T cell activation response construct for enhanced CAR-T cell therapy

Abstract

The present disclosure generally relates to a gene circuit comprising a response element engineered from NR4A1 that increases the activation of chimeric antigen receptors by the delivery of bioactive molecules. In particular, the present disclosure provides a nucleic acid construct comprising a response element operably linked to a nucleic acid sequence of interest that responds to the activation of CAR-T cells. The present disclosure also relates to vectors and cells comprising the gene circuit and the nucleic acid construct. Also provided are methods of inducing an immune response, methods of treating a subject, and methods for delivering a bioactive molecule by activated T cells having a chimeric antigen receptor and a response element construct of the present disclosure.

Description

Technical Field

[0001] Statement Regarding Federally Sponsored Research This invention was made with government support under grant number OD025751 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 342,578, filed May 16, 2022. The disclosure of the above - referenced application is hereby expressly incorporated herein by reference in its entirety, including any drawings.

[0003] Incorporation of Sequence Listing The accompanying sequence listing is hereby incorporated by reference into this application. The accompanying XML file of the sequence listing, named 2023 - 05 - 15 Sequence_Listing_ST26 048536 - 728001WO.xml, was created on May 15, 2023 and is 178,800 bytes in size.

[0004] Field This disclosure generally relates to the fields of immunology and medicine. More particularly, this disclosure relates to compositions and methods comprising T - cell activation response constructs. This disclosure also relates to enhanced CAR - T cell therapies comprising said constructs.

Background Art

[0005] Background An important problem limiting the development of engineered cell therapies in humans is the regulation of therapeutic gene expression to reduce or eliminate interactions that cause significant side effects of chimeric antigen receptor T - cell (CAR - T) administration, such as the inability to modulate or stop CAR - T activity when needed.

[0006] High CAR expression can cause antigen-independent CAR signaling, which can result in T cell exhaustion and suboptimal antitumor responses. Uncontrolled high CAR expression can also lead to inappropriate recognition of tumor antigens on self-tissues. Controlling CAR-T cell signaling is also important for the formation of appropriate memory cells.

[0007] Transcriptional regulatory regions containing promoters, enhancers, and / or response elements are extremely important for expressing the transgene at optimal levels in CAR-T cells for the production of functional proteins or non-coding RNAs. Since surface expression of CAR can be limited by mRNA levels, the selection of transcriptional regulatory elements is also important.

[0008] Furthermore, in the process of developing antibody-based therapies using CAR-T, various assays are required to bring them into clinical trials and ultimately screen and identify the best candidates for market release. In particular, T cell activity reporters, and specifically T cell activation response circuits, as well as constructs with tunable promoters and response elements are required.

[0009] It is also desirable to generate T cell activity reporters using natural response elements found within T cells for early response transcription factors after T cell activation.

[0010] Constructs with transcriptional regulatory elements such as response elements for use in CAR-T in vivo and in vitro assays are needed. Also needed are constructs containing response elements that can be modified in a precisely tunable manner for gene expression and / or cell behavior. Also needed are gene circuits that can combine T cell activity with the production of therapeutic payloads (e.g., bioactive molecules). Summary of the Invention Means for Solving the Problems

[0011] Summary The present disclosure generally relates to, among other things, a genetic circuit and nucleic acid construct comprising a series of transcriptional regulatory regions having response elements from the NR4A1 locus that selectively turn on customizable genetic programs in primary human T cells in response to CAR receptor or TCR ligation. In particular, provided herein is (i) a first nucleic acid construct having a transcriptional regulatory region comprising a response element (RE) operably linked to a nucleic acid sequence of interest (NAS); and (ii) a second nucleic acid construct having a nucleic acid sequence encoding a first chimeric antigen receptor (CAR) having specificity for a target antigen. The genetic circuit of the present disclosure is such that activation of the CAR by the target antigen (i.e., the antigen to which the CAR has specificity) activates the T cell, thereby resulting in expression of the NAS under the influence of the RE and delivery of the bioactive molecule encoded by the NAS.

[0012] In one aspect, provided herein is a nucleic acid construct comprising (i) a nucleic acid sequence of interest (NAS), and (ii) a transcriptional regulatory region having a response element (RE) operably linked to a nucleic acid sequence encoding a CAR.

[0013] Non-limiting exemplary embodiments of the genetic circuit or construct according to the present disclosure include one or more of the following features. In some embodiments, the transcriptional regulatory region comprises any one of SEQ ID NOs: 2-12, or a functional variant thereof comprising a sequence having about 85% to about 99% sequence identity to SEQ ID NOs: 2-12.

[0014] In some embodiments, the disclosed gene circuits or nucleic acid constructs include at least one copy of a response element. In some embodiments, the NAS encodes a bioactive molecule. In some embodiments, the bioactive molecule includes an antibody, a nanobody, a diabody, a triabody, a minibody, an F(ab)2 fragment, an F(ab)v fragment, a single-chain variable fragment (scFv), a single-domain antibody (sdAb), or a functional fragment thereof. In some embodiments, the bioactive molecule includes a ligand, a short hairpin RNA (shRNA), or a microRNA (miRNA). In some embodiments, the bioactive molecule is an anti-programmed cell death-1 (anti-PD1) or an anti-programmed cell death-1 ligand 1 (anti-PDL1). In some embodiments of the constructs of the present disclosure, the ligand is a secreted ligand, or CD40L or a derivative thereof.

[0015] In another aspect, the gene circuits or constructs of the present disclosure include a NAS that encodes a second CAR. In some embodiments, the second CAR includes a signaling domain different from the first CAR. In some embodiments, the different signaling domains are 4-1BB, CD28, ICOS, CD2, BAFFR, TACI, CD30, NTB-A.

[0016] In some embodiments of the gene circuits or constructs of the present invention, the NAS encodes a reporter molecule. In some embodiments, the reporter molecule is GFP, enhanced green fluorescent protein (EGFP), Cherry, BFP, luciferase, Nanoluc™, herpes simplex virus thymidine kinase, or a variant thereof.

[0017] In one aspect, the target antigen for the CAR is HER-2, CD-19, GD2, PSMA, CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placenta-like 2 (ALPPL2), B cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), signal regulatory protein alpha (SIRPα), or CTLA-4.

[0018] In some embodiments, activation of the response element results in the expression of NAS.

[0019] In some embodiments, the transcriptional regulatory region includes a promoter. In some embodiments, the promoter is a minimal TATA promoter, a minimal CMV promoter, a minimal IL-2 promoter, a synthetic inducible promoter, a natural inducible promoter, a pGK promoter, an SFFV or EF1α promoter, or a functional variant thereof.

[0020] In one aspect, the present disclosure provides a nucleic acid construct comprising SEQ ID NOs: 6 to 12 or a functional variant thereof.

[0021] In some embodiments, the gene circuit is encoded by SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 27, or a functional variant of any of them.

[0022] In some embodiments of the gene circuit of the present disclosure, the first nucleic acid construct and the second nucleic acid construct are present tandemly on a single nucleic acid molecule. In some embodiments of the gene circuit of the present disclosure, the first nucleic acid construct and the second nucleic acid construct are present on separate nucleic acid molecules.

[0023] In one aspect, the present disclosure provides a vector comprising the gene circuit or nucleic acid construct of the present disclosure. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a lentiviral vector.

[0024] In another aspect, the present disclosure provides a recombinant cell comprising the gene circuit or construct of the present disclosure. In some embodiments, the recombinant cell is transduced by the vector provided herein. In some embodiments, the recombinant cell is an immune cell. In some embodiments, the cell is a regulatory T cell, a helper T cell, a cytotoxic T cell, a CAR-expressing reporter T (CAR-T) cell, a CD4+ T cell, a CD8+ T cell, or other T cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a non-human primate cell.

[0025] Also provided by the present disclosure is a method for inducing an immune response in a subject, the method comprising administering to the subject a) the gene circuit of the present disclosure, b) a vector, or c) a recombinant cell.

[0026] Also provided herein is a method for treating a health condition in a subject in need of treatment of the health condition, the method comprising administering to the subject a) the gene circuit of the present disclosure, or b) a vector or c) a recombinant cell.

[0027] Further provided herein is a method of treating a subject in need thereof using a combination therapy, the method comprising a T cell therapy and a second therapy, the second therapy comprising administering to the subject a) a gene circuit or vector or c) a recombinant cell of the present disclosure.

[0028] In some embodiments, the subject has cancer or an autoimmune disease. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is multiple myeloma.

[0029] In another aspect, provided herein is a method for delivering a bioactive molecule by T cells, the T cells comprising a) a CAR having specificity for a target antigen and b) a construct comprising at least one response element operably linked to a nucleic acid sequence encoding the bioactive molecule, wherein activation of the T cells by binding of the CAR to the target antigen results in expression of the nucleic acid sequence and delivery of the bioactive molecule by the T cells.

[0030] In some embodiments, the response element of the present disclosure comprises SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or a functional variant of any of them. In some embodiments, the CAR is constitutively expressed.

[0031] In some embodiments, the bioactive molecule is an antibody, nanobody, diabody, triabody, minibody, F(ab)2 fragment, F(ab)v fragment, single-chain variable fragment (scFv), single-domain antibody (sdAb), or a functional fragment thereof. In some embodiments of the method, the bioactive molecule is a ligand, short hairpin RNA (shRNA), or microRNA (miRNA). In some embodiments, the ligand is a secreted ligand, or CD40L or a derivative thereof. In some embodiments, the bioactive molecule is anti-programmed cell death-1 (anti-PD1) or anti-programmed cell death-1 ligand 1 (anti-PDL1).

[0032] In some embodiments, the bioactive molecule comprises a second CAR against a second target antigen. In some embodiments, activation of the T cell by binding of the CAR to the target antigen results in expression of the second CAR, thereby enabling the T cell to target cells expressing the second target antigen. In some embodiments, the second CAR comprises a signaling domain different from that of the CAR of a).

[0033] Another aspect of the disclosure relates to a T cell comprising: a) a first nucleic acid construct having a first promoter operably linked to a nucleic acid sequence encoding a CAR having specificity for a target antigen; and b) a second nucleic acid construct having a transcriptional regulatory region comprising a response element (RE) operably linked to a second promoter and a nucleic acid sequence of interest (NAS) encoding a bioactive molecule, wherein activation of the T cell by binding of the CAR to the target antigen results in expression of the NAS. In some embodiments of the T cells of the disclosure, the first promoter is a constitutive promoter.

[0034] In one aspect, the T cells of the disclosure comprise the first nucleic acid construct and the second nucleic acid construct tandemly on the same nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 27, or a functional variant of any of them.

[0035] The foregoing summary is illustrative only and is not intended to be limiting in any way. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become fully apparent from the drawings and the detailed description and claims.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0042] Detailed Description of the Present Disclosure The present disclosure provides, among other things, a gene circuit and a nucleic acid construct comprising a series of transcriptional regulatory regions having response elements from the NR4A1 locus that selectively turn on a customizable gene program in primary human T cells in response to CAR receptor or TCR ligation.

[0043] The gene circuit of the present disclosure is engineered such that activation of the CAR in the gene circuit by a target antigen activates the host T cell, thereby resulting in the expression of a nucleic acid sequence of interest in the gene circuit under the control of the response element and the delivery of an active molecule encoded by the sequence of interest.

[0044] The present disclosure also provides constructs comprising a transcriptional regulatory region having response elements operably linked to a CAR(s) and a nucleic acid sequence of interest, which can span various activation kinetics and sensitivity levels that can be selected for a particular output requirement. The constructs of the present disclosure can be efficiently engineered and delivered to T cells via a single lentiviral vector or by a dual vector system. The present disclosure also provides constructs having a transcriptional regulatory region comprising any one of SEQ ID NOs: 2-12, or a functional variant thereof having a sequence identity of about 85% to about 99% to SEQ ID NOs: 2-12. The present disclosure also provides vectors and recombinant cells comprising the constructs of the present disclosure, methods useful for inducing an immune response in a subject, methods for preventing and / or treating various health conditions, and methods for delivering a bioactive molecule by T cells.

[0045] The present disclosure provides T cells comprising a CAR having specificity for a target antigen such that when the CAR binds to the target antigen, the T cells are activated. Activation of the T cells results in the expression of the nucleic acid of interest under the control of the response element.

[0046] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like numerals generally identify like components throughout the several views, unless context dictates otherwise. The exemplary alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented herein. Aspects described herein in their entirety and illustrated in the drawings may be arranged, substituted, combined, and designed in a variety of different configurations, all of which are explicitly contemplated and will be readily understood to form part of this application.

[0047] I. Definitions Unless otherwise defined, all technical terms, notations, and other scientific or technical terms or specialized terms used in this specification are intended to have the meanings commonly understood by those skilled in the art to which this disclosure pertains. In some cases, terms having commonly understood meanings are defined in this specification for the purposes of clarification and / or for ready reference, and including such definitions in this specification should not necessarily be construed as representing a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced in this specification are well understood and commonly employed by those skilled in the art using conventional methodologies.

[0048] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "cell" includes one or more cells, including mixtures thereof. As used herein, "A and / or B" is used to include all of the following alternatives: "A," "B," "A or B," and "A and B."

[0049] When a range of values is provided, unless the context clearly indicates otherwise, each intervening value between the upper and lower limits of that range, to one tenth of the unit of the lower limit, as well as any other recited value or intervening value within the recited range, is understood to be included within the present disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also included within the present disclosure, subject to any specifically excluded limitations within the recited range. When the recited range includes one or both of the limits, ranges excluding one or both of those included limits are also included in the present disclosure. A particular range is presented herein with the numerical value preceded by the term “about,” and when used herein, this term has its ordinary meaning of approximately. The term “about” is used to provide literal support for the exact number that it precedes, as well as numbers that are near to or approximate the number that the term precedes. In determining whether a number is near to or approximate a specifically recited number, an unrecited number that is near to or approximate may be a number that provides substantially the same thing as the specifically recited number in the context in which it is presented. When the degree of approximation is not clear from the context, “about” means within plus or minus 10% of the provided value, or rounded to the nearest significant digit, in all cases in which the provided value is included. In some embodiments, the term “about” indicates plus or minus 10%, plus or minus 5%, or plus or minus 1% of the specified value.

[0050] As used herein, the terms “administer” and “administering” refer to the delivery of a bioactive composition or formulation by a route of administration including, but not limited to, oral, intranasal, transdermal, intravenous, intraarterial, intramuscular, intraarticular, intraperitoneal, subcutaneous, and intramuscular administration, or combinations thereof. The term includes, but is not limited to, administration by a medical professional and self-administration.

[0051] As used herein, the terms “recombinant” or “engineered” nucleic acid molecule, polypeptide, or cell refer to a nucleic acid molecule, polypeptide, or cell that has been altered by human intervention.

[0052] The terms "cell", "cell culture", and "cell line" refer not only to a particular target cell, cell culture, or cell line, but also to the progeny or potential progeny of such a cell, cell culture, or cell line, regardless of the number of transfers or passages of the culture. It should be understood that not all progeny are exactly identical to the parental cells. This is because certain modifications may occur in subsequent generations due to either mutations (e.g., intentional or unintentional mutations) or environmental effects (e.g., methylation or other epigenetic modifications), and as a result, the progeny may not actually be identical to the parental cells, but are still included within the scope of the terms used herein as long as the progeny retain the same functionality as the original cell, cell culture, or cell line.

[0053] The aspects and embodiments of the present disclosure described herein are understood to include the plurality of aspects and embodiments "comprising", "consisting of", and "consisting essentially of". As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising", particularly in the description of the components of a composition or the steps of a method, is understood to encompass compositions and methods consisting essentially of, and consisting of, the recited components or steps.

[0054] The term "cancer" refers to the presence of cells having characteristics typical of cancer-causing cells, such as uncontrolled growth, immortality, metastatic potential, rapid growth and proliferation rates, and certain characteristic morphological features. Some types of cancer cells can aggregate into masses like tumors, while some cancer cells can exist alone within a subject. Tumors can be solid tumors, soft tissue tumors, or metastatic lesions. As used herein, the term "cancer" also includes other types of non-tumor cancers. Non-limiting examples include blood cancers or hematological malignancies such as leukemia, lymphoma, and myeloma. Cancers can include pre-malignant, and malignant cancers.

[0055] The term "construct" refers to a recombinant molecule, e.g., a recombinant nucleic acid or polypeptide, that contains one or more nucleic acid sequences or amino acid sequences from disparate sources. For example, a polypeptide construct can be a chimeric polypeptide molecule in which two or more amino acid sequences of different origin are operably linked to one another in a single polypeptide construct. Similarly, a nucleic acid construct can be a chimeric nucleic acid molecule in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule. Representative nucleic acid constructs include any recombinant nucleic acid molecule, linear or circular, single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, such as plasmids, cosmids, viruses, autonomously replicating polynucleotide molecules, phages, etc., that contain a nucleic acid molecule in which one or more nucleic acid sequences are operably linked and are capable of genomic integration or autonomous replication. Two or more nucleic acid constructs can be contained within a single nucleic acid molecule, such as a single vector, or within two or more separate nucleic acid molecules, such as two or more separate vectors.

[0056] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein are applicable. Accordingly, the term includes, but is not limited to, genes and gene products from human and mouse. When a gene or gene product from a particular species is disclosed, it is intended that the disclosure be exemplary only and not be construed as limiting unless the context in which it appears clearly indicates otherwise. Thus, for example, in some embodiments, for genes or gene products disclosed herein with respect to mammalian nucleic acid and amino acid sequences, homologs and / or orthologous genes and gene products from other animals, including but not limited to other mammals, fish, amphibians, reptiles, and birds, are intended to be encompassed. In some embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides, and proteins are human. The term "gene" is also intended to include variants thereof.

[0057] As used herein, the term "nucleic acid" is used with respect to either DNA or RNA, or a molecule containing deoxynucleotides and / or ribonucleotides. Nucleic acids may be naturally occurring or synthetically produced, and thus include analogs of naturally occurring polynucleotides in which one or more nucleotides are modified relative to the naturally occurring nucleotides.

[0058] As used herein, the term "promoter" generally refers to a DNA molecule that is involved in the recognition and binding of other proteins, such as RNA polymerase II and trans-acting transcription factors, to initiate transcription. A promoter may be derived from the 5' untranslated region (5'UTR) of a gene. Alternatively, a promoter may be a synthetically produced or engineered DNA molecule. A promoter may also be chimeric. A chimeric promoter is produced by the fusion of two or more heterologous DNA molecules. Promoters useful in the practice of the present invention include, but are not limited to, the minimal TATA promoter, the minimal CMV promoter, the minimal IL-2 promoter, synthetic inducible promoters, natural inducible promoters, the pGK promoter, the SFFV or EF1α promoter, or functional variants thereof.

[0059] As used herein, the term "operably linked" indicates a physical or functional linkage between two or more elements, such as polypeptide sequences or polynucleotide sequences, that enables them to operate in the intended manner.

[0060] As used herein in the context of two or more nucleic acid sequences or proteins, the term "percent identity" refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same when measured, for example, using the BLAST or BLAST 2.0 sequence comparison algorithms of the National Center for Biotechnology (NCBI) with the default parameters described below, or by manual alignment and visual inspection (e.g., over a particular region having, for example, about 50% or higher sequence identity, such as about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region). Thus, such sequences are said to be "substantially identical." This definition also refers to, or can apply to, the complement of a sequence. This definition also includes sequences having deletions and / or additions, as well as sequences having substitutions. Sequence identity can be calculated using published techniques and generally available computer programs such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol. Biol. 215:403, 1990 (which is hereby incorporated by reference in its entirety)), etc. Sequence identity can be measured using the default parameters of sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group of the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705).For example, an amino acid sequence that is "substantially identical" to a reference sequence has at least about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity to the reference amino acid sequence, including all values between these. In the case of polypeptides, the length of the comparison sequence is generally at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 50, at least 75, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, or at least 350 contiguous amino acids (e.g., the full-length sequence), including all values between these. In the case of nucleic acids, the length of the comparison sequence is generally at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or 25 contiguous nucleotides, including all values between these (e.g., the full-length nucleotide sequence).

[0061] As used herein, "subject" or "individual" includes animals such as humans (e.g., human individuals) and non-human animals. In some embodiments, the "subject" or "individual" is a patient under the care of a physician. Thus, a subject can be a human patient or individual who has, is at risk of having, or is suspected of having a target disease (e.g., cancer) and / or one or more symptoms of the disease. A subject can also be an individual diagnosed with having a risk of the target condition at the time of diagnosis or thereafter. The term "non-human animal" includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non-mammals, e.g., non-human primates, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0062] When used in reference to a nucleic acid, the term "recombinant" means that the nucleic acid has been altered or produced by human intervention, such as, for example, being modified or the result of being modified by laboratory methods. Thus, for example, recombinant nucleic acids include viral genomes and nucleic acids produced by laboratory methods. A recombinant protein or polypeptide produced by a recombinant construct may contain amino acid residues not found within the native (non-recombinant or wild-type) form of the protein, or may be modified, for example, may contain labeled amino acid residues. The term may include any modification to a peptide, protein, or nucleic acid sequence. Such modifications include, for example: any chemical modification of a peptide, protein, or nucleic acid sequence that includes one or more amino acids, deoxyribonucleotides, or ribonucleotides; addition, deletion, and / or substitution of one or more amino acids in a peptide or protein; production of a fusion protein, such as a fusion protein that includes an antibody fragment; and addition, deletion, and / or substitution of one or more nucleic acids in a nucleic acid sequence.

[0063] As used herein, the term "recombinant" polypeptide refers to a polypeptide that has been altered by human intervention. By way of non-limiting example, an engineered polypeptide can be one that has been synthesized or modified in vitro, for example, using chemical or enzymatic techniques; one that includes linked polypeptide sequences that are not linked in nature; one that has been engineered using molecular cloning techniques such that one or more amino acids are missing relative to a naturally occurring polypeptide sequence; and / or one that has been engineered using molecular cloning techniques such that it has one or more sequence changes or rearrangements relative to a naturally occurring polypeptide.

[0064] For purposes of describing the positions of two constructs or nucleic acid sequences of the same molecule, as used herein, the term "tandem" refers to constructs that are arranged adjacent to one another.

[0065] As will be understood by those skilled in the art, for all purposes, such as providing an explanation in writing, all ranges disclosed herein include all possible sub-ranges and combinations of sub-ranges of that range. Any recited range can be fully described and enabled as being divisible into at least equal halves, thirds, fourths, fifths, tenths, etc. of the same range. By way of non-limiting example, each range discussed herein can be readily divided into lower thirds, middle thirds, and upper thirds, etc. Also, as will be understood by those skilled in the art, all language such as "up to", "at least", "greater than", "less than", etc. includes the recited number and refers to a range that can later be divided into sub-ranges as described above. Finally, as will be understood by those skilled in the art, a range includes each of its individual members. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0066] It is understood that, for clarity, certain features of the present disclosure that are described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the present disclosure that are described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of embodiments related to the present disclosure are specifically encompassed by the present disclosure and are disclosed herein as if each such combination were individually and explicitly disclosed. In addition, all sub-combinations of various embodiments and their elements are also specifically encompassed by the present disclosure and are disclosed herein as if each such sub-combination were individually and explicitly disclosed herein.

[0067] The various features of the present disclosure may be described in the context of a single embodiment, but the features may also be provided separately or in any suitable combination. Conversely, the present disclosure may be described herein in the context of separate embodiments for clarity, but the present disclosure may also be implemented in a single embodiment.

[0068] II. Compositions A. Gene Circuits and Constructs The present disclosure provides a gene circuit comprising: (i) a first nucleic acid construct having a transcriptional regulatory region comprising a response element (RE) operably linked to a nucleic acid sequence of interest (NAS); and (ii) a second nucleic acid construct having a nucleic acid sequence encoding a first chimeric antigen receptor (CAR) having specificity for a target antigen. The gene circuit of the present disclosure is such that activation of the CAR by the target antigen, which is specific, activates T cells, thereby resulting in the expression of the NAS under the influence of the RE and the delivery of a bioactive molecule encoded by the NAS.

[0069] In some embodiments of the gene circuit of the present disclosure, the first nucleic acid construct and the second nucleic acid construct are in proximity to each other on a single nucleic acid molecule. In some embodiments, the gene circuit of the present disclosure can be delivered to cells via a single vector system.

[0070] In some embodiments of the gene circuit of the present disclosure, the first nucleic acid construct and the second nucleic acid construct are not in proximity and are on separate nucleic acid molecules. In some embodiments, the gene circuit can be delivered via a dual vector system.

[0071] The present disclosure also provides, inter alia, constructs having a transcriptional regulatory region operably linked to a downstream sequence comprising, for example, a nucleic acid sequence (NAS) and a nucleic acid encoding a chimeric antigen receptor (CAR).

[0072] An operable linkage between a nucleic acid molecule or coding sequence described herein and a response element and / or promoter sequence can be a linkage such that the sequences are in-frame and appropriately spatially and distantly separated to allow for the effect of the respective binding by a transcription factor or RNA polymerase on transcription.

[0073] The transcriptional regulatory region of the present disclosure is a region upstream of a nucleic acid sequence encoding a polypeptide or nucleic acid sequence of interest. The transcriptional regulatory region can include one or more elements that regulate the transcription of the downstream sequence. The transcriptional regulatory region can include response elements, enhancers, promoters, mini-promoters (e.g., mini TATA promoters), and / or other regulatory elements that can direct or assist in directing the transcription of sequences downstream of the transcriptional regulatory region in a construct. Thus, the transcriptional regulatory region of the present disclosure can direct the transcription of downstream sequences in the constructs of the present disclosure.

[0074] In some embodiments, the transcriptional regulatory region of the constructs of the present disclosure includes a promoter. The promoter can be constitutive, inducible, activatable, repressible, tissue-specific, cell-specific, cell-state specific, or any combination thereof. Any promoter and / or promoter / operator known in the art can be used to control the expression of the output array(s). Non-limiting examples of promoters include the minimal TATA promoter, the minimal CMV promoter, the minimal IL-2 promoter, synthetic inducible promoters, natural inducible promoters, the pGK promoter, the SFFV or EF1α promoter, the NR4A1 promoter (also referred to as the Nur77 promoter), or functional variants thereof. A functional variant of a nucleic acid retains the same function as the nucleic acid. When used in reference to a nucleic acid sequence, the term "variant" refers to a nucleic acid sequence that is different from another, usually related nucleotide acid sequence by one or more nucleotides. Thus, the term "variant" can refer to a change in one or more nucleotides of a reference nucleic acid that includes the insertion of one or more new nucleotides, the deletion of one or more nucleotides, and the substitution of one or more existing nucleotides. Variants also include point mutations, multiple mutations, single nucleotide polymorphisms (SNPs), deletions, insertions, and translocations. A functional variant of a response element or promoter is a variant that retains the same function as the promoter or response element. A functional variant of a nucleic acid encoding a polypeptide allows for different nucleotides that encode one or more "conservative amino acid substitutions" in the polypeptide. A functional variant of a polypeptide can include a coding sequence of a polypeptide having an amino acid sequence that is the same as or essentially the same as the amino acid sequence of a reference polypeptide, except that at least one amino acid has been modified, e.g., deleted, inserted, or replaced. The amino acid replacement can preferably be a conservative amino acid substitution at a non-essential amino acid residue in the protein. A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art.These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Variants of the protein can have an amino acid sequence that is at least about 80%, 90%, 95%, or 99% identical to the amino acid sequence of the protein, preferably at least about 90%, more preferably at least about 95%. Preferably, the variant is a functional variant of the protein that retains the same function as the protein.

[0075] In some embodiments, the transcriptional regulatory region of the constructs of the disclosure comprises an NR4A1 promoter or a portion thereof that contains an element recognized by a response element or a transcription factor.

[0076] The transcriptional regulatory region can include a response element operably linked or ligated to another sequence. In some embodiments, the transcriptional regulatory region includes a response element operably linked to a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid sequence of interest (NAS). In some embodiments, the NAS is a coding sequence encoding a polypeptide. In some embodiments, the NAS can be a coding sequence (gene) having 5' and 3' sequences. In some embodiments, the NAS encodes an RNA molecule.

[0077] The response elements of the disclosure can be from various species. In some embodiments, the response element is from a mouse. In some embodiments, the response element is from a rat. In some embodiments, the response element is from a human.

[0078] As used herein, the term "response element" refers to a nucleic acid sequence or DNA sequence within a gene promoter or enhancer that can bind to a specific transcription factor that regulates gene transcription. The transcription factor can be a gene repressor or activator. The response element derived from the NR4A1 locus described herein can be a sequence of any length derived from the NRAA1 promoter or enhancer region that can bind to the transcription factor(s). The response element can be a member of the NR4A1 family of transcription factors, referred to as the "response element of the NR4A1 family" or simply "response element(s).

[0079] In some embodiments, the response element is or comprises NR4A1 (SEQ ID NO: 2). In some embodiments, the response element is human NR4A1.

[0080] The response element may be "NIR ABC". In some embodiments, the response element is rat "NIR ABC". In some embodiments, rat "NIR ABC" is or comprises SEQ ID NO: 3. In some embodiments, the constructs of the present disclosure comprise a NIR ABC response element as shown in FIGS. 4A and 4B.

[0081] The response element may be the response element "496". In some embodiments, the response element is "hu496". In some embodiments, "hu496" is or comprises SEQ ID NO: 4.

[0082] The response element may be the response element "319". In some embodiments, the response element is "hu319". In some embodiments, "hu319" is or comprises SEQ ID NO: 5. In some embodiments, the constructs of the present disclosure comprise a hu319 response element as shown in FIG. 6.

[0083] The response element may be or may include array numbers 2, 3, 4, 5, 6, 7, or 8, 9, 10, 11, or 12.

[0084] The response element may include functional variants of array numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. A functional variant of a response element of array numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 may be any sequence that functions in a manner similar to array numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 such that when linked to a nucleic acid of interest, for example, it results in transcriptional expression or enhanced transcriptional expression of the nucleic acid of interest.

[0085] Functional variants may include sequences having about 85% to about 99% sequence identity to array numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the functional variant has about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or any value in between sequence identity to array numbers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0086] The constructs of the present disclosure can have any number of copies of the response element. In some embodiments, the construct comprises one copy of the response element. In some embodiments, the construct comprises two copies of the response element. In some embodiments, the construct comprises three copies of the response element. In some embodiments, the construct comprises four copies of the response element.

[0087] In some embodiments, the construct comprises a combination of different response elements. In some embodiments, the gene circuit or construct comprises a cleavage or deletion of a response element of the present disclosure.

[0088] The constructs of the present disclosure can have a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a transcriptional regulatory region operably linked to a nucleic acid sequence of interest (NAS). In some embodiments, the NAS is one gene.

[0089] The nucleic acid of interest can be a gene encoding a bioactive molecule. In some embodiments, the bioactive molecule is an antibody. In some embodiments, the bioactive molecule is a nanobody. In some embodiments, the bioactive molecule is a diabody. In some embodiments, the bioactive molecule is a triabody. In some embodiments, the bioactive molecule is a minibody. In some embodiments, the bioactive molecule is an F(ab)2 fragment. In some embodiments, the bioactive molecule is an F(ab)v fragment. In some embodiments, the bioactive molecule is a single-chain variable fragment (scFv). In some embodiments, the bioactive molecule is a single-domain antibody (sdAb) or a functional fragment thereof.

[0090] In one aspect, the constructs of the present disclosure can comprise a bioactive molecule that is a ligand. In some embodiments, the ligand is a secreted ligand. In some embodiments, the secreted ligand is CD40L or a derivative thereof.

[0091] In another aspect, the constructs of the present disclosure can comprise a bioactive molecule that is a short hairpin RNA (shRNA) or a microRNA (miRNA).

[0092] In yet another aspect, the constructs of the present disclosure can include a bioactive molecule that is an anti-programmed cell death-1 (anti-PD1) or anti-programmed cell death-1 ligand 1 (anti-PDL1).

[0093] The constructs of the present disclosure can include a CAR. In some embodiments, the constructs of the present disclosure can include two CARs. In certain embodiments, the NAS can encode a second CAR. The second CAR can include a signaling domain different from the first CAR. Non-limiting examples of signaling domains include 4-1BB, CD28, ICOS, CD2, BAFFR, TACI, CD30, NTB-A, and others.

[0094] Non-limiting examples of CARs include HER-2, CD-19, GD2, PSMA, CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placenta-like 2 (ALPPL2), B cell maturation antigen (BCMA), signal regulatory protein alpha (SIRPα), or a CAR against CTLA-4.

[0095] The constructs of the present disclosure may include a constitutive CAR, for example, as shown in FIG. 6. In some embodiments, the constitutive CAR is a Myc-ALPPL2 CAR.

[0096] In certain aspects of the present disclosure, the nucleic acid sequence of interest (NAS) can be a gene encoding a reporter molecule. In some embodiments, the reporter molecule is green fluorescent protein (GFP). In some embodiments, the reporter molecule is enhanced green fluorescent protein (eGFP). In some embodiments, the reporter molecule is Cherry. In some embodiments, the reporter molecule is blue fluorescent protein (BFP). In some embodiments, the reporter molecule is luciferase. In some embodiments, the reporter molecule is Nanoluc™. In some embodiments, the reporter molecule is herpes simplex virus thymidine kinase or a variant thereof.

[0097] In some embodiments, the construct includes a BFP reporter gene and a constitutive PGK-driven mCherry marker gene, as shown in FIG. 1A. In some embodiments, the construct is or comprises SEQ ID NO: 1.

[0098] The constructs of the present disclosure can be chimeric or recombinant molecules comprising one or more isolated nucleic acid sequences from heterologous sources. For example, a construct can be a chimeric nucleic acid molecule in which two or more nucleic acid sequences of different origins are assembled into a single nucleic acid molecule. Thus, representative nucleic acid constructs include (1) nucleic acid sequences that contain regulatory and coding sequences that are not found to be adjacent to each other in nature (e.g., at least one of the nucleotide sequences is heterologous to at least one of the other nucleotide sequences), or (2) sequences that encode a portion of a functional nucleic acid molecule that is not adjacent in nature, or (3) any construct that contains a portion of a promoter that is not adjacent in nature. Representative nucleic acid constructs include any chimeric nucleic acid molecule, linear or circular, single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, etc., that contains a nucleic acid molecule in which one or more nucleic acid sequences are operably linked and are capable of genomic integration or autonomous replication.

[0099] In some embodiments, the constructs of the present disclosure are, or comprise, SEQ ID NO: 1, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 27, or functional variants thereof.

[0100] The constructs of the present disclosure can be used as T cell activity reporters in in vitro and in vivo assays. The constructs of the present disclosure can be, for example, part of a gene circuit (also referred to as a response element circuit) designed to cooperate with a CAR-T cell receptor to enhance T cell therapy in hematological malignancies such as multiple myeloma and solid tumors. The constructs can, for example, combine T cell activation with the production of a therapeutic payload (e.g., a bioactive molecule), or drive cell fate in different lineages, and thus combine T cell activation by a primary CAR with a secondary CAR to optimize T cell function after encounter with an antigen. For example, as shown in FIG. 5, the construct can comprise a constitutively expressed Myc-ALPPL2 CAR, which can disrupt the ALPPL2 target, activate T cells, and induce a payload when engaged with the ALPPL2 target antigen. The payload can be a CAR with mesothelin co-stimulation only without CD3z, or a mesothelin complete CAR containing CD28 co-stimulation and CD3z. Then, expression of this CAR payload should enable T cells to target cells expressing mesothelin.

[0101] B. Vector The present disclosure also provides vectors incorporating the gene circuits and / or constructs of the present disclosure.

[0102] In some embodiments, the gene circuit and / or construct is incorporated into an expression vector designed for movement between host cells and can be used for the purpose of transformation, e.g., introduction of heterologous DNA into a host cell. Thus, in some embodiments, the vector can be a replicon such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to effect replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector.

[0103] In addition to the components of the gene or construct, the vector may include, for example, one or more selectable markers, one or more origins of replication such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and / or elements to facilitate stable integration of the construct into the genome of the cell. Two or more constructs may be incorporated into a single nucleic acid molecule such as a single vector (Figures 4A and 5), or may be contained within two or more separate nucleic acid molecules such as two or more separate vectors (Figures 1A and 4B).

[0104] In some embodiments, the expression vector can be a viral vector. As will be understood by those skilled in the art, the term "viral vector" generally refers to a nucleic acid molecule (e.g., a transfer plasmid) that contains viral-derived nucleic acid elements that facilitate the transfer or integration of nucleic acid molecules into the genome of a cell, or to a viral particle that mediates nucleic acid transfer. Viral particles generally contain, in addition to the nucleic acid(s), various viral components and sometimes also host cell components. The term "viral vector" can refer to either a virus or viral particle that can transfer nucleic acid into a cell, or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional gene elements mainly derived from viruses.

[0105] The constructs of the present disclosure may be incorporated into a retroviral vector. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional gene elements or portions thereof mainly derived from a retrovirus. In some embodiments, the constructs of the present disclosure may be incorporated into a lentiviral vector. A lentiviral vector may include structural and functional gene elements or portions thereof that include LTRs mainly derived from lentiviruses, which are members of the retrovirus genus.

[0106] Viral vectors that can be used in the present disclosure include, for example, adenoviral vectors, adeno-associated viral vectors, herpesviruses, simian virus 40 (SV40), and bovine papillomavirus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). For example, the constructs disclosed herein can be produced in eukaryotic hosts such as mammalian cells (e.g., K562 cells, COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). Care should be taken to ensure that the components are compatible with each other when selecting an expression system. A person skilled in the art can make such a determination. Further, if guidance is needed when selecting an expression system, a person skilled in the art can refer to P. Jones, "Vectors: Cloning Applications", John Wiley and Sons, New York, N.Y., 2009).

[0107] The constructs of the present disclosure can be contained, for example, within a vector that can direct their expression in cells transformed / transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or can be readily prepared by those skilled in the art.

[0108] DNA vectors can be introduced into eukaryotic cells by conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection, can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals.

[0109] C. Recombinant cells The gene circuits, constructs and / or vectors of the present disclosure can be introduced or transduced into host cells or recombinant cells, such as human T lymphocytes, for example, to produce recombinant cells containing nucleic acid molecules. Accordingly, some embodiments of the present disclosure relate to recombinant cells containing the constructs or vectors of the present disclosure.

[0110] Introduction of the gene circuits, constructs or vectors of the present disclosure into cells can be achieved by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0111] Thus, in some embodiments, the construct can be delivered by a viral or non-viral delivery vehicle known in the art. For example, the construct can be stably integrated into the host genome, or replicated as an episome, or present in a recombinant host cell as a mini-circle expression vector for transient expression. Thus, in some embodiments, the construct is maintained and replicated as an episomal unit in a recombinant host cell. In some embodiments, the construct is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genomic recombination techniques, or more precise techniques such as guide RNA-guided CRISPR / Cas9 genome editing, or DNA-guided endonuclease genome editing by NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nuclease). In some embodiments, the construct is present in a recombinant host cell as a mini-circle expression vector for transient expression.

[0112] The construct can be encapsulated within a viral capsid or lipid nanoparticles, or delivered by viral or non-viral delivery means and methods known in the art such as electroporation. For example, introduction of nucleic acids into cells can be achieved by viral transduction. In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver the construct to target cells by viral transduction. Several AAV serotypes have been described, and all known serotypes can infect cells from a plurality of diverse tissue types. AAV is non-toxic in vivo, capable of transducing various species and tissues, and elicits a relatively mild innate and adaptive immune response.

[0113] Lentivirus-derived vector systems are also useful for the delivery of constructs and gene therapy by viral transduction. Lentiviral vectors offer several attractive properties as gene delivery vehicles, including: (i) sustained gene delivery by stable integration of the vector into the host genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropism, including important genes and target cell types for cell therapy; (iv) the lack of expression of viral proteins after vector transduction; (v) the ability to deliver complex gene elements such as polycistronic sequences or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0114] In some embodiments, the recombinant host cell can be, for example, a viral vector or a vector for homologous recombination, comprising a nucleic acid sequence homologous to a portion of the genome of the host cell, or an expression vector for the expression of a polypeptide of interest, for example, genetically engineered (e.g., transduced or transformed or transfected) with a vector construct of the present application. The host cell can be either an untransformed cell or a cell already transfected with at least one nucleic acid molecule.

[0115] In some embodiments, the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the mammalian cell is an immune cell, or a stem cell, or other. In some embodiments, the recombinant cell is an immune system cell, such as a lymphocyte (e.g., a T cell or an NK cell), or a dendritic cell. In some embodiments, the immune cell is a B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell (Tx), a cytotoxic T cell (Tcm), or other T cells. In some embodiments, the immune system cell is a T lymphocyte. In some embodiments, the cell is a chimeric antigen receptor-expressing reporter T (CAR-T) cell.

[0116] In some embodiments, the cell is a stem cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is a progenitor T cell or a T regulatory (Treg) cell. In some embodiments, the cell is a CD34+, CD8+, or CD4+ cell. In some embodiments, the cell is a CD8+ T cell cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments, the cell is a CD4+ T helper lymphocyte cell selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the cell can be obtained by leukapheresis performed on a sample obtained from a subject. In some embodiments, the subject is a human patient.

[0117] In some embodiments, the recombinant cells of the present disclosure comprise a construct having a transcriptional regulatory region operably linked to a nucleic acid sequence of interest and a nucleic acid sequence encoding a chimeric antigen receptor (CAR). In some embodiments, the second CAR comprises a signaling domain different from the first CAR. Non-limiting examples of signaling domains include 4-1BB, CD28, ICOS, CD2, BAFFR, TACI, CD30, NTB-A, and others.

[0118] In some embodiments, the nucleic acid sequence of interest is heterologous to the recombinant cell. In some embodiments, the nucleic acid sequence of interest encodes a heterologous protein. A heterologous protein is a protein that is not normally found intracellularly, for example, a protein that is not normally produced by a cell. In principle, there is no particular limitation with respect to suitable proteins that can be modulated in expression by the chimeric receptor transcriptional regulator. Exemplary types of proteins suitable for use with the compositions and methods disclosed herein include cytokines, cytotoxins, chemokines, immunomodulatory factors, apoptosis-promoting factors, anti-apoptosis factors, hormones, differentiation factors, dedifferentiation factors, immune cell receptors, or reporters. In some embodiments, the immune cell receptor is a T cell receptor (TCR). In some embodiments, the immune cell receptor is a chimeric antigen receptor (CAR). In some embodiments, the expression cassette encoding the protein of interest is incorporated into the same nucleic acid molecule encoding the chimeric receptor of the present disclosure. In some embodiments, the expression cassette encoding the protein of interest is incorporated into a second expression vector separate from the nucleic acid molecule encoding the chimeric receptor of the present disclosure. In another aspect, provided herein is a cell culture comprising at least one recombinant cell disclosed herein and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming the above-described diverse host cells and species are known in the art and are described in technical and scientific literature. Accordingly, a cell culture comprising at least one recombinant cell disclosed herein is also within the scope of this application.

[0119] In some embodiments, the recombinant cells of the present disclosure comprise a construct having a transcriptional regulatory region operably linked to nucleic acid sequences encoding a first chimeric antigen receptor (CAR) and a second CAR. In some embodiments, the second CAR comprises a signaling domain different from the first CAR. In some embodiments, the recombinant cells further comprise an expression cassette encoding a second CAR operably linked to a promoter, and the expression of the second CAR is modulated by the chimeric receptor transcriptional regulator of the first CAR.

[0120] Also provided herein are cells for delivering a bioactive molecule, wherein the cells comprise: a) a CAR having specificity for a target antigen; and b) a construct having at least one response element operably linked to a nucleic acid sequence encoding the bioactive molecule, wherein activation of the T cell by binding of the CAR to the target antigen results in expression of the nucleic acid sequence and delivery of the bioactive molecule by the T cell.

[0121] Also provided herein are: a) a first nucleic acid construct comprising a first promoter operably linked to a nucleic acid sequence encoding a CAR having specificity for a target antigen; and b) a second nucleic acid construct comprising a transcriptional regulatory region having a second promoter and a response element (RE) operably linked to a nucleic acid sequence (NAS) of interest encoding a bioactive molecule, wherein activation of the T cell by binding of the CAR to the target antigen results in expression of the NAS. In some embodiments, the first promoter is a constitutive promoter. In some embodiments, the first nucleic acid construct and the second nucleic acid construct are present tandemly on the same nucleic acid molecule. In some embodiments, the nucleic acid molecule is, or comprises, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 27, or a functional variant thereof.

[0122] Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0123] III. Methods of the Present Disclosure A. Methods for Inducing an Immune Response or Treatment The present disclosure provides, inter alia, a method for inducing an immune response in a subject by administering to the subject a vector of the present disclosure or a recombinant cell of the present disclosure.

[0124] Non-limiting examples of immune responses include a cytotoxic T lymphocyte (CTL) response, a B cell response (e.g., antibody production), an NK cell response, or any combination thereof when administered to an immunocompetent subject.

[0125] The present disclosure also provides, inter alia, a method for treating a health condition in a subject by administering to the subject a vector of the present disclosure or a recombinant cell of the present disclosure.

[0126] Administration of any one of the vectors or cells described herein can be used to treat a patient for a related health condition or disease such as cancer, autoimmune disease or infectious disease. In some embodiments, the vectors or cells of the present disclosure can be incorporated into a composition, e.g., a pharmaceutical composition or a therapeutic composition, for use in a method of treating an individual having, suspected of having, or at high risk of developing one or more autoimmune disorders or diseases associated with checkpoint inhibition. Exemplary autoimmune disorders and diseases can include, but are not limited to, celiac disease, type I diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0127] The present disclosure further provides a method of treating a subject in need thereof using a combination therapy comprising a T cell therapy and a second therapy, wherein the second therapy comprises administering to the subject a recombinant cell or construct of the present disclosure. In some embodiments, the second therapy inhibits target cells. For example, the target cells can be inhibited when their proliferation is reduced, when their pathological or pathogenic behavior is reduced, when the target cells are destroyed or killed, etc. The inhibition comprises at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% measured reduction in pathological or pathogenic behavior. In some embodiments, the method comprises administering to an individual an effective number of recombinant cells disclosed herein, wherein the recombinant cells inhibit the activity of target cells in the individual. Generally, the target cells of the disclosed methods can be any cell type within an individual, such as cells derived from hematological malignancies, multiple myeloma cells, solid tumor cells, acute myeloid leukemia cells, undifferentiated lymphoma cells, astrocytoma cells, B cell cancer cells, breast cancer cells, colon cancer cells, epithelioma cells, esophageal cancer cells, glioblastoma cells, glioma cells, leiomyosarcoma cells, liposarcoma cells, liver cancer cells, lung cancer cells, mantle cell lymphoma cells, melanoma cells, neuroblastoma cells, non-small cell lung cancer cells, oligodendroglioma cells, ovarian cancer cells, pancreatic cancer cells, peripheral T cell lymphoma cells, kidney cancer cells, sarcoma cells, stomach cancer cells, carcinoma cells, mesothelioma cells, or sarcoma cells. In some embodiments, the target cells are pathogenic cells.

[0128] In some embodiments, the method of the present disclosure involves administering to an individual in need of such treatment an effective amount of the recombinant somatic cells of the present disclosure. This administration step can be accomplished using any method of transplantation delivery in the art. For example, the recombinant cells of the present disclosure can be directly injected into the bloodstream of the individual or administered to the individual by other means.

[0129] In some embodiments, the methods disclosed herein involve administering recombinant cells to an individual by a method or route that results in at least partial localization of the introduced cells at a desired site such that a desired effect(s) occurs. (This term is used interchangeably with the terms “introducing,” “implanting,” and “transplanting.”) The recombinant cells or their differentiated progeny can be administered by any suitable route that delivers them to a desired location in the individual where at least a portion of the administered cells or cell components remain viable. The survival period of the cells after administration to the individual can range from a short period of hours, e.g., 24 hours, to days, years, or even the lifetime of the individual, i.e., long-term engraftment.

[0130] When provided prophylactically, the recombinant cells described herein can be administered to an individual prior to any symptoms of the disease or condition to be treated. Thus, in some embodiments, prophylactic administration of a recombinant cell population prevents the occurrence of symptoms of the disease or condition.

[0131] In some embodiments, when provided therapeutically, the recombinant cells are provided at the onset (or after the onset) of symptoms or signs of the disease or condition, e.g., at the onset of the disease or condition.

[0132] A therapeutically effective amount includes an amount of recombinant cells sufficient to promote a particular beneficial effect when administered to an individual such as an individual having a disease, an individual suspected of having a disease, or an individual at risk of a disease. In some embodiments, the effective amount includes an amount sufficient to prevent or delay the onset of symptoms of the disease, alter the course of symptoms of the disease (e.g., but not limited to, slow the progression of symptoms of the disease), or reverse the symptoms of the disease. In any case, it is understood that an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0133] For use in the various embodiments described herein, an effective amount of the recombinant cells disclosed herein is at least 10 2 cells, at least 5 × 102 cells, at least 10 3 cells, at least 5×10 3 cells, at least 10 4 cells, at least 5×10 4 cells, at least 10 5 cells, at least 2×10 5 cells, at least 3×10 5 cells, at least 4×10 5 cells, at least 5×10 5 cells, at least 6×10 5 cells, at least 7×10 5 cells, at least 8×10 5 cells, at least 9×10 5 cells, at least 1×10 6 cells, at least 2×10 6 cells, at least 3×10 6 cells, at least 4×10 6 cells, at least 5×10 6 cells, at least 6×10 6 cells, at least 7×10 6 cells, at least 8×10 6 cells, at least 9×10 6 cells, or may be a multiple thereof. The recombinant cells may be derived from one or more donors or may be obtained from an autologous source. In some embodiments, the recombinant cells are expanded in culture prior to administration to an individual in need thereof.

[0134] In some embodiments, at least partial localization of the cell composition at a desired site is achieved by delivering a recombinant cell composition (e.g., a composition comprising a plurality of recombinant cells by any of the cells described herein) to an individual by a method or route. The composition comprising the recombinant cells can be administered by any suitable route that provides an effective treatment in the individual, e.g., the administration is at least a portion of the delivered composition, e.g., at least 1×10 4Individual cells effect delivery to a desired location in an individual, where the cells are delivered to the desired location for a period of time. Modes of administration include injection, infusion, and intravenous drip. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For delivery of cells, delivery by injection or infusion is the preferred mode of administration.

[0135] In some embodiments, the recombinant cells are administered systemically, e.g., by infusion or injection. For example, a population of recombinant cells is administered other than directly to a target site, tissue, or organ such that it enters the circulatory system of an individual and is thus subject to metabolism and other similar biological processes.

[0136] The effectiveness of a treatment comprising any of the compositions provided herein for the treatment of a disease or condition can be determined by a skilled clinician. However, one of ordinary skill in the art will understand that a treatment is considered effective if any one or all of the signs or symptoms or markers of the disease are improved or ameliorated. Effectiveness can also be measured by the individual not worsening (e.g., the progression of the disease is stopped or at least slowed) when evaluated by a decrease in the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those of ordinary skill in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or mammals), and includes (1) inhibiting the disease, e.g., stopping or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the onset of symptoms.

[0137] Diseases suitable for treatment by the compositions and methods of the present disclosure include, but are not limited to, cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. In some embodiments, the disease is cancer or a chronic infection. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the disease is a solid tumor.

[0138] In some embodiments of the disclosed methods, the individual is a mammal. In some embodiments, the mammal is a human. In some embodiments, the individual has or is suspected of having a disease associated with inhibition of cell signaling mediated by a cell surface ligand or antigen.

[0139] Method for delivering a bioactive molecule by B and T cells The present disclosure also provides a method for delivering a bioactive molecule (i.e., a payload) by T cells having a chimeric antigen receptor (CAR) specific for a target antigen; and a construct having at least one response element operably linked to a nucleic acid sequence encoding the bioactive molecule, wherein activation of the T cells by binding of the CAR to the target antigen results in expression of the nucleic acid sequence and delivery of the bioactive molecule or payload by the T cells.

[0140] The bioactive molecule can be an antibody, nanobody, diabody, triabody, minibody, F(ab)2 fragment, F(ab)v fragment, single-chain variable fragment (scFv), single-domain antibody (sdAb), or a functional fragment thereof. In some embodiments, the bioactive molecule is a ligand (e.g., a secreted ligand or CD40L or a derivative thereof), short hairpin RNA (shRNA), or microRNA (miRNA). In some embodiments, the bioactive molecule is an anti-programmed cell death-1 (anti-PD1) or anti-programmed cell death-1 ligand 1 (anti-PDL1).

[0141] In some embodiments, the bioactive molecule is a chimeric antigen receptor (second CAR) that binds to a target antigen different from the target antigen to which the first CAR binds (i.e., has specificity for the target antigen). In some embodiments, the first CAR is constitutively expressed.

[0142] Non-limiting examples of CARs include CARs against the following target antigens: HER-2, CD-19, GD2, PSMA, CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placental-like 2 (ALPPL2), B cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), signal regulatory protein alpha (SIRPα), or CTLA-4.

[0143] Accordingly, provided herein is a method for delivering a bioactive molecule (i.e., payload) by T cells having a chimeric antigen receptor (CAR) specific for a target antigen; and a construct having at least one response element operably linked to a nucleic acid sequence encoding a second CAR specific for a second target antigen, wherein activation of the T cells by binding of the CAR to the target antigen results in expression of the nucleic acid sequence encoding the second CAR, thereby enabling the T cells to target cells expressing the second target antigen. In some embodiments, the second CAR has a signaling domain different from that of the CAR of (a). In some embodiments, the first CAR is a Myc-ALPPL2 CAR. In some embodiments, the second CAR is a mesothelin CAR comprising a CD28 co-stimulatory molecule and CD3z. In some embodiments, the mesothelin CAR comprises a mesothelin co-stimulation-only CAR that does not include CD3z.

[0144] An exemplary workflow for a method for delivering a bioactive molecule can be as follows. In some embodiments, when the first CAR engages the ALPPL2 target antigen, the ALPPL2 target is killed and the T cells are activated. Activation of the T cells induces expression of the second CAR (mesothelin). Expression of the second CAR causes the T cells to exhibit toxicity (e.g., destroy, suppress, kill) against target cells expressing mesothelin.

[0145] In some embodiments, the first CAR is a Myc-ALPPL2 CAR and the bioactive molecule is a reporter molecule. In some embodiments, when the first CAR engages the ALPPL2 target antigen, the ALPPL2 target is killed and the T cells are activated. Activation of the T cells induces expression of the reporter molecule. Such embodiments can be used to test the expression, intensity, or functionality of a CAR of interest in in vitro or in vivo assays.

[0146] The discussion of the general methods provided in this specification is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those skilled in the art upon consideration of the present disclosure, and they are intended to be included within the spirit and scope of this application.

[0147] Throughout this specification, various patents, patent applications, and other types of publications (e.g., academic papers, electronic database entries, etc.) are referenced. The disclosures of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0148] None of the references cited herein are admitted to constitute prior art. The discussion of the references states what their authors assert, and the inventors reserve the right to dispute the accuracy and validity of the cited documents. Although several information sources including scientific academic papers, patent documents, and textbooks are mentioned herein, it will be clearly understood that this reference does not admit that any of these documents form part of the common general knowledge in the art.

Examples

[0149] Unless otherwise indicated, the practice of the present invention utilizes conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art, Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as "Sambrook"); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements from 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology.New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements from 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., etc. are well described in the literature, and these disclosures are hereby incorporated by reference into this specification.

[0150] Additional embodiments are disclosed in more detail in the following examples, which are provided for illustration purposes and are in no way intended to limit the scope of the present disclosure or the claims.

[0151] The following examples demonstrate the generation of constructs according to the present disclosure.

[0152] (Example 1) Design of constructs for receptors and response elements This example shows the design of the CAR and RE of the present disclosure. The pHR’SIN:CSW vector (Thrasher, 2004. Human Gene Therapy; Vol. 13, No. 7) was modified to generate a response element (RE) plasmid. The promoter fragment was cloned 5' to the minimal pybTATA promoter except for the human NR4A1 response element (RE). All cloned cassettes were present upstream of the inducible BFP, and downstream of this inducible cassette, there was a PGK promoter that constitutively drove mCherry expression to appropriately identify the transduced T cells. All constructs were cloned by In-fusion cloning (Clontech #ST0345).

[0153] (Example 2) Isolation and culture of primary human T cells This example describes the isolation and culture of primary human T cells. Primary CD4+ and CD8+ T cells were isolated from the blood of anonymous donors after apheresis by negative selection (STEMCELL Technologies #15062 & 15063). The blood was obtained from Blood Centers of the Pacific (San Francisco, CA) with approval from the University Institutional Review Board. The T cells were cryopreserved in RPMI-1640 (UCSF cell culture core) containing 20% human AB serum (Valley Biomedical Inc., #HP1022) and 10% DMSO. After thawing, the T cells were cultured in human T cell medium consisting of X-VIVO15 (Lonza #04-418Q), 5% human AB serum, and 10 mM neutralizing N-acetyl-L-cysteine (Sigma-Aldrich #A9165), and supplemented with 30 units / mL of IL-2 (NCI BRB Preclinical Repository) for all experiments.

[0154] (Example 3) Lentiviral transduction of human T cells This example shows the lentiviral transduction of human T cells. A pantropic VSV-G pseudotyped lentivirus was generated by transfection of Lenti-X 293T cells (Clontech #11131D) with the pHR’SIN:CSW transgene expression vector and the viral packaging plasmids pCMVdR8.91 and pMD2.G using Mirus TransIT-Lenti (Mirus #MIR 6606). Primary T cells were thawed on the same day and, after 24 hours of culture, were stimulated with human T-activator CD3 / CD28 Dynabeads (Life Technologies #11131D) at a 1:3 cell:bead ratio. At 48 hours, the viral supernatant was harvested and the primary T cells were exposed to the virus for 24 hours. On day 5 post-T cell stimulation, the Dynabeads were removed and the T cells were rested and expanded until day 14 when they were ready for assay. T cells were sorted for assay using a Beckton Dickinson (BD) FACs ARIA II.

[0155] (Example 4) Cancer cell lines This example shows the various cell lines used in the examples. The cancer cell line used was the K562 myelogenous leukemia cell (ATCC #CCL-243). K562 was transduced with lentivirus to stably express human CD19 at a level equivalent to the Daudi tumor or to express HER2 via a doxycycline-inducible system. CD19 levels were determined by staining the cells with α-CD19 APC (BioLegend #302212) and HER2 levels were determined by staining the cells with α-HER2 AF647 (BioLegend #324412). All cell lines were sorted for transgene expression.

[0156] (Example 5) In vitro stimulation of primary T cells For all in vitro T cell stimulations, 1×10 5Individual T cells were co-cultured with target cells at a 1:1 ratio in a U-bottom 96-well tissue culture plate. Cultures were analyzed using a BD Fortessa X-50 at 24 hours or at the time point indicated for reporter activation. All flow cytometry analyses were performed using FlowJo software (TreeStar).

[0157] (Example 6) Design and construction of various response element constructs a) Constructs of BFP and mCherry under the control of RE (Figure 1A) The construction of the complete plasmid sequences is as follows. The main backbone (excluding the RE element) is SEQ ID NO: 1, which was constructed by standard cloning using linkers and cloning sites between elements. NR4A1 was constructed by taking SEQ ID NO: 2 and adding overhangs containing EcoRI and BamHI cloning sites, and assembled by In-fusion cloning into the linearized vector of SEQ ID NO: 1, which contains the BFP reporter gene and the constitutive PGK-driven mCherry marker gene (SEQ ID NO: 9). For NIR ABC, hu496, and hu319 (SEQ ID NOs: 3, 4, and 5 respectively), each was fused to the minimal TATA promoter (SEQ ID NO: 13) by In-fusion cloning to generate SEQ ID NOs: 6, 7, and 8. These were then assembled by In-fusion cloning into the linearized vector of SEQ ID NO: 1, which contains the BFP reporter gene and the constitutive PGK-driven mCherry marker gene, to obtain SEQ ID NOs: 10, 11, and 12 respectively.

[0158] b) Construction of the complete plasmid sequence of SFFV promoter-α-HER2-BBZ-t2a-GFP: Array numbers 17 - 20 were assembled into the linearized vector by In-fusion to generate the plasmid array number 16 of the full-length plasmid sequence (the construct shown in Figure 1C). Similarly, the other constructs presented in Figures 6A, 6B, and 6C were assembled from individual components using In-fusion cloning into the linearized vector.

Claims

1. (i) a first nucleic acid construct comprising a transcriptional regulatory region comprising a response element (RE) operably linked to a target nucleic acid sequence (NAS); (ii) A second nucleic acid construct comprising a nucleic acid sequence encoding a first chimeric antigen receptor (CAR) having specificity for a target antigen, A gene circuit that includes this.

2. The gene circuit according to claim 1, wherein the transcriptional regulatory region includes a functional variant thereof that includes one of sequence numbers 2 to 8, or a sequence having approximately 85% to approximately 99% sequence identity with sequence numbers 2 to 12.

3. The aforementioned NAS encodes a bioactive molecule, and the bioactive molecule is used as needed. (a) comprising antibodies, nanobodies, diabodies, triabodies, minibodies, F(ab)2 fragments, F(ab)v fragments, single-strand variable fragments (scFv), single-domain antibodies (sdAb), or functional fragments thereof; (b) comprising a ligand, short hairpin RNA (shRNA), or microRNA (miRNA), wherein the ligand is optionally a secretory ligand, or CD40L or a derivative thereof; or (c) comprising anti-programmed cell death-1 (anti-PD1) or anti-programmed cell death-1 ligand 1 (anti-PDL1), The gene circuit according to claim 1.

4. The aforementioned NAS, (a) encoding a second CAR, wherein the second CAR optionally includes a different signaling domain than the first CAR; or (b) Encoding a reporter molecule, wherein the reporter molecule is, if necessary, GFP, enhanced green fluorescent protein (EGFP), Cherry, BFP, luciferase, Nanoluc™, herpesvirus thymidine kinase, or a variant thereof. The gene circuit according to claim 1.

5. The target antigen for the first CAR is HER-2, CD-19, GD2, PSMA, CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD1 The gene circuit according to claim 1, wherein the gene is 81 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FFFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), signal regulatory protein α (SIRPα), or CTLA-4.

6. (a) The activation of the RE results in the expression of the NAS; (b) The transcriptional regulatory region comprises a first promoter that drives the expression of the NAS, wherein the promoter optionally comprises a minimal TATA promoter, a minimal CMV promoter, a minimal IL-2 promoter, a synthetically inducible promoter, a naturally inducible promoter, a pGK promoter, an SFFV or EF1α promoter, or a functional variant thereof; or (c) The gene circuit is encoded by SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 27, or a functional variant thereof. The gene circuit according to claim 1.

7. The gene circuit according to claim 1, wherein the second nucleic acid construct comprises a second promoter that controls the expression of the first CAR, and optionally the second promoter comprises a pGK promoter.

8. (a) The transcriptional regulatory region comprises any of SEQ ID NOs: 6 to 12, or a functional variant thereof; (b) The first nucleic acid construct and the second nucleic acid construct are present in tandem on a single nucleic acid molecule; and / or (c) The activation of the CAR by the target antigen results in the expression of the NAS. The gene circuit according to claim 1.

9. A vector comprising a gene construct according to any one of claims 1 to 8, wherein the vector is optionally a retroviral vector or a lentiviral vector.

10. Recombinant cells transduced by a vector comprising a gene circuit according to any one of claims 1 to 8, or a gene construct according to any one of claims 1 to 8, wherein the cells are optionally immune cells, and the immune cells are optionally regulatory T cells, helper T cells, cytotoxic T cells, CAR expression reporter T (CAR-T) cells, CD4+ T cells, CD8+ T cells, or other T cells, and the cells are optionally animal cells, mammalian cells, human cells, or non-human primate cells.

11. A composition for use in a method for inducing an immune response in a subject or for treating a health condition, comprising recombinant cells transduced by a gene circuit according to any one of claims 1 to 8, a vector comprising a gene construct according to any one of claims 1 to 8, or a vector comprising a gene circuit according to any one of claims 1 to 8 or a gene construct according to any one of claims 1 to 8.

12. A composition for use in a method of treating a target requiring treatment in combination with T cell therapy, comprising recombinant cells transduced by a gene circuit according to any one of claims 1 to 8, a vector comprising a gene construct according to any one of claims 1 to 8, or a vector comprising a gene circuit according to any one of claims 1 to 8 or a gene construct according to any one of claims 1 to 8.

13. A composition for use in a method for inducing an immune response in a subject or for treating a health condition, or for use in a method for treating a subject in need of treatment in combination with T-cell therapy, wherein the composition comprises recombinant cells transduced by a gene circuit according to any one of claims 1 to 8, a vector comprising a gene construct according to any one of claims 1 to 8, or a vector comprising a gene circuit according to any one of claims 1 to 8 or a gene construct according to any one of claims 1 to 8, wherein the subject has cancer or an autoimmune disease, and optionally the cancer is a solid tumor or a hematological malignancy, and optionally the hematological malignancy is multiple myeloma.

14. A composition for delivering bioactive molecules by T cells, wherein the composition comprises T cells, and the T cells a) A first chimeric antigen receptor (CAR) having specificity for the target antigen; b) A construct comprising at least one response element (RE) operably linked to a nucleic acid sequence encoding a bioactive molecule The RE optionally includes SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or functional variants thereof, the CAR optionally constitutively expresses, and the bioactive molecule optionally delivers (i) an antibody, nanobody, diabody, triabody, minibody, F(ab)2 fragment, F(ab)v fragment, single-strand variable fragment (scFv), single-domain antibody (sdAb), or functional fragment thereof; (ii) a ligand, short hairpin RNA (shRNA), or microRNA (miRNA), wherein the ligand is, if applicable, a secretory ligand, or CD40L or a derivative thereof; or (iii) Anti-programmed cell death-1 (anti-PD1) or anti-programmed cell death-1 ligand 1 (anti-PDL1), A method wherein the bioactive molecule optionally comprises a second CAR for a second target antigen, the binding of the first CAR to the target antigen optionally results in the expression of the second CAR, thereby enabling the T cell to target a cell expressing the second target antigen, and the second CAR optionally comprises a signaling domain different from that of the first CAR.

15. a) A first nucleic acid construct comprising a first promoter operably linked to a nucleic acid sequence encoding a chimeric antigen receptor (CAR) having specificity for a target antigen; b) A second nucleic acid construct comprising a transcriptional regulatory region including a response element (RE) operably linked to a second promoter and a target nucleic acid sequence (NAS) encoding a bioactive molecule. T cells, including T cells wherein the binding of the CAR to the target antigen results in the expression of the NAS, the first promoter is optionally a constitutive promoter, the first nucleic acid construct and the second nucleic acid construct are optionally present in tandem on the same nucleic acid molecule, and the nucleic acid molecule optionally contains SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 27, or functional variants thereof.

16. The gene circuit according to claim 1, wherein the response element is NR4A1, NIR ABC, Hu496, or Hu319.

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