A system for inducible expression of adaptors in immune cells

Abstract

The present invention provides a system for inducible expression of an adaptor in immune cells, comprising: a) an inducible gene expression system, I) a first nucleic acid comprising an inducible promoter operably linked to a second nucleic acid; and II) the second nucleic acid encoding an adaptor, i) a first (poly)peptide comprising an antigen-binding domain that specifically binds to an antigen, and ii) a second nucleic acid comprising a second (poly)peptide that binds to the antigen-binding domain of a chimeric antigen receptor (CAR). an inducible gene expression system comprising: b) a third nucleic acid encoding the CAR specific for the second polypeptide of the adapter; The CAR is i) the antigen-binding domain specific for the second (poly)peptide of the adapter; ii) a transmembrane domain, and iii) providing a system comprising an intracellular signaling domain. The gene expression system can be antigen-activated or drug-induced. The system can be a one-cell or two-cell approach.

Description

[Technical Field]

[0001] The present invention relates generally to the field of chimeric antigen receptors (CARs) expressed on immune cells, and in particular to the combination of adapter CAR (anti-tag CAR) technology with the regulated expression of corresponding tagged adapter molecules. [Background technology]

[0002] Adoptive transfer of CAR immune cells, such as CAR T cells, has demonstrated remarkable success in the treatment of hematological malignancies. However, dysregulation of CAR immune cell function in patients and the resulting excessive inflammation can cause severe side effects, especially when targeting tumor-associated antigens rather than tumor-specific antigens. Therefore, the temporal, adjustable, and spatial control of CAR activity is of great importance.

[0003] Adapter CAR immune cells, such as adapter CAR T cells, are based on the separation of target antigen recognition and activation domains into two complementary parts, offering an additional strategy for enhancing the safety and versatility of CAR immune cell systems. Anti-tag CAR immune cells do not directly recognize tumor antigens, but rather rely on a tag fused to an antigen-binding molecule called an adapter or adapter molecule to confer immune cell specificity. Such "universal" CAR systems (or adapter CAR (adCAR) systems), which indirectly bind to target cells via an adapter, are described, for example, in WO2012082841A2, WO2013044225A1, and WO2016030414A1. Because antigen recognition strictly depends on the presence of the adapter molecule, it is conceivable that attenuating the administration of the adapter molecule may enable the control and temporal on / off switching of CAR-mediated functions. Furthermore, the magnitude of the response can be fine-tuned by adjusting the adapter concentration. However, because functionality depends on the presence of the adapter molecule, patients must undergo routine injections of the adapter molecule, which can be associated with side effects and increase patient stress. Furthermore, centralized GMP-compliant production, formulation, storage, and transportation of adapter molecules are time-consuming and costly. Challenges limiting the efficacy of adapter CAR immune cells are the limited tissue penetration and uneven distribution of adapter molecules in tumors upon systemic administration.

[0004] Ambrose et al. developed CAR-CD19 T cells that constitutively secrete a CD19-anti-Her2 cross-linking protein. This cell therapy strategy exploits the ability of CD19-targeted CAR T cells to interact with CD19 on normal B cells, leading to their proliferation, persistence, and adaptation. The secreted cross-linking protein potently binds to Her2-positive tumor cells and mediates the cytotoxicity of CAR-CD19 T cells in vitro and in vivo (Doi:10.1101 / 2020.03.25.007658).

[0005] WO2017075537A1 discloses cells comprising a constitutive expression construct encoding (a) an antigen-binding protein or fragment that binds to a tumor antigen and (b) a fusion protein comprising a polypeptide target of a cell preparation, antibody, or antibody-drug conjugate.

[0006] WO2018156802A1 discloses cells comprising a constitutive expression construct encoding (a) an antigen-binding protein or fragment that binds to a tumor antigen and (b) a fusion protein comprising an anti-idiotype antibody or fragment, or an anti-idiotype peptide, that binds to the antigen-binding domain of a cell preparation, antibody, or antibody-drug conjugate.

[0007] WO2019199689A1 discloses modified immune cells comprising a single viral vector that includes both a first polynucleotide comprising a constitutive promoter operably linked to a nucleic acid encoding at least one transgene, e.g., a CAR, and a second polynucleotide comprising an inducible promoter operably linked to a nucleic acid encoding an effector.

[0008] There is a need in the art for improved or alternative adaptor CAR immune cell systems that include adaptor CARs and adaptors expressed by immune cells. Summary of the Invention

[0009] To overcome the limitations associated with centralized GMP production and local administration and injection of adapter molecules into patients, tumor-homing immune cells, such as CAR T cells or tumor-infiltrating T cells (TILs), can be engineered to function as in situ adapter delivery vehicles by inducible-dependently expressing an adapter suitable for the adapter CAR. The inducible gene expression system may be an "antigen-activated inducible gene expression system," i.e., the inducible expression system can be activated in cells harboring the inducible gene expression system when the cell's receptor, e.g., a CAR or TCR, binds to an antigen / ligand, either directly or upon MHC presentation. The binding of the antigen / ligand to the receptor can induce a signal cascade in the cell, which can then lead to the induction of expression of the introduced gene (or transgene), herein the adapter. Alternatively, the inducible gene expression system may be a drug-inducible gene expression system, i.e., the inducible gene expression system can be activated in cells harboring the inducible gene expression system when a drug, e.g., a synthetic drug, e.g., tamoxifen, is introduced into the cells. The drug within the cell may bind to a synthetic transcription factor, which may then cause induction of expression of the transgene, herein the adapter. Both types of inducible gene expression systems may be used in one-cell and / or two-cell systems. While constitutive expression of the adapter by a cell vehicle does not allow for regulating adapter expression and thereby controlling adapter CAR cell function in patients, drug-dependent control of the expression of the adapter molecule by a "vehicle cell" allows for temporal and tunable control of adapter molecule secretion, thereby enabling, for example, I) Control of the on-off switching of CAR immune cell activity by restricting CAR cell immune activity to the temporal control of adaptor secretion, ii) Fine-tuning CAR immune cell activity by precisely adjusting adapter concentrations at the tumor site This becomes possible.

[0010] Furthermore, in the case of locally restricted antigens, the "antigen activation-inducible gene expression system" offers the advantage that because activation-induced signaling relies on T cell recruitment by the cognate antigen (in TCR / CD3 pathway signaling), adaptor secretion is restricted to the area where the antigen is expressed (e.g., solid tumors), thereby providing improved spatial regulation of adaptor concentration. In contrast, constitutive expression of adaptors results in systemic release, which may increase the risk of undesirable systemic toxicity. [Brief explanation of the drawings]

[0011] [Figure 1] Schematic diagram of an activation-inducible system for adaptor secretion. Immune cells are modified with an activation-inducible cassette, i.e., a gene expression system (called activation-inducible cells), which drives the expression of adaptor molecules via a promoter responsive to TCR / CAR-mediated signaling. These cells can be selected based on their reactivity to tumor antigens, oncovirus antigens, or tumor neoantigens. Alternatively, T cells can be transduced with CARs or TCRs specific for any of the above antigen categories. Upon TCR / CAR recognition of a TAA, oncovirus, or tumor neoantigen, the TCR-responsive promoter drives the expression of anti-tumor adaptor molecules. These tagged adaptor molecules are specific for tumor antigens. The tag portion of the adaptor molecule (e.g., a 6xHis tag in this case) is recognized by the extracellular recognition domain of an adaptor CAR (in this case, an anti-His6-specific CAR) expressed by a second immune cell. The tagged adaptor molecule serves as a bridging molecule that redirects the adaptor CAR cells to the tumor. Consequently, the tumor cells are lysed by the adaptor CAR cells. [Figure 2]Schematic diagram of a drug-inducible system for adaptor secretion. Immune cells are modified with a drug-inducible gene expression cassette (referred to as drug-inducible cells). In the "off" state, these inducible cells are indistinguishable from unmodified immune cells. Upon administration of an inducer drug, the inducible expression cassette is activated, and a tagged adaptor molecule is expressed and secreted by the immune cells. The concentration of the secreted tagged adaptor molecule can be adjusted by the concentration of the inducing drug. Such tagged adaptor molecules are specific for tumor antigens. The tag portion of the adaptor molecule (in this case, a 6xHis tag) is recognized by the extracellular recognition domain of an adaptor CAR (in this case, an anti-His6-specific CAR) expressed by a second immune cell. The tagged adaptor molecule serves as a bridging molecule that redirects the adaptor CAR cells to tumors. Consequently, tumor cells are lysed by the adaptor CAR cells. The antitumor activity of His-adapter CAR T cells depends on the presence of a His-tagged tumor-specific adaptor molecule. The adaptor molecule is produced by inducible anti-CD19 Fab T cells upon induction at the tumor site. [Figure 3] Induction of anti-CD19 Fab-His6 secretion by primary T cells. T cells were transduced with an inducible anti-CD19 Fab construct and cultured for 48 hours in the presence of different concentrations of 4-OHT. CD19+ Raji cells were then stained using T cell supernatants and anti-His-APC as secondary antibodies. The determined mean fluorescence intensity was correlated with the Fab concentration by extrapolation from a standard curve. The induction of His6-tagged anti-CD19 Fab secretion by inducible T cells strictly depends on the presence of the inducing drug 4-OHT. With increasing concentrations of 4-OHT, increasing amounts of His6-tagged anti-CD19 Fab are detected in the supernatants of T cell cultures. [Figure 4]The cytolytic activity of anti-His adaptor CAR T cells in coculture with Raji cells depends on the presence of inducible anti-CD19 Fab T cells and the inducing drug 4-OHT. Anti-His adaptor CAR T cells were cocultured with CD19+ Raji cells at an E:T ratio of 2:1 in the presence of 1 x 104 anti-CD19 Fab-inducible T cells that secrete the adaptor molecule, His-tagged CD19 Fab, upon induction with the inducing drug 4-OHT (● symbol). Specific lysis of CD19+ Raji cells was only detected in cocultures of CD19+ Raji cells, adaptor CAR T cells, and inducible T cells in the presence of at least 1 nM 4-OHT. Maximum specific lysis was obtained after the addition of at least 10 nM 4-OHT. Cocultures of untransduced T cells and CD19+ Raji in the presence of anti-CD19 Fab-inducible T cells (■ symbols) and cocultures of inducible anti-CD19 Fab T cells and CD19+ Raji (▲ symbols) served as negative controls, demonstrating that tumor cell lysis cannot be induced by the adapter molecule itself but requires the simultaneous presence of anti-His adapter CAR T cells. Specific tumor cell lysis was determined by quantification of viable Raji cells on a MACSQuant® Analyzer 6 days after the start of the assay. [Figure 5]Activation of adaptor CAR T cells in coculture with Raji cells was dependent on the presence of inducible anti-CD19 Fab T cells and the induction drug 4-OHT. Anti-His adaptor CAR T cells were cocultured with CD19+ Raji cells at an E:T ratio of 2:1 in the presence of 1 x 104 inducible T cells that secrete the adaptor molecule His-tagged CD19 Fab upon induction with the induction drug 4-OHT (● symbol). PD-1 expression on T cells was only detected in cocultures of CD19+ Raji cells, adaptor CAR T cells, and induced T cells in the presence of at least 10 nM 4-OHT, and the frequency of PD-1-positive T cells increased with the concentration of 4-OHT. The maximum frequency of PD-1-positive cells was obtained after the addition of at least 50 nM 4-OHT. Cocultures of untransduced T cells and CD19+ Raji in the presence of anti-CD19 Fab-induced T cells (■ symbols) and cocultures of inducible anti-CD19 Fab T cells and CD19+ Raji (▲ symbols) served as negative controls, demonstrating that T cell activation (as indicated by PD-1 expression) cannot be induced by the adapter molecule itself but requires the co-presence of anti-His adapter CAR T cells. The frequency of PD-1-expressing T cells was determined by staining the cocultures with anti-PD-1-PE-Vio770 on day 2 after the start of the assay. DETAILED DESCRIPTION OF THE INVENTION

[0012] In a first aspect, the present invention provides a system (or combination of nucleic acids) for the inducible expression of an adaptor (or adaptor molecule or tagged polypeptide) in an immune cell, comprising: a) an inducible gene expression system, I) a first nucleic acid comprising an inducible promoter operably linked to a second nucleic acid; and II) the second nucleic acid encoding an adaptor, i) a first (poly)peptide comprising an antigen-binding domain that specifically binds to an antigen, and ii) a second (poly)peptide that binds to the antigen-binding domain of a chimeric antigen receptor (CAR). an inducible gene expression system comprising: b) a third nucleic acid encoding the CAR specific for the second polypeptide of the adapter; The CAR is i) the antigen-binding domain specific for the second (poly)peptide of the adapter; ii) a transmembrane domain, and iii) Intracellular signaling domain The present invention provides a system comprising:

[0013] The CAR specific for the second polypeptide of the adapter can be constitutively expressed in a cell, or the expression can be inducible in a cell.

[0014] The antigen may be an antigen expressed on the surface of a target cell.

[0015] The antigen may be a tumor-associated antigen (TAA) and the target cell may be a tumor cell.

[0016] The antigen may be expressed by cells of the tumor microenvironment.

[0017] The antigen may be an infectious pathogen-associated antigen (eg, from human immunodeficiency virus or other viruses), and the target cell may be a pathogen-infected cell.

[0018] The second (poly)peptide of an adapter that can bind to the antigen-binding domain of a chimeric antigen receptor (CAR) may also be called a "tag," and the CAR specific for the second polypeptide of the adapter may also be called an anti-tag CAR or adapter CAR (adCAR) or universal CAR.

[0019] The tag may be a (poly)peptide comprising at least four amino acids.

[0020] The tag can be a peptide containing at least 4 amino acids up to 8, 10, 15, 20, 25, or 30 amino acids.

[0021] The tag may comprise an epitope of a protein.

[0022] The tag may comprise an epitope of a protein that is naturally present in the subject or in the subject's circulatory system.

[0023] The tag may comprise an epitope of a neoantigen, wherein said epitope comprises a mutation of the antigen.

[0024] The tag may comprise an epitope of a neoantigen that arises in tumorigenesis, wherein said epitope comprises a mutation of the antigen.

[0025] The tag may comprise an epitope of a neoantigen, wherein said epitope comprises a mutation of an antigen, said antigen may be a protein naturally occurring in the subject or in the subject's circulatory system.

[0026] The tag may be a (poly)peptide that does not naturally occur in the subject.

[0027] The tag may be a (poly)peptide that does not naturally occur in the circulatory system of a subject.

[0028] The tag may be a (poly)peptide that does not naturally occur in the circulatory system of a healthy subject.

[0029] The tag can be, for example, the following peptide tags: c-Myc tag, Strep tag II, Flag tag, polyhistidine tag, Avi tag, calmodulin binding protein tag, Yol tag (derived from alpha-tubulin), E tag, HA tag, S tag, SBP tag or V5 tag.

[0030] The c-Myc tag may comprise SEQ ID NO:1.

[0031] Strep tag II may comprise SEQ ID NO:2 or SEQ ID NO:3.

[0032] The Flag tag may comprise SEQ ID NO:4.

[0033] The polyhistidine tag may comprise SEQ ID NO:5.

[0034] The polyhistidine tag may comprise SEQ ID NO:5.

[0035] The Avi tag may comprise SEQ ID NO:6.

[0036] The calmodulin-binding protein tag may comprise SEQ ID NO:7.

[0037] The E-tag may comprise SEQ ID NO:8.

[0038] The HA tag may comprise SEQ ID NO:9.

[0039] The S tag may comprise SEQ ID NO:10.

[0040] The SBP tag can comprise SEQ ID NO:11.

[0041] The V5 tag may comprise SEQ ID NO:12.

[0042] The tag may be a Yol tag (derived from alpha-tubulin). The Yol tag may comprise SEQ ID NO: 13.

[0043] In the system for inducible expression of an adaptor, the inducible gene expression system is an antigen-activated inducible gene expression system, and when a cell having the inducible gene expression system can be activated by the antigen, the inducible promoter can be an antigen-activated inducible promoter capable of causing expression of the adaptor.

[0044] The antigen that induces the antigen-activation-inducible gene expression system / antigen-activation-inducible promoter is not the same antigen as the antigen that binds to the first (poly)peptide of the adapter. The antigen that induces the antigen-activation-inducible gene expression system / antigen-activation-inducible promoter can be a different antigen compared to the antigen that binds to the first (poly)peptide of the adapter.

[0045] In the system for inducible expression of an adapter, the antigen that binds to the first (poly)peptide of the adapter and the antigen that induces the antigen activation inducible promoter are different.

[0046] Said activation of a cell by an antigen, which may be a cell surface antigen, a soluble antigen or an MHC-presented antigen, may be activation of a signaling domain of a receptor of said cell, for example activation of the intracellular signaling domain of a CAR or TCR.

[0047] Said antigen activation inducible promoter, which is activated / induced by a cell signaling pathway / cascade, can be a cell surface protein promoter (e.g., CD69 promoter), a cytokine promoter (e.g., TNF promoter, IL-2 promoter), a cell activation protein promoter (e.g., CTLA4, OX40, CD40L) or a cell surface adhesion protein promoter, or a functional part of such promoters.

[0048] The antigen activation-inducible promoter (first nucleic acid comprising an inducible promoter) that can be operably linked to the second nucleic acid encoding the adapter can be any promoter responsive to a transcription factor whose activation increases when immune cells are specifically activated, such as SP1, BATF, AP-1, IRF4, RUNX, NFAT, NF-κB, STAT5, or STAT3 sensing promoter, and a minimal promoter operably linked to an inducible enhancer, for example, as described in WO2019199689A1. The NFAT-inducible promoter can be replaced with any inducible promoter that specifically binds to a particular transcription factor. Without wishing to be bound by theory, if an NFAT-responsive element is present, it requires a signal from a TCR / CAR or other immune receptor to induce NFAT signaling.

[0049] The antigen activation-inducible promoter can include a minimal promoter (PMIN). Alternative minimal promoters, such as a minimal TATA box promoter, a minimal CMV promoter, or a minimal IL-2 promoter, can also be used. In some embodiments, the minimal promoter can be optimized for a desired transcription level or rate.

[0050] In the system for inducible expression of an adaptor, the inducible gene expression system is a drug-inducible expression system, the inducible promoter is a drug-inducible promoter, and the inducible gene expression system further comprises a nucleic acid encoding a synthetic transcription factor of the drug-inducible promoter, and when a drug is administered to a cell having the inducible gene expression system, the gene expression system is induced to express the adaptor.

[0051] The drug may be a synthetic drug.

[0052] The synthetic transcription factor may comprise a DNA binding domain, a drug binding domain and an activation domain, wherein the synthetic transcription factor can be activated by binding to the drug.

[0053] The nucleic acid encoding the synthetic transcription factor can be operably linked to a constitutive promoter.

[0054] The constitutive promoter can be, for example, the EF-1 alpha promoter, or any other constitutive promoter that causes constitutive expression in immune cells (e.g., MSCV, PGK-1, UBC, CMV, CAGG, SV40, or a pan-hemopoietic promoter, e.g., vav).

[0055] The synthetic transcription factor may, for example, comprise a DNA-binding protein or DNA-binding domain of a transcription factor (wild-type or modified domain, e.g., a zinc finger protein or POU domain), a nuclear receptor, and an activation domain, wherein the drug may be a ligand of the nuclear receptor.

[0056] The nuclear receptor can be, for example, the estrogen receptor (ER), progesterone (PR), retinoid X, or Drosophila ecdysone receptor.

[0057] Suitably, the synthetic transcription factor may comprise a zinc finger protein, a nuclear receptor and an activation domain, wherein the drug may be a ligand for the nuclear receptor.

[0058] The synthetic transcription factor may comprise a zinc finger protein, an estrogen receptor (ER) and an activation domain, wherein the drug may be tamoxifen or a tamoxifen metabolite.

[0059] The activation domain can be, for example, derived from the herpes simplex virus protein VP16, tetrameric repeats of the VP16 minimal activation domain VP64, the p65 domain of the human endogenous transcription factor NFκB, or a fusion protein comprising a sequence portion of the p65 domain of the human endogenous transcription factor NFκB and a sequence portion of human heat shock factor 1.

[0060] The tamoxifen metabolite can be endoxifen or 4-hydroxytamoxifen (4-OHT).

[0061] The ER may be an ER having a point mutation, for example, mouse ER (G525R or G521R), human ER (G400V, M543A, L540A), or human ER (G400V, M543A, L544A).

[0062] The drug-inducible promoter may be a hybrid promoter comprising a zinc finger binding motif and a minimal promoter, for example, a minimal promoter selected from the group consisting of E1b, TK, IL2, CMV, SV40, or any minimal TATA box promoter.

[0063] In the system for inducible expression of an adaptor, the synthetic transcription factor is a zinc finger protein.

[0064] In this system for inducible expression of an adapter, the synthetic transcription factor is a zinc finger protein, and the expression level of the adapter depends on the amount of drug administered to the cells and / or the number of binding sites (zinc finger binding motifs) for which the synthetic transcription factor binds to DNA in a drug-inducible promoter, thereby allowing tunable control of adapter expression.

[0065] In the system for inducible expression of an adaptor, the inducible gene expression system and the nucleic acid encoding the CAR specific for the second polypeptide of the adaptor can be present in one (or the same) immune cell.

[0066] In the system for inducible expression of an adaptor, the inducible gene expression system may be present in a first immune cell and the nucleic acid encoding the CAR specific for the second polypeptide of the adaptor may be present in a second immune cell (a different immune cell).

[0067] In the system for inducible expression of an adaptor, the inducible gene expression system is an antigen activation-inducible gene expression system disclosed herein, and the antigen activation-inducible gene expression system may be present in a first immune cell, and the first immune cell may comprise a CAR specific for an additional antigen, wherein the CAR comprises: i) said antigen-binding domain specific for said further antigen; ii) a transmembrane domain, and iii) Intracellular signaling domain and / or the first immune cell may comprise a TCR specific for a further antigen, and the nucleic acid encoding the CAR specific for the second polypeptide of the adapter may be present in a second immune cell (a different immune cell).

[0068] Said further antigen may be a further antigen expressed on the surface of a further target cell or on the surface of said (first) target cell, or may be a soluble antigen.

[0069] The soluble antigen is I) soluble antigens of the tumor microenvironment (TME), or II) a soluble antigen specifically associated with an autoimmune disease, or III) a soluble antigen specifically associated with an allergic disease, or IV) a soluble antigen specifically associated with an infectious disease, or V) Soluble antigens specifically associated with graft rejection in a subject It could be.

[0070] The further antigen may be a further TAA and the target cell may be a tumor cell.

[0071] Further said antigens may be expressed by cells of the tumor microenvironment, for example, tumor-associated fibroblasts.

[0072] Further such antigens may be antigens derived from human infectious pathogens associated with tumor cells (e.g., oncoviral antigens derived from Epstein-Barr virus, human papillomavirus, human cytomegalovirus, etc.) or more generally, antigens derived from human infectious pathogens in infectious diseases (e.g., human immunodeficiency virus).

[0073] The first immune cell and the second immune cell can be of the same type of immune cell, for example, a T cell (e.g., a CD4 T and / or CD8 T cell) or an NK cell.

[0074] The first immune cell and the second immune cell may be different types of immune cells, for example, the first immune cell may be a T cell and the second immune cell may be an NK cell, or the first immune cell may be, for example, a tumor-infiltrating lymphocyte (TIL) and the second immune cell may be a T cell or an NK cell.

[0075] In another aspect, the present invention provides an immune cell comprising: a) an inducible gene expression system, I) a first nucleic acid comprising an inducible promoter operably linked to a second nucleic acid; and II) the second nucleic acid encoding an adaptor, i) a first (poly)peptide comprising an antigen-binding domain that specifically binds to an antigen, and ii) a second (poly)peptide that binds to the antigen-binding domain of a chimeric antigen receptor (CAR). an inducible gene expression system comprising: b) a third nucleic acid encoding the CAR specific for the second polypeptide of the adapter; The CAR is i) the antigen-binding domain specific for the second (poly)peptide of the adapter; ii) a transmembrane domain, and iii) Intracellular signaling domain The present invention provides immune cells comprising:

[0076] The adaptor can be a secreted adaptor.

[0077] The adaptor may be secreted by the immune cell.

[0078] The adapter may include a signal peptide for secretion.

[0079] The immune cells can be T cells or NK cells.

[0080] In the immune cells, the inducible expression system can be an antigen-activated inducible expression system, and when the cell having the inducible gene expression system can be activated by the antigen, the inducible promoter can be an antigen-regulated inducible promoter capable of causing expression of the adapter.

[0081] In the immune cells, the inducible gene expression system may be a drug-inducible expression system, the inducible promoter may be a drug-inducible promoter, and the inducible gene expression system may further comprise a nucleic acid encoding a synthetic transcription factor of the drug-inducible promoter, and when a drug is administered to the immune cells, the gene expression system may be activated / induced and an adapter may be expressed.

[0082] In the immune cell, the synthetic transcription factor may comprise a DNA-binding domain, a drug-binding domain and an activation domain, and the synthetic transcription factor may be activated by binding to the drug.

[0083] The immune cells disclosed herein are immune cells for use in drug therapy.

[0084] The immune cells are immune cells for use in the treatment of cancer.

[0085] The immune cells are for use in the treatment of leukemia.

[0086] The immune cells are for use in the treatment of solid tumors.

[0087] The solid tumors include adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain / CNS tumors in children and adults, breast cancer, cervical cancer, colon / rectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, liver cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, lung cancer, pulmonary The cancer may be carcinoid tumor, lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity or oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, sarcoma, basal skin cancer, squamous cell skin cancer, melanoma, Merkel cell skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, or Wilms' tumor.

[0088] The immune cells are for use in the treatment of infectious diseases.

[0089] In another aspect, the present invention provides a composition comprising: a) a first immune cell comprising an inducible gene expression system, wherein the inducible gene expression system comprises: I) a first nucleic acid comprising an inducible promoter operably linked to a second nucleic acid; and II) the second nucleic acid encoding an adaptor, i) a first (poly)peptide comprising an antigen-binding domain that specifically binds to an antigen, and ii) a second (poly)peptide that binds to the antigen-binding domain of a chimeric antigen receptor (CAR). a first immune cell comprising: b) a second immune cell comprising a nucleic acid encoding the CAR specific for the second polypeptide of the adapter; The CAR is i) the antigen-binding domain specific for the second (poly)peptide of the adapter; ii) a transmembrane domain, and iii) Intracellular signaling domain A composition comprising:

[0090] In the composition, the inducible gene expression system may be a drug-inducible expression system, the inducible promoter may be a drug-inducible promoter, the inducible gene expression system may further comprise a nucleic acid encoding a synthetic transcription factor of the drug-inducible promoter, and when a drug may be administered to the immune cells, the gene expression system may be activated / induced and an adapter may be expressed, and the composition may comprise c) the drug.

[0091] In the compositions disclosed herein, the synthetic transcription factor may comprise a DNA-binding domain, a drug-binding domain, and an activation domain, and the synthetic transcription factor may be activated by binding to the drug.

[0092] In one embodiment of the invention, the composition comprises: a) a first immune cell comprising an inducible gene expression system, wherein the inducible gene expression system comprises: I) a first nucleic acid comprising an inducible promoter operably linked to a second nucleic acid; and II) the second nucleic acid encoding an adaptor, i) a first (poly)peptide comprising an antigen-binding domain that specifically binds to an antigen, and ii) a second (poly)peptide that binds to the antigen-binding domain of a chimeric antigen receptor (CAR). a first immune cell comprising: b) a second immune cell comprising a nucleic acid encoding the CAR specific for the second polypeptide of the adapter; wherein the CAR is i) the antigen-binding domain specific for the second (poly)peptide of the adapter; ii) a transmembrane domain, and iii) Intracellular signaling domain and the first immune cell may comprise a CAR specific for a further antigen, wherein the CAR specific for the further antigen comprises: i) an antigen-binding domain specific for a further antigen; ii) a transmembrane domain, and iii) Intracellular signaling domain wherein the inducible promoter can be an antigen activation inducible promoter capable of causing expression of the adapter when an antigen binding domain of the CAR specific for the further antigen binds to the further antigen, thereby activating the first immune cell.

[0093] In another embodiment of the present invention, the composition comprises: a) a first immune cell comprising an inducible gene expression system, wherein the inducible gene expression system comprises: I) a first nucleic acid comprising an inducible promoter operably linked to a second nucleic acid; and II) the second nucleic acid encoding an adaptor, i) a first (poly)peptide comprising an antigen-binding domain that specifically binds to an antigen, and ii) a second (poly)peptide that binds to the antigen-binding domain of a chimeric antigen receptor (CAR). a first immune cell comprising: b) a second immune cell comprising a nucleic acid encoding the CAR specific for the second polypeptide of the adapter; wherein the CAR is i) the antigen-binding domain specific for the second (poly)peptide of the adapter; ii) a transmembrane domain, and iii) Intracellular signaling domain wherein the first immune cell may comprise a TCR specific for a further antigen, and the inducible promoter may be an antigen activation inducible promoter capable of causing expression of the adapter when the TCR specific for the further antigen binds to the further antigen, thereby activating the first immune cell.

[0094] The compositions disclosed herein are for use in medical therapy.

[0095] The composition is for use in the treatment of cancer.

[0096] The composition is for use in the treatment of leukemia.

[0097] The composition is for use in the treatment of solid tumors.

[0098] The composition is for use in the treatment of an infectious disease.

[0099] In a further aspect, the present invention provides a pharmaceutical composition comprising a composition disclosed herein and, optionally, a pharmaceutically acceptable carrier.

[0100] Pharmaceutically acceptable carriers, diluents, or excipients may include buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0101] In a further aspect, the present invention provides a method for treating a subject suffering from cancer, comprising: A) administering immune cells to a subject, wherein the immune cells a) an inducible gene expression system, I) a first nucleic acid comprising an inducible promoter operably linked to a second nucleic acid; and II) the second nucleic acid encoding an adaptor, i) a first (poly)peptide comprising an antigen-binding domain that specifically binds to an antigen, and ii) a second (poly)peptide that binds to the antigen-binding domain of a chimeric antigen receptor (CAR). an inducible gene expression system comprising: b) a nucleic acid encoding the CAR specific for the second polypeptide of the adapter, The CAR is i) the antigen-binding domain specific for the second (poly)peptide of the adapter; ii) a transmembrane domain, and iii) Intracellular signaling domain The present invention provides a method comprising:

[0102] In the method, the inducible promoter is an antigen-activated inducible promoter capable of causing expression of the adapter when the immune cell is activated by the antigen.

[0103] In the method, the method B) administering a drug to said subject. wherein the inducible gene expression system is a drug-inducible expression system, the inducible promoter is a drug-inducible promoter, and the inducible gene expression system further comprises a nucleic acid encoding a synthetic transcription factor of the drug-inducible promoter, and when the drug is administered to the immune cells, the gene expression system is induced to express the adapter.

[0104] In the method, the synthetic transcription factor comprises a DNA-binding domain, a drug-binding domain, and an activation domain, and the synthetic transcription factor is activated by binding to the drug.

[0105] In the method, the agent may be administered simultaneously with, before, or after administration of the immune cells.

[0106] In another aspect, the present invention provides a method for treating a subject suffering from cancer, comprising: A) administering first immune cells to a subject, wherein the first immune cells a) an inducible gene expression system, I) a first nucleic acid comprising an inducible promoter operably linked to a second nucleic acid; and II) the second nucleic acid encoding an adaptor, i) a first (poly)peptide comprising an antigen-binding domain that specifically binds to an antigen, and ii) a second (poly)peptide that binds to the antigen-binding domain of a chimeric antigen receptor (CAR). and an inducible gene expression system comprising: B) administering to the subject second immune cells comprising a nucleic acid encoding the CAR specific for the second polypeptide of the adapter; The CAR is i) the antigen-binding domain specific for the second (poly)peptide of the adapter; ii) a transmembrane domain, and iii) Intracellular signaling domain The present invention provides a method comprising:

[0107] In said method, the method comprises: C) administering a drug to the subject. wherein the inducible gene expression system is a drug-inducible expression system, the inducible promoter is a drug-inducible promoter, and the inducible gene expression system further comprises a nucleic acid encoding a synthetic transcription factor of the drug-inducible promoter, and when the drug is administered to the immune cells, the gene expression system is induced to express the adapter.

[0108] In the method, the synthetic transcription factor comprises a DNA-binding domain, a drug-binding domain, and an activation domain, and the synthetic transcription factor is activated by binding to the drug.

[0109] In the method, the agent may be administered simultaneously with, before, or after administration of the immune cells.

[0110] In the method, the inducible promoter is capable of causing expression of the adaptor when the first immune cell is activated.

[0111] All definitions, properties and embodiments defined herein with respect to the first aspect of the invention disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention disclosed herein.

[0112] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0113] As used herein, the term "comprising" or "comprise" is used in reference to compositions, methods, and components that are essential to the respective methods or compositions, but is not limited to including non-specified elements, whether essential or not.

[0114] The term "system for inducible expression of adaptors in immune cells" refers to a combination of nucleic acids in a one-cell or two-cell constellation as disclosed herein. In this context, the term "system" may be used interchangeably with "combination of nucleic acids" or "composition of nucleic acids."

[0115] Generally, a CAR may comprise an extracellular domain (portion outside the cell) comprising an antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (intracellular signaling domain). The extracellular domain may be connected to the transmembrane domain by a linker or spacer. The extracellular domain may also comprise a signal peptide. In some embodiments of the present invention, the antigen-binding domain of the CAR binds to a tag conjugated to a (poly)peptide (a "tagging" polypeptide, or an adapter comprising said first (poly)peptide disclosed herein and said second (poly)peptide disclosed herein), wherein the tagged polypeptide may bind to a disease-associated antigen, for example, a tumor-associated antigen (TAA) that may be expressed on the surface of cancer cells, or an antigen expressed by cells in the tumor microenvironment, or an infection-associated antigen that may be expressed by pathogen-infected cells.

[0116] Said second (poly)peptide of an adapter capable of binding to the antigen-binding domain of a chimeric antigen receptor (CAR) disclosed herein may also be referred to as a "tag" (of a tagged polypeptide), and a CAR specific for said second polypeptide of said adapter is also referred to as an anti-tag CAR or adapter CAR (adCAR) or "universal CAR".

[0117] Such anti-tag CARs are disclosed, for example, in US Pat. No. 9,233,125 (B2).

[0118] Generally, the tag of the anti-tag CAR may be directly or indirectly conjugated to a polypeptide (tagged polypeptide), where the polypeptide can bind to said disease-associated antigen expressed on the target (cell) surface. The tag can often be, for example, a dextran or a hapten, such as biotin or fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or thiamine. However, the tag as part of the adapter of the present invention will usually be a (poly)peptide (the second (poly)peptide of the adapter disclosed herein) that can be produced in immune cells via transcription and translation of an exogenous nucleic acid sequence introduced into said immune cells.

[0119] The tag may be a (poly)peptide comprising at least four amino acids.

[0120] The tag may be a (poly)peptide comprising at least 4 amino acids up to and including 8, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or 200 amino acids.

[0121] Tags may have higher proteolytic stability and lower immunogenicity in humans compared to common (poly)peptides.

[0122] The tag can be located at or near the N-terminus or C-terminus of the first polypeptide.

[0123] A tag may also be incorporated into the sequence of said first polypeptide and therefore not be located at or near the N-terminus or C-terminus of said first polypeptide.

[0124] The tag may comprise an epitope of a protein.

[0125] The tag may comprise an epitope of a protein that is naturally present in the subject or in the subject's circulatory system.

[0126] The tag can be, for example, a hormone, cytokine, chemokine, growth factor, cell adhesion molecule, signaling peptide, receptor, cell surface peptide, or fragment thereof. The tag can be a ligand or fragment thereof. The ligand can be a hormone ligand. The ligand can be a peptide ligand. The tag can be an antigen, an epitope, including linear and non-linear epitopes. The tag can be a tumor-associated antigen. Alternatively, the tag may not be a tumor-associated antigen.

[0127] The tag may comprise an epitope of a neoantigen, wherein said epitope comprises a mutation of the antigen.

[0128] The tag may comprise an epitope of a neoantigen that may arise in tumorigenesis, wherein said epitope comprises a mutation of the antigen.

[0129] The tag may comprise an epitope of a neoantigen, wherein said epitope comprises a mutation of an antigen, said antigen may be a protein naturally occurring in the subject or in the subject's circulatory system.

[0130] The tag may be a (poly)peptide that does not naturally occur in the subject.

[0131] The tag may be a (poly)peptide that does not naturally occur in the circulatory system of a subject.

[0132] The tag may be a (poly)peptide that does not naturally occur in the circulatory system of a healthy subject.

[0133] The tag can be, for example, the following peptide tags: c-Myc tag, Strep tag II, Flag tag, polyhistidine tag, Avi tag, calmodulin binding protein tag, Yol tag (derived from alpha-tubulin), E tag, HA tag, S tag, SBP tag or V5 tag.

[0134] A "signal peptide" refers to a peptide sequence that directs the transport and localization of an intracellular protein, for example, to a particular organelle (eg, the endoplasmic reticulum) and / or to the cell surface.

[0135] Generally, "antigen-binding domain" refers to the region of a CAR that specifically binds to an antigen, for example, a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). More specifically, the "antigen-binding domain" of an anti-tag CAR may specifically bind to a tag present on a tagged polypeptide, where the polypeptide can bind to said disease-associated antigen expressed on the target (cell) surface. A CAR may comprise one or more antigen-binding domains (e.g., tandem CARs). Generally, the target region on a CAR is extracellular. The antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof. The antigen-binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a diabody. Any molecule that specifically binds to a given antigen, for example, an affibody or a ligand-binding domain derived from a naturally occurring receptor, may be used as the antigen-binding domain. The antigen-binding domain is often an scFv. Typically, in an scFv, the variable regions of the immunoglobulin heavy and light chains are fused by a flexible linker to form the scFv. Such a linker can be, for example, a "(G4 / S)3 linker."

[0136] In some cases, it is advantageous for the antigen-binding domain to be derived from the same species as the CAR will be used in. For example, when planning its therapeutic use in humans, it may be advantageous for the antigen-binding domain of the CAR to comprise a human or humanized antibody or an antigen-binding fragment thereof. Human or humanized antibodies or antigen-binding fragments thereof can be produced by a variety of methods well known in the art.

[0137] As used herein, "spacer" or "hinge" refers to a hydrophilic region between the antigen-binding domain and the transmembrane domain. The CAR of the present invention may include an extracellular spacer domain, but may also exclude such a spacer. The spacer may include, for example, an Fc fragment or fragment thereof of an antibody, a hinge region or fragment thereof of an antibody, a CH2 or CH3 region of an antibody, an accessory protein, an artificial spacer sequence, or a combination thereof. A notable example of a spacer is the CD8 alpha hinge.

[0138] The transmembrane domain of a CAR can be derived from any desired natural or synthetic source for such a domain. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domain can be derived from, for example, CD8 alpha or CD28. If the key signaling and antigen recognition modules (domains) are present on two (or even more) polypeptides, the CAR can have two (or more) transmembrane domains. Due to the small molecule-dependent heterodimerization domains in each polypeptide of the CAR, the division of the key signaling and antigen recognition modules allows for small molecule-dependent and reversible control of CAR cell expression (e.g., WO2014127261A1).

[0139] When the respective CAR is an activating CAR (unless explicitly indicated as an inhibitory CAR (iCAR), the CARs described herein generally refer to activating CARs), the cytoplasmic signaling domain (intracellular signaling domain or activation endodomain) of the CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" refers to a specialized function of a cell; for example, in T cells, effector function can be cytolytic activity or helper activity, including secretion of cytokines. The intracellular signaling domain refers to the portion of a protein that transmits an effector function signal and directs the cell expressing the CAR to perform a specialized function. The intracellular signaling domain can include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein sufficient to transmit a signal that initiates or blocks an immune cell effector function.

[0140] Prominent examples of intracellular signaling domains for use in CARs include the cytoplasmic signaling sequences of T cell receptors (TCRs) and co-receptors that initiate signaling after antigen receptor engagement.

[0141] Generally, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences: first, sequences that initiate antigen-dependent primary activation by the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domains), and second, sequences that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences, costimulatory signaling domains). Thus, the intracellular signaling domain of a CAR can comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0142] The primary cytoplasmic signaling domain that acts in a stimulatory manner may contain an ITAM (immunoreceptor tyrosine-based activation motif).

[0143] Examples of ITAMs containing primary cytoplasmic signaling domains that are often used in CARs are those derived from TCRζ (CD3ζ), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most notable are sequences derived from CD3ζ.

[0144] The cytoplasmic domain of a CAR can be designed to contain a CD3ζ signaling domain alone or in combination with any other desired cytoplasmic domain. The cytoplasmic domain of a CAR can include a CD3ζ chain portion and a costimulatory signaling region (domain). The costimulatory signaling region refers to a portion of a CAR that contains the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors, or their ligands, that are required for efficient lymphocyte responses to antigens. Examples of costimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0145] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specific order, with or without a linker. The linkages can be formed by short oligo- or polypeptide linkers, preferably 2-10 amino acids in length. A prominent linker is the glycine-serine double linker.

[0146] As an example, the cytoplasmic domain may comprise the signaling domain of CD3ζ and the signaling domain of CD28. In another example, the cytoplasmic domain may comprise the signaling domain of CD3ζ and the signaling domain of CD137. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3ζ, the signaling domain of CD28, and the signaling domain of CD137.

[0147] As mentioned above, either the extracellular portion or the transmembrane or cytoplasmic domain of the CAR may also contain a heterodimerization domain for the purpose of separating the key signaling and antigen recognition modules of the CAR.

[0148] The CAR may be further modified to include one or more elements at the level of the nucleic acid encoding the CAR that are effective for the elimination of CAR-expressing immune cells by a suicide switch. The suicide switch may include, for example, a drug that induces apoptosis or cell death, which induces a signaling cascade. In one embodiment, the nucleic acid that expresses and encodes the CAR may be further modified to express an enzyme, such as thymidine kinase (TK) or cytosine deaminase (CD). The CAR may also be part of a gene expression system that allows for the control of CAR expression in immune cells. Such a gene expression system may be an inducible gene expression system, where, when an inducer is administered to a cell transduced with the inducible gene expression system, the gene expression system is induced and the CAR is expressed on the surface of the transduced cell.

[0149] In some embodiments, the endodomain may comprise a primary cytoplasmic signaling domain or a costimulatory region, but not both.

[0150] In some embodiments of the invention, the CAR may be a "SUPRA" (split, universal, programmable) CAR, in which the "zipCAR" domain may be linked to an intracellular costimulatory domain and an extracellular leucine zipper (WO2017 / 091546). This zipper may be tagged with a complementary zipper fused to, for example, an scFv region, making the SUPRA CAR T cells tumor-specific. This approach is useful in part for generating universal CAR T cells for various tumors, where adapter molecules can be engineered to be tumor-specific, providing the option to alter specificity after adoptive transfer, which is key in situations such as selective pressure and antigen escape.

[0151] The CARs of the present invention can be designed to comprise any portion or subset of the above domains described herein, in any order and / or combination, resulting in a functional CAR, i.e., a CAR that mediates an immune effector response of immune effector cells expressing a CAR disclosed herein.

[0152] The term "tagged polypeptide," as used herein, refers to a polypeptide directly or indirectly linked to at least one additional component, i.e., a tag. A tagged polypeptide, as used herein, is capable of binding to an antigen expressed on a target cell. The polypeptide can be an antibody or an antigen-binding fragment thereof that binds to an antigen expressed on the surface of a target cell, for example, a tumor-associated antigen on a cancer cell. Alternatively, the polypeptide of the tagged polypeptide can be a cytokine or growth factor, or another soluble polypeptide capable of binding to an antigen on a target cell. Alternatively, the polypeptide of the tagged polypeptide can be an affibody or a ligand-binding domain derived from a naturally occurring receptor.

[0153] As used herein, "T cell receptor" or "TCR" refers to an antigen-recognition molecule present on the surface of a T cell. In normal T cell development, each of the four TCR genes, α, β, γ, and δ, can rearrange to result in highly diverse TCR proteins.

[0154] The term "antibody," as used herein, is used broadly to encompass various forms of antibody structures that specifically recognize (i.e., bind to) an antigen, including, but not limited to, monoclonal and polyclonal antibodies (including full-length antibodies), multispecific antibodies (e.g., bispecific antibodies), antibody fragments, i.e., antigen-binding fragments of antibodies, immunoadhesins, and antibody-immunoadhesin chimeras. An "antigen-binding fragment" comprises a portion of a full-length antibody, preferably the variable domain thereof, or at least the antigen-binding site thereof ("antigen-binding fragment of an antibody"). Examples of antigen-binding fragments include Fab (antigen-binding fragment), scFv (single-chain variable fragment), single-domain antibodies (nanobodies), diabodies, dsFv, FAb', diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. The antibody or antibody fragment may be human, fully human, humanized, human-engineered, nonhuman, and / or chimeric antibodies or antibody fragments. Non-human antibodies or antibody fragments can be humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. A chimeric antibody can refer to an antibody produced by combining two or more antibody genes that originally encoded separate antibodies.

[0155] The terms "having specificity for," "specifically binds," or "specific for," with respect to an antigen-binding domain of an antibody, fragment thereof, or CAR, refer to an antigen-binding domain that recognizes and binds to a particular antigen but does not substantially recognize or bind to other molecules in a sample. An antigen-binding domain that specifically binds to an antigen from one species may also bind to an antigen from another species. This cross-species reactivity does not violate the definition of an antigen-binding domain being specific. An antigen-binding domain that specifically binds to an antigen may also bind to different allelic forms of the antigen (allelic variants, splice variants, isoforms, etc.). This cross-reactivity does not violate the definition of an antigen-binding domain being specific.

[0156] As used herein, the term "antigen" is intended to include substances that bind to or induce the production of one or more antibodies, and may include, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates such as dextrans, haptens, and combinations thereof, such as glycosylated proteins or glycolipids. The term "antigen" as used herein refers to a molecular entity that may be expressed, for example, on the surface of a target cell, and that can be recognized by the adaptive immune system, including, but not limited to, antibodies or TCRs, or engineered molecules, including, but not limited to, endogenous or transgenic TCRs, CARs, scFvs or multimers thereof, Fab fragments or multimers thereof, antibodies or multimers thereof, single-chain antibodies or multimers thereof, or any other molecule capable of binding to the structure with high affinity.

[0157] The term "soluble antigen" as used herein refers to an antigen that is not immobilized on a surface such as a bead or cell membrane.

[0158] The terms "immune cell" or "immune effector cell" are used interchangeably and may refer to cells that are part of the immune system and can perform specific effector functions, such as alpha-beta T cells, NK cells, NKT cells, B cells, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, gamma-delta T cells, regulatory T cells (Tregs), monocytes, or macrophages. Preferably, such immune cells are human immune cells. Preferred immune cells are cells with cytotoxic effector function, such as alpha-beta T cells, NK cells, NKT cells, ILCs, CIK cells, LAK cells, or gamma-delta T cells. Most preferred immune effector cells are T cells and NK cells. Tumor-infiltrating lymphocytes (TILs) are T cells that migrate from a subject's blood to a tumor. Such TILs can be removed from a patient's tumor by methods well known in the art, such as enzymatic and mechanical tumor disruption followed by density centrifugation and / or cell marker-specific enrichment. The TILs can be genetically modified as disclosed herein and then returned to the patient. "Effector function" refers to the specialized function of a cell; for example, in T cells, effector function can be cytolytic activity or helper activity, including secretion of cytokines.

[0159] Immunotherapy is a medical term defined as "the treatment of disease by inducing, enhancing, or suppressing an immune response." Immunotherapies designed to induce or amplify an immune response are classified as activating immunotherapies, while those that decrease or suppress the immune response are classified as suppressing immunotherapies. Cancer immunotherapy, as an activating immunotherapy, attempts to stimulate the immune system to reject and destroy tumors. Adoptive cell transfer uses a cytotoxic response based on cells, preferably T cells or NK cells, to attack cancer cells. T cells that are naturally or genetically modified to be reactive against the patient's cancer are generated in vitro and then transferred back into the cancer patient. For this reason, immunotherapy is referred to as "CAR cell immunotherapy," or when using only T cells, "CAR T cell therapy" or "CAR T cell immunotherapy."

[0160] The term "treating" as used herein means reducing the frequency or severity of at least one sign or symptom of a disease.

[0161] The term "autologous" as used herein refers to any substance originating from the same subject to which it is subsequently reintroduced.

[0162] The term "allogeneic" as used herein refers to any substance that originates from a different subject of the same species as the subject into which the substance is being reintroduced.

[0163] The term "therapeutically effective amount" or "therapeutically effective population" refers to an amount of a cell population that confers a therapeutic benefit in a subject.

[0164] As used herein, the term "subject" refers to an animal. Preferably, the subject is a mammal, such as a mouse, rat, cow, pig, goat, chicken, dog, monkey, or human. More preferably, the subject is a human. The subject may be a subject (patient) suffering from a disease such as cancer, or an autoimmune disease, or an allergic disease, or an infectious disease, or transplant rejection.

[0165] The term "expression", as used herein, is defined as the transcription and / or translation of a particular nucleotide sequence caused by its promoter in a cell.

[0166] The terms "engineered cell" and "genetically modified cell" can be used interchangeably herein. This term refers to a cell that contains and / or expresses a foreign gene or nucleic acid sequence, which then modifies the genotype or phenotype of the cell or its progeny. In particular, this term refers to a cell, preferably a T cell, that can be engineered by recombinant methods well known in the art to stably or transiently express a peptide or protein not naturally expressed in such a cell. For example, T cells, preferably human T cells, are engineered to express an artificial construct, e.g., a chimeric antigen receptor, on their cell surface.

[0167] The term "cancer" is medically known as malignant neoplasia. Cancer is a broad group of diseases involving unregulated cell growth and includes all forms of leukemia. In cancer, cells (cancer cells) divide and grow uncontrollably, forming malignant tumors that invade nearby parts of the body. Cancer can also spread to more distant parts of the body via the lymphatic system or bloodstream. There are over 200 different known types of cancer that affect humans.

[0168] The terms "nucleic acid" or "polynucleotide," when used interchangeably herein, refer to a polymer of nucleotides. A polynucleotide is one that can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, the term "polynucleotide" encompasses all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cellular genomes using conventional cloning techniques and PCR, etc., and synthetic means.

[0169] A peptide is a short chain of 2 to 50 amino acids linked by peptide bonds. Chains of less than 10 or 15 amino acids may also be called oligopeptides.

[0170] Polypeptides are longer, continuous, unbranched peptide chains of up to 50 or more amino acids. Polypeptides containing more than 50 amino acids are also called proteins.

[0171] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous, where necessary to join two protein-coding regions in the same reading frame.

[0172] As used herein, the term "promoter" or "regulatory sequence" refers to a nucleic acid sequence operably linked to the promoter / regulatory sequence and required for transcription of a gene product. In some cases, this sequence may be the core promoter sequence, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements required for transcription of the gene product. The promoter / regulatory sequence may be, for example, a sequence that provides tissue-specific expression of the gene product.

[0173] The term "minimal promoter (PMIN)" as used herein refers to the smallest genetic element capable of inducing transcription of a gene located downstream of the minimal promoter. Eukaryotic promoters of protein-coding genes have one or more of three conserved sequences in this region (i.e., the TATA box, the initiation region, and the downstream promoter element). A minimal promoter allows for reduced basal leakage in the absence of specific transcriptional activators and high expression when the transcriptional activator binds at a specific DNA-binding site upstream of the minimal promoter. Alternative minimal promoters, such as a minimal TATA box promoter, a minimal CMV promoter, or a cellular IL-2 promoter, can also be used.

[0174] The minimal promoter may be altered / modified by the introduction of binding sites for specific transcription factors (e.g., required for drug-inducible as well as antigen-activated inducible systems; the promoter may contain several repeats of, for example, NFAT binding sites).

[0175] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological cellular conditions.

[0176] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only in the presence or absence of a particular condition, such as when an inducer (e.g., an inducing signal or inducer, e.g., a drug, metal ion, alcohol, oxygen, etc.) is present in the cell. The inducer can be, for example, activation of the intracellular signaling domain of a CAR.

[0177] The constitutive promoter operably linked to the transgene can be, for example, the EF-1 alpha promoter, or any other constitutive promoter that drives constitutive expression in immune cells (e.g., MSCV, PGK-1, UBC, CMV, CAGG, SV40, or a pan-hemopoietic promoter, e.g., vav).

[0178] For example, the inducible promoter operably linked to the polynucleotide encoding the adapter disclosed herein can be any promoter responsive to a transcription factor whose activation increases when immune cells are specifically activated or localized in a given microenvironment (e.g., a tumor microenvironment), such as SP1, BATF, AP-1, IRF4, RUNX, NFAT, NF-κB, STAT5, or STAT3 sensing promoter, and a minimal promoter operably linked to an inducible enhancer, for example, as described in WO2019199689A1. The inducible promoter may further include a minimal promoter operably linked to an effector. The NFAT inducible promoter can be replaced with any inducible promoter that specifically binds to a particular transcription factor. Without wishing to be bound by theory, if an NFAT responsive element is present, it requires a signal from a TCR / CAR or other immune receptor to induce NFAT signaling.

[0179] Inducible promoters can include minimal promoters (PMIN). Alternative minimal promoters, such as a minimal TATA box promoter, a minimal CMV promoter, or a minimal IL-2 promoter, can also be used. In some embodiments, the minimal promoter can be optimized for a desired transcription level or rate.

[0180] In certain variants, the inducible promoter may be a drug-inducible promoter.

[0181] Such systems may include nucleic acids containing promoters that are inducible by drugs, e.g., synthetic drugs. By utilizing drug-inducible promoters, transgene expression can be turned on and off to avoid toxic side effects and / or allow cell quiescence in remission. Many such systems use chimeric transcriptional regulators (e.g., synthetic transcription factors).

[0182] In some variants, the inducible promoter can be inducible by a drug, i.e., a drug-inducible promoter. The drug is selected based on its safety record, favorable pharmacokinetic profile, tissue distribution, low partition coefficient between the extracellular space and the cytosol, low immunogenicity, low toxicity, and / or high expression in lymphocytes. In some alternatives, the inducible promoter is activated by a transcriptional activator (e.g., a synthetic transcription factor) that interacts with the drug. The transcriptional activator is activated in the presence of the drug or is capable of binding to and activating the inducible promoter. A particular alternative to the drug is a drug that binds to the estrogen receptor ligand-binding domain of the transcriptional activator. In some alternatives, the drug includes tamoxifen, its metabolites, analogs, and pharmaceutically acceptable salts and / or hydrates or solvates thereof.

[0183] The term "synthetic transcription factor," as used herein, may comprise a DNA-binding domain, a drug-inducible domain (drug-binding domain), and an effector (activation) domain that can be linked and / or fused together, thereby allowing the individual domains to be arranged in any order.

[0184] The DNA-binding domain of a synthetic transcription factor can be a protein or a portion of a protein that specifically recognizes the DNA-binding motif of a drug-inducible promoter and mediates the binding of the synthetic transcription factor to this DNA sequence. In addition, zinc finger proteins, TALEs (transcription activator-like effectors), and Cas9 (clustered regulatory interspaced short palindromic repeat systems) can be engineered to recognize specific DNA sequences. Furthermore, the DNA-binding domain of a naturally occurring transcription factor (e.g., a POU homeodomain) can be utilized.

[0185] The DNA-binding domain can be, for example, a zinc finger protein (or a DNA-binding domain thereof) or a protein comprising or consisting of a POU domain.

[0186] The DNA-binding motif of a drug-inducible promoter is a specific DNA sequence that is recognized directly or indirectly (in the case of Cas9) by the DNA-binding domain of a synthetic transcription factor. For example, since each zinc finger domain specifically recognizes a 3-bp DNA sequence, a three-finger zinc finger protein can be designed to recognize a 9-bp sequence.

[0187] A drug-binding domain of a synthetic transcription factor refers to a protein or portion of a protein that binds to a drug or ligand of the domain. Upon drug binding, the drug-binding domain allows the synthetic transcription factor to transition from an inactive to an active state. This transition may involve the release of the inactivated factor and / or translocation of the synthetic transcription factor from the cytoplasm to the nucleus. Examples of drug-binding domains are nuclear receptors, extracellular domains of receptors, antigen / substance-binding proteins (including dimerization factors), and / or the active sites of enzymes.

[0188] An activation domain of a synthetic transcription factor refers to a protein or portion of a protein that autonomously promotes recruitment of the transcription machinery to initiate mRNA transcription. Examples of activation domains are VP16, VP64, fragments of NFkB p65, heat shock factor 1, and combinations thereof.

[0189] For example, the synthetic transcription factor may include a zinc finger protein, an estrogen receptor (ER), and an activation domain, where the drug may be tamoxifen or a tamoxifen metabolite. The activation domain may be, for example, tetrameric repeats of the herpes simplex virus protein VP16, the VP16 minimal activation domain VP64, a portion of the p65 domain of human endogenous transcription factor NFκB, or a fusion protein containing a fragment of human NFκB p65 and heat shock factor 1. The tamoxifen metabolite may be endoxifen or 4-OHT. The ER may be an ER with a point mutation, such as mouse ER (G525R or G521R), human ER (G400V, M543A, L540A), or human ER (G400V, M543A, L544A).

[0190] The drug-inducible promoter may be a hybrid promoter comprising a DNA-binding motif for the DNA-binding domain of a synthetic transcription factor and a minimal promoter.

[0191] The drug-inducible promoter may be a hybrid promoter comprising a zinc finger binding motif and a minimal promoter selected from the group consisting of E1b, TK, IL2, CMV, and SV40.

[0192] The term "inducible (gene) expression system" refers to the expression of an exogenous polypeptide (transgene), herein generally an adapter as disclosed herein, in an immune cell. An inducible (gene) expression system may also be an "antigen-activated inducible gene expression system," i.e., the inducible expression system can be activated in a cell harboring said inducible gene expression system when an antigen / ligand binds to a receptor of the cell, e.g., a CAR or TCR, either directly or upon MHC presentation. Said binding of the antigen / ligand to the receptor can induce a signal cascade in the cell, which can then lead to the induction of expression of the introduced gene (or transgene), herein generally an adapter. Said antigen / ligand, which can induce a signal cascade in a cell when it binds to its cognate receptor, is also referred to as an "inducible signal."

[0193] Alternatively, the inducible gene expression system may be a drug-inducible gene expression system, i.e., the inducible gene expression system may be activated in a cell having said inducible gene expression system when a drug, e.g., a synthetic drug, e.g., tamoxifen, may be introduced into the cell, where said drug may bind to a synthetic transcription factor, which may then cause induction of expression of the transgene, herein the adapter.

[0194] Such drugs may also be referred to as "inducers."

[0195] Both types of inducible gene expression systems can be used in the one-cell and / or two-cell systems disclosed herein.

[0196] In the presence of an inducing signal or inducer, an inducible expression system causes expression of the exogenous polypeptide. In an induced system, withdrawal of the inducing signal or inducer can reduce and / or stop expression of the exogenous polypeptide. Upon reintroduction of the inducing signal or inducer, the system is re-induced and expression of the exogenous polypeptide, i.e., adapter, begins. An inducible (gene) expression system can be inducible by an inducing signal, for example, by activation of the intracellular signaling domain of the CAR or TCR upon antigen / ligand binding as disclosed herein ("activation" induction), or by an inducer, for example, a (synthetic) drug as disclosed herein ("drug induction").

[0197] In some embodiments, the inducible (gene) expression system disclosed herein can also provide tunable control of adapter expression. As used herein, the term "tunable control" refers to the ability to control the expression level of an adapter disclosed herein. For example, the level of induced adapter expression can depend on the amount of inducer or induction signal present. For example, the presence of a higher amount of inducer, e.g., a synthetic drug, can result in a higher level of induction of adapter expression. For example, the presence of a high amount of inducer, e.g., a synthetic drug, can induce a higher level of adapter expression compared to the presence of a low amount of inducer. Thus, inducible or tunable expression of an adapter can be dose-dependent with respect to the amount of inducer present.

[0198] In addition to the inducing drug dose, in some embodiments, the inducible (gene) expression system disclosed herein can also provide adjustable control of adapter expression depending on the number of response elements for the synthetic transcription factor. As used herein, the term "adjustable control" refers to the ability to regulate the expression level of the adapter disclosed herein. For example, the level of induced adapter expression can depend on the number of response elements, or in other words, the number of binding sites, for the synthetic transcription factor in the inducible promoter. For example, binding of five synthetic transcription factor molecules to an inducible promoter containing five binding sites will induce transcriptional output, i.e., a higher level of adapter expression, compared to a construct containing two response elements in the inducible promoter. Thus, inducible or adjustable expression of the adapter can be dependent on the number of response elements for the synthetic transcription factor.

[0199] Embodiment In one embodiment of the present invention, the immune cells disclosed herein expressing an adapter CAR and an inducible adapter in the same cell are immune cells for use in treating cancer in a subject suffering from cancer. The adapter may be specifically bound by the adapter CAR, and the adapter may specifically bind to an antigen of the cancer. The subject's immune cells, such as T cells or NK cells, are isolated. The subject may be suffering from the cancer or may be a healthy subject. These cells are genetically modified in vitro or in vivo to inducibly express the CAR and the adapter. These modified cells may be activated and expanded in vitro or in vivo. For cell therapy, these modified cells are injected into a recipient in need thereof. These cells may be a pharmaceutical composition (the cells plus a pharmaceutically acceptable carrier). When the expression of the adapter is induced by the injected cells, it is possible to kill (or at least stop the proliferation of) cancer cells in the recipient. When the inducible expression system is a drug-inducible system disclosed herein, induction of expression can be caused by the application of a synthetic drug, such as tamoxifen. The recipient can be the same subject from which the cells were obtained (autologous cell therapy) or can be from another subject of the same species.

[0200] Immune cells, preferably T cells or NK cells, engineered to express the CAR and the adapter can be administered either alone or as a pharmaceutical composition in combination with diluents and / or other components, such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present invention can comprise a cell population of genetically modified cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0201] Preferably, the compositions of the present invention are formulated for intravenous administration.Administration of the cell composition to a subject may be carried out by any convenient method known in the art.

[0202] The pharmaceutical composition of the present invention can be administered by a method appropriate for the disease to be treated. The appropriate dosage can be determined by clinical trials. However, the amount and frequency of administration will also be determined and influenced by factors such as the patient's condition and the type and severity of the patient's disease.

[0203] The pharmaceutical compositions disclosed herein, comprising immune cells, preferably T cells or NK cells, are 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6 The cell composition may be administered in a dose of cells / kg body weight. The cell composition may also be administered multiple times at such dosages. The cell composition may be injected directly into the bloodstream, tumor, lymph node, or site of infection.

[0204] The drug for inducing the expression of the adapter can be administered by a method appropriate for the disease to be treated. The appropriate dosage can be determined by clinical trials. However, the amount and frequency of administration will also be determined and influenced by factors such as the patient's condition and the type and severity of the patient's disease. The drug can be injected directly into the bloodstream, applied to the skin, or taken orally. The drug can also be administered multiple times in variable dosages. The drug is formulated according to the route of administration.

[0205] The cells may be activated and expanded to a therapeutically effective amount using methods known in the art.

[0206] The cells of the invention may be used in combination with, for example, chemotherapy, radiation, immunosuppressants, antibodies, or antibody therapy.

[0207] In another embodiment of the present invention, the immune cell composition disclosed herein is for use in treating cancer in a subject suffering from cancer or for use in treating a viral infection in a patient. The composition may comprise immune cells expressing an inducible adapter disclosed herein, such as a TIL, or immune cells expressing an adapter CAR disclosed herein. The adapter may specifically bind to an antigen of the cancer or an antigen of a pathogen associated with a viral infection. The subject's immune cells, such as T cells, TIL, or NK cells, are isolated. The immune cells may be further isolated based on TCR reactivity to oncoviral antigens or tumor neoantigens (e.g., isolation of cytomegalovirus-reactive T cells using PepTivator™ CMV p65 peptide activation (Miltenyi Biotec) and isolation of activated T cells using CliniMACs™ Cytokine Capture System (Miltenyi Biotec)). The subject may be suffering from cancer or may be a healthy subject. Such cells are genetically modified in vitro or in vivo to express the CAR and, inducibly, the adapter. Such modified cells can be activated and expanded in vitro or in vivo. In cell therapy, such modified cells are infused into a recipient in need thereof. Such cells can be a pharmaceutical composition (the cells plus a pharmaceutically acceptable carrier). When the expression of the adapter is induced by the infused immune cells expressing the CAR, it can kill (or at least stop the proliferation of) cancer cells in the recipient. If the inducible expression system is a drug-inducible system as disclosed herein, induction of expression can be triggered by application of a synthetic drug, e.g., tamoxifen. The recipient can be the same subject from whom the cells were obtained (autologous cell therapy) or can be from another subject of the same species.

[0208] Immune cells, preferably T cells, TILs, or NK cells, engineered to express the CAR and the adapter on two different cells can be administered either alone or as a pharmaceutical composition in combination with a diluent and / or other components, such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present invention can comprise a cell population of genetically modified cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0209] Preferably, the compositions of the present invention are formulated for intravenous administration.Administration of the cell composition to a subject may be carried out by any convenient method known in the art.

[0210] The pharmaceutical composition of the present invention can be administered by a method appropriate for the disease to be treated. The appropriate dosage can be determined by clinical trials. However, the amount and frequency of administration will also be determined and influenced by factors such as the patient's condition and the type and severity of the patient's disease.

[0211] The pharmaceutical compositions disclosed herein comprising immune cells, preferably T cells, TIL or NK cells, are 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6 The cell composition may be administered in a dose of cells / kg body weight. The cell composition may also be administered multiple times at such dosages. The cell composition may be injected directly into a tumor, lymph node, or site of infection.

[0212] The drug for inducing adapter expression can be administered by a method appropriate for the disease to be treated. The appropriate dosage can be determined through clinical trials. However, the amount and frequency of administration will also be determined and influenced by factors such as the patient's condition and the type and severity of the patient's disease.

[0213] Drugs can be injected directly into the bloodstream, applied to the skin, or taken orally. Drugs can also be administered multiple times in variable dosages. Drugs are formulated according to the route of administration. Cells can be activated and expanded to a therapeutically effective amount using methods known in the art.

[0214] The cells of the invention may be used in combination with, for example, chemotherapy, radiation, immunosuppressants, antibodies, or antibody therapy.

[0215] The CAR-expressing immune cells and the inducible adaptor-expressing immune cells can be administered simultaneously, or the CAR-expressing immune cells can be administered before the inducible adaptor-expressing immune cells, or vice versa. A drug to induce expression of the adaptor can be administered to the patient simultaneously with the immune cells or after administration of the immune cells.

[0216] In a preferred embodiment, the immune cells disclosed herein that express inducible adapters are TILs that migrate into (solid) tumors and can serve as vehicles for adapter delivery in situ.

[0217] In another embodiment of the invention, immune cells can be modified to express "activation-inducible" or "drug-inducible" adaptors that target human pathogen-infected cells (e.g., HIV-1-infected cells). Such cells can be isolated, for example, by isolation of activated T cells with the CliniMACs™ Cytokine Capture System (Miltenyi Biotec) or other isolation modalities, following the use of pathogen-associated peptide pools to activate T cells based on their TCR reactivity to pathogen-associated antigens or tumor neoantigens. Such cells can use an "activation-inducible" promoter to drive expression of an adaptor specific for a pathogen antigen upon migration to the site of infection and TCR recognition of a pathogen-infected cell. Alternatively, drug-inducible expression of the adaptor can be used to controllably produce an adaptor specific for a pathogen antigen, specifically at the site of infection. Additionally, such engineered immune cells can contain an adaptor CAR specific for the adaptor that binds to the pathogen antigen, or other engineered immune cells can contain a CAR that does not express an adaptor.

[0218] In one embodiment of the present invention, immune cells are modified with an activation-inducible cassette (referred to as activation-inducible cells), which drives the expression of an adapter molecule via a promoter responsive to TCR / CAR-mediated signaling. Such cells can be selected based on their reactivity to tumor antigens by isolating cells from specific regions of the body (e.g., isolating TILs from tumor sites) or by selecting T cells with TCRs reactive to oncovirus antigens (e.g., CMV PepTivator-activated T cells) or tumor neoantigens (e.g., tumor neoantigen peptide pool-activated T cells). Alternatively, T cells can be transduced with a CAR or TCR specific for any of the above antigen categories. Upon TCR / CAR recognition of a TAA, oncovirus, or tumor neoantigen, a TCR-responsive promoter drives the expression of an anti-tumor adapter molecule. Such tagged adapter molecules are specific for tumor antigens. The tag portion of the adapter molecule (in this case, a 6xHis tag) is recognized by the extracellular recognition domain of an adapter CAR (in this case, an anti-His6-specific CAR) expressed by a second immune cell. The tagged adapter molecule serves as a bridging molecule that redirects the adapter CAR cells to the tumor, resulting in tumor cell lysis by the adapter CAR cells.

[0219] In a further embodiment of the present invention, immune cells are modified with a drug-inducible expression cassette (referred to as drug-inducible cells). In the "off" state, such inducible cells are indistinguishable from unmodified immune cells. Upon administration of an inducible drug, the inducible expression cassette is activated, and a tagged adapter molecule is transcribed and secreted by the immune cells. Such tagged adapter molecules are specific for tumor antigens. The tag portion of the adapter molecule (in this case, a 6xHis tag) is recognized by the extracellular recognition domain of an adapter CAR (in this case, an anti-His6-specific CAR) expressed by a second immune cell. The tagged adapter molecule serves as a bridging molecule that redirects the adapter CAR cells to the tumor. Consequently, the tumor cells are lysed by the adapter CAR cells. [Example]

[0220] Generation of anti-tag CAR T cells and drug-induced T cells 1.1 Design of the structure The adaptor CAR T cells contain an anti-His6 scFv as the binding moiety. This scFv binds to the human CD8 transmembrane domain via the hIgG4 hinge domain. The signaling domain consists of 4-1BB, CD28, and CD3ζ. A truncated LNGFR following a furin P2A site is 3' of the CAR construct. LNGFR is used as a transduction marker.

[0221] Inducible T cells constitutively express a synthetic transcription factor driven by the PGK promoter. The synthetic transcription factor consists of a three-finger zinc finger protein called N1, the mouse estrogen receptor (G525R), and the activation domain VP64. The synthetic transcription factor sequence binds to LNGFR, used as a transduction marker, via the furin P2A site. The inducible gene expression cassette also contains the sequence of a tagged adapter molecule (in this case, His6-tagged anti-CD19 Fab), whose transcription is regulated by the inducible promoter. The inducible promoter consists of a binding site for the N1 zinc finger (five repeats) that binds to the E1b minimal promoter.

[0222] 1.2 LV particle generation and titration Lentiviral vector particles were produced by transient transfection of HEK-293T cells. The lentiviral vector particles were pseudotyped with VSV-G. For transfection, HEK-293T cells were seeded in DMEM (Biowest) supplemented with 2 mM L-glutamine (Lonza) and 10% FCS (Biochrom) in a T175 culture flask 3 days prior to transfection. On the day of transfection, the culture medium was removed and replaced with DMEM (Biowest) supplemented with 2 mM L-glutamine (Lonza). The cells were transfected with a three-plasmid system encoding VSV-G, gag / pol / rev, and a psi-positive transfer vector (anti-tag CAR or inducible cassette). After 48 hours, the supernatant was collected and centrifuged at 1000 rpm for 10 minutes to remove cell debris. Additionally, the supernatant was filtered through a 0.45 μm filter. The pellet was resuspended in ice-cold PBS and stored at -80°C.

[0223] The functional titer of VSV-G pseudotyped lentiviral vector particles was determined by titration on Sup-T1 cells. 2E5 cells were seeded in 100 μL of RPMI (Biowest) supplemented with 2 mM L-glutamine (Lonza) in a 96-well round-bottom plate. For transduction, 100 μL of serially diluted lentiviral vector particles was added to the seeded cells. 24 h later, 90 μL of RPMI (Biowest) supplemented with 2 mM L-glutamine (Lonza) and 10% FCS (Biowest) was added. After 96 h, the frequency of transduced cells was quantified by flow cytometry using an LNGFR APC conjugate (Miltenyi Biotec). Based on the frequency of LNGFR-positive cells, the number of seeded cells and the amount of lentiviral particles used for transduction, and the titer were calculated. The titer was expressed as transducing units per mL.

[0224] 1.3 Transduction, culture and analysis of anti-tag CAR T cells and induced T cells Anti-tag CAR T cells and induced T cells were generated using primary T cells from healthy donors. T cells were isolated from PBMCs using a PAN T cell isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. Prior to transduction, 2E6 T cells were seeded in 2 mL of TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec), IL-15 (Miltenyi Biotec), and TransAct (Miltenyi Biotec) in a 24-well plate. After 24 hours, T cells were transduced at an MOI of 5 by adding the corresponding amount of lentiviral vector particles. On day 3 postactivation, the culture medium was removed and replaced with TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec). On day 6 after transduction, the frequencies of anti-tag CAR-positive T cells and induced T cells were indirectly analyzed by determining LNGFR expression by flow cytometry using an LNGFR APC conjugate (Miltenyi Biotec). On day 7 after transduction, transduced T cells were enriched for LNGFR-positive cells using MACSelect LNGFR MicroBeads (Miltenyi Biotec). The enrichment procedure was performed according to the supplier's protocol. Transduced T cells were used for functional assays on day 13 after activation. [Example]

[0225] Analysis of induction of anti-CD19 Fab-His6 secretion and adapter concentration Lentiviral particles encoding the drug-inducible cassette were produced as described in Example 1.2. Drug-inducible T cells were generated as described in Example 1.3. 1E4 LNGFR-positive drug-inducible T cells were seeded in 100 μL of TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec) in a 96-round-bottom well plate. Secretion of His6-tagged anti-CD19 Fab was induced by adding various concentrations of 4-OHT (0–500 nM, Sigma-Aldrich) to each well in 100 μL of TexMACS medium (Miltenyi Biotec) supplemented with IL-7 (Miltenyi Biotec) and IL-15 (Miltenyi Biotec). Non-transduced T cells served as a negative control. Cells were incubated at 37°C and 5% CO2. 48 hours after induction, the supernatant containing His6-tagged anti-CD19 Fab was collected. + Raji cells were stained using T cell supernatant and anti-His-APC as secondary antibodies. + 1E5 Raji cells were seeded in a 96-round-bottom well plate. The cells were pelleted at 300g for 5 minutes and stained with 50 μL of the collected T cell supernatant for 10 minutes at 4°C. CD19 + Raji cells were washed twice with 200 μL of CliniMACS buffer (Miltenyi Biotec) supplemented with 0.5% BSA (Miltenyi Biotec) (referred to as PEB) and incubated for 10 min at 4 °C in 50 μL of secondary staining mixture consisting of the secondary antibody anti-HisAPC (Miltenyi Biotec) diluted in PEB. +Raji cells were washed with 200 μL of PEB (300 g, 5 min). Cells were resuspended in 100 μL of PEB for subsequent flow cytometry analysis. To exclude dead cells, propidium iodide (PI; Miltenyi Biotec) was added to stained cells immediately before sample acquisition on a MACSQuant® Analyzer 10 (Miltenyi Biotec). The mean fluorescence intensity determined in the APC channel was correlated with Fab concentration by extrapolation from a standard curve. A defined concentration of His6-tagged anti-CD19-Fab (Miltenyi Biotec) was used to generate the standard curve.

[0226] The induction of His6-tagged anti-CD19-Fab secretion by induced T cells strictly depends on the presence of the inducing drug 4-OHT (Figure 3). + As determined by staining of Raji cells and extrapolation with a standard curve, increasing amounts of His6-tagged anti-CD19-Fab are detected in the supernatants of T cell cultures with increasing concentrations of 4-OHT. [Example]

[0227] Cytolytic activity of anti-His adaptor CAR T cells in co-culture with Raji cells in the presence of inducible anti-CD19 Fab T cells and the inducing drug 4-OHT Cytotoxicity against tumor cells mediated by anti-His adaptor CAR T cells was assessed by quantification of viable target cells using a MACSQuant® Analyzer 10 (Miltenyi Biotec). Thus, GFP was added to 50 μL of TexMACS medium (Miltenyi Biotec) in a 96-well round-bottom plate. +1E4 Raji cells were seeded. Anti-His adaptor CAR T cells were then added to 50 μL of TexMACS medium (Miltenyi Biotec) at an E:T ratio of 2:1. T cell numbers were adjusted for LNGFR expression to correlate with equal transduced and total T cell numbers in each well. The number of untransduced T cells was adjusted for total cell number. 1E4 anti-CD19 Fab-induced T cells secreting the adaptor molecule, His-tagged anti-CD19 Fab, were added to each well. His-tagged anti-CD19 Fab secretion was induced by adding 100 nM 4-OHT (Sigma-Aldrich) in 50 μL of TexMACS (Miltenyi Biotec) at the start of the assay. Plates were centrifuged at 300 g for 1 minute and incubated at 37°C and 5% CO2. Specific lysis of target cells was determined 6 days after co-culture setup. The plates were then incubated at 4°C for 20 minutes to stop further target cell lysis. Live target cells were then quantified by flow cytometry. Propidium iodide (PI; Miltenyi Biotec) was automatically added to the co-cultures using the MACSQuant® autolabeling function, and 70 μL was obtained from each well using high acquisition mode. Live Raji tumor cells were quantified by flow cytometry. - GFP is defined as + Cell and specific lysis were calculated according to the following formula: % specific lysis = (1 − (Raji cell number [sample] / Raji cell number [target cells])) · 100%.

[0228] CD19 + Specific lysis of Raji cells was observed in CD19 cells in the presence of at least 1 nM 4-OHT. + It was only detected in co-cultures of Raji cells, adaptor CAR T cells, and induced T cells (● symbol) (Figure 4). Maximum specific lysis was obtained after the addition of at least 10 nM 4.OHT. The cytotoxicity of non-transduced T cells and CD19 Fab-induced T cells in the presence of anti-CD19 Fab was also observed. + Co-culture of Raji (■ symbol) and induced anti-CD19 Fab T cells and CD19 +Raji co-cultures (▲ symbols) served as negative controls, demonstrating that tumor cell lysis cannot be induced by the adapter molecule itself but requires the simultaneous presence of anti-His adapter CAR T cells. [Example]

[0229] Analysis of PD-1 expression by anti-His adaptor CAR T cells after co-culture with Raji cells in the presence of inducible anti-CD19 Fab T cells and the inducing drug 4-OHT To set up the co-culture, add 50 μL of TexMACS medium (Miltenyi Biotec) to GFP cells in a 96-well round-bottom plate. +1E4 Raji cells were seeded. Anti-His adaptor CAR T cells were then added to 50 μL of TexMACS medium (Miltenyi Biotec) at an E:T ratio of 2:1. T cell numbers were adjusted for LNGFR expression to correlate with equal transduced and total T cell numbers in each well. The number of untransduced T cells was adjusted for total cell number. 1E4 anti-CD19 Fab-induced T cells secreting the adaptor molecule, His-tagged anti-CD19 Fab, were added to each well. His-tagged anti-CD19 Fab secretion was induced by the addition of 100 nM 4-OHT (Sigma-Aldrich) in 50 μL of TexMACS (Miltenyi Biotec) at the start of the assay. Plates were centrifuged at 300 g for 1 minute and incubated at 37°C, 5% CO2 for 2 days. T cell activation was analyzed by staining cocultured cells with anti-PD-1-PEVio770, CD3-VioBlue, CD8-VioGreen, and LNGFR-APC conjugates (Miltenyi Biotec). Therefore, the coculture plates were centrifuged at 300 g for 5 minutes, and cells were stained with 50 μL of a staining mixture consisting of PD-1PE07 conjugates (Miltenyi Biotec) diluted in PEB for 10 minutes at 4°C. Cells were washed twice with 200 μL of PEB (300 g, 5 minutes). Cells were resuspended in 100 μL of PEB for subsequent flow cytometry analysis. To exclude dead cells, propidium iodide (PI; Miltenyi Biotec) was added to the stained cells, and samples were immediately taken on a MACSQuant® Analyzer 10 (Miltenyi Biotec).

[0230] PD-1 expression on T cells was significantly elevated in the presence of at least 10 nM 4-OHT compared with CD19. + The frequency of PD-1-positive T cells was detected only in cocultures of Raji cells, adaptor CAR T cells, and induced T cells and increased with the concentration of 4-OHT (● symbol) (Figure 4). The maximum frequency of PD-1-positive cells was obtained after the addition of at least 50 nM of 4-OHT. The frequency of PD-1-positive T cells was significantly higher in non-transduced T cells and CD19 cells in the presence of anti-CD19 Fab-induced T cells. +Co-culture of Raji (■ symbol) and induced anti-CD19 Fab T cells and CD19 + Raji co-cultures (▲ symbols) served as negative controls, and both demonstrated that T cell activation (as indicated by PD-1 expression) cannot be induced by the adapter molecule itself but requires the simultaneous presence of anti-His adapter CAR T cells.

Claims

1. 1. A system for inducible expression of an adaptor in an immune cell, comprising: a) an inducible gene expression system, I) a first nucleic acid comprising an inducible promoter operably linked to a second nucleic acid; and II) the second nucleic acid encoding an adaptor, i) a first (poly)peptide comprising an antigen-binding domain that specifically binds to an antigen; and ii) a second (poly)peptide that binds to the antigen-binding domain of a chimeric antigen receptor (CAR). an inducible gene expression system comprising: b) a third nucleic acid encoding the CAR specific for the second polypeptide of the adapter; The CAR is i) the antigen-binding domain specific for the second (poly)peptide of the adapter; ii) a transmembrane domain, and iii) Intracellular signaling domain A system including:

2. 2. The system of claim 1, wherein the inducible gene expression system is an antigen-activated inducible gene expression system, and the antigen-activated inducible promoter is an antigen-activated promoter capable of causing expression of the adapter when a cell having the inducible gene expression system is activated by the antigen.

3. 2. The system of claim 1, wherein the inducible gene expression system is a drug-inducible expression system, the inducible promoter is a drug-inducible promoter, the inducible gene expression system further comprises a nucleic acid encoding a synthetic transcription factor of the drug-inducible promoter, and when a drug is administered to a cell having the inducible gene expression system, the gene expression system is induced and the adapter is expressed.

4. The system of claim 3 , wherein the synthetic transcription factor comprises a DNA-binding domain, a drug-binding domain, and an activation domain, and the synthetic transcription factor is activated by binding to the drug.

5. 5. The system of claim 3 or 4, wherein the expression level of the adaptor depends on the amount of drug administered to the cells, thereby allowing tunable control of adaptor expression.

6. 6. The system of any one of claims 1 to 5, wherein the inducible gene expression system and the nucleic acid encoding the CAR specific for the second polypeptide of the adaptor are present in one immune cell.

7. 6. The system of any one of claims 1 to 5, wherein the inducible gene expression system is present in a first immune cell and the nucleic acid encoding the CAR specific for the second polypeptide of the adaptor is present in a second immune cell.

8. the first immune cell comprises a CAR specific for an additional antigen, the CAR comprising: i) said antigen-binding domain specific for said further antigen; ii) a transmembrane domain, and iii) Intracellular signaling domain and / or the first immune cell comprises a TCR specific for a further antigen.

9. 9. The system of claim 7 or 8, wherein the first immune cell and the second immune cell are of the same type.

10. 9. The system of claim 7 or 8, wherein the first immune cell and the second immune cell are different types of immune cells.

11. The system described in claim 7 or 8, wherein both the first immune cell and the second immune cell are T cells or NK cells.

12. The system described in claim 7 or 8, wherein the first immune cell is a T cell and the second immune cell is a NK cell.

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