Compositions and methods for treating cancer with self-driving chimeric antigen receptors

Abstract

To provide novel compositions for treatment of multiple myeloma and chronic lymphocytic leukemia using an approach that can exhibit specific and efficacious anti-tumor effect.SOLUTION: The invention provides an isolated nucleic acid molecule encoding a therapeutic payload operably connected to a surface antigen-regulated inducible promoter comprising a nucleotide sequence comprising multiple specific sequences or a combination thereof, and where the surface antigen-regulated inducible promoter adjusts its level of transcription dependent upon the level of expression of the surface antigen on a target cell thereby achieving a T-cell response precisely regulated to the level of the target antigen present in the tumor milieu. Also provided are a vector, a cell, and a pharmaceutical composition.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] Cross - Reference to Related Applications This PCT patent application claims priority based on U.S. Provisional Patent Application No. 62 / 954,161, filed on December 27, 2019, and U.S. Provisional Patent Application No. 62 / 814,759, filed on March 6, 2019, and incorporates by reference the entire disclosure of each of them herein.

[0002] Field of the Disclosure This application relates to the field of cancer, and in particular, to inducible promoters conjugated to therapeutic payloads and methods of using the same.

Background Art

[0003] Background Cancer is one of the most lethal threats to human health. In the United States alone, nearly 1.3 million people are newly diagnosed with cancer each year, making it the second leading cause of death after cardiovascular disease, and one in four deaths is due to cancer. Most of these deaths are caused by solid tumors. Although medical treatments for some specific cancers have advanced significantly, the 5 - year survival rate for all cancers combined has only improved by about 10% over the past 20 years. The metastasis and proliferation of cancer or malignant tumors are rapid and uncontrollable, making treatment extremely difficult.

[0004] Multiple myeloma ("MM") is a debilitating disease, often incurable, 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 lymphoma ("NHL") (Smith L, McCourt O, Henrich M et al. Multiple myeloma and physical activity: a scoping review. BMJ Open. 2015;5:e009576). MM affects plasma cells in the bone marrow and 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, kidney dysfunction, and neuropathy (Mayo Clinic staff. Diseases and conditions: multiple myeloma: treatments and drugs. December 4, 2015).

[0005] The first-line treatments for MM include proteasome inhibitors, immunomodulatory drugs, steroids, histone deacetylase ("HDAC") inhibitors, and chemotherapy. These approaches aim to kill MM cells, but many of them are also associated with extensive immunosuppression and systemic toxicity.

[0006] Chimeric antigen receptor T cell (CAR T) technology has brought great progress to the treatment of blood malignancies such as MM and is very promising for the treatment of solid tumors. However, this technology currently has two unresolved drawbacks. One is that the efficacy of CAR T is insufficient and the response is suboptimal. The other is that there are harmful side effects such as cytokine release syndrome caused by overactivation of CAR T. The object of the present invention is to provide a CAR T site and other therapeutic payloads that can be naturally fine-tuned based on the tumor burden and inflammatory environment in a patient at any given time during treatment to any local microenvironment By producing them in the context, we can address these two problems.

[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 generally engineered to mimic the single chain Fragment variable (ScFv), the smallest binding domain of immunoglobulin (Ig) molecules. Other antigen-binding motifs such as receptor ligands (e.g., IL-13 designed to bind to the IL-13 receptor expressed in tumors), full-length immunoreceptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D) have also been designed. Other cell targets (such as NK or gamma-delta T cells) for expressing CARs are also under development (Brown CE et al. Clin Cancer Res. 2012;18(8):2199-209; Lehner M et al. PLoS One. 2012;7(2):e31210). Considerable additional effort must be expended to identify the most highly active T cell populations to transduce with CAR vectors, find optimal culture and expansion techniques, and elucidate the structure of the CAR protein itself in detail at the molecular level.

[0008] The binding motif of the CAR may be a relatively stable structural domain such as the constant domain of IgG, or it can be designed as a long flexible linker. Using a structural motif such as a structural motif derived from the IgG constant domain, the ScFv binding domain can be extended to a position far from the cell membrane surface of T cells. This may be important for some tumor targets where the binding domain is particularly close to the surface membrane of tumor cells (such as disialoganglioside GD2; Orentas et al., this observation is unpublished). All of the signaling motifs used in CARs to date include the CD3-zeta chain. This is because this core motif is an important signal for T cell activation. The first reported second-generation CARs were characterized by the CD28 signaling domain and the CD28 transmembrane sequence. This motif was used in third-generation CARs that further contained the CD137 (4-1BB) signaling motif (Zhao Y et al. J Immunol. 2009;183(9):5563-74). With the emergence of new technologies, the need for T cell activation by the presence of beads bound to anti-CD3 and anti-CD28 antibodies and the classical "signal 2" from CD28 to be encoded by the CAR itself has disappeared. Third-generation vectors using bead activation were found not to exceed second-generation vectors in in vitro assays, and furthermore, did not provide a clear benefit over second-generation vectors in a leukemia mouse model (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 pertains to CD19 in the context of second-generation CD28 / CD3-zeta (Lee DW et al., American Society of Hematology Annual Meeting, New Orleans, LA; December 7 - 10, 2013) and CD137 / CD3-zeta signaling modalities (Porter DL et al., N Engl J Med. 2011;365(8):725 - 33). This is demonstrated by the clinical success of specific CARs. In addition to CD137, other members of the tumor necrosis factor receptor superfamily, such as OX40, can also provide important persistent 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 cutting-edge area in synthetic biology. Multiple promoters and gene products have been conceived for the purpose of guiding these highly potent cells to the tumor microenvironment, where T cells can mediate effective tumor killing by avoiding negative regulatory signals. The approach of removing unwanted T cells by dimerization of an inducible caspase 9 construct using AP1903 (drug-induced dimerization) presents one strategy that can pharmacologically initiate a powerful switch for controlling T cell populations (Di Stasi A et al., N Engl J Med. 2011;365(18):1673 - 83). Furthermore, the method of creating an effector T cell population that does not respond to the negative regulatory action of transforming growth factor β by expressing a dominant negative receptor indicates the extent to which effector T cells can be manipulated to obtain optimal anti-tumor 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 the endogenous T cell receptor, at present, the in vivo +The limited proliferation of T cells, the immediate disappearance of these cells after infusion, and the poor clinical activity have been major obstacles, and the clinical application of this technology has not advanced.

[0010] To date, prior art CAR therapy approaches have used CAR T constructs under the control of constitutive promoters such as the human elongation factor 1 alpha (EF1α), phosphoglycerate kinase (PGK), murine leukemia virus (MuLV), murine stem cell virus (MSCV), or other constitutive promoters well-known in the art, and such constitutive promoters are often expressed at high levels or artificially induced (via small molecules or soluble components). These approaches are not well-suited to the dynamics of antigen expression, and as a result, effector cell responses can be excessive or insufficient at the cellular level and can lead to inadequate treatment or overtreatment and toxicity for the patient at the organismal level.

Summary of the Invention

Problems to be Solved by the Invention

[0011] Therefore, it has been an urgent and long-standing need in the art to discover new compositions and methods for treating MM and CLL using an approach that can provide a specific and effective anti-tumor effect without exhibiting the problems described above (such as high toxicity, insufficient efficacy, etc.).

Means for Solving the Problems

[0012] The present invention addresses the above-described need by providing compositions and methods of treatment that can be used for the treatment of cancer and other diseases and / or conditions. In particular, the present invention disclosed and described herein can be used for the treatment of diseases, disorders, or conditions associated with dysregulated expression of various antigens, such as, but not limited to, mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38, or combinations thereof, and provides an inducible promoter - therapeutic payload construct that contains an antigen-specific binding domain with high surface expression on transduced T cells, has a high degree of cell lysis, and allows the transduced T cells to proliferate and persist in vivo. Further, the present invention disclosed and described herein provides self-driven control of such therapeutic payloads by utilizing a surface antigen-regulated inducible promoter that regulates or modulates the expression of a therapeutic payload construct by a surface antigen-regulated promoter, which is directly correlated with the activity of the therapeutic payload and thus the expression level of the surface antigen in the target cell environment. The present invention provides self-driven control of such therapeutic payloads by utilizing a surface antigen-regulated inducible promoter that adjusts the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells.

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

[0014] Disclosed and described herein is a novel self-driven inducible promoter - payload therapeutic construct that includes a therapeutic payload operably linked to a surface antigen-regulated inducible promoter that adjusts the expression level of one or more therapeutic payloads depending on the expression level of surface antigens on target cells.

[0015] Without being limited to any particular mechanism of action, as used herein, a "self-driven" surface antigen-controlled inducible promoter utilizes a surface antigen-controlled inducible promoter to drive a therapeutic payload and provides for surface expression of a low basal level of the therapeutic payload in the absence of expression of a tumor target antigen. When an activating target antigen is present on the surface of a target cell, the therapeutic payload is activated, thereby triggering activation of an appropriate signaling pathway, which in turn activates the signal mediators of the surface antigen-controlled inducible promoter, resulting in an increase in the expression of the therapeutic payload above the basal level of expression.

[0016] In this way, a positive feedback loop is created such that as the expression of a given target antigen increases, the expression of the therapeutic payload also increases, and vice versa, so that the expression of the therapeutic payload is efficiently controlled and an accurate T cell response is achieved that is tailored to the level of the target present at a particular site and time. 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, an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-controlled inducible promoter is provided herein, where the surface antigen-controlled inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of the surface antigen on the target cell, thereby achieving an accurately controlled T cell response according to the level of the target antigen present in the tumor environment.

[0018] In one embodiment, an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising a nucleotide sequence of SEQ ID NO: 137 and / or 138 or a combination thereof is provided herein, wherein the surface antigen-regulated inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of the surface antigen on the target cells, thereby achieving a precisely controlled T cell response according to the level of the target antigen present in the tumor environment.

[0019] In one aspect, at least one therapeutic payload comprising 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-apoptosis proteins, shRNA, or proteases, or combinations thereof, is 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 to form a self-driven surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the surface antigen-regulated inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of the surface antigen on the target cells, is provided herein, and also provided are host cells (e.g., T cells) expressing the surface antigen-regulated inducible promoter-therapeutic payload construct, and nucleic acid molecules encoding the surface antigen-regulated inducible promoter-therapeutic payload construct.

[0020] In one embodiment, one or more therapeutic payloads (e.g., based on chimeric antigen receptors (CARs), cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulatory receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptosis proteins, shRNA, or proteases, among others) 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 aspect, the surface antigen-regulated inducible promoter-therapeutic payload constructs disclosed herein may include, for example, but not limited to, a CAR, which may include a single molecule expressed on the effector cell surface, or may include a signaling module expressed by the effector cell and a soluble targeting module, such that the soluble targeting module binds to the cell-expressed signaling module to form a fully functional CAR. This CAR has high surface expression on transduced T cells, a high degree of cell lysis, and the transduced T cells proliferate and persist in vivo. 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 the expression of the therapeutic payload.

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

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

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

[0025] In one aspect, there is provided an isolated polynucleotide encoding a surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the therapeutic payload is operably linked to the surface antigen-regulated inducible promoter, and the therapeutic payload comprises, from the N-terminus to the C-terminus, at least one extracellular binding domain comprising the 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, and a CAR.

[0026] Thus, in one embodiment, there is provided an isolated polynucleotide 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, there is provided an isolated polynucleotide 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, there is provided an isolated polynucleotide encoding a surface antigen-regulated inducible promoter-CAR-based therapeutic payload construct, wherein the therapeutic payload is operably linked to the surface antigen-regulated inducible promoter and the therapeutic payload comprises a CAR comprising at least one extracellular binding domain 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 combinations thereof, at least one transmembrane domain, and at least one intracellular signaling domain, from N-terminus to C-terminus.

[0029] In one embodiment, there is provided an isolated polynucleotide encoding a surface antigen-regulated inducible promoter-therapeutic payload construct based on a chimeric antigen receptor (CAR), wherein the therapeutic payload is operably linked to the surface antigen-regulated inducible promoter, and the therapeutic payload comprises, from N-terminus 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, wherein the therapeutic payload comprises a CAR.

[0030] In one embodiment, the targeting domain of the surface antigen-regulated inducible promoter-therapeutic payload construct based on a CAR is expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2, and comprises at least one 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, which binds to a further binding tag or epitope. The CAR contains a mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38 antigen targeting domain, and the component of the CAR expressed by the effector cell contains a binding domain specifically directed to bind to a tag or epitope expressed on the soluble CAR module, whereby the soluble component of the CAR and the cell-binding component of the CAR specifically bind 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 expressed separately in the form of a monoclonal antibody, ScFv Fab, Fab'2, and contains at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38 antigen targeting domain selected from the group consisting of the nucleic acid sequences 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, and a further ScFv. The component of this CAR expressed by the effector cell contains a tag or epitope that specifically responds to the further ScFv expressed on the soluble CAR module, whereby the soluble component of the CAR and the cell-binding component of the CAR specifically bind 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 (antikaryin) that binds to a 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 encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the extracellular mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen-binding domain encoded is bound to the transmembrane domain by a linker domain.

[0034] 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 extracellular mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen-binding domain is located downstream of a sequence encoding a leader or signal peptide.

[0035] In yet another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct comprising 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 is provided, wherein the CAR targets an antigen comprising (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 and further encodes an extracellular antigen-binding domain. The

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

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

[0038] In one embodiment, an isolated nucleic acid molecule encoding a surface antigen-regulated inducible promoter-therapeutic payload construct based on a CAR is provided, wherein the extracellular antigen-binding domain, 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 the transmembrane domain by a linker or spacer domain.

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

[0040] In another embodiment, an isolated nucleic acid molecule encoding a surface antigen-regulated inducible promoter-therapeutic payload construct based on a CAR is provided, wherein the encoded CAR further comprises a transmembrane domain comprising the 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 combinations thereof.

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

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

[0043] In another embodiment, an isolated nucleic acid molecule encoding a surface antigen-regulated inducible promoter-therapeutic payload construct based on a CAR is provided, wherein the at least one encoded intracellular signaling domain comprises a co-stimulatory 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 at least one co-stimulatory domain encoded 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 encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, which further contains a leader sequence or a signal peptide, and 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 aspect, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided herein, which comprises, from the N-terminus to the 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 surface antigen-regulated inducible promoter-therapeutic payload construct based on a CAR 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 combinations thereof.

[0050] In some embodiments, a surface antigen-regulated inducible promoter-therapeutic payload construct based on a CAR is provided, wherein the CAR comprises 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 a TCR, a MAGE A3 TCR, or an extracellular antigen-binding domain encoding an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or any combination thereof.

[0051] In one embodiment, a surface antigen-regulated inducible promoter-therapeutic payload construct based on a CAR 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, 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 having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity 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 TCR VH antigen-binding domain, anti-MAGE A3 TCR VH antigen-binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity 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 that can specifically bind to a target antigen, which is anti-CD19 P antigen-binding domain, anti-CD20 P antigen-binding domain, anti-CD22 P antigen-binding domain, anti-ROR1 P antigen-binding domain, anti-mesothelin P antigen-binding domain, anti-CD33 P antigen-binding domain, anti-CD38 P antigen-binding domain, anti-CD123 (IL3RA) P antigen-binding domain, anti-CD138 P antigen-binding domain, anti-BCMA (CD269) P antigen-binding domain, anti-GPC2 P antigen-binding domain, anti-GPC3 P antigen-binding domain, anti-FGFR4 P antigen-binding domain, anti-c-Met P antigen-binding domain, anti-PMSA It may be derived from a natural or synthetic sequence comprising a 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 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 at least one intracellular signaling domain comprises a co-stimulatory domain and a primary signaling domain.

[0054] In yet another embodiment, a surface antigen-regulated inducible promoter-based on CAR- A therapeutic payload construct is provided, wherein at least one intracellular signaling domain comprises a co-stimulatory 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 the surface antigen-regulated inducible promoter-based on CAR-therapeutic payload construct comprises the nucleic acid sequence of SEQ ID NO: 139. In one embodiment, the nucleic acid sequence encodes a promoter-therapeutic payload construct based on a CAR comprising the amino acid sequence of SEQ ID NO: 78.

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

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

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

[0059] In one embodiment, the nucleic acid molecule encoding the surface antigen-regulated inducible promoter-therapeutic payload construct based on CAR may be contained in a vector such as a viral vector. The vector is 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, the vector expressing the surface antigen-regulated inducible promoter-therapeutic payload construct based on CAR may be further modified to include one or more operable elements for controlling the expression of CAR T cells or for removing 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 vector expressing CAR may be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0061] In another aspect, there is further provided a host cell comprising a nucleic acid molecule encoding a 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 cell.

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

[0063] In another aspect, provided herein are transduced CAR T cells 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 enables these transduced CAR T cells to develop an anti-tumor response depending on the expression level of the corresponding surface antigen on the target cells, thereby achieving a CAR T cell response that is precisely controlled according to the level of the target surface antigen present in the tumor environment, where: 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) when an activating target surface antigen is present on the surface of the target cells, the CAR is activated, thereby triggering the activation of the appropriate signaling pathway, which in turn activates the signal mediator of the surface antigen-regulated inducible promoter, resulting in an increase in CAR expression beyond the basal level of expression; iii) as the expression of a given target surface antigen increases, the expression of the CAR also increases, and vice versa, such that CAR expression is efficiently regulated and an accurately adapted CAR T cell response is achieved according to the level of the target present at a specific site and time point; iv) the elevated CAR expression leads to optimal anti-tumor activity and rapid elimination of the target tumor cells; and v) as the tumor cells are quantitatively decreased / eliminated, the level of CAR expression returns to its basal level of expression.

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

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

[0066] In yet another aspect, there is provided a pharmaceutical composition comprising a population of human T cells in an anti-tumor effective amount, wherein the T cells comprise a nucleic acid sequence encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, and 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 the T cells are human T cells having cancer. The cancer includes, among others, blood cancers such as leukemia (e.g., chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML)), lymphoma (e.g., mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma), or multiple myeloma (MM), or combinations thereof.

[0067] In one embodiment, there is provided a pharmaceutical composition, 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 combinations thereof.

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

[0069] In yet another embodiment, a pharmaceutical composition is provided that comprises a population of human T cells from a human having cancer in an anti-tumor effective amount, wherein the cancer is a refractory cancer that does not respond to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), adult B cell malignancies including non-Hodgkin lymphoma (NHL), pediatric B cell malignancies (including B-line ALL (acute lymphoblastic leukemia)), multiple myeloma (MM), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other blood 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 native effector cell pathways based on native cell activation, proliferation-promoting states, or combinations thereof.

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

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

[0073] In yet another aspect, provided is a method for diagnosing a disease, disorder, or condition associated with the expression 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 in a cell, which includes: a) 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 a fragment thereof, or a combination 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 or a combination thereof; and b) antigen mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123, or CD38, or these A step of detecting the presence of a combination, wherein when mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123, or CD38, or a combination thereof is present, diagnosing that it is a disease, disorder, or condition associated with the 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 the expression of mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38, or a combination thereof is hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumor, minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), adult B-cell malignancies including non-Hodgkin lymphoma (NHL), pediatric B-cell malignancies (including B-line ALL (acute lymphoblastic leukemia)), multiple myeloma (MM), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma, or other blood cancers and solid tumors, or any combination thereof.

[0075] In another embodiment, a method for diagnosing, prognosticating, or determining the risk of a disease associated with mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 (or combinations thereof) in a mammal is provided, which comprises the step of detecting the expression of mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38, or combinations thereof, in a sample derived from the mammal, the step comprising: a) contacting the sample 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 combinations 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, and diagnosing that the mammal has a mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38-related disease if the antigen is present.

[0076] In another embodiment, a method of redirecting a CAR antigen target is provided, which comprises 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 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. In one embodiment, the cell is 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 inducing anti-tumor immunity in a mammal is provided, which comprises administering to the mammal, in a therapeutically effective amount, 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, such as, but not limited to, a CAR, cytokine, chemokine, transporter receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulatory receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptosis protein, shRNA, or protease, or a combination thereof. This includes the step of administration.

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

[0079] In yet another embodiment, a method for treating a mammal having a disease, disorder, or condition associated with increased expression of a tumor antigen is provided, the method comprising administering to the subject a pharmaceutical composition comprising one or more of the disclosed surface antigen-regulated inducible promoter-therapeutic payload constructs and comprising a population of anti-tumor effective amount of T cells, the T cells comprising a nucleic acid sequence encoding a CAR, the CAR comprising at least one extracellular mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen-binding domain comprising the amino acid sequences 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 any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the T cells are T cells of a subject having cancer.

[0080] In yet another embodiment, a method for treating cancer in a subject in need thereof is provided, which comprises administering to the subject a pharmaceutical composition comprising one or more of the disclosed surface antigen-regulated inducible promoter-therapeutic payload constructs and a population of anti-tumor effective amount of T cells, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and the CAR comprises at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen-binding domain comprising the amino acid sequences 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 any combination thereof, at least one linker or spacer domain, at least one transmembrane domain, and at least one intracellular signaling domain, and the T cells are T cells of a subject having cancer. In some embodiments of the methods described above, the at least one transmembrane domain comprises the 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 for generating a population of persistently genetically engineered T cells in a human diagnosed with cancer is provided. In one embodiment, the method comprises (e.g., CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptosis protein, shRNA, protease-based) surface antigen-regulated pro Administering to a human genetically engineered T cells that express a motor-therapeutic payload construct, wherein the self-driven surface antigen-regulated promoter-therapeutic payload construct comprises at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen-binding domain comprising the amino acid sequences 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 any combination thereof, at least one transmembrane domain, and at least one intracellular signaling domain, and the population of persistently genetically engineered T cells, or the population of progeny of T cells, persists in the human 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 comprise 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 cancer, disease, disorder, or condition associated with increased expression of a tumor antigen, as described above, can be treated or prevented or remitted using one or more of the surface antigen-regulated promoter-therapeutic payload constructs disclosed herein.

[0084] To avoid misunderstanding, the specification and claims disclosed herein exclude, inter alia, conditionally, the Syn-Notch constructs described in U.S. Patent No. 9,670,281 (inventor name "Binding-triggered transcriptional switches and methods of use thereof") issued on June 6, 2017 and U.S. Patent No. 9,834,608 (Wendell A. Lim et al.) issued on December 5, 2017, and the expression of IL-12 controlled by NFAT described in U.S. Patent No. 8,556,882 (Richard A. Morgan et al.) issued on October 15, 2013.

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

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

[0087]

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Modes for Carrying Out the Invention

[0088] Detailed Description Definitions As used herein, the singular forms "a", "an", and "the" refer to both the singular and plural forms unless the context clearly dictates otherwise. For example, the term "an antigen" can include 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 phrase "and / or" means "and" or "or". Further, unless otherwise specified, 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 values 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 case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are provided only to illustrate and are not intended to limit. To facilitate identification of various embodiments, explanations of terms are provided below. For purposes of illustration and not limitation.

[0089] The term "about", when referring to measurable values such as amounts and durations, means a variation 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. This is because such variations are appropriate for the practice of the disclosed methods.

[0090] Unless otherwise specified, scientific terms in this specification are used in their conventional sense. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), 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 reference materials.

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

[0092] The present disclosure provides a novel self-driven inducible promoter-therapeutic payload construct comprising one or more therapeutic payloads operably linked to a surface antigen-regulated inducible promoter, wherein the surface antigen-regulated inducible promoter regulates the expression level of the one or more therapeutic payloads depending on the expression level of the surface antigen in the environment of the target cell, and also provides a host cell (e.g., a T cell) expressing the surface antigen-regulated inducible promoter-therapeutic payload construct, and a nucleic acid molecule encoding the surface antigen-regulated inducible promoter-therapeutic payload construct.

[0093] Without being limited to any particular mechanism of action, as used herein, a "self-driven" surface antigen-controlled inducible promoter utilizes a surface antigen-controlled inducible promoter to drive a therapeutic payload and provides a low basal level of surface expression of the therapeutic payload in the absence of expression of a tumor target antigen. When an activating target antigen is present on the surface of a target cell, the therapeutic payload is activated, thereby triggering the activation of an appropriate signaling pathway, which in turn activates the signal mediators of the surface antigen-controlled inducible promoter, resulting in an increase in the expression of the therapeutic payload beyond its basal level of expression. In this way, a positive feedback loop is created such that as the expression of a given target antigen increases, the expression of the therapeutic payload also increases, and vice versa, so that the expression of the therapeutic payload is efficiently controlled and an accurate T cell response is achieved that is precisely tailored to the level of the target present at a particular site and time. 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.

[0094] This self-driven mode of activation can then be repeated upon subsequent re-exposure to the antigen (such as may occur due to tumor recurrence, metastatic events, tumor migration / spread, etc., but is not limited thereto). As a result, the timing and magnitude of the T cell response of the self-driven therapeutic payload can thus be controlled, enabling improvement of treatment efficacy, reduction of the risk of tumor escape, and reduction of the toxicity associated with the treatment. This is analogous to biologically or intrinsically adjusting the dosage of a treatment according to the state of the disease at the cellular level. This self-driven mode of activation can then be repeated upon subsequent re-exposure to the antigen (such as may occur due to tumor recurrence, metastatic events, tumor migration / spread, etc., but is not limited thereto). As a result, the timing and magnitude of the T cell response of the self-driven therapeutic payload can thus be controlled, enabling improvement of treatment efficacy, reduction of the risk of tumor escape, and reduction of the toxicity associated with the treatment. This is analogous to biologically or intrinsically adjusting the dosage of a treatment according to the state of the disease at the cellular level.

[0095] Next, the self-driven surface antigen-controlled promoter-therapeutic payload construct will be described in detail, and this description includes an explanation of its surface antigen-controlled inducible promoter, its therapeutic payload, and the self-driven surface antigen-controlled promoter-therapeutic payload construct based on a CAR, antibodies and their antigen-binding fragments, conjugates, nucleotides, expression, vectors, and host cells, as well as further detailed descriptions of treatment methods, compositions, and kits using the disclosed self-driven surface antigen-controlled promoter-therapeutic payload construct based on a CAR.

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

[0097] In one embodiment, an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-controlled inducible promoter comprising the nucleotide sequence of SEQ ID NO: 137 and 138 or combinations thereof is provided herein, wherein the surface antigen-controlled inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of the surface antigen on the target cells, thereby achieving a precisely controlled T cell response according to the level of the target antigen present in the tumor environment.

[0098] In one embodiment, an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising the nucleotide sequence of SEQ ID NO: 137 and 138 or a combination thereof, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, is provided herein, wherein the surface antigen-regulated inducible promoter regulates the expression level of one or more therapeutic payloads depending on the expression level of the surface antigen on the target cell, thereby achieving an accurately controlled T cell response according to the level of the target antigen present in the tumor environment.

[0099] In another embodiment, an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising the nucleotide sequence of SEQ ID NO: 137 and 138 or a combination thereof is provided herein, wherein the surface antigen-regulated inducible promoter upregulates the expression level of one or more therapeutic payloads depending on the expression level of the surface antigen on the target cell, thereby achieving an accurately controlled T cell response according to the level of the target antigen present in the tumor environment.

[0100] In another embodiment, an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising the nucleotide sequence of SEQ ID NO: 137 and 138 or a combination thereof, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, is provided herein, wherein the surface antigen-regulated inducible promoter upregulates the expression level of one or more therapeutic payloads depending on the expression level of the surface antigen on the target cell, thereby achieving an accurately controlled T cell response according to the level of the target antigen present in the tumor environment.

[0101] In yet another embodiment, the expression level of the surface antigen-regulated inducible promoter therapeutic payload construct described herein may be upregulated, for example, by about 10-100%, 200%, 300%, 400%, and 500% (not limited thereto). The ranges listed herein clearly include all integer values contained therein as if they were explicitly listed.

[0102] In one embodiment, an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising the nucleotide sequence of SEQ ID NO: 137 and 138 or a combination thereof is provided herein, wherein the surface antigen-regulated inducible promoter downregulates the expression level of one or more therapeutic payloads depending on the expression level of the surface antigen on the target cells, thereby achieving an accurately controlled T cell response according to the level of the target antigen present in the tumor environment.

[0103] In another embodiment, an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter comprising the nucleotide sequence of SEQ ID NO: 137 and 138 or a combination thereof or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto is provided herein, wherein the surface antigen-regulated inducible promoter downregulates the expression level of one or more therapeutic payloads depending on the expression level of the surface antigen on the target cells, thereby achieving an accurately controlled T cell response according to the level of the target antigen present in the tumor environment.

[0104] In yet another embodiment, the expression level of the surface antigen-regulated inducible promoter therapeutic payload construct described herein may be downregulated, for example, by about 10-100%, 200%, 300%, 400%, and 500% (not limited thereto). The ranges listed herein clearly include all integer values contained therein as if they were explicitly listed.

[0105] B. Therapeutic Payload In its broadest aspect, a self - driving surface antigen - regulated inducible promoter - therapeutic payload construct comprising at least one therapeutic payload comprising 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, shRNAs, or proteases, 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 - kappaB 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 the surface antigen on the target cell, is provided herein, as well as host cells (e.g., T cells) expressing the surface antigen - regulated inducible promoter - therapeutic payload construct, and nucleic acid molecules encoding the surface antigen - regulated inducible promoter - therapeutic payload construct.

[0106] In one aspect, a self - driving 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 NF - kappaB response element, or combinations thereof Provided herein is a surface antigen-controlled inducible promoter that regulates the expression levels of a therapeutic CAR and a 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-controlled inducible promoter-therapeutic payload construct, and nucleic acid molecules encoding the surface antigen-controlled inducible promoter-therapeutic payload construct.

[0107] In one embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-controlled inducible promoter-therapeutic payload construct further comprises a cytokine comprising 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, or any combination thereof.

[0108] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-controlled inducible promoter-therapeutic payload construct further comprises a chemokine (including but not limited to) comprising, 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-controlled inducible promoter-therapeutic payload construct further comprises a transport 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, and this transport receptor plays a role in assisting the transport of CAR T cells 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, for example, a bispecific antibody (including but not limited to) a bispecific T cell engager antibody (BiTE) that includes, for example, but is 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, or other multi-targeting antibodies.

[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 (including but not limited to) against, for example, 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 IgG, or scFvFc, or VHH, or F(ab), or F(ab)2, or a native ligand-binding domain or other configuration, and this neutralizing / blocking antibody plays a role in enhancing T cell lysis function, cytokine release, persistence, proliferation ability and preventing T cell checkpoint blockade, exhaustion, apoptosis, 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 stimulating receptor (including but not limited to) such as IL-2R alpha, IL-15R alpha, IL-7R alpha, CXCR5, etc., and this stimulating receptor plays a role in enhancing the anti-tumor function of T cells. Strengthen.

[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., and this truncated inhibitory receptor plays a role in preventing the 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 extracellular domain of PD-1 fused to the endodomain of CD28, the extracellular domain of TGF beta receptor II fused to the endodomain of gp130, the extracellular domain of IL-10 receptor fused to the endodomain of 4-1BB, or the extracellular domain of IL-4 receptor fused to the endodomain of IL-7 receptor, etc., and this hybrid inhibitory / activating receptor plays a role in converting T cell inhibitory signals into T cell activating signals.

[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, etc., and this anti-apoptotic protein plays a role in prolonging the persistence of T cells and preventing apoptosis.

[0116] In another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-regulated inducible promoter-therapeutic payload construct further comprises, for example, shRNA for PD-1, CTLA-4, KLRG-1, CD160, TGF beta receptor II, IL-10R, etc. (not limited thereto), and this shRNA plays a role in downregulating T cell suppressors 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 proteases such as MMP2, MMP4, etc., and this protease plays a role in digesting the tumor stroma to increase the invasion of T cells 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 the iRGD peptide, and this peptide plays a role in increasing the invasion of anti-cancer agents into the tumor.

[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, and this CAR T construct targets a second tumor antigen or an antigen expressed on suppressor cells present in the tumor microenvironment (for example, PD-L1, PD-L2, TRAIL receptor CD33, CD138, etc. present on MDSC and inhibitory B cells, but not limited thereto).

[0120] In yet another embodiment, one or more therapeutic payloads (for example, 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-apoptosis proteins, shRNAs, or proteases (not limited to these) are expressed under the control of a surface antigen-regulated inducible promoter, and the one or more therapeutic payloads are separated by a 2A ribosome 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 further comprises cytokines such as 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; chemokines such as CCR4, CCR6, CXCR5; transport receptors such as cytokine receptors including, but not limited to, CCR4, CCR7, CCR2; bispecific antibodies including, but not limited to, bispecific T cell-engaging antibodies (BiTE) such as anti-CD3 and anti-CD19 targeting or other multi-targeting antibodies; neutralizing / blocking antibodies against, for example, PD-L1, IL-6R, IL-1R (not limited to these), expressed as scFv or IgG or in other configurations; T cell-stimulating receptors; truncated inhibitory receptors; hybrid inhibitory / activating receptors such as the extracellular domain of PD-1 fused to the endodomain of CD28 (not limited to this); anti-apoptosis proteins such as, but not limited to, BCL-2 or BCL-XL; shRNAs; proteases; a second CAR T construct; or any combination thereof (each having 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 the regulation or modulation of the expression of a therapeutic payload construct by a surface antigen-regulated promoter can be directly correlated with the activity of the therapeutic payload and thus the level of surface antigen expression in the target cell environment.

[0123] Unlike previously existing CAR T constructs whose expression is controlled by a constitutive promoter, the surface antigen-regulated inducible promoter-therapeutic payload constructs based on CARs described herein have several advantages over the prior art, including but not limited to, for example: i) the ability to optimally execute the anti-tumor CAR function by adjusting or modulating the timing and magnitude of the anti-tumor response according to the specific amount of antigen being expressed by the tumor at that time; ii) the ability to prevent harmful T cell overactivation (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-related cytokine release syndrome (CRS); or v) the ability to reduce or eliminate CAR-related neurotoxicity, or any combination thereof.

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

[0125] In one embodiment, the self-driven surface antigen-regulated promoter-therapeutic payload construct based on CAR provided herein is, for example, surface antigen-regulated Comprising one or more CAR-based therapeutic payloads (including, but not limited to) operably linked to an inducible promoter, the CAR-based therapeutic payloads comprising, from the N-terminus towards the C-terminus, at least one extracellular binding domain comprising mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123, or CD38 antigen binding domains, or combinations thereof, at least one transmembrane domain, and at least one intracellular signaling domain, and a CAR comprising the same.

[0126] A CAR is an artificially constructed hybrid protein or polypeptide containing an antigen-binding domain of an antibody (e.g., a single-chain variable fragment (ScFv)) linked to a T cell signaling domain via a transmembrane domain. The characteristics of a CAR include the ability to redirect the specificity and reactivity of T cells to a selected target in a manner not restricted by MHC and to utilize the antigen-binding properties of monoclonal antibodies. Since it can recognize antigens without MHC restriction, T cells expressing CARs have the ability to recognize antigens independently of antigen processing and, as a result, to avoid a major mechanism of tumor escape. Also, 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 sites from different protein domains is an innovative feature of CARs. The choice of which of these protein domains to select is as important a design feature as the manner in which they specifically bind. Individual design domains are essential components that can be used in any heterologous CAR platform for the purpose of manipulating lymphocyte function. For example, the selection of an extracellular binding domain can render a CAR that would otherwise be ineffective, effective.

[0128] The physicochemical properties of the immunoglobulin-derived protein sequences used to generate the extracellular antigen-binding domain of a surface antigen-controlled promoter-therapeutic payload construct based on a self-driven CAR may be completely neutral or may be such that they self-associate and cause T cell metabolic exhaustion, significantly reducing the effectiveness of therapeutic T cells expressing this surface antigen-controlled promoter-therapeutic payload construct based on a self-driven CAR. This phenomenon occurs independently of the antigen-binding function of this CAR domain. Furthermore, the choice of intracellular signaling domain can also govern the activity and durability of the therapeutic lymphocyte population used in immunotherapy. Here, the ability to bind a target antigen and the ability to transmit an activation signal to a T cell via each of the extracellular and intracellular domains described above are important CAR design aspects, but it has also become clear that the choice of source of the extracellular antigen-binding fragment can have a significant effect on the efficacy of a surface antigen-controlled promoter-therapeutic payload construct based on a self-driven CAR and, therefore, may have a decisive role in the function and clinical utility of a surface antigen-controlled promoter-therapeutic payload construct based on a self-driven CAR.

[0129] As disclosed herein, the intracellular T cell signaling domain of a surface antigen-controlled promoter-therapeutic payload construct based on a self-driven CAR may include, for example, a T cell receptor signaling domain, a T cell co-stimulatory signaling domain, or both. The T cell receptor signaling domain refers to a portion of a surface antigen-controlled promoter-therapeutic payload construct that includes an intracellular domain of a T cell receptor, such as (but not limited to) the intracellular portion of the CD3 zeta protein. The co-stimulatory signaling domain refers to a portion of a surface antigen-controlled promoter-therapeutic payload construct that includes an intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand that is required for lymphocytes to efficiently respond to an antigen.

[0130] Next, the surface antigen-regulated promoter-therapeutic payload construct based on the self-driving CAR according to the present invention will be described in detail. This description includes an explanation of its extracellular antigen-binding domain, transmembrane domain, and intracellular domain, as well as further explanations of the surface antigen-regulated promoter-therapeutic payload construct based on the self-driving CAR, antibodies and their antigen-binding fragments, conjugates, nucleotides, expression, vectors, and host cells, and treatment methods, compositions, and kits using the disclosed surface antigen-regulated promoter-therapeutic payload construct based on the self-driving CAR.

[0131] 1. Extracellular domain In one embodiment, the surface antigen-regulated promoter-therapeutic payload construct based on a CAR includes 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 act 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 diseases, and cancer cells.

[0132] In one embodiment, a surface antigen-regulated promoter-therapeutic payload construct can be designed to target a desired tumor antigen by designing a desired antigen-binding domain that specifically binds to an antigen on a tumor cell. A tumor antigen is a protein produced by a tumor cell that elicits an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding domain may depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, 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 carcinoma 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 merely by way of example. The listing is not intended to be limiting, and other examples will be readily recognized by those skilled in the art.

[0133] In one embodiment, the tumor antigen comprises one or more antigen cancer epitopes associated with a malignant tumor. 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 prostate 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 cancer gene HER-2 / Neu / ErbB-2. Further, as another group of target antigens, there are cancer fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotype immunoglobulins correspond to truly tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens such as CD19, CD20, and CD37 are also candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) are used as targets for passive immunotherapy using monoclonal antibodies, but have not achieved sufficient success.

[0134] The type of tumor antigen may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is unique to tumor cells and does not occur on other cells of the body. TAA is not unique to tumor cells; instead, it is expressed on normal cells under conditions where immune tolerance to this antigen is not induced. The expression of this antigen in tumors can occur under conditions that allow an immune system response to this antigen. TAA can 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 TAA can be an antigen that is normally present at very low levels on normal cells but is expressed at a significantly higher level 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 multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed cancer genes and mutant tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, etc.; 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, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, 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] Also, in certain embodiments, a human extracellular antigen-binding domain is used in place of the mouse-derived binding domain, resulting in the generation of a surface antigen-regulated promoter-therapeutic payload construct based on a self-driven CAR that functions better in vivo, while simultaneously avoiding the induction of anti-CAR immunity in the host immune response and the death of the CAR-T cell population associated with the mouse-based antigen-binding domain.

[0137] A surface antigen-regulated promoter-therapeutic payload construct based on a self-driven CAR that expresses a fully human extracellular ScFv antigen-binding domain exhibits excellent activity, including: i) preventing poor CAR-T persistence and function as seen in mouse-derived binding sequences; ii) eliminating the need to deliver the self-driven CAR-based surface antigen-regulated promoter-therapeutic payload construct to a specific region for efficacy; and iii) enabling the design of CAR-T cells based on both high-affinity and low-affinity binders for individual antigens. Due to the latter property, researchers can better regulate the efficacy-to-toxicity and / or tissue specificity of CAR-T products because low-affinity binders can have higher specificity for tumors than for normal tissues, given the higher expression of specific antigens in tumors than in 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 the CAR-based surface antigen-regulated promoter-therapeutic payload construct is, for example, as described in the issued U.S. Patent No. 10,183,993 to the same applicant (title of the invention "Compositions and" May include an scFv binder as disclosed in "Methods for Treating Cancer with Anti-Mesothelin Immunotherapy", originally filed as Provisional Patent Application No. 62 / 444,201 on January 9, 2017, issued on January 22, 2019, and assigned Event Number LEN_017 by Lentigen Technology.

[0140] In a preferred embodiment, the isolated nucleic acid molecule encoding the extracellular mesothelin antigen-binding domain comprises the nucleotide sequence of SEQ ID NO: 87, 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: 88, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity 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 the scFv sequence that can be used as an extracellular antigen-binding domain in a surface antigen-regulated promoter-therapeutic payload construct based on the CAR, a single-chain antigen binder (as against the scFv) can be incorporated into the functional CAR.

[0142] For example, co-pending non-provisional patent application No. 15 / 934,770 (Provisional Patent No. 62 / 476,438) by the same applicant (title of the invention "Compositions and Methods For Treating Cancer With Anti-CD33 A CD33-specific heavy chain-only binder as disclosed in "Immunotherapy", filed on March 24, 2018, and assigned Lentigen Technology case number LEN_018.

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

[0144] In another embodiment, a nucleic acid sequence encoding a functional CAR LTG1906 targeting CD33-expressing malignancies comprises the nucleic acid sequence of SEQ ID NO: 93 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: 94 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0145] In one embodiment, the extracellular antigen-binding domain in a CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise, for example, an scFV binder as disclosed in co-pending Provisional Patent Application No. 62 / 773,940 by the same applicant (inventive name "Compositions and Methods for Treating Cancer with Anti-CD38 Immunotherapy", filed on November 30, 2018, and assigned Lentigen Technology case number LEN_026).

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

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

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

[0149] In one embodiment, an isolated nucleic acid molecule encoding an extracellular CD38 antigen-binding domain M3811 comprises the nucleotide sequence of SEQ ID NO: 150 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 CD38 antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 151 or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 151. In another embodiment, a nucleic acid sequence encoding a functional CAR LTG 2091 targeting CD38-expressing malignancies comprises the nucleic acid sequence of SEQ ID NO: 7 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: 8 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0150] In another embodiment, a nucleic acid sequence encoding a functional CAR LTG 2092 targeting CD38-expressing malignancies comprises the nucleic acid sequence of SEQ ID NO: 9 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: 10 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0151] In another embodiment, a nucleic acid sequence encoding a functional CAR LTG 2095 targeting CD38-expressing malignancies comprises the nucleic acid sequence of SEQ ID NO: 11 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: 12 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0152] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2097 targeting CD38-expressing malignancies comprises the nucleic acid sequence of SEQ ID NO: 15 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: 16 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

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

[0154] In one embodiment, the 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 to the amino acid sequence of SEQ ID NO: 153.

[0155] In one embodiment, an isolated nucleic acid molecule encoding an 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 to the amino acid sequence of SEQ ID NO: 155.

[0156] In one embodiment, an isolated nucleic acid molecule encoding an 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 to the amino acid sequence of SEQ ID NO: 157.

[0157] In another embodiment, a nucleic acid sequence encoding a functional CAR LTG 1941 targeting ROR1-expressing malignancies comprises the nucleic acid sequence of SEQ ID NO: 17 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: 18 or a sequence having 85%, 90%, 95%, 96% , 97%, 98%, or 99% identity thereto.

[0158] In another embodiment, a nucleic acid sequence encoding a functional CAR LTG 1942 targeting ROR1-expressing malignancies comprises the nucleic acid sequence of SEQ ID NO: 19 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: 20 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0159] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 1943 targeting ROR1-expressing malignant tumors comprises the nucleic acid sequence of SEQ ID NO: 21 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: 22 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0160] In one embodiment, the extracellular antigen-binding domain in the CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise, for example, an scFV binder as disclosed in co-pending Provisional Patent Application No. 62 / 734,106 by the same applicant (entitled "Compositions and Methods for Treating Cancer with Anti-CD123 Immunotherapy", filed on September 20, 2018, assigned case number LEN_024 of Lentigen Technology).

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

[0162] In one embodiment, an isolated nucleic acid molecule encoding an 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 to the amino acid sequence of SEQ ID NO: 161.

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

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

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

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

[0167] In one embodiment, the 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 to the amino acid sequence of SEQ ID NO: 171.

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

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

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

[0171] In another embodiment, a functional CAR LTG targeting CD123-expressing malignancies The nucleic acid sequence encoding 2075 includes the nucleic acid sequence of SEQ ID NO: 23 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR including the amino acid sequence of SEQ ID NO: 24 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0172] In another embodiment, a functional CAR LTG targeting CD123-expressing malignancies The nucleic acid sequence encoding 2076 includes the nucleic acid sequence of SEQ ID NO: 25 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR including the amino acid sequence of SEQ ID NO: 26 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0173] In another embodiment, a functional CAR LTG targeting CD123-expressing malignancies The nucleic acid sequence encoding 2077 includes the nucleic acid sequence of SEQ ID NO: 115 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR including the amino acid sequence of SEQ ID NO: 116 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0174] In another embodiment, a functional CAR LTG targeting CD123-expressing malignancies The nucleic acid sequence encoding 2078 includes the nucleic acid sequence of SEQ ID NO: 117 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR including the amino acid sequence of SEQ ID NO: 118 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0175] In another embodiment, a functional CAR LTG targeting CD123-expressing malignancies The nucleic acid sequence encoding 2079 includes the nucleic acid sequence of SEQ ID NO: 119 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR including the amino acid sequence of SEQ ID NO: 120 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0176] In another embodiment, a functional CAR LTG targeting CD123-expressing malignancies The nucleic acid sequence encoding 2082 includes the nucleic acid sequence of SEQ ID NO: 121 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR including the amino acid sequence of SEQ ID NO: 122 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0177] In another embodiment, a functional CAR LTG targeting CD123-expressing malignancies The nucleic acid sequence encoding 2083 includes the nucleic acid sequence of SEQ ID NO: 123 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR including the amino acid sequence of SEQ ID NO: 124 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0178] In another embodiment, a functional CAR LTG targeting CD123-expressing malignancies The nucleic acid sequence encoding 2085 includes the nucleic acid sequence of SEQ ID NO: 125 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR including the amino acid sequence of SEQ ID NO: 126 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0179] In another embodiment, a functional CAR LTG targeting CD123-expressing malignancies The nucleic acid sequence encoding 2087 includes the nucleic acid sequence of SEQ ID NO: 127 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR including the amino acid sequence of SEQ ID NO: 128 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0180] In another embodiment, a functional CAR LTG targeting CD123-expressing malignancies The nucleic acid sequence encoding 2088 includes the nucleic acid sequence of SEQ ID NO: 129 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and encodes a CAR including the amino acid sequence of SEQ ID NO: 130 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0181] In one embodiment, the extracellular antigen-binding domain in a surface antigen-regulated promoter-therapeutic payload construct based on a CAR may include, for example, an scFV binder as disclosed in co-pending Provisional Patent Application No. 62 / 736,955 by the same applicant (entitled "Compositions and Methods for Treating Cancer with Human Anti-CD19 / 22 Immunotherapy", filed on September 26, 2018, assigned Lentigen Technology docket number LEN_025).

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

[0183] In another embodiment, a functional CAR targeting CD19 / CD22-expressing malignancies The nucleic acid sequence encoding LTG 2737 comprises the nucleic acid sequence of SEQ ID NO: 131 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: 132 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0184] In one embodiment, the extracellular antigen-binding domain in a CAR-based surface antigen-regulated promoter-therapeutic payload construct is, for example, an scFV binder as disclosed in 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, assigned Attorney Docket No. LEN_021 of Lentigen Technology).

[0185] In one embodiment, the 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 to the amino acid sequence of SEQ ID NO: 181.

[0186] In one embodiment, the 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 to the amino acid sequence of SEQ ID NO: 183.

[0187] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2209 targeting CD22-expressing malignancies comprises the nucleic acid sequence of SEQ ID NO: 133 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: 134 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0188] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2219 targeting CD22-expressing malignancies comprises the nucleic acid sequence of SEQ ID NO: 135 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: 136 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

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

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

[0191] In one embodiment, an isolated nucleic acid molecule encoding an 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 to the amino acid sequence of SEQ ID NO: 114.

[0192] In another embodiment, a functional CAR targeting CD19 / CD20-expressing malignancies The nucleic acid sequence encoding LTG 1496 comprises the nucleic acid sequence of SEQ ID NO: 95 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: 96 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0193] In another embodiment, a functional CAR targeting CD19 / CD20-expressing malignancies The nucleic acid sequence encoding LTG 1497 comprises the nucleic acid sequence of SEQ ID NO: 97 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: 98 or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

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

[0195] Thus, in one embodiment, variant single-specific CAR constructs known to be adaptable in the context of DuoCAR by the same applicant can also be utilized to create a surface antigen-regulated promoter-therapeutic payload construct based on a CAR. Specific examples of single-specific therapeutic surface antigen-regulated promoter-payload constructs (e.g., CAR-based) that can underlie DuoCAR-based surface antigen-regulated promoter-therapeutic payload construct technology include the single CD20-targeted vector LTG1495 alone (nucleotide sequence SEQ ID NO: 142, amino acid sequence SEQ ID NO: 143). A second example is the single-specific CAR LTG2200 specific for CD22 (nucleotide sequence SEQ ID NO: 69, amino acid sequence SEQ ID NO: 70).

[0196] In yet another embodiment, variant CAR structures known to be suitable for the DuoCAR context can also be utilized to create surface antigen-regulated promoter-therapeutic payload constructs based on the CARs within the scope of the present 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) that binds the heavy and light chains of the scFv. In some cases, for example, in the CD19 CAR format, the (GGGGS)n linker (SEQ ID NO: 185) was substituted by the Whitlow linker 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 created. The anti-CD19 CAR LTG1562 (nucleotide sequence SEQ ID NO: 75, amino acid sequence SEQ ID NO: 76) utilizes the CD4 (not CD8) transmembrane domain. Similarly, the anti-CD19 CAR LTG1563 has another transmembrane type derived from TNFRSF19 (nucleotide sequence SEQ ID NO: 77, amino acid sequence SEQ ID NO: 78).

[0197] In yet another embodiment, another example of therapeutic use can be the treatment of leukemia expressing the CD19, CD20, and TSLPR antigens with the surface antigen-regulated promoter-therapeutic payload construct according to the DuoCAR of the present invention. In particular, the surface antigen-regulated promoter-therapeutic payload construct based on DuoCAR comprises LTG1496 or LTG1497 (SEQ ID NOs: 95, 97 respectively) conjugated to the TSLPR-specific CAR (LTG1789) (SEQ ID NO: 10 1, amino acid sequence SEQ ID NO: 102) created from the 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 surface antigen-regulated promoter-therapeutic payload constructs based on the DuoCAR described above, each individual CAR construct is self-driven by a single surface antigen-regulated promoter, and these CAR constructs are separated by a ribosome 2A skip site.

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

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

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

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

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

[0204] Without intending to be limited to any particular mechanism of action, the reasons for the improved therapeutic function in relation to an exemplary surface antigen-regulated promoter-therapeutic payload construct according to the present invention include, for example, a) signal transduction became more efficient due to improved lateral movement in the cell membrane, b) the ability to interact with transmembrane signal transduction cascades related to T cell activation was improved due to excellent positioning in cell membrane microdomains (such as lipid rafts), c) the position in the cell membrane was excellent due to a preferential movement away from inhibitory or downregulatory interactions, for example, the distance from a phosphatase such as CD45 was relatively far or the interaction with the phosphatase was relatively small, and d) the assembly into the T cell receptor signal transduction complex (such as an immune synapse) was excellent, or any combination of these is considered, but not limited thereto.

[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), when CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as the antigen-binding domain incorporated into the surface antigen-regulated promoter-therapeutic payload construct.

[0206] In an exemplary embodiment, the antigen-binding domain portion of the surface antigen-regulated promoter-therapeutic payload construct based on a CAR targets CD19. Preferably, the extracellular antigen-binding domain in the surface antigen-regulated promoter-therapeutic payload construct is an anti-CD19 scFV, where 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 aspect of the invention, for example, an antigen from the Retroviridae family (such as human immunodeficiency viruses like HIV-1 and HIV-LP), Picornaviridae family (such as 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 family, Herpesviridae family (such as herpes simplex virus type 1 and 2 (HSV), varicella-zoster virus, cytomegalovirus (CMV), and herpesvirus), Poxviridae family (such as variola virus, vaccinia virus, and poxvirus), or hepatitis C virus, or any combination thereof (not limited thereto) is provided, a surface antigen-regulated promoter-therapeutic payload construct capable of binding to other than TSA or TAA.

[0208] In another aspect of the present invention, a surface antigen-regulated promoter-therapeutic payload construct capable of binding to an antigen derived from a bacterial species such as Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas, or Salmonella is provided. In particular, for example, Helicobacter pylori, Legionella pneumophilia, Mycobacterium species (e.g., Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae, or Clostridium tetani, or a surface antigen-regulated promoter-therapeutic payload construct capable of binding to an antigen derived from an infectious bacterium such as a combination thereof is provided.

[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 The son contains 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 contains the amino acid sequence of SEQ ID NO: 52 or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 52.

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

[0213] Transmembrane regions particularly useful in the surface antigen-regulated promoter-therapeutic payload constructs described herein may be derived from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 (i.e., including at least their transmembrane regions). Alternatively, the transmembrane domain may be synthetic and may predominantly contain 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 to 10 amino acids in length, may form a linkage between the transmembrane domain and the cytoplasmic signaling domain of the surface antigen-regulated promoter-therapeutic payload construct. A doublet of glycine and serine 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 examples, the transmembrane domain can be selected by amino acid substitution so that the domain avoids binding to the transmembrane domains of the same or different surface membrane proteins in order to minimize the interaction between the domain and other receptor complex components.

[0216] In one embodiment, the transmembrane domain in the surface antigen-regulated promoter-therapeutic payload construct according to the present invention is the 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 an amino acid sequence with at least 1, 2, or 3 modifications (e.g., substitutions) that are 95-99% identical to the amino acid sequence of SEQ ID NO: 28, provided that the modifications (e.g., substitutions) are 20, 10, or 5 or fewer.

[0218] In some examples, the transmembrane domain of the CAR comprises the 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 that is 95-99% identical 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 binds to the transmembrane CD8 domain, the transmembrane CD28 domain, or a combination thereof.

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

[0221] In one embodiment, the transmembrane domain in the CAR according to the present invention is the 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 having at least 1, 2, or 3 modifications (e.g., substitutions) with 95-99% identity to the amino acid sequence of SEQ ID NO: 52, provided that the number of modifications (e.g., substitutions) is 20, 10, or 5 or less.

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

[0224] In some embodiments, the linker may contain a spacer element, and when the spacer element is present, the spacer element enlarges the linker and increases the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Specific examples of spacers are well known to those skilled in the art and include those listed in U.S. Pat. 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, 5,521,284, 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, as well as those listed in U.S. Patent Publications 20110212088 and 20110070248 (which are hereby incorporated by reference in their entirety).

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

[0226] As this spacer domain, all or part of amino acid numbers 118 - 178 (SEQ ID NO: 31) of the hinge region of CD8 alpha (NCBI RefSeq: NP__001759.3), amino acid numbers 135 - 195 of CD8 beta (GenBank: AAA35664.1), amino acid numbers 315 - 396 of CD4 (NCBI RefSeq: NP__000607.1), or amino acid numbers 137 - 152 of CD28 (NCBI RefSeq: NP__006130.1) can be used. Also, as this spacer domain, a part of the constant region of the H chain or L chain of an antibody (CH1 region or CL region, for example, a peptide having the amino acid sequence of SEQ ID NO: 32) can be used. Furthermore, this spacer domain may be an artificially synthesized sequence.

[0227] In addition, all or part of the amino acids including the constant region of human IgG4 (UniProt ID: P01861), including CH1 (amino acid numbers 1 - 98), hinge, SEQ ID NO: 80, and the corresponding nucleotide SEQ ID NO: 79, (amino acid numbers 99 - 110), CH2, amino acid SEQ ID NO: 81 and the corresponding nucleotide SEQ ID NO: 80, (amino acid numbers 111 - 220) and CH3, SEQ ID NO: 84 and the 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 the 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 domains comprise the nucleic acid sequence of SEQ ID NO: 49. In one embodiment, the TNFRSF19 hinge and transmembrane domains comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50. In another embodiment, the TNFRSF19 hinge and transmembrane domains comprise 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, in the surface antigen-regulated promoter-therapeutic payload construct, a signal peptide sequence (also referred to as a leader peptide) may be attached to the N-terminus. This signal peptide sequence is present at the N-terminus of many secreted and membrane proteins and has a length of 15 to 30 amino acids. 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 the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44. In another embodiment, the CD8a hinge domain is fused to the 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 the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40. In another embodiment, the CD8a hinge domain is fused to the 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 the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42. In another embodiment, the CD8a hinge domain is fused to the 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 the cleaved form of the epidermal growth factor receptor (tEGFR) comprises the nucleic acid sequence of SEQ ID NO: 67. In one embodiment, 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 95-99% identity thereto.

[0237] In one embodiment, the furin recognition site and the downstream T2A self-cleaving peptide sequence designed for the simultaneous bicistronic expression of the tag sequence and the 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, the T2A self-cleaving peptide sequence, and the furin recognition downstream site designed for the simultaneous bicistronic expression of the tag sequence and the 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 the CAR-based surface antigen-regulated 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, but is expressed by effector cells of the surface antigen-regulated promoter-therapeutic payload construct. The presented components contain a binding domain specifically directed to bind to a tag or epitope expressed on the soluble CAR module, whereby the soluble component of the CAR and the cell-binding component of the CAR specifically bind to form a fully functional CAR structure.

[0240] 4. Intracellular Domain The cytoplasmic domain or intracellular signaling domain of the CAR is responsible for activating at least one of the normal effector functions of the immune cell into which the CAR has been introduced. The term "effector function" refers to the special functions of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the protein portion that transmits effector function signals and directs the cell to perform special functions. Usually, the entire intracellular signaling domain can be used, but often it is not necessary to use the entire chain. When a truncated portion of the intracellular signaling domain is used, this truncated portion may be used instead of the full chain as long as it can transmit effector function signals. Thus, the meaning of the term "intracellular signaling domain" includes any truncated portion of the intracellular signaling domain sufficient to transmit effector function signals.

[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 act in concert to initiate signaling after binding of the antigen to the receptor, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional ability.

[0242] Signals issued only through the TCR are known to be insufficient to fully activate T cells, and a second or co-stimulatory signal is further required. Therefore, T cell activation involves two separate types of cytoplasmic signaling sequences: one that initiates antigen-dependent first activation via the TCR (the first cytoplasmic signaling sequence), and one that acts in an antigen-independent manner to provide a second or co-stimulatory signal (the second cytoplasmic signaling sequence).

[0243] The first cytoplasmic signaling sequence controls the first activation of the TCR complex in either a stimulatory or inhibitory manner. The first cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor activation tyrosine 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), amino acids 402-495 of CD5 (NCBI RefSeq: NP__055022.2), amino acids 707-847 of 0022 (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), and peptides having the sequences thereof, and variants having the same functions as these peptides, but not limited thereto. The amino acid numbers based on the NCBI RefSeq ID or GenBank amino acid sequence information described herein are numbered based on the full length of the precursor (including the signal peptide sequence, etc.) of each protein. 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 itself include a CD3-zeta signaling domain or may be combined with any other desirable cytoplasmic domain useful in the context of the CAR. For example, the intracellular domain of the CAR may include a CD3 zeta chain portion and a co-stimulatory signaling region. The co-stimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for lymphocytes to efficiently respond to an antigen. Examples of such co-stimulatory 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, among others. Specific examples of such co-stimulatory molecules include peptides having the sequences of amino acids 236 to 351 of CD2 (NCBI RefSeq:NP__001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq:NP__000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq:NP__055022.2), amino acids 207 to 235 of CD8 alpha (NCBI RefSeq:NP__001759.3), amino acids 196 to 210 of CD83 (GenBank:AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq:NP__006130.1), amino acids 214 to 255 of CD137 (4-1BB, NCBI RefSeq:NP__001552.2), amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq:NP__003318.1), and amino acids 166 to 199 of ICOS (NCBI RefSeq:NP__036224.1), as well as variants having the same function as these peptides, but are not limited thereto. Thus, although the present disclosure has so far been exemplified mainly using 4-1BB as a co-stimulatory signaling element, other co-stimulatory elements are also within the scope of the present disclosure.

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

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

[0248] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes 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 includes 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 include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes the 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 includes the 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 include the signaling domain of 4-1BB and the signaling domain of CD3-zeta, where the signaling domain of 4-1BB includes the amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 46, or SEQ ID NO: 60, and the signaling domain of CD3-zeta includes 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 include the signaling domain of CD28 and the signaling domain of CD3-zeta, where the signaling domain of CD28 includes the nucleic acid sequence of SEQ ID NO: 45 or SEQ ID NO: 59, respectively, and the signaling domain of CD3-zeta includes 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 include the signaling domain of CD28 and the signaling domain of CD3-zeta, where the signaling domain of CD28 includes the 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 includes the 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 include the signaling domain of CD28 and the signaling domain of CD3-zeta, where the signaling domain of CD28 includes the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3-zeta includes 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-controlled inducible promoter-therapeutic payload construct comprising additional auxiliary components including any dominant negative receptor of TGFBRII (TGFBRIIdn) and / or PD1 (PD1dn) lacking an intracellular signaling domain co-expressed with a CAR LTG1563 construct via a ribosome skipping site (P2A) is constructed, which contains the nucleic acid sequences of SEQ ID NO: 103, 105, 107 or combinations thereof, and contains the amino acid sequences of SEQ ID NO: 104, 106, 108 or combinations thereof. The preparation of a surface antigen-controlled inducible promoter-therapeutic payload construct encoding a CAR-based promoter-therapeutic payload construct is described in Example 2 below.

[0255] In another embodiment, a surface antigen-controlled inducible promoter-therapeutic payload construct comprising additional auxiliary components including a dominant negative receptor of TGFBRII (TGFBRIIdn) or PD1 (PD1dn) lacking an intracellular signaling domain co-expressed with a CAR LTG1563 construct via a skipping site (P2A) is constructed, which contains the nucleic acid sequences of SEQ ID NO: 103, 105, 107 or combinations thereof, and contains the amino acid sequences of SEQ ID NO: 104, 106, 108 or combinations thereof. The preparation of a surface antigen-controlled inducible promoter-therapeutic payload construct encoding a CAR-based promoter-therapeutic payload construct is carried out. By using the dominant negative receptors dnTGFb and dnPD1, the autoimmune toxicity typically associated with the constitutive activation of dominant negative receptors is prevented, and at the same time, when the CAR is expressed and becomes functional, the immunosuppression of T cell function is reduced. Alternatively, compared with each dn receptor alone or combinations thereof, the resistance to immunosuppression of CAR T cells by the tumor microenvironment is increased.

[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 a CAR LTG1563 having a dominant negative inhibitory TGF-beta receptor comprising the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence having 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 a CAR LTG1563 having 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-therapeutic payload construct having an AP1-NFκB_RE promoter, in which a dominant negative inhibitory TGF-beta receptor and a dominant negative PD1 are co-expressed with a CAR LTG1563 construct separated by a ribosome 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 a CAR LTG1563 construct separated by a ribosome 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-controlled inducible promoter-therapeutic payload construct having the STAT5_RE promoter comprises the nucleic acid sequence of SEQ ID NO: 109 and encodes a CAR LTG1563 having a dominant negative inhibitory TGF-beta comprising the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0260] In yet another embodiment, an isolated nucleic acid molecule is provided, wherein the surface antigen-controlled inducible promoter-therapeutic payload construct having the STAT5_RE promoter comprises the nucleic acid sequence of SEQ ID NO: 110 and encodes a CAR LTG1563 having 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-controlled inducible promoter-therapeutic payload construct having the STAT5_RE promoter, in which a dominant negative inhibitory TGF-beta receptor and a dominant negative PD1 are co-expressed with a CAR LTG1563 construct separated by a ribosome 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 co - expresses a dominant negative inhibitory TGF - beta receptor with a CAR LTG1563 construct, and the constitutive EF1a promoter is used to express additional proteins together 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 co - expresses a dominant negative PD1 with a CAR LTG1563 construct, and the constitutive EF1a promoter is used to express additional proteins together 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 co - expresses a dominant negative inhibitory TGF - beta receptor and a dominant negative PD1 with a CAR LTG1563 construct and is separated by a ribosome - skipping site (P2A), and the constitutive EF1a promoter is used to successfully express additional proteins together with the co - expressed CAR LTG1563 construct.

[0265] 6. Further Explanation about CAR The functional portions of the 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-apoptosis proteins, shRNAs, protease-based) disclosed herein are also clearly encompassed within the scope of the present invention. As used herein with reference to a CAR, the term "functional portion" 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, CAR portions that retain the ability to recognize target cells, or detect, treat, or prevent disease, to a similar extent as, to the same extent as, or to a greater extent than the parent CAR. With respect to a parent CAR, a functional portion can include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0266] A functional portion can include additional amino acids not found in the amino acid sequence of the parent CAR at the amino terminus or carboxy terminus or both termini of the portion. Desirably, these additional amino acids do not interfere with the biological function of the functional portion (e.g., recognition of target cells, detection of cancer, treatment or prevention of cancer). More desirably, these additional amino acids enhance 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 the present disclosure. As used herein, the term "functional variant" refers to a CAR, polypeptide, or protein having a substantial or significant sequence identity or similarity to a parent CAR, which 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, or detect, treat, or prevent disease, to a similar extent as, to the same extent as, or to a greater extent than the parent CAR. Those that retain the ability to recognize target cells to a greater extent than R are included. For a parent CAR, a functional variant may have, for example, an amino acid sequence identity with the parent CAR of at least about 30%, 50%, 75%, 80%, 90%, 98%, or more.

[0268] A functional variant may include, for example, one having at least one conservative amino acid substitution added to the amino acid sequence of the parent CAR. Alternatively or additionally, a functional variant may include one having at least one non-conservative amino acid substitution added to the amino acid sequence of the parent CAR. In this case, it is preferred that this non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. This 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] Amino acid substitutions of the 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 certain physical and / or chemical properties is replaced with another amino acid having the same or similar chemical or physical properties. For example, a conservative amino acid substitution may be replacing an acidic / negatively charged polar amino acid (e.g., Asp or Glu) with another acidic / negatively charged polar amino acid, replacing 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, replacing a basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.) with another basic / positively charged polar amino acid, replacing 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, replacing a beta-branched side chain-containing amino acid (e.g., He, Thr, and Val) with another beta-branched side chain-containing amino acid, replacing an aromatic side chain-containing amino acid (e.g., His, Phe, Trp, and Tyr) with another aromatic side chain-containing amino acid, etc.

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

[0271] A CAR (including functional portions and functional variants) may be of any length, i.e., may contain any number of amino acids, as long as the CAR (or its functional portion or functional variant) retains a biological activity, such as the ability to specifically bind to an antigen, the ability to detect diseased cells in a mammal, or the ability to treat or prevent a disease in a mammal. For example, a CAR may be about 50 to about 5000 amino acids in length, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acids in length.

[0272] A CAR (including functional portions and functional variants according to the present invention) may contain synthetic amino acids in place of one or more natural amino acids. Such synthetic amino acids are well known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-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 It includes '-benzyl-N'-methyl-lysine, Ν',Ν'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0273] CAR (including functional moieties and functional variants) may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., by disulfide bridging), or converted to an acid addition salt, and / or optionally dimerized or polymerized or conjugated.

[0274] A CAR (including its functional portions and functional variants) can be obtained by methods well known in the art. The CAR can be made by any suitable polypeptide or protein production method. Suitable methods for newly synthesizing 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. Also, the polypeptides and proteins may be recombinantly produced using the nucleic acids described herein and standard recombinant methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, 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. Further, some of the CARs (including their functional portions and functional variants) 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 (including their functional portions and functional variants) described herein may be those commercially synthesized by companies. In this regard, the CAR may be synthetic, recombinant, isolated, and / or purified.

[0275] In each of the descriptions of Section A described above, in addition to the surface antigen-regulated promoter-therapeutic payload construct based on the CAR described above, the surface antigen-regulated promoter-therapeutic payload construct includes cytokines such as 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, chemokines such as CCR4, CCR6, CXCR5, transport receptors such as cytokine receptors including, but not limited to, CCR4, CCR7, CCR2, bispecific antibodies including, but not limited to, bispecific T cell engager antibodies (BiTE) such as anti-CD3 and anti-CD19 targeting or other multi-targeting antibodies, neutralizing / blocking antibodies against, for example, PD-L1, IL-6R, IL-1R (not limited to these), expressed as scFv or IgG or in other configurations, T cell stimulatory receptors, truncated inhibitory receptors, for example, hybrid inhibitory / activating receptors including, but not limited to, the extracellular domain of PD-1 fused to the endodomain of CD28, anti-apoptosis proteins including, but not limited to, BCL-2 or BCL-XL may further include one or more of various other therapeutic modalities or auxiliary components including, but not limited to, shRNA, protease, a second CAR T construct, or any combination thereof (each of these 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., a T cell expressing a CAR, an antibody, or an antigen-binding domain or portion thereof, but not limited thereto) that specifically binds to one or more of the antigens disclosed herein. As used herein, "T cell expressing a CAR" or "CAR T cell" means a T cell expressing a CAR and has antigen specificity, e.g., determined by the antibody-derived targeting domain of the CAR.

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

[0278] A "monoclonal antibody" is an antibody obtained from a substantially homogeneous population of antibodies. That is, the individual antibodies that make up this population are identical except for natural mutations that may be present in very small amounts. Monoclonal antibodies are highly specific and are directed against a single antigen epitope. The modifier "monoclonal" indicates the property that the antibody is obtained from a substantially homogeneous population of antibodies and should not be interpreted to mean that the antibody must be produced by any particular method. In some instances, monoclonal antibodies are antibodies produced by a single clone of B lymphocytes or by cells transfected with nucleic acids encoding the light and heavy chain variable regions of the antibody (or antigen-binding fragments thereof) and their progeny. In some instances, monoclonal antibodies are isolated from a subject. Monoclonal antibodies 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, an immunoglobulin has heavy (H) and light (L) chains that are linked to each other by disulfide bonds. Immunoglobulin genes include 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 main classes (or isotypes) of heavy chains, which determine the functional activity of the antibody molecule (IgM, IgD, IgG, IgA, and IgE).

[0280] The heavy and light chains each contain 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., page 91 (2007)). In some embodiments, the heavy and light chain variable regions combine to specifically bind an antigen. In additional embodiments, only the heavy chain variable region is required. For example, natural camelid antibodies consisting of only heavy chains are functional and stable without a light chain (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). Reference to "VH", or "VH", refers 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. Reference to "VL", or "VL", refers to the variable domain of an antibody light chain, including those of Fv, ScFv, dsFv, or Fab. For example, see Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996.

[0281] The variable regions of the light and heavy chains contain "framework" regions and three hypervariable regions that interrupt them (also called "complementary determining regions" or "CDRs") (see, e.g., 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 antigen epitopes. The boundaries of the amino acid sequences of a given CDR can be readily determined using any of a number of well-known 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 (from N-terminus to C-terminus), and are further typically identified by the chain in which the CDR is located. Thus, VH CDR3 is the CDR3 from the variable domain of the heavy chain of an antibody that contains it, and VL CDR1 is the CDR1 from the variable domain of the light chain of an antibody that contains it. Light chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs may be referred to as HCDR1, HCDR2, and HCDR3.

[0283] "Antigen-binding fragment" refers to a portion of a full-length antibody that retains the ability to specifically recognize cognate antigens, as well as various combinations of such portions. 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 made by modification of whole antibodies or newly synthesized using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Ed), Antibody Engineering, Vols. 1-2, 2nd Edition, Springer Press, 2010).

[0284] A single-chain antibody (ScFv) is a genetically engineered molecule that contains the VH domain and VL domain of one or more antibodies joined by a suitable polypeptide linker to form a gene fusion 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 domain and VL domain within an ScFv typically does not determine the ScFv. Thus, ScFvs having both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) may be used.

[0285] In dsFv, the variable chains of the heavy and light chains have disulfide bonds introduced by mutation to stabilize the binding of both chains. Also included is the diabody, which is a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to connect the two domains into one chain is used, so the two domains are connected to the complementary domains of another chain, forming two antigen-binding sites (see, for example, 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 mouse 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 produced recombinantly, or obtained, for example, by screening a combinatorial library consisting of variable heavy and variable light chains as described by Huse et al., Science 246:1275-1281 (1989) (incorporated herein by reference). These methods, as well as other methods for making, 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 Edition (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 an antagonistic assay, and conversely, the reference antibody inhibits the binding of this antibody to its antigen by 50% or more in an antagonistic assay. Antibody antagonistic assays are well known and exemplary antagonistic assays are provided herein.

[0289] A "humanized" antibody or antigen-binding fragment thereof comprises a human framework region and one or more CDRs from a non-human (such as mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment that provides the CDR is called the "donor", and the human antibody or antigen-binding fragment that provides the framework is called the "acceptor". In one embodiment, all of the CDRs are from the donor immunoglobulin in the humanized immunoglobulin. The constant region may be absent, but if present, it may be substantially identical to the human immunoglobulin constant region, for example, at least about 85-90% (such as about 95% or more) identical. Thus, all parts of the humanized antibody or antigen-binding fragment (presumably except for the CDRs) are substantially identical to the corresponding parts of the native human antibody sequence.

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

[0291] A "fully human antibody" or "human antibody" is an antibody that contains sequences from the human genome (or derived therefrom) 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 the human genome. Human antibodies can be identified and isolated, for example, by using antibody production techniques based on human genome-derived sequences, such as by phage display or the use of genetically engineered animals (for example, Barbas et al., Phage display: A Laboratory Manuel. 1st Edition, New York: Cold Spring Harbor Laboratory Press, 2004. Print.; see 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. When there is more than one binding site, these binding sites may be the same as each other or different. For example, a natural 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 antibody ability to bind to any functional part of a CAR are well known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assay (see, for example, Janeway et al. below, US Patent Application Publication No. 2002 / 0197266 Al, and US Patent No. 7,338,929).

[0294] Also, a CAR, a T cell expressing a CAR, an antibody, or an antigen-binding portion thereof may be modified to include 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 elemental particles (e.g., gold particles).

[0295] In each of the descriptions of Section B described above, in addition to the surface antigen-regulated promoter-therapeutic payload construct based on the CAR described above, the surface antigen-regulated promoter-therapeutic payload construct includes cytokines such as 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, chemokines such as CCR4, CCR6, CXCR5, transport receptors such as cytokine receptors including, but not limited to, CCR4, CCR7, CCR2, bispecific antibodies including, but not limited to, bispecific T cell engager antibodies (BiTE) such as anti-CD3 and anti-CD19 targeting or other multi-targeting antibodies, neutralizing / blocking antibodies against, for example, PD-L1, IL-6R, IL-1R (not limited to these), expressed as scFv or IgG or other constructs, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors such as, but not limited to, the extracellular domain of PD-1 fused to the endodomain of CD28, anti-apoptosis proteins including, but not limited to, BCL-2 or BCL-XL, shRNA, proteases, a second CAR T construct, or one or more of any combination thereof, each of which has biological properties as described above). It may further include various other therapeutic modalities including one or more of those not limited to these, such as anti-apoptosis proteins, 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 that express, for example, a CAR or a monoclonal antibody or an antigen-binding fragment thereof specific for one or more of the antigens disclosed herein (e.g., CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell-stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptosis protein, shRNA, protease-based) may be conjugated to agents such as effector molecules or detectable markers using any of a number of means well known to those of skill in the art. Either covalent or 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 to one or more of the antigens disclosed herein is covalently linked to an effector molecule or detectable marker. It will be understood by those of skill in the art that a variety of effector molecules and detectable markers can be used, including, but not limited to, chemotherapeutic agents, anti-angiogenic agents, toxins, 125 I, 32 P, 14 C, 3 H, and 35 radioactive agents such as S, as well as other labels, target sites, and ligands, etc.

[0297] The choice of a particular effector molecule or detectable marker depends 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 specific target cells (such as tumor cells).

[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 (-NH2), or sulfhydryl (-SH) groups, which can be utilized in reactions with suitable functional groups on the antibody, resulting in the attachment of the effector molecule or detectable marker. Alternatively, the antibody or antigen-binding fragment is derivatized to expose or attach additional reactive functional groups. Derivatization may be associated with the attachment of any of a plurality of well-known linker molecules such as those available from Pierce Chemical Company (Rockford, IL). A linker may be any molecule used to couple 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 linker may be attached to the constituent amino acids via its side chain (e.g., to cysteine via a disulfide bond), or to the amino and carboxy groups of the alpha carbon of the terminal amino acids.

[0299] In some embodiments, the linker may include a spacer element, and when the spacer element is present, the linker is enlarged by the spacer element, increasing the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment. Specific 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, 5,66 including those listed in U.S. Patent Nos. 5,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725, 5,530,097, 5,521,284, 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, as well as U.S. Patent Publications Nos. 20110212088 and 20110070248 (each of which is incorporated herein by reference in its entirety).

[0300] In some embodiments, the linker is cleavable under intracellular conditions, and cleavage of the linker releases an effector molecule or detectable marker from the antibody or antigen-binding fragment in the intracellular environment. In yet another embodiment, the linker is not cleavable, and the effector molecule or detectable marker is released, for example, by degradation of the antibody. In some embodiments, the linker is cleavable by a cleaving agent present within the intracellular environment (e.g., within a lysosome or endosome or caveolea). The linker may be, for example, a peptide linker cleaved by an intracellular protease, or a protease enzyme including but not limited to lysosomal or endosomal proteases. 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 may 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, 1 - 15 amino acids in length. The proteases may include cathepsin B and D, as well as plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug in target cells (e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics (see 83:67-123). For example, a peptide linker cleavable by a 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 hereby incorporated by reference herein. 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, which describes the synthesis of doxorubicin and a valine-citrulline linker).

[0301] In another embodiment, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, such pH-sensitive linkers hydrolyze under acidic conditions. For example, acid-labile linkers that can hydrolyze within lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amide, orthoesters, acetals, or ketals, etc.) can be used. (See, e.g., 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 unstable at pH 5.5, or less than 5.0, which is the approximate pH of lysosomes. In a particular embodiment, the hydrolyzable linker is a thioether linker (e.g., a thioether linked to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Patent No. 5,622,929, etc.)).

[0302] In another embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are well known in the art and include, 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, for example, 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. 68:92809290, 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 U.S. Publication No. 2005 / 0238649, which is hereby incorporated by reference in its entirety).

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

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

[0307] Maytansinoid compounds suitable for use as the maytansinoid toxin moiety are well known in the art and can be isolated from natural sources according to well-known methods, prepared using genetic engineering techniques (see Yu et al. (2002) PNAS 99:7968 - 7973), or prepared synthetically from maytansinol and maytansinol analogs according to well-known methods. Maytansinoids act by inhibiting tubulin polymerization. It is a mitotic inhibitor. Maytansine was first isolated from Maytenus serrata, a shrub native to East Africa (U.S. Patent No. 3,896,111). Subsequently, it was further discovered that certain microorganisms produce maytansinoids such as maytenol and C-3 maytenol esters (U.S. Patent No. 4,151,042). Synthetic maytenol and its derivatives and analogs are disclosed, for example, in U.S. Patent 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, 4,313,946, 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. Conjugates containing maytansinoids, methods for their preparation, and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020, 5,416,064, 6,441,163, and European Patent No. EP0425235 B1, the disclosures of which are expressly incorporated herein by reference.

[0308] Additional toxins can be used in combination with, for example, CARs, T cells 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-apoptosis proteins, shRNAs, protease-based) surface antigen-regulated promoter-therapeutic payload constructs. Examples of 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 of them are readily available from commercial suppliers (e.g., Sigma Chemical Company, St. Louis, MO). The intended toxins also include variants of such toxins (see, e.g., U.S. 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 does not have a mechanism for specifically entering cells and thus needs to be conjugated to an antibody or antigen-binding fragment that recognizes an endogenous cell surface protein in order to efficiently enter cells.

[0310] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Typically, for use in immunotoxins, diphtheria toxin has been mutated to reduce or eliminate nonspecific toxicity. A mutant known as CRM107 has sufficient enzymatic activity but significantly reduced nonspecific toxicity, has been well-known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976), and has been used in human clinical trials. See U.S. Patent No. 5,792,458 and U.S. Patent No. 5,208,021.

[0311] Ricin is the lectin RCA60 obtained from Ricinus communis (castor bean). Examples of ricin are described in U.S. Patent No. 5,079,163 and U.S. Patent No. 4,689,401. Ricinus communis agglutinin (RCA) has two forms, referred to as RCA 60 and RCA 120 due to their respective molecular weights of approximately 65 kD and approximately 120 kD (Nicholso n & Blaustein, J. Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible for inactivation of protein synthesis and cell death. The B chain binds ricin to cell surface galactose residues and facilitates 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 binding 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 It is described in Pt 2:247-54,2000. Calicheamicin was first isolated from Micromonospora echinospora and is a member of the enediyne antitumor antibiotic family that causes DNA double-strand breaks and induces apoptosis (see, for example, Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic site of immunotoxins in clinical trials (see, for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0313] Abrin contains a toxic lectin obtained from Abrus precatorius. Its toxic components, abrin a, b, c, and d, have a molecular weight of about 63-67 kD and are composed of two polypeptide chains A and B linked by disulfide bonds. Chain A inhibits protein synthesis, and chain B (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 that express, for example, CARs, monoclonal antibodies, antigen-binding fragments thereof, that are specific for one or more of the antigens disclosed herein (for example, CARs, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell-stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptosis proteins, shRNAs, protease-based) may also be conjugated to a detectable marker, for example, a detectable marker detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging diagnostic techniques (computed tomography (CT), computerized axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination, etc.). Specific examples of detectable markers include, but are not limited to, fluorophores, chemiluminescent agents, enzyme conjugates, radioisotopes, and heavy metals or compounds (for example, superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and lanthanide phosphors. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also useful. CARs, T cells expressing CARs, 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 a CAR, T cell expressing a CAR, antibody, or antigen-binding portion thereof is conjugated to a detectable enzyme, additional reagents are added that produce a reaction product distinguishable from that used for the enzyme It can be detected in this way. For example, when horseradish peroxidase, which is an agent, is present, a colored reaction product can be obtained by adding hydrogen peroxide and diaminobenzidine, and this can be detected visually. CAR, T cells expressing CAR, an antibody, or an antigen-binding portion thereof may also be bound to biotin and may be detected by indirectly measuring the binding of avidin or streptavidin. It should be noted that avidin itself may be bound to an enzyme or a fluorescent label.

[0315] For example, a surface antigen-regulated promoter-therapeutic payload construct expressing CAR, an antibody, or an antigen-binding portion thereof (such as CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell-stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptosis protein, shRNA, protease-based) may be bound to a paramagnetic agent such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also useful as labels. Antibodies may also be bound to lanthanides (such as europium and dysprosium) and manganese. An antibody or antigen-binding fragment may also be labeled with a predetermined polypeptide epitope recognized by a second reporter (such as a leucine zipper sequence pair, a binding site for a secondary antibody, a metal-binding domain, an epitope tag).

[0316] For example, surface antigen-regulated promoter-therapeutic payload constructs expressing a CAR, antibody, or antigen-binding portion thereof (e.g., CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulatory receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptosis protein, shRNA, protease-based) may also be conjugated to radiolabeled amino acids. The radiolabels may be used for both diagnostic and therapeutic purposes. For example, the radiolabels may be used to detect one or more of the antigens disclosed herein and antigen-expressing cells by X-ray, luminescence spectroscopy, or other diagnostic techniques. Additionally, the radiolabels may be used as toxins in therapy to treat tumors in a subject, for example, 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 radioisotopes or radiolabeled nucleotides such as I, but are not limited thereto.

[0317] Means for detecting such detectable markers are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or a scintillation counter, and fluorescent markers may be detected by detecting emitted light using a light detector. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction product generated 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 of Section C described above, in addition to the surface antigen-regulated promoter-therapeutic payload conjugate construct based on the CAR described above, the surface antigen-regulated promoter-therapeutic payload conjugate construct may further include various other therapeutic modalities including cytokines such as 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, chemokines such as CCR4, CCR6, CXCR5, transport receptors such as cytokine receptors including, but not limited to, CCR4, CCR7, CCR2, bispecific antibodies including, but not limited to, bispecific T cell engager antibodies (BiTE) such as anti-CD3 and anti-CD19 targeting or other multi-targeting antibodies, neutralizing / blocking antibodies against, for example, PD-L1, IL-6R, IL-1R (not limited to these) expressed as scFv or IgG or in other configurations, T cell stimulatory receptors, truncated inhibitory receptors, for example hybrid inhibitory / activating receptors including, but not limited to, the extracellular domain of PD-1 fused to the endodomain of C

[0319] F. Nucleotides, Expression, Vectors, and Host Cells According to one embodiment of the present invention, there is further provided 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., CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulatory receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptosis protein, shRNA, protease-based). The nucleic acid according to the present 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 have modified codons. Without being bound by any theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing native codons with other codons that encode the same amino acid but can be translated by tRNAs that are more readily available intracellularly, and thus, translation efficiency may be increased. Optimization of the nucleotide sequence may also be one that can reduce secondary mRNA structures that can interfere with translation, and thus, translation efficiency may be increased.

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

[0322] As used herein, "nucleic acid" includes "polynucleotide", "oligonucleotide", and "nucleic acid molecule", and generally may be single-stranded or double-stranded, may be obtained from synthetic or 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 found between nucleotides of unmodified oligonucleotides), and means a polymer of DNA or RNA. In some embodiments, the nucleic acid contains no insertions, deletions, inversions, and / or substitutions. However, as described herein, in some instances, it may be suitable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0323] A recombinant nucleic acid may have a sequence that does not occur naturally or may have a sequence in which two regions that are distant in the sequence are artificially combined. This artificial combination is often achieved by chemical synthesis or, more generally, by artificially manipulating distant nucleic acid regions by genetic engineering techniques such as those described in the references of Sambrook et al. mentioned above. Nucleic acids may be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures well known in the art. See, for example, the references of Sambrook et al. and Ausubel et al. mentioned above. For example, nucleic acids may be constructed using natural nucleotides or the biological safety of the molecule Chemically synthesized nucleotides modified in various ways (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) may be used that are designed to increase the identity or increase the physical stability of the double-stranded formed by hybridization. Examples of modified nucleotides that can be used in nucleic acid production include, but are not limited to, 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 adenine, 7-methylguanine, 5-methylaminomethyluracil, 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, methyl ester of uracil-5-oxyacetic acid, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids according to the present invention may be purchased from companies such as Integrated DNA Technologies (Coralville, IA, USA).

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

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

[0326] The nucleotide sequence that hybridizes under stringent conditions may hybridize under highly stringent conditions. "Highly stringent conditions" means that the nucleotide sequence specifically hybridizes to the target sequence (the nucleotide sequence of any of the nucleic acids described herein), and the amount thereof is detectably more than non-specific hybridization. Highly stringent conditions include conditions that can distinguish a polynucleotide having a strictly complementary sequence or having only 2 to 3 scattered mismatches from a random sequence that accidentally has 2 to 3 small regions (for example, 3 to 10 bases) matching the nucleotide sequence. Such small complementary regions are more easily melted than the full-length complementary regions of 14 to 17 bases or more in length, and these can be easily distinguished by highly stringent hybridization. Relatively highly stringent conditions may include, for example, low salt and / or high temperature conditions such as about 0.02 to 0.1 M NaCl or equivalent thereto at a temperature of about 50 to 70 °C. Thus, highly stringent conditions allow very little, if any, mismatch between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any of the CARs of the present invention. Generally, for It is understood that the conditions can be made more stringent by increasing the amount of the muamide added.

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

[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 the purposes described herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and when the vector and a host cell are contacted under conditions sufficient for the mRNA, protein, polypeptide, or peptide to be expressed in the host cell, the host cell is capable of expressing the mRNA, protein, polypeptide, or peptide. Such vectors are generally not naturally occurring.

[0329] However, some of such vectors may occur naturally. 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, may be synthetic or obtained in part from natural sources, and may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may contain natural or non-natural nucleotide linkages, or both types of linkages. Preferably, the non-natural or altered nucleotides or nucleotide linkages 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 can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for purposes of propagation and growth, or for expression, or for both purposes (such as plasmids and viruses). 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 λΤΙΟ, λΤΙ 1, λZapII (Stratagene), EMBL4, and λΝΜΙ 149 can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, for example, a retroviral vector or a 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 can be used in a clinic include, for example, the LENTIVECTOR® gene delivery technology from Oxford BioMedica plc, the LENTIMAX™ vector system from Lentigen, etc., but are not limited thereto. Non-clinical type lentiviral vectors are also available and may be well known to those skilled in the art.

[0332] Multiple transfection techniques are generally well 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 co-precipitation (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 transfection (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid introduction using a high-velocity particle gun (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0334] In one embodiment, the recombinant expression vector may be prepared using standard recombinant DNA techniques as described, for example, in Sambrook et al. supra and Ausubel et al. supra. The circular or linear construct of the expression vector may be prepared to contain a replication mechanism that functions in prokaryotic or eukaryotic host cells. The replication mechanism may be derived from, for example, ColE1, 2μ plasmid, λ, SV40, and bovine papillomavirus, etc.

[0335] The recombinant expression vector is appropriately specific for the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is introduced, and may contain regulatory sequences such as transcription codons, translation initiation codons, and stop codons, taking into account whether the vector is based on DNA or RNA. The recombinant expression vector may contain restriction sites for facilitating cloning.

[0336] The recombinant expression vector may contain one or more marker genes that enable the selection of transformed or transfected host cells. Marker genes include, for example, biocide resistance such as resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic hosts for prototrophy. Marker genes suitable 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 contain a natural or non-natural promoter operably linked to a nucleotide sequence encoding a CAR (including its functional portions and functional variants), or a nucleotide sequence complementary to or hybridizing to the nucleotide sequence encoding a CAR. The selection of the promoter (e.g., strong, weak, inducible, tissue-specific, and developmental-specific, etc.) is within the ordinary knowledge of those skilled in the art. Similarly, the binding of the nucleotide sequence to the promoter is also within the ordinary knowledge of those skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, or the promoter found in the terminal repeat sequence of the murine stem cell virus.

[0338] The recombinant expression vector may be designed for either transient expression, stable expression, or both. Further, the recombinant expression vector may be prepared for constitutive expression or inducible expression.

[0339] Furthermore, the recombinant expression vector may be prepared to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of cells expressing the suicide gene. A suicide gene may confer sensitivity to an agent, such as a drug, on the cells expressing the gene, causing the death of the cells when the cells are contacted or exposed to the agent. Suicide genes are well known in the art (see, e.g., 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, the 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 the recombinant expression vector according to the present invention. The host cell may be a eukaryotic cell such as, for example, a plant, animal, fungus, or alga, or may be a prokaryotic cell such as, for example, a bacterium or protozoan. The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism such as, for example, a human). The 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 Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, and HEK293 cells. When the purpose is amplification or replication of the recombinant expression vector, the host cell may be a prokaryotic cell, such as DH5a cells. When the purpose is production of 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 developmental stage, 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 the purposes described herein, the T cell may be any T cell, may be a cultured T cell (such as, for example, a primary T cell), or a T cell from a cultured T cell line (such as, for example, Jurkat, SupTl, etc.), or a T cell obtained from a mammal. When obtained from a mammal, the T cell may be obtained from a very diverse source including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or body fluids. The T cell may be enriched or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell may be of any type and may be at any developmental stage, CD4 + / CD8 + double positive T cell, CD4 + helper T cell, such as Th1 and Th2 cells, CD8+ Including, but 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, etc. T cells are CD8 + T cells or CD4 + T cells may be.

[0342] In one embodiment, the surface antigen-regulated promoter-therapeutic payload construct described herein can be used in suitable cells that are not T cells. Such cells are, for example, immune effector cells such as NK cells and T-like cells generated from pluripotent stem cells having a factor function.

[0343] Also, one embodiment provides a population of cells comprising at least one host cell described herein. This population of cells, in addition to host cells containing any of the recombinant expression vectors described, includes at least one other cell, for example, a host cell that does not contain any of the recombinant expression vectors (e.g., a T cell), or a cell other than a T cell, for example, a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc., and may be a heterogeneous population. Alternatively, the population of cells may be a substantially homogeneous population mainly comprising (e.g., consisting essentially of) host cells containing the recombinant expression vector. Also, the population may be a clonal cell population in which all cells of the population are clones of a single host cell containing the recombinant expression vector, and thus all cells of the population contain this recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells containing the recombinant expression vector described herein.

[0344] A CAR (including its functional portion and variants), nucleic acid, recombinant expression vector, host cell (including its population), and antibody (including its antigen-binding portion) may be isolated and / or purified. For example, in a preparation of purified (or isolated) host cells, the purity of the host cells is higher than that in the natural environment in vivo. Such host cells may be prepared, for example, by standard purification techniques. In some embodiments, the preparation of host cells is purified such that the host cells comprise at least about 50%, such as at least about 70%, of the total cell content of the preparation. For example, the purity may be at least about 50%, or may exceed about 60%, about 70%, or about 80%, or may be about 100%.

[0345] In each of the descriptions of Section D described above, in addition to the surface antigen-regulated promoter-therapeutic payload construct nucleotides, expression, vectors, and host cells based on the CARs described above, the surface antigen-regulated promoter-therapeutic payload construct may further include various other therapeutic modalities including one or more of 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 cytokines, chemokines including CCR4, CCR6, CXCR5, transport receptors such as cytokine receptors including but not limited to CCR4, CCR7, CCR2, bispecific antibodies including but not limited to bispecific T cell engager antibodies (BiTE) such as anti-CD3 and anti-CD19 targeting or other multi-targeting antibodies, neutralizing / blocking antibodies against, for example, PD-L1, IL-6R, IL-1R (not limited to these), T cell stimulatory receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors such as the extracellular domain of PD-1 fused to the endodomain of CD28 (not limited to this), anti-apoptosis proteins such as BCL-2 or BCL-XL (not limited to these), shRNA, proteases, a second CAR T construct, or any combination thereof (each of which has biological properties as described above).

[0346] G. Treatment Methods It is contemplated that the surface antigen-regulated promoter-therapeutic payload constructs (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-apoptosis proteins, shRNA, proteases) disclosed herein may be used in methods for treating or preventing diseases in mammals. In this regard , one embodiment provides a method for treating or preventing cancer in a mammal, which comprises administering to the mammal 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, and / or a pharmaceutical composition in an amount effective for treating or preventing cancer in the mammal.

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

[0348] For the purposes of the method in which the host cell or population of cells is administered, the cell may be an allogeneic cell from the mammal or an autologous cell thereof. Preferably, the cell may be an autologous cell of the mammal. As used herein, "allogeneic" means any material derived from an animal that is of the same species as, but a different individual from, the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other if the genes at one or more loci are not identical. In some embodiments, allogeneic materials from individuals of the same species may be genetically different enough to interact antigenically with each other. As used herein, "autologous" means any material derived from the same individual as the individual into which the material will later be reintroduced.

[0349] As used herein, the mammal 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, including the families Felidae (cats) and Canidae (dogs). The mammal may be of the order Artiodactyla, including the subfamily Bovinae (cattle) and swine (pigs), or of the order Perissodactyla, including the family Equidae (horses). The mammal may be of the order Primates, Ceboids, or Simoids (monkeys), or of the family Hominidae (humans and apes). Preferably, the mammal is a human.

[0350] With respect to the methods described above, the cancer may be any cancer, including but not limited to acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., urothelial carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nasal, nasal cavity, or middle ear cancer, oral cavity cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mastocytoma, melanoma, multiple myeloma, oropharyngeal cancer, non-Hodgkin lymphoma, B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omentum, and mesentery cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer, and ureteral cancer.

[0351] As used herein, the terms "treatment" and "prevention" and their derivatives do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that would be recognized as beneficial or having a therapeutic effect by one of ordinary skill in the art. 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 method may include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented (e.g., cancer). Also, for the purposes described herein, "prevention" may include delaying the onset of a disease or its symptoms or conditions.

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

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

[0355] Regarding one embodiment of a method for detecting the presence of a proliferative disorder, such as cancer, in a mammal, a sample containing mammalian cells may be a sample containing whole cells, their lysates, or whole cell lysate fractions, such as nuclear or cytoplasmic fractions, total protein fractions, or nucleic acid fractions. When the sample contains whole cells, these cells may be any cells of a mammal, such as cells of any organ or tissue (including blood cells or endothelial cells).

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

[0357] Also, the detection of the complex may be performed by any of a plurality of methods well known in the art. For example, the CARs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or antibodies, or antigen-binding portions thereof, disclosed herein may be labeled with detectable labels such as, for example, radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles) as disclosed above.

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

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

[0360] Any method of administration, including local and systemic administration, may be used for the disclosed therapeutic agents. For example, topical, oral, intravascular such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administrations may be used. The specific mode of administration and dosing regimen may be selected by the attending clinician taking into account the details of the case (e.g., the subject, the disease, the associated disease state, and whether the treatment is prophylactic). If more than one agent or composition is administered, more than one route of administration may be used, e.g., a chemotherapeutic agent may be administered orally and an antibody or antigen-binding fragment or conjugate or composition may be administered intravenously. The method of administration includes 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, a non-volatile 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 an area suspected of having a tendency to develop tumors. In some embodiments, sustained intratumoral (or peritumoral) 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 as an eye drop to the cornea or into the vitreous humor of the eye.

[0361] The disclosed therapeutic agent may be formulated in unit dosage forms suitable for administering the exact dosage each time. In addition, the disclosed therapeutic agent may be administered according to a schedule of single or multiple administrations. The multiple - administration schedule may be such that in the first series of treatments, more than one administration (e.g., 1 to 10 administrations) is carried out separately each time, and subsequently, if necessary, the remaining administrations may be carried out at time intervals to maintain or increase the action of the composition. The treatment may involve administering the compound once or multiple times a day (multi - daily doses) over a period of 2 to 3 days to several months, or even several years. Thus, the dosing regimen may be determined based at least in part on the specific requirements of the subject to be treated and may depend 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, such as from about 0.1 to about 10 mg / kg.

[0363] In a specific example, the subject is administered a therapeutic composition comprising one or more of a conjugate, an antibody, a composition, a CAR, a CAR T - cell, or a further agent according to a multiple - daily dosing schedule, such as for at least 2 consecutive days and up to 10 consecutive days, over a period of, for example, several weeks, several months, or several years. In one example, the subject is administered a conjugate, an antibody, a composition, or a further agent over a period of at least 30 days, such as over 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 to a subject surgery, radiation therapy, and / or chemotherapy, in combination with (e.g., sequentially, substantially simultaneously, or simultaneously) the disclosed antibodies, antigen-binding fragments, conjugates, CARs, or T cells expressing a CAR. 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. Preparations and dosing schedules for additional agents may be used according to the manufacturer's instructions or based on the judgment of a skilled physician's experience. Also, such chemotherapy preparations and dosing schedules are described in Chemotherapy Service, (1992) Ed., M.C. Perry, Williams & Wilkins, Baltimore, Md as described therein.

[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 cross-linkers, 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-angiogenic agent may be administered in combination with a CAR, CAR-T cell, antibody, antigen-binding fragment, or conjugate disclosed herein. Methods and therapeutic dosages for such agents are well known to those of skill in the art and may be determined by a skilled clinician.

[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 folic acid (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 podophyllum (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca (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 including, but not limited to, 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, vemurafinib, vandetanib, and tretinoin. The selection and therapeutic dosage of such agents are well known to those skilled in the art and may be determined by a skilled clinician.;

[0367] Combination therapies can be synergistic and can be demonstrated to be synergistic, i.e., the effect achieved when multiple active ingredients are used together is greater than the sum of the effects obtained when the same compounds are used separately. Synergy can occur when multiple active ingredients are (1) formulated together and administered or delivered simultaneously as a combined unit dosage preparation, (2) delivered as separate preparations, alternately or in parallel, or (3) some other regimen is used. When delivered alternately, for example, by separate injection with separate syringes, synergy can occur when the compounds are administered or delivered sequentially. Generally, in the alternate case, the effective dosage of each active ingredient is administered sequentially, i.e., continuously, while in combination therapy, the 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 of the antigens disclosed herein is administered to a subject having a tumor after anti-cancer treatment. After sufficient time has elapsed for the administered antibody or antigen-binding fragment or conjugate to form an immune complex with the antigen expressed on the respective cancer cells, the immune complex is detected. The presence (or absence) of the immune complex indicates the effectiveness of the treatment. For example, if the immune complex is increased compared to a control obtained prior to the treatment, it is shown that the treatment is ineffective, and if the immune complex is decreased compared to a control obtained prior to the treatment, it is shown that the treatment is effective.

[0369] In each of the above-described descriptions of Section E, in addition to the treatment method using the surface antigen-regulated promoter-therapeutic payload construct based on the above-described CAR, the surface antigen-regulated promoter-therapeutic payload construct includes cytokines such as 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, chemokines such as CCR4, CCR6, CXCR5, transport receptors such as cytokine receptors including, but not limited to, CCR4, CCR7, CCR2, bispecific antibodies including, but not limited to, bispecific T cell engager antibodies (BiTE) such as anti-CD3 and anti-CD19 targeting or other multi-targeting antibodies, neutralizing / blocking antibodies against, for example, PD-L1, IL-6R, IL-1R (not limited to these), expressed as scFv or IgG or in other configurations, T cell stimulatory receptors, truncated inhibitory receptors, for example, hybrid inhibitory / activating receptors including, but not limited to, the extracellular domain of PD-1 fused to the endodomain of CD28, anti-apoptosis proteins including, but not limited to, BCL-2 or BCL-XL, shRNA, proteases, a second CAR T construct, or various other therapeutic modalities including one or more of any combination thereof (each of these has biological properties as described above).

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

[0371] This composition for administration may comprise a solution in which a CAR, or a T cell expressing a CAR, conjugate, antibody, or antigen-binding fragment is 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 substances. 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 modifiers, and adjuvants, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, as necessary to approximate physiological conditions. The concentration of the CAR, or the T cell expressing a CAR, antibody, or antigen-binding fragment, or conjugate in the preparation may vary widely and may be selected according to the particular mode of administration and requirements of the subject, mainly based on factors such as the volume of the fluid, viscosity, and body weight. The actual method of preparing such dosage forms for use in gene therapy, immunotherapy, and / or cell therapy is well-known or will be apparent to those skilled in the art.

[0372] A typical composition for intravenous administration comprises from about 0.01 to about 30 mg / kg per day per subject of an antibody or antigen-binding fragment or conjugate (or the corresponding dosage of a CAR, or a T cell expressing a CAR, antibody or antigen-binding fragment-containing conjugate). The actual method of preparing the composition for administration may be well-known or apparent to those skilled in the art and is described in more detail in publications such as Remington’s Pharmaceutical Science, 19th Edition, Mack Publishing Company, Easton, PA (1995).

[0373] A CAR, or a T cell expressing a CAR, an antibody, an antigen-binding fragment, or a conjugate may be provided in lyophilized form and reconstituted with sterile water for administration, but may also be provided in solution dissolved in a sterile solution of a known concentration. The solution of the CAR, or a T cell expressing a CAR, an antibody, or an antigen-binding fragment, or a conjugate is then filled into an infusion bag containing 0.9% sodium chloride (USP) and, optionally, administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience can be found in the art regarding the administration of drugs of antibodies or antigen-binding fragments and conjugates. For example, antibody drugs have been on the US market since the approval of Rituxan® in 1997. The CAR, or a T cell expressing a CAR, an antibody, its antigen-binding fragment, and a conjugate may be administered by slow infusion rather than by intravenous push or bolus. In one example, a higher loading dose is administered, followed by a maintenance dose administered at a lower level. For example, an antibody or antigen-binding fragment is infused at an initial loading dose of 4 mg / kg (or the corresponding dose of a conjugate containing the antibody or antigen-binding fragment) over about 90 minutes, and if this initial dose is well tolerated, subsequently, a maintenance dose of 2 mg / kg once a week is infused over 30 minutes each for 4 to 8 weeks.

[0374] Controlled-release parenteral preparations may be prepared as implants, oily injections, or particulate systems. A comprehensive overview of protein delivery systems can be found in Banga, A.J., 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 therapeutic proteins, such as cytotoxins or drugs, as a central core. In microspheres, the therapeutic agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Since 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. For example, see Kreuter, J., Colloidal Drug De livery 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] The polymer may be used for ion-controlled release of the CARs, or T cells expressing CARs, antibodies, or antigen-binding fragments, or conjugates 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 polaxamer 407 exists as a mobile liquid with viscosity at low temperatures but forms a semi-solid gel at body temperature. This 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 aspect, liposomes are used for the controlled release of lipid-encapsulated drugs and drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). In addition to these, a very large number of systems for the controlled delivery of therapeutic proteins are well known (U.S. Patent No. 5,055,303, U.S. Patent No. 5,188,837, U.S. Patent No. 4,235,871, U.S. Patent No. 4,501,728, U.S. Patent No. 4,837,028, U.S. Patent No. 4,957,735, U.S. Patent No. 5,019,369, U.S. Patent No. 5,055,303, U.S. Patent No. 5,514,670, U.S. Patent No. 5,413,797, U.S. Patent No. 5,268,164, U.S. Patent No. 5,004,697, U.S. Patent No. 4,902,505, U.S. Patent No. 5,506,206, U.S. Patent No. 5,271,961, U.S. Patent No. 5,254,342, and U.S. Patent No. 5,534,496).

[0376] In each of the descriptions of Section F described above, in addition to the surface antigen-regulated promoter-therapeutic payload construct composition based on the CAR described above, the surface antigen-regulated promoter-therapeutic payload construct may further include various other therapeutic modalities including one or more of 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, chemokines including CCR4, CCR6, CXCR5, transport receptors such as cytokine receptors including, but not limited to, CCR4, CCR7, CCR2, bispecific antibodies including bispecific T cell engager antibodies (BiTE) including, but not limited to, anti-CD3 and anti-CD19 targeting or other multi-targeting antibodies, neutralizing / blocking antibodies against, for example, PD-L1, IL-6R, IL-1R (not limited to these), expressed as scFv or IgG or in other configurations, T cell stimulatory receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors including, but not limited to, the extracellular domain of PD-1 fused to the endodomain of CD28, anti-apoptosis proteins including, but not limited to, BCL-2 or BCL-XL, shRNA, proteases, a second CAR T construct, or any combination thereof (each of these having biological properties as described above).

[0377] I. Kit In one aspect, there is further provided a kit that uses a surface antigen-regulated promoter-therapeutic payload construct (e.g., based on CAR, cytokine, chemokine, transporter receptor, bispecific antibody, neutralizing / blocking antibody, T cell-stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptosis protein, shRNA, protease). For example, a kit for treating a tumor in a subject or for generating CAR T cells that express one or more of the CARs disclosed herein. Such kits may typically include an antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cell that expresses a CAR, as disclosed herein. More than one of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells that express a CAR may be included in the kit.

[0378] The kit may include a container and a label or package insert attached to 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 contains a composition comprising one or more of the disclosed antibodies, antigen-binding fragments, conjugates, nucleic acid molecules, CARs, or T cells that express a CAR. 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 to be used for treating a particular condition.

[0379] The label or package insert may typically further include, for example, a description of the use of the disclosed antibody, antigen-binding fragment, conjugate, nucleic acid molecule, CAR, or T cell expressing a CAR in a method of treating or preventing a tumor, or a method of making CAR T cells. The package insert typically includes the instructions customarily included in the commercial package of a therapeutic product, which instructions include information regarding the indications, usage, dosage, administration, contraindications, and / or warnings associated with the use of the therapeutic product. The content of the instructions may be described in electronic form (such as a floppy disk or compact disk) or visual form (such as a video file). The kit may further include additional components for facilitating a particular use for which the kit is designed. Thus, for example, the kit may further include means for detecting a label (such as an enzyme substrate for an enzyme label, a filter set for detecting a fluorescent label, or a suitable 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 ordinary skill in the art.

[0380] In each of the descriptions of the G segment described above, in addition to the surface antigen-controlled promoter-therapeutic payload construct based on the CAR described above, the surface antigen-controlled promoter-therapeutic payload construct includes cytokines such as 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, chemokines such as CCR4, CCR6, CXCR5, transport receptors such as cytokine receptors including but not limited to CCR4, CCR7, CCR2, bispecific antibodies including but not limited to bispecific T cell engager antibodies (BiTE) such as anti-CD3 and anti-CD19 targeting or other multi-targeting antibodies, neutralizing / blocking antibodies against, for example, PD-L1, IL-6R, IL-1R (not limited to these) expressed as scFv or IgG or other constructs, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors including but not limited to the extracellular domain of PD-1 fused to the endodomain of CD28, anti-apoptosis proteins including but not limited to BCL-2 or BCL-XL, shRNA, proteases, a second CAR T construct, or various other therapeutic modalities including one or more of any combination thereof (each of these having 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 present invention. On the contrary, reliance must be placed on various other embodiments, modifications, and equivalents, and it is clearly understood 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 and / or scope of the appended claims.

[0382] Example 1 Generation of a self-driven CAR construct targeting the CD19 antigen Despite the clinical success of cancer treatments based on anti-CD19 (such as CAR LTG1563), suboptimal CAR activation and persistence, on the one hand, and excessive CAR activation and related toxicities (CRS, neurotoxicity), on the other hand, have been problems. To improve the safety and efficacy of CAR T therapy, a self-driven CAR was generated.

[0383] This example describes CAR T cells that use CAR signaling to control their own expression and the expression of co-introduced genes that are co-regulated by two mechanisms, and the use of these mechanisms outside of CAR expression.

[0384] 1. Positive regulation of CAR T expression by a STAT5 response element (STAT5_RE) driven by a cytokine (such as IL-2 / IL-15 / IL-7), inducing the expression of the CAR molecule (such as anti-CD19 CAR LTG1563). When CAR T detects a tumor antigen, a slow positive feedback loop is activated, resulting in an increase in CAR expression on the CAR T cell. When the tumor is removed, this positive feedback loop gradually subsides and stops, and CAR surface expression naturally decreases and returns to a monitoring 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 elements (AP1 / NFκB_RE) and that driven by CAR / TCR, and the expression of CAR molecules (e.g., CAR LTG1563) is induced. When CAR T detects a tumor antigen, a rapid positive feedback loop is activated, which increases CAR expression on CAR-T cells. Over time, compared with constitutively expressed CAR, the CD19-dependent killing activity of AP1 / NFκB-driven CAR LTG1563 becomes greater, cytokine secretion increases, exhaustion is reduced, and CAR T cells are generally in a better state. When the tumor is removed, this positive feedback loop gradually subsides, stops, and CAR surface expression naturally decreases and returns to a monitoring state (Figure 1).

[0386] 3. Expression of additional proteins that regulate CAR T function in the presence of CAR antigen (including, but not limited to, avoidance of negative T cell control (dominant negative TGFBRII receptor, anti-PD1 antibody, etc.), positive regulators of T cell proliferation (IL15, IL12, etc.), T cell homing to tumors (chemokine receptors), and promotion of 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 e.g., the inducible CAR is expressed as a result of activation of the constitutive CAR. The second CAR can target a second tumor antigen, or an antigen expressed on myeloid-derived suppressor cells (MDSC) (e.g., CD33, mesothelin), or an antigen expressed on inhibitory B cells (e.g., CD19). In any case, these inducible proteins can be expressed in the presence of CAR stimulation and can be downregulated when CAR signaling stops.

[0387] 4. STAT5_RE can regulate the self-expression of either the CAR protein or the T cell function regulatory protein in a defined manner using the AP1 / NFκB_RE promoter. Taking advantage of the greater cytokine (IL2)-dependent response of STAT5_RE (Figure 3), a protein that does not respond directly to an antigen can be expressed in a paracrine manner near the tumor in CAR-T cells. Alternatively, in particular in CAR T cells, a protein can be expressed in response to a tumor via AP1 / NFκB_RE (CAR signaling is required to maximize the response via this element) (Figure 4).

[0388] Materials and methods: Preparation of chimeric antigen receptor (CAR) expression vectors To generate a novel transmembrane domain of CAR LTG1563, a single-chain variable fragment (scFv) derived from FMC-63 mouse hybridoma (FMC-63: AA 1-267, GenBank ID: HM852952.1), linked by a (GGGGS)3 flexible linker (SEQ ID NO: 187) in the VL to VH orientation, was used. Subsequently, the resulting targeting domain was ligated in-frame to a human CD8 hinge (AA 138-179, RefSeq ID: NP_001759.3), a human TNF receptor superfamily 19 transmembrane domain (TNFRSF19, AA 167-196, Uniprot ID: Q9NS68), a human 4-1BB co-stimulatory domain (CD137, AA 214-255, Uniprot ID: Q07011), and a human CD3 zeta signaling domain (CD247, AA 52-163, RefSeq ID: NP_000725.1.). To facilitate 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 the surface antigen-regulated inducible promoter described below, or the constitutive promoter MSCV or EF1α as a control.

[0389] Cell lines used to demonstrate CAR activity Unless otherwise specified, the Burkitt lymphoma cell line Raji cell line, the chronic myelogenous leukemia cell line K562 cell line, and the reagents were purchased from the American Tissue Culture Collection (ATCC, Manassass, VA). The 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 cell line 293T was purchased from ATCC and grown in CD FortiCho medium (Gibco / Thermo Fisher Scientific, Grand Island, NY). A lentiviral vector encoding firefly luciferase (Lentigen Technology, Gaithersburg, MD) was used to stably transduce wild-type tumor cell lines, and then single-cell clones of luciferase-expressing cell lines were generated by cloning and selecting luciferase-positive clones.

[0390] Primary human T cells used to demonstrate CAR activity At the Oklahoma Blood Institute (OBI), whole blood was collected from healthy volunteers with written consent from the donors. Processed buffy coats were purchased from OBI (Oklahoma City, OK). From the buffy coats, CD4 ma CD4-positive and CD8-positive human T cells were purified by positive selection using a 1:1 mixture of CD4 microbeads 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 6Cultured at [[ID=]], activated on day 0 using CD3 / CD28 MACS® GMP T Cell TransAct reagent (all reagents were obtained from Miltenyi Biotec), transduced overnight on day 1 / 2 using LV encoding the CAR construct, and the medium was changed on day 2 / 3. As described, cytokines (IL-2, TNFα; Miltenyi Biotec, Bergisch Gladbach, Germany) were added. After growing the cultures, they were harvested on days 5 - 6 and used for co-incubation analysis.

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

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

[0394] A positive regulatory loop of the CAR was designed by placing CAR LTG1563 expression under the control of a tandem STAT5 response element followed by a minimal promoter array. When IL-2 binds to the native IL-2 receptor on the T cell surface, Jak / STAT signaling is initiated, and STAT5 translocates to the regulatory region of the CAR promoter, increasing CAR expression. A CAR LTG1563 driven by the constitutively expressed EF1α promoter was constructed as a control and tested in parallel with the STAT-5- and AP1 / NFκB-controlled CARs. The positive regulation of the CAR by (1) IL-2-driven Stat5 and (2) the mechanism involving IL-2 / TNFα and CAR-driven AP1 / NFκB is illustrated in Figure 1, and the structure of each of these constructs is shown in Figure 2.

[0395] T cells were purified by immunogenic selection using a 1:1 mixture of CD4 beads and CD8 beads (Miltenyi Biotec) from the blood of two healthy donors not related to each other. Without adding cytokines, the CAR LTG1563 construct was transduced into the cells. The experimental groups contained a CD19 CAR driven by the EF1α promoter at an MOI of 10 or had the self-driven CARs STAT5_RE CAR LTG1563 and AP1 / NFκB_RE CAR LTG1563, which are under positive regulation, at 0.25% vol / (vol of the LV preparation) (Figure 3). Cells not transduced (UTD) cultured under the same conditions were used as a negative control. On day 5, IL-2 or TNFα was added to the selected groups, and 20 hours later, CAR expression was measured by flow cytometry. The present was detected. The expression of CAR driven by EF1α was 30 - 40% depending on the transduction MOI and did not change with the addition of IL-2 / TNFα. In contrast, the expression of STAT5_RE CAR increased significantly with the addition of IL-2 as expected but not with TNFα (Figure 3). The expression of AP1 / NFκB_RE CAR increased with both IL-2 and TNFα, but to a lesser extent compared to the expression of STAT5_RE CAR LTG1563 driven by IL-2.

[0396] Furthermore, in this experiment, the self-driving property of CAR LTG1563, which is possessed by both the STAT5 response element and the AP1 / NFκB response element, to drive the expression of CAR LTG1563 was examined by CD19 antigen stimulation (Figure 4). This was carried out using CAR T cells from donor A and donor B co-cultured with CD19-positive Raji NHL tumor cells that stably express GFP. Notably, within 20 hours after stimulation, CAR expression was strongly induced in CAR LTG1563 T cells driven by AP1 / NFκB_RE (Figure 4B), which indicates that when driving CAR expression using these response elements, both CAR signaling and CAR-dependent cytokine expression are incorporated. This stimulation was significantly greater than that which could be observed when simply stimulating these cells with cytokines, indicating that the response to CAR signaling is stronger and more direct. This rapid AP1 / NFκB-dependent upregulation of CAR is in contrast to the case of CAR LTG1563 driven by constitutively expressed EF1α, which transiently decreased upon stimulation of the CAR via the CD19 antigen (presumably due to internalization of these receptors). On the other hand, CAR LTG1563 driven by STAT5_RE was induced by co-culture with CD19+ Raji cells, but the time scale was significantly longer compared to that observed for CAR LTG1563 driven by AP1 / NFκB. This is presumably thought to reflect the intermediate steps necessary for the functionalization of the STAT5 response element, the production of IL2 by CAR T cells, and the accompanying STAT5 signaling. When IL2 was added to these cells at D3 - D5 after activation, the induction of CAR LTG1563 expression via the STAT5 element was significantly increased, presumably due to priming of STAT-mediated signaling.

[0397] In particular, when CD19+ Raji cells are stimulated multiple times (from D6 to D22 after activation), the expression of AP1-NFκB CAR LTG1563 can be re-induced, indicating that CAR T cells retain the ability to respond to AP1 / NFκB signaling during long-term ex vivo culture (data not shown). Importantly, there is a transient apparent correlation between the degree of CAR expression under the control of the AP1 / NFκB promoter and the number of Raji cells in co-culture (data not shown). These results indicate that the AP1 / NFκB element rapidly drives the expression of CAR LTG1563 in the presence of the cognate CD19 antigen, and that CAR LTG1563 is rapidly downregulated in the absence of the antigen.

[0398] In the next experiment, T cells were purified by immunogenic selection using a 1:1 mixture of CD4 beads and CD8 beads (Miltenyi Biotec) from the blood of three healthy donors not related to each other. At D1 after activation, without adding cytokines, the CAR LTG1563 construct was transduced into the cells as described in Materials and Methods. The experimental groups included CD19 CAR driven by a constitutive EF1α-derived CAR, and STAT5 RE CAR LTG1563 and AP1-NFκB CAR LTG1563 were transduced at an MOI of 10 using LV (Figure 3). In a subset of samples, IL2 (30 IU / mL) was added from D3 to D6 after activation to prime the expression of both STAT5 RE CAR LTG1563 and AP1 / NFκB CAR LTG1563. CAR LTG1563-dependent cytotoxicity was determined by co-culturing CAR T cells with CD19+ Raji NHL cells stably expressing GFP It was examined by using a very low effector target ratio (1:3 CAR T:Raji cells) and counting GFP+ Raji cells by flow cytometry at D6 - D13 (co - culture 1) after activation to examine CD19 - dependent cytotoxicity. The long - term function and proliferation of CAR T cells were examined by restimulating the cells at D13 - D20 (co - culture 2) after activation with the same effector target ratio (1:3 co - culture 1:Raji cells).

[0399] The co - incubation of CAR T and target cells was examined for a total of 14 days (co - culture 1 & 2), during which the number of viable Raji cells and T cells was counted by flow cytometry analysis (Figure 5). Raji cells in the Raji alone group and UTD control group continued to proliferate unhindered until day 8 of the experiment, while CAR LTG1563 T cells strongly suppressed Raji proliferation in all groups, regardless of IL2 priming. Notably, when measured at 1, 2, 3, 6, and 7 days after Raji addition, both STAT5 RE and AP1 / NFκB CAR LTG1563 showed better tumor killing than EF1α CAR LTG1563 (Figure 5B). Significantly, although the expression of CAR LTG1563 in IL2 culture was initially low, both STAT5RE and AP1 / NFκB were superior to the EF1α constitutive expression CAR LTG1563 construct (Figure 5B). This may indicate that constitutive expression of CAR in the absence of cognate antigen may have a negative impact on the efficacy of CAR T cells, perhaps for sustained signaling, even over a short period (D2 - D6 after activation).

[0400] By restimulating CAR T cells with CD19+ Raji cells, further long-term analysis of CAR LTG1563-dependent cytotoxicity was performed. All CAR groups again showed effective suppression of Raji proliferation (data not shown), and AP1 / NFκB-driven CAR LTG1563 showed superior tumor killing function compared to constitutively expressed EF1α_CAR LTG1563 (Figure 5B). T cells expressing AP1 / NFκB_CAR LTG1563 had a significantly greater degree of proliferation throughout the culture period than T cells expressing STAT5RE_CAR LTG1563 or EF1α_CAR LTG1563 (Figure 5C). This is presumably because CAR signaling exists only when target CD19+ cells are present together, thereby maintaining the overall health of these cells and concomitantly increasing T cell proliferation.

[0401] Overall, the results described above indicate the superiority of the inducible self-driven AP1 / NFκB RE CAR LTG1563, which is manifested by low unstimulated CAR expression, low sensitivity to exhaustion, low levels of priming by cytokine addition, 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 fact that upon antigen stimulation, these AP1 / NFκB CAR T cells proliferated more overall than all other constructs tested.

[0402] Example 2 Expansion to Auxiliary Proteins with Self-Driven Expression In response to the successful self-driven expression of high levels of CAR LTG1563 by the inducible AP1 / NFκB promoter, especially in the presence of the cognate CAR antigen (CD19), it was desired to expand the functionality of this promoter to auxiliary proteins to provide additional functionality to CAR-T cells. CAR antigen-inducible AP1 / NFκB promoter The advantage of is that any protein under the control of this promoter can be highly expressed only when the CAR tumor antigen is present. Thus, proteins that enable immune escape of CAR-T cells, such as dominant-negative TGF-beta receptor (TGFBRIIdn) and dominant-negative programmed cell death protein 1 (PD1dn), were expressed using the AP1 / NFκB promoter. These dominant-negative receptors were thought to enable immune escape avoidance of CAR-T cells by TGF-β or PD-L1, respectively. The expected advantage regarding the use of the AP1 / NFκB promoter is that TGF-β / PD-L1 immunosuppression plays an important role in preventing the occurrence of T cell-mediated autoimmunity in patients under normal conditions. However, th...

Claims

**Claim 1** 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 NO: 137, SEQ ID NO: 138, or a combination thereof, wherein said surface antigen-regulated inducible promoter regulates its own transcription level depending on the expression level of the surface antigen on the target cell, thereby achieving a precisely controlled T cell response according to the level of the target antigen present in the tumor environment. **Claim 2** The isolated nucleic acid molecule of claim 1, wherein the therapeutic payload comprises at least one extracellular antigen-binding domain, at least one transmembrane domain, and at least one intracellular signaling domain of a chimeric antigen receptor (CAR) comprising a mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38 antigen-binding domain, or a combination thereof. **Claim 3** The isolated nucleic acid molecule of claim 2, wherein the at least one antigen-binding domain encoded comprises at least one single-chain variable fragment of an antibody that binds to one or more antigens comprising mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38, or a combination thereof. **Claim 4** The isolated nucleic acid molecule of claim 2, wherein the at least one antigen-binding domain encoded comprises at least one heavy-chain variable region of an antibody that binds to one or more antigens comprising mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38, or a combination thereof. **Claim 5** The isolated nucleic acid molecule of claim 2, wherein the at least one antigen-binding domain encoded, the at least one intracellular signaling domain, or both are bound to the transmembrane domain by a linker or spacer domain. **Claim 6** The isolated nucleic acid molecule of claim 5, wherein the linker or spacer domain encoded is derived from the extracellular domain of CD8, TNFRSF19, or CD28 and is bound to the transmembrane domain. **Claim 7** The extracellular mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38 antigen-binding domain to be encoded is located behind the leader nucleotide sequence encoding the leader peptide, and the isolated nucleic acid molecule according to claim 2.

8. The leader nucleotide sequence includes a nucleotide sequence including SEQ ID NO: 13 encoding the leader amino acid sequence of SEQ ID NO: 14, or SEQ ID NO: 39 encoding the leader amino acid sequence of SEQ ID NO: 40, or SEQ ID NO: 41 encoding the leader amino acid sequence of SEQ ID NO: 42, or SEQ ID NO: 43 encoding the leader amino acid sequence of SEQ ID NO: 44, and the isolated nucleic acid molecule according to claim 7.

9. The transmembrane domain includes the transmembrane domain of a protein including the alpha, beta, or zeta chain of the T cell receptor, CD8, 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, and the isolated nucleic acid molecule according to claim 2.

10. The surface antigen-regulated inducible promoter includes a nucleotide sequence including SEQ ID NO: 137 or 138, or a sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the isolated nucleic acid molecule according to claim 2.

11. The at least one intracellular signaling domain to be encoded further includes the CD3 zeta intracellular domain, and the isolated nucleic acid molecule according to claim 2.

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

13. The at least one intracellular signaling domain to be encoded includes a costimulatory domain, a primary signaling domain, or any combination thereof, and the isolated nucleic acid molecule according to claim 2.

14. At least one of the costimulatory domains to be encoded 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, the isolated nucleic acid molecule according to claim 13.

15. A chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule according to claim 2.

16. The CAR according to claim 15, wherein the antigen-binding domain comprises at least one single-chain variable fragment of an antibody that binds to one or more antigens comprising mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38, or a combination thereof.

17. The CAR according to claim 15, wherein the antigen-binding domain comprises at least one heavy-chain variable region of an antibody that binds to one or more antigens comprising mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123, or CD38, or a combination thereof.

18. The CAR according to claim 15, wherein the transmembrane domain comprises the transmembrane domain of a protein comprising an alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and TNFRSF19, or any combination thereof.

19. The CAR according to claim 18, wherein the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO:

28.

20. The 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, and the at least one intracellular signaling domain, or both, are bound to the transmembrane domain by a linker or spacer domain, the CAR according to claim 15.

21. The CAR according to claim 20, wherein the linker or spacer domain is derived from the extracellular domain of CD8, TNFRSF19, IgG4, or CD28 and is bound to the transmembrane domain.

22. The CAR according to claim 17, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

23. The CAR according to claim 22, wherein the 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.

24. A vector comprising the nucleic acid molecule according to claim 2.

25. The vector according to claim 24, 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.

26. A cell comprising the vector according to claim 24.

27. The cell according to claim 26, wherein the cell is a T cell.

28. The T cells are CD8 ＋ T cells, and the cell according to claim 26.

29. The cell according to claim 26, wherein the cell is a human cell.

30. A method for producing a cell, comprising the step of transducing the vector according to claim 24 into a T cell.

31. A method for generating a population of RNA-engineered cells, comprising the step of introducing in vitro transcribed RNA or synthetic RNA into cells, wherein the RNA comprises the nucleic acid molecule according to claim 2.

32. A method for providing anti-tumor immunity to a mammal, comprising the step of administering to the mammal an effective amount of the cell according to claim 26.

33. A method for treating or preventing cancer in a mammal, comprising the step of administering to the mammal the CAR according to claim 15 in an amount effective for treating or preventing cancer in the mammal.

34. A pharmaceutical composition comprising a population of human T cells in an anti-tumor effective amount, wherein said T cells are operably linked to a surface antigen-regulated inducible promoter encoded by a nucleic acid sequence comprising SEQ ID NO: 137, 138, or a combination thereof, and contain a therapeutic payload, said surface antigen-regulated inducible promoter adjusting its own transcription level depending on the expression level of the surface antigen on the target cell, thereby achieving a precisely controlled T cell response according to the level of the target antigen present in the tumor environment, said therapeutic payload comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), said CAR comprising 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 linker domain, at least one transmembrane domain, at least one intracellular signaling domain, and wherein said T cells are human T cells having cancer. A pharmaceutical composition according to claim 34, wherein said at least one transmembrane domain comprises a transmembrane domain of a protein comprising an alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof. Claim 35 The pharmaceutical composition according to claim 34, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein comprising an alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof. Claim 36 The pharmaceutical composition according to claim 34, wherein the T cells are human T cells having a blood cancer. Claim 37 The pharmaceutical composition according to claim 36, wherein the blood cancer is leukemia or lymphoma. Claim 38 The pharmaceutical composition according to claim 37, 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). Claim 39 The pharmaceutical composition according to claim 37, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin lymphoma, or Hodgkin lymphoma. Claim 40 The pharmaceutical composition according to claim 36, wherein the blood cancer is multiple myeloma. Claim 41 The human cancers include adult cancers such as oral and pharyngeal cancers (tongue, mouth, pharynx, head and neck), digestive system cancers (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung, and bronchus), bone and joint cancers, soft tissue cancers, skin cancers (melanoma, basal cell carcinoma, and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing sarcoma), central nervous system tumors (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, genital system (cervix, uterine body, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), brain and other nervous systems, or any combination thereof, the pharmaceutical composition according to claim 36.

42. A method for treating a mammal having a disease, disorder, or condition associated with increased expression of a tumor antigen, the method comprising administering to the subject a pharmaceutical composition comprising a population of T cells in an anti-tumor effective amount, the T cells comprising an isolated nucleic acid molecule encoding a therapeutic payload operably linked to a surface antigen-regulated inducible promoter nucleotide sequence comprising SEQ ID NO: 137, 138, or a combination thereof, the surface antigen-regulated inducible promoter adjusting its own transcription level depending on the expression level of the surface antigen on the target cell, thereby achieving a precisely controlled T cell response according to the level of the target antigen present in the tumor environment.

43. The therapeutic payload 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, a chimeric antigen receptor (CAR), the method according to claim 42.

44. A method of treating a mammal having a disease, disorder, or condition associated with increased expression of a tumor antigen, said method comprising administering to the subject a pharmaceutical composition comprising a population of T cells in an anti-tumor effective amount, said T cells comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), said CAR comprising at least one extracellular antigen-binding domain comprising an antigen-binding domain comprising a 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 TCR, or an antigen-binding domain comprising 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: 137, 138, or a combination thereof, said surface antigen-regulated inducible promoter regulating its own transcription level depending on the expression level of the surface antigen on the target cell, thereby achieving a precisely controlled T cell response according to the level of the target antigen present in the tumor environment, method.

45. A method of treating cancer in a subject in need thereof, said method comprising administering to the subject a pharmaceutical composition comprising a population of T cells in an anti-tumor effective amount, said T cells comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), said CAR comprising at least one extracellular antigen-binding domain comprising 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: 137, 138, or a combination thereof, said CAR comprising the amino acid sequence of SEQ ID NO: 78, said T cells being the T cells of said subject having cancer, method.

46. The method according to claim 42, 43, 44, or 45, wherein the at least one transmembrane domain comprises the transmembrane domain of a protein comprising the alpha, beta, or zeta chain of a T cell receptor, CD8, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154, or any combination thereof.

47. A process for producing chimeric antigen receptor-expressing cells, the process comprising the step of introducing the isolated nucleic acid according to claim 2 into a cell.

48. The process for producing chimeric antigen receptor-expressing cells according to claim 47, wherein the cell is a T cell or a cell population containing T cells.

49. A therapeutic CAR T cell comprising an isolated nucleic acid molecule encoding a CAR operably linked to a surface antigen-regulated inducible promoter comprising the nucleotide sequence of SEQ ID NO: 137, 138, or a combination thereof, wherein the surface antigen-regulated inducible promoter CAR construct enables the transduced CAR T cells to develop an anti-tumor response depending on the expression level of the corresponding surface antigen in the target cells, thereby achieving a CAR T cell response that is precisely controlled according to the level of the target surface antigen present in the tumor environment, where: i) in the absence of expression of the tumor target surface antigen, the surface antigen-regulated inducible promoter gives a low basal level of CAR expression; ii) when an activating target surface antigen is present on the surface of the target cells, the CAR is activated, thereby enabling appropriate signal transduction pathways iii) The activation of the pathway triggers the activation of the signal mediators of the surface antigen-regulated inducible promoter, thereby increasing the expression of the CAR beyond the basal level of expression. iv) As the expression of a given target surface antigen increases, the expression of the CAR also increases, and vice versa, so that the CAR expression is efficiently regulated and an accurate CAR T cell response adapted to the level of the target present at a specific site and time point is achieved. v) The increased CAR expression leads to optimal anti-tumor activity and rapid elimination of the target tumor cells. And vi) As the tumor cells are quantitatively reduced / eliminated, the level of CAR expression returns to its basal level of expression, or any combination thereof, a therapeutic CAR T cell.

50. The therapeutic payload according to claim 49, comprising one or more 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, MAGE A3 TCR, or any combination thereof.

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