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

Abstract

The invention relates to compositions and methods for treating cancer with a self-driven chimeric antigen receptor. A self-driven surface antigen modulated promoter-therapeutic loading construct comprising an antigen binding domain is disclosed. Nucleic acids, recombinant expression vectors, host cells, antigen binding fragments and pharmaceutical compositions associated with the promoter-therapeutic load constructs modulated by surface antigens are also disclosed. Also disclosed are methods of treating or preventing cancer in a subject, as well as methods of preparing self-driven surface antigen modulated promoter-therapeutic load constructs in T cells.

Description

[0001] This application is a divisional application of a Chinese patent application with an application date of March 6, 2020, application number 202080034145.7, and invention name “Compositions and methods for treating cancer with self-driven chimeric antigen receptors”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This PCT patent application claims priority to 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, the entire contents of each of which are incorporated herein by reference. Technical Field

[0004] The present invention relates to the field of cancer, and in particular to inducible promoters linked to therapeutic payloads and methods of using the same. Background Art

[0005] Cancer is one of the most deadly threats to human health. In the United States alone, cancer affects nearly 1.3 million new patients each year and is the second leading cause of death after cardiovascular disease, accounting for about a quarter of deaths. Solid tumors are responsible for most of these deaths. Despite significant advances in the medical treatment of some cancers, the overall 5-year survival rate for all cancers has only increased by about 10% in the past 20 years. Cancer or malignant tumors metastasize and grow rapidly in an uncontrolled manner, which makes treatment extremely difficult.

[0006] Multiple myeloma ("multiple myeloma, MM") is a debilitating and often incurable disease with more than 30,000 new cases diagnosed in the United States each year (Source: MM Research Foundation). In the United States, MM is the second most common blood cancer after non-Hodgkin's lymphoma ("Non-Hodgkin's Lymphoma, NHL") (Smith L, McCourt O, Henrich M et al., Multiple myeloma and physical activity: a scoping review. BMJ Open. 2015; 5: e009576). MM affects plasma cells in the bone marrow and can lead to bone marrow failure and patient death (National Cancer Institute. Asnapshot of myeloma. November 5, 2014. See cancer.gov on the World Wide Web). Complications of myeloma include bone pain, bone loss, anemia, immunosuppression, renal dysfunction, and neuropathy (Mayo Clinic staff. Diseases and conditions: multiple myeloma: treatments and drugs. December 4, 2015).

[0007] First-line treatment for MM includes proteasome inhibitors, immunomodulatory drugs, steroids, histone deacetylase (HDAC) inhibitors and chemotherapy. These approaches aim to kill MM cells, however, many of them are also associated with extensive immunosuppression and systemic toxicity.

[0008] Chimeric antigen receptor T-cell (CAR T) technology has brought great progress to the treatment of malignant hematological diseases (like MM) and provides broad prospects for the treatment of solid tumors. However, two shortcomings of this technology that have not yet been solved are: on the one hand, CAR T is not effective enough and the response is poor, and on the other hand, harmful side effects such as cytokine release syndrome are caused by excessive CAR T activation. The purpose of the present invention is to solve these two problems by generating CAR T parts and other therapeutic payloads that can be fine-tuned naturally based on the patient's tumor load and inflammatory environment at any given time during the treatment process and in any local microenvironment.

[0009] Chimeric antigen receptor (CAR) is a hybrid molecule comprising three basic units: (1) extracellular antigen binding motif, (2) junction / transmembrane motif, and (3) 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 CAR is usually formed after the single chain variable fragment (ScFv) of the minimal binding domain of the immunoglobulin (Ig) molecule. Alternative antigen binding motifs have also been modified, such as receptor ligands (e.g., IL-13 has been modified to bind to IL-13 receptors expressed by tumors), complete immune receptors, library-derived peptides, and innate immune system effector molecules (e.g., NKG2D). Alternative cell targets for CAR expression (e.g., NK or γ-δ T cells) 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). There is still important work to be done to define the most active T cell populations for transduction with CAR vectors, determine the optimal culture and expansion techniques, and define the molecular details of the CAR protein structure itself.

[0010] The connection motif of CAR can be a relatively stable structural domain, such as the constant domain of IgG, or a flexible linker designed to be extended. Structural motifs (such as those derived from IgG constant domains) can be used to extend the ScFv binding domain away from the T cell plasma membrane surface. This can be important for some tumor targets (such as disialoganglioside GD2; Orentas et al., unpublished observations) in which the binding domain is particularly close to the surface membrane of tumor cells. So far, the signaling motif used in CAR always includes the CD3-ζ chain because the core motif is a key signal for T cell activation. The second-generation CAR reported for the first time is characterized by the CD28 signaling domain and the CD28 transmembrane sequence. This motif is also used in the third-generation CAR containing the CD137 (4-1BB) signaling motif (Zhao Y et al., J Immunol. 2009; 183 (9): 5563–74). With the advent of new technologies that utilize beads connected to anti-CD3 and anti-CD28 antibodies to activate T cells, the presence of the classic "signal 2" from CD28 no longer needs to be encoded by the CAR itself. Using beads for activation, it was found that the third-generation vectors were not superior to the second-generation vectors in in vitro assays, and they did not provide a clear benefit over the second-generation vectors in mouse models of leukemia (Haso W, Lee DW, Shah NN, Stetler-Stevenson M, Yuan CM, Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL, Orentas RJ. Anti-CD22-chimeric antigen receptors targeting B cell precursor acute lymphoblastic leukemia. Blood. 2013; 121 (7): 1165–74; Kochenderfer JN et al., Blood. 2012; 119 (12): 2709–20). This is confirmed by the clinical success of CD19-specific CARs in the second-generation CD28 / CD3-ζ (Lee DW et al., American Society of Hematology Annual Meeting. New Orleans, LA; December, 7-10, 2013) and CD137 / CD3-ζ signaling formats (Porter DL et al., N Engl J Med. 2011; 365(8):725–33).In addition to CD137, other tumor necrosis factor receptor superfamily members (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). The culture conditions for culturing CAR T cell populations are also important.

[0011] T cell-based immunotherapy has become a new frontier in synthetic biology; a variety of promoters and gene products are being considered to direct these highly potent cells to the tumor microenvironment, where they can both escape negative regulatory signals and mediate effective tumor killing. Elimination of unwanted T cells by drug-induced dimerization of an inducible caspase 9 construct with AP1903 illustrates a way in which a powerful switch that can control T cell populations can be pharmacologically activated (Di Stasi A et al., N Engl J Med. 2011; 365(18): 1673–83). Generation of effector T cell populations immune to the negative regulatory effects of transforming growth factor-β by expression of a dominant negative receptor further illustrates the extent to which effector T cells can be engineered to achieve optimal antitumor activity (Foster AE et al., J Immunother. 2008; 31(5): 500–5). Thus, although CARs appear to be able to trigger T cell activation in a manner similar to endogenous T cell receptors, the major obstacle to the clinical application of this technology to date has been the limitation of CARs to the extent that they can be used to target T cells. + The in vivo expansion of T cells, the rapid disappearance of cells after infusion, and the disappointing clinical activity.

[0012] So far, the CAR treatment methods of the prior art use CAR T constructs under the control of the following constitutive promoters: for example, human elongation factor 1alpha (elongation factor 1alpha, EF1α), phosphoglycerate kinase (phosphoglycerate kinase, PGK), murine leukemia virus (murine leukemia virus, MuLV), murine stem cell virus (murine stem cell virus, MSCV) or other constitutive promoters known in the art, which are usually expressed at high levels or artificially induced (by small molecules or soluble components). These methods are not suitable for the dynamics of antigen expression, which can lead to excessive or insufficient effector cell responses at the cellular level, and insufficient or excessive treatment and toxicity to patients at the organism level.

[0013] Therefore, there is an urgent and long-felt need in the art to discover new compositions and methods for treating MM and CLL using approaches that can exhibit specific and effective anti-tumor effects without the above-mentioned disadvantages (e.g., high toxicity, insufficient efficacy).

[0014] The present invention addresses these needs by providing compositions and methods of treatment that can be used to treat cancer and other diseases and / or conditions. In particular, as disclosed and described herein, the present invention provides inducible promoter-therapeutic payload constructs that can be used to treat diseases, disorders or conditions associated with dysregulated expression of multiple antigens, such as, but not limited to, mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123 or CD38, or a combination thereof, and the inducible promoter-therapeutic payload constructs comprise antigen-specific binding domains that exhibit high surface expression on transduced T cells and exhibit a high degree of cell lysis and amplification and persistence of transduced T cells in vivo. In addition, as disclosed and described herein, the present invention provides self-driving regulation of such therapeutic payloads by utilizing an inducible promoter regulated by a surface antigen, wherein the promoter regulates the expression level of one or more therapeutic payloads according to the expression level of the surface antigen on the target cell. Summary of the invention

[0015] As disclosed and described herein, the present invention is based on the unexpected discovery that modulation or regulation of expression of a surface antigen-regulated promoter of a therapeutic cargo construct can be directly correlated with the activity of the therapeutic cargo and, therefore, with the expression level of the surface antigen in the target cell environment.

[0016] Provided herein are novel self-driven inducible promoter-loaded therapeutic constructs comprising a therapeutic load operably linked to an inducible promoter regulated by a surface antigen, which regulates the expression level of one or more therapeutic loads depending on the expression level of the surface antigen on a target cell.

[0017] Without being limited to any particular mechanism of action, the "self-driven" inducible promoter regulated by surface antigen as used herein refers to the use of an inducible promoter regulated by surface antigen to drive a therapeutic load to provide a low basal level of surface expression of a therapeutic load in the absence of tumor target antigen expression. In the presence of target antigen activation on the surface of target cells, the therapeutic load is activated, which triggers the activation of applicable signal transduction pathways, thereby activating the signal regulator of the inducible promoter regulated by surface antigen, thereby causing the expression of the therapeutic load to be increased above the basal expression level.

[0018] In this way, a positive feedback loop is created whereby higher expression of a given target antigen leads to higher expression of the therapeutic payload and vice versa, resulting in effective regulation of the therapeutic payload expression to achieve a T cell response precisely tuned to the target level present at a specific site and time. Increased expression of the therapeutic payload leads to optimal anti-tumor activity and rapid elimination of target tumor cells. As the amount of tumor cells decreases / removes, the therapeutic payload expression level returns to its basal expression level.

[0019] In one aspect, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, and wherein the inducible promoter regulated by the surface antigen regulates the expression level of one or more therapeutic payloads according to the expression level of the surface antigen on the target cell, thereby achieving a T cell response precisely regulated to the level of the target antigen present in the tumor environment.

[0020] In one embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen comprising a nucleotide sequence comprising SEQ ID NO: 137 and 138 or a combination thereof, and wherein the inducible promoter regulated by the surface antigen regulates the expression level of one or more therapeutic payloads according to the expression level of the surface antigen on the target cell, thereby achieving a T cell response that is precisely regulated to the level of target antigen present in the tumor environment.

[0021] In one aspect, the present invention provides a self-driven inducible promoter-therapeutic payload construct regulated by surface antigen, a host cell (e.g., T cell) expressing the inducible promoter-therapeutic payload construct regulated by surface antigen, and a nucleic acid molecule encoding the inducible promoter therapeutic payload construct regulated by surface antigen, wherein the self-driven inducible promoter-therapeutic payload construct comprises at least one therapeutic payload operably linked to an inducible promoter regulated by surface antigen, the therapeutic payload comprising a chimeric antigen receptor (CAR), a cytokine, a chemokine, a trafficking receptor, a bispecific antibody, a neutralizing / blocking antibody, a T cell stimulating receptor, a truncated inhibitory receptor, a hybrid inhibitory / activating receptor, an anti-apoptotic protein, shRNA or a protease, or a combination thereof, the inducible promoter regulated by surface antigen comprises a STAT5 response element, an AP-1 response element or a NFκB response element, or a combination thereof, and the inducible promoter regulated by surface antigen regulates the expression level of one or more therapeutic payloads according to the expression level of the surface antigen on the target cell.

[0022] In one embodiment, one or more therapeutic cargoes (e.g., but not limited to, based on a chimeric antigen receptor (CAR), a cytokine, a chemokine, a trafficking receptor, a bispecific antibody, a neutralizing / blocking antibody, a T cell stimulating receptor, a truncated inhibitory receptor, a hybrid inhibitory / activating receptor, an anti-apoptotic protein, a shRNA, or a protease) are expressed under the control of an inducible promoter regulated by a surface antigen, wherein the one or more therapeutic cargoes are separated by a 2A ribosome skipping sequence or an internal ribosome entry sequence (IRES), or a combination thereof.

[0023] In one aspect, the inducible promoter-therapeutic load construct regulated by surface antigen disclosed herein may include, for example, but not limited to, CAR, which may include a single molecule expressed on the surface of effector cells, or may include a signal transduction module and a soluble targeting module expressed by effector cells, so that when the soluble targeting module is combined with the signal transduction module expressed by the cell, a complete functional CAR is formed. CAR shows high surface expression on transduced T cells, with a high degree of cell lysis and amplification and persistence of transduced T cells in vivo. Also provided are methods for treating cancer in a subject using the disclosed CAR, host cells, and nucleic acid molecules, for example. Also provided are methods for regulating the expression of therapeutic loads using the disclosed inducible promoter-therapeutic load constructs regulated by surface antigens, host cells, and nucleic acid molecules, for example.

[0024] In one aspect, an isolated polynucleotide encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload is provided, wherein the therapeutic payload is operably linked to an inducible promoter regulated by a surface antigen, and wherein the therapeutic payload comprises a CAR containing a fragment selected from the group consisting of a Fab fragment, a F(ab') 2 Fragments, Fv fragments and single-chain Fv (ScFv).

[0025] In one embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload is provided, wherein the encoded extracellular antigen binding domain comprises at least one single-chain variable fragment of an antibody that binds to: mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123 or CD38 antigen binding domain, or a combination thereof.

[0026] In another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload is provided, wherein the encoded extracellular antigen binding domain comprises at least one heavy chain variable region of an antibody that binds to: mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123 or CD38 antigen binding domain, or a combination thereof.

[0027] In one aspect, an isolated polynucleotide encoding an inducible promoter-therapeutic payload construct regulated by a surface antigen is provided, wherein the therapeutic payload is operably linked to an inducible promoter regulated by a surface antigen, and wherein the therapeutic payload comprises a CAR comprising, from N-terminus to C-terminus, at least one extracellular binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the at least one extracellular binding domain comprises a mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38 antigen binding domain, or a combination thereof.

[0028] Thus, in one embodiment, an isolated polynucleotide encoding a CAR-based therapeutic payload operably linked to an inducible promoter regulated by a surface antigen is provided, 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 SEQ ID NO: 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.

[0029] In one embodiment, an isolated polynucleotide encoding a CAR-based therapeutic payload operably linked to at least one inducible promoter regulated by a surface antigen is provided, 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 SEQ ID NO: 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.

[0030] In one embodiment, an isolated polynucleotide encoding a CAR-based inducible promoter-therapeutic payload construct regulated by a surface antigen is provided, wherein the therapeutic payload is operably linked to an inducible promoter regulated by a surface antigen, and wherein the therapeutic payload comprises a CAR comprising, from N-terminus to C-terminus, at least one extracellular binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the at least one extracellular binding domain comprises a nucleic acid sequence selected from SEQ ID NO: 7, 9, 11, 15, 17, 19, 21, 23, 25, 87, 89, 91, 93, 95, 97, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, and 135, or a combination thereof.

[0031] In one embodiment, an isolated polynucleotide encoding a CAR-based inducible promoter-therapeutic payload construct regulated by a surface antigen is provided, wherein the therapeutic payload is operably linked to an inducible promoter regulated by a surface antigen, and wherein the therapeutic payload comprises a CAR comprising, from N-terminus to C-terminus, at least one extracellular binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the at least one extracellular binding domain comprises an amino acid sequence selected from SEQ ID NO: 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.

[0032] In one embodiment, the targeting domain of the CAR-based surface antigen-regulated inducible promoter-based therapeutic payload construct is expressed alone in the form of a monoclonal antibody, ScFv Fab, Fab'2 and comprises at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123 or CD38 antigen targeting domain coupled to an additional binding tag or epitope, the antigen targeting domain comprising a motif selected from SEQ ID NO: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 nucleic acid sequences, wherein the component expressed by the effector cell of CAR comprises a binding domain, which is specifically targeted to bind to a tag or epitope expressed on a soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component of CAR to form a complete functional CAR structure.

[0033] In another embodiment, the targeting domain of the CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is expressed alone in the form of a monoclonal antibody, ScFv Fab, Fab'2 and comprises at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123 or CD38 antigen targeting domain and an additional ScFv, wherein the antigen targeting domain comprises a protein selected from SEQ ID NO: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 nucleic acid sequences, and the component expressed by the effector cell of CAR comprises a tag or epitope that specifically reacts with the additional ScFv expressed on the soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component of CAR to form a complete functional CAR structure.

[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 CAR extracellular antigen binding domain further comprises at least one lipocalin-based antigen binding antigen (anticalin) that binds to mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38 antigen binding domains or a combination thereof.

[0035] In one 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 connected to the transmembrane domain via a linker domain.

[0036] 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 preceded by a sequence encoding a leader or signal peptide.

[0037] In another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, the 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 NO: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 wherein the CAR further encodes an extracellular antigen binding domain targeting an antigen including but not limited to: CD19, CD20, CD22, CD33, CD123, CD5, CD7, CD138, BCMA (CD269), ROR1, TSLPR, TEM-1, TEM-7, TEM-8, TEM-9, CD371, CD276, CD99, GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1 TCR, MAGE A3 TCR, PRAME TCR, KRAS TCR, or any combination thereof.

[0038] In certain embodiments, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the additionally encoded extracellular antigen binding domain comprises 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, anti-PMSAScFv antigen binding domain, anti-glycolipid F77 ScFv antigen binding domain, anti-EGFRvIII ScFv antigen binding domain, anti-GD-2ScFv antigen binding domain, anti-NY-ESo-1 TCR ScFv antigen binding domain, anti-MAGE A3 TCRScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0039] In one aspect, the CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct provided herein further comprises a linker or spacer domain.

[0040] 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 antigen binding domain, the intracellular signaling domain, or both of mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38 or a combination thereof are connected to the transmembrane domain by a linker or spacer domain.

[0041] In one embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8, TNFRSF19 or CD28 and is connected to the transmembrane domain.

[0042] In another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded CAR further comprises a transmembrane domain, the transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFRSF19 and CD154, or a combination thereof.

[0043] In another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded intracellular signaling domain further comprises a CD3ζ intracellular domain.

[0044] In one embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded intracellular signaling domain is arranged on the C-terminal side relative to the CD3ζ intracellular domain.

[0045] In another embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded at least one intracellular signaling domain comprises a co-stimulatory domain, a primary signaling domain, or a combination thereof.

[0046] In other embodiments, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the encoded at least one co-stimulatory domain comprises a functional signaling domain of: OX40, CD70, CD27, CD28, CD5, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), DAP10, DAP12 and 4-1BB (CD137), or a combination thereof.

[0047] In one embodiment, an isolated nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, which further comprises a leader sequence or signal peptide, wherein the leader or signal peptide nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 13, SEQ ID NO: 39, SEQ ID NO: 41 or SEQ ID NO: 43.

[0048] 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 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.

[0049] In one aspect, provided herein is a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct comprising, from N-terminus to C-terminus, at least one antigen binding domain, at least one transmembrane domain, and at least one intracellular signaling domain.

[0050] In one embodiment, a CAR-based inducible promoter-therapeutic payload construct regulated by a surface antigen 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.

[0051] In another embodiment, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the at least one transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0052] In some embodiments, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the CAR further encodes an extracellular antigen binding domain comprising: CD19, CD20, CD22, CD19 / 22, ROR1, mesothelin, CD33, CD38, CD123 (IL3RA), CD138, BCMA (CD269), GPC2, GPC3, FGFR4, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, NY-ESO-1TCR, MAGE A3TCR, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0053] In one embodiment, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the extracellular antigen binding domain comprises 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, anti-PMSAScFv antigen binding domain, anti-glycolipid F77ScFv antigen binding domain, anti-EGFRvIII ScFv antigen binding domain, anti-GD-2ScFv antigen binding domain, anti-NY-ESo-1 TCR ScFv antigen binding domain, anti-MAGE A3TCR ScFv antigen binding domain, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof.

[0054] In another embodiment, a CAR-based inducible promoter-therapeutic payload construct regulated by a surface antigen is provided, wherein the extracellular antigen binding domain comprises only an immunoglobulin variable heavy chain (VH) anti-CD19 antigen binding domain, an anti-CD20 VH antigen binding domain, an anti-CD22 VH antigen binding domain, an anti-ROR1 VH antigen binding domain, an anti-mesothelin VH antigen binding domain, an anti-CD33 VH antigen binding domain, an anti-CD38 VH antigen binding domain, an anti-CD123 (IL3RA) VH antigen binding domain, an anti-CD138 VH antigen binding domain, an anti-BCMA (CD269) VH antigen binding domain, an anti-GPC2 VH antigen binding domain, an anti-GPC3 VH antigen binding domain, an anti-FGFR4 VH antigen binding domain, an anti-c-Met VH antigen binding domain, an anti-PMSA VH antigen binding domain, an anti-glycolipid F77 VH antigen binding domain, an 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.

[0055] In another embodiment, a CAR-based inducible promoter-therapeutic payload construct regulated by a surface antigen is provided, wherein the extracellular antigen binding domain comprises a protein or peptide (P) sequence capable of specifically binding to a target antigen, which may be derived from a natural or synthetic sequence comprising: an anti-CD19 P antigen binding domain, an anti-CD20 P antigen binding domain, an anti-CD22 P antigen binding domain, an anti-ROR1 P antigen binding domain, an anti-mesothelin P antigen binding domain, an anti-CD33 P antigen binding domain, an anti-CD38 P antigen binding domain, an anti-CD123 (IL3RA) P antigen binding domain, an anti-CD138 P antigen binding domain, an anti-BCMA (CD269) P antigen binding domain, an anti-GPC2 P antigen binding domain, an anti-GPC3 P antigen binding domain, an anti-FGFR4 P antigen binding domain, an anti-c-Met P antigen binding domain, an anti-PMSAP antigen binding domain, an anti-glycolipid F77 P antigen binding domain, anti-EGFRvIIIP antigen binding domain, anti-GD-2P antigen binding domain, anti-NY-ESO-1TCR P antigen binding domain, anti-MAGE A3 TCR P antigen binding domain, or an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, or any combination thereof. In another embodiment, a CAR is provided, wherein the at least one intracellular signaling domain comprises a costimulatory domain and a primary signaling domain.

[0056] In another embodiment, a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is provided, wherein the at least one intracellular signaling domain comprises a co-stimulatory domain, and the co-stimulatory domain comprises 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.

[0057] In another embodiment, the nucleic acid sequence encoding the CAR-based inducible promoter-therapeutic payload construct regulated by the surface antigen comprises the nucleic acid sequence of SEQ ID NO: 139. In one embodiment, the nucleic acid sequence encodes a CAR-based promoter-therapeutic payload construct comprising the amino acid sequence of SEQ ID NO: 78.

[0058] In another embodiment, the nucleic acid sequence encoding the CAR-based inducible promoter-therapeutic payload construct regulated by the surface antigen comprises the nucleic acid sequence of SEQ ID NO: 140. In one embodiment, the nucleic acid sequence encodes a CAR-based promoter-therapeutic payload construct comprising the amino acid sequence of SEQ ID NO: 78.

[0059] In another embodiment, the nucleic acid sequence encoding the CAR-based inducible promoter-therapeutic payload construct regulated by the surface antigen comprises the nucleic acid sequence of SEQ ID NO: 77. In one embodiment, the nucleic acid sequence encodes a CAR-based promoter-therapeutic payload construct comprising the amino acid sequence of SEQ ID NO: 78.

[0060] In one aspect, the surface antigen-regulated inducible promoter-based therapeutic payload constructs disclosed herein are modified to express or contain a detectable marker for diagnosis, monitoring and / or prediction of treatment outcomes (e.g., progression-free survival of cancer patients) or for monitoring the progress of such treatment.

[0061] In one embodiment, the nucleic acid molecule encoding the inducible promoter-therapeutic load construct based on CAR regulated by surface antigen can be included 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 lentiviral vector, an adenoviral vector or a retroviral vector, or a combination thereof.

[0062] In another embodiment, the vector expressing the inducible promoter-therapeutic load construct based on CAR regulated by surface antigen can also be modified to include one or more control CAR T cell expression or eliminate CAR-T cell manipulation elements by suicide switch. The suicide switch may include, for example, a drug that induces apoptosis-inducing signaling cascade or induces cell death. In a preferred embodiment, the vector expressing CAR can also be modified to express enzymes, such as thymidine kinase (thymidine kinase, TK) or cytosine deaminase (cytosine deaminase, CD).

[0063] In another aspect, a host cell comprising a nucleic acid molecule encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct is also provided. In some embodiments, the host cell is a T cell, such as a primary T cell obtained from a subject. In one embodiment, the host cell is a CD8 + T cells.

[0064] In one aspect, provided herein are transduced T cells comprising an isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, wherein the inducible promoter therapeutic payload construct regulated by a surface antigen confers upon the transduced CAR T cells the ability to mount an anti-tumor response based on the expression level of the surface antigen on the target cell, thereby achieving a T cell response precisely regulated to the level of the target antigen present in the tumor environment.

[0065] In another aspect, provided herein are transduced CAR T cells comprising an isolated nucleic acid molecule encoding a CAR operably linked to an inducible promoter regulated by a surface antigen, wherein the inducible promoter regulated by a surface antigen CAR construct confers upon the transduced CAR T cells the ability to: increase anti-tumor responses according to the expression levels of the corresponding surface antigen on target cells, thereby achieving a CAR T cell response that is precisely regulated to the target surface antigen levels present in the tumor environment, wherein i) in the absence of tumor target surface antigen expression, the inducible promoter regulated by the surface antigen confers a low basal level of CAR expression; ii) in the presence of target surface antigen activation on the surface of the target cell, the CAR is activated, which triggers activation of applicable signal transduction pathways, thereby activating the signal regulator of the inducible promoter regulated by the surface antigen, thereby resulting in increased expression of the CAR above the basal expression level; iii) higher expression of a given target surface antigen results in higher expression of the CAR and vice versa, resulting in effective regulation of CAR expression to achieve a CAR that is precisely adjusted to the target levels present at a specific site and time. T cell response; iv) increased CAR expression leads to optimal anti-tumor activity and rapid elimination of target tumor cells; and v) as the amount of tumor cells is reduced / eliminated, the expression level of CAR returns to its basal expression level.

[0066] In one embodiment, transduced T cells with increased CAR expression result in optimal anti-tumor activity and rapid elimination of target tumor cells, such that as the amount of tumor cells is reduced / eliminated, the expression level of the therapeutic payload is restored to the basal expression level (see, pre-anti-tumor response expression level).

[0067] In one embodiment, the transduced T cells are autologous. In another embodiment, the transduced T cells are allogeneic.

[0068] In another aspect, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population is provided, wherein the T cell comprises a nucleic acid sequence encoding a CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct, wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, and the at least one extracellular antigen binding domain comprises an antigen binding domain comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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; wherein the T cell is a T cell of a person suffering from cancer. The cancer particularly includes hematological cancer, 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's lymphoma or Hodgkin's lymphoma) or multiple myeloma (MM), or a combination thereof.

[0069] In one embodiment, a pharmaceutical composition is provided, wherein at least one transmembrane domain of the CAR-based surface antigen-regulated inducible promoter-therapeutic payload construct comprises a transmembrane domain of a protein selected from the group consisting of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0070] In another embodiment, a pharmaceutical composition is provided wherein the human cancer comprises adult carcinoma, including oral and pharynx cancer (tongue, mouth, pharynx, head and neck), digestive system cancer (esophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancer (larynx, lung and bronchus), bone and joint cancer, soft tissue cancer, skin cancer (melanoma, basal and squamous cell carcinoma), pediatric tumors (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumors of the central nervous system (brain, astrocytoma, glioblastoma, glioma), as well as cancers of the breast, reproductive system (cervix, uterine body, ovary, vulva, vagina, prostate, testis, penis, endometrium), urinary system (bladder, kidney and renal pelvis, ureter), eye and orbit, endocrine system (thyroid), and brain and other nervous systems, or any combination thereof.

[0071] In another embodiment, a pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population of a human suffering from cancer is provided, wherein the cancer is a refractory cancer that is not responsive to one or more chemotherapeutic agents. The cancer includes hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B-cell malignancies (including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL)), minimal residual disease (MRD) in pediatric B-cell malignancies (including B-lineage ALL (acute lymphoblastic leukemia)), multiple myeloma (MM), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0072] In another embodiment, a pharmaceutical composition is provided wherein an inducible promoter-therapeutic cargo construct regulated by a surface antigen modulates the activity of the therapeutic cargo by utilizing natural effector cell pathways based on natural cell activation, mitogenic state, or a combination thereof.

[0073] In another aspect, a method for preparing a T cell comprising a CAR (hereinafter referred to as a "CAR-T cell") is provided. The method includes transducing T cells with a vector or nucleic acid molecule encoding a disclosed CAR containing an inducible promoter-therapeutic load regulated by a surface antigen, thereby preparing a CAR-T cell, wherein the disclosed CAR specifically binds to mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38, or a combination thereof.

[0074] In another aspect, a method for producing an RNA-modified cell population is provided, comprising introducing in vitro transcribed RNA or synthetic RNA comprising an inducible promoter-therapeutic payload regulated by a surface antigen, encoding a nucleic acid molecule of the disclosed CAR, into the cells of a subject, thereby producing CAR T cells.

[0075] In another aspect, a method for diagnosing a disease, disorder or condition associated with expression of an inducible promoter-therapeutic payload construct regulated by a surface antigen in a cell is provided, the construct comprising a nucleotide sequence selected from SEQ ID NO: 139, 140 and 141 or a combination thereof, the method comprising contacting the cell with a human anti-mesothelin, anti-CD33, anti-CD19, anti-CD19 / CD20, anti-CD22, anti-ROR1, anti-CD123 or anti-CD38 antibody or fragment thereof, or a combination thereof, wherein the antibody or fragment thereof comprises a nucleotide sequence selected from SEQ ID NO: 139, 140 and 141 or a combination thereof. NO: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) detecting the presence of the antigen mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123 or CD38 or a combination thereof, wherein the presence of mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123 or CD38 or a combination thereof is diagnostic of a disease, disorder or condition associated with the expression of mesothelin, CD33, CD19, CD19 / CD20, CD22, ROR1, CD123 or CD38 or a combination thereof.

[0076] 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 cancer, including hematopoietic cancer, myelodysplastic syndrome, pancreatic cancer, head and neck cancer, skin tumors, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adult B-cell malignancies including CLL (chronic lymphocytic leukemia), CML (chronic myeloid leukemia), non-Hodgkin lymphoma (NHL)), minimal residual disease (MRD) in pediatric B-cell malignancies including B-lineage ALL (acute lymphoblastic leukemia), multiple myeloma (MM), lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, melanoma or other hematological cancers and solid tumors, or any combination thereof.

[0077] In another embodiment, a method for diagnosing, prognosing or determining the risk of a mesothelin-, CD33-, CD19-, CD19 / CD20-, CD22-, CD19 / 22-, ROR1-, CD123- or CD38- (or a combination thereof)-related disease in a mammal is provided, comprising detecting the expression of mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38 or a combination thereof in a sample derived from the mammal, 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, (or a combination thereof) antibody or fragment thereof, wherein the antibody or fragment thereof comprises a protein selected from SEQ ID NO: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 amino acid sequences; and b) detecting the presence of one or more antigens, wherein the presence of the antigens is diagnostic for a mesothelin, CD33-, CD19, CD19 / CD20-, CD22-, CD19 / 22-, ROR1-, CD123- or CD38-related disease in a mammal.

[0078] In another embodiment, a method for redirecting a CAR antigen target is provided, comprising contacting a cell with an inducible promoter-therapeutic payload construct regulated by a surface antigen 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 SEQ ID NO: 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 a tumor cell expressing mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38, a tumor associated macrophage, and any combination thereof.

[0079] In another aspect, a method for inducing anti-tumor immunity in a mammal is provided, comprising administering to the mammal a therapeutically effective amount of T cells transduced with an inducible promoter-therapeutic payload construct regulated by a surface antigen, wherein the T cells comprise a vector or nucleic acid molecule encoding a disclosed therapeutic payload, such as, but not limited to, a CAR, a cytokine, a chemokine, a trafficking receptor, a bispecific antibody, a neutralizing / blocking antibody, a T cell stimulating receptor, a truncated inhibitory receptor, a hybrid inhibitory / activating receptor, an anti-apoptotic protein, a shRNA or a protease, or a combination thereof.

[0080] In another embodiment, a method for treating or preventing cancer in a mammal is provided, comprising administering to the mammal one or more disclosed promoter-therapeutic payload constructs regulated by surface antigens in an amount effective to treat or prevent cancer in the mammal. The method comprises administering to the subject a therapeutically effective amount of a host cell expressing a disclosed CAR specifically binding to mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38 and / or one or more of the foregoing antigens, under the following conditions, the conditions being sufficient to form an immune complex of the antigen binding domain on the CAR with the extracellular domain of mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38 and / or one or more of the foregoing antigens in the subject.

[0081] In another embodiment, a method for treating a mammal suffering from a disease, disorder or condition associated with elevated expression of a tumor antigen is provided, the method comprising administering to a subject a pharmaceutical composition comprising one or more disclosed surface antigen-regulated inducible promoter-therapeutic payload constructs, the pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises at least one extracellular mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the antigen binding domain comprises SEQ ID NO: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, and wherein the T cell is a T cell of a subject having cancer.

[0082] In another embodiment, a method for treating cancer in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising one or more of the disclosed surface antigen-regulated inducible promoter-therapeutic payload constructs, the pharmaceutical composition comprising an anti-tumor effective amount of a T cell population, wherein the T cells comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38 antigen binding domain, at least one linker or spacer domain, at least one transmembrane domain, at least one intracellular signaling domain, and the antigen binding domain comprises SEQ IDNO: 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, wherein the T cell is a T cell of a subject suffering from cancer. In some embodiments of the aforementioned methods, the at least one transmembrane domain comprises the following transmembrane domains: α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, mesothelin, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, or a combination thereof.

[0083] In another embodiment, a method for producing a persistent population of genetically modified T cells in a person diagnosed with cancer is provided. In one embodiment, the method includes administering to a person a T cell genetically modified to express a promoter-therapeutic load construct regulated by a surface antigen (e.g., based on CAR, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases), wherein the self-driven promoter-therapeutic load construct regulated by a surface antigen comprises at least one mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38 antigen binding domain, at least one transmembrane domain and at least one intracellular signaling domain, and the antigen binding domain comprises SEQ ID NO: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, wherein after administration, the persistent population of genetically modified T cells or the progeny population of T cells persists in humans for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years or 3 years.

[0084] In one embodiment, the progeny T cells in the human comprise memory T cells. In another embodiment, the T cells are autologous T cells.

[0085] In all aspects and embodiments of the methods described herein, any of the aforementioned cancers, diseases, disorders or conditions associated with elevated tumor antigen expression can be treated or prevented or ameliorated using one or more surface antigen-regulated promoter-therapeutic payload constructs disclosed herein.

[0086] For the avoidance of doubt, the description and claims disclosed herein specifically exclude in a proviso manner the Syn-Notch constructs as described in U.S. Pat. No. 9,670,281 (issued on June 6, 2017, entitled Binding-triggered transcriptional switches and methods of use thereof) and U.S. Pat. No. 9,834,608 (issued on December 5, 2017) (Wendell A. Lim et al.), respectively, and the NFAT-regulated expression of IL-12 as described in U.S. Pat. No. 8,556,882 (issued on October 15, 2013) (Richard A. Morgan et al.).

[0087] In another aspect, a kit is provided for preparing a promoter-therapeutic payload construct regulated by a surface antigen in a T cell as described above, or for preventing, treating or ameliorating any cancer, disease, disorder or condition associated with elevated tumor antigen expression as described above in a subject, the kit comprising a container containing any one of the nucleic acid molecules, vectors, host cells or compositions disclosed above, or any combination thereof, and instructions for using the kit.

[0088] It should be understood that the promoter-therapeutic payload constructs, host cells, nucleic acids and methods regulated by surface antigens are applicable outside the specific aspects and embodiments described in detail herein. The foregoing features and advantages of the present disclosure will become more apparent from the following detailed description with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 Schematic diagram depicting the positive regulation of self-driven STAT5 CAR via the IL-2 pathway and AP1 / NFκB CAR via the cytokine / CAR stimulation pathway.

[0090] Figure 2 The structures of STAT5- and AP1 / NFκB-inducible CAR LTG1563 and constitutive EF1α-driven CAR LTG1563 are depicted.

[0091] Figure 3 Depicted is the induction of CAR expression by a cytokine-driven positive feedback loop in the STAT5 and AP1 / NFκB inducible CAR LTG1563 in one healthy donor (representative of 2 donors). T cells were treated with different concentrations of IL2 and TNFα for 18 hours 5 days after activation (4 days after transduction).

[0092] Figure 4Depicts the rapid recovery of CAR expression after antigen exposure by AP1 / NFκB-regulated self-driven CAR LTG1563 compared to the constitutive CAR driven by EF1α. In 2 healthy donors, after initial co-culture with Raji cells, the expression of self-driven CAR constructs and constitutive CAR constructs driven by EF1α was regulated by inducible STAT5 & AP1 / NFκB promoters. Cells were activated with TransAct reagents at D0, transduced with LV vectors with an MOI of 10 at D1, and washed and treated with 0IU / mL IL-2 or 30IU / mL IL2 from D2 to D6 after activation. Starting from D6 after activation, CAR LTG1563 T cells were co-cultured with Raji-GFP (CD19+) cells with effectors: target ratios of 1:3. CAR expression before co-culture (D6) and 1 day (D7) after co-culture of a representative donor in 2 is shown.

[0093] Figures 5A to 5C Depicts superior Raji tumor killing and T cell expansion in the context of CAR LTG1563 T cells regulated by an AP1-NFκB-positive feedback loop, ( Figure 5A ) Long-term killing assay: co-culture time course diagram. Cells were activated with TransAct reagent at D0, transduced with LV vectors at an MOI of 10 at D1, and washed and treated with 0 IU / mL IL-2 or 30 IU / mL IL2 from D2 to D6 after activation. From D6 to D13 after activation, CAR LTG1563 T cells were co-cultured with Raji-GFP (CD19+) cells at an effector: target ratio of 1:3 (co-culture 1), and from D13 to D20, the cells were restimulated with Raji-GFP cells. ( Figure 5B ) CAR LTG1563-dependent Raji cell cytotoxicity during co-culture 1. ( Figure 5C )CAR LTG1563-dependent T cell expansion during co-culture 1&2. ( Figure 5B , Figure 5C ). n=3 donors (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001; two-way ANOVA with Tukey correction).

[0094] Figure 6 The structures of AP1 / NFκB-inducible and constitutive EF1α-driven CARs with additional auxiliary components (e.g., dominant negative receptors) are depicted: CAR LTG1563-TGFBRIIdn, CAR LTG1563-PD1dn, and CAR LTG1563-PD1dn-TGFBRIIdn.

[0095] Fig. 7A , Figure 7B and Figure 7C Depicted compared to the constitutive CAR driven by EF1α, the self-driven CAR LTG 1563 regulated by AP1 / NFKB with additional auxiliary components exhibited rapid induction of load (CAR and related auxiliary components: TGFBRIIdn, PD1dn) expression after antigen exposure. In 2 healthy donors, after initial co-culture with Raji cells, the expression of the self-driven CAR construct and the constitutive CAR construct driven by EF1α was regulated by the inducible AP1 / NFκB promoter. The cells were activated with the TransAct reagent at D0, transduced with an LV vector with an MOI of 10 at D1, and washed and treated with 30IU / mL IL2 from D2 to D5 after activation. Starting from D5 after activation, CAR LTG1563 T cells were co-cultured with Raji-GFP (CD19+) cells at an effector: target ratio of 1:3. Shown are CAR, TGFBRII, and PD1 expression before co-culture (D5) and 1 day after co-culture (D6) for one representative donor out of 2.

[0096] Figures 8A to 8C Depicts superior Raji tumor killing and T cell expansion in the presence of CAR LTG 1563 (with additional helper components) T cells protected from TGF-β inhibition. Fig. 8A ) Co-culture time course of long-term killing assay. Cells were activated with TransAct reagent at D0, transduced with LV vector (0.5% v / v) at D1, and washed and treated with 0 IU / mL IL-2 or 30 IU / mL IL2 from D2 to D5 after activation. From D5 to D9 after activation, CAR LTG1563 T cells were co-cultured with Raji-GFP (CD19+) cells at an effector:target ratio of 1:3 in the presence or absence of 10 ng / mL TGF-β (co-culture 1); from D9 to D13, cells were restimulated with Raji-GFP cells in the presence or absence of 10 ng / mL TGF-β (co-culture 2), and from D13 to 19, cells were restimulated with Raji-GFP cells in the presence or absence of 10 ng / mL TGF-β (co-culture 3). ( Figure 8B )CAR LTG1563-dependent T cell expansion during co-culture 1 to 3. ( Figure 8C ) CAR LTG1563-dependent Raji cell cytotoxicity during co-culture 1 to 3. ( Figure 8B , Figure 8C ). n = 2 donors.

[0097] Figures 9A to 9C Depicts superior Raji tumor killing and less T cell exhaustion when CAR LTG 1563 (with additional helper components) T cells are protected from TGF-β inhibition. Fig. 9A ) Co-culture time course of long-term killing assay. Cells were activated with TransAct reagent at D0, transduced with LV vector (0.5% v / v) at D1, and washed and treated with 30 IU / mL IL2 from D0 to D8 after activation. From D8 to D14 after activation, CAR LTG1563 T cells were co-cultured with Raji-GFP (CD19+) cells at an effector:target ratio of 1:3 in the presence or absence of 10 ng / mLTGF-β (co-culture 1); from D14 to D20, cells were restimulated with Raji-GFP cells at an effector:target ratio of 1:3 in the presence or absence of 10 ng / mLTGF-β (co-culture 2). ( Fig. 9B ) CAR LTG1563-dependent Raji cell cytotoxicity during co-culture 1 to 2. ( Fig. 9C ) Quantification of CAR T exhaustion marker expression (PD1) by flow cytometry at D20 after activation (co-culture 2, D8) at different time points before and after co-culture with CD19+Raji-GFP cells (co-culture 1&2). ( Fig. 9B , Fig. 9C ). *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. UTD = untransduced T cells. n = 3 independent donors.

[0098] Figures 10A to 10E Depicted are superior Raji tumor killing, enhanced T cell expansion, and enhanced T cell cytokine production when CAR LTG 1563 (with additional adjuvant components) T cells are protected from both TGF-β inhibition and PD-L1 inhibition. ( Fig. 10A ) PD-L1 expression on transduced and magnetically-sorted Raji-GFP-PDL1 NHL B cell line. ( Fig. 10B) Co-culture time course of long-term killing assay. Cells were activated with TransAct reagent at D0, transduced with LV vector (0.5% v / v) at D1, and washed and treated with 30 IU / mL IL2 from D0 to D8 after activation. From D8 to D14 after activation, CAR LTG1563 T cells were co-cultured with Raji-GFP (CD19+, PDL1-) or Raji-GFP-PDL1 (CD19+, PDL1+) cells at an effector:target ratio of 1:3 in the presence or absence of 10 ng / mL TGF-β (co-culture 1); from D14 to D20, cells were restimulated with Raji-GFP or Raji-GFP-PDL1 cells at an effector:target ratio of 1:3 in the presence or absence of 10 ng / mL TGF-β (co-culture 2). ( Fig. 10C ) CAR LTG1563-dependent Raji cell cytotoxicity at the final time points analyzed in co-culture 1 and co-culture 2 (D14 after activation, D20 after activation, respectively). ( Fig. 10D )CAR LTG1563-dependent T cell expansion during the complete T cell culture time (including co-culture 1 to 2). Fig. 10E ) Quantification of CAR T IL-2 production by flow cytometry 24 hours after co-culture with CD19+Raji-GFP or Raji-GFP-PDL1 cells (co-culture 1&2). ( Figures 10B to 10E ). *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001. UTD = untransduced T cells. n = 3 independent donors. DETAILED DESCRIPTION

[0099] definition

[0100] Unless the context clearly indicates otherwise, the nouns used in this article without quantifier modification represent one / kind or more / kind. For example, the term "antigen" includes single / kind or multiple / kind antigens, and can be considered to be equivalent to the phrase "at least one / kind antigen". The term "comprising" used in this article means "including". Therefore, "comprising antigens" means "comprising antigens" without excluding other elements. The phrase "and / or" means "and" or "or / or". It should be further understood that, unless otherwise indicated, any and all base sizes or amino acid sizes and all molecular weights or molecular mass values ​​given for nucleic acids or polypeptides are approximate, and are provided for the purpose of description. Although many methods and materials similar or equivalent to the methods and materials described herein can be used, specific suitable methods and materials are described below. In the event of a conflict, this specification (including the explanation of the term) shall prevail. In addition, materials, methods and examples are only illustrative and are not intended to be restrictive. For ease of access to multiple embodiments, the following explanation of terms is provided:

[0101] The term "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is meant to encompass variations of +-.20%, or in some cases +-.10%, or in some cases +-.5%, or in some cases +-.1%, or in some cases +-.0.1% from the particular value, as such variations are suitable for performing the disclosed methods.

[0102] Unless otherwise indicated, technical terms herein are used according to conventional usage. Definitions of commonly used 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 Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references.

[0103] As disclosed and described herein, the present invention is based on the unexpected discovery that modulation or regulation of expression of a surface antigen-regulated promoter of a therapeutic cargo construct can be directly correlated with the activity of the therapeutic cargo and, therefore, with the expression level of the surface antigen in the target cell environment.

[0104] The present disclosure provides novel self-driven inducible promoter-therapeutic payload constructs, host cells (e.g., T cells) expressing the surface antigen-regulated inducible promoter-therapeutic payload constructs, and nucleic acid molecules encoding the surface antigen-regulated inducible promoter-therapeutic payload constructs, wherein the self-driven inducible promoter-therapeutic payload constructs comprise one or more therapeutic payloads operably linked to an inducible promoter regulated by a surface antigen, wherein the inducible promoter regulated by a surface antigen regulates the expression level of one or more therapeutic payloads according to the expression level of the surface antigen in the target cell environment.

[0105] Without being limited by any specific mechanism of action, the "self-driven" inducible promoter regulated by surface antigen used herein refers to the use of an inducible promoter regulated by surface antigen to drive a therapeutic load, so as to provide a low basal level of therapeutic load surface expression in the absence of tumor target antigen expression. In the case of target antigen activation on the surface of the target cell, the therapeutic load is activated, which triggers the activation of applicable signal transduction pathways, thereby activating the signal regulator of the inducible promoter regulated by the surface antigen, thereby causing the expression of the therapeutic load to be increased to higher than the basal expression level. In this way, a positive feedback loop is generated, whereby the higher expression of a given target antigen leads to the higher expression of the therapeutic load and vice versa, thereby resulting in effective regulation of the expression of the therapeutic load to achieve a T cell response accurately adjusted for the target level present at a specific site and time. The increase in the expression of the therapeutic load leads to the best anti-tumor activity and the rapid elimination of target tumor cells. With the reduction / removal of the amount of tumor cells, the therapeutic load expression level is restored to its basal expression level.

[0106] This self-driven activation pattern will be repeated upon subsequent re-exposure to the antigen (e.g., but not limited to, occurring through tumor recurrence, metastatic events, tumor migration / spread, etc.). As a result, this control over the timing and magnitude of the self-driven therapeutically loaded T cell response provides improved therapeutic efficacy, reduced risk of tumor escape, and reduced therapeutic toxicity. This is similar to biologically or endogenously adjusting the therapeutic dose to the disease state at the cellular level.

[0107] The following is a detailed description of a self-driven surface antigen-regulated promoter-therapeutic payload construct, including a description of an inducible promoter regulated by a surface antigen, a therapeutic payload, and a more detailed description of a self-driven CAR-based surface antigen-regulated promoter-therapeutic payload construct, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression, vectors and host cells, treatment methods, compositions, and kits for using the disclosed self-driven CAR-based surface antigen-regulated promoter-therapeutic payload construct.

[0108] A. Inducible promoter regulated by surface antigen

[0109] In one aspect, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, and wherein the inducible promoter regulated by the surface antigen regulates the expression level of one or more therapeutic payloads according to the expression level of the surface antigen on the target cell, thereby achieving a T cell response precisely regulated to the level of the target antigen present in the tumor environment.

[0110] In one embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, wherein the inducible promoter regulated by a surface antigen comprises a nucleotide sequence comprising SEQ ID NOs: 137 and 138 or a combination thereof, and wherein the inducible promoter regulated by a surface antigen regulates the expression level of one or more therapeutic payloads according to the expression level of the surface antigen on the target cell, thereby achieving a T cell response that is precisely regulated to the level of the target antigen present in the tumor environment.

[0111] In one embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, wherein the inducible promoter regulated by a surface antigen comprises a nucleotide sequence comprising SEQ ID NO: 137 and 138 or a combination thereof, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and wherein the inducible promoter regulated by a surface antigen regulates the expression level of one or more therapeutic payloads according to the expression level of the surface antigen on the target cell, thereby achieving a T cell response precisely regulated to the level of the target antigen present in the tumor environment.

[0112] In another embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, wherein the inducible promoter regulated by a surface antigen comprises a nucleotide sequence comprising SEQ ID NOs: 137 and 138 or a combination thereof, and wherein the inducible promoter regulated by a surface antigen upregulates the expression level of one or more therapeutic payloads based on the expression level of the surface antigen on the target cell, thereby achieving a T cell response precisely regulated to the level of the target antigen present in the tumor environment.

[0113] In another embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, wherein the inducible promoter regulated by a surface antigen comprises a nucleotide sequence comprising SEQ ID NO: 137 and 138 or a combination thereof, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and wherein the inducible promoter regulated by a surface antigen upregulates the expression level of one or more therapeutic payloads based on the expression level of the surface antigen on the target cell, thereby achieving a T cell response precisely regulated to the level of the target antigen present in the tumor environment.

[0114] In another embodiment, the expression level of the inducible promoter therapeutic cargo construct regulated by the surface antigen described herein can be up-regulated, for example, but not limited to, from about 10% to 100%, 200%, 300%, 400% and 500%. The ranges recited herein specifically include all integer numbers therein, as if they were specifically recited.

[0115] In one embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, wherein the inducible promoter regulated by a surface antigen comprises a nucleotide sequence comprising SEQ ID NOs: 137 and 138 or a combination thereof, and wherein the inducible promoter regulated by a surface antigen downregulates the expression level of one or more therapeutic payloads based on the expression level of the surface antigen on the target cell, thereby achieving a T cell response precisely regulated to the level of the target antigen present in the tumor environment.

[0116] In another embodiment, provided herein is an isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, wherein the inducible promoter regulated by a surface antigen comprises a nucleotide sequence comprising SEQ ID NO: 137 and 138 or a combination thereof, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and wherein the inducible promoter regulated by a surface antigen downregulates the expression level of one or more therapeutic payloads based on the expression level of the surface antigen on the target cell, thereby achieving a T cell response precisely regulated to the level of the target antigen present in the tumor environment.

[0117] In another embodiment, the expression level of the inducible promoter therapeutic cargo construct regulated by the surface antigen described herein can be down-regulated, for example, but not limited to, from about 10% to 100%, 200%, 300%, 400% and 500%. The ranges recited herein specifically include all integer numbers therein, as if they were specifically recited.

[0118] B. Therapeutic Load

[0119] In its broadest aspect, provided herein are self-driven surface antigen-regulated inducible promoter-therapeutic payload constructs, host cells (e.g., T cells) expressing the surface antigen-regulated inducible promoter therapeutic payload constructs, and nucleic acid molecules encoding the surface antigen-regulated inducible promoter-therapeutic payload constructs, wherein the self-driven surface antigen-regulated inducible promoter-therapeutic payload constructs comprise at least one therapeutic payload operably linked to the surface antigen-regulated inducible promoter, the therapeutic payload comprising a chimeric antigen receptor (CAR), a cytokine, a chemokine, a trafficking receptor, a bispecific antibody, a neutralizing / blocking antibody, a T cell stimulating receptor, a truncated inhibitory receptor, a hybrid inhibitory / activating receptor, an anti-apoptotic protein, a shRNA or a protease, or a combination thereof, the surface antigen-regulated inducible promoter comprises a STAT5 response element, an AP-1 response element or a NFκB response element, or a combination thereof, and the surface antigen-regulated inducible promoter regulates the expression level of the therapeutic payload according to the expression level of the surface antigen on the target cell.

[0120] In one aspect, provided herein are self-driven inducible promoter-therapeutic payload constructs regulated by surface antigens, host cells (e.g., T cells) expressing the inducible promoter-therapeutic payload constructs regulated by surface antigens, and nucleic acid molecules encoding the inducible promoter-therapeutic payload constructs regulated by surface antigens, wherein the self-driven inducible promoter-therapeutic payload constructs comprise a therapeutic CAR operably linked to an inducible promoter regulated by surface antigens and at least one therapeutic payload, wherein the therapeutic payload comprises CAR, cytokine, chemokine, trafficking receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptotic protein, shRNA or protease, or a combination thereof, wherein the inducible promoter regulated by surface antigens comprises a STAT5 response element, an AP-1 response element or a NFκB response element, or a combination thereof, and the inducible promoter regulated by surface antigens regulates the expression level of the therapeutic CAR and the therapeutic payload according to the expression level of the surface antigen on the target cell.

[0121] In one embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises a cytokine comprising IL-2, IL-15, IL-7, TNFa, IFNγ, IFNβ, IFNα, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGFβ, IL-17, IL-18, or any combination thereof.

[0122] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises a cytokine such as, but not limited to, CCL2, CCL3, CCL4, CCL5, CCL19, CCL21, CCL25, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, or any combination thereof.

[0123] In another embodiment, an isolated nucleic acid molecule is provided wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises a trafficking receptor, such as a cytokine receptor, such as but not limited to CCR2, CCR3, CCR4, CCR7, CCR8, CCR9, CXCR3, CXCR4, CXCR6, SIP 1 , or any combination thereof, the transport receptor is used to help CART cells be transported to the tumor site.

[0124] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises, for example but not limited to, a bispecific antibody, including a bispecific T cell engager (BiTE), for example but not limited to, an anti-CD3 and anti-CD19 targeting antibody, or an anti-CD3 and anti-CD22 targeting antibody, or an anti-CD3 and anti-CD20 targeting antibody, or an anti-CD3 and anti-CD33 targeting antibody, or an anti-CD3 and anti-CD123 targeting antibody, or an anti-CD3 and anti-CD38 targeting antibody, other multi-targeted antibodies.

[0125] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises a neutralizing / blocking antibody, such as, but not limited to, against PD-L1, PD-L2, CD95L, TRAIL receptor, IL-6R, IL-1R, TGFβ receptor, PD-1, LAG-3, Tim-3, TGFβ, IL-10, CTLA-4, VISTA, TIGIT, IL-1, IL-1R, expressed as scFv, or IgG, or scFvFc, or VHH, or F(ab), or F(ab)2, or a natural ligand binding domain, or in other configurations, and the neutralizing / blocking antibody is used to enhance T cell lytic function, cytokine release, persistence, proliferation potential, prevent T cell checkpoint blockade, exhaustion, apoptosis, activation-induced cell death.

[0126] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises a T cell stimulatory receptor, such as but not limited to IL-2Rα, IL-15Rα, IL-7Rα, CXCR5, which is used to enhance the anti-tumor function of T cells.

[0127] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises a truncated inhibitory receptor (dominant negative; "dn"), such as, but not limited to, dn-TGFβ receptor II, dn-PD-1, dn-CTLA-4, dn-IL-10 receptor, dn-KLRG1, dn-CD160, dn-TIM3, dn-LAG3, dn-BTLA, dn-VISTA, which is used to prevent inhibition of T cell function.

[0128] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises a hybrid inhibitory / activating receptor, such as, but not limited to, the extracellular domain (ectodomain) of PD-1 fused to the intracellular domain (endodomain) of CD28, the extracellular domain of TGFβ receptor II fused to the intracellular domain of gp130, the extracellular domain of IL-10 receptor fused to the intracellular domain of 4-1BB, or the IL-4 receptor fused to the intracellular domain of IL-7 receptor, which hybrid inhibitory / activating receptor is used to convert T cell inhibitory signals into T cell activation signals.

[0129] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises an anti-apoptotic protein, such as but not limited to BCL-2, MCL-1, CED9, Bfl-1, Brag-1, A-1 or BCL-XL, which is used to prolong T cell persistence and prevent apoptosis.

[0130] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises shRNA, such as but not limited to PD-1, CTLA-4, KLRG-1, CD160, TGFβ receptor II, IL-10R, which shRNA is used to downregulate T cell inhibitory factors and enhance T cell anti-tumor function.

[0131] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises a protease, such as MMP2, MMP4, which is used to digest the tumor matrix and increase the infiltration of T cells into the tumor.

[0132] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises a peptide, such as an iRGD peptide, for enhancing tumor penetration of an anticancer drug.

[0133] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen further comprises a second CAR T construct for targeting a second tumor antigen, or targeting an antigen expressed on suppressive cells present in the tumor microenvironment, such as, but not limited to, PD-L1, PD-L2, TRAIL receptors CD33, CD138 present on MDSCs and suppressive B cells.

[0134] In another embodiment, one or more therapeutic cargoes (e.g., but not limited to, based on a chimeric antigen receptor (CAR), a cytokine, a chemokine, a trafficking receptor, a bispecific antibody, a neutralizing / blocking antibody, a T cell stimulating receptor, a truncated inhibitory receptor, a hybrid inhibitory / activating receptor, an anti-apoptotic protein, a shRNA, or a protease) are expressed under the control of an inducible promoter regulated by a surface antigen, wherein the one or more therapeutic cargoes are separated by a 2A ribosomal skipping sequence or an internal ribosome entry sequence (IRES), or a combination thereof.

[0135] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by the surface antigen further comprises: cytokines, including IL-2, IL-15, IL-7, TNFa, IFNγ, IFNβ, IFNα, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGFβ, IL-17, IL-18; chemokines, including CCR4, CCR6, CXCR5; trafficking receptors, such as cytokine receptors, such as but not limited to CCR4, CCR7, CCR2 ; bispecific antibodies, including bispecific T cell engagers (BiTEs), such as but not limited to anti-CD3 and anti-CD19 targeting antibodies or other multi-targeted antibodies; neutralizing / blocking antibodies, such as but not limited to those against PD-L1, IL-6R, IL-1R, expressed as scFv or IgG, or in other configurations; 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 intracellular domain of CD28; anti-apoptotic proteins, such as but not limited to BCL-2 or BCL-XL; shRNA; protease; a second CAR T construct; or any combination thereof; each having its aforementioned biological properties as listed above.

[0136] C. Chimeric Antigen Receptor (CAR)

[0137] In a narrower aspect, the invention, as disclosed and described herein, is based on the unexpected discovery that modulation or regulation of expression of a surface antigen-regulated promoter of a therapeutic cargo construct can be directly correlated with the activity of the therapeutic cargo and, therefore, with the expression level of the surface antigen in the target cell environment.

[0138] Compared to pre-existing CAR T constructs whose expression is regulated by a constitutive promoter, the CAR-based surface antigen-regulated inducible promoter-therapeutic payload constructs described herein have certain advantages over the prior art, including, for example but not limited to: i) adjusting or modulating the timing and amplitude of the anti-tumor response to the specific amount of antigen expressed by the tumor at the time, thereby achieving optimal execution of the anti-tumor CAR function; ii) preventing harmful T cell overactivation (exhaustion, activation-induced cell death, reduced metabolic capacity, rapid terminal differentiation); iii) reducing or eliminating the risk of toxicity associated with inappropriate CAR activation or overactivation; iv) reducing or eliminating CAR-related cytokine release syndrome (CRS); or v) reducing or eliminating CAR-related neurotoxicity; or any combination thereof.

[0139] Therefore, as disclosed and described herein, in one aspect of the present invention, at least one CAR-based therapeutic payload construct self-driven by an inducible promoter regulated by a surface antigen provided herein comprises at least one chimeric antigen receptor operably linked to an inducible promoter regulated by a surface antigen, which inducible promoter regulated by a surface antigen regulates the expression level of one or more therapeutic payloads according to the expression level of the surface antigen on the target cell.

[0140] In one embodiment, the self-driven CAR-based surface antigen-regulated promoter-therapeutic payload construct provided herein comprises, for example but not limited to, one or more CAR-based therapeutic payloads operably linked to an inducible promoter regulated by a surface antigen, and wherein the CAR-based therapeutic payload comprises a CAR that comprises, from N-terminus to C-terminus, at least one extracellular binding domain, at least one transmembrane domain, and at least one intracellular signaling domain, wherein the extracellular binding domain comprises a mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / 22, ROR1, CD123 or CD38 antigen binding domain, or a combination thereof.

[0141] CAR is an artificially constructed hybrid protein or polypeptide, which comprises an antigen binding domain (e.g., a single-chain variable fragment (ScFv)) of an antibody connected to a T cell signaling domain via a transmembrane domain. The characteristics of CAR include its ability to redirect T cell specificity and reactivity toward a selected target in a non-MHC restricted manner, as well as the antigen binding properties of monoclonal antibodies. Non-MHC restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independently of antigen processing, thus bypassing the main tumor escape mechanism. In addition, when expressed in T cells, CAR advantageously does not dimerize with endogenous T cell receptors (T cell receptor, TCR) α and β chains.

[0142] The unique ability to combine functional parts derived from different protein domains is a key innovative feature of CAR. The selection of each of these protein domains is a key design feature, as is the way in which its specificity is combined. Each design domain is an essential component that can be used between different CAR platforms to transform the function of lymphocytes. For example, the choice of an extracellular binding domain can make CARs that are ineffective in other cases effective.

[0143] The physicochemical properties of the immunoglobulin-derived protein sequence of the extracellular antigen binding domain used to generate the self-driven CAR-based promoter-therapeutic load construct regulated by surface antigen can be completely neutral, or it can self-associate and drive T cells to a state of metabolic exhaustion, thereby making the effect of the therapeutic T cells expressing the self-driven CAR-based promoter-therapeutic load construct regulated by surface antigen much lower. This occurs independently of the antigen binding function of the CAR domain. In addition, the selection of intracellular signaling domains can also control the activity and persistence of therapeutic lymphocyte populations for immunotherapy. Although the ability to bind to target antigens and the ability to transmit activation signals to T cells through these extracellular and intracellular domains, respectively, are important CAR design aspects, it is still obvious that the selection of the source of the extracellular antigen binding fragment can have a significant effect on the efficacy of the self-driven CAR-based promoter-therapeutic load construct regulated by surface antigen, and thus have a limiting effect on the function and clinical utility of the self-driven CAR-based promoter-therapeutic load construct regulated by surface antigen.

[0144] As disclosed herein, the intracellular T cell signaling domain of the self-driven CAR-based promoter-therapeutic load construct regulated by surface antigens 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 part of a promoter-therapeutic load construct regulated by surface antigens comprising an intracellular domain of a T cell receptor (e.g., such as, but not limited to, the intracellular portion of a CD3ζ protein). A co-stimulatory signaling domain refers to a part of a promoter-therapeutic load construct regulated by surface antigens comprising an intracellular domain of a co-stimulatory molecule, which is a cell surface molecule other than an antigen receptor or its ligand required for an effective response of lymphocytes to antigens.

[0145] The following is a detailed description of the self-driven CAR-based surface antigen-regulated promoter-therapeutic payload construct of the present invention, including a description of its extracellular antigen binding domain, transmembrane domain and intracellular domain, as well as other descriptions of the self-driven CAR-based surface antigen-regulated promoter-therapeutic payload construct, antibodies and antigen-binding fragments thereof, conjugates, nucleotides, expression vectors and host cells, treatment methods, compositions and kits using the disclosed self-driven CAR-based surface antigen-regulated promoter-therapeutic payload construct.

[0146] 1. Extracellular domain

[0147] In one embodiment, the CAR-based surface antigen-regulated promoter-therapeutic load construct comprises a target-specific binding element, which is also referred to as an antigen binding domain or portion. The choice of domain depends on the type and number of ligands that define the surface of the target cell. For example, an antigen binding domain can be selected to identify a ligand that acts as a cell surface marker associated with a specific disease state on the target cell. Therefore, some examples of cell surface markers that can serve as ligands of antigen binding domains of therapeutic surface antigen target-specific promoter-load constructs include those associated with viruses, bacteria and parasitic infections, autoimmune diseases and cancer cells.

[0148] In one embodiment, a promoter-therapeutic payload construct regulated by a surface antigen can be engineered to target a tumor antigen of interest by engineering a desired antigen binding domain that specifically binds to an antigen on a tumor cell. Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. The choice of antigen binding domain will depend on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, 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-1), ... In some embodiments, the tumor antigens disclosed herein are TNF-α, ...

[0149] In one embodiment, tumor antigens include one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can serve as target antigens for immune attack. These molecules include but are not limited to tissue-specific antigens, such as MART-1, tyrosinase and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as oncogenes HER-2 / Neu / ErbB-2. Another group of target antigens is onco-fetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute the unique real tumor-specific immunoglobulin antigens of individual tumors. B cell differentiation antigens (such as CD19, CD20 and CD37) are other candidates for target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, but with limited success.

[0150] The type of tumor antigen may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not appear on other cells in the body. TAAs are not unique to tumor cells and are instead expressed on normal cells under conditions where a state of immune tolerance to the antigen cannot be induced. Expression of an antigen on a tumor may occur under conditions that enable the immune system to respond to the antigen. A TAA may be an antigen that is expressed on normal cells during fetal development when the immune system is immature and unable to respond, or it may be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0151] Some non-limiting examples of TSAs or TAAs include the following: differentiation antigens, such as MART-1 / MelanA (MART-1), 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 oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER-2 / neu; unique tumor antigens caused by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; 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, CA19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, α-fetoprotein, β-HCG, BCA225, BTAA, CA125, CA15-3\CA27.29\BCAA, CA195, CA242, 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.

[0152] Furthermore, in certain embodiments, the use of a human extracellular antigen binding domain in place of a mouse-derived binding domain results in the generation of a self-driven CAR-based promoter-therapeutic payload construct regulated by a surface antigen that functions better in vivo while avoiding the induction of anti-CAR immunity and killing of CAR-T cell populations associated with the murine-based antigen binding domain in the host immune response.

[0153] The self-driven CAR-based surface antigen-regulated promoter-therapeutic payload construct expressing a fully human extracellular ScFv antigen-binding domain exhibits excellent activity / properties, including i) preventing poor CAR-T persistence and function as seen in the case of mouse-derived binding sequences; ii) validating the lack of regional delivery of the self-driven CAR-based surface antigen-regulated promoter-therapeutic payload construct; and iii) enabling the generation of CAR-T cell designs based on both high-affinity and low-affinity binders for the corresponding antigen. The latter property allows researchers to better adjust the potency and toxicity of CAR-T products, and / or tissue specificity, because due to the higher expression of certain antigens on tumors than normal tissues, lower affinity binders can have higher specificity for tumors relative to normal tissues, which can prevent on-target off tumor toxicity and bystander cell killing.

[0154] 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 the following: CD19, CD20, CD22, ROR1, mesothelin, CD33, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1TCR, MAGE A3 TCR, etc.

[0155] In one embodiment, the extracellular antigen binding domain in the CAR-based surface antigen-regulated promoter-therapeutic payload construct may include, for example, a scFV conjugate as disclosed in U.S. Patent No. 10,183,993, entitled Compositions and Methods for Treating Cancer with Anti-Mesothelin Immunotherapy, first filed as Provisional Patent Application No. 62 / 444,201 on January 9, 2017 and issued on January 22, 2019, and assigned to Lentigen Technology, Inc. (Case No. LEN_017).

[0156] 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 thereto.

[0157] In another embodiment, the nucleic acid sequence encoding the CAR comprises a 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 an amino acid sequence as shown in SEQ ID NO: 90, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In addition to scFv sequences that can be used as extracellular antigen binding domains in CAR-based surface antigen-regulated promoter-therapeutic payload constructs, the extracellular antigen binding domains in CAR-based surface antigen-regulated promoter-therapeutic payload constructs can comprise single-chain antigen binders (as opposed to scFv) that can be incorporated into functional CARs.

[0158] For example, CD33-specific heavy chain-only binders as disclosed in Applicant's co-pending non-provisional patent application No. 15 / 934,770 (Provisional Patent No. 62 / 476,438), entitled Compositions and Methods For Treating Cancer With Anti-CD33 Immunotherapy, filed on March 24, 2018, and assigned to Lentigen Technology, Inc. (Case No. LEN_018).

[0159] In one embodiment, the isolated nucleic acid molecule encoding the 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 thereto.

[0160] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG1906 targeting a malignant tumor expressing CD33 comprises the nucleic acid sequence of SEQ ID NO:93, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:94, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0161] In one embodiment, the extracellular antigen binding domain in the CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise, for example, a scFV conjugate as disclosed in the applicant's co-pending provisional patent application No. 62 / 773,940, entitled Compositions and Methods for Treating Cancer with Anti-CD38 Immunotherapy, filed on November 30, 2018, and assigned to Lentigen Technology, Inc. (Event No. LEN_026).

[0162] 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 thereto.

[0163] 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 thereto.

[0164] 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 thereto.

[0165] In one embodiment, the isolated nucleic acid molecule encoding the 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 thereto. In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2091 targeting a malignant tumor expressing CD38 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 shown in SEQ ID NO:8, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0166] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2092 targeting a malignant tumor expressing CD38 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 shown in SEQ ID NO:10, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0167] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2095 targeting a malignant tumor expressing CD38 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 shown in SEQ ID NO: 12, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0168] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2097 targeting a malignant tumor expressing CD38 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 shown in SEQ ID NO: 16, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0169] In one embodiment, the extracellular antigen binding domain in the CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise, for example, a scFV conjugate as disclosed in the applicant's co-pending non-provisional patent application No. 16 / 179,364, entitled Compositions and Methods for Treating Cancer with Anti-ROR1 Immunotherapy, filed on November 2, 2018, and assigned to Lentigen Technology, Inc. (Case No. LEN_022).

[0170] 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 thereto.

[0171] In one embodiment, the isolated nucleic acid molecule encoding the extracellular ROR1 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 154, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 155, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0172] In one embodiment, the isolated nucleic acid molecule encoding the extracellular ROR1 antigen binding domain comprises the nucleotide sequence of SEQ ID NO: 156, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular ROR1 antigen binding domain comprises the amino acid sequence of SEQ ID NO: 157, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0173] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 1941 targeting a malignant tumor expressing ROR1 comprises the nucleic acid sequence of SEQ ID NO:17, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:18, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0174] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 1942 targeting a malignant tumor expressing ROR1 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 shown in SEQ ID NO:20, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0175] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 1943 targeting a malignant tumor expressing ROR1 comprises the nucleic acid sequence of SEQ ID NO:21, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO:22, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0176] In one embodiment, the extracellular antigen binding domain in the CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise, for example, a scFV binder as disclosed in the applicant's co-pending provisional patent application No. 62 / 734,106, entitled Compositions and Methods for Treating Cancer with Anti-CD123 Immunotherapy, filed on September 20, 2018 and assigned to Lentigen Technology, Inc. (Event No. LEN_024).

[0177] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 antigen binding domain M12303 includes SEQ ID NO:158 nucleotide sequence, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain includes SEQ ID NO:159 amino acid sequence or with SEQ ID NO:159 amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity amino acid sequence.

[0178] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 antigen binding domain M12304 includes SEQ ID NO:160 nucleotide sequence, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain includes SEQ ID NO:161 amino acid sequence or with SEQ ID NO:161 amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity amino acid sequence.

[0179] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 antigen binding domain M12305 includes SEQ ID NO:162 nucleotide sequence, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain includes SEQ ID NO:163 amino acid sequence or with SEQ ID NO:163 amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity amino acid sequence.

[0180] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 antigen binding domain M12306 includes SEQ ID NO:164 nucleotide sequence, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain includes SEQ ID NO:165 amino acid sequence or with SEQ ID NO:165 amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity amino acid sequence.

[0181] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 antigen binding domain M12308 includes SEQ ID NO:166 nucleotide sequence, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain includes SEQ ID NO:167 amino acid sequence or with SEQ ID NO:167 amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity amino acid sequence.

[0182] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 antigen binding domain M12311 includes SEQ ID NO:168 nucleotide sequence, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain includes SEQ ID NO:169 amino acid sequence or with SEQ ID NO:169 amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity amino acid sequence.

[0183] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 antigen binding domain M12313 includes SEQ ID NO:170 nucleotide sequence, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain includes SEQ ID NO:171 amino acid sequence or with SEQ ID NO:171 amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity amino acid sequence.

[0184] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 antigen binding domain M12315 includes SEQ ID NO:172 nucleotide sequence, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain includes SEQ ID NO:173 amino acid sequence or with SEQ ID NO:173 amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity amino acid sequence.

[0185] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 antigen binding domain M12317 includes SEQ ID NO:174 nucleotide sequence, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain includes SEQ ID NO:175 amino acid sequence or an amino acid sequence with SEQ ID NO:175 amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity.

[0186] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD123 antigen binding domain M12318 includes SEQ ID NO:176 nucleotide sequence, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular CD123 antigen binding domain includes SEQ ID NO:177 amino acid sequence or with SEQ ID NO:177 amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% homogeneity amino acid sequence.

[0187] In another embodiment, the nucleic acid sequence encoding a functional CAR LTG 2075 targeting a malignant tumor expressing CD123 comprises a 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 comprising an amino acid sequence shown in SEQ ID NO: 24, 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 2076 targeting a malignant tumor expressing CD123 comprises the nucleic acid sequence of SEQ ID NO: 25, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 26, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0189] In another embodiment, the nucleic acid sequence encoding a functional CAR LTG 2077 targeting a malignant tumor expressing CD123 comprises a 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 comprising an amino acid sequence shown in SEQ ID NO: 116, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0190] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2078 targeting a malignant tumor expressing CD123 comprises 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 comprising the amino acid sequence shown in SEQ ID NO: 118, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0191] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2079 targeting a malignant tumor expressing CD123 comprises the nucleic acid sequence of SEQ ID NO: 119, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 120, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0192] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2082 targeting a malignant tumor expressing CD123 comprises 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 comprising the amino acid sequence shown in SEQ ID NO: 122, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0193] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2083 targeting a malignant tumor expressing CD123 comprises the nucleic acid sequence of SEQ ID NO: 123, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 124, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0194] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2085 targeting a malignant tumor expressing CD123 comprises 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 comprising the amino acid sequence shown in SEQ ID NO: 126, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0195] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2087 targeting a malignant tumor expressing CD123 comprises the nucleic acid sequence of SEQ ID NO: 127, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 128, or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0196] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2088 targeting a malignant tumor expressing CD123 comprises 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 comprising the amino acid sequence shown in SEQ ID NO: 130, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0197] In one embodiment, the extracellular antigen binding domain in the CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise, for example, a scFV conjugate as disclosed in the applicant's co-pending provisional patent application No. 62 / 736,955, entitled Compositions and Methods for Treating Cancer with Human Anti-CD19 / 22 Immunotherapy, filed on September 26, 2018 and assigned to Lentigen Technology, Inc. (Event No. LEN_025).

[0198] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 / CD22 antigen binding domain comprises the nucleotide sequence 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 thereto.

[0199] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG2737 targeting a malignant tumor expressing CD19 / CD22 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 shown in SEQ ID NO: 132, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0200] In one embodiment, the extracellular antigen binding domain in the CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise, for example, a scFV conjugate as disclosed in the applicant's co-pending non-provisional patent application No. 16 / 161,542, entitled Compositions and Methods for Treating Cancer with Human Anti-CD22 Immunotherapy, filed on October 16, 2018 and assigned to Lentigen Technology, Inc. (Case No. LEN_021).

[0201] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 antigen binding domain 16P17 comprises the nucleotide sequence SEQ ID NO: 180, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular mesothelin antigen binding domain comprises the amino acid sequence of SEQ ID NO: 181, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0202] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD22 antigen binding domain 16P13 comprises the nucleotide sequence SEQ ID NO: 182, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular mesothelin antigen binding domain comprises the amino acid sequence of SEQ ID NO: 183, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0203] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2209 targeting a CD22-expressing malignant tumor comprises the nucleic acid sequence of SEQ ID NO: 133, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 134, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0204] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG 2219 targeting a CD22-expressing malignant tumor comprises the nucleic acid sequence of SEQ ID NO: 135, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 136, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereof.

[0205] In one embodiment, the extracellular antigen binding domain in the CAR-based surface antigen-regulated promoter-therapeutic payload construct may comprise, for example, a scFV binder as disclosed in the applicant's co-pending non-provisional patent application No. 16 / 050,754, entitled Compositions and Methods for Treating Cancer with Anti-CD19 / 20 Immunotherapy, filed on July 31, 2018, and assigned to Lentigen Technology, Inc. (Event No. LEN_019).

[0206] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 / CD20 antigen binding domain comprises the nucleotide sequence 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 thereto.

[0207] In one embodiment, the isolated nucleic acid molecule encoding the extracellular CD19 / CD20 antigen binding domain comprises the nucleotide sequence SEQ ID NO: 113, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded extracellular mesothelin antigen binding domain comprises the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0208] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG1496 targeting a malignant tumor expressing CD19 / CD20 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 shown in SEQ ID NO:96, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0209] In another embodiment, the nucleic acid sequence encoding the functional CAR LTG1497 targeting a malignant tumor expressing CD19 / CD20 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 shown in SEQ ID NO:98, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0210] In another embodiment, the nucleic acid sequence encoding the CAR-based surface antigen-regulated promoter-therapeutic payload construct comprises one or more nucleic acid sequences disclosed in applicant's co-pending continuation-in-part patent application No. 16 / 134,735, filed on September 18, 2018, entitled Compositions and Methods for Treating Cancer with DuoCARs, which claims priority to PCT Application No. PCT / US17 / 49923, filed on September 1, 2017, which in turn claims the benefit of priority under 35 U.S.C. Section 119(e) to U.S. Provisional Patent Application No. 62 / 382,791, filed on September 2, 2016, the entire contents of each of which are incorporated herein by reference.

[0211] Therefore, in one embodiment, it is known that variant monospecific CAR structures compatible in the DuoCAR environment of the applicant can also be used to generate CAR-based surface antigen-regulated promoter-therapeutic load constructs. Based on DuoCAR surface antigen-regulated promoter-therapeutic load construct technology, some specific examples of monospecific therapeutic surface antigen-regulated promoter-load constructs (e.g., CAR-based) can be based on single CD20 targeting vector LTG1495, which has a nucleotide sequence of SEQ ID NO: 142 and an amino acid sequence of SEQ ID NO: 143. The second example is a monospecific CAR LTG2200 specific for CD22, which has a nucleotide sequence of SEQ ID NO: 69 and an amino acid sequence of SEQ ID NO: 70.

[0212] In another embodiment, variant CAR structures known to be compatible in the DuoCAR environment can also be used to generate promoter-therapeutic load constructs based on CARs regulated by surface antigens included within the scope of the present disclosure. These include CD19 specific CAR LTG1494 described in nucleotide sequence SEQ ID NO:71 and amino acid sequence SEQ ID NO:72, respectively. The sequence includes a well-described joint connecting the heavy chain and light chain of scFv, which is referred to as Whitlow joint (amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO:184), see Whitlow M., et al., 1993, ProteinEng.6:989-995). In some cases, Whitlow joints are replaced by (GGGGS) n joints (SEQ ID NO:185), for example, with CD19 CAR formats, such as in LTG1538, which have nucleotide sequence SEQ ID NO:73 and amino acid sequence SEQID NO:74, respectively. In another example, a CAR with an alternative transmembrane domain is produced. The anti-CD19 CAR LTG1562, which has a nucleotide sequence of SEQ ID NO: 75 and an amino acid sequence of SEQ ID NO: 76, respectively, utilizes a CD4 (as opposed to CD8) transmembrane domain. Similarly, the anti-CD19 CAR LTG1563, which has a nucleotide sequence of SEQ ID NO: 77 and an amino acid sequence of SEQ ID NO: 78, respectively, has an alternative transmembrane domain derived from TNFRSF19.

[0213] In another embodiment, another example of therapeutic application is to treat leukemia expressing CD19, CD20 and TSLPR antigens with a promoter-therapeutic load construct based on DuoCAR of the present invention regulated by surface antigen. In particular, the promoter-therapeutic load construct based on DuoCAR regulated by surface antigen comprises LTG1496 or LTG1497 (having SEQ ID NO: 95, 97, respectively) combined with TSLPR specific CAR (LTG1789) (having SEQ ID NO: 101 and amino acid sequence SEQ ID NO: 102, respectively), and the TSLPR specific CAR (LTG1789) is produced by a TSLPR specific scFV domain (having nucleotide sequence SEQ ID NO: 99 and amino acid sequence SEQ ID NO: 100).

[0214] In one embodiment, in each of the aforementioned DuoCAR-based surface antigen-regulated promoter-therapeutic payload constructs, the respective CAR construct is self-driven by a single surface antigen-regulated promoter, wherein the CAR constructs are separated by a ribosomal 2A skipping site therebetween.

[0215] In another embodiment, each of the aforementioned DuoCAR-based surface antigen-regulated promoter-therapeutic payload constructs, the respective CAR construct is self-driven by an independent surface antigen-regulated promoter.

[0216] In certain embodiments, some non-limiting examples of anti-CD19 CAR constructs used herein include an anti-CD19 CAR construct encoded by a nucleotide sequence referred to herein as LTG1563 (see, SEQ ID NO: 77), and the nucleotide sequence encodes an anti-CD19 CAR construct identified herein as CAR-LTG1563 (see, SEQ ID NO: 78).

[0217] In one embodiment, the construction of the following surface antigen-regulated inducible promoter-therapeutic payload construct is described in Example 1 below: it comprises a nucleic acid sequence of SEQ ID NO: 139 and encodes a CAR-based promoter-therapeutic payload construct (CAR LTG1563) comprising an amino acid sequence of SEQ ID NO: 78.

[0218] In one embodiment, the construction of the following surface antigen-regulated inducible promoter-therapeutic payload construct is described in Example 1 below: it comprises a nucleic acid sequence of SEQ ID NO: 140 and encodes a CAR-based promoter-therapeutic payload construct (CAR LTG1563) comprising an amino acid sequence of SEQ ID NO: 78.

[0219] In one embodiment, the construction of the following surface antigen-regulated inducible promoter-therapeutic payload construct is described in Example 1 below: it comprises a nucleic acid sequence of SEQ ID NO: 77 and encodes a CAR-based promoter-therapeutic payload construct (CAR LTG1563) comprising an amino acid sequence of SEQ ID NO: 78.

[0220] Without intending to be limited to any particular mechanism of action, possible reasons for the enhanced therapeutic function associated with the exemplary surface antigen-regulated promoter-therapeutic payload constructs of the invention are believed to include, for example, but not limited to: a) improved lateral movement within the plasma membrane allowing more efficient signal transduction, b) superior location within the plasma membrane microdomains (e.g., lipid rafts) and greater ability to interact with transmembrane signaling cascades associated with T cell activation, c) superior location within the plasma membrane by preferentially moving away from inhibitory (dampening) or downregulatory interactions (e.g., lower proximity or interaction with phosphatases (e.g., CD45)), and d) superior assembly into T cell receptor signaling complexes (e.g., immunological synapses), or any combination thereof.

[0221] Depending on the desired antigen to be targeted, the surface antigen-regulated promoter-therapeutic payload construct can be engineered to contain a suitable antigen binding domain specific for the desired antigen target. For example, but not limited to, if CD19 is the desired antigen to be targeted, an antibody against CD19 can be used as an antigen binding domain incorporated into the surface antigen-regulated promoter-therapeutic payload construct.

[0222] In an exemplary embodiment, the antigen binding domain portion of the CAR-based surface antigen-regulated promoter-therapeutic load construct targets CD19. Preferably, the extracellular antigen binding domain in the surface antigen-regulated promoter-therapeutic load construct is an anti-CD19scFV, wherein the nucleic acid sequence of the extracellular anti-CD19 scFV comprises a sequence shown in SEQ ID NO: 37 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, the extracellular anti-CD19 scFV comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 38 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In another embodiment, the extracellular anti-CD19 scFV portion of CAR comprises an amino acid sequence shown in SEQ ID NO: 38 or a sequence with 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0223] In one aspect of the invention, a surface antigen-regulated promoter-therapeutic payload construct is provided that is capable of binding to a non-TSA or non-TAA, including, for example, but not limited to, an antigen derived from the family Retroviridae (e.g., human immunodeficiency virus, such as HIV-1 and HIV-LP), the family Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, and echovirus), rubella virus, coronavirus, vesicular stomatitis virus, rabies virus, Ebola virus, parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus, influenza virus, hepatitis B virus, parvovirus, adenoviridae, herpesviridae (e.g., herpes simplex virus type 1 and type 2, varicella-zoster virus, cytomegalovirus (CMV) and herpes virus), poxviridae (e.g., smallpox virus, vaccinia virus, and poxvirus), or hepatitis C virus, or any combination thereof.

[0224] In another aspect of the invention, a surface antigen-regulated promoter-therapeutic payload construct is provided that is capable of binding to an antigen derived from a bacterial strain of Staphylococci, Streptococcus, Escherichia coli, Pseudomonas or Salmonella. In particular, surface antigen-regulated promoter-therapeutic payload constructs are provided that are capable of binding to antigens derived from infectious bacteria such as Helicobacter pyloris, Legionella pneumophilia, bacterial strains of the genus Mycobacteria sps. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansaii, or M. gordonea), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitides, Listeria monocytogenes, Streptococcus pyogenes, Group A Streptococcus, A Streptococcus), Group B Streptococcus (Streptococcus agalactiae), Streptococcus pneumoniae or Clostridium tetani, or a combination thereof.

[0225] 2. Transmembrane domain

[0226] 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.

[0227] 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.

[0228] In one embodiment, the isolated nucleic acid molecule encoding the TNFRSF19 transmembrane domain comprises the nucleotide sequence of SEQ ID NO: 51, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto. In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded TNFRSF19 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0229] The transmembrane domain may be derived from natural or synthetic sources. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0230] Transmembrane regions particularly useful for the surface antigen-regulated promoter-therapeutic payload constructs described herein may be derived from (i.e., at least comprising the transmembrane region thereof): 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. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise primarily hydrophobic residues, such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be present at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) may form a connection between the transmembrane domain of the surface antigen-regulated promoter-therapeutic payload construct and the cytoplasmic signaling domain. Glycine-serine doublets provide particularly suitable linkers.

[0231] In one embodiment, in addition to the above-mentioned transmembrane domains, a transmembrane domain naturally associated with one of the domains in the promoter-therapeutic cargo construct regulated by a surface antigen is used.

[0232] In some cases, transmembrane domains may be selected by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0233] In one embodiment, the transmembrane domain in the surface antigen-regulated promoter-therapeutic payload construct of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO: 27. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 28. In another embodiment, the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 28.

[0234] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO:28, or a sequence with 95% to 99% identity to an amino acid sequence of SEQ ID NO:28.

[0235] In some cases, the transmembrane domain of CAR comprises a CD8.α. hinge domain. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence of SEQ ID NO:29. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:30. In another embodiment, the CD8 hinge domain comprises an amino acid sequence of SEQ ID NO:30, or a sequence having 95% to 99% identity thereto.

[0236] In one embodiment, an isolated nucleic acid molecule is provided, wherein the encoded linker domain is derived from the extracellular domain of CD8 and is linked to a transmembrane CD8 domain, a transmembrane CD28 domain, or a combination thereof.

[0237] In one embodiment of the patient-specific autologous anti-tumor lymphocyte cell population as disclosed herein, some non-limiting exemplary transmembrane domains for the CAR-based surface antigen-regulated promoter-therapeutic payload constructs disclosed herein include TNFRSF16 and TNFRSF19 transmembrane domains, which can be used to obtain TNFRSF transmembrane domains and / or linker or spacer domains as disclosed in the applicant's co-pending patent application No. 15 / 767,076, entitled CHIMERIC ANTIGEN RECEPTORS AND METHODS OF USE, filed on April 9, 2018 and assigned to Lentigen Technology, Inc. (Case No. LEN_015) (US), particularly including those other TNFRSF members listed within the tumor necrosis factor receptor superfamily as listed in Table 1.

[0238] In one embodiment, the transmembrane domain in the CAR of the present invention is a TNFRSF19 transmembrane domain. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO:51. In one embodiment, the TNFRSF19 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:52. In another embodiment, the TNFRSF19 transmembrane domain comprises an amino acid sequence of SEQ ID NO:52.

[0239] In one embodiment, the encoded transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO:52, or a sequence with 95% to 99% identity to an amino acid sequence of SEQ ID NO:52.

[0240] 3. Spacer domain

[0241] In a CAR-based surface antigen-regulated promoter-therapeutic payload construct, a spacer domain may be arranged between the extracellular domain and the TNFRSF transmembrane domain, or between the intracellular domain and the TNFRSF transmembrane domain. A spacer domain means any oligopeptide or polypeptide for connecting a TNFRSF transmembrane domain to an extracellular domain and / or a TNFRSF transmembrane domain to an intracellular domain. The spacer domain comprises up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.

[0242] In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker such that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill and include those listed in the following: U.S. Patent Nos. 7,964,5667, 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 and U.S. Patent Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0243] The spacer domain preferably has a sequence that promotes the binding of the CAR-based surface antigen-regulated promoter-therapeutic payload construct to the antigen and enhances signal transduction into the cell. Some examples of amino acids that are expected to promote binding include cysteine, charged amino acids, and serine and threonine in potential glycosylation sites, and these amino acids can be used as amino acids constituting the spacer domain.

[0244] As the spacer domain, all or part of the following can be used: amino acids 118 to 178 (which is the hinge region) of CD8.α. (NCBI RefSeq: NP.sub.--001759.3) (SEQ ID NO: 31), amino acids 135 to 195 of CD8.β. (GenBank: AAA35664.1), amino acids 315 to 396 of CD4 (NCBI RefSeq: NP.sub.--000607.1), or amino acids 137 to 152 of CD28 (NCBI RefSeq: NP.sub.--006130.1). In addition, as the spacer domain, a part of the constant region of the antibody H chain or L chain (CH1 region or CL region, for example, a peptide having the amino acid sequence shown in SEQ ID NO: 32) can be used. In addition, the spacer domain may be an artificially synthesized sequence.

[0245] In addition, all or a portion of the amino acids comprising the constant region of human IgG4 (UniProt ID: P01861) may be used, which includes CH1 (amino acids 1 to 98), hinge (SEQ ID NO: 80, and corresponding nucleotides SEQ ID NO: 79) (amino acids 99 to 110), CH2 (amino acids SEQ ID NO: 81 and corresponding nucleotides SEQ ID NO: 80) (amino acids 111 to 220) and CH3 (SEQ ID NO: 84 and corresponding nucleotides SEQ ID NO: 83) (amino acids 221 to 327) or a combination thereof, such as the IgG4 hinge CH2 CH3 domain (SEQ ID NO: 86 and corresponding nucleotides SEQ ID NO: 85).

[0246] In one embodiment, the spacer domain of the CAR comprises a TNFRSF19 hinge domain comprising a nucleic acid sequence of SEQ ID NO: 53. In one embodiment, the TNFRSF19 hinge domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 54. In another embodiment, the TNFRSF19 hinge domain comprises an amino acid sequence of SEQ ID NO: 54, or a sequence having 95% to 99% identity thereto.

[0247] In one embodiment, the spacer domain of the CAR comprises a truncated hinge domain of TNFRSF19 comprising a nucleic acid sequence of SEQ ID NO: 55. In one embodiment, the truncated hinge domain of TNFRSF19 comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 56. In another embodiment, the truncated hinge domain of TNFRSF19 comprises an amino acid sequence of SEQ ID NO: 56, or a sequence having 95% to 99% identity thereto.

[0248] In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 49. In one embodiment, the TNFRSF19 hinge and transmembrane domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 50. In another embodiment, the TNFRSF19 hinge and transmembrane domain comprises the amino acid sequence of SEQ ID NO: 50, or a sequence with 95% to 99% identity thereof.

[0249] In one embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising a 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 an amino acid sequence of SEQ ID NO: 58. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising an amino acid sequence of SEQ ID NO: 58 or a sequence having 95% to 99% identity thereto.

[0250] In addition, in the promoter-therapeutic payload construct regulated by the surface antigen, a signal peptide sequence (also referred to as a leader peptide) can be connected to the N-terminus. The signal peptide sequence is present at the N-terminus of many secreted proteins and membrane proteins and is 15 to 30 amino acids in length. Since many of the protein molecules mentioned above as intracellular domains have signal peptide sequences, these signal peptides can be used as signal peptides for promoter-therapeutic payload constructs regulated by the surface antigen. In one embodiment, the signal peptide comprises the amino acid sequence shown in SEQ ID NO: 14).

[0251] In one embodiment, the CD8α leader peptide comprises the nucleic acid sequence of SEQ ID NO: 43. In one embodiment, the CD8α leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 44. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 44 or a sequence having 95% to 99% identity thereto.

[0252] In another embodiment, the GMCSF leader peptide comprises the nucleic acid sequence of SEQ ID NO: 39. In one embodiment, the GMCSF leader peptide comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 40. In another embodiment, the CD8a hinge domain is fused to a TNFRSF19 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 40, or a sequence having 95% to 99% identity thereof.

[0253] 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 CD8α leader peptide comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 42. In another embodiment, the CD8a hinge domain is fused to the TNFRSF19 transmembrane domain, which comprises the amino acid sequence of SEQ ID NO: 42 or a sequence having 95% to 99% identity thereto.

[0254] In one embodiment, the tag sequence encoding the truncated sequence of 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% to 99% identity thereto.

[0255] In one embodiment, the furin recognition site and downstream T2A self-cleaving peptide sequence designed for 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% to 99% identity thereto.

[0256] In one embodiment, the upstream furin recognition site and 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% to 99% identity thereto.

[0257] 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 is contained in a binding tag or epitope, while the effector cell expression component of the surface antigen-regulated promoter-therapeutic payload construct comprises a binding domain that is specifically directed to bind to a tag or epitope expressed on a soluble CAR module, such as the specific binding of the soluble component of CAR to the cell binding component to form a complete functional CAR structure.

[0258] 4. Intracellular domain

[0259] The cytoplasmic domain or other intracellular signaling domain of the CAR is responsible for activating at least one normal effector function of the immune cell into which the CAR has been placed. The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be a cell lytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specialized function. Although the entire intracellular signaling domain can usually be used, the entire chain does not have to be used in many cases. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used instead of a complete chain as long as it transduces an effector function signal. Therefore, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.

[0260] Some preferred examples of intracellular signaling domains for CAR include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors, which function together to initiate signal transduction after antigen receptor engagement; and any derivatives or variants of these sequences and any synthetic sequences having the same functional capabilities.

[0261] It is known that the signal generated by TCR alone is insufficient to fully activate T cells and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two different types of cytoplasmic signaling sequences: those that trigger antigen-dependent primary activation through TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0262] The primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0263] Some examples of ITAMs comprising primary cytoplasmic signaling sequences that are particularly useful in the CARs disclosed herein include those derived from: TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Some specific non-limiting examples of ITAMs include peptides having the following sequences: amino acids 51 to 164 of CD3.ζ. (NCBI RefSeq: NP.sub.--932170.1), amino acids 45 to 86 of Fc.ε.RI.γ (NCBI RefSeq: NP.sub.--004097.1), amino acids 201 to 244 of Fc.ε.RI.β (NCBI RefSeq: NP.sub.--000130.1), amino acids 139 to 182 of CD3.γ. (NCBI RefSeq: NP.sub.--000064.1), amino acids 128 to 171 of CD3.δ. (NCBI RefSeq: NP.sub.--000723.1), amino acids 140 to 145 of CD3.ε. (NCBI RefSeq: NP.sub.--00064.1). RefSeq: NP.sub.--000724.1), amino acids 153 to 207 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 707 to 847 of 0022 (NCBI RefSeq: NP.sub.--001762.2), amino acids 166 to 226 of CD79a (NCBI RefSeq: NP.sub.--001774.1), amino acids 182 to 229 of CD79b (NCBI RefSeq: NP.sub.--000617.1), and amino acids 177 to 252 of CD66d (NCBI RefSeq: NP.sub.--001806.2), and variants thereof having the same functions as these peptides. The amino acid number based on the amino acid sequence information of the NCBI RefSeq ID or GenBank described herein is numbered based on the full length of the precursor of each protein (including the signal peptide sequence, etc.). In one embodiment, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0264] In a preferred embodiment, the intracellular domain of CAR can be designed to include the CD3-ζ signaling domain itself, or it can be combined with any other desired cytoplasmic domain available in the case of CAR. For example, the intracellular domain of CAR may include a CD3ζ chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a part of a CAR that includes an intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for the effective response of lymphocytes to antigens. Some examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (lymphocyte function-associated antigen-1, LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, etc. Some specific non-limiting examples of such costimulatory molecules include peptides having the following sequences: amino acids 236 to 351 of CD2 (NCBI RefSeq: NP.sub.--001758.2), amino acids 421 to 458 of CD4 (NCBI RefSeq: NP.sub.--000607.1), amino acids 402 to 495 of CD5 (NCBI RefSeq: NP.sub.--055022.2), amino acids 207 to 235 of CD8.α. (NCBI RefSeq: NP.sub.--001759.3), amino acids 196 to 210 of CD83 (GenBank: AAA35664.1), amino acids 181 to 220 of CD28 (NCBI RefSeq: NP.sub.--006130.1), amino acids 181 to 220 of CD137 (4-1BB, NCBI RefSeq: NP.sub.--001552.2) amino acids 214 to 255, amino acids 241 to 277 of CD134 (OX40, NCBI RefSeq: NP.sub.--003318.1) and amino acids 166 to 199 of ICOS (NCBI RefSeq: NP.sub.--036224.1), and variants thereof having the same functions as these peptides. Therefore, although the disclosure herein is mainly illustrated by 4-1BB as a costimulatory signaling element, other costimulatory elements are also within the scope of the disclosure.

[0265] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of CAR can be interconnected in a random or specific order. Optionally, a short oligopeptide linker or polypeptide linker (preferably 2 to 10 amino acids in length) can form a connection. Glycine-serine doublets provide particularly suitable linkers.

[0266] In one embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of 4-1BB. In another embodiment, the intracellular domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28 and 4-1BB.

[0267] 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-ζ, wherein the signaling domain of 4-1BB includes the nucleic acid sequence shown in SEQ ID NO: 33, SEQ ID NO: 45 or SEQ ID NO: 59, respectively, and the signaling domain of CD3-ζ includes the nucleic acid sequence shown in SEQ ID NO: 35, SEQ ID NO: 47 or SEQ ID NO: 61, respectively.

[0268] 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-ζ, wherein the signaling domain of 4-1BB comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 48 or SEQ ID NO: 62.

[0269] 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-ζ, wherein the signaling domain of 4-1BB comprises the amino acid sequence shown in SEQ ID NO: 34, SEQ ID NO: 46, or SEQ ID NO: 60, respectively, and the signaling domain of CD3-ζ comprises the amino acid sequence shown in SEQ ID NO: 36, SEQ ID NO: 48, or SEQ ID NO: 62, respectively.

[0270] In one embodiment, the intracellular domain in the CAR is designed to include the signaling domain of CD28 and the signaling domain of CD3-ζ, wherein the signaling domain of CD28 comprises the nucleic acid sequence shown in SEQ ID NO:45 or SEQ ID NO:59, respectively, and the signaling domain of CD3-ζ comprises the nucleic acid sequence shown in SEQ ID NO:35, SEQ ID NO:47 or SEQ ID NO:61, respectively.

[0271] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-ζ, wherein the signaling domain of CD28 comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 60, respectively, and the signaling domain of CD3-ζ comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 36, or SEQ ID NO: 48, or SEQ ID NO: 62.

[0272] In one embodiment, the intracellular domain in the CAR is designed to comprise the signaling domain of CD28 and the signaling domain of CD3-ζ, wherein the signaling domain of CD28 comprises the amino acid sequence shown in SEQ ID NO:46 or SEQ ID NO:60, respectively, and the signaling domain of CD3-ζ comprises the amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:48, or SEQ ID NO:62, respectively.

[0273] 5. CAR with additional auxiliary components

[0274] In another embodiment, the construction of an inducible promoter-therapeutic payload construct regulated by a surface antigen comprising additional auxiliary components is described in Example 2 below, wherein the additional auxiliary components include a dominant negative receptor lacking the intracellular signaling domain of TGFBRII (TGFBRIIdn) and / or PD1 (PD1dn), and the above-mentioned inducible promoter-therapeutic payload construct is co-expressed with the CAR LTG1563 construct through a ribosomal skipping site (P2A) to produce the following inducible promoter-therapeutic payload construct regulated by a surface antigen: a nucleic acid sequence comprising SEQ ID NO: 103, 105, 107 or a combination thereof, and encoding a CAR-based promoter-therapeutic payload construct comprising an amino acid sequence comprising SEQ ID NO: 104, 106, 108 or a combination thereof.

[0275] In another embodiment, an inducible promoter-therapeutic payload construct regulated by a surface antigen comprising an additional auxiliary component (comprising a dominant negative receptor lacking the intracellular signaling domain of TGFBRII (TGFBRIIdn) or PD1 (PD1dn)) is co-expressed with the CAR LTG1563 construct through a skipping site (P2A) to produce the following inducible promoter-therapeutic payload construct regulated by a surface antigen: a nucleic acid sequence comprising SEQ ID NO: 103, 105, 107 or a combination thereof, and encoding an amino acid sequence comprising SEQ ID NO: 104, 106, 108 or a combination thereof. A CAR-based promoter-therapeutic payload construct, such that the use of the dominant negative receptors dnTGFb and dnPD1 results in the prevention of autoimmune toxicity typically associated with the use of constitutively activated dominant negative receptors, while benefiting from reduced immunosuppression of T cell function when the CAR is expressed and functional, or generating greater resistance to immunosuppression of CAR T cells by the tumor microenvironment of each dn receptor alone or in combination.

[0276] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen comprises the nucleic acid sequence of SEQ ID NO: 103 and encodes CAR LTG1563 comprising the amino acid sequence shown in SEQ ID NO: 104 or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and having a dominant negative form of an inhibitory TGF-β receptor.

[0277] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen comprises the nucleic acid sequence of SEQ ID NO: 105 and encodes CAR LTG1563 comprising the amino acid sequence shown in SEQ ID NO: 106 or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and having a dominant negative form of PD1.

[0278] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by the surface antigen has an AP1-NFκB_RE promoter, has a dominant negative form of the inhibitory TGF-β receptor and a dominant negative form of PD1, is co-expressed with the CAR LTG1563 construct, is separated by a ribosomal skipping site (P2A), and comprises a nucleic acid sequence of SEQ ID NO: 107 (the construct has a dominant negative form of the inhibitory TGF-β receptor and a dominant negative form of PD1, is co-expressed with the CAR LTG1563 construct, is separated by a ribosomal skipping site (P2A), and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 108 or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0279] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen having a STAT5_RE promoter comprises the nucleic acid sequence of SEQ ID NO: 109, and encodes a CAR LTG1563 comprising the amino acid sequence shown in SEQ ID NO: 104 or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and having a dominant negative form of inhibitory TGF-β.

[0280] In another embodiment, an isolated nucleic acid molecule is provided, wherein the inducible promoter-therapeutic payload construct regulated by a surface antigen having a STAT5_RE promoter comprises the nucleic acid sequence of SEQ ID NO: 110, and encodes CAR LTG1563 comprising the amino acid sequence shown in SEQ ID NO: 106 or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and having a dominant negative form of PD1.

[0281] In another embodiment, an isolated nucleic acid molecule is provided, in which an inducible promoter-therapeutic payload construct regulated by a surface antigen having a STAT5_RE promoter, an inhibitory TGF-β receptor having a dominant negative form, and a dominant negative form of PD1 is co-expressed with a CAR LTG1563 construct, separated by a ribosomal skipping site (P2A), comprises a nucleic acid sequence of SEQID NO: 111, and encodes a CAR comprising the amino acid sequence shown in SEQ ID NO: 108, or an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0282] In another embodiment, an isolated nucleic acid molecule is provided wherein a constitutive promoter-therapeutic payload construct having an EF1a promoter, an inhibitory TGF-β receptor in a dominant negative form, is co-expressed with a CAR LTG1563 construct, wherein the constitutive EF1a promoter is used to express an additional protein with the co-expressed CAR LTG1563 construct.

[0283] In another embodiment, an isolated nucleic acid molecule is provided, wherein a constitutive promoter-therapeutic payload construct having an EF1a promoter, having a dominant negative form of PD1 is co-expressed with a CAR LTG1563 construct, wherein the constitutive EF1a promoter is used to express an additional protein with the co-expressed CAR LTG1563 construct.

[0284] In another embodiment, an isolated nucleic acid molecule is provided in which a constitutive promoter-therapeutic payload construct having an EF1a promoter, an inhibitory TGF-β receptor having a dominant negative form, and a dominant negative form of PD1 are co-expressed with a CARLTG1563 construct and separated by a ribosomal skipping site (P2A), wherein the constitutive EF1a promoter is used to successfully express an additional protein with the co-expressed CAR LTG1563 construct.

[0285] 6. Additional description of CAR

[0286] The present invention also explicitly includes the functional part of the promoter-therapeutic load construct (e.g., based on CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralization / blocking antibody, T cell stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activation receptor, anti-apoptotic protein, shRNA, protease) disclosed herein.When referring to the use of CAR, the term "functional part" refers to any part or fragment of one or more CAR disclosed herein, which retains the biological activity of the CAR (parent CAR) as part thereof. The functional part covers, for example, those CAR parts that retain the ability to recognize target cells or detect, treat or prevent diseases to a similar degree or to the same degree or to a higher degree as the parent CAR. According to the parent CAR, the functional part may include, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% or more of the parent CAR.

[0287] The functional part may include additional amino acids at the amino or carboxyl end of the part or at both ends, and the additional amino acids are not present in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological functions of the functional part (e.g., identifying target cells, detecting cancer, treating or preventing cancer, etc.). More desirably, compared with the biological activity of the parent CAR, the additional amino acids enhance the biological activity.

[0288] The scope of the present disclosure includes functional variants of CAR disclosed herein. The term "functional variant" used herein refers to a CAR, polypeptide or protein having substantial or significant sequence identity or similarity with a parent CAR, and the functional variant retains the biological activity of the CAR as a variant. Functional variants encompass, for example, those variants of the CAR (parent CAR) described herein that retain the ability to recognize target cells to a similar degree, the same degree or a higher degree as the parent CAR. According to the parent CAR, the functional variant may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 98% or more identity with the parent CAR in terms of amino acid sequence.

[0289] Functional variants may, for example, comprise the amino acid sequence of a parent CAR having at least one conservative amino acid substitution. As an alternative or supplement, functional variants may comprise the amino acid sequence of a parent CAR having at least one non-conservative amino acid substitution. In this case, it is preferred that non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of functional variants. Non-conservative amino acid substitutions can enhance the biological activity of functional variants so that the biological activity of functional variants is improved compared to parent CAR.

[0290] The amino acid substitutions of CAR are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be acidic / negatively charged polar amino acids replacing another acidic / negatively charged polar amino acid (e.g., Asp or Glu), amino acids having non-polar side chains replacing another amino acid having non-polar side chains (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), basic / positively charged polar amino acids replacing another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), uncharged amino acids having polar side chains replacing another uncharged amino acids having polar side chains (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), amino acids having β-branched side chains replacing another amino acid having β-branched side chains (e.g., He, Thr and Val), amino acids having aromatic side chains replacing another amino acid having aromatic side chains (e.g., His, Phe, Trp and Tyr), etc.

[0291] The CAR may consist essentially of the specific amino acid sequence described herein, such that other components (e.g., other amino acids) do not substantially alter the biological activity of the functional variant.

[0292] CAR (including functional parts and functional variants) can have any length, that is, can contain any number of amino acids, provided that CAR (or its functional part or functional variant) retains its biological activity, for example, the ability to specifically bind to an antigen, detect diseased cells in a mammal, or treat or prevent a disease in a mammal, etc. For example, the length of CAR can be about 50 to about 5000 amino acids, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.

[0293] CAR (including functional parts and functional variants of the present invention) may include synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, -amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxylphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, Indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, -aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, γ-diaminobutyric acid, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

[0294] CAR (including functional parts and functional variants) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (via, for example, a disulfide bridge) or converted into an acid addition salt and / or optionally dimerized or multimerized or conjugated.

[0295] CAR (including functional parts and functional variants thereof) can be obtained by methods known in the art. CAR can be prepared by any suitable method for preparing a polypeptide or protein. Suitable methods for synthesizing polypeptides and proteins from scratch are described in references, such as Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, editor, Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, editor, Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001; and U.S. Patent No. 5,449,752. In addition, the nucleic acids described herein can be used to recombinantly produce polypeptides and proteins using 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. In addition, some CARs (including functional parts and functional variants thereof) can be separated and / or purified from sources such as plants, bacteria, insects, mammals (e.g., rats, humans), etc. Methods for separation and purification are well known in the art. Alternatively, the CARs described herein (including functional parts and functional variants thereof) can be commercially synthesized by a company. In this regard, CARs can be synthetic, recombinant, separated and / or purified.

[0296] In each of the foregoing descriptions in Section A above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload constructs described above, the surface antigen-regulated promoter-therapeutic payload constructs may also include a variety of other therapeutic modalities or auxiliary components including one or more of the following: cytokines, including IL-2, IL-15, IL-7, TNFa, IFNγ, IFNβ, IFNα, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGFβ, IL-17, IL-18; chemokines, including CCR4, CCR6, CXCR5; transport receptors, such as cell Cytokine receptors, such as but not limited to CCR4, CCR7, CCR2; bispecific antibodies, including bispecific T cell engagers (BiTEs), such as but not limited to anti-CD3 and anti-CD19 targeting antibodies or other multi-targeted antibodies; neutralizing / blocking antibodies, such as but not limited to those against PD-L1, IL-6R, IL-1R, expressed as scFv or IgG, or in other configurations; 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 intracellular domain of CD28; anti-apoptotic proteins, such as but not limited to BCL-2 or BCL-XL; shRNA; protease; a second CAR T construct; or any combination thereof; each having the aforementioned biological properties as listed above.

[0297] D. Antibodies and Antigen-binding Fragments

[0298] One embodiment also provides a CAR-based surface antigen-regulated promoter-therapeutic payload construct specifically bound to one or more antigens disclosed herein, such as, but not limited to, a T cell expressing CAR, an antibody, or an antigen binding domain or portion thereof. "CAR-expressing T cell" or "CAR T cell" as used herein means a T cell expressing CAR and having an antigen specificity determined by, for example, a targeting domain derived from an antibody of CAR.

[0299] As used herein, "antigen binding domain" may include antibodies and antigen binding fragments thereof. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antigen binding fragments thereof, as long as they exhibit the desired antigen binding activity. Some non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain binding affinity for an antigen.

[0300] "Monoclonal antibody" is an antibody obtained from a group of substantially homogeneous antibodies, that is, except for naturally occurring mutations that may be present in small amounts, the individual antibodies comprising the group are identical. Monoclonal antibodies are highly specific, directed against a single antigenic epitope. The modifier "monoclonal" represents the characteristics of an antibody such as obtained from a substantially homogeneous antibody group, and is not interpreted as requiring the production of antibodies by any particular method. In some instances, a monoclonal antibody is an antibody produced by a monoclonal B lymphocyte or a cell or its progeny transfected with nucleic acid encoding a single antibody (or its antigen-binding fragment) of an antibody light chain and a variable region of a heavy chain. In some instances, a monoclonal antibody is separated from an object. A monoclonal antibody may have a conservative amino acid substitution, which has substantially no effect on antigen binding or other immunoglobulin functions. Exemplary methods for producing monoclonal antibodies are known, for example, referring to Harlow & Lane, Antibodies, A Laboratory Manual, 2nd edition. Cold Spring Harbor Publications, New York (2013).

[0301] Generally speaking, immunoglobulins have heavy chains (H) and light chains (L) interconnected by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε and μ constant region genes and numerous immunoglobulin variable domain genes. There are two types of light chains, lambda (λ) and kappa (κ). There are five major heavy chain species (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA and IgE.

[0302] Each heavy chain and light chain comprises a constant region (or constant domain) and a variable region (or variable domain; see, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007)). In several embodiments, the heavy chain and light chain variable regions combine to specifically bind to an antigen. In some other embodiments, only the heavy chain variable region is required. For example, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). Reference to "VH" or "VH" refers to the variable region of an antibody heavy chain, including an antigen-binding fragment, such as a variable region of an Fv, ScFv, dsFv or Fab. Reference to "VL" or "VL" refers to the variable domain of an antibody light chain, including the variable domain of an Fv, ScFv, dsFv or Fab.

[0303] The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions (also called "complementarity determining regions" or "CDRs") (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within species. The framework region of an antibody (i.e., the combined framework regions of the component light and heavy chains) is used to position and align the CDRs in three-dimensional space.

[0304] The CDR is primarily responsible for binding to the epitope of the antigen. The amino acid sequence boundaries 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 Edition. 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 usually referred to as CDR1, CDR2, and CDR3 (from N-terminus to C-terminus), and are also usually identified by the chain in which the specific CDR is located. Thus, VH CDR3 is the CDR3 of the variable domain of the heavy chain of the antibody in which it is found, and VL CDR1 is the CDR1 of the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0305] "Antigen binding fragments" are portions of full-length antibodies that retain the ability to specifically recognize a cognate antigen, as well as various combinations of such portions. Some non-limiting examples of antigen binding fragments include 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 produced by modifying intact antibodies or those synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dubel (eds.), Antibody Engineering, Vols. 1-2, 2nd ed., Springer Press, 2010).

[0306] Single-chain antibodies (ScFv) are genetically engineered molecules comprising the VH and VL domains of one or more antibodies connected by a suitable polypeptide linker as a genetically fused single-chain molecule (see, e.g., Bird et al., Science, 242: 423 426, 1988; Huston et al., Proc. Natl. Acad. Sci., 85: 5879 5883, 1988; Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155 / 2012 / 980250; Marbry, ID Rugs, 13: 543-549, 2010). The intramolecular orientation of the VH and VL domains in ScFv is generally not critical for ScFv. Therefore, ScFvs with both possible arrangements (VH domain-linker domain-VL domain; VL domain-linker domain-VH domain) can be used.

[0307] In dsFv, the heavy and light variable chains have been mutated to introduce disulfide bonds to stabilize the association of the chains. Also included are diabodies, which are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci., 90:64446448, 1993; Poljak et al., Structure, 2:1121 1123, 1994).

[0308] Antibodies also include genetically engineered forms, such as chimeric antibodies (eg, humanized murine antibodies) and heteroconjugate antibodies (eg, bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., WH Freeman & Co., New York, 1997.

[0309] Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced, or can be obtained, for example, by screening a combinatorial library consisting of a variable heavy chain and a variable light chain as described in Huse et al., Science 246: 1275-1281 (1989), which is incorporated herein by reference. These and other methods for preparing, for example, chimeric, humanized, CDR-grafted, single-chain and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14: 243-246 (1993); Ward et al., Nature 341: 544-546 (1989); Harlow and Lane, supra, 1988; Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2nd ed. (Oxford University Press 1995); each of which is incorporated herein by reference).

[0310] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks 50% or more of the binding of the reference antibody to its antigen in a competition assay, and conversely, the reference antibody blocks 50% or more of the binding of the antibody to its antigen in a competition assay. Antibody competition assays are known, and exemplary competition assays are provided herein.

[0311] "Humanized" antibodies or antigen-binding fragments include human framework regions and one or more CDRs from non-human (e.g., mouse, rat, or synthetic) antibodies or antigen-binding fragments. The non-human antibody or antigen-binding fragment providing the CDR is referred to as a "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as an "acceptor." In one embodiment, in a humanized immunoglobulin, all CDRs are from a donor immunoglobulin. The constant region does not need to be present, but if present, it may be substantially identical to a human immunoglobulin constant region, e.g., having at least about 85% to 90%, e.g., about 95% or higher identity. Therefore, except for possible CDRs, all parts of a humanized antibody or antigen-binding fragment are substantially identical to corresponding parts of a natural human antibody sequence.

[0312] A "chimeric antibody" is an antibody comprising sequences derived from two different antibodies (which are usually from different species). In some examples, a chimeric antibody comprises one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from other human antibodies.

[0313] "Fully human antibodies" or "human antibodies" are antibodies that contain sequences from (or derived from) the human genome, but do not contain sequences from other species. In some embodiments, human antibodies contain CDRs, framework regions, and Fc regions (if present) from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques for producing antibodies based on sequences derived from the human genome, such as by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1st edition. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20: 450-459, 2008).

[0314] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to each other or may be different. For example, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, and a bispecific or bifunctional antibody has two different binding sites.

[0315] Methods for testing the ability of an antibody to bind to any functional portion of a CAR are known in the art and include any antibody-antigen binding assay, such as, for example, radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, and competitive inhibition assays (see, e.g., Janeway et al., infra, U.S. Patent Application Publication No. 2002 / 0197266A1 and U.S. Patent No. 7,338,929).

[0316] In addition, CAR, CAR-expressing T cells, antibodies, or antigen-binding portions thereof can be modified to include detectable labels, such as radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0317] In each of the foregoing descriptions in Section B above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload constructs described above, the surface antigen-regulated promoter-therapeutic payload constructs may also include a variety of other therapeutic modalities including one or more of the following: cytokines, including IL-2, IL-15, IL-7, TNFa, IFNγ, IFNβ, IFNα, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGFβ, IL-17, IL-18; chemokines, including CCR4, CCR6, CXCR5; transport receptors, such as cytokines. Receptors, such as but not limited to CCR4, CCR7, CCR2; bispecific antibodies, including bispecific T cell engagers (BiTEs), such as but not limited to anti-CD3 and anti-CD19 targeting antibodies or other multi-targeted antibodies; neutralizing / blocking antibodies, such as but not limited to those against PD-L1, IL-6R, IL-1R, expressed as scFv or IgG, or in other configurations; 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 intracellular domain of CD28; anti-apoptotic proteins, such as but not limited to BCL-2 or BCL-XL; shRNA; protease; a second CAR T construct; or any combination thereof; each having the aforementioned biological properties as listed above.

[0318] E. Conjugates

[0319] A promoter-therapeutic payload construct (e.g., based on CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptotic protein, shRNA, protease) or a monoclonal antibody or its antigen-binding fragment that has specific expression for one or more antigens disclosed herein, such as CAR, can be conjugated with an agent such as an effector molecule or a detectable marker using any number of methods known to those skilled in the art. Both covalent and non-covalent attachment methods can be used. Conjugates include, but are not limited to, molecules in which there are effector molecules or detectable markers covalently attached to antibodies or antigen-binding fragments, and the antibodies or antigen-binding fragments specifically bind to one or more antigens disclosed herein. It will be appreciated by those skilled 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, radioactive agents (e.g. 125 I. 32 P. 14 C. 3 H and 35 S) and other labels, target moieties and ligands, etc.

[0320] 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 can be a cytotoxin used to cause the death of a particular target cell (e.g., a tumor cell).

[0321] The methods used to attach effector molecules or detectable markers to antibodies or antigen-binding fragments vary depending on the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; for example, carboxylic acids (COOH), free amines (-NH 2 ) or sulfhydryl (-SH) groups, which can be used to react with suitable functional groups on the antibody to cause binding of effector molecules or detectable markers. Alternatively, the antibody or AF is derivatized to expose or attach additional reactive functional groups. Derivatization may involve attachment of any of many known linker molecules (e.g., those available from Pierce Chemical Company, Rockford, IL). The linker can be any molecule used to connect the antibody or AF to an effector molecule or detectable marker. The linker can form a covalent bond with both the antibody or AF 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 or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody or AF and the effector molecule or detectable marker are polypeptides, the linker can be connected to the constituent amino acids (e.g., connected to cysteine ​​by a disulfide bond) or to the alpha carbon amino and carboxyl groups of the terminal amino acids through its side groups.

[0322] In several embodiments, the linker may include a spacer element that, when present, increases the size of the linker so that the distance between the effector molecule or detectable marker and the antibody or antigen-binding fragment is increased. Exemplary spacers are known to those of ordinary skill in the art and include those listed in the following: U.S. Patent Nos. 7,964,5667, 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, 4,879,278; 4,816,444; and 4,486,414, and U.S. Patent Nos. 20110212088 and 20110070248, each of which is incorporated herein by reference in its entirety.

[0323] In some embodiments, the joint is cleavable under intracellular conditions, so that the cutting of the joint in the intracellular environment releases the effector molecule or detectable marker from the antibody or Fab. In other embodiments, the joint is not cleavable, and the effector molecule or detectable marker is released, for example, by antibody degradation. In some embodiments, the joint can be cut by a cutting agent present in the intracellular environment (for example, in a lysosome or endosome or caveolea). The joint can be, for example, a peptide joint, which is cut by an intracellular peptidase or protease (including but not limited to a lysosomal protease or an endosomal protease). In some embodiments, the peptide joint is at least two amino acids long or at least three amino acids long. However, the joint can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids long, for example, 1 to 2, 1 to 3, 2 to 5, 3 to 10, 3 to 15, 1 to 5, 1 to 10, 1 to 15 amino acids long. Proteases can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs within target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). For example, a peptide linker that can be cleaved by the sulfhydryl-dependent protease cathepsin B (e.g., phenylalanine-leucine or glycine-phenylalanine-leucine-glycine linker (SEQ ID NO: 186)) can be used. Other examples of such linkers are described, e.g., in U.S. Pat. No. 6,214,345, which is incorporated herein by reference. In a specific embodiment, the peptide linker that can be cleaved by an intracellular protease is a valine-citrulline linker or a phenylalanine-lysine linker (see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker).

[0324] In other embodiments, the cleavable linker is pH sensitive, i.e., sensitive to hydrolysis at certain pH values. Generally speaking, pH sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes 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 (e.g., conditions in blood), but are unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., such as a thioether attached to the therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0325] In other embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(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 (CW Vogel, ed., Oxford U. Press, 1987); Phillips et al., Cancer Res. 68:9280-9290, 2008). See also U.S. Pat. No. 4,880,935).

[0326] In other specific embodiments, 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).

[0327] In other embodiments, 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 incorporated herein by reference in its entirety).

[0328] In several embodiments, the linker is resistant to cleavage in the extracellular environment. For example, when the conjugate is present in the extracellular environment (e.g., in plasma), no more than about 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linker in a sample of the conjugate is cleaved. Whether the linker is resistant to cleavage in the extracellular environment can be determined, for example, by incubating the conjugate containing the linker of interest with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours) and then quantifying the amount of free effector molecules or detectable markers present in the plasma. A variety of exemplary linkers that can be used in the conjugate are described in WO 2004-010957, U.S. Publication No. 2006 / 0074008, U.S. Publication No. 20050238649, and U.S. Publication No. 2006 / 0024317, each of which is incorporated herein by reference in its entirety.

[0329] In several embodiments, a promoter-therapeutic payload construct conjugate expressing, for example, a CAR regulated by a surface antigen (e.g., based on CAR, cytokines, chemokines, trafficking receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases), an antibody or antigen binding portion thereof, and one or more small molecule toxins (e.g., calicheamicin, maytansinoids, dolastatin, auristatin, trichothecene, and CC1065) and derivatives of these toxins having toxin activity is provided.

[0330] Maytansine compounds suitable for use as maytansine compounds toxin part are well known in the art, and can be separated from natural sources according to known methods, produced using genetic engineering techniques (see Yu et al. (2002) PNAS 99: 7968-7973), or maytansinol and maytansinol analogs are prepared synthetically according to known methods. Maytansine compounds are mitotic inhibitors that play a role by inhibiting tubulin polymerization. Maytansine was first separated from the East African shrub Maytenus serrata (US Pat. No. 3,896,111). Subsequently, it was found that some microorganisms also produced maytansine compounds, such as maytansinol and C-3 maytansinol esters (US Pat. No. 4,151,042). and 4,371,533, each of which is incorporated herein by reference. Conjugates comprising maytansinoids, methods for their preparation and therapeutic uses thereof are disclosed, for example, in US Pat. Nos. 5,208,020, 5,416,064, 6,441,163 and European Patent EP 0 425235 Bl, the disclosures of which are expressly incorporated herein by reference.

[0331] Additional toxins can be used with promoter-therapeutic load construct conjugates (e.g., based on CAR, cytokines, chemokines, transport receptors, bispecific antibodies, neutralization / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases) expressing CAR, T cells, antibodies, or their antigen-binding portions. Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin, diphtheria toxin and its subunits, ribotoxins, ribonucleases, saporin, and calicheamicin, as well as botulinum toxins A to F. These toxins are well known in the art, and many are readily available from commercial sources (e.g., Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins (see, e.g., U.S. Pat. Nos. 5,079,163 and 4,689,401).

[0332] Saponin is a toxin from soapwort (Saponaria officinalis) that disrupts protein synthesis by inactivating the 60S portion of the ribosome complex (Stirpe et al., Bio / Technology, 10:405-412, 1992). However, the toxin does not have a mechanism for specific entry into cells and therefore needs to be conjugated to an antibody or antigen-binding fragment that recognizes a cell surface protein that is internalized for efficient uptake by the cell.

[0333] Diphtheria toxin is isolated from Corynebacterium diphtheriae. Generally speaking, diphtheria toxins used for immunotoxins are mutated to reduce or eliminate non-specific toxicity. A mutant called CRM107 with full enzyme activity but significantly reduced non-specific toxicity has been known since the 1970s (Laird and Groman, J. Virol. 19:220, 1976) and has been used in human clinical trials. See U.S. Patent No. 5,792,458 and U.S. Patent No. 5,208,021.

[0334] Ricin is a lectin RCA60 from Ricinus communis (castor bean). For examples of ricin, see U.S. Pat. No. 5,079,163 and U.S. Pat. No. 4,689,401. Based on their molecular weights, Ricinus communis agglutinin (RCA) is referred to as RCA in the form of approximately 65 and 120 kD, respectively.60 and RCA 120 There are two forms of ricin (Nicholson & Blaustein, J. Biochim. Biophys. Acta 266: 543, 1972). The A chain is responsible for inactivating protein synthesis and killing cells. The B chain binds ricin to galactose residues on the cell surface and helps the A chain to be transported into the cytosol (Olsnes et al., Nature 249: 627-631, 1974 and U.S. Patent No. 3,060,165).

[0335] Ribonucleases are also conjugated to targeting molecules for use as immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999). Some exemplary ribotoxins (e.g., α-sarcin and restrictocin) are discussed, for example, in Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra, Biochem. 345 Pt 2:247-54, 2000. Calicheamicin was first isolated from Micromonospora echinospora and is a member of the enediyne family of antitumor antibiotics that cause double-strand breaks in DNA, leading to apoptosis (see, e.g., Lee et al., J. Antibiot. 42:1070-87, 1989). This drug is the toxic portion of an immunotoxin in clinical trials (see, eg, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

[0336] Abrins comprise toxic lectins from Abrus precatorius. Toxin mechanism, abrin a, b, c and d have a molecular weight of about 63 to 67 kD and are composed of two disulfide-linked polypeptide chains, A and B. Chain A inhibits protein synthesis; 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).

[0337] A surface antigen-regulated promoter-therapeutic payload construct (e.g., based on CAR, cytokine, chemokine, trafficking receptor, bispecific antibody, neutralizing / blocking antibody, T cell stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptotic protein, shRNA, protease), monoclonal antibody, antigen-binding fragment thereof, which has specific expression for one or more antigens disclosed herein, such as CAR, can also be conjugated to a detectable marker; for example, a detectable marker that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., computed tomography (CT), computed axial tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoscopy, and laparoscopy). Some specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, available detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent markers are also useful, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). CAR, T cells expressing CAR, antibodies, or antigen-binding portions thereof may also be conjugated with enzymes that can be used for detection (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc.). When CAR, T cells expressing CAR, antibodies, or antigen-binding portions thereof are conjugated with a detectable enzyme, it can be detected by adding additional reagents, which the enzyme uses to produce a distinguishable reaction product. For example, when the reagent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine produces a visually detectable colored reaction product. CAR, T cells expressing CAR, antibodies, or antigen-binding portions thereof can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label.

[0338] Expressing, for example, a promoter-therapeutic payload construct regulated by a surface antigen of a CAR (e.g., based on CAR, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases), antibodies or their antigen-binding portions can be conjugated with paramagnetic agents (e.g., gadolinium). Paramagnetic agents (e.g., superparamagnetic iron oxides) can also be used as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies or antigen-binding fragments can also be labeled with a predetermined polypeptide epitope (e.g., a leucine zipper pair sequence, a binding site of a second antibody, a metal binding domain, an epitope tag) recognized by a second reporter.

[0339] A promoter-therapeutic payload construct (e.g., based on CAR, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases) expressing, for example, CAR may also be conjugated to radiolabeled amino acids. Radiolabels may be used for both diagnostic and therapeutic purposes. For example, radiolabels may be used to detect one or more antigens and cells expressing antigens disclosed herein by x-rays, emission spectroscopy, or other diagnostic techniques. In addition, radiolabels may be used therapeutically as toxins for treating tumors in subjects, for example, for treating neuroblastoma. Some examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H. 14 C. 15 N. 35 S. 90 Y. 99 Tc, 111 In, 125 I. 131 I.

[0340] Means for detecting such detectable markers are well known to those skilled in the art. Thus, for example, radioactive labels can be detected using photographic film or scintillation counters, and fluorescent labels can be detected using photodetectors to detect emitted radiation. Enzyme labels are usually detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0341] In each of the foregoing descriptions in Section C above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload constructs described above, the surface antigen-regulated promoter-therapeutic payload constructs may also include a variety of other therapeutic modalities including one or more of the following: cytokines, including IL-2, IL-15, IL-7, TNFa, IFNγ, IFNβ, IFNα, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGFβ, IL-17, IL-18; chemokines, including CCR4, CCR6, CXCR5; transport receptors, such as cytokines. Receptors, such as but not limited to CCR4, CCR7, CCR2; bispecific antibodies, including bispecific T cell engagers (BiTEs), such as but not limited to anti-CD3 and anti-CD19 targeting antibodies or other multi-targeted antibodies; neutralizing / blocking antibodies, such as but not limited to those against PD-L1, IL-6R, IL-1R, expressed as scFv or IgG, or in other configurations; 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 intracellular domain of CD28; anti-apoptotic proteins, such as but not limited to BCL-2 or BCL-XL; shRNA; protease; a second CAR T construct; or any combination thereof; each having the aforementioned biological properties as listed above.

[0342] F. Nucleotides, expression, vectors and host cells

[0343] One embodiment of the present invention also provides a nucleic acid comprising a nucleotide sequence encoding any promoter-therapeutic payload construct regulated by surface antigens described herein (e.g., based on CAR, cytokines, chemokines, transport receptors, bispecific antibodies, neutralization / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases), antibodies or their antigen binding portions (including functional portions and functional variants thereof). The nucleic acid of the present invention may include a nucleotide sequence encoding any leader sequence, antigen binding domain, transmembrane domain, and / or intracellular T cell signaling domain described herein.

[0344] In some embodiments, the nucleotide sequence can be codon-modified. Without being bound by a particular theory, it is believed that codon optimization of nucleotide sequences improves the translation efficiency of mRNA transcripts. Codon optimization of nucleotide sequences can involve replacing natural codons with another codon that encodes the same amino acid but can be translated by tRNA that is more easily available in the cell, thereby improving translation efficiency. Optimization of nucleotide sequences can also reduce secondary mRNA structures that interfere with translation, thereby improving translation efficiency.

[0345] In one embodiment of the invention, nucleic acid may include a codon-modified nucleotide sequence encoding the antigen binding domains of the CAR of the present invention. In another embodiment of the invention, nucleic acid may include a codon-modified nucleotide sequence encoding any CAR described herein (including its functional parts and functional variants).

[0346] "Nucleic acid" as used herein includes "polynucleotide", "oligonucleotide" and "nucleic acid molecule", and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and / or purified) from a natural source, which can contain natural, non-natural or altered nucleotides, and which can contain natural, non-natural or altered internucleotide connections, such as phosphoramidate connections or phosphorothioate connections, rather than phosphodiester between nucleotides present in unmodified oligonucleotides. In some embodiments, the nucleic acid does not include any insertion, deletion, inversion and / or replacement. However, as discussed herein, in some cases, it may be appropriate for the nucleic acid to include one or more insertions, deletions, inversions and / or replacements.

[0347] Recombinant nucleic acid can be a nucleic acid having a non-naturally occurring sequence or having a sequence prepared by an artificial combination of two otherwise separated sections of a sequence. This artificial combination is usually by chemical synthesis, or more commonly by the artificial manipulation of separated nucleic acid sections, such as by genetic engineering techniques (such as those described in Sambrook et al. (ibid)). Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation using methods known in the art. See, for example, Sambrook et al. (ibid) and Ausubel et al. (ibid). For example, naturally occurring nucleotides or a variety of modified nucleotides can be used to chemically synthesize nucleic acids, and the modified nucleotides are designed to improve the biological stability of the molecule or improve the physical stability of the duplex formed after hybridization (such as nucleotides substituted by thiophosphate derivatives and acridine). Some examples of modified nucleotides that can be used to generate nucleic acids 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, β-D-galactosylquenoside, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenine In some embodiments, the nucleic acid of the present invention can be purine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl quercetin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, quercetin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the present invention can be purchased from a company such as Integrated DNA Technologies (Coralville, IA, USA).

[0348] The nucleic acid may comprise any isolated or purified nucleotide sequence encoding any CAR or a functional part or functional variant thereof. Alternatively, the nucleotide sequence may comprise a nucleotide sequence that is degenerate to any sequence or a combination of degenerate sequences.

[0349] One embodiment also provides an isolated or purified nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0350] Nucleotide sequences hybridized under stringent conditions can hybridize under high stringency conditions. "High stringency conditions" means that the nucleotide sequence specifically hybridizes with a target sequence (nucleotide sequence of any nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides with precise complementary sequences or polynucleotides containing only a few scattered mismatches from random sequences that happen to have a few small regions (e.g., 3 to 10 bases) that match the nucleotide sequence. Such complementary small regions are more easily melted than the full-length complementary sequences of 14 to 17 or more bases, and highly stringent hybridization makes them easy to distinguish. Relatively high stringency conditions include, for example, low salt and / or high temperature conditions, such as provided by about 0.02M to 0.1M NaCl or equivalents at a temperature of about 50°C to 70°C. Such high stringency conditions allow small (if any) mismatches between nucleotide sequences and templates or target chains, and are particularly suitable for detecting the expression of any CAR of the present invention. It is generally recognized that conditions can be made more stringent by adding an increased amount of formamide.

[0351] Also provided are nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein.

[0352] In one embodiment, nucleic acid can be incorporated into a recombinant expression vector. In this regard, one embodiment provides a recombinant expression vector comprising any nucleic acid. For purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that allows host cells to express mRNA, protein, polypeptide or peptide when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide and when the vector is contacted with a cell under conditions sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector as a whole is not naturally occurring.

[0353] However, portions of the vector may be naturally occurring. The recombinant expression vector may contain any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from a natural source, and which may contain natural, non-natural or altered nucleotides. The recombinant expression vector may contain naturally occurring or non-naturally occurring internucleotide linkages or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector.

[0354] In one embodiment, the recombinant expression vector can be any suitable recombinant expression vector, and can be used for transformation or transfection of any suitable host cell. Suitable vectors include vectors designed for propagation and amplification or for expression or for both, such as plasmids and viruses. Vectors can be selected from pUC series (Fermentas Life Sciences, GlenBurnie, MD), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA).

[0355] Phage vectors can also be used, e.g. 1, λZapII (Stratagene), EMBL4 and λNMI149. Some examples of plant expression vectors include pBIOl, pBI101.2, pBHOl.3, pBI121 and pBIN19 (Clontech). Some examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral vector or a lentiviral vector. A lentiviral vector is a vector derived from at least a portion of a lentiviral genome, particularly including a self-inactivating lentiviral vector, such as Milone et al., Mol. Ther. 17 (8): 1453-1464 (2009) provided. Other examples of lentiviral vectors that can be used for clinical use include, for example, but not limited to, LENTIVECTOR.RTM. gene delivery technology from Oxford BioMedica plc, LENTIMAX.TM. vector system from Lentigen, etc. Non-clinical types of lentiviral vectors are also available and are known to those skilled in the art.

[0356] Many transfection techniques are generally known in the art (see, e.g., Graham et al., Virology, 52:456-467 (1973); Sambrook et al., supra; Davis et al., Basic Methods in Molecular Biology, Elsevier (1986); and Chu et al., Gene, 13:97 (1981).

[0357] Transfection methods include calcium phosphate coprecipitation (see, e.g., Graham et al., supra), direct microinjection into cultured cells (see, e.g., Capecchi, Cell, 22:479-488 (1980)), electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:742-751 (1988)), liposome-mediated gene transfer (see, e.g., Mannino et al., BioTechniques, 6:682-690 (1988)), lipid-mediated transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)), and nucleic acid delivery using high-speed microprojectiles (see, e.g., Klein et al., Nature, 327:70-73 (1987)).

[0358] In one embodiment, recombinant expression vectors can be prepared using standard recombinant DNA techniques as described in, for example, Sambrook et al., supra, and Ausubel et al., supra. Circular or linear expression vector constructs can be prepared to contain a replication system that is functional in a prokaryotic or eukaryotic host cell. The replication system can be derived from, for example, ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0359] Recombinant expression vectors may include regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, as appropriate, and taking into account whether the vector is DNA or RNA based. Recombinant expression vectors may include restriction sites to facilitate cloning.

[0360] The recombinant expression vector may include one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc., complementation in auxotrophic hosts to provide prototrophy, etc. Suitable marker genes for expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0361] The recombinant expression vector may include a natural or non-natural promoter, which is operably connected to a nucleotide sequence encoding CAR (including its functional part and functional variant) or to a nucleotide sequence complementary to or hybridized with the nucleotide sequence encoding CAR. The selection of promoters (e.g., strong, weak, inducible, tissue-specific and developmentally specific) is within the ordinary technical scope of the technician. Similarly, the combination of nucleotide sequence and promoter is also within the technical scope of the technician. Promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter or a promoter found in the long terminal repeat of a mouse stem cell virus.

[0362] The recombinant expression vector can be designed to be used for transient expression, for stable expression or for both. In addition, the recombinant expression vector can be used for constitutive expression or for inducible expression.

[0363] In addition, the recombinant expression vector can be prepared to include suicide genes. The term "suicide gene" used herein refers to a gene that causes cell death of the suicide gene. Suicide genes can be genes that give cells expressing genes sensitivity to reagents (e.g., drugs), and cause cell death when cells are contacted or exposed to reagents. Suicide genes are known in the art (see, e.g., Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004), and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) genes, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0364] One embodiment also provides a host cell comprising any recombinant expression vector described herein. The term "host cell" used herein refers to any type of cell that may contain the recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, such as a plant, an animal, a fungus or an algae, or may be a prokaryotic cell, such as a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism (e.g., a human)). The host cell may be an adherent cell or a suspension cell (i.e., a cell grown in suspension). Suitable host cells are known in the art, and include, for example, DH5a Escherichia coli (E.coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5a cell. For the purpose of producing a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. Although the host cell may be any cell type, may be derived from any type of tissue, and may be at any developmental stage, 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.

[0365] For purposes herein, T cells can be any T cells, such as cultured T cells, such as primary T cells, or T cells from cultured T cell lines (e.g., Jurkat, SupT1, etc.), or T cells obtained from mammals. If obtained from mammals, T cells can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. T cells can be human T cells. T cells can be T cells isolated from humans. T cells can be any type of T cell and can be at any stage of development, including but not limited to CD4 T cells. + / CD8 + Double positive T cells, CD4 + Helper T cells (such as Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells (i.e., Tscm), naive T cells, etc. T cells can be CD8 + T cells or CD4 + T cells.

[0366] In one embodiment, the surface antigen-regulated promoter-therapeutic payload constructs described herein can be used in suitable non-T cells. Such cells are those with immune effector functions, such as NK cells and T-like cells generated from pluripotent stem cells.

[0367] One embodiment also provides a cell group comprising at least one host cell described herein. The cell group can be a heterogeneous group, which includes a host cell containing any recombinant expression vector described, and in addition includes at least one other cell (e.g., host cell (e.g., T cell)) that does not contain any recombinant expression vector, or cells other than T cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, myocytes, brain cells, etc. Alternatively, the cell group can be a substantially homogeneous group, wherein the group mainly includes (e.g., is substantially composed of) host cells containing recombinant expression vectors. The group can also be a clonal group of cells, wherein all cells of the group are clones of a single host cell containing a recombinant expression vector, so that all cells of the group include the recombinant expression vector. In one embodiment of the invention, the cell group is a clonal group comprising a host cell containing a recombinant expression vector described herein.

[0368] CAR (including its functional parts and variants), nucleic acids, recombinant expression vectors, host cells (including its groups) and antibodies (including its antigen binding portions) can be separated and / or purified. For example, a purified (or separated) host cell preparation is a preparation in which the host cell has a higher purity than cells in the natural environment in vivo. Such host cells can be produced, for example, by standard purification techniques. In some embodiments, the preparation of the host cell is purified so that the host cell accounts for at least about 50%, for example, at least about 70% of the total cell content of the preparation. For example, the purity can be at least about 50%, can be greater than about 60%, about 70% or about 80%, or can be about 100%.

[0369] In each of the foregoing descriptions in Section D above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload construct nucleotides, expression vectors, and host cells described above, the surface antigen-regulated promoter-therapeutic payload construct may also include a variety of other therapeutic modalities including one or more of the following: cytokines, including IL-2, IL-15, IL-7, TNFa, IFNγ, IFNβ, IFNα, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGFβ, IL-17, IL-18; chemokines, including CCR4, CCR6, CXCR5; transport receptors , such as cytokine receptors, such as but not limited to CCR4, CCR7, CCR2; bispecific antibodies, including bispecific T cell engagers (BiTEs), such as but not limited to anti-CD3 and anti-CD19 targeting antibodies or other multi-targeted antibodies; neutralizing / blocking antibodies, such as but not limited to PD-L1, IL-6R, IL-1R, expressed as scFv or IgG, or in other configurations; 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 intracellular domain of CD28; anti-apoptotic proteins, such as but not limited to BCL-2 or BCL-XL; shRNA; protease; a second CAR T construct; or any combination thereof; each having the aforementioned biological properties as listed above.

[0370] G. Treatment methods

[0371] It is expected that the promoter-therapeutic load constructs (e.g., based on CAR, cytokines, chemokines, transport receptors, bispecific antibodies, neutralization / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases) disclosed herein that are regulated by surface antigens can be used in mammals for treating or preventing disease methods. In this regard, one embodiment provides a method for treating or preventing cancer in a mammal, comprising administering to a mammal CAR, nucleic acid, recombinant expression vector, host cell, cell group, antibody and / or its antigen binding portion and / or pharmaceutical composition in an amount that is effective for treating or preventing cancer in a mammal.

[0372] One embodiment also includes lymphodepleting the mammal prior to administering the CAR disclosed herein. Some examples of lymphodepleting include, but are not limited to, non-myeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, whole body irradiation, etc.

[0373] For methods in which host cells or cell populations are administered, cells can be mammalian allogeneic or autologous cells. Preferably, cells are mammalian autologous. Allogeneic as used herein means any material derived from different animals of the same species as the individual of the introduced material. When the genes of one or more loci are different, two or more individuals are considered to be allogeneic to each other. In some aspects, allogeneic materials from individuals of the same species can be genetically different enough to interact antigenically. "Autologous" as used herein means any material from the same individual of an individual of which material is subsequently reintroduced.

[0374] Mammals mentioned herein can be any mammals. The term "mammal" as used herein refers to any mammal, including but not limited to mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. Mammals can be from the order Carnivora, including felines (cats) and canines (dogs). Mammals can be from the order Artiodactyla, including Bovidae (cows) and Suidae (pigs), or the order Perissodactyla, including Equine (horses). Mammals can be Primates, Ceboid or Simoid (monkeys), or the order Anthropoids (humans and apes). Preferably, mammals are humans.

[0375] For the method, the cancer can be any cancer, including any of the following: acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder cancer), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, or pleural cancer, nasal cancer, nasal cavity cancer, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumors, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer , kidney cancer, laryngeal cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer and lung adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, B chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer and ureteral cancer.

[0376] As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would consider to have a potential benefit or therapeutic effect. In this regard, the method can provide any amount or any level of treatment or prevention of cancer in a mammal.

[0377] In addition, the treatment or prevention provided by the method may include treating or preventing one or more disorders or symptoms of the disease (e.g., cancer) being treated or prevented. Moreover, for purposes herein, "prevention" may encompass delaying the onset of a disease or its symptoms or disorders.

[0378] Another embodiment provides a method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells from a mammal with a CAR, a nucleic acid, a recombinant expression vector, a host cell, a cell population, an antibody and / or an antigen binding portion thereof, or a pharmaceutical composition to form a complex, (b) and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0379] The sample can be obtained by any suitable method (e.g., biopsy or autopsy). A biopsy is the removal of tissue and / or cells from an individual. Such removal can be the collection of tissue and / or cells from an individual in order to perform experiments on the removed tissue and / or cells. The experiment may include an experiment to determine whether an individual has and / or suffers from a certain condition or disease state. The condition or disease can be, for example, cancer.

[0380] In one embodiment of a method for detecting the presence of a proliferative disorder (e.g., cancer) in a mammal, the sample comprising mammalian cells can be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate (e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction). If the sample comprises whole cells, the cell can be any cell of a mammal, such as a cell of any organ or tissue, including a blood cell or an endothelial cell.

[0381] The contacting can occur in vitro or in vivo with respect to the mammal. Preferably, the contacting is in vitro.

[0382] In addition, the detection of the complex can be performed by any number of methods known in the art. For example, a detectable label (e.g., a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)) as disclosed above), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and an element particle (e.g., gold particles)) can be used to label the CAR disclosed herein, the polypeptide described herein, the protein, the nucleic acid, the recombinant expression vector, the host cell, the cell group or the antibody or its antigen binding portion thereof.

[0383] The method for testing the ability of CAR to recognize target cells and antigen specificity is known in the art. For example, Clay et al., J.Immunol, 163: 507-513 (1999) teach measuring cytokines (e.g., interferon-γ, granulocyte / monocyte colony stimulating factor (granulocyte / monocyte colony stimulating factor, GM-CSF), tumor necrosis factor a (tumor necrosis factor a, TNF-a) or interleukin 2 (interleukin 2, IL-2)) release method. In addition, CAR function can be evaluated by measuring the cytotoxicity of cells, such as Zhao et al., J.Immunol. 174: 4415-4423 (2005) described in.

[0384] Another embodiment provides the use of CAR, nucleic acid, recombinant expression vector, host cell, cell group, antibody or antigen binding portion thereof and / or pharmaceutical composition of the present invention for treating or preventing proliferative disorders (e.g., cancer) in mammals. Cancer can be any cancer described herein.

[0385] Any method of administration can be used for the disclosed therapeutic agent, including topical administration and systemic administration. For example, topical, oral, intravascular (e.g., intravenous), intramuscular, intraperitoneal, intranasal, intradermal, intrathecal, and subcutaneous administration can be used. The specific mode of administration and dosage regimen will be selected by the attending clinician considering the details of the case (e.g., object, disease, disease state involved, and whether the treatment is preventive). In the case where more than one agent or composition is administered, one or more routes of administration can be used; for example, a chemotherapeutic agent can be administered orally, and an antibody or antigen binding fragment or conjugate or composition can be administered intravenously. The method of administration includes injection, wherein CAR, CAR T cells, conjugates, antibodies, antigen binding fragments, or compositions are provided in a non-toxic pharmaceutically acceptable carrier (e.g., water, saline, Ringer's solution, glucose solution, 5% human serum albumin, fixed oil, ethyl oleate, or liposomes). In some embodiments, local administration of the disclosed compound can be used, for example, by applying an antibody or antigen binding fragment to a tissue area where a tumor is removed or an area suspected of being prone to tumors. In some embodiments, sustained intratumoral (or peritumoral) release of a pharmaceutical formulation comprising a therapeutically effective amount of an antibody or antigen-binding fragment may be beneficial. In other examples, the conjugate is topically applied to the cornea as eye drops, or intravitreally administered into the eye.

[0386] The disclosed therapeutic agents can be formulated into unit dosage forms suitable for separate administration of precise doses. In addition, the disclosed therapeutic agents can be administered in single doses or multiple dose regimens. A multiple dose regimen is a regimen in which the main course of treatment may have more than one separate dose (e.g., 1 to 10 doses), and then other doses are given at subsequent time intervals as needed to maintain or enhance the effect of the composition. Treatment may involve a daily dose or multiple daily doses of the compound over a period of days to months or even years. Therefore, the dosage regimen will also be determined at least in part based on the specific needs of the subject to be treated, and will depend on the judgment of the administering operator.

[0387] A typical dosage of the antibody or conjugate may be about 0.01 to about 30 mg / kg, such as about 0.1 to about 10 mg / kg.

[0388] In some specific examples, a therapeutic composition comprising one or more of a conjugate, an antibody, a composition, a CAR, a CAR T cell, or another agent is administered to a subject with multiple daily dosing regimens (e.g., at least two consecutive days, 10 consecutive days, etc., for example, for a period of weeks, months, or years). In one example, a conjugate, an antibody, a composition, or another agent is administered to a subject for at least 30 days, e.g., 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.

[0389] In some embodiments, the disclosed method includes combining with disclosed antibodies, antigen binding fragments, conjugates, CARs or T cells expressing CARs, providing surgery, radiotherapy and / or chemotherapeutic agents (e.g., sequentially, substantially simultaneously or simultaneously) to the subject. Such agents and treatment methods and therapeutic doses are known to those skilled in the art and can be determined by skilled clinicians. The preparation and dosing regimen of additional agents can be used according to the manufacturer's instructions or determined empirically by skilled practitioners. Such chemotherapeutic preparation and dosing regimens are also described in Chemotherapy Service, (1992) editor, MCPerry, Williams & Wilkins, Baltimore, MD.

[0390] In some embodiments, the combination therapy may include administering to the subject a therapeutically effective amount of an additional cancer inhibitor. Some non-limiting examples of other therapeutic agents that can be used with the combination therapy include microtubule binders, DNA intercalators or crosslinkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (administered in a therapeutically effective amount) and treatments can be used alone or in combination. For example, any suitable anticancer agent or anti-angiogenic agent can be administered in combination with CAR, CAR-T cells, antibodies, antigen binding fragments, or conjugates disclosed herein. The methods and therapeutic doses of such agents are known to those skilled in the art and can be determined by a skilled clinician.

[0391] Additional chemotherapeutic agents include, but are not limited to, alkylating agents such as nitrogen mustards (e.g., chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (e.g., carmustine, fotemustine, lomustine, and streptozotocin), platinum compounds (e.g., carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites such as folates (e.g., methotrexate, pemetrexed, and raltitrexed), purines (e.g., cladribine, clofarabine, fludarabine, mercaptopurine, and thioguanine), pyrimidines (e.g., capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophyllotoxins (e.g., etoposide and teniposide), taxanes (e.g., docetaxel and paclitaxel), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor 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 trastuzumab. sodium) and verteporfin; and other agents, such as alitretinoin, hexamethylmelamine, amridine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxyurea, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib and tretinoin. The selection and therapeutic dosage of such agents are known to those skilled in the art and can be determined by a skilled clinician.

[0392] Combination therapy can provide synergy and prove to be synergistic, that is, the effect achieved when the active ingredients are used together is greater than the sum of the effects produced by the compounds used alone. Synergy can be obtained when the active ingredients are: (1) co-formulated in a combined unit dosage form and administered or delivered simultaneously; (2) delivered in alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation, synergy can be obtained when the compounds are administered or delivered sequentially, for example, by different injections in separate syringes. Generally, during alternation, an effective dose of each active ingredient is administered sequentially (i.e., in sequence), whereas in combination therapy, effective doses of two or more active ingredients are administered together.

[0393] In one embodiment, an effective amount of an antibody or antigen-binding fragment or conjugate thereof that specifically binds to one or more antigens disclosed herein is administered to a subject with a tumor after anti-cancer treatment. After a sufficient amount of time has passed to allow the administered antibody or antigen-binding fragment or conjugate to form immune complexes with the antigens expressed on each cancer cell, immune complexes are detected. The presence (or absence) of immune complexes indicates the effectiveness of treatment. For example, an increase in immune complexes compared to a control taken before treatment indicates that treatment is ineffective, while a decrease in immune complexes compared to a control taken before treatment indicates that treatment is effective.

[0394] In each of the foregoing descriptions in Section E above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload construct treatment methods described above, the surface antigen-regulated promoter-therapeutic payload construct may also include a variety of other therapeutic modalities containing one or more of the following: cytokines, including IL-2, IL-15, IL-7, TNFa, IFNγ, IFNβ, IFNα, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGFβ, IL-17, IL-18; chemokines, including CCR4, CCR6, CXCR5; transport receptors, such as cell Cytokine receptors, such as but not limited to CCR4, CCR7, CCR2; bispecific antibodies, including bispecific T cell engagers (BiTEs), such as but not limited to anti-CD3 and anti-CD19 targeting antibodies or other multi-targeted antibodies; neutralizing / blocking antibodies, such as but not limited to those against PD-L1, IL-6R, IL-1R, expressed as scFv or IgG, or in other configurations; 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 intracellular domain of CD28; anti-apoptotic proteins, such as but not limited to BCL-2 or BCL-XL; shRNA; protease; a second CAR T construct; or any combination thereof; each having the aforementioned biological properties as listed above.

[0395] H. Biopharmaceutical Compositions

[0396] Provided herein are biopharmaceutical or biologic compositions (hereinafter referred to as "compositions") for gene therapy, immunotherapy, and / or cell therapy, comprising a disclosed promoter-therapeutic payload construct regulated by a surface antigen in a carrier (e.g., a pharmaceutically acceptable carrier) (e.g., based on CAR, cytokines, chemokines, transport receptors, bispecific antibodies, neutralizing / blocking antibodies, T cell stimulating receptors, truncated inhibitory receptors, hybrid inhibitory / activating receptors, anti-apoptotic proteins, shRNA, proteases), or T cells expressing CAR, antibodies, antigen binding fragments, conjugates, CAR, or one or more of the T cells expressing CAR specifically bound to one or more antigens disclosed herein. The composition can be prepared in unit dosage form for administration to a subject. The amount and time of administration are determined by the treating clinician to achieve the desired outcome. The composition can be formulated for systemic (e.g., intravenous) or local (e.g., intratumoral) administration. In one example, the disclosed CAR, or T cells expressing CAR, antibodies, antigen binding fragments, and conjugates are formulated for parenteral administration, such as intravenous administration. Compositions comprising CAR as disclosed herein, or T cells expressing CAR, conjugates, antibodies or antigen binding fragments are used, for example, to treat and detect tumors, such as but not limited to neuroblastoma. In some examples, the composition can be used to treat or detect cancer. Compositions comprising CAR as disclosed herein, or T cells expressing CAR, conjugates, antibodies or antigen binding fragments are also used, for example, to detect pathological angiogenesis.

[0397] The composition for administration may include a solution of CAR, or T cells expressing CAR, conjugates, antibodies or antigen binding fragments dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers may be used, such as buffered saline, etc. These solutions are sterile and generally do not contain undesirable substances. These compositions can be sterilized by conventional, well-known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH regulators and buffers, toxicity regulators, adjuvants, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. In these preparations, the concentration of CAR, or T cells expressing CAR, antibodies or antigen binding fragments or conjugates may vary widely, and will be selected mainly based on the specific mode of administration selected and the needs of the subject based on fluid volume, viscosity, body weight, etc. The actual method of preparing such a dosage form for gene therapy, immunotherapy and / or cell therapy is known or will be obvious to those skilled in the art.

[0398] Typical compositions for intravenous administration include about 0.01 to about 30 mg / kg antibody or antigen binding fragment or conjugate (or a corresponding dose of CAR, or T cells expressing CAR, or a conjugate comprising an antibody or antigen binding fragment) per subject per day. Actual methods for preparing administrable compositions will be known or obvious to those skilled in the art, and are described in more detail in publications such as Remington's Pharmaceutical Science, 19th edition, Mack Publishing Company, Easton, PA (1995).

[0399] CAR, or T cells expressing CAR, antibodies, antigen binding fragments, or conjugates can be provided in a lyophilized form and rehydrated with sterile water before administration, but they can also be provided as a sterile solution of known concentration. The solution of CAR, or T cells expressing CAR, antibodies, antigen binding fragments, or conjugates is then added to an infusion bag containing 0.9% sodium chloride (USP) and in some cases administered at a dose of 0.5 to 15 mg / kg body weight. There is considerable experience in the art in administering antibodies or antigen binding fragments and conjugate drugs; for example, since the approval of the FDA in 1997, the FDA has been approved for use in the treatment of CAR-related diseases. Since 2005, antibody drugs have been listed in the United States. CAR, or T cells expressing CAR, antibodies, antigen binding fragments thereof, and conjugates can be administered by slow infusion rather than by intravenous push or rapid bolus injection (bolus). In one example, a higher loading dose is administered, and the administered dose is subsequently maintained at a lower level. For example, an initial loading dose of 4 mg / kg antibody or antigen binding fragment (or a conjugate comprising an antibody or antigen binding fragment of a corresponding dose) can be infused within about 90 minutes, and then if the previous dose is well tolerated, a maintenance dose of 2 mg / kg per week is infused within about 30 minutes for 4 to 8 weeks.

[0400] Controlled release parenteral formulations can be prepared as implants, oily injections or as particle systems. For a broad overview of protein delivery systems, see Banga, AJ, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particle systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres and nanoparticles. Microcapsules contain therapeutic proteins (e.g., cytotoxins or drugs) as a central core. In microspheres, therapeutic agents are dispersed throughout the particles. Particles, microspheres and microcapsules less than about 1 μm are usually referred to as nanoparticles, nanospheres and nanocapsules, respectively. Capillaries have a diameter of about 5 μm, so only nanoparticles are administered intravenously. The diameter of microparticles is generally about 100 μm, and is administered subcutaneously or intramuscularly. See, e.g., Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).

[0401] Polymers can be used for CAR disclosed herein or T cells expressing CAR, antibodies or antigen binding fragments or conjugate compositions for controlled release of ions. A variety of degradable and non-degradable polymer matrices for controlled drug delivery are known in the art (Langer, Accounts Chem. Res. 26: 537-542, 1993). For example, block copolymer poloxamer 407 exists as a viscous flowing liquid at low temperatures, but forms a semisolid gel at body temperature. It has been shown to be an effective carrier 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 controlled release of proteins (Ijntema et al., Int. J. Pharm. 112: 215-224, 1994). In another aspect, liposomes are used for controlled release of lipid-encapsulated drugs as well as drug targeting (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Many additional systems for controlled delivery of therapeutic proteins are known (see U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,188,837, U.S. Pat. No. 4,235,871, U.S. Pat. No. 4,501,728, U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,957,735, U.S. Pat. No. 5,019,369, U.S. Pat. No. 5,055,303, U.S. Pat. No. 5,514,670, U.S. Pat. No. 5,413,797, U.S. Pat. No. 5,268,164, U.S. Pat. No. 5,004,697, U.S. Pat. No. 4,902,505, U.S. Pat. No. 5,506,206, U.S. Pat. No. 5,271,961, U.S. Pat. No. 5,254,342, and U.S. Pat. No. 5,534,496).

[0402] In each of the foregoing descriptions of Section F above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload construct composition described above, the surface antigen-regulated promoter-therapeutic payload construct may also include a variety of other therapeutic modalities including one or more of the following: cytokines, including IL-2, IL-15, IL-7, TNFa, IFNγ, IFNβ, IFNα, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGFβ, IL-17, IL-18; chemokines, including CCR4, CCR6, CXCR5; transport receptors, such as cell factor receptors, such as but not limited to CCR4, CCR7, CCR2; bispecific antibodies, including bispecific T cell engagers (BiTEs), such as but not limited to anti-CD3 and anti-CD19 targeting antibodies or other multi-targeted antibodies; neutralizing / blocking antibodies, such as but not limited to those against PD-L1, IL-6R, IL-1R, expressed as scFv or IgG, or in other configurations; 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 intracellular domain of CD28; anti-apoptotic proteins, such as but not limited to BCL-2 or BCL-XL; shRNA; protease; a second CAR T construct; or any combination thereof; each having the aforementioned biological properties as listed above.

[0403] I.Medicine Box

[0404] In one aspect, a promoter-therapeutic load construct (e.g., based on CAR, cytokine, chemokine, transport receptor, bispecific antibody, neutralization / blocking antibody, T cell stimulating receptor, truncated inhibitory receptor, hybrid inhibitory / activating receptor, anti-apoptotic protein, shRNA, protease) regulated by surface antigen disclosed herein is also provided. For example, a kit for treating a tumor in a subject or preparing a kit for expressing one or more CARs disclosed herein. The kit will generally include a disclosed antibody, Fab, conjugate, nucleic acid molecule, CAR or T cell expressing CAR as disclosed herein. More than one disclosed antibody, Fab, conjugate, nucleic acid molecule, CAR or T cell expressing CAR may be included in the kit.

[0405] The medicine box may include a container and a label or package insert associated with the container or on the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be formed of a variety of materials (e.g., glass or plastic). The container typically contains one or more compositions comprising the disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs, or T cells expressing CARs. In several embodiments, the container may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial having a plug that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition.

[0406] The label or package insert will also generally include instructions for using disclosed antibodies, antigen binding fragments, conjugates, nucleic acid molecules, CARs or T cells expressing CARs, for example, in methods for treating or preventing tumors or preparing CAR T cells. The package insert generally includes instructions customarily included in the commercial packaging of the therapeutic product, which includes information about the indications, usage, dosage, administration, contraindications and / or warnings for using such therapeutic products. The guidance material may be written in electronic form (e.g., a computer floppy disk or CD), or may be visual (e.g., a video file). The medicine box may also include additional components to facilitate the specific application of the medicine box designed for it. Therefore, for example, the medicine box may additionally include means for detecting the label (e.g., enzyme substrates for enzyme labeling, filtering devices for detecting fluorescent labels, suitable secondary labels (e.g., secondary antibodies), etc.). The medicine box may additionally include buffers and other reagents conventionally used to practice a particular method. Such medicine boxes and suitable contents are well known to those skilled in the art.

[0407] In each of the foregoing descriptions of Section G above, in addition to the CAR-based surface antigen-regulated promoter-therapeutic payload constructs described above, the surface antigen-regulated promoter-therapeutic payload constructs may also include a variety of other therapeutic modalities including one or more of the following: cytokines, including IL-2, IL-15, IL-7, TNFa, IFNγ, IFNβ, IFNα, IL-21, IL-33, IL-22, IL-6, IL-10, IL-9, IL-4, IL-12, TGFβ, IL-17, IL-18; chemokines, including CCR4, CCR6, CXCR5; transport receptors, such as cytokines. Receptors, such as but not limited to CCR4, CCR7, CCR2; bispecific antibodies, including bispecific T cell engagers (BiTEs), such as but not limited to anti-CD3 and anti-CD19 targeting antibodies or other multi-targeted antibodies; neutralizing / blocking antibodies, such as but not limited to those against PD-L1, IL-6R, IL-1R, expressed as scFv or IgG, or in other configurations; 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 intracellular domain of CD28; anti-apoptotic proteins, such as but not limited to BCL-2 or BCL-XL; shRNA; protease; a second CAR T construct; or any combination thereof; each having the aforementioned biological properties as listed above.

[0408] Example

[0409] The present invention is further illustrated by the following examples, which should not be construed in any way as limiting the scope of the invention. On the contrary, it should be clearly understood that a variety of other embodiments, modifications and equivalents thereof may be adopted without departing from the spirit of the present invention and / or the scope of the appended claims, which, after reading the description herein, are themselves suggested to those skilled in the art.

[0410] Example 1

[0411] Generation of self-driving CAR constructs targeting CD19 antigen

[0412] Despite the clinical success of anti-CD19 (e.g., CAR LTG1563)-based cancer therapy, suboptimal CAR activation and persistence on one hand and CAR overactivation and associated toxicities (CRS, neurotoxicity) on the other hand have raised questions. To improve the safety and efficacy of CAR T therapy, self-driving CARs were generated.

[0413] In this example, CAR T cells that utilize CAR signaling to regulate their own expression and the expression of co-regulated transgenes through two mechanisms are described, as well as the use of these mechanisms beyond CAR expression:

[0414] 1. Positive regulation of CART expression by STAT5 response elements (STAT5_RE) driven by cytokines (e.g. IL-2 / IL-15 / IL-7), which induce the expression of CAR molecules (e.g. anti-CD19CAR LTG1563). When CAR T detects tumor antigens, a slow positive feedback loop is activated, which increases CAR expression on CAR T cells. Once the tumor is eliminated, the positive feedback loop gradually decreases and closes, naturally reducing CAR surface expression and returning it to the monitoring state ( Figure 1 ).

[0415] 2. Positive regulation of CAR T expression by a combination of cytokine (e.g. IL-2 / IL-15 / IL-7 / TNFα)-driven and CAR / TCR-driven AP1 / NFκB response elements (AP1 / NFκB_RE), which induces the expression of CAR molecules (e.g. CAR LTG1563). When CAR T detects tumor antigens, a rapid positive feedback loop is activated, which increases CAR expression on CAR-T cells. Over time, AP1 / NFκB-driven CAR LTG1563 exhibits greater CD19-dependent killing activity, increased cytokine secretion, reduced exhaustion, and overall adaptability of CAR T cells relative to constitutively expressed CARs. Once the tumor is eliminated, the positive feedback loop gradually decreases and closes, naturally reducing CAR surface expression and returning it to a monitoring state ( Figure 1 ).

[0416] 3. In the presence of CAR antigens, additional proteins are expressed to regulate CAR T function, including but not limited to: escape of negative T cell regulation (dominant negative TGFBRII receptor, anti-PD1 antibody, etc.), positive regulators of T cell growth (IL15, IL12, etc.), T cell homing to tumors (chemokine receptors), and closer to the tumor microenvironment (matrix metalloproteinases), or a constitutive CAR and a second inducible CAR, for example, both CARs are encoded by the same lentiviral vector, and for example, inducible CAR is expressed due to the activation of constitutive CAR. The second CAR can target a second tumor antigen, or an antigen (e.g., CD33, mesothelin) expressed on myeloid-derived suppressor cells (MDSC) or an antigen (e.g., CD19) expressed on suppressive B cells. In each case, these inducible proteins will be expressed in the presence of CAR stimulation and downregulated after CAR signaling is terminated.

[0417] 4. STAT5_RE and AP1 / NFκB_RE promoters can be used to regulate the self-expression of CAR protein or T cell function regulatory protein in a defined manner. Greater cytokine (IL2)-dependent response of STAT5_RE ( Figure 3 ) can be used to express proteins that do not directly respond to antigens in a paracrine manner in CAR-T cells near the tumor. Alternatively, since CAR signaling is required for maximum response through this element, proteins can be expressed specifically through AP1 / NFκB_RE in CAR T cells responding to tumors ( Figure 4 ).

[0418] Materials and methods:

[0419] Generation of vectors expressing chimeric antigen receptors (CARs)

[0420] To generate the novel CAR LTG1563 transmembrane domain, a single-chain variable fragment (scFv) derived from FMC-63 mouse hybridoma (FMC-63: AA 1 to 267, GenBank ID: HM852952.1) oriented VL to VH and connected by a (GGGGS)3 flexible intrachain linker (SEQ ID NO: 187) was used. The resulting targeting domain was then linked in frame to a human CD8 hinge (AA 138 to 179, reference sequence ID NP_001759.3), a human TNF receptor superfamily 19 transmembrane domain (TNFRSF19, AA 167 to 196, UntProt sequence ID: Q9NS68), a human 4-1BB co-stimulatory domain (CD137, AA 214 to 255, UniProt sequence ID: Q07011), and a human CD3ζ signaling domain (CD247, AA 52 to 163, reference sequence ID: NP_000725.1.) A leader sequence from the human granulocyte macrophage colony stimulating factor receptor alpha subunit (AA 1 to 22, GenBank ID: EAW98673.1) was included to promote CAR expression at the cell surface. The CAR sequence was codon-optimized and cloned into a third-generation lentiviral plasmid backbone under the regulation of the following inducible promoter regulated by surface antigen or the constitutive MSCV or EF1α promoter as a control.

[0421] Cell lines used to demonstrate CAR activity

[0422] Unless otherwise specified, the Burkitt lymphoma cell line Raji cell line, chronic myeloid leukemia line K562 line and 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 2mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney line 293T was purchased from ATCC and propagated in CD FortiCho medium (Gibco / ThermoFisher Scientific, Grand Island, NY). Single cell clones of cell lines expressing luciferase were generated by stably transducing wild-type tumor lines with lentiviral vectors encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), followed by cloning and selecting luciferase-positive clones.

[0423] Primary human T cells used to demonstrate CAR activity

[0424] Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (Oklahoma Blood Institute, OBI) with written consent from the donors. Processed buffy coats were purchased from OBI (Oklahoma City, OK). CD4- and CD8-microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to purify CD4- and CD8-positive human T cells from buffy coats by positive selection according to the manufacturer's protocol.

[0425] Primary T cell transduction

[0426] Human primary CD4+ and CD8+ T cells from normal donors were cultured in TexMACS medium at 1×10 6 Cells / ml were cultured at a density of 10 cells / ml and CD3 / CD28 GMP T cells were activated with TransAct reagent (all reagents from Miltenyi Biotec) and transduced overnight with LV encoding CAR constructs on day 1 / 2, and the medium was changed on day 2 / 3. Cytokine (IL-2, TNFα; Miltenyi Biotec, Bergisch Gladbach, Germany) supplementation was performed as described. Cultures were propagated until harvested on day 5 to 6 for co-incubation analysis. Immune effector assays (CTL and cytokines)

[0427] For long-term co-incubation assays, target cells expressing CAR T effectors and GFP were propagated as above, and flow cytometric analysis was used to determine the extent of target cell population killing and CAR T population survival and expansion. Cells were gated based on forward and side scatter and live (7AAD-negative) cells. The percentage of surviving cells after co-culture was determined based on GFP positivity for Raji targets and CD3 for CAR T effectors. In addition, CAR T expression in live CD3-positive cells was determined by staining with CD19 Fc peptide followed by anti-Fc (Fab') 2-FL reagent.

[0428] result:

[0429] A fully human CART construct targeting the CD19 antigen was designed by combining a leader peptide sequence derived from GMCSFR, a fully human anti-human CD19 ScFv sequence, a CD8 hinge, a TNFRSF19 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3z activation domain in frame.

[0430] A CAR positive regulatory loop was designed by placing CAR LTG1563 expression under the control of a tandem STAT5 response element followed by a minimal promoter sequence. When IL-2 binds to the natural IL-2 receptor on the surface of T cells, Jak / STAT signaling is initiated, STAT5 translocates to the regulatory region of the CAR promoter and enhances CAR expression. A constitutively expressed EF1α promoter-driven CAR LTG1563 was constructed as a control and tested in parallel with CARs regulated by STAT-5 and AP1 / NFκB. Figure 1 A scheme of positive CAR regulation via (1) IL-2-driven Stat5 and (2) IL-2 / TNFα and CAR-driven AP1 / NFκB-mediated mechanisms is depicted in and in Figure 2 The structure of each of these constructs is depicted in .

[0431] T cells were purified from the blood of two unrelated healthy donors by immunogenic selection using a 1:1 mixture of CD4 and CD8 beads (Miltenyi Biotec). Cells were transduced with the CAR LTG1563 construct in the absence of cytokine supplementation as described in Materials and Methods. Experimental groups included CD19 CAR driven by the EF1α promoter at MOI 10, or self-driven CARs with STAT5_RE CAR LTG1563 and AP1 / NFκB_RE CAR LTG1563 (positively regulated at 0.25% volume / volume LV preparation) ( Figure 3 ). Untransduced cells (UTD) cultured under the same conditions were used as negative controls. On day 5, select groups received IL-2 or TNFα supplementation, and CAR expression was detected by flow cytometry 20 hours later. EF1α-driven CAR expression was 30% to 40% depending on the MOI of transduction and did not change with IL-2 / TNFα supplementation. In contrast, STAT5_RE CAR expression was significantly enhanced by IL-2 supplementation, but not by TNFα supplementation, as expected ( Figure 3 ). AP1 / NFκB_RE CAR expression was enhanced by both IL-2 and TNFα, but to a lesser extent than IL-2-driven expression of STAT5_RE CAR LTG1563.

[0432] In addition, in this experiment, the self-driving properties of both STAT5 and AP1 / NFκB response elements driving the expression of CAR LTG1563 were evaluated by CD19 antigen stimulation ( Figure 4This was performed using CAR T cells from donor A and donor B co-cultured with CD19-positive Raji NHL tumor cells stably expressing GFP. Strikingly, CAR expression was strongly induced in AP1 / NFκB_RE-driven CAR LTG1563 T cells within 20 hours after stimulation ( Figure 4 ), demonstrating the integration of CAR signaling and CAR-dependent cytokine expression when these response elements are used to drive CAR. This stimulation is much greater than what can be observed by simply stimulating these cells with cytokines, indicating a stronger direct response to CAR signaling. This rapid AP1 / NFκB-dependent CAR upregulation is in sharp contrast to the constitutively expressed EF1α-driven CAR LTG1563, which is temporarily reduced by CD19 antigen-mediated CAR stimulation (possibly due to the internalization of these receptors). In contrast, STAT5_RE-driven CAR LTG1563 is induced by co-culturing with CD19+Raji cells, but the time scale is much longer than that observed for AP1 / NFκB-driven CAR LTG1563. This may reflect the functionality of the STAT5 response element, the intermediate steps required for the production of IL2 and the accompanying STAT5 signaling by CAR T cells. The IL2 supplementation of these cells from D3 to D5 after activation significantly increased the induction of CAR LTG1563 expression by the STAT5 element, which may be carried out by triggering STAT-mediated signaling.

[0433] Notably, expression of AP1-NFκB CAR LTG1563 can be re-induced during multiple stimulations with CD19+Raji cells (D6 to D22 after activation), indicating that CAR T cells maintain AP1 / NFκB signaling responsiveness, even during long-term ex vivo culture (data not shown). Importantly, the extent of CAR expression under the control of the AP1 / NFκB promoter temporally correlated well with the number of Raji cells in the co-culture (data not shown). These results indicate that in the presence of cognate CD19 antigen, the AP1 / NFκB element rapidly drives CAR LTG1563 expression, while in the absence of antigen, CAR LTG1563 is rapidly downregulated.

[0434] In the following experiments, T cells were purified from the blood of three unrelated healthy donors by immunogenic selection using a 1:1 mixture of CD4 and CD8 beads (Miltenyi Biotec). Cells were transduced with the CAR LTG1563 construct at D1 after activation in the absence of cytokine supplementation as described in Materials and Methods. Experimental groups included CD19 CAR driven by a constitutive EF1α-derived CAR, STAT5 RE CAR LTG1563, and AP1-NFκB CAR LTG1563 transduced with LV at an MOI of 10 ( Figure 3 ). In a subset of samples, IL2 supplementation (30 IU / mL) was performed from D3 to D6 after activation to induce the expression of both STAT5 RE CAR LTG1563 and AP1 / NFκBCAR LTG1563. CAR LTG1563-dependent cytotoxicity was evaluated by co-culturing CAR T cells with CD19 + Raji NHL cells stably expressing GFP. Very low effector to target ratios (1: 3CAR T: Raji cells) were used, and CD19-dependent cytotoxicity was evaluated by flow cytometry counting of GFP + Raji cells from D6 to D13 (co-culture 1) after activation. The long-term function and expansion of CAR T cells were evaluated by re-stimulating cells with similar effector to target ratios (1: 3 co-culture 1: Raji cells) from D13 to D20 (co-culture 2) after activation.

[0435] CAR T cells co-incubated with target cells were examined for a total of 14 days (co-culture 1 & 2), during which the number of viable Raji and T cells was counted by flow cytometry analysis ( Figure 5A , 5B 5C). Although Raji cells in the Raji-only and UTD control groups continued to grow unhindered until day 8 of the experiment, CAR LTG1563 T cells strongly suppressed Raji expansion in all groups regardless of IL2 priming. Notably, both STAT5 RE and AP1 / NFκB CAR LTG1563 outperformed EF1α CAR LTG1563 in tumor killing throughout the initial co-culture (co-culture 1), as measured on days 1, 2, 3, 6, and 7 after Raji addition ( Figure 5B Strikingly, despite low initial CAR LTG1563 expression in IL2- cultures, both STAT5RE and AP1 / NFκB outperformed the CAR LTG1563 construct constitutively expressing EF1α ( Figure 5BThis potentially suggests that constitutive expression of CAR in the absence of cognate antigen, even for a short period (D2 to D6 after activation), can negatively impact the potency of CAR T cells, possibly due to tonic signaling.

[0436] In addition, long-term analysis of CAR LTG1563-dependent cytotoxicity was evaluated by restimulating CAR T cells with CD19+Raji cells. All CAR groups again demonstrated effective inhibition of Raji expansion (data not shown), with AP1 / NFκB-driven CAR LTG1563 showing superior tumor killing function compared to the constitutively expressed EF1α_CAR LTG1563 ( Figure 5B ). T cells expressing AP1 / NFκB_CAR LTG1563 expanded to a greater extent than T cells expressing STAT5RE_CAR LTG1563 or EF1α_CAR LTG1563 throughout the culture period ( Figure 5C This is likely due to CAR signaling being present only when target CD19+ cells are also present, thereby maintaining the overall health of these cells and leading to concomitant greater T cell expansion.

[0437] Overall, the results demonstrate the superiority of the inducible self-driven AP1 / NFκB RE CAR LTG1563, which is manifested by lower unstimulated CAR expression and lower susceptibility to exhaustion, low-level priming caused by cytokine supplementation, and rapid upregulation and sustained expression in the presence of tumor Raji cells. In addition, in terms of initial activity, superior CTL function was observed compared to the same CAR construct under the control of the constitutive EF1α promoter. This was associated with greater overall expansion of these AP1 / NFκB CAR T cells due to antigen stimulation relative to all other tested constructs.

[0438] Example 2

[0439] Extension of self-driven expression to auxiliary proteins

[0440] In view of the success of the inducible AP1 / NFκB promoter in self-driving high-level CAR LTG1563 expression, especially in the presence of the cognate CAR antigen (CD19), it is expected that the function of the promoter will be extended to auxiliary proteins to provide additional functions for CAR-T cells. The advantage of the CAR antigen-inducible AP1 / NFκB promoter is that any protein under the control of the promoter is highly expressed only when the CAR tumor antigen is present. Therefore, the AP1 / NFκB promoter is used to express proteins that allow CAR-T cells to escape immunosuppression, such as the dominant negative TGF-β receptor (TGFBRIIdn) and the dominant negative programmed cell death protein 1 (PD1dn). These dominant negative receptors are expected to allow CAR-T cells to escape immunosuppression through TGF-β or PD-L1, respectively. The expected advantage of using the AP1 / NFκB promoter is that TGF-β / PD-L1 immunosuppression plays an important role in patients under normal conditions to prevent the occurrence of T cell-mediated autoimmunity. However, the microenvironment of many cancers is characterized by overexpression of TGF-β and / or PD-L1, which can prevent T cell or CAR-T cell-mediated anti-tumor immune responses. Therefore, expressing these dominant negative receptors (especially in the presence of tumor cells) would be highly advantageous.

[0441] Materials and methods:

[0442] Generation of vectors expressing dominant negative receptors

[0443] To generate dominant negative receptors TGFBRIIdn and PD1dn, the proteins were linked in frame to a vector expressing CAR LTG1563 (LTG1563). Downstream of the CAR LTG1563 sequence, a cleaved ribosomal skipping site (FP2AF) consisting of: a consensus furin cleavage site (amino acid: RAKR (SEQ ID NO: 188)) fused to a ribosomal skipping site (P2A) derived from porcine Teschovirus-1 polyprotein (976 to 997 AA, GenBank ID: CAB40546.1, mutated residue P977S), the ribosomal skipping site (P2A) fused to a consensus furin cleavage sequence (amino acid: RAKR (SEQ ID NO: 188)). The resulting CAR LTG1563-FP2AF sequence was co-expressed with a dominant negative TGF-β receptor II sequence (TGFBRII: AA 1 to 191, Uniprot ID: P37173) in the TGFBRIIdn single expression vector (LTG2864 and LTG2867). The resulting CAR LTG1563-FP2AF sequence was co-expressed with a dominant negative PD1 receptor (PD1: AA 1 to 199, Uniprot ID: Q15116) in the PD1dn single expression vector (LTG2864 and LTG2867).

[0444] In the CAR-FP2AF-PD1dn-P2AF-TGFBRIIdn expression vector, CAR LTG1563-FP2AF was co-expressed with the dominant negative PD1 receptor (PD1: AA 1 to 199, Uniprot ID: Q15116) followed by a consensus furin cleavage sequence (amino acids: RAKR (SEQ ID NO: 188)) fused to the ribosomal skipping site (P2A) derived from porcine teschovirus-1 polyprotein (AA 976 to 997, GenBank ID: CAB40546.1, mutated residue P977S), and it was in turn co-expressed with the dominant negative TGF-β receptor II sequence (TGFBRII: AA 1 to 191, Uniprot ID: P37173). The protein sequences were codon-optimized and cloned into a third-generation lentiviral plasmid backbone under the regulation of the inducible promoter AP1 / NFκB regulated by surface antigens or the constitutive EF1α promoter as a control.

[0445] Cell lines used to demonstrate CAR activity

[0446] Unless otherwise specified, the Burkitt lymphoma cell line Raji cell line, chronic myeloid leukemia line K562 line and 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 2mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney line 293T was purchased from ATCC and propagated in CDFortiCho medium (Gibco / Thermo Fisher Scientific, Grand Island, NY). Single cell clones of cell lines expressing luciferase were generated by stably transducing wild-type tumor lines with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), followed by cloning and selecting luciferase-positive clones.

[0447] Primary human T cells used to demonstrate CAR activity

[0448] Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI) with written consent from the donors. Processed buffy coats were purchased from OBI (Oklahoma City, OK). CD4- and CD8-microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to purify CD4- and CD8-positive human T cells from buffy coats by positive selection according to the manufacturer's protocol.

[0449] Primary T cell transduction

[0450] Human primary CD4+ and CD8+ T cells from normal donors were cultured in TexMACS medium at 1×10 6 Cells / ml were cultured at a density of 10 cells / ml and CD3 / CD28 GMP T cells were activated with TransAct reagent (all reagents from Miltenyi Biotec) and transduced overnight with LV encoding CAR constructs on day 1 / 2, and the medium was changed on day 2 / 3. Cytokine (IL-2, TNFβ; Miltenyi Biotec, Bergisch Gladbach, Germany) supplementation was performed as described. Cultures were propagated until harvested on day 5 to 6 for co-incubation analysis. Immune effector assays (CTL and cytokines)

[0451] For long-term co-incubation assays, target cells expressing CAR T effectors and GFP were proliferated as above, and flow cytometric analysis was used to determine the extent of target cell population killing and CAR T population survival and expansion. Cells were gated based on forward and side scatter and live (7AAD-negative) cells. Based on GFP positivity of Raji targets and CD3 of CAR T effectors, the percentage of surviving cells after co-culture was determined. In addition, CAR T expression in live CD3-positive cells was determined by staining with CD19 Fc peptide followed by anti-Fc (Fab') 2-FL reagents. PD1 and TGFBRII expression (covering both transgenic PD1dn and TGFBRIIdn as well as natural PD1 and TGFBRII proteins) was also determined.

[0452] result:

[0453] The self-driving capability of the CAR LTG1563 construct was expanded to include auxiliary components to add additional functionality to the CAR-T cell or CAR. In this case, dominant negative receptors lacking the intracellular signaling domain of TGFBRII (TGFBRIIdn) or PD1 (PD1dn) were co-expressed with the CAR LTG1563 construct via a ribosomal skipping site (P2A). A constitutively expressed EF1α promoter-driven CAR LTG1563 was constructed as a control and tested in parallel with an AP1 / NFκB-regulated CAR with auxiliary components. Figure 6 The structure of each of these CAR constructs with accessory components is depicted in .

[0454] T cells were purified from the blood of two unrelated healthy donors by immunogenic selection using a 1: 1 mixture of CD4 and CD8 beads (Miltenyi Biotec). As described in the materials and methods, cells were transduced with a CAR LTG1563 construct with auxiliary components in the absence of cytokine supplementation. The experimental group included CAR LTG1563, CAR LTG1563-TGFBRIIdn, CAR LTG1563-PD1dn or CAR LTG1563-PD1dn-TGFBRIIdn driven by the EF1α promoter, or CAR LTG1563 driven by AP1 / NFκB_RE, which was a self-driven CAR positively regulated by CAR LTG1563-TGFBRIIdn, CAR LTG1563-PD1dn or CAR LTG1563-PD1dn-TGFBRIIdn, with auxiliary components ( Figure 6). Untransduced cells (UTD) cultured under the same conditions were used as negative controls. The expression of CARLTG1563 and auxiliary components (TGFBRII, PD1) was assessed by flow cytometry; however, the expression of auxiliary components also reflects the expression of the native full-length forms of these proteins on CAR-T cells. Under the control of the constitutive EF1α promoter, significant increases in the expression of these TGFBRII and PD1 proteins were observed for the EF1a-CAR LTG1563-TGFBRIIdn and EF1a-CAR LTG1563-PD1dn constructs, respectively ( Figure 6 ), demonstrating that expression of helper transgenes can be detected above the native expression of these proteins.

[0455] In addition, in this experiment, the self-driving properties of AP1 / NFκB response elements driving CARLTG1563 expression were evaluated by CD19 antigen stimulation ( Figure 6 This was performed using CAR T cells from donor A and donor B co-cultured with CD19-positive Raji NHL tumor cells stably expressing GFP. Remarkably, CAR was strongly induced in AP1 / NFκB_RE-CAR LTG1563-TGFBRIIdn T cells within 20 hours after stimulation ( Figure 6 ) and TGFBRII( Figure 6 This suggests that the AP1 / NFκB promoter can be used to induce proteins other than CAR through the cognate CAR receptor in the presence of antigen signaling.

[0456] In addition, in this experiment, CAR LTG1563-dependent cytotoxicity was evaluated by co-culturing CAR T cells with CD19+Raji NHL cells stably expressing GFP in the presence or absence of the immunosuppressive cytokine TGFβ (10ng / mL). Very low effector to target ratios (1:3CAR T:Raji cells) were used, and CD19-dependent cytotoxicity was evaluated by flow cytometry counting of GFP+Raji cells from D5 to D9 (co-culture 1) after activation. Cells were re-stimulated twice with similar effector to target ratios (1:3 co-culture 1:Raji cells) from D9 to D13 (co-culture 2) after activation, and cells were re-stimulated three times from D13 to D19 (1:3 co-culture 2:Raji cells) after activation to evaluate the long-term function and expansion of CAR T cells.

[0457] CAR T cells co-incubated with target cells were examined for a total of 15 days (co-culture 1 to 3), during which the number of viable Raji and T cells was counted by flow cytometry analysis ( Fig. 8A). Although Raji cells in the Raji-only group and the UTD control group continued to grow unhindered, in the absence of TGFβ, CAR LTG1563 T cells strongly inhibited Raji expansion in all vector conditions, while the AP1NFKB-CAR LTG1563 and EF1a-CAR LTG1563 vectors exhibited poor cytotoxicity against Raji cells in the presence of TGFβ. Strikingly, in the vector EF1a-CAR LTG1563-TGFBRIIdn, the dominant negative receptor TGFBRIIdn largely restored CAR-dependent cytotoxicity in the presence of TGFβ ( Figure 8B ). Similar restoration of CAR-dependent cytotoxicity was observed for the AP1NFKB-CAR LTG1563-TGFBRIIdn vectors, despite lower initial TGFBRII expression in these vectors ( Figure 7B ). This suggests that the inducible nature of the AP1-NFKB promoter can be used to successfully express other proteins besides CAR in a manner regulated by antigen signaling through the CAR. For both the EF1a-CAR LTG1563-TGFBRIIdn and AP1NFKB-CAR LTG1563-TGFBRIIdn vectors, restoration of cytotoxicity in the presence of TGFβ was also associated with a significant increase in CAR LTG1563-dependent T cell expansion throughout co-cultures 1 to 3 ( Figure 8C ). Overall, this demonstrates the successful use and induction of CAR with accessory components as a single vector system using the inducible AP1-NFKB promoter.

[0458] Example 3

[0459] Characterization of CAR19LTG1563 with TGFBRIIdn and PD-1dn decoy components in the presence of TGFβ or PD-L1 ligands

[0460] Materials and methods:

[0461] Cell lines used to demonstrate CAR activity

[0462] Unless otherwise specified, the Burkitt lymphoma cell line Raji cell line, chronic myeloid leukemia line K562 line and 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 2mM L-Glutamax (Thermo Fisher Scientific, Grand Island, NY). The human embryonic kidney line 293T was purchased from ATCC and propagated in CDFortiCho medium (Gibco / Thermo Fisher Scientific, Grand Island, NY). Single cell clones of cell lines expressing luciferase were generated by stably transducing wild-type tumor lines with a lentiviral vector encoding firefly luciferase (Lentigen Technology, Inc., Gaithersburg, MD), followed by cloning and selecting luciferase-positive clones. Cell lines expressing GFP and PDL1 were generated by stably transducing luciferase-activated tumor lines with lentiviral vectors encoding GFP and / or PD-L1 (PDL1: AA 1 to 290, Uniprot ID: Q9NZQ7), followed by magnetic or FACS-based separation of GFP+ and PD-L1+ cells.

[0463] Primary human T cells used to demonstrate CAR activity

[0464] Whole blood was collected from healthy volunteers at the Oklahoma Blood Institute (OBI) with written consent from the donors. Processed buffy coats were purchased from OBI (Oklahoma City, OK). CD4- and CD8-microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were used to purify CD4- and CD8-positive human T cells from buffy coats by positive selection according to the manufacturer's protocol.

[0465] Primary T cell transduction

[0466] Human primary CD4+ and CD8+ T cells from normal donors were cultured in TexMACS medium at 1×10 6 Cells / ml were cultured at a density of 10 cells / ml and CD3 / CD28 GMP T cells were activated with TransAct reagent (all reagents from Miltenyi Biotec) and transduced overnight with LV encoding CAR constructs on day 1, and the medium was changed on day 3. Cytokine (IL-2, TNFβ; Miltenyi Biotec, Bergisch Gladbach, Germany) supplementation was performed as described. Cultures were propagated until harvested on day 8 for co-incubation analysis. Immune effector assays (CTL and cytokines)

[0467] For long-term co-incubation assays, target cells expressing CAR T effectors and GFP were proliferated as above, and flow cytometric analysis was used to determine the extent of target cell population killing and CAR T population survival and expansion. Cells were gated based on forward and side scatter and live (7AAD-negative) cells. Based on GFP positivity for Raji targets and CD3 for CAR T effectors, the percentage of surviving cells after co-culture was determined. In addition, CAR T expression in live CD3-positive cells was determined by staining with CD19 Fc peptide followed by anti-Fc (Fab') 2-FL reagent. PD1 and TGFBRII expression were also determined by flow cytometry.

[0468] result:

[0469] Previous experiments with the CAR LTG1563 construct expressing the accessory component TGFBRIIdn ( Fig. 8A , 8B And 8C) demonstrated significant immunity to the anti-inflammatory effects of the immunosuppressive cytokine TGFβ. Therefore, these results were extended to include additional T cell donors and analyze other parameters (cytokine production, activation marker expression, and exhaustion marker expression) of CAR T efficacy. In brief, a 1:1 mixture of CD4 and CD8 beads (Miltenyi Biotec) was used to purify T cells from the blood of three unrelated healthy donors by immunogenic selection. As described in Materials and Methods, in the presence of IL2 supplementation, cells were transduced with a CAR LTG1563 construct with auxiliary components. The experimental group included CAR LTG1563 or CAR LTG1563-TGFBRIIdn driven by the EF1α promoter, or CAR LTG1563 driven by AP1 / NFκB_RE, which was a self-driven CAR regulated by CAR LTG1563-TGFBRIIdn, with auxiliary components ( Fig. 9AUntransduced cells (UTD) cultured under the same conditions were used as negative controls. The expression of CAR LTG1563 and auxiliary components (TGFBRIIdn) was evaluated by flow cytometry before and after stimulation with CD19 antigen on Raji NHL tumor cells, which showed similar characteristics to previous experiments ( Fig. 8A , 8B and 8C and data not shown).

[0470] In addition, in this experiment, CAR LTG1563-dependent cytotoxicity was evaluated by co-culturing CAR T cells with CD19+ Raji NHL cells stably expressing GFP in the presence or absence of the immunosuppressive cytokine TGFβ (10 ng / mL). A very low effector-to-target ratio (1:3 CAR T: Raji cells) was used and the cells were cultured from D8 to D14 after activation (co-culture 1, Fig. 9A ) Flow cytometry counts of GFP+Raji cells were used to assess CD19-dependent cytotoxicity. The long-term function and expansion of CAR T cells were assessed by secondary restimulation of cells with a similar effector to target ratio (1:3 co-culture 1:Raji cells) from D14 to D20 after activation (co-culture 2). CAR T co-incubated with target cells was examined for a total of 14 days (co-culture 1 to 2), during which the number of live Raji and T cells was counted by flow cytometric analysis ( Fig. 9B ). Although Raji cells in the Raji-only group and the UTD control group continued to grow unhindered, in the absence of TGFβ, CAR LTG1563 T cells strongly inhibited Raji expansion in all vector conditions, while in the presence of TGFβ, T cells transduced with AP1NFKB-CAR LTG1563 and EF1a-CAR LTG1563 exhibited poor cytotoxicity against Raji cells. Strikingly, in the vector EF1a-CAR LTG1563-TGFBRIIdn, the dominant negative receptor TGFBRIIdn largely restored CAR-dependent cytotoxicity in the presence of TGFβ ( Figure 8B ). Similar restoration of CAR-dependent cytotoxicity was also observed for the AP1NFKB-CAR LTG1563-TGFBRIIdn vectors, despite lower initial TGFBRII expression in these vectors (data not shown). These results extend previous findings that the inducible nature of the AP1-NFκB promoter can be used to successfully express other proteins besides CAR in a manner regulated by antigen signaling through the CAR.

[0471] Several other T cell parameters were assessed during the experiment. In the presence of TGFβ, expression of the accessory component TGFBRIIdn restored high levels of proinflammatory cytokines (e.g., IL2) and expression of activation markers (e.g., CD25) (data not shown). TGFβ supplementation of CAR T co-cultures resulted in significantly higher expression levels of the exhaustion marker PD1 ( Fig. 9C ), as measured at the end of co-culture 2 (D20 after activation). Of note, in the presence of TGFβ, a significant decrease in the expression of the exhaustion marker PD1 was observed in both AP1NFKB-CAR LTG1563-TGFBRIIdn and EF1a-CAR LTG1563-TGFBRIIdn CAR T cells relative to CAR T cells expressing AP1NFKB-CAR LTG1563 and EF1a-CAR LTG1563 under the same conditions ( Fig. 9C ). The TGFβ-dependent reduction in PD-1 expression led to the hypothesis that TGFBRIIdn expression protects CAR-T cells not only from the anti-inflammatory effects of TGFβ but also from the negative effects of PD-1 signaling.

[0472] Therefore, the ability of the auxiliary CAR component TGFBRIIdn to protect against the immunosuppressive effects of PD-1 / PD-L1 signaling was evaluated by transducing CD19+ target Raji-GFP NHL cells with a lentiviral vector stably expressing PD-L1 to generate the cell line Raji-GFP-PDL1. Magnetic selection of PDL1 cells was performed, and stable expression of PDL1 on Raji-GFP-PDL1 cells over several weeks was assessed by flow cytometry (data not shown). T cells were purified from the blood of three unrelated healthy donors by immunogenic selection using a 1:1 mixture of CD4 and CD8 beads (Miltenyi Biotec) as described previously. Cells were transduced with the CAR LTG1563 construct with auxiliary components in the presence of IL2 supplementation as described in Materials and Methods. The experimental groups included CAR LTG1563 driven by EF1α promoter or CAR LTG1563-TGFBRIIdn, or CAR LTG1563 with AP1 / NFκB_RE driver, which was a self-driven CAR regulated by CAR LTG1563-TGFBRIIdn, with auxiliary components at 0.5% v / v LV preparation ( Fig. 9A). Untransduced cells (UTD) cultured under the same conditions were used as negative controls. CAR LTG1563-dependent cytotoxicity was evaluated by co-culturing CAR T cells with CD19+Raji-GFP or CD19+Raji-GFP-PDL1 NHL cells in the presence or absence of the immunosuppressive cytokine TGFβ (10 ng / mL). A very low effector-to-target ratio (1:3 CAR T:Raji cells) was used and the cells were cultured from D8 to D14 after activation (co-culture 1, Fig. 10C ) Flow cytometry counts of GFP+Raji cells were used to evaluate CD19-dependent cytotoxicity. The long-term function and expansion of CART cells were evaluated by re-stimulating cells with a similar effector to target ratio (1:3 co-culture 1: Raji cells) from D14 to D20 after activation (co-culture 2). CAR T co-incubated with target cells was examined for a total of 14 days (co-culture 1 to 2), during which the number of live Raji and T cells was counted by flow cytometry analysis ( Fig. 10C ).

[0473] Although PD-L1 expression alone on target Raji NHL cells was not sufficient to significantly affect CAR LTG1563-dependent cytotoxicity ( Fig. 10A ), but...

Claims

1. An isolated nucleic acid molecule encoding a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, the inducible promoter regulated by a surface antigen comprising a nucleotide sequence comprising SEQ ID NO: 138, or a sequence having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto, and wherein the therapeutic payload comprises a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain and at least one intracellular signaling domain, the at least one extracellular antigen binding domain comprising a mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123 or CD38 antigen binding domain, and wherein the inducible promoter regulated by a surface antigen regulates its transcription level according to the expression level of the surface antigen on the target cell, thereby achieving a T cell response precisely regulated to the level of the target antigen present in the tumor environment.

2. A chimeric antigen receptor (CAR) encoded by the isolated nucleic acid molecule of claim 1. A vector comprising the nucleic acid molecule according to claim 1.

4. The vector of claim 3, wherein the vector is selected from a DNA vector, an RNA vector, a plasmid vector, a cosmid vector, a herpes virus vector, a measles virus vector, a lentiviral vector, an adenoviral vector and a retroviral vector, or a combination thereof.

5. An isolated cell comprising the vector of claim 3.

6. The cell of claim 5, wherein the isolated cell is a T cell.

7. The cell of claim 5, wherein the T cell is a CD8 + T cells.

8. The cell of claim 5, wherein the isolated cell is a human cell.

9. A method for preparing cells, comprising transducing T cells with the vector according to claim 3.

10. A method for producing an RNA-engineered cell population, comprising introducing in vitro transcribed RNA or synthetic RNA into cells, wherein the RNA comprises the nucleic acid molecule of claim 1.

11. A pharmaceutical composition comprising an anti-tumor effective amount of a human T cell population, wherein the human T cell population comprises a therapeutic payload operably linked to an inducible promoter regulated by a surface antigen, the inducible promoter regulated by a surface antigen being encoded by a nucleic acid sequence comprising SEQ ID NO: 138 or a combination thereof, wherein the inducible promoter regulated by a surface antigen regulates its transcription level according to the expression level of the surface antigen on the target cell, thereby achieving a T cell response precisely regulated to the level of the target antigen present in the tumor environment, wherein the therapeutic payload comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR), and wherein the CAR comprises at least one extracellular antigen binding domain, at least one linker domain, at least one transmembrane domain, at least one intracellular signaling domain, the at least one extracellular antigen binding domain comprising mesothelin, CD33, CD19, CD19 / CD20, CD22, CD19 / CD22, ROR1, CD123 or CD38 antigen binding domain, or a combination thereof, and wherein the human T cell population is a T cell of a person suffering from cancer.

12. The pharmaceutical composition of claim 11, wherein the T cells are T cells from a human suffering from a hematological cancer.

13. The pharmaceutical composition of claim 12, wherein the hematological cancer is leukemia or lymphoma.

14. The pharmaceutical composition of claim 13, wherein the leukemia is acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), acute lymphocytic T-cell leukemia (T-ALL), or acute lymphocytic B-cell leukemia (B-ALL).

15. The pharmaceutical composition of claim 13, wherein the lymphoma is mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma.

16. The pharmaceutical composition of claim 12, wherein the hematological cancer is multiple myeloma.

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