147] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD38 antigen-binding domain M3804 comprises the nucleotide sequence of SEQ ID NO: 146, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 147, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0148] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD38 antigen-binding domain M3809 comprises the nucleotide sequence of SEQ ID NO: 148, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 149, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0149] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD38 antigen-binding domain M3811 comprises the nucleotide sequence of SEQ ID NO: 150, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD38 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 151, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2091 that targets CD38-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 7 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 8 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0150] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2092 that targets CD38-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 9 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 10 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0151] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2095 that targets CD38-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 11 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the amino acid sequence of SEQ ID NO: 12 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0152] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2097 that targets CD38-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 15 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 16 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0153] In one embodiment, the extracellular antigen-binding domain in a CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise an scFV binder, for example, as disclosed in commonly owned, co-pending non-provisional patent application Ser. No. 16 / 179,364, entitled "Compositions and Methods for Treating Cancer with Anti-ROR1 Immunotherapy," filed Nov. 2, 2018, and assigned Docket No. LEN_022 to Lentigen Technology, Inc.

[0154] In one embodiment, an isolated nucleic acid molecule encoding the extracellular ROR1 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 152, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 153, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0155] In one embodiment, an isolated nucleic acid molecule encoding the extracellular ROR1 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 154, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0156] In one embodiment, an isolated nucleic acid molecule encoding the extracellular ROR1 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 156, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 157, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0157] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 1941 that targets ROR1-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 17 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the amino acid sequence of SEQ ID NO: 18 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0158] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 1942 that targets ROR1-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 19 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the amino acid sequence of SEQ ID NO: 20 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0159] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 1943 that targets ROR1-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 21 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the amino acid sequence of SEQ ID NO: 22 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0160] In one embodiment, the extracellular antigen-binding domain in a CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise an scFV binder, for example, as disclosed in commonly-assigned, co-pending provisional patent application Ser. No. 62 / 734,106, entitled "Compositions and Methods for Treating Cancer with Anti-CD123 Immunotherapy," filed Sep. 20, 2018, and assigned Docket No. LEN_024 to Lentigen Technology, Inc.

[0161] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 antigen-binding domain M12303 comprises the nucleotide sequence of SEQ ID NO: 158, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 159, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0162] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 antigen-binding domain M12304 comprises the nucleotide sequence of SEQ ID NO: 160, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 161, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0163] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 antigen-binding domain M12305 comprises the nucleotide sequence of SEQ ID NO: 162, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 163, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0164] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 antigen-binding domain M12306 comprises the nucleotide sequence of SEQ ID NO: 164, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 165, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0165] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 antigen-binding domain M12308 comprises the nucleotide sequence of SEQ ID NO: 166, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 167, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0166] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 antigen-binding domain M12311 comprises the nucleotide sequence of SEQ ID NO: 168, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 169, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0167] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 antigen-binding domain M12313 comprises the nucleotide sequence of SEQ ID NO: 170, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 171, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0168] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 antigen-binding domain M12315 comprises the nucleotide sequence of SEQ ID NO: 172, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 173, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0169] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 antigen-binding domain M12317 comprises the nucleotide sequence of SEQ ID NO: 174, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 175, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0170] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD123 antigen-binding domain M12318 comprises the nucleotide sequence of SEQ ID NO: 176, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 177, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0171] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2075 that targets CD123-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 23 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 24 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0172] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2076 that targets CD123-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 25 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 26 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0173] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2077 that targets CD123-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 115 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 116 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0174] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2078 that targets CD123-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 117 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the amino acid sequence of SEQ ID NO: 118 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0175] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2079 that targets CD123-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 119 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 120 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0176] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2082 that targets CD123-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 121 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 122 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0177] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2083 that targets CD123-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 123 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the amino acid sequence of SEQ ID NO: 124 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0178] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2085 that targets CD123-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 125 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 126 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0179] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2087 that targets CD123-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 127 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 128 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0180] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2088 that targets CD123-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 129 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and the amino acid sequence of SEQ ID NO: 130 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0181] In one embodiment, the extracellular antigen-binding domain in a CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise an scFV binder, for example, as disclosed in commonly-assigned, co-pending provisional patent application Ser. No. 62 / 736,955, entitled "Compositions and Methods for Treating Cancer with Human Anti-CD19 / 22 Immunotherapy," filed Sep. 26, 2018, and assigned Lentigen Technology Inc., Case No. LEN_025.

[0182] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD19 / CD22 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 178, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular mesothelin antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 179, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0183] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2737 that targets CD19 / CD22-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 131 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 132 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0184] In one embodiment, the extracellular antigen-binding domain in a CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise an scFV binder, for example, as disclosed in commonly owned, co-pending non-provisional patent application Ser. No. 16 / 161,542, entitled "Compositions and Methods for Treating Cancer with Human Anti-CD22 Immunotherapy," filed Oct. 16, 2018, and assigned Docket No. LEN_021 to Lentigen Technology, Inc.

[0185] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD22 antigen-binding domain 16P17 comprises the nucleotide sequence of SEQ ID NO: 180, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular mesothelin antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 181, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0186] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD22 antigen-binding domain 16P13 comprises the nucleotide sequence of SEQ ID NO: 182, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular mesothelin antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 183, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0187] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2209 that targets CD22-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 133 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 134 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0188] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2219 that targets CD22-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 135 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 136 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0189] In one embodiment, the extracellular antigen-binding domain in a CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise an scFV binder, for example, as disclosed in commonly owned, co-pending non-provisional patent application Ser. No. 16 / 050,754, entitled "Compositions and Methods for Treating Cancer with Anti-CD19 / 20 Immunotherapy," filed Jul. 31, 2018, and assigned Docket No. LEN_019 to Lentigen Technology, Inc.

[0190] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD19 / CD20 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 141, or a sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular mesothelin antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0191] In one embodiment, an isolated nucleic acid molecule encoding the extracellular CD19 / CD20 antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 113, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular mesothelin antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0192] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 1496 that targets CD19 / CD20-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 95 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 96 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0193] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 1497 that targets CD19 / CD20-expressing malignancies encodes a CAR comprising the nucleic acid sequence of SEQ ID NO: 97 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto, and comprising the amino acid sequence of SEQ ID NO: 98 or a sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0194] In yet another embodiment, the nucleic acid sequence encoding the CAR-based surface antigen-regulated promoter-therapeutic payload construct comprises one or more of the nucleic acid sequences disclosed in commonly-owned, co-pending continuation-in-part patent application Ser. No. 16 / 134,735, filed Sep. 18, 2018, entitled "Compositions and Methods for Treating Cancer with DuoCARs," which claims priority to PCT Application No. PCT / US17 / 49923, filed Sep. 1, 2017, which claims the benefit of priority pursuant to U.S. Provisional Patent Application No. 62 / 382,791, filed Sep. 2, 2016, pursuant to 35 U.S.C. § 119(e) (the entire contents of each of these applications are incorporated herein by reference).

[0195] Thus, in one embodiment, variant monospecific CAR structures known to be compatible with the applicant's DuoCAR platform can also be utilized to generate CAR-based surface antigen-regulated promoter-therapeutic payload constructs. A specific example of a monospecific therapeutic surface antigen-regulated promoter-payload construct (e.g., CAR-based) on which DuoCAR-based surface antigen-regulated promoter-therapeutic payload construct technology can be based includes the single CD20-targeting vector LTG1495 (nucleotide sequence SEQ ID NO: 142, amino acid sequence SEQ ID NO: 143). A second example is the monospecific CAR LTG2200 (nucleotide sequence SEQ ID NO: 69, amino acid sequence SEQ ID NO: 70) specific for CD22.

[0196] In yet another embodiment, variant CAR structures known to be compatible with the DuoCAR field can also be utilized to generate CAR-based surface antigen-regulated promoter-therapeutic payload constructs within the scope of this disclosure. These include the CD19-specific CAR LTG1494 (nucleotide sequence SEQ ID NO: 71, amino acid sequence SEQ ID NO: 72). This sequence includes a well-described linker called the Whitlow linker (amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 184); Whitlow M. et al., 1993, Protein Eng. 6:989-995), which connects the heavy and light chains of the scFv. In some cases, for example, in the CD19 CAR format, the Whitlow linker replaced the (GGGGS)n linker (SEQ ID NO: 185), as in LTG1538 (nucleotide sequence SEQ ID NO: 73, amino acid sequence SEQ ID NO: 74). In another example, CARs with other transmembrane domains were generated. Anti-CD19 CAR LTG1562 (nucleotide sequence SEQ ID NO:75, amino acid sequence SEQ ID NO:76) utilizes the CD4 (but not CD8) transmembrane domain. Similarly, anti-CD19 CAR LTG1563 has the other transmembrane form derived from TNFRSF19 (nucleotide sequence SEQ ID NO:77, amino acid sequence SEQ ID NO:78).

[0197] In yet another embodiment, another example of a therapeutic application may be the treatment of leukemias expressing CD19, CD20, and TSLPR antigens using the DuoCAR-based surface antigen-regulated promoter-therapeutic payload construct of the present invention. In particular, the DuoCAR-based surface antigen-regulated promoter-therapeutic payload construct comprises LTG1496 or LTG 1497 (SEQ ID NOs: 95 and 97, respectively) linked to a TSLPR-specific CAR (LTG1789) (SEQ ID NO: 101, amino acid sequence SEQ ID NO: 102) made from a TSLPR-specific scFV domain (nucleotide sequence SEQ ID NO: 99, amino acid sequence SEQ ID NO: 100).

[0198] In one embodiment, for each of the DuoCAR-based surface antigen-regulated promoter-therapeutic payload constructs described above, the individual CAR constructs are self-driven by a single surface antigen-regulated promoter and are separated by ribosomal 2A skip sites.

[0199] In another embodiment, for each of the DuoCAR-based surface antigen-regulated promoter-therapeutic payload constructs described above, the individual CAR constructs are self-driven by separate surface antigen-regulated promoters.

[0200] In certain embodiments, as used herein, non-limiting examples of anti-cd19 CAR constructs include an anti-cd19 CAR construct encoded by the nucleotide sequence designated herein as LTG1563 (see SEQ ID NO: 77), which encoded anti-cd19 CAR construct is identified herein as CAR-LTG1563 (see SEQ ID NO: 78).

[0201] In one embodiment, construction of a surface antigen-regulated inducible promoter-therapeutic payload construct comprising the nucleic acid sequence of SEQ ID NO: 139 and encoding a CAR-based promoter-therapeutic payload construct comprising the amino acid sequence of SEQ ID NO: 78 (CAR LTG1563) is described in Example 1 below.

[0202] In one embodiment, construction of a surface antigen-regulated inducible promoter-therapeutic payload construct comprising the nucleic acid sequence of SEQ ID NO: 140 and encoding a CAR-based promoter-therapeutic payload construct comprising the amino acid sequence of SEQ ID NO: 78 (CAR LTG1563) is described in Example 1 below.

[0203] In one embodiment, construction of a surface antigen-regulated inducible promoter-therapeutic payload construct comprising the nucleic acid sequence of SEQ ID NO: 77 and encoding a CAR-based promoter-therapeutic payload construct comprising the amino acid sequence of SEQ ID NO: 78 (CAR LTG1563) is described in Example 1 below.

[0204] While not intending to be limited to any particular mechanism of action, reasons for improved therapeutic function associated with exemplary surface antigen-regulated promoter-therapeutic payload constructs of the present invention may include, but are not limited to, for example, a) more efficient signaling due to improved lateral movement in the plasma membrane, b) improved ability to interact with transmembrane signaling cascades associated with T cell activation due to superior location in plasma membrane microdomains (e.g., lipid rafts), c) superior location in the plasma membrane due to preferential movement away from reducing or down-regulating interactions, e.g., greater distance from or less interaction with phosphatases such as CD45, and d) superior assembly into the T cell receptor signaling complex (e.g., immune synapse), or any combination thereof.

[0205] Depending on the desired antigen to be targeted, the surface antigen-regulated promoter-therapeutic payload construct can be designed to include an appropriate antigen binding domain specific for the desired antigen target. For example (but not limited to), if CD19 is the desired antigen to be targeted, an antibody to CD19 can be used as the antigen binding domain to be incorporated into the surface antigen-regulated promoter-therapeutic payload construct.

[0206] In one exemplary embodiment, the antigen-binding domain portion of a CAR-based surface antigen-regulated promoter-therapeutic payload construct targets CD19. Preferably, the extracellular antigen-binding domain in the surface antigen-regulated promoter-therapeutic payload construct is an anti-CD19 scFV, wherein the nucleic acid sequence of the extracellular anti-CD19 scFV comprises the sequence of SEQ ID NO: 37 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, the extracellular anti-CD19 scFV comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 38 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In another embodiment, the extracellular anti-CD19 scFV portion of the CAR comprises the amino acid sequence of SEQ ID NO: 38 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0207] In one embodiment of the present invention, surface antigen-regulated promoter-therapeutic payload constructs are provided that can bind to a non-TSA or a non-TAA, including, for example, but not limited to, antigens derived from Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, Adenoviridae, Herpesviridae (e.g., herpes simplex virus types 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpes viruses), Poxviridae (e.g., smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus, or any combination thereof.

[0208] In another aspect of the present invention, surface antigen-regulated promoter-therapeutic payload constructs are provided that can bind to antigens derived from bacterial species such as Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella. In particular, surface antigen-regulated promoter-therapeutic payload constructs are provided that can bind to antigens derived from infectious bacteria such as Helicobacter pylori, Legionella pneumophila, mycobacterial species (e.g., Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or combinations thereof.

[0209] 2. Transmembrane domain With respect to the transmembrane domain, the CAR-based surface antigen-regulated promoter-therapeutic payload construct comprises one or more TNFRSF transmembrane domains fused to the extracellular domain of the surface antigen-regulated promoter-therapeutic payload construct.

[0210] In one embodiment, the TNFRSF transmembrane domain comprises at least one TNFRSF19 transmembrane domain. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded TNFRSF transmembrane domain comprises a TNFRSF19 transmembrane domain.

[0211] In one embodiment, an isolated nucleic acid molecule encoding the TNFRSF19 transmembrane domain comprises the nucleotide sequence of SEQ ID NO:51 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded TNFRSF19 transmembrane domain comprises the amino acid sequence of SEQ ID NO:52 or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0212] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0213] Particularly useful transmembrane regions in the surface antigen-controlled promoter-therapeutic payload constructs described herein may be derived from (i.e., comprise at least the transmembrane region of) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic and, in this case, may comprise primarily hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the cytoplasmic signaling domain of the surface antigen-controlled promoter-therapeutic payload construct. A glycine and serine doublet provides a particularly suitable linker.

[0214] In one embodiment, a transmembrane domain originally associated with one of the domains in the surface antigen-regulated promoter-therapeutic payload construct is used in addition to the transmembrane domains described above.

[0215] In some instances, the transmembrane domain can be selected by amino acid substitution to prevent the domain from binding to the transmembrane domain of the same or a different surface membrane protein, in order to minimize interactions of the domain with other receptor complex components.

[0216] In one embodiment, the transmembrane domain in a surface antigen-regulated promoter-therapeutic payload construct of the invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 27. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 28.

[0217] In one embodiment, the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO: 28, or a sequence that is 95-99% identical to the amino acid sequence of SEQ ID NO: 28 with at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions).

[0218] In some examples, the transmembrane domain of the CAR comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 29. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 30 or a sequence having 95-99% identity thereto.

[0219] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0220] In one embodiment of the patient-specific autologous anti-tumor lymphocyte cell populations disclosed herein, non-limiting examples of transmembrane domains for use in the CAR-based surface antigen-regulated promoter-therapeutic payload constructs disclosed herein include the TNFRSF16 and TNFRSF19 transmembrane domains, which can be used to derive TNFRSF transmembrane domains and / or linker or spacer domains (including, in particular, other TNFRSF members listed in the tumor necrosis factor receptor superfamily listed in Table I thereof) as disclosed in commonly-owned, co-pending patent application Ser. No. 15 / 767,076 (entitled "CHIMERIC ANTIGEN RECEPTORS AND METHODS OF USE," filed Apr. 9, 2018, to Lentigen Technology, Inc., and assigned Docket No. LEN_015(US)).

[0221] In one embodiment, the transmembrane domain in a CAR of the present invention is a TNFRSF19 transmembrane domain. In one embodiment, the TNFRSF19 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 51. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 52. In another embodiment, the TNFRSF19 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 52.

[0222] In one embodiment, the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO: 52, or a sequence that is 95-99% identical to the amino acid sequence of SEQ ID NO: 52 with at least one, two, or three modifications (e.g., substitutions), but not more than 20, 10, or 5 modifications (e.g., substitutions).

[0223] 3. Spacer domain In CAR-based surface antigen-regulated promoter-therapeutic payload constructs, a spacer domain may be located between the extracellular domain and the TNFRSF transmembrane domain or between the intracellular domain and the TNFRSF transmembrane domain. A spacer domain refers to any oligopeptide or polypeptide that functions to connect the TNFRSF transmembrane domain to the extracellular domain and / or the TNFRSF transmembrane domain to the intracellular domain. The spacer domain may contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0224] In some embodiments, the linker may include a spacer element, which, when present, increases the size of the linker and the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Examples of spacers are well known to those skilled in the art and are described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,2 84, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, and those listed in U.S. Patent Publication Nos. 20110212088 and 20110070248, which are incorporated by reference in their entireties.

[0225] The spacer domain preferably has a sequence that promotes binding between the CAR-based surface antigen-regulated promoter-therapeutic payload construct and the antigen, thereby increasing signal transduction into the cell. Examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine at potential glycosylation sites, and these amino acids can be used as amino acids constituting the spacer domain.

[0226] The spacer domain can be the entire or a portion of amino acids 118-178 (SEQ ID NO: 31) of the hinge region of CD8 alpha (NCBI RefSeq: NP_001759.3), amino acids 135-195 of CD8 beta (GenBank: AAA35664.1), amino acids 315-396 of CD4 (NCBI RefSeq: NP_000607.1), or amino acids 137-152 of CD28 (NCBI RefSeq: NP_006130.1). The spacer domain can also be a portion of the constant region of an antibody heavy or light chain (CH1 region or CL region, e.g., a peptide having the amino acid sequence of SEQ ID NO: 32). Furthermore, the spacer domain may be an artificially synthesized sequence.

[0227] Additionally, all or part of the amino acids comprising the constant region of human IgG4 (UniProt ID: P01861), including CH1 (amino acid numbers 1-98), hinge, SEQ ID NO: 80, and corresponding nucleotide SEQ ID NO: 79, (amino acid numbers 99-110), CH2, amino acid number 81 and corresponding nucleotide SEQ ID NO: 80, (amino acid numbers 111-220), and CH3, SEQ ID NO: 84 and corresponding nucleotide SEQ ID NO: 83, (amino acid numbers 221-327), or combinations thereof (such as the IgG4 hinge CH2 CH3 domain, SEQ ID NO: 86, and corresponding nucleotide SEQ ID NO: 85) can also be used.

[0228] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 hinge domain comprising the nucleic acid sequence of SEQ ID NO: 53. In one embodiment, the TNFRSF19 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 54. In another embodiment, the TNFRSF19 hinge domain comprises the amino acid sequence of SEQ ID NO: 54, or a sequence having 95-99% identity thereto.

[0229] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 truncated hinge domain comprising the nucleic acid sequence of SEQ ID NO: 55. In one embodiment, the TNFRSF19 truncated hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 56. In another embodiment, the TNFRSF19 truncated hinge domain comprises the amino acid sequence of SEQ ID NO: 56, or a sequence having 95-99% identity thereto.

[0230] In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 49. In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50. In another embodiment, the TNFRSF19 hinge and transmembrane domain comprises the amino acid sequence of SEQ ID NO: 50, or a sequence having 95-99% identity thereto.

[0231] In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the nucleic acid sequence of SEQ ID NO: 57. In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 58. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 58, or a sequence having 95-99% identity thereto.

[0232] Furthermore, the surface antigen-regulated promoter-therapeutic payload construct may have a signal peptide sequence (also referred to as a leader peptide) attached to its N-terminus. This signal peptide sequence is present at the N-terminus of many secretory proteins and membrane proteins and is 15 to 30 amino acids in length. Since many of the protein molecules described above as intracellular domains have a signal peptide sequence, this signal peptide can be used as the signal peptide for the surface antigen-regulated promoter-therapeutic payload construct. In one embodiment, the signal peptide comprises the amino acid sequence of SEQ ID NO: 14.

[0233] In one embodiment, the CD8 alpha leader peptide comprises the nucleic acid sequence of SEQ ID NO: 43. In one embodiment, the CD8 alpha leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 44, or a sequence having 95-99% identity thereto.

[0234] In another embodiment, the GMCSF leader peptide comprises the nucleic acid sequence of SEQ ID NO: 39. In one embodiment, the GMCSF leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 40, or a sequence having 95-99% identity thereto.

[0235] In another embodiment, the TNFRSF19 leader peptide comprises the nucleic acid sequence of SEQ ID NO: 41. In one embodiment, the TNFRSF19 leader peptide and the CD8 alpha leader peptide comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 42, or a sequence having 95-99% identity thereto.

[0236] In one embodiment, the tag sequence encoding a truncated sequence of epidermal growth factor receptor (tEGFR) comprises the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the tEGFR comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68, or a sequence having 95 to 99% identity thereto.

[0237] In one embodiment, the furin recognition site and downstream T2A self-cleaving peptide sequence designed for simultaneous bicistronic expression of the tag sequence and therapeutic payload sequence comprise the nucleic acid sequence of SEQ ID NO: 65. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 66. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 66, or a sequence having 95-99% identity thereto.

[0238] In one embodiment, the upstream furin recognition site, T2A self-cleaving peptide sequence, and downstream furin recognition site designed for simultaneous bicistronic expression of the tag sequence and CAR sequence comprise the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, the furin and T2A sequences comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 68. In another embodiment, the tEGFR tag comprises the amino acid sequence of SEQ ID NO: 68, or a sequence having 95-99% identity thereto.

[0239] In one embodiment, the targeting domain of a CAR-based surface antigen-controlled promoter-therapeutic payload construct is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2 and contains a binding tag or epitope, while the effector cell-expressed component of the surface antigen-controlled promoter-therapeutic payload construct contains a binding domain specifically directed to bind to the tag or epitope expressed on the soluble CAR module, thereby specifically binding the soluble component of the CAR with the cell-binding component of the CAR to form a fully functional CAR structure.

[0240] 4. Intracellular domain The cytoplasmic domain or intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of an immune cell into which the CAR is introduced. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a protein portion that transmits an effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, it can be used in place of the entire chain, as long as the truncated portion is capable of transmitting the effector function signal. Thus, the meaning of the term "intracellular signaling domain" includes any truncated portion of the intracellular signaling domain that is sufficient to transmit the effector function signal.

[0241] Preferred examples of intracellular signaling domains for use in CARs include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that cooperate to initiate signal transduction following binding of an antigen to the receptor, as well as any derivatives or variants of these sequences, and any synthetic sequence with the same functional capability.

[0242] It is known that signals emitted through the TCR alone are insufficient to fully activate T cells, and that a secondary or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct types of cytoplasmic signaling sequences: one that initiates antigen-dependent primary activation via the TCR (first cytoplasmic signaling sequence), and one that acts in an antigen-independent manner to provide a secondary or costimulatory signal (second cytoplasmic signaling sequence).

[0243] The first cytoplasmic signaling sequence controls the primary activation of the TCR complex in either a stimulatory or inhibitory manner. A first cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM.

[0244] Examples of ITAMs containing a first cytoplasmic signaling sequence that are particularly useful in the CARs disclosed herein include those derived from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Specific examples of ITAMs include amino acids 51-164 of CD3 zeta (NCBI RefSeq: NP__932170.1), amino acids 45-86 of Fc epsilon RI gamma (NCBI RefSeq: NP__004097.1), amino acids 201-244 of Fc epsilon RI beta (NCBI RefSeq: NP__000130.1), amino acids 139-182 of CD3 gamma (NCBI RefSeq: NP__000064.1), amino acids 128-171 of CD3 delta (NCBI RefSeq: NP__000723.1), amino acids 153-207 of CD3 epsilon (NCBI RefSeq: NP__000724.1), and CD5 (NCBI RefSeq: NP__000724.1). These peptides include, but are not limited to, peptides having amino acids 402-495 of CD79a (NCBI RefSeq:NP__055022.2), amino acids 707-847 of CD79a (NCBI RefSeq:NP__001762.2), amino acids 166-226 of CD79a (NCBI RefSeq:NP__001774.1), amino acids 182-229 of CD79b (NCBI RefSeq:NP__000617.1), and amino acids 177-252 of CD66d (NCBI RefSeq:NP__001806.2), as well as variants having the same functions as these peptides. The amino acid numbers based on the NCBI RefSeq IDs or GenBank amino acid sequence information described herein are numbered based on the full length of the precursor of each protein (including the signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0245] In a preferred embodiment, the intracellular domain of the CAR may be designed to contain a CD3-zeta signaling domain by itself, or may be combined with any other desired cytoplasmic domain useful in the context of a CAR. For example, the intracellular domain of a CAR may contain a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of a CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is necessary for lymphocytes to efficiently respond to antigens. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83. Specific examples of such costimulatory molecules include amino acids 236-351 of CD2 (NCBI RefSeq: NP__001758.2), amino acids 421-458 of CD4 (NCBI RefSeq: NP__000607.1), amino acids 402-495 of CD5 (NCBI RefSeq: NP__055022.2), amino acids 207-235 of CD8 alpha (NCBI RefSeq: NP__001759.3), amino acids 196-210 of CD83 (GenBank: AAA35664.1), amino acids 181-220 of CD28 (NCBI RefSeq: NP__006130.1), and CD137 (4-1BB, NCBI The costimulatory signaling element includes, but is not limited to, peptides having amino acids 214-255 of CD134 (OX40, NCBI RefSeq:NP_001552.2), amino acids 241-277 of CD134 (OX40, NCBI RefSeq:NP_003318.1), and amino acids 166-199 of ICOS (NCBI RefSeq:NP_036224.1), as well as variants having the same function as these peptides. Thus, although the present disclosure has thus far been exemplified primarily using 4-1BB as a costimulatory signaling element, other costimulatory elements are also within the scope of the present disclosure.

[0246] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specific order. Optionally, a short oligo- or polypeptide linker, preferably 2-10 amino acids in length, can form this linkage. A glycine and serine doublet provides a particularly suitable linker.

[0247] In one embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domains of CD28 and 4-1BB.

[0248] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence of SEQ ID NO:33, SEQ ID NO:45, or SEQ ID NO:59, respectively, and the signaling domain of CD3-zeta comprises the nucleic acid sequence of SEQ ID NO:35, SEQ ID NO:47, or SEQ ID NO:61, respectively.

[0249] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:34, SEQ ID NO:46, or SEQ ID NO:60, respectively, and the signaling domain of CD3-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:48 or SEQ ID NO:62.

[0250] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the amino acid sequence of SEQ ID NO:34, SEQ ID NO:46, or SEQ ID NO:60, respectively, and the signaling domain of CD3-zeta comprises the amino acid sequence of SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62, respectively.

[0251] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, wherein the signaling domain of CD28 comprises the nucleic acid sequence of SEQ ID NO:45 or SEQ ID NO:59, respectively, and the signaling domain of CD3-zeta comprises the nucleic acid sequence of SEQ ID NO:35, SEQ ID NO:47, or SEQ ID NO:61, respectively.

[0252] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, wherein the signaling domain of CD28 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:46 or SEQ ID NO:60, respectively, and the signaling domain of CD3-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62.

[0253] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-zeta, wherein the signaling domain of CD28 comprises the amino acid sequence of SEQ ID NO:46 or SEQ ID NO:60, respectively, and the signaling domain of CD3-zeta comprises the amino acid sequence of SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62, respectively.

[0254] 5. CAR with additional auxiliary components In another embodiment, a surface antigen-regulated inducible promoter-therapeutic payload construct is constructed comprising additional ancillary components including a dominant-negative receptor of either TGFBRII (TGFBRIIdn) and / or PD1 (PD1dn) lacking an intracellular signaling domain co-expressed with the CAR LTG1563 construct via a ribosomal skipping site (P2A) to generate a surface antigen-regulated inducible promoter-therapeutic payload construct encoding a CAR-based promoter-therapeutic payload construct comprising the nucleic acid sequence of SEQ ID NO: 103, 105, 107, or a combination thereof, and the amino acid sequence of SEQ ID NO: 104, 106, 108, or a combination thereof, as described in Example 2 below.

[0255] In another embodiment, a surface antigen-regulated inducible promoter-therapeutic payload construct is constructed that includes additional ancillary components, including dominant negative receptors for TGFBRII (TGFBRIIdn) or PD1 (PD1dn) lacking the intracellular signaling domain, co-expressed with the CAR LTG1563 construct via a skipping site (P2A), to generate a surface antigen-regulated inducible promoter-therapeutic payload construct that encodes a CAR-based promoter-therapeutic payload construct comprising the nucleic acid sequence of SEQ ID NO: 103, 105, 107, or a combination thereof, and the amino acid sequence of SEQ ID NO: 104, 106, 108, or a combination thereof, wherein the dominant negative receptors dnTGFb and dnPD1 are used to prevent autoimmune toxicity typically associated with the use of constitutively activated dominant negative receptors, while providing the advantage of reduced immunosuppression of T cell function when the CAR is expressed and functional, or to prevent the autoimmune toxicity of the CAR by the tumor microenvironment compared to each dn receptor alone or in combination. T cells become more resistant to immunosuppression.

[0256] In yet another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct comprises the nucleic acid sequence of SEQ ID NO: 103 and encodes CAR LTG1563 with a dominant-negative inhibitory TGF-beta receptor comprising the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0257] In yet another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct comprises the nucleic acid sequence of SEQ ID NO: 105 and encodes CAR LTG1563 with a dominant negative PD1 comprising the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0258] In yet another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter with AP1-NFκB_RE promoter, in which a dominant negative inhibitory TGF-beta receptor and a dominant negative PD1 are co-expressed with the CAR LTG1563 construct separated by a ribosomal skipping site (P2A), comprises the nucleic acid sequence of SEQ ID NO: 107 (a construct in which a dominant negative inhibitory TGF-beta receptor and a dominant negative PD1 are co-expressed with the CAR LTG1563 construct separated by a ribosomal skipping site (P2A)), and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 108, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0259] In yet another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct with STAT5_RE promoter comprises the nucleic acid sequence of SEQ ID NO: 109 and encodes CAR LTG1563 with dominant negative inhibitory TGF-beta comprising the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0260] In yet another embodiment, an isolated nucleic acid molecule is provided, wherein a surface antigen-regulated inducible promoter-therapeutic payload construct with a STAT5_RE promoter comprises the nucleic acid sequence of SEQ ID NO: 110 and encodes CAR LTG1563 with a dominant negative PD1 comprising the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0261] In another embodiment, an isolated nucleic acid molecule is provided, wherein a surface antigen-regulated inducible promoter-therapeutic payload construct with a STAT5_RE promoter, in which a dominant negative inhibitory TGF-beta receptor and a dominant negative PD1 are co-expressed with the CAR LTG1563 construct separated by a ribosomal skipping site (P2A), comprises the nucleic acid sequence of SEQ ID NO: 111 and encodes a CAR comprising the amino acid sequence of SEQ ID NO: 108, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0262] In yet another embodiment, an isolated nucleic acid molecule is provided, wherein a constitutive promoter-therapeutic payload construct having an EF1a promoter is co-expressed with a dominant negative inhibitory TGF-beta receptor with a CAR LTG1563 construct, and the constitutive EF1a promoter is used to express an additional protein along with the co-expressed CAR LTG1563 construct.

[0263] In yet another embodiment, an isolated nucleic acid molecule is provided, wherein a constitutive promoter-therapeutic payload construct having an EF1a promoter is co-expressed with a dominant negative PD1 with a CAR LTG1563 construct, and the constitutive EF1a promoter is used to express an additional protein along with the co-expressed CAR LTG1563 construct.

[0264] In yet another embodiment, an isolated nucleic acid molecule is provided, wherein a constitutive promoter-therapeutic payload construct having an EF1a promoter, a dominant negative inhibitory TGF-beta receptor and a dominant negative PD1 are co-expressed with the CAR LTG1563 construct and separated by a ribosomal skipping site (P2A), and the constitutive EF1a promoter is used to successfully express additional proteins along with the co-expressed CAR LTG1563 construct.

[0265] 6. Further explanation of CAR Functional portions of the surface antigen-regulated promoter-therapeutic payload constructs disclosed herein (e.g., based on CARs, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulatory receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNAs, and proteases) are also expressly included within the scope of the present invention. The term "functional portion," when used in reference to a CAR, refers to any portion or fragment of one or more of the CARs disclosed herein, which portion or fragment retains the biological activity of the CAR (parent CAR). Functional portions include, for example, portions of a CAR that retain the ability to recognize target cells or detect, treat, or prevent disease to a similar extent as, or to a greater extent than, the parent CAR. With respect to a parent CAR, the functional portion can comprise, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0266] A functional portion may contain additional amino acids at the amino or carboxy terminus, or both, of the portion that are not found in the amino acid sequence of the parent CAR. Desirably, these additional amino acids do not interfere with the biological function of the functional portion (e.g., target cell recognition, cancer detection, cancer treatment or prevention, etc.). More desirably, these additional amino acids improve such biological activity over the biological activity of the parent CAR.

[0267] Functional variants of the CARs disclosed herein are included within the scope of this disclosure. The term "functional variant," as used herein, refers to a CAR, polypeptide, or protein that has substantial or significant sequence identity or similarity with a parent CAR, and the functional variant retains the biological activity of the CAR from which the variant is derived. Functional variants include, for example, variants of the CARs (parent CARs) described herein that retain the ability to recognize target cells to a similar extent as, or to a greater extent than, the parent CAR. With respect to the parent CAR, the functional variant may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98%, or more amino acid sequence identity with the parent CAR.

[0268] A functional variant may, for example, comprise at least one conservative amino acid substitution in the amino acid sequence of the parent CAR. Alternatively, or in addition, a functional variant may comprise at least one non-conservative amino acid substitution in the amino acid sequence of the parent CAR. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may improve the biological activity of the functional variant, such that the biological activity of the functional variant is superior to that of the parent CAR.

[0269] The amino acid substitutions in CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are well known in the art and include amino acid substitutions in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be the substitution of an acidic / charged polar amino acid (e.g., Asp or Glu) with another acidic / charged polar amino acid, the substitution of a non-polar side chain-containing amino acid (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.) with another non-polar side chain-containing amino acid, the substitution of a basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.) with another basic / positively charged polar amino acid, the substitution of a polar side chain-containing uncharged amino acid (e.g., Asn, Gin, Ser, Thr, Tyr, etc.) with another polar side chain-containing uncharged amino acid, the substitution of a beta-branched side chain-containing amino acid (e.g., He, Thr, and Val) with another beta-branched side chain-containing amino acid, the substitution of an aromatic side chain-containing amino acid (e.g., His, Phe, Trp, and Tyr) with another aromatic side chain-containing amino acid, and the like.

[0270] A CAR can consist essentially of one or more of the specified amino acid sequences described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0271] CARs (including functional portions and functional variants) can be of any length, i.e., contain any number of amino acids, so long as the CAR (or functional portion or functional variant thereof) retains biological activity, such as, for example, the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal. For example, a CAR can be from about 50 to about 5,000 amino acids in length, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or more amino acids in length.

[0272] CARs (including functional portions and functional variants according to the invention) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid. acid), a-aminocyclohexanecarboxylic acid, a-aminocycloheptanecarboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0273] CARs (including functional portions and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., by disulfide bridges), or converted into acid addition salts, and / or optionally dimerized or polymerized or conjugated.

[0274] CAR (including its functional part and functional variant) can be obtained by methods well known in the art. CAR can be produced by any suitable polypeptide or protein production method. Suitable methods for de novo synthesis of polypeptides and proteins are described in prior art documents such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. Polypeptides and proteins can also be produced recombinantly using the nucleic acids described herein using standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some CARs (including functional portions and functional variants thereof) may be isolated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans, etc.). Isolation and purification methods are well known in the art. Alternatively, the CARs described herein (including functional portions and functional variants thereof) may be commercially synthesized by companies. In this regard, CARs may be synthetic, recombinant, isolated, and / or purified.

[0275] In each of the descriptions in Section A above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload constructs described above, the surface antigen-regulated promoter-therapeutic payload constructs may also be directed against cytokines, including IL-2, IL-15, IL-7, TNFα, IFN gamma, IFN beta, IFN alpha, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGF beta, IL-17, IL-18; transport receptors, such as cytokine receptors, including, but not limited to, chemokines, including CCR4, CCR6, CXCR5, e.g., CCR4, CCR7, CCR2; and / or a variety of other therapeutic modalities or adjunctive components, including one or more of: bispecific antibodies, including bispecific T cell engaging antibodies (BiTEs), such as but not limited to, anti-CD19 targeted or other multi-targeting antibodies; neutralizing / blocking antibodies against, for example, but not limited to, PD-L1, IL-6R, IL-1R, expressed as scFvs or as IgG or in other configurations; T cell stimulatory receptors; truncated inhibitory receptors, hybrid inhibitory / activating receptors, such as but not limited to, the ectodomain of PD-1 fused to the endodomain of CD28; anti-apoptotic proteins, such as but not limited to, BCL-2 or BCL-XL; shRNA; proteases; second CAR T constructs; or any combination thereof (each of which has biological properties as described above).

[0276] D. Antibodies and Antigen-Binding Fragments One embodiment further provides a CAR-based surface antigen-regulated promoter-therapeutic payload construct (e.g., but not limited to, a CAR-expressing T cell, an antibody, or an antigen-binding domain or portion thereof) that specifically binds to one or more of the antigens disclosed herein. As used herein, "CAR-expressing T cell" or "CAR T cell" means a T cell that expresses a CAR and has antigen specificity, e.g., as determined by the antibody-derived targeting domain of the CAR.

[0277] As used herein, an "antigen-binding domain" may include antibodies and antigen-binding fragments thereof. The term "antibody" is used herein in its broadest sense and encompasses a variety of antibody structures, including (but not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments thereof, so long as they exhibit the desired antigen-binding activity. Examples of antibodies include, but are not limited to, intact immunoglobulins and variants and fragments thereof well known in the art that retain binding affinity for an antigen.

[0278] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical except for possible natural mutations, which may be present in trace amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic epitope. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. In some instances, a monoclonal antibody is produced by a single clone of B lymphocytes or by cells transfected with nucleic acid encoding the light and heavy chain variable regions of a single antibody (or antigen-binding fragment thereof), or their progeny. In some instances, a monoclonal antibody is isolated from a subject. A monoclonal antibody may have conservative amino acid substitutions that do not substantially affect antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are well known, see, for example, Harlow & Lane, Antibodies, A Laboratory Manual, 2nd ed. Cold Spring Harbor Publications, New York (2013).

[0279] Typically, immunoglobulins have heavy (H) chains and light (L) chains linked together by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable domain genes. There are two types of light chains: lambda (λ) and kappa (κ). There are five major classes (or isotypes) of heavy chains, which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0280] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). In some embodiments, the heavy and light chain variable regions combine to specifically bind to an antigen. In additional embodiments, only the heavy chain variable region is required. For example, natural camelid antibodies consisting only of heavy chains are functional and stable even in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). References to "VH," or "VH," refer to the variable region of an antibody heavy chain, including the variable region of an antigen-binding fragment, such as Fv, ScFv, dsFv, or Fab. References to "VL," or "VL," refer to the variable domain of an antibody light chain, including that of an Fv, ScFv, dsFv or Fab.

[0281] The variable regions of light and heavy chains contain a "framework" region interrupted by three hypervariable regions (also called "complementarity-determining regions" or "CDRs") (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved among species. The framework regions of an antibody, i.e., the framework regions of the constituent light and heavy chains, together position and align the CDRs in three-dimensional space.

[0282] CDRs are primarily responsible for binding to antigenic epitopes. The amino acid sequence boundaries of a given CDR can be readily determined using any of several well-known numbering schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (N- to C-terminus), and are further typically identified by the chain in which the CDR is located. Thus, a VH CDR3 is the CDR3 from the variable domain of the heavy chain of the antibody that contains it, and a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody that contains it. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0283] "Antigen-binding fragments" are portions of full-length antibodies, and various combinations of such portions, that retain the ability to specifically recognize their cognate antigen. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., ScFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments generated by modification of whole antibodies or synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010).

[0284] Single-chain antibodies (ScFv) are genetically engineered molecules containing the VH and VL domains of one or more antibodies joined by a suitable polypeptide linker into a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry, IDrugs, 13:543-549, 2010). The intramolecular orientation of the VH and VL domains within an ScFv is typically not determinative of the ScFv. Thus, ScFvs with both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) may be used.

[0285] In dsFvs, the heavy and light variable chains are mutated to introduce disulfide bonds to stabilize the bond between the two chains. Also included are diabodies, which are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but are connected to complementary domains on another chain using a linker that is too short to connect the two domains into a single chain, thereby forming two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

[0286] Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., W.H. Freeman & Co., New York, 1997.

[0287] Non-naturally occurring antibodies can be constructed using solid-phase peptide synthesis, or can be made recombinantly, or can be obtained by screening combinatorial libraries composed of variable heavy and light chains, for example, as described by Huse et al., Science 246:1275-1281 (1989), incorporated herein by reference. These methods, as well as other methods for producing, for example, chimeric, humanized, CDR-grafted, single-chain, and bifunctional antibodies, are well known to those of skill in the art (Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

[0288] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody inhibits the binding of this antibody to its antigen by 50% or more in a competition assay. Antibody competition assays are well known, and exemplary competition assays are provided herein.

[0289] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. Constant regions may be absent, but if present, may be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90% (e.g., about 95% or more) identical. Thus, all parts of the humanized antibody or antigen-binding fragment (possibly with the exception of the CDRs) are substantially identical to corresponding parts of natural human antibody sequences.

[0290] A "chimeric antibody" is an antibody that contains sequences derived from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and the CDRs and / or framework regions from another human antibody.

[0291] A "fully human antibody" or "human antibody" is an antibody that contains sequences from (or derived from) the human genome and does not contain sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated by using antibody production techniques based on sequences from the human genome, for example, by phage display or the use of transgenic animals (see, e.g., Barbas et al., Phage display: A Laboratory Manuel. 1st ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23:1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0292] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For example, a native immunoglobulin has two identical binding sites, a single-chain antibody or a Fab fragment has one binding site, and a bispecific or bifunctional antibody has two different binding sites.

[0293] Methods for testing the ability of an antibody to bind to any functional portion of a CAR are well known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., U.S. Patent Application Publication No. 2002 / 0197266 A1, and U.S. Patent No. 7,338,929, below).

[0294] Additionally, the CAR, CAR-expressing T cell, antibody, or antigen-binding portion thereof may be modified to contain a detectable label, such as, for example, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., a gold particle).

[0295] In each of the descriptions in Section B above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload constructs described above, the surface antigen-regulated promoter-therapeutic payload constructs may also be directed against cytokines, including IL-2, IL-15, IL-7, TNFα, IFN gamma, IFN beta, IFN alpha, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGF beta, IL-17, IL-18; transport receptors, such as cytokine receptors, including, but not limited to, chemokines, including CCR4, CCR6, CXCR5, e.g., CCR4, CCR7, CCR2; The therapeutic compositions may further comprise one or more of a variety of other therapeutic modalities, including bispecific antibodies, including bispecific T cell engaging antibodies (BiTEs), such as but not limited to, anti-CD19 targeted or other multi-targeting antibodies; neutralizing / blocking antibodies against, for example, but not limited to, PD-L1, IL-6R, IL-1R, expressed as scFvs or as IgG or in other configurations; T cell stimulatory receptors; truncated inhibitory receptors, hybrid inhibitory / activating receptors, such as but not limited to, the ectodomain of PD-1 fused to the endodomain of CD28; anti-apoptotic proteins, such as but not limited to, BCL-2 or BCL-XL; shRNA; proteases; a second CAR T construct; or any combination thereof (each of which has biological properties as described above).

[0296] E. Conjugates Surface antigen-regulated promoter-therapeutic payload constructs (e.g., based on CARs, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases) expressing, for example, a CAR or monoclonal antibody or antigen-binding fragment thereof specific for one or more of the antigens disclosed herein may be conjugated to an agent such as an effector molecule or detectable marker using any of a number of means well known to those of skill in the art. Both covalent and non-covalent means may be used. Conjugates include, but are not limited to, molecules in which an antibody or antigen-binding fragment that specifically binds one or more of the antigens disclosed herein is covalently linked to an effector molecule or detectable marker. Those skilled in the art will recognize that the conjugates described herein are well known for their ability to conjugate chemotherapeutic agents, anti-angiogenic agents, toxins, and the like. 125 I, 32 P, 14 C. 3 H, and 35 It will be understood that a wide variety of effector molecules and detectable markers can be used, including, but not limited to, radioactive agents such as S, as well as other labels, targeting moieties, and ligands.

[0297] The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or cell and the desired biological effect. Thus, for example, the effector molecule may be a cytotoxin used to cause the death of a particular target cell (such as a tumor cell).

[0298] The procedure for attaching an effector molecule or detectable marker to an antibody or antigen-binding fragment varies depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH), free amine (-NH), or sulfhydryl (-SH) groups, which are available for reaction with suitable functional groups on an antibody, resulting in the attachment of an effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment can be derivatized to expose or attach additional reactive functional groups. The derivatization can involve the attachment of any of several well-known linker molecules, such as those available from Pierce Chemical Company (Rockford, IL). The linker can be any molecule used to connect an antibody or antigen-binding fragment to an effector molecule or detectable marker. The linker can form covalent bonds to both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers are well known to those skilled in the art and include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or antigen-binding fragment and the effector molecule or detectable marker are polypeptides, the linkers may be attached to constituent amino acids through their side chains (e.g., to cysteine ​​via a disulfide bond) or to the amino and carboxy groups of the alpha carbon of the terminal amino acid.

[0299] In some embodiments, the linker may comprise a spacer element, which, when present, increases the size of the linker and the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Examples of spacers are well known to those skilled in the art and are described in U.S. Patent Nos. 7,964,566, 7,498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065, 5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,28 Nos. 4, 5,504,191, 5,410,024, 5,138,036, 5,076,973, 4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, and those listed in U.S. Patent Publication Nos. 20110212088 and 20110070248, each of which is incorporated by reference in its entirety.

[0300] In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet other embodiments, the linker is not cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., within a lysosome, endosome, or caveolea). The linker can be, for example, a peptide linker cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptide linker is at least two amino acids in length, or at least three amino acids in length. However, the linker can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, e.g., 1-2, 1-3, 2-5, 3-10, 3-15, 1-5, 1-10, or 1-15 amino acids in length. Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release active drugs in target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). For example, a peptide linker cleavable by the thiol-dependent protease cathepsin-B can be used (e.g., a phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker (SEQ ID NO: 186)). Other examples of such linkers are described, for example, in U.S. Patent No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker cleavable by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Patent No. 6,214,345).This document describes the synthesis of doxorubicin with a valine-citrulline linker.

[0301] In another embodiment, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a certain pH value. Typically, such pH-sensitive linkers are hydrolyzed under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, or ketals, etc.) that can be hydrolyzed in lysosomes can be used. (See, for example, U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions (such as in blood), but are unstable below pH 5.5 or 5.0, the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929)).

[0302] In another embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Patent No. 4,880,935.

[0303] In yet another specific embodiment, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0304] In yet another embodiment, the linker is not cleavable, and the effector molecule or detectable marker is released by degradation of the antibody (see US Publication No. 2005 / 0238649, incorporated herein by reference in its entirety).

[0305] In some embodiments, the linker is resistant to cleavage in an extracellular environment. For example, when the conjugate is present in an extracellular environment (e.g., plasma), about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 3% or less, or about 1% or less of the linkers in a sample of the conjugate are cleaved. Whether a linker is resistant to cleavage in an extracellular environment can be determined, for example, by incubating a conjugate containing the desired linker with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of effector molecule or detectable marker free in the plasma. A variety of exemplary linkers that can be used in the conjugates are described in WO2004-010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 20050238649, and U.S. Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0306] In some embodiments, surface antigen-regulated promoter-therapeutic payload construct conjugates (e.g., based on CARs, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNAs, proteases) are provided that express, for example, a CAR, an antibody, or antigen-binding portion thereof, and one or more small molecule toxins such as calicheamicin, maytansinoids, dolastatins, auristatins, trichothecenes, and CC1065, as well as derivatives of these toxins that have toxin activity.

[0307] Maytansine compounds suitable for use as maytansinoid toxin moieties are well known in the art and can be isolated from natural sources according to well-known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968-7973), or maytansinol and maytansinol analogs can be prepared synthetically according to well-known methods. Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, certain microorganisms were further discovered to produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Pat. Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, and 4, Nos. 313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533, each of which is incorporated herein by reference. Maytansinoid-containing conjugates, methods for their preparation, and therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, and 6,441,163, and European Patent No. EP 0 425 235 B1, the disclosures of which are expressly incorporated herein by reference.

[0308] Additional toxins can be used with surface antigen-regulated promoter-therapeutic payload constructs, for example, expressing a CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof (e.g., based on a CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptotic protein, shRNA, protease). Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribotoxin, ribonuclease, saporin, and calicheamicin, as well as botulinum toxins A-F. Such toxins are well known in the art, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of such toxins (see, eg, US Pat. Nos. 5,079,163 and 4,689,401).

[0309] Saporin is a toxin derived from Saponaria officinalis that inhibits protein synthesis by inactivating the 60S portion of the ribosomal complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, this toxin lacks a mechanism for specific intracellular entry and therefore must bind to an antibody or antigen-binding fragment that recognizes an endogenous cell surface protein for efficient entry into cells.

[0310] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, diphtheria toxin for use in immunotoxins has been mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107, which has full enzymatic activity but significantly reduced nonspecific toxicity, has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent Nos. 5,792,458 and 5,208,021.

[0311] Ricin is the lectin RCA60 obtained from Ricinus communis (Castor bean). For examples of ricin, see U.S. Patent Nos. 5,079,163 and 4,689,401. Ricinus communis agglutinin (RCA) exists in two forms, with molecular weights of approximately 65 kD and 120 kD, respectively, and hence RCA. 60 and RCA 120 (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivating protein synthesis and killing the cell. The B chain binds lysine to cell surface galactose residues, facilitating transport of the A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Patent No. 3,060,165).

[0312] Ribonucleases have also been used as immunotoxins by conjugating them to target molecules (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Exemplary ribotoxins, such as α-sarcin and restrictocin, are described, for example, in Rathore et al., Gene 190:31-5, 1997, and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicin, originally isolated from Micromonospora echinospora, is a member of the enediyne antitumor antibiotic family that causes DNA double-strand breaks, leading to apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic moiety of immunotoxins in clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0313] Abrin includes toxic lectins obtained from Abrus precatorius. The toxic components, abrins a, b, c, and d, have molecular weights of approximately 63-67 kD and consist of two disulfide-linked polypeptide chains, A and B. The A chain inhibits protein synthesis, while the B chain (abrin-b) binds to D-galactose residues (see Funatsu et al., Agr. Biol. Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

[0314] Surface antigen-regulated promoter-therapeutic payload constructs (e.g., CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptotic protein, shRNA, protease-based) expressing, for example, a CAR, monoclonal antibody, antigen-binding fragment thereof specific for one or more of the antigens disclosed herein may also be linked to a detectable marker, for example, a detectable marker detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques such as computed tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiber optic examination, and laparoscopy. Specific examples of detectable markers include, but are not limited to, fluorophores, chemiluminescent agents, enzyme conjugates, radioactive isotypes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, and lanthanide illuminators. Bioluminescent markers, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP), are also useful. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof, may also be conjugated to enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, and glucose oxidase. When the CAR, CAR-expressing T cell, antibody, or antigen-binding portion thereof is conjugated to a detectable enzyme, it can be detected by adding additional reagents that, when used with the enzyme, produce a reaction product that can be identified.For example, in the presence of the agent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected visually. CARs, CAR-expressing T cells, antibodies, or antigen-binding portions thereof may also be conjugated to biotin and detected by indirectly measuring avidin or streptavidin binding. Of note, avidin itself may be conjugated to an enzyme or fluorescent label.

[0315] For example, surface antigen-regulated promoter-therapeutic payload constructs expressing CARs, antibodies, or antigen-binding portions thereof (e.g., CARs, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases) may be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also useful as labels. Antibodies may also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments may also be labeled with a predetermined polypeptide epitope recognized by a second reporter (e.g., leucine zipper sequence pair, secondary antibody binding site, metal binding domain, epitope tag, etc.).

[0316] For example, surface antigen-regulated promoter-therapeutic payload constructs (e.g., based on CARs, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases) expressing a CAR, antibody, or antigen-binding portion thereof may also be conjugated to a radiolabeled amino acid. Radiolabels may be used for both diagnostic and therapeutic purposes. For example, radiolabels may be used to detect one or more of the antigens disclosed herein and antigen-expressing cells by X-ray, emission spectroscopy, or other diagnostic techniques. Furthermore, radiolabels may be used in therapy as toxins to treat tumors in a subject, e.g., to treat neuroblastoma. Examples of labels for polypeptides include: 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 These include, but are not limited to, radioisotopes such as I or radionucleotides.

[0317] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radioactive labels may be detected using photographic film or a scintillation counter, fluorescent markers may be detected by detecting emitted light using a photodetector. Enzyme labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected simply by visualizing the colored label.

[0318] In each of the descriptions in Section C above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload conjugate constructs described above, the surface antigen-regulated promoter-therapeutic payload conjugate constructs may also be directed against cytokines, including IL-2, IL-15, IL-7, TNFα, IFN gamma, IFN beta, IFN alpha, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGF beta, IL-17, IL-18; transport receptors, such as cytokine receptors, including, but not limited to, chemokines, including CCR4, CCR6, CXCR5, e.g., CCR4, CCR7, CCR2; and anti-CD19 targeted or other multi-targeting antibodies; neutralizing / blocking antibodies against, for example, but not limited to, PD-L1, IL-6R, IL-1R expressed as scFv or as IgG or in other configurations; T cell stimulatory receptors; truncated inhibitory receptors, hybrid inhibitory / activating receptors, for example, but not limited to, the ectodomain of PD-1 fused to the endodomain of CD28; anti-apoptotic proteins, for example, but not limited to, BCL-2 or BCL-XL; shRNA; proteases; second CAR T constructs; or any combination thereof (each of which has the biological properties as described above).

[0319] F. Nucleotides, Expression, Vectors, and Host Cells Further provided according to one embodiment of the present invention is a nucleic acid comprising a nucleotide sequence encoding any of the surface antigen-regulated promoter-therapeutic payload constructs, antibodies, or antigen-binding portions thereof (including functional portions and functional variants thereof) described herein (e.g., CARs, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNAs, protease-based). Nucleic acids of the invention may comprise a nucleotide sequence encoding any of the leader sequences, antigen-binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein.

[0320] In some embodiments, the nucleotide sequence may be modified with codons.Without being bound by any theory, it is believed that the codon optimization of nucleotide sequence can increase the translation efficiency of mRNA transcripts.The codon optimization of nucleotide sequence can involve replacing the native codon with another codon that encodes the same amino acid but can be translated by tRNA that is more easily utilized in cells, thus increasing translation efficiency.The optimization of nucleotide sequence can also reduce the secondary mRNA structure that can interfere with translation, thus increasing translation efficiency.

[0321] In one embodiment of the invention, a nucleic acid may comprise a codon-modified nucleotide sequence encoding the antigen-binding domain of a CAR of the invention. In another embodiment of the invention, a nucleic acid may comprise a codon-modified nucleotide sequence encoding any of the CARs described herein (including functional portions and functional variants thereof).

[0322] As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA that may be single-stranded or double-stranded, may be synthetic or obtained from natural sources (e.g., by isolation and / or purification), may contain natural, non-natural, or altered nucleotides, and may contain natural, non-natural, or altered internucleotide linkages (such as phosphoramidate or phosphorothioate linkages instead of the phosphodiester linkages found between nucleotides in unmodified oligonucleotides). In some embodiments, a nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, as described herein, in some instances, it may be preferred for a nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0323] Recombinant nucleic acids may have sequences that do not occur in nature or sequences that are an artificial combination of two separate regions of a sequence. This artificial combination is often achieved by chemical synthesis or, more commonly, by artificially manipulating separate nucleic acid regions using genetic engineering techniques, such as those described in Sambrook et al., supra. Nucleic acids may be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures well known in the art. See, for example, Sambrook et al., supra, and Ausubel et al., supra. For example, nucleic acids may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or the physical stability of the duplex formed by hybridization.Examples of modified nucleotides that can be used in nucleic acid generation include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenines, 7-methylguanine, 5-methylaminomethyl Examples of nucleic acids include, but are not limited to, uracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention may be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0324] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any of the above-described CARs, or a functional portion or variant thereof. Alternatively, the nucleotide sequence may comprise a nucleotide sequence degenerate to any of the above-described sequences, or a combination of degenerate sequences.

[0325] One embodiment further provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein, or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0326] Nucleotide sequences that hybridize under stringent conditions may also hybridize under highly stringent conditions. "Highly stringent conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount detectably greater than nonspecific hybridization. Highly stringent conditions include conditions under which polynucleotides with exactly complementary sequences or those with only a few scattered mismatches can be distinguished from random sequences that coincidentally contain a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. These small regions of complementarity are more easily melted than full-length regions of complementarity of 14-17 bases or more, and can be easily distinguished by highly stringent hybridization. Relatively highly stringent conditions can include low salt and / or high temperature conditions, such as about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C. Such highly stringent conditions tolerate very little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the CARs of the present invention. It is generally understood that conditions can be made more stringent by adding increasing amounts of formamide.

[0327] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more identical to any of the nucleic acids described herein, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical.

[0328] In one embodiment, the nucleic acid may be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any of the nucleic acids described above. For purposes herein, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and that is capable of causing a host cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with a host cell under conditions sufficient for the expression of the mRNA, protein, polypeptide, or peptide in the host cell. Such vectors generally do not occur in nature.

[0329] However, some of these vectors may be naturally occurring. The recombinant expression vector may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or derived in part from natural sources, and may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may contain natural or non-natural internucleotide bonds, or both types of bonds. Preferably, the non-natural or altered nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector.

[0330] In one embodiment, the recombinant expression vector may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those (such as plasmids and viruses) designed for propagation and propagation, or for expression, or both. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).

[0331] Bacteriophage vectors such as λυTIO, λυTΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBHO1.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral or lentiviral vector. Lentiviral vectors are vectors derived from at least a portion of the lentiviral genome, including, in particular, self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used in the clinic include, but are not limited to, for example, LENTIVECTOR® gene transfer technology from Oxford BioMedica plc, the LENTIMAX™ vector system from Lentigen, etc. Non-clinical versions of lentiviral vectors are also available and will be known to those skilled in the art.

[0332] Several transfection techniques are generally known in the art (see, e.g., Graham et al., Virology, 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:97 (1981)).

[0333] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid transfer using high-velocity particle bombardment (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0334] In one embodiment, recombinant expression vectors may be prepared using standard recombinant DNA techniques, e.g., as described in Sambrook et al., supra, and Ausubel et al., supra. Circular or linear constructs of expression vectors may be prepared to contain replication mechanisms that function in prokaryotic or eukaryotic host cells. Replication mechanisms may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, and bovine papilloma virus.

[0335] Recombinant expression vectors may contain control sequences, such as transcription and translation initiation and termination codons, that are appropriate for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector is to be introduced, and taking into account whether the vector is DNA or RNA based. Recombinant expression vectors may contain restriction sites to facilitate cloning.

[0336] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic hosts for prototrophy. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0337] The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding the CAR (including functional portions and functional variants thereof) or a nucleotide sequence complementary to or hybridizing to the CAR-encoding nucleotide sequence. The selection of a promoter (e.g., strong, weak, inducible, tissue-specific, developmental-specific, etc.) is within the ordinary skill of those in the art. Similarly, the association of a nucleotide sequence with a promoter is also within the ordinary skill of those in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long terminal repeat of murine stem cell virus.

[0338] Recombinant expression vectors may be designed for either transient expression, stable expression, or both, and may be made for constitutive or inducible expression.

[0339] Furthermore, the recombinant expression vector may be engineered to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell expressing the suicide gene. A suicide gene may be a gene that sensitizes a cell in which the gene is expressed to an agent, such as a drug, causing the cell to die when the cell comes into contact with or is exposed to the agent. Suicide genes are well known in the art (see, for example, Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004) and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0340] One embodiment further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector of the present invention. A host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protozoan. A host cell may be a cultured cell or a primary cell (i.e., isolated directly from an organism, such as a human). A host cell may be an adherent cell or a suspension cell (i.e., a cell that grows in suspension). Suitable host cells are well known in the art and include, for example, DH5a E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, and HEK293 cells. When the purpose is to amplify or replicate a recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5a cell. When the purpose is to produce a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. The host cell may be of any cell type, may be derived from any type of tissue, and may be at any stage of development, but the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell.

[0341] For purposes described herein, a T cell may be any T cell, including cultured T cells (e.g., primary T cells), or T cells from a cultured T cell line (e.g., Jurkat, SupTl, etc.), or T cells obtained from a mammal. If obtained from a mammal, the T cells may be obtained from a wide variety of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells may be enriched or purified. The T cells may be human T cells. The T cells may be T cells isolated from a human. The T cells may be any type of T cell, at any stage of development, and may be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, such as Th1 and Th2 cells, CD8+ These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor-infiltrating cells, memory T cells, memory stem cells (i.e., Tscm), and naive T cells. T cells include CD8 + T cells or CD4 + It may be a T cell.

[0342] In one embodiment, the surface antigen-regulated promoter-therapeutic payload constructs described herein can be used in suitable cells other than T cells, such as those with immune effector function, such as NK cells and T-like cells developed from pluripotent stem cells.

[0343] One embodiment also provides a population of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population, comprising host cells comprising any of the described recombinant expression vectors, as well as at least one other cell type, e.g., host cells that do not comprise any of the recombinant expression vectors (e.g., T cells), or cells other than T cells, e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the population of cells may be a substantially homogeneous population, comprising primarily (e.g., consisting essentially of) host cells comprising the recombinant expression vector. The population may also be a clonal cell population, in which all cells in the population are clones of a single host cell comprising the recombinant expression vector and therefore all cells in the population comprise this recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vector described herein.

[0344] CARs (including functional portions and variants thereof), nucleic acids, recombinant expression vectors, host cells (including populations thereof), and antibodies (including antigen-binding portions thereof) may be isolated and / or purified. For example, in a preparation of purified (or isolated) host cells, the host cells are more pure than they are in their natural environment in the body. Such host cells may be produced, for example, by standard purification techniques. In some embodiments, a preparation of host cells is purified such that the host cells represent at least about 50%, e.g., at least about 70%, of the total cell content of the preparation. For example, the purity may be at least about 50%, or may be greater than about 60%, about 70%, or about 80%, or may be about 100%.

[0345] In each of the descriptions in Section D above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload construct nucleotides, expression vectors, and host cells described above, the surface antigen-regulated promoter-therapeutic payload construct may also be a surface antigen-regulated promoter-therapeutic payload construct that is capable of transporting a cytokine, including IL-2, IL-15, IL-7, TNFα, IFN gamma, IFN beta, IFN alpha, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGF beta, IL-17, IL-18; a transport receptor, such as a cytokine receptor, including, but not limited to, CCR4, CCR6, CXCR5, e.g., CCR4, CCR7, CCR2; The therapeutic agent may further comprise one or more of a variety of other therapeutic modalities, including bispecific antibodies, including, but not limited to, bispecific T cell engagers (BiTEs), such as, for example, anti-CD3 and anti-CD19 targeted or other multi-targeting antibodies; neutralizing / blocking antibodies against, for example, but not limited to, PD-L1, IL-6R, IL-1R, expressed as scFvs or as IgG or in other configurations; T cell stimulatory receptors; truncated inhibitory receptors; hybrid inhibitory / activating receptors, such as, but not limited to, the ectodomain of PD-1 fused to the endodomain of CD28; anti-apoptotic proteins, such as, but not limited to, BCL-2 or BCL-XL; shRNA; proteases; a second CAR T construct; or any combination thereof (each of which has biological properties as described above).

[0346] G. Treatment Method It is contemplated that the surface antigen-regulated promoter-therapeutic payload constructs disclosed herein (e.g., based on CARs, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNAs, proteases) may be used in methods of treating or preventing disease in a mammal. In this regard, one embodiment provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody and / or antigen-binding portion thereof, and / or a pharmaceutical composition in an amount effective to treat or prevent cancer in the mammal.

[0347] One embodiment further comprises lymphodepleting the mammal prior to administering a CAR disclosed herein. Examples of lymphodepletion may include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0348] For purposes of this method, in which a host cell or population of cells is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells may be autologous to the mammal. As used herein, allogeneic refers to any material derived from an animal of the same species as the individual into which the material is introduced, but from a different individual. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci for those individuals are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically different to be able to interact antigenically. As used herein, "autologous" refers to any material derived from the same individual into whom the material will later be reintroduced.

[0349] The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals such as mice and hamsters, and lagomorph mammals such as rabbits. The mammal may be of the order Carnivora, which includes Felidae (cats) and Canidae (dogs). The mammal may be of the order Artiodactyla, which includes Bovinae (cattle) and Porcinae (pigs), or Perissodactyla, which includes Equidae (horses). The mammal may be of the order Primates, Ceboids, or Simoids (monkeys), or Apes (humans and apes). Preferably, the mammal is a human.

[0350] For the methods described above, the cancer may be selected from acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, The cancer may be any cancer, including any of laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, gastric cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0351] The terms "treatment" and "prevention," and derivatives thereof, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would recognize as potentially beneficial or therapeutic. In this regard, the method can provide any amount or level of cancer treatment or prevention in a mammal.

[0352] Furthermore, the treatment or prevention provided by the present methods may include treatment or prevention of one or more conditions or symptoms of the disease (e.g., cancer) being treated or prevented. For purposes herein, "prevention" may also encompass delaying the onset of the disease or its symptoms or conditions.

[0353] Another embodiment provides a method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells from the mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a population of cells, an antibody, and / or an antigen-binding portion thereof, or a pharmaceutical composition, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0354] The sample may be obtained by any suitable method, such as, for example, biopsy or autopsy. A biopsy is the removal of tissue and / or cells from an individual. Such removal may involve the collection of tissue and / or cells from the individual for subjecting the removed tissue and / or cells to an experimental procedure. The experimental procedure may include an experiment to determine whether the individual has and / or is suffering from a particular condition or disease state. The condition or disease may be, for example, cancer.

[0355] For one embodiment of the method for detecting the presence of a proliferative disorder, e.g., cancer, in a mammal, the sample containing mammalian cells can be a sample containing whole cells, a lysate thereof, or a whole cell lysate fraction, e.g., a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction. When the sample contains whole cells, the cells can be any cells of the mammal, e.g., cells of any organ or tissue, including blood cells or endothelial cells.

[0356] The contacting described above may occur in vitro or in vivo in a mammal. Preferably, the contacting occurs in vitro.

[0357] In addition, detection of the complex may be performed by any of several methods known in the art. For example, the CAR disclosed herein, the polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or antibody described herein, or antigen-binding portion thereof, may be labeled with a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., gold particle), as disclosed above.

[0358] The method for testing the target cell recognition ability and antigen specificity of CAR is well known in the art.For example, Clay et al., J.Immunol,163:507-513(1999) teaches the method for measuring the release of cytokines (for example, interferon-γ, granulocyte / monocyte colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNF-α) or interleukin 2 (IL-2)).In addition, the function of CAR can be evaluated by measuring the cytotoxicity of cells, as described in Zhao et al., J.Immunol,174:4415-4423(2005).

[0359] Another embodiment provides the use of a CAR, nucleic acid, recombinant expression vector, host cell, population of cells, antibody, or antigen-binding portion thereof, and / or pharmaceutical composition of the invention to treat or prevent a proliferative disorder, such as cancer, in a mammal, which may be any of the cancers described herein.

[0360] Any administration method, including local and systemic administration, may be used for the disclosed therapeutic agents. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration may be used. The specific mode of administration and dosing regimen may be selected by the attending clinician, taking into consideration the specifics of the case (e.g., the subject, the disease, any associated disease states, and whether the treatment is prophylactic). When more than one agent or composition is administered, one or more routes of administration may be used; for example, a chemotherapeutic agent may be administered orally, and an antibody or antigen-binding fragment or conjugate or composition may be administered intravenously. Administration methods include injection, in which the CAR, CAR T cell, conjugate, antibody, antigen-binding fragment, or composition is provided in a non-toxic and pharmaceutically acceptable carrier, such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds may be used, for example, by applying an antibody or antigen-binding fragment to a tissue area after tumor removal or to an area suspected of being prone to tumor development. In some embodiments, sustained intratumoral (or near-tumor) release of a pharmaceutical preparation containing a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is applied topically to the cornea as eye drops or intravitreously to the eye.

[0361] The disclosed therapeutic agents may be formulated in unit dosage forms suitable for administering precise dosage amounts one at a time. Additionally, the disclosed therapeutic agents may be administered in a single-dose or multiple-dose schedule. A multiple-dose schedule may involve an initial series of treatment in which more than one dose (e.g., 1-10 doses) may be administered separately, followed by subsequent doses at intervals, as needed, to maintain or enhance the effect of the composition. Treatment may involve administering the compound once daily or multiple times daily (multi-daily doses) for a period ranging from two to three days to several months or even years. Accordingly, the dosage regimen may be determined, at least in part, based on the specific requirements of the subject being treated and may be dependent on the judgment of the administering physician.

[0362] Typical dosages of the antibody or conjugate may range from about 0.01 to about 30 mg / kg, for example, from about 0.1 to about 10 mg / kg.

[0363] In a specific example, a subject is administered a therapeutic composition comprising one or more of a conjugate, antibody, composition, CAR, CAR T cell, or additional agent on a multiple daily dosing schedule, such as at least 2 consecutive days and at least 10 consecutive days, for a period of, e.g., weeks, months, or years. In one example, a subject is administered a conjugate, antibody, composition, or additional agent for a period of at least 30 days, for example, for a period of at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.

[0364] In some embodiments, the disclosed methods include providing a subject with surgery, radiation therapy, and / or chemotherapy in combination (e.g., sequentially, substantially simultaneously, or simultaneously) with the disclosed antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells. Methods and therapeutic dosages for such agents and treatments are well known to those of skill in the art and may be determined by a skilled clinician. Preparation and administration schedules for additional agents may be used according to manufacturer's instructions or according to the experienced judgment of a skilled physician. Preparation and administration schedules for such chemotherapy are also described in Chemotherapy Service, (1992) Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md.

[0365] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Examples of additional therapeutic agents that can be used in combination therapy include, but are not limited to, microtubule binding agents, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in therapeutically effective amounts) and treatments may be used alone or in combination. For example, any suitable anti-cancer or anti-angiogenesis agent may be administered in combination with the CAR, CAR-T cells, antibodies, antigen-binding fragments, or conjugates disclosed herein. The methods and therapeutic dosages for such agents are well known to those skilled in the art and can be determined by skilled clinicians.

[0366] Additional chemotherapeutic agents include alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folates (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids such as benzodiazepines (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), and vincas (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antineoplastic antibiotics such as members of the anthracycline family (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors such as topotecan and irinotecan; monoclonal antibodies such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfin;and other agents such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafenib, vandetanib, and tretinoin. The selection and therapeutic dosage of such agents are well known to those skilled in the art and can be determined by a skilled clinician.

[0367] Combination therapy can produce synergistic effects and can be proven to be synergistic, i.e., the effect achieved when multiple active ingredients are used together is greater than the combined effect achieved when the same compounds are used separately. Synergistic effects can occur when multiple active ingredients are (1) formulated together and administered or delivered simultaneously as a combined unit-dose preparation, (2) delivered alternately or in parallel as separate preparations, or (3) used in some other regimen. In the case of alternate delivery, synergistic effects can occur when the compounds are administered or delivered sequentially, for example, by separate injections in separate syringes. Generally, in the case of alternate administration, an effective dosage of each active ingredient is administered sequentially, i.e., consecutively, while in combination therapy, effective dosages of two or more active ingredients are administered together.

[0368] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to one or more antigens disclosed herein is administered to a tumor-bearing subject after anti-cancer treatment. After a sufficient time has passed, the administered antibody or antigen-binding fragment or conjugate forms an immune complex with the antigen expressed on each cancer cell, and the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, an increase in immune complexes compared to a control obtained before the treatment indicates that the treatment is ineffective, and a decrease in immune complexes compared to a control obtained before the treatment indicates that the treatment is effective.

[0369] In each of the descriptions in Section E above, in addition to the methods of treatment with the CAR-based surface antigen-regulated promoter-therapeutic payload constructs described above, the surface antigen-regulated promoter-therapeutic payload constructs can be used to treat a variety of conditions, including but not limited to cytokines, including IL-2, IL-15, IL-7, TNFα, IFN gamma, IFN beta, IFN alpha, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGF beta, IL-17, IL-18; transport receptors, such as cytokine receptors, including but not limited to, chemokines, including CCR4, CCR6, CXCR5, e.g., CCR4, CCR7, CCR2; and anti-CD19 or other multi-targeting antibodies; neutralizing / blocking antibodies against, for example, but not limited to, PD-L1, IL-6R, IL-1R expressed as scFvs or as IgG or in other configurations; T cell stimulatory receptors; truncated inhibitory receptors; hybrid inhibitory / activating receptors, for example, but not limited to, the ectodomain of PD-1 fused to the endodomain of CD28; anti-apoptotic proteins, for example, but not limited to, BCL-2 or BCL-XL; shRNA; proteases; a second CAR T construct; or any combination thereof (each of which has the biological properties as described above).

[0370] H. Biopharmaceutical Compositions Provided herein are biopharmaceutical or biological compositions (hereinafter "compositions") for use in gene therapy, immunotherapy, and / or cell therapy, comprising one or more of the disclosed surface antigen-regulated promoter-therapeutic payload constructs (e.g., CARs, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulatory receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNAs, protease-based) that specifically bind to one or more antigens disclosed herein, or CAR-expressing T cells, antibodies, antigen-binding fragments, conjugates, CARs, or CAR-expressing T cells, in a carrier (e.g., a pharmaceutically acceptable carrier). The compositions may be prepared in a unit dosage form for administration to a subject. The amount and timing of administration are determined by the treating clinician to achieve the desired outcome. The compositions may be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, are formulated for parenteral administration, such as intravenous administration. Compositions comprising the disclosed CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, are useful, for example, for the treatment and detection of tumors (e.g., but not limited to, neuroblastoma). In some examples, the compositions are useful for the treatment or detection of cancer. Compositions comprising the disclosed CARs, or CAR-expressing T cells, conjugates, antibodies, or antigen-binding fragments, are also useful, for example, for the detection of pathological angiogenesis.

[0371] The administration composition may comprise a solution of the CAR, or CAR-expressing T cells, conjugate, antibody, or antigen-binding fragment, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers may be used, such as buffered saline. Such solutions are sterile and generally free of undesirable material. The composition may be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, and adjuvants, as needed to approximate physiological conditions, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. The concentration of the CAR, or CAR-expressing T cells, antibody, or antigen-binding fragment, or conjugate in the preparation may vary widely and may be selected depending primarily on fluid volume, viscosity, and body weight, depending on the particular mode of administration selected and the requirements of the subject. Actual methods for preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy are well known or will be apparent to those skilled in the art.

[0372] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of antibody or antigen-binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing a CAR, or a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Actual methods for preparing compositions for administration will be well known or apparent to those skilled in the art and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0373] CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugates, may be provided in lyophilized form and administered by reconstitution with sterile water, or may be provided dissolved in a sterile solution of known concentration. The CAR, or CAR-expressing T cells, antibodies, antigen-binding fragments, or conjugate solutions are then loaded into infusion bags containing 0.9% sodium chloride (USP) and, in some cases, administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience can be found in the art in administering antibody, antigen-binding fragment, and conjugate drugs; for example, antibody drugs have been available on the U.S. market since the approval of Rituxan® in 1997. CARs, or CAR-expressing T cells, antibodies, antigen-binding fragments, and conjugates thereof, may be administered by slow infusion rather than intravenous push or bolus. In one example, a higher loading dose is administered, followed by a lower maintenance dose. For example, an initial loading dose of 4 mg / kg of the antibody or antigen-binding fragment (or the corresponding dose of a conjugate comprising the antibody or antigen-binding fragment) may be infused over approximately 90 minutes, and if this initial dose is well tolerated, then weekly maintenance doses of 2 mg / kg may be infused over 30 minutes for 4-8 weeks.

[0374] Controlled-release parenteral preparations may be prepared as implants, oily injections, or particulate systems. For a comprehensive overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain a therapeutic protein, such as a cytotoxin or drug, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are commonly referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Because capillaries are about 5 μm in diameter, only nanoparticles are administered intravenously. Microparticles are typically about 100 μm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, edited by J. Kreuter, Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, edited by A. Kydonieus, Marcel Dekker, Inc., New York, NY, pp. 315-339, (1992).

[0375] Polymers may be used for ion-controlled release of the CAR, or CAR-expressing T cells, antibodies, or antigen-binding fragments, or conjugate compositions disclosed herein. A variety of degradable and non-degradable polymer matrices for use in controlled drug delivery are well known in the art (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer poloxamer 407 exists as a viscous, mobile liquid at low temperatures but forms a semi-solid gel at body temperature. It has been shown to be an effective vehicle for the preparation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yet another embodiment, liposomes are used for controlled release and drug targeting of lipid-encapsulated drugs (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous other systems for controlled delivery of therapeutic proteins are known (U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,188,837, U.S. Pat. No. 4,235,871, U.S. Pat. No. 4,501,728, U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,957,735, U.S. Pat. No. 5,019,369, U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,514,670, U.S. Pat. No. 5,413,797, U.S. Pat. No. 5,268,164, U.S. Pat. No. 5,004,697, U.S. Pat. No. 4,902,505, U.S. Pat. No. 5,506,206, U.S. Pat. No. 5,271,961, U.S. Pat. No. 5,254,342, and U.S. Pat. No. 5,534,496).

[0376] In each of the descriptions in Section F above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload construct compositions described above, the surface antigen-regulated promoter-therapeutic payload construct may also be a surface antigen-regulated promoter that is capable of transporting a cytokine, including, but not limited to, IL-2, IL-15, IL-7, TNFα, IFN gamma, IFN beta, IFN alpha, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGF beta, IL-17, IL-18; a chemokine, including, but not limited to, CCR4, CCR6, CXCR5, e.g., CCR4, CCR7, CCR2; a transport receptor, such as a cytokine receptor, including, but not limited to, anti-CD3 and anti-CD3 receptors; and anti-CD19 targeted or other multi-targeting antibodies; neutralizing / blocking antibodies against, for example, but not limited to, PD-L1, IL-6R, IL-1R expressed as scFv or as IgG or in other configurations; T cell stimulatory receptors; truncated inhibitory receptors, hybrid inhibitory / activating receptors, for example, but not limited to, the ectodomain of PD-1 fused to the endodomain of CD28; anti-apoptotic proteins, for example, but not limited to, BCL-2 or BCL-XL; shRNA; proteases; second CAR T constructs; or any combination thereof (each of which has biological properties as described above).

[0377] I. Kit In one embodiment, kits are further provided that use the surface antigen-regulated promoter-therapeutic payload constructs disclosed herein (e.g., based on CARs, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulatory receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNAs, proteases). For example, kits for treating tumors in a subject or for generating CAR T cells expressing one or more of the CARs disclosed herein. Such kits may typically include the antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells disclosed herein. More than one of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells may be included in the kit.

[0378] The kit may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container typically holds a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or CAR-expressing T cells. In some embodiments, the container may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used for treating a particular condition.

[0379] The label or package insert may typically further include instructions for using the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or CAR-expressing T cells, for example, in a method for treating or preventing tumors or in a method for generating CAR T cells. The package insert typically includes instructions customarily included in commercial packaging for therapeutic products, which include information about the indications, usage, dosage, administration, contraindications, and / or warnings associated with the use of the therapeutic product. The contents of the instructions may be written in electronic format (e.g., a floppy disk or compact disk) or in visual format (e.g., a video file). The kit may further include additional components to facilitate the particular use for which the kit is designed. Thus, for example, the kit may further include label detection means (e.g., an enzyme substrate for an enzymatic label, a filter set for detecting a fluorescent label, or an appropriate secondary label such as a secondary antibody). The kit may further include buffers and other reagents customarily used in the practice of a particular method. Such kits and suitable contents are well known to those of skill in the art.

[0380] In each of the descriptions in Section G above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload constructs described above, the surface antigen-regulated promoter-therapeutic payload constructs may also be directed against cytokines, including IL-2, IL-15, IL-7, TNFα, IFN gamma, IFN beta, IFN alpha, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGF beta, IL-17, IL-18; transport receptors, such as cytokine receptors, including, but not limited to, chemokines, including CCR4, CCR6, CXCR5, e.g., CCR4, CCR7, CCR2; The therapeutic compositions may further comprise one or more of a variety of other therapeutic modalities, including bispecific antibodies, including bispecific T cell engaging antibodies (BiTEs), such as but not limited to, anti-CD19 targeted or other multi-targeting antibodies; neutralizing / blocking antibodies against, for example, but not limited to, PD-L1, IL-6R, IL-1R, expressed as scFvs or as IgG or in other configurations; T cell stimulatory receptors; truncated inhibitory receptors, hybrid inhibitory / activating receptors, such as but not limited to, the ectodomain of PD-1 fused to the endodomain of CD28; anti-apoptotic proteins, such as but not limited to, BCL-2 or BCL-XL; shRNA; proteases; a second CAR T construct; or any combination thereof (each of which has biological properties as described above). [Example]

[0381] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. On the contrary, it is clearly understood that various other embodiments, modifications, and equivalents must be relied upon, and that such other embodiments, modifications, and equivalents may occur to those skilled in the art after reading the description herein without departing from the spirit of the invention and / or the scope of the appended claims.

[0382] Example 1 Generation of a self-driving CAR construct targeting the CD19 antigen Despite the clinical success of anti-CD19-based cancer therapies (e.g., CAR LTG1563), suboptimal CAR activation and persistence, on the one hand, and excessive CAR activation and associated toxicities (CRS, neurotoxicity), on the other hand, remain problems. To improve the safety and efficacy of CAR T therapy, we engineered self-powered CARs.

[0383] This example describes CAR T cells that utilize CAR signaling to regulate their own expression and the expression of a co-regulated transgene by two mechanisms, and the use of these mechanisms beyond CAR expression.

[0384] 1. Cytokine (e.g., IL-2 / IL-15 / IL-7)-driven positive regulation of CAR T expression by the STAT5 response element (STAT5_RE) induces expression of the CAR molecule (e.g., anti-CD19 CAR LTG1563). When the CAR T detects tumor antigens, a slow positive feedback loop is activated, which increases CAR expression on the CAR T cells. Once the tumor is eliminated, this positive feedback loop gradually declines and ceases, resulting in a natural decline in CAR surface expression and a return to a surveillance state (Figure 1).

[0385] 2. Positive regulation of CAR T expression by a combination of cytokine (e.g., IL-2 / IL-15 / IL-7 / TNFα)-driven AP1 / NFκB response element (AP1 / NFκB_RE) and CAR / TCR-driven expression of the CAR molecule (e.g., CAR LTG1563). When the CAR T detects tumor antigens, a rapid positive feedback loop is activated, leading to increased CAR expression on the CAR-T cells. Over time, compared to constitutively expressed CARs, the AP1 / NFκB-driven CAR LTG1563 exhibits greater CD19-dependent killing activity, increased cytokine secretion, reduced exhaustion, and overall better CAR T cells. Once the tumor is eliminated, this positive feedback loop gradually declines and ceases, resulting in a natural decline in CAR surface expression and a return to a surveillance state (Figure 1).

[0386] 3. Expression of additional proteins that modulate CAR T function in the presence of the CAR antigen (including, but not limited to, evasion of negative T cell regulation (such as dominant-negative TGFBRII receptors, anti-PD1 antibodies), positive regulators of T cell proliferation (such as IL15, IL12), T cell homing to tumors (chemokine receptors), and facilitating access to the tumor microenvironment (matrix metalloproteinases)), or expression of one constitutive CAR and a second, inducible CAR, e.g., both CARs are encoded by the same lentiviral vector, and the inducible CAR is expressed, e.g., as a result of activation of the constitutive CAR. The second CAR may target a second tumor antigen, or an antigen expressed on myeloid-derived suppressor cells (MDSCs) (e.g., CD33, mesothelin), or an antigen expressed on suppressor B cells (e.g., CD19). In either case, these inducible proteins may be expressed in the presence of CAR stimulation and downregulated when CAR signaling ceases.

[0387] 4. The STAT5_RE versus AP1 / NFκB_RE promoter can be used to regulate the self-expression of either CAR proteins or T cell function-regulating proteins in a defined manner. The greater cytokine (IL2)-dependent response of STAT5_RE (Figure 3) can be used to express proteins in CAR-T cells in a paracrine manner near the tumor that do not directly respond to antigens. Alternatively, proteins can be expressed via the AP1 / NFκB_RE in response to tumors, particularly in CAR T cells, because CAR signaling is required to maximize the response via this element (Figure 4).

[0388] material and method: Construction of chimeric antigen receptor (CAR) expression vector To generate the novel transmembrane domain of CAR LTG1563, single-chain variable fragments (scFv) from the FMC-63 murine hybridoma (FMC-63:AA 1-267, GenBank ID: HM852952.1) linked by a (GGGGS)3 flexible intrachain linker (SEQ ID NO: 187) in the VL to VH orientation were used. The resulting targeting domain was then linked in frame to a human CD8 hinge (AA 138-179, Reference SEQ ID NO: NP_001759.3), a human TNF receptor superfamily 19 transmembrane domain (TNFRSF19, AA 167-196, UntProt Sequence ID: Q9NS68), a human 4-1BB costimulatory domain (CD137, AA 214-255, UniProt Sequence ID: Q07011), and a human CD3 zeta signaling domain (CD247, AA 52-163, Reference SEQ ID NO: NP_000725.1). To promote CAR expression on the cell surface, a leader sequence from the human granulocyte-macrophage colony-stimulating factor receptor alpha subunit (AA 1-22, GenBank ID: EAW98673.1) was included. The CAR sequence was codon-optimized and cloned into a third-generation lentiviral plasmid backbone under the control of a surface antigen-regulated inducible promoter described below, or the constitutive promoters MSCV or EF1α as controls.

[0389] Cell lines used to demonstrate CAR activity Unless otherwise noted, the Burkitt lymphoma cell line Raji, the chronic myeloid leukemia cell line K562, and reagents were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA). Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney line 293T was purchased from ATCC and grown in CD FortiCho medium (Gibco / Thermo Fisher Scientific, Grand Island, NY). Single-cell clones of luciferase-expressing cell lines were generated by stably transducing wild-type tumor lines with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Gaithersburg, MD), followed by cloning and selection of luciferase-positive clones.

[0390] Primary human T cells used to demonstrate CAR activity Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI) with the donor's written consent. Processed buffy coats were purchased from OBI (Oklahoma City, OK). CD4+ and CD8+ human T cells were purified from the buffy coats by positive selection using a 1:1 mixture of CD4 and CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol.

[0391] Transduction of primary T cells CD4+ and CD8+ human primary T cells from normal donors were cultured in TexMACS medium at a cell density of 1 × 10 6Cells were cultured at 1000 / ml and activated with CD3 / CD28 MACS® GMP T Cell TransAct reagent on day 0 (all reagents obtained from Miltenyi Biotec), transduced overnight with LV encoding the CAR construct on days 1 / 2, and medium was changed on days 2 / 3. Cytokines (IL-2, TNFα; Miltenyi Biotec, Bergisch Gladbach, Germany) were added as indicated. Cultures were expanded and harvested on days 5-6 for co-incubation analysis.

[0392] Immune effector assays (CTL and cytokines) For long-term co-incubation assays, CAR T effector and GFP-expressing target cells were grown as described above, and flow cytometry analysis was used to examine the extent of target cell death and CAR T population survival and proliferation. Cells were gated based on forward and side scatter and live (7AAD-negative) cells. The percentage of viable cells after co-culture was examined based on GFP positivity for Raji targets and CD3 positivity for CAR T effectors. In addition, CAR T expression in live CD3-positive cells was examined by staining with CD19 Fc peptide followed by anti-Fc(Fab')2-FL reagent.

[0393] result: A fully human CAR T construct targeting the CD19 antigen was designed by combining in-frame the sequences of a leader peptide derived from GMCSFR, a fully human anti-human CD19 ScFv sequence, a CD8 hinge, a TNFRSF19 transmembrane domain, a 4-1BB costimulatory domain, and a CD3z activation domain.

[0394] We engineered a positive regulatory loop for the CAR by placing expression of the CAR LTG1563 under the control of tandem STAT5 response elements followed by a minimal promoter sequence. Binding of IL-2 to the native IL-2 receptor on the T cell surface initiates Jak / STAT signaling, leading to STAT5 translocation to the regulatory region of the CAR promoter and increasing CAR expression. A constitutively expressed EF1α promoter-driven CAR LTG1563 was constructed as a control and tested in parallel with the STAT-5 and AP1 / NFκB-regulated CAR. Positive regulation of the CAR by (1) IL-2-driven Stat5 and (2) IL-2 / TNFα- and CAR-driven AP1 / NFκB-mediated mechanisms is illustrated in Figure 1, and the structures of each of these constructs are shown in Figure 2.

[0395] T cells were purified from the blood of two unrelated healthy donors by immunogenic selection using a 1:1 mixture of CD4 and CD8 beads (Miltenyi Biotec). Cells were transduced with the CAR LTG1563 construct as described in Materials and Methods without the addition of cytokines. Experimental groups contained either the EF1α promoter-driven CD19 CAR at an MOI of 10 or the positively regulated self-driven CARs STAT5_RE CAR LTG1563 and AP1 / NFκB_RE CAR LTG1563 at 0.25% vol / (vol of LV preparation) (Figure 3). Untransduced cells (UTD) cultured under the same conditions served as a negative control. On day 5, selected groups were supplemented with IL-2 or TNFα, and CAR expression was detected by flow cytometry 20 hours later. Expression of EF1α-driven CARs was 30–40% dependent on the transduction MOI and was unchanged by the addition of IL-2 / TNFα. In contrast, expression of STAT5_RE CARs was significantly increased by the addition of IL-2, but not by TNFα, as expected (Figure 3). Expression of AP1 / NFκB_RE CARs was increased by both IL-2 and TNFα, but to a lesser extent than that of the IL-2-driven STAT5_RE CAR LTG1563.

[0396] Furthermore, in this experiment, the self-driving properties of both the STAT5 and AP1 / NFκB response elements to drive expression of the CAR LTG1563 were investigated by CD19 antigen stimulation (Figure 4). This was performed using CAR T cells from donor A and donor B co-cultured with CD19-positive Raji NHL tumor cells stably expressing GFP. Notably, within 20 hours of stimulation, CAR expression was robustly induced in AP1 / NFκB_RE-driven CAR LTG1563 T cells (Figure 4B), indicating that using these response elements to drive CAR expression integrates both CAR signaling and CAR-dependent cytokine expression. This stimulation was significantly greater than that observed when simply stimulating these cells with cytokines, indicating a stronger and more direct response to CAR signaling. This rapid AP1 / NFκB-dependent CAR upregulation contrasts with that of the constitutively expressed EF1α-driven CAR LTG1563, which was transiently reduced upon stimulation of the CAR via the CD19 antigen (presumably due to internalization of these receptors). On the other hand, the STAT5_RE-driven CAR LTG1563 was induced by coculture with CD19+ Raji cells, although the timescale was significantly longer than that observed for the AP1 / NFκB-driven CAR LTG1563. This likely reflects an intermediate step required for STAT5 response element function, IL2 production by CAR T cells, and the accompanying STAT5 signaling. Addition of IL2 to these cells between D3 and D5 after activation significantly enhanced STAT5 element-mediated induction of CAR LTG1563 expression, likely by priming STAT-mediated signaling.

[0397] Notably, multiple stimulations of CD19+ Raji cells (from D6 to D22 after activation) were able to re-induce expression of the AP1-NFκB CAR LTG1563, indicating that CAR T cells retain their ability to respond to AP1 / NFκB signaling even during long-term ex vivo culture (data not shown). Importantly, there was a clear temporal correlation between the degree of CAR expression under the control of the AP1 / NFκB promoter and the number of Raji cells in coculture (data not shown). These results indicate that the AP1 / NFκB element rapidly drives CAR LTG1563 expression in the presence of cognate CD19 antigen and that CAR LTG1563 is rapidly downregulated in the absence of antigen.

[0398] In the following experiment, T cells were purified from the blood of three unrelated healthy donors by immunogenic selection using a 1:1 mixture of CD4 and CD8 beads (Miltenyi Biotec). On Day 1 postactivation, cells were transduced with the CAR LTG1563 construct as described in Materials and Methods without the addition of cytokines. The experimental group included a CD19 CAR driven by a constitutive EF1α-derived CAR, while the STAT5 RE CAR LTG1563 and AP1-NFκB CAR LTG1563 were transduced with LV at an MOI of 10 (Figure 3). In a subset of samples, IL2 (30 IU / mL) was added on Days 3–6 postactivation to prime the expression of both the STAT5 RE CAR LTG1563 and AP1 / NFκB CAR LTG1563. CAR LTG1563-dependent cytotoxicity was examined by coculture of CAR T cells with CD19+ Raji NHL cells stably expressing GFP. CD19-dependent cytotoxicity was examined by counting GFP+ Raji cells by flow cytometry using a very low effector-target ratio (1:3 CAR T:Raji cells) between days 6 and 13 after activation (coculture 1). Long-term CAR T cell function and proliferation were examined by restimulating the cells at a similar effector-target ratio (1:3 Coculture 1:Raji cells) between days 13 and 20 after activation (coculture 2).

[0399] Co-incubation of CAR T with target cells was examined for a total of 14 days (co-culture 1 & 2), during which time the number of viable Raji cells and T cells was counted by flow cytometry analysis (Figure 5). While Raji cells in the Raji-alone and UTD control groups continued to proliferate unimpeded until experimental day 8, CAR LTG1563 T cells strongly suppressed Raji proliferation in all groups, regardless of IL2 priming. Notably, both the STAT5 RE and AP1 / NFκB CAR LTG1563 exhibited superior tumor killing compared with the EF1α CAR LTG1563 throughout the initial co-culture (co-culture 1) period, as measured on days 1, 2, 3, 6, and 7 after Raji addition (Figure 5B). Notably, although expression of CAR LTG1563 in IL2 cultures was initially low, both STAT5RE and AP1 / NFκB were dominant over the EF1α constitutive expression CAR LTG1563 construct (Figure 5B), potentially indicating that constitutive expression of CAR in the absence of cognate antigen, even for a short period (D2-D6 after activation), may have a negative impact on CAR T cell potency, possibly due to persistent signaling.

[0400] Further longitudinal analysis of CAR LTG1563-dependent cytotoxicity was performed by restimulating CAR T cells with CD19+ Raji cells. All CAR groups again demonstrated effective suppression of Raji proliferation (data not shown), with the AP1 / NFκB-driven CAR LTG1563 demonstrating superior tumor-killing function compared with the constitutively expressed EF1α_CAR LTG1563 (Figure 5B). T cells expressing AP1 / NFκB_CAR LTG1563 proliferated to a significantly greater extent than T cells expressing STAT5RE_CAR LTG1563 or EF1α_CAR LTG1563 throughout the culture period (Figure 5C). This is likely because CAR signaling is only present in the presence of target CD19+ cells, thereby preserving the overall health of these cells and concomitantly increasing T cell proliferation.

[0401] Overall, the results described above demonstrate the superiority of the inducible, self-driven AP1 / NFκB RE CAR LTG1563, as reflected by its low unstimulated CAR expression, reduced susceptibility to exhaustion, reduced levels of cytokine-induced priming, and rapid upregulation and sustained expression in the presence of tumor Raji cells. Furthermore, superior CTL function was observed in terms of initial activity compared to the same CAR construct under the control of the constitutive EF1α promoter. This correlated with the overall greater proliferation of these AP1 / NFκB CAR T cells upon antigen stimulation than all other constructs tested.

[0402] Example 2 Extending self-driven expression to auxiliary proteins Given the success of the inducible AP1 / NFκB promoter in self-driving high-level expression of the CAR LTG1563, particularly in the presence of the cognate CAR antigen (CD19), we sought to extend the functionality of this promoter to auxiliary proteins to provide additional functionality to CAR-T cells. The advantage of the CAR antigen-inducible AP1 / NFκB promoter is that any protein under its control can be highly expressed only in the presence of the CAR tumor antigen. Therefore, we used the AP1 / NFκB promoter to express proteins that enable CAR-T cells to escape immunosuppression, such as the dominant-negative TGF-beta receptor (TGFBRIIdn) and the dominant-negative programmed cell death protein 1 (PD1dn). These dominant-negative receptors were thought to enable CAR-T cells to evade immunosuppression by TGF-β or PD-L1, respectively. An expected advantage of using the AP1 / NFκB promoter is that TGF-β / PD-L1 immunosuppression plays an important role in preventing the development of T cell-mediated autoimmunity in patients under normal conditions. However, the microenvironment of many cancers is characterized by overexpression of TGF-β and / or PD-L1, which can interfere with antitumor immune responses mediated by T cells or CAR-T cells. Therefore, expressing these dominant-negative receptors, especially in the presence of tumor cells, may be highly advantageous.

[0403] material and method: Construction of dominant-negative receptor expression vectors To generate the dominant-negative receptors TGFBRIIdn and PD1dn, these proteins were linked in-frame to the CAR LTG1563 expression vector (LTG1563). Downstream of the CAR LTG1563 sequence, a cleaved ribosomal skip site (FP2AF) was introduced, which consisted of a consensus furin cleavage site (amino acids: RAKR (SEQ ID NO: 188)) fused to a ribosomal skip site (P2A) derived from the porcine teschovirus-1 polyprotein (AA 976-997, GenBank ID: CAB40546.1, mutated residue P977S) and fused to a consensus furin cleavage sequence (amino acids: RAKR (SEQ ID NO: 188)). The resulting CAR LTG1563-FP2AF sequence was co-expressed with a dominant-negative TGF-beta receptor II sequence (TGFBRII:AA 1-191, Uniprot ID: P37173) in the TGFBRIIdn alone expression vector (LTG2864 and LTG2867). The resulting CAR LTG1563-FP2AF sequence was co-expressed with a dominant-negative PD1 receptor (PD1:AA 1-199, Uniprot ID: Q15116) in the PD1dn alone expression vector (LTG2864 and LTG2867).

[0404] In a vector expressing CAR-FP2AF-PD1dn-P2AF-TGFBRIIdn, CAR LTG1563-FP2AF was co-expressed with a dominant-negative PD1 receptor (PD1:AA 1-199, Uniprot ID: Q15116), followed by a consensus furin cleavage sequence (amino acids: RAKR (SEQ ID NO: 188)) fused to a ribosomal skip site (P2A) derived from the porcine teschovirus-1 polyprotein (AA 976-997, GenBank ID: CAB40546.1, mutated residue P977S), which was then co-expressed with a dominant-negative TGF-beta receptor II sequence (TGFBRII:AA 1-191, Uniprot ID: P37173). The protein sequence was codon-optimized and cloned into a third-generation lentiviral plasmid backbone under the control of the surface antigen-regulated inducible promoter AP1 / NFκB or, as a control, the constitutive EF1α promoter.

[0405] Cell lines used to demonstrate CAR activity Unless otherwise noted, the Burkitt lymphoma cell line Raji, the chronic myeloid leukemia cell line K562, and reagents were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA). Cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Logan, UT) and 2 mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney line 293T was purchased from ATCC and grown in CD FortiCho medium (Gibco / Thermo Fisher Scientific, Grand Island, NY). Single-cell clones of luciferase-expressing cell lines were generated by stably transducing wild-type tumor lines with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Gaithersburg, MD), followed by cloning and selection of luciferase-positive clones. 【...

Claims

1. 1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) operably linked to a surface antigen-regulated inducible promoter comprising a nucleotide sequence comprising SEQ ID NO:

138.

2. 2. The isolated nucleic acid molecule of claim 1, wherein the CAR comprises at least one extracellular antigen-binding domain comprising a mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38 antigen-binding domain, or a combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain.

3. 3. The isolated nucleic acid molecule of claim 2, wherein the encoded at least one antigen binding domain comprises at least one single chain variable fragment of an antibody that binds to one or more antigens including mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38, or a combination thereof.

4. 3. The isolated nucleic acid molecule of claim 2, wherein the encoded at least one antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to one or more antigens including mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38, or a combination thereof.

5. 3. The isolated nucleic acid molecule of claim 2, wherein the encoded at least one antigen binding domain, the at least one intracellular signaling domain, or both, are linked to the transmembrane domain by a linker or spacer domain.

6. 6. The isolated nucleic acid molecule of claim 5, wherein the encoded linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, or CD28 and is linked to a transmembrane domain.

7. 3. The isolated nucleic acid molecule of claim 2, wherein the encoded extracellular mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38 antigen binding domain is preceded by a leader nucleotide sequence encoding a leader peptide.

8. The isolated nucleic acid molecule of claim 7, wherein the leader nucleotide sequence comprises a nucleotide sequence comprising SEQ ID NO:13, which encodes the leader amino acid sequence of SEQ ID NO:14, or SEQ ID NO:39, which encodes the leader amino acid sequence of SEQ ID NO:40, or SEQ ID NO:41, which encodes the leader amino acid sequence of SEQ ID NO:42, or SEQ ID NO:43, which encodes the leader amino acid sequence of SEQ ID NO:

44.

9. 3. The isolated nucleic acid molecule of claim 2, wherein the transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD19 / CD22, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

10. 3. The isolated nucleic acid molecule of claim 2, wherein the surface antigen-regulated inducible promoter comprises a nucleotide sequence comprising SEQ ID NO: 138, or a sequence having 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

11. The isolated nucleic acid molecule of claim 2, wherein the encoded at least one intracellular signaling domain further comprises a CD3 zeta intracellular domain.

12. The isolated nucleic acid molecule of claim 11, wherein the encoded at least one intracellular signaling domain is located C-terminal to the CD3 zeta intracellular domain.

13. 3. The isolated nucleic acid molecule of claim 2, wherein the encoded at least one intracellular signaling domain comprises a costimulatory domain, a primary signaling domain, or any combination thereof.

14. 14. The isolated nucleic acid molecule of claim 13, wherein the encoded at least one costimulatory domain comprises a functional signaling domain of OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12, and 4-1BB (CD137), or any combination thereof.

15. A vector comprising the nucleic acid molecule of claim 2.

16. 16. The vector of claim 15, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentivirus vector, an adenovirus vector, and a retrovirus vector, or a combination thereof.

17. A cell comprising the vector of claim 15.

18. The cell of claim 17 , wherein the cell is a T cell.

19. The T cells are CD8 ＋ The cell of claim 18 which is a T cell.

20. The cell of claim 17 , wherein the cell is a human cell.

21. 20. Use of the cells of claim 17 for the manufacture of a pharmaceutical composition for providing anti-tumor immunity to a mammal.

22. 1. A pharmaceutical composition comprising an anti-tumor effective amount of a population of human T cells, wherein the T cells comprise a chimeric antigen receptor (CAR) operably linked to a surface antigen-regulated inducible promoter encoded by a nucleic acid sequence comprising SEQ ID NO: 138, the CAR comprising at least one extracellular antigen-binding domain comprising mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38 antigen-binding domain, or a combination thereof, at least one linker domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein the T cells are human T cells having cancer.

23. 23. The pharmaceutical composition of claim 22, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.

24. 23. The pharmaceutical composition of claim 22, wherein the T cells are T cells of a human with a hematological cancer.

25. 25. The pharmaceutical composition of claim 24, wherein the hematological cancer is leukemia or lymphoma.

26. 26. The pharmaceutical composition of claim 25, wherein the leukemia is acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphoblastic T-cell leukemia (T-ALL), or acute lymphoblastic B-cell leukemia (B-ALL).

27. 26. The pharmaceutical composition of claim 25, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma.

28. 25. The pharmaceutical composition of claim 24, wherein the hematological cancer is multiple myeloma.

29. The human cancers include oral and pharyngeal cancer (tongue, mouth, pharynx, head and neck), gastrointestinal cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, etc.) 25. The pharmaceutical composition of claim 24, comprising cancers of the following organs: adult cancers including: human, pulmonary, esophageal, gallbladder, pancreas), respiratory tract (larynx, lung, and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell carcinoma, and squamous cell carcinoma), childhood tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, reproductive system (cervix, uterus, ovary, vulva, vagina, prostate, testes, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), brain and other nervous system, or any combination thereof.

30. 1. Use of a population of T cells for the manufacture of a pharmaceutical composition for treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, wherein the T cells comprise an isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) operably linked to a surface antigen-regulated inducible promoter nucleotide sequence comprising SEQ ID NO:

138.

31. 31. The use of claim 30, wherein the CAR comprises at least one extracellular antigen-binding domain comprising a CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38 antigen-binding domain, or a combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain.

32. 1. Use of a population of T cells for the manufacture of a pharmaceutical composition for treating a mammal having a disease, disorder, or condition associated with elevated expression of a tumor antigen, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), the CAR being selected from the group consisting of CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, CD38, CD138, BCMA (CD269), TSLPR, TEM-1, TEM-7, TEM-8, TEM-9, CD371, CD276, CD99, GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, or MAGE A3.

20. The method of claim 19, further comprising administering to said subject a method of administering to said subject at least one extracellular antigen-binding domain, comprising administering to said subject an antigen-binding domain comprising an antigen-binding domain of a TCR, or any combination thereof, or an antigen-binding domain comprising a combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, operably linked to a surface antigen-regulated inducible promoter nucleotide sequence comprising SEQ ID NO:

138.

33. 1. Use of a population of T cells for the manufacture of a pharmaceutical composition for treating cancer, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one extracellular antigen binding domain, including an antigen binding domain comprising a CD19 antigen binding domain or a combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, operably linked to a surface antigen-regulated inducible promoter nucleotide sequence comprising SEQ ID NO: 138, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 78, and wherein the T cells are T cells of a subject with cancer.

34. 34. The use of claim 31, 32, or 33, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising the alpha, beta, or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.

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