WO2025174812A1 - Chimeric antigen receptor and/or synnotch receptor systems and cells and methods of their use - Google Patents

Abstract

The present disclosure relates generally to adaptors, systems, and cells for the use with chimeric antigen receptors and/or synNotch receptors. Disclosed herein is a chimeric antigen receptor (CAR) system, a synthetic notch (synNotch) receptor system, and the method of use thereof in treating a disease or disorder (such as, for example, a cancer, an autoimmune disease, an autoinflammatory disease, a viral infection, a bacterial infection, or a fungal infection) in a subject. Further disclosed herein is an engineered cell comprising a chimeric antigen receptor (CAR) system and/or a synthetic notch (synNotch) receptor system.

Description

CHIMERIC ANTIGEN RECEPTOR AND/OR SYNNOTCH RECEPTOR

SYSTEMS AND CELLS AND METHODS OF THEIR USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63/552,308, filed February 12, 2024, which is incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Government support under GM 142007 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

REFERENCE TO SEQUENCE LISTING

[0003] The sequence listing submitted on February 12, 2025 as an .XML entitled “10504- 099WOl_ST26.xml” created on January 31, 2025, and having a file size of 72,462 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1 .52(e)(5).

BACKGROUND OF THE INVENTION

[0004] Despite the promise that chimeric antigen receptor (CAR) T cells might have in treating cancer patients, there are several limitations to the generalized clinical application of CAR T cells. First, since no single tumor antigen is universally expressed by all cancer types, scFv in CAR needs to be constructed for each tumor antigen to be targeted. Second, the financial cost and labor-intensive tasks associated with identifying and engineering scFvs against a variety of tumor antigens pose a major challenge. Third, tumor antigens targeted by CAR could be down-regulated or mutated in response to treatment resulting in tumor evasion.

[0005] Antibody drug conjugates (ADCs) are compositions that deliver a cytotoxic agent or an immunostimulatory agent to a cell. For example, some ADCs target cancer cells for killing of the cancer cells. Using ADCs has several disadvantages, however. Target cells can be resistant to ADC killing when antigens targeted by the ADCs are down-regulated or mutated or when there are changes or issues with the cell’s trafficking of the cytotoxic payload.

[0006] Accordingly, what is needed in the art is adaptors, systems, and cells that can be used with CARs and/or synNotch receptors to address and overcome the hurdles currently preventing the development of the systems into effective means of in vivo treatment. SUMMARY

The present disclosure relates generally to applicable adaptors, systems, and cells for the use with chimeric engineered antigen receptors.

[0007] Accordingly, in one aspect disclosed herein is a chimeric antigen receptor (CAR) system, comprising an adaptor comprising an antigen binding element, a small molecule therapeutic and a tag ligand, and a CAR comprising a receptor that binds the tag ligand and a signaling domain.

[0008] In one aspect, disclosed herein is a chimeric antigen receptor (CAR) system, comprising an adaptor comprising an antigen binding element and a small molecule therapeutic, and a CAR comprising a receptor that binds the small molecule therapeutic and a signaling domain.

[0009] In one aspect, disclosed herein is a synthetic notch (synNotch) receptor system, comprising an adaptor molecule comprising an antigen binding element, a small molecule therapeutic and a tag ligand, and a synNotch receptor comprising a receptor that binds the tag ligand, a notch core comprising one or more cleavage sites, and one or more transcription factors.

[0010] In some embodiments, the antigen binding element comprises an antibody or antigen binding fragment thereof. In some embodiments, the antigen binding element comprises brentuximab, inotuzumab, polatuzumab, trastuzumab, enfortumab, Sacituzumab, belantamab, tisotumab, loncastuximab, disitamab, rituximnab, FMC63, herceptin, cetuximab, nimotuzumab, panitumumab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacuzimab or an antigen-binding fragment of any one thereof.

[0011 ] In some embodiments, the adaptor molecule comprises gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotzuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, trastuzumab deruxtecan, sacituzumab govitecan, belantamab mafodotin, tisotumab vedotin, loncastuximab tesirine, disitamab vedotin, mirvetuximab soravtansine, trastuzumab duocarmazine. In some embodiments, the adaptor molecule comprises mirvetuximab soravtansine.

[0012] In some embodiments, the small molecule therapeutic is a cytotoxic compound.

[0013] In some embodiments, the cytotoxic compound comprises a maytansinoid, an auristatin, a tubulysin, a calicheamicin, a duocarmycin, an exatecan, a pyrrolobenzodiazepine. a TLR agonist, or a STING agonist. In some embodiments, the cytotoxic compound comprises a maytansinoid. In some embodiments, the cytotoxic compound comprises a DM1 or a DM4. In some embodiments, the small molecule therapeutic is linked to the antigen recognition element by a cleavable linker.

[0014] In some embodiments, the cleavable linker is cleaved by exposure to a glutathione, an enzyme, hypoxia, or hydrolysis at acidic pH. In some embodiments, the cleavable linker comprises a disulfide bond cleavable by glutathione. In some embodiments, the cleavable linker comprises an acid-sensitive moiety.

[0015] In some embodiments, the enzyme comprises cathepsin, glycosidase, phosphatase, sulfatase, legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member Al, (ALDH7A1), lipase C, hepatic type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase-A (KHK-A), hexokinases (HK), nucleoside diphosphate kinase (NDPK or NDK), and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), mitochondrial a-ketoglutarate dehydrogenase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short-chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), a- ketoglutarate dehydrogenase (α-KGDH), CD39, CD73, or fumarase.

[0016] In some embodiments, the tag ligand comprises a benzylguanine (BG), benzylcytosine (BC), chloroalkane (CA), fluoresceine (FITC), SpyTag, leucine-zipper, La- SS-B, CD19, anti-folate receptor antibody, Fc domain, peptide neoepitope (PNE), or biotin. In some embodiments, the tag ligand comprises a benzylguanine. In some embodiments, the tag ligand comprises a biotin.

[0017] In some embodiments, the CAR or synNotch receptor is comprised on a CAR T cell, a CAR NK cell, a CAR NK T cell, a CAR B cell, or a CAR macrophage.

[0018] In one aspect, disclosed herein is a method of treating disease or disorder in a subject comprising administering to the subject the CAR system or synNotch receptor system, comprising an adaptor comprising an antigen binding element, a small molecule therapeutic and a tag ligand, and a CAR comprising a receptor that binds the tag ligand and a signaling domain.

[0019] In some embodiments, the disease or disorder comprises a cancer, an autoimmune disease, an autoinflammatory disease, a viral infection, a bacterial infection, or a fungal infection. In some embodiments, the disease is a cancer selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers, small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, rectal cancer, prostatic cancer, and pancreatic cancer. In some embodiments, the disease is a cancer selected from the group consisting of ovarian cancer, breast cancer, and lung cancer. In some embodiments, the disease is an ovarian cancer. In some embodiments, the disease is a triple- negative breast cancer.

[0020] In one aspect, disclosed herein is an engineered cell comprising a receptor that binds a cytotoxic compound or a tag ligand and a signaling domain and further comprising a channel forming protein or a binding protein. In some embodiments, the binding protein comprises a tubulin protein. In some embodiments, the channel forming protein comprises an MDR1 protein.

BRIEF DESCRIPTION OF FIGURES

[0021] Figure l(A-B) is a schematic showing the interaction of elements of a CAR system and related cells wherein the CAR system includes 1) an adaptor molecule including an antibody bound to a tag ligand, benzylguanine (BG), and a cytotoxic payload and 2) a CAR that targets the tag ligand (A) or the cytotoxic pay load (B).

[0022] Figure 2 is a schematic showing an adaptor molecule including a mirvetuximab antibody, a sulfo-SPDB linker and a cytotoxic compound, DM4.

[0023] Figure 3 shows quantification of glutathione (GSH) levels per million of the indicated primary human cells or cell lines. N=3 biological replicates per sample and significance was calculated using a 1-way AN OVA.

[0024] Figure 4(A-B) shows (A) a mass spec, analysis of the ADC-BG to evaluate the DAR for DM4 and (B) the number of BG moieties per antibody.

[0025] Figure 5(A-B) shows plots of percent (%) live (A) K562 HER2 or (B) K562+FOLR1 (K562 FR A) target cells after a 72-hour incubation with the indicated concentrations of adaptor antibodies and controls. MIRV is anti-FOLRl antibody (line with open triangular markers). MIRV-BG is MIRV conjugated to benzylguanine (BG) (line with closed triangular markers). ADC is an antibody drug conjugate (line with open circular markers). ADC-BG is BG conjugated to an antibody drug conjugate (line with closed circular markers). No T cells were added. The dashed line indicates the no adaptor condition. N=2 replicates and error bars indicated std. dev. from the mean. [0026] Figure 6(A-C) shows data from co-incubations of K562+FOLR1 target cells with SNAP-CAR T cells (Fig. 6A) or MOCK (untransduced) T cells (Fig. 6C) and the indicated antibody adaptors and controls for 72 hours and assayed for target cell lysis and CD69 activation marker expression (Fig. 6B) on CAR T cells. The dashed line indicates the no adaptor condition. N=2 replicates and error bars indicated std. dev. from the mean. ADC is an antibody drug conjugate (line with open circular markers). ADC-BG is BG conjugated to an antibody drug conjugate (line with closed circular markers). MIRV is anti-FOLRl antibody (line with open triangular markers). MIRV-BG is MIRV conjugated to benzylguanine (BG) (line with closed triangular markers).

[0027] Figure 7(A-C) shows data from co-incubations of K562 (FOLR1 antigen negative) target cells and SNAP-CAR T cells (Fig. 7A) or MOCK (untransduced) T cells (Fig. 7C) with the indicated antibody adaptors and controls for 72 hours and assayed for target cell lysis and CD69 activation marker expression (Fig. 7B) on CAR T cells. The dashed line indicates the no adaptor condition. N=2 replicates and error bars indicated std. dev. from the mean. ADC is an antibody drug conjugate (line with open circular markers). ADC-BG is BG conjugated to an antibody drug conjugate (line with closed circular markers). MIRV is anti-FOLRl antibody (line with open triangular markers). MIRV-BG is MIRV conjugated to benzylguanine (BG) (line with closed triangular markers).

[0028] Figure 8(A-C) shows data from co-incubations of various FOLR1+ target cell lines (Fig. 8A, SKOV3, Fig. 8B, OVCAR3, Fig. 8C, IBROV-1) with SNAP-CAR T cells and the indicated antibody adaptors and controls for 72 hours and assayed for target cell lysis. The dashed line indicates the no adaptor condition. N=2 replicates and error bars indicated std. dev. from the mean. ADC is an antibody drug conjugate (line with open circular markers). ADC-BG is BG conjugated to an antibody drug conjugate (line with closed circular markers). MIRV is anti-FOLRl antibody (line with open triangular markers). MIRV-BG is MIRV conjugated to benzylguanine (BG) (line with closed triangular markers).

[0029] Figure 9(A-B) shows a schematic representation (Fig. 9A) and data (Fig. 9B) for T cell outcomes in adaptor and target cell co-incubation experiments of K562 (FOLR1) target cells with SNAP-CAR T cells and the indicated antibody adaptors and controls for 72hrs. T cells were analyzed for viability by Ghost Dye staining (Tonbo Biosciences). The dashed line indicates the no adaptor condition. N=2 replicates and error bars indicated std. dev. from the mean. ADC is an antibody drug conjugate (line with open circular markers). ADC-BG is BG conjugated to an antibody drug conjugate (line with closed circular markers). MIRV is anti-FOLRl antibody (line with open triangular markers). MIRV-BG is MIRV conjugated to benzylguanine (BG) (line with closed triangular markers).

[0030] Figure 10(A-D) shows data for T cell outcomes in adaptor and target cell coincubation experiments of K562 (FOLR1) target cells (Figs. 10A and 10B) or K562 off- target (Figs. 10C and 10D) with SNAP-CAR T cells and the indicated antibody adaptors and controls for 72 hrs. T cells were analyzed for viability by Ghost Dye staining (Tonbo Biosciences) and LNGFR CAR (Figs. 10B and 10D) marker expression by fluorescent antibody staining. The dashed line indicates the no adaptor condition. N=2 replicates and error bars indicated std. dev. from the mean. ADC is an antibody drug conjugate (line with open circular markers). ADC-BG is BG conjugated to an antibody drug conjugate (line with closed circular markers). MIRV is anti-FOLRl antibody (line with open triangular markers). MIRV-BG is MIRV conjugated to benzylguanine (BG) (line with closed triangular markers).

[0031 ] Figure 11 (A-E) shows a schematic of the target cells co-incubated in the same well for the bystander assay (Fig. 11 A) and data for specific lysis of FOLR1+ target cells and FOLR1 negative bystander cells co-incubated in the same wells with SNAP-CAR T cells (Figs. 1 IB and 11C) or MOCK T cells (Figs. 1 ID and 1 IE) and the indicated adaptor or antibody control. The dashed line indicates the no adaptor condition. N=2 replicates per sample and error bars indicated std. dev. from the mean. ADC is an antibody drug conjugate (line with open circular markers). ADC-BG is BG conjugated to an antibody drug conjugate (line with closed circular markers). MIRV is anti-FOLRl antibody (line with open triangular markers). MIRV-BG is MIRV conjugated to benzylguanine (BG) (line with closed triangular markers).

[0032] Figure 12 is a schematic showing the interaction of elements of a CAR system and related cells wherein the CAR system includes 1 ) am adaptor molecule including an antibody bound to a cytotoxic payload, 2) a second adaptor molecule including an antibody that targets the cytotoxic payload and that is bound to a tag ligand, and 3) a CAR that targets the tag ligand.

[0033] Figure 13(A-B) shows that BG-modified trastuzumab ADCs can function as an adaptor for universal SNAP-CAR T cells. SNAP-CAR T cells are co-incubated with K562+HER2 target cells for 72 hours and assayed for target cell viability. Figure 13(A-B) shows specific lysis of HER2+ target cells by SNAP-CAR T cells and trastuzumab- emtansine conjugated to benzylguanine (T-DM1-BG) (line with closed circular markers) as in Figure 13A or trastuzumab deruxtecan conjugated to benzylguanine (T-Dxd-BG) (line with closed squared markers) as in Figure 13B. Note, the data for trastuzumab (T) (line with open rhomboid markers) and trastuzumab-BG (T-BG) (line with closed rhomboid markers) are duplicated for both panels as all adaptors were characterized in the same assay, n = 2 independent exp ± s.d. Other controls were trastuzumab-emtansine (T- DM1) (line with open circular markers) and trastuzumab-deruxtecan (T-Dxd) (line with open squared markers).

[0034] Figure 14(A-C) shows anti-DMl/DM4 CAR design and expression. Figure 14A shows a schematic of a gene expression construct and Figure 14B shows a schematic of anti-DMl/DM4 payload binding CAR. Figure 14C shows flow cytometry of CAR expression on primary human T cells by staining with anti-LNGFR-BV421 antibody (marker) and M-DM4 (mirvetuximab-soravtansine) followed by anti-human IgG-Fc- AF647(CAR).

[0035] Figure 15(A-D) shows flow cytometry analysis of CD69T cell activation marker expression (MFI) on anti-DMl/DM4 CAR T cells (A, C) and specific lysis of FOLR1+ target cells with mirvetuximab adaptors (B, D) as in Figure 15A-B (mirvetuximab (M) and mirvetuximab-soravtansine (M-DM4)) and coincubations with HER2+ target cells and with trastuzumab adaptors as shown in 15C-D (trastuzumab (T) and trastuzumab- emtansine (T-DM1)). Incubations were done at an E:T ratio of 1 :1 with target cell lines and the indicated antibodies for 72 h. n = 2 independent exp ± s.d.

[0036] Figure 16(A-B) shows anti-DMl/DM4-CAR in an in vivo mouse model. Figure 16A shows IVIS imaging of NSG mice challenged on day 0 with FF-Luc+ SK-OV-3 tumor cells, on day 3 with SNAP-CAR T cells (SNAP-CAR) (black dashed line in Figure 16B), anti-DMl/DM4 CAR T cells (DM1/DM4-CAR), and on days 3 and 4 with 25pg of trastuzumab-emtansine (T-DM1) (grey line in Figure 16B) or PBS, SNAP-CAR + T-DM1 (black line in Figure 16B) and DM1/DM4-CAR (grey dashed line in Figure 16B). Figure 16B shows individual tumor growth of treated mice. n=3 mice per group.

[0037] Figure 17(A-H) shows anti-SNAP-CAR in an in vivo mouse model. Figure 17A shows IVIS imaging of NSG mice challenged on day 0 with FF-Luc-i- SK-OV-3 tumor cells, on day 3 with SNAP-CAR T cells (SNAP-CAR only) (Figure 17B), trastuzumab-BG (T-BG) (Figure 17C), SNAP CAR + T-BG (Figure 17D), trastuzumab-emtansine (T- DM1) (Figure 17E), SNAP CAR + T-DM1 (Figure 17F), T-DM1-BG (Figure 17G) and SNAP CAR + T-DM1-BG (Figure 17H). Figure 17(B-H) shows individual tumor growth of treated mice. n=3 mice per group.

[0038] Figure 18(A-E) shows data for DM1 cytotoxicity (Figure 18A) and percent (%) specific lysis of H2030 + HER2 target cells co-incubated in the same wells with SNAPCAR T cells (Figure 18D) or no T cells (Figure. 18B) and percent (%) specific lysis of H1975 + HER2, DM 1 -resistant target cells co-incubated in the same wells with SNAPCAR T cells (Figure 18E) or no T cells (Figure 18C) and treated with trastuzumab (T) (line with open rhomboid markers), benzylguanine (BG) (line with closed rhomboid markers), trastuzumab-emtansine (T-DM1) (line with open circular markers) or T-DM1- BG (line with closed circular markers).

[0039] Figure 19(A-F) shows data for DM1 cytotoxicity (Figure 19 A) and percent (%) specific lysis of H2030 + HER2 target cells co-incubated in the same wells with anti- DM1/DM4 CAR (DM1/DM4 CAR) T cells (Figure 19D) or no T cells (Figure.19B) and percent (%) specific lysis of H1975 + HER2, DMl-resistant target cells co-incubated in the same wells with anti-DMl/DM4 CAR (DM1/DM4 CAR) T cells (Figure 19E) or no T cells (Figure 19C) and with SNAP-CAR off-target T cells (Figure 19F) treated with trastuzumab (T) (line with open rhomboid markers), trastuzumab-emtansine (T-DM1) (line with open circular markers).

DETAILED DESCRIPTION

[0040] Provided herein is a chimeric antigen receptor system that includes an adaptor molecule and a chimeric antigen receptor (CAR). In some embodiments, the CAR system includes 1 ) an adaptor molecule comprising an antigen binding element, a small molecule therapeutic, and a tag ligand, and 2) a CAR comprising a signaling domain and a receptor that binds the tag ligand. Also provided herein is a CAR system that includes 1) an adaptor molecule comprising an antigen binding element and a small molecule therapeutic and 2) a CAR comprising a receptor that binds the small molecule therapeutic and a signaling domain. Also provided herein is synthetic Notch (synNotch) receptor system that includes an adaptor molecule and a synNotch receptor. In some embodiments, the synNotch system includes 1) an adaptor molecule comprising an antigen binding element, a small molecule therapeutic and a tag ligand, and 2) a synNotch receptor comprising a receptor that binds the tag ligand, a notch core comprising one or more cleavage sites, and one or more transcription factors.

[0041] DEFINITIONS

[0042] Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicants desire that the following terms be given the particular definition as provided below.

[0043] As used in the specification and claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0044] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non- limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0045] The term “administering” refers to an administration that is oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra- arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intra- articular, intra- synovial, intrastemal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.

[0046] The term "antibody" is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end. As used herein, the term “antibody” or “antibodies” can also refer to a human antibody and/or a humanized antibody. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans, and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.

[0047] The term "antibody fragment" refers to a portion of a full-length antibody, that includes the target, or antigen, binding or variable region. Examples of antibody fragments include Fab, Fab’, F(ab')2 and Fv fragments. The “antibody fragment” is a compound having qualitative biological activity in common with a full-length antibody. As used herein, "antibody fragment" with respect to antibodies, includes Fv, F(ab) and F(ab')2 fragments. An "Fv" fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define an target binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer target binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) has the ability to recognize and bind target, although at a lower affinity than the entire binding site. "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for target binding. The Fab fragment contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.

[0048] The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.

[0049] The term “cancer” is used to address any neoplastic disease and is not limited to epithelial neoplasms (surface and glandular cancers; such a squamous cancers or adenomas)). It is used here to describe both solid tumors and hematologic malignancies, including epithelial (surface and glandular) cancers, soft tissue and bone sarcomas, angiomas, mesothelioma, melanoma, lymphomas, leukemias and myeloma.

[0050] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of" when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.

[0051 ] The term “cytotoxic” as used herein refers to the ability to kill a target cell.

[0052] The term "identity" shall be construed to mean the percentage of nucleotide bases or amino acid residues in the candidate sequence that are identical with the bases or residues of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions shall be construed as reducing identity or homology. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) that has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned over their full lengths, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In one embodiment, default parameters are used for alignment. In one embodiment a BLAST program is used with default parameters. In one embodiment, BLAST programs BLASTN and BLASTP are used with the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR.

[0053] "Mammal" for purposes of treatment refers to any animal classified as a mammal, including human, domestic and farm animals, nonhuman primates, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.

[0054] The terms “specific for,” “specific binding,” “specifically binds,” “selective binding,” and “selectively binds” mean that a polypeptide such as an antibody or antigen binding element exhibits appreciable affinity for a particular binding partner polypeptide such as an antigen. Appreciable binding affinity includes binding with an affinity of at least 10⁶ M ¹, specifically at least 10⁷ M ¹, more specifically at least 10⁸ M ¹, yet more specifically at least 10⁹ M ¹, or even yet more specifically at least 10¹⁰ M ¹. A binding affinity can also be indicated as a range of affinities, for example, 10⁶ M ¹ to 10¹⁰ M ¹, specifically 10⁷ M^-1 to 10¹⁰ M’¹, more specifically 10⁸ M^-1to 10¹⁰ M^-1. Specific binding can be determined according to any art-recognized means for determining such binding. In some embodiments, specific binding is determined according to Scatchard analysis and/or competitive binding assays.

[0055] The terms “pharmaceutically effective amount”, “therapeutically effective amount” or “therapeutically effective dose” refer to the amount of a compound or compounds such as a CAR system that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. In some embodiments, a desired response is treatment of a cancer. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years. The terms “pharmaceutically effective amount”, “therapeutically effective amount” or “therapeutically effective dose” include that amount of a compound or compounds such as a CAR system that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the condition or disorder being treated. The therapeutically effective amount will vary depending on the compound or compounds such as CAR system, the disorder or conditions and its severity, the route of administration, time of administration, rate of excretion, drug combination, judgment of the treating physician, dosage form, and the age, weight, general health, sex and/or diet of the subject to be treated. In the context of the present method, a pharmaceutically or therapeutically effective amount or dose of a CAR system includes an amount that is sufficient to prevent development of, reduce the size or numbers of, or reduce metastasis of a cancer.

[0056] The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the invention may be applied palliatively or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a cancer. In some instances, the terms “treat”, “treating”, “treatment” and grammatical variations thereof, include partially or completely reducing the size of a tumor and reducing the number of tumors as compared with prior to treatment of the subject or as compared with the incidence of such symptom in a general or study population.

[0057] COMPOSITIONS [0058] Adoptive cell therapy using antigen receptor engineered T cells is a highly promising therapeutic approach, in which T cells are genetically modified to express an antigen receptor protein and are then adoptively transferred into the patient. These cells then act as a “living drug” that can elicit potent and therapeutic effects in response to sensing a target antigen on a neighboring cell anywhere in the body.

[0059] The most clinically advanced antigen receptor technology is chimeric antigen receptors (CARs) that consist of an antigen binding region, an extracellular spacer domain, a transmembrane domain, and T cell signaling domains. Upon antigen binding, the CARs cluster and activate the T cell receptor signaling pathway leading to lysis of target cells, cell proliferation, and production of cytokines that can amplify the immune response. CAR T cell therapy targeting the CD 19 protein, an antigen found on the surface of leukemia B cells and normal B cells, is FDA-approved, showing remarkable levels of cancer remission in 85% of patients. CAR T cells targeting new antigens are being developed to treat a wide variety of diseases, including solid tumors, viral infections, and autoimmune diseases.

100601 An even more versatile class of antigen receptors in development are synthetic Notch (synNotch) receptors that upon binding to a target antigen can regulate the expression of one or more therapeutic genes of interest without affecting endogenous cell signaling pathways. Modified from the Notch/Delta signaling pathway, synNotch receptors consist of an extracellular antigen binding domain, the Notch core protein, and an intracellular transcription factor. Upon antigen binding, the receptor is stretched by mechanical force, exposing an enzyme (such as a protease) cleavage site and releasing a transcription factor upon proteolysis in order to regulate gene expression. SynNotch receptors can be engineered to sense a wide range of antigens and to express a plethora of therapeutic output genes in response, including cytokines, toxins, chemokines, other receptors, and entire gene circuits. This highly programmable platform technology has been applied to various cell types, in addition to T cells, and is of great interest to the fields of immunotherapy and tissue engineering.

[0061 ] A universal adaptor CAR system as well as the first universal adaptor synNotch system using the SNAPtag-targeting domain was previously created. Universal adaptor receptors are a new generation of antigen receptors that instead of directly binding to an antigen on a target cell, bind to a tag molecule fused or conjugated to an antigen- specific antibody or antibody fragment; this tag-antibody /antibody fragment fusion is referred to as the “adaptor”. These systems are designed such that a patient is infused with an adaptor that forms the physical link between the target cells and the receptor T cells. Adaptor receptors are termed “universal” as one receptor can target multiple tumor antigens within a single patient or across different disease indications simply by administering different adaptors sequentially or simultaneously. The universal SNAP receptors are unique, since instead of merely transiently interacting with the adaptor, they form a covalent bond. Through a bio-orthogonal benzylguanine (BG) tag and the SNAP receptors, several different antigens can be targeted and highly potent receptor activation and signaling can be achieved. The importance of adaptor CAR systems has been recognized in the development of generating adaptors based on antibodies modified with biotin, fluorescein, peptide neo-epitopes, Fey, and leucine zippers, and the first adaptor CAR system is currently in clinical trials.

[0062] The engineered receptor system described herein is, in some embodiments, a surprisingly effective combination of the above-described adaptor CAR systems with antibody drug conjugate (ADC) technology. ADC technology employs an antibody or antibody fragment linked to a small molecule therapeutic. The present disclosure demonstrates that the combination of these two technologies is synergistic. These synergistic results are particularly surprising given the assumption that the cytotoxic nature of the ADC would decrease the effectiveness of the CAR system element of the combination.

[0063] Accordingly, provided herein is a chimeric antigen receptor system that includes an adaptor molecule and a chimeric antigen receptor (CAR). In some embodiments, the CAR system includes 1) an adaptor molecule comprising an antigen binding element and a small molecule therapeutic and 2) a CAR comprising a receptor that binds the small molecule therapeutic and a signaling domain. Also provided herein is a CAR system that includes 1) an adaptor molecule comprising an antigen binding element, a small molecule therapeutic and a tag ligand, and 2) a CAR comprising a receptor that binds the tag ligand and a signaling domain. In some aspects, the CAR is a synthetic intramembrane proteolysis receptor (SNIPR). In some aspects, the CAR further comprises a hinge domain. It should be understood that the term “CAR” refers to any receptor having an intracellular signaling domain that has an extracellular region that binds to a portion of the adaptor molecule — that portion of the adaptor molecule being the “antigen” bound by the Chimeric Antigen Receptor. In some embodiments, the CAR is a T cell receptor or a modified T cell receptor. In some embodiments, the CAR is a human T cell receptor or a humanized T cell receptor. As used herein, the term “signaling domain” refers to any polypeptide having an intracellular portion that can affect or initiate one or more signaling cascades or signaling pathways inside a cell.

[0064] Also provided herein is a synthetic Notch (synNotch) receptor system that includes an adaptor molecule and a synNotch receptor. The term “synNotch receptor” refers to a polypeptide that is a modified Notch receptor. In some embodiments, the synNotch receptor is a modified human Notch receptor. In some embodiments, an extracellular portion of a Notch receptor is modified. In other or further embodiments, an intracellular portion of a Notch receptor is modified.

[0065] Notch receptors are a family of proteins involved in the Notch signaling pathway that are usually characterized by N-terminal EGF-like repeats followed by LNR domains which form a complex with ligands to prevent signaling. In some embodiments, the Notch receptor comprises a heterodimer. In some embodiments a human Notch receptor or a part of a human Notch receptor heterodimer is encoded by a NOTCH1, NOTCH2, NOTCH3 or NOTCH4 gene.

[0066] In some embodiments, the Notch 1 polypeptide or polynucleotide is that identified in one or more publicly available databases as follows: HGNC (7881), NCB1 Gene (4851), Ensembl (ENS G00000148400), OMIM® (190198), UniProtKB/Swiss-Prot (P46531) and Open Targets Platform (ENSG00000148400).

[0067] In some embodiments, the Notchl polypeptide comprises the sequence of SEQ ID NO: 28 or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 28, or a polypeptide comprising a portion of SEQ ID NO: 28. The Notchl polypeptide of SEQ ID NO: 28 may represent an immature or pre-processed form of mature Notchl, and accordingly, included herein are mature or processed portions of the Notchl polypeptide in SEQ ID NO: 28. In some embodiments, the Notch2 polypeptide or polynucleotide is that identified in one or more publicly available databases as follows: HGNC: 7882, NCBI Gene: 4853, Ensembl: ENSG00000134250, OMIM®: 600275, and UniProtKB/Swiss-Prot: Q04721. In some embodiments, the Notch2 polypeptide comprises the sequence of SEQ ID NO: 29, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 29, or a polypeptide comprising a portion of SEQ ID NO: 29. The Notch2 polypeptide of SEQ ID NO: 29 may represent an immature or pre-processed form of mature Notch2, and accordingly, included herein are mature or processed portions of the Notch2 polypeptide in SEQ ID NO: 29. In some embodiments, the Notch3 polypeptide or polynucleotide is that identified in one or more publicly available databases as follows: HGNC: 7883, NCBI Gene: 4854, Ensembl: ENSG00000074181, OMIM®: 600276 and UniProtKB/Swiss-Prot: Q9UM47. In some embodiments, the Notch3 polypeptide comprises the sequence of SEQ ID NO: 30, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 30, or a polypeptide comprising a portion of SEQ ID NO: 30. The Notch3 polypeptide of SEQ ID NO: 30 may represent an immature or pre-processed form of mature Notch3, and accordingly, included herein are mature or processed portions of the Notch3 polypeptide in SEQ ID NO: 30. In some embodiments, the Notch4 polypeptide or polynucleotide is that identified in one or more publicly available databases as follows: HGNC: 7884, NCBI Gene: 4855, Ensembl: ENSG00000204301, OMIM®: 164951, and UniProtKB/Swiss-Prot: Q99466. In some embodiments, the Notch4 polypeptide comprises the sequence of SEQ ID NO: 31, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 31, or a polypeptide comprising a portion of SEQ ID NO: 31. The Notch4 polypeptide of SEQ ID NO: 31 may represent an immature or pre-processed form of mature Notch4, and accordingly, included herein are mature or processed portions of the Notch4 polypeptide in SEQ ID NO: 31.

[0068] In some embodiments, the synNotch system includes 1) an adaptor molecule comprising an antigen binding element, a small molecule therapeutic and a tag ligand, and 2) a synNotch receptor comprising a receptor that binds the tag ligand, a notch core comprising one or more cleavage sites, and one or more transcription factors. As used herein, the term “notch core” refers to a transmembrane region of a Notch polypeptide. In some embodiments, the one or more transcription factors comprise Gal4-VP64, Gal4- VP16, TetR-VP64, or LacI-VP64. In some embodiments, the one or more transcription factors activate expression of one or more native cell response genes (such as, for example, IL-4, IL- 10, FASL, IFN-γ, TNF-α, granzyme A. granzyme B, granulysin, and/or perforin).

[0069] The molecules disclosed herein facilitate the formation of a binding interaction between an antigen binding element (such as for example, an antibody or antibody fragment) that is a part of the adaptor and a CAR or synNotch receptor. The binding interaction can be a covalent bond, non-covalent bond, or other interaction such as a receptor- ligand or antibody-antigen/peptide/protein binding interaction (such as, for example FITC and anti-FITC or biotin/avidin). When covalently bonded, covalent bonding can occur through pi-clamp; ligand directed electrophilic chemistry; recombinant antibodies with tag ligand incorporation (such as, for example, BG or BC incorporation) through short peptide tags, sortase mediated labeling; unnatural amino acid mutagenesis followed by 'click' chemistry, [3+2] cycloaddition, split inteins, THIOMABs, tetrazine ligation, Staudinger ligation, imine formation, thiol-ene reaction, native chemical ligation; biotin ligase mediated labeling; lipoic acid ligase mediated labeling, mTGase mediated labeling, NHS-ester conjugation, conjugation to cysteine, disulfide re-stapling via bissulfones or other reagents, glycan conjugating chemistry, or formyl glycine conversion. Covalent bond formation can also occur through the use of C ARs that comprise polypeptide “tags” that covalently bind a target modification or tag ligand that is a part of the adaptor. Examples of polypeptide tags include, but are not limited to SNAP-tag (which covalently bonds to an O⁶-benzylguanine tag ligand which can be inserted into the adaptor or antigen recognition element), CLIP-tag (which covalently bonds to an O²- benzylcytosine tag ligand which can be inserted into the adaptor or antigen recognition element), Halo-tag (which covalently bonds to a chloroalkane linker tag ligand which can be inserted into the adaptor or antigen recognition element), SpyTag (which covalently bonds to a Spy catcher peptide sequence tag ligand which can be inserted into the adaptor or antigen recognition element), SnoopTag (which covalently bonds to a Snoop catcher peptide sequence tag ligand which can be inserted into the adaptor or antigen recognition element), or Isopep-tag (which covalently bonds to its biding partner tag ligand which can be inserted into the adaptor or antigen recognition element). The formation of a covalent bond is a key improvement over other adaptor CAR T cell systems which rely on weak interactions and which is sufficiently strong such that the bond is not broken upon antigen binding allowing for the Notch cleavage site(s) to be revealed in the synNotch receptor. In some embodiments, the adaptor is a conditional adaptor.

[0070] Accordingly, included herein is a CAR system wherein the adaptor molecule comprises an antigen binding element, a small molecule therapeutic and a tag ligand. In some aspects the tag ligand comprises a benzylguanine (BG), benzylcytosine (BC), chloroalkane, fluoresceine (FITC), SpyTag, leucine-zipper, La-SS-B, CD19, anti-folate receptor antibody, Fc domain, peptide neoepitope (PNE), or biotin. In some embodiments, the tag ligand comprises a benzylguanine. In some embodiments, the tag ligand comprises a biotin.

[0071 ] In the embodiments wherein the adaptor molecule includes or comprises a tag ligand, the CAR system comprises a receptor that binds the tag ligand, a hinge domain, and a signaling domain. Non-limiting examples of a hinge domain are a CD8a domain, CD28 hinge, or modified IgG hinges with a deletion or modification of the CH2 and CH3 domain. Non-limiting examples of a signaling domain are a DAP 10, NKG2D, NKG2C, NKp44, CD28, CD27, Megl O, CD32, CD16, 2B4, and CD3^ signaling domain.

[0072] In some aspects, the antigen binding element of the adaptor molecule comprises an antibody or fragment thereof. As described above, the term "fragment thereof" when referencing an antibody refers to a portion of a full-length antibody, that includes the target, or antigen, binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments. The “antibody fragment” is a compound having qualitative biological activity in common with a full-length antibody. As used herein, "antibody fragment" with respect to antibodies, includes Fv, F(ab) and F(ab')2 fragments. An "Fv" fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer target binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) has the ability to recognize and bind target, although at a lower affinity than the entire binding site. "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for target binding. The Fab fragment contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art.

[0073] In one aspect, the antigen binding element can comprise an anti-cancer-based monoclonal antibody such as cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), panitumumab (anti-EGFR), retuximab (anti-CD20), omalizumab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-Her2), herceptin (anti-Her2), gemtuzumab (anti-CD33), alemtuzumab (anti-CD52), FMC63 (anti-CD19), bevacuzimab (anti-VEGF) or antigen recognizing fragment thereof. In some aspects, the antigen binding element can comprise protein binding domains (such as, for example, nanobodies and single domain antibodies (e.g., monobodies), lectins, DNA aptamers, RNA aptamers, any small molecule ligands for cell surface receptors (e.g., folic acid which is bound by the folic acid receptor), peptide/protein ligands for natural protein receptors (such as, for example, NKG2D and/or cytokines which can be bound to their natural receptors).

[0074] In some embodiments, the antigen binding element comprises brentuximab, inotuzumab, polatuzumab, trastuzumab, enfortumab, Sacituzumab, belantamab, tisotumab, loncastuximab, disitamab,rituximnab, FMC63, herceptin, cetuximab, nimotuzumab, panitumumab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacuzimab or an antigen-binding fragment of any one thereof.

[0075] As described herein, the adaptor molecule further comprises a small molecule therapeutic compound. Small molecule therapeutics are chemical compounds with a low molecular weight. In some embodiments, the small molecule therapeutic contains 20 to 100 atoms. In some embodiments, the small molecule therapeutic compound is an immunostimulatory compound. In some embodiments, the small molecule therapeutic compound is a cytotoxic compound. As used herein, the term “cytotoxic” refers to the ability of a compound to kill a cell. In some embodiments, the cytotoxic compound comprises a maytansinoid, an auristatin, a tubulysin, a calicheamicin, a duocarmycin, an exatecan, a pyrrolobenzodiazepine, a TLR agonist, a STING agonist, a SN-38, a SG3199, or a pharmaceutically acceptable salt or derivative thereof. Exemplary chemical formulas of one or more of a maytansinoid, an auristatin, a tubulysin, a calicheamicin, a duocarmycin, an exatecan, a pyrrolobenzodiazepine (PBD), a TLR agonist, a STING agonist, a SN-38, and a SG3199 are provided below. In other embodiments, the cytotoxic compound comprises a radionuclide and a metal (e.g., a chelate). In some aspects, the radionuclide is actinium-225 (225Ac), astatine-211 (211 At), bismuth-213 (213Bi), indium-111 ( 111 In), iodine-123 (1231), iodine-124 (124I), iodine-131 (1311), lead-212 (212Pb), lutetium-177 (177Lu), technetium-99m (99mTc), copper-64 (64Cu), gallium-68 (68Ga), yttrium-86 (86Y), yttrium-90 (90Y), and zirconium-89 (89Zr). However, it should be understood that the disclosure is not limited to these specific cytotoxic compounds.

[0088] SN-38

[0089] SG3199

[0090] In some embodiments, the cytotoxic compound comprises a maytansinoid. In some embodiments, the maytansinoid is selected from a DM1 and a DM4, or a pharmaceutically acceptable salt or derivative thereof. In some embodiments, the cytotoxic compound is a DM4 or a pharmaceutically acceptable salt or derivative thereof. Exemplary chemical formulas for DM 1 and DM4 are provided below.

[0094] Accordingly, the CAR system disclosed herein includes an adaptor molecule that comprises an antigen recognition element and a cytotoxic compound. In certain aspects, the adaptor molecule comprises gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotzuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, trastuzumab deruxtecan, sacituzumab govitecan, belantamab mafodotin, tisotumab vedotin, loncastuximab tesirine, disitamab vedotin, mirvetuximab soravtansine, or trastuzumab duocarmazine. In some embodiments, the adaptor molecule comprises mirvetuximab soravtansine. In some embodiments, the adaptor molecule comprises trastuzumab emtansine. In some embodiments, the adaptor molecule comprises trastuzumab deruxtecan.

[0095] In some embodiments, the adaptor molecule comprises a small molecule therapeutic that is linked to an antigen recognition element by a cleavable linker. In certain aspects, the cleavable linker is comprised so that it can be cleaved inside a target cell such as a cancer cell for separation of the small molecule therapeutic compound from the antigen recognition element. In some embodiments, the cleavable linker is not cleaved outside a target cell after administration of the adaptor molecule to the subject. In other embodiments, the cleavable linker is preferably cleaved inside a target cell after administration of the adaptor molecule and CAR and/or synNotch receptor to the subject. Included herein are embodiments where the cleavable linker is cleaved outside of the target cell after administration to the subject in an amount of less than about 1%, 5%, 10%, 15%, 20%, 25% or 30% as compared to cleavage inside the target cell, on average.

[0096] In some aspects, the cleavable linker is comprised or structured for cleavage by exposure to a small molecule such as glutathione, an enzyme such as cathepsin B, a hydrolysis at acidic pH or hypoxia. For example: a linker comprised of a Sulfo-SPDB- based compound can be cleaved by glutathione; a linker comprised of valine and citrulline can be cleaved by cathepsin B ; a linker comprised of an acid-sensitive hydrolysable compound can be cleaved by a hydrolysis at acidic pH. Accordingly, in some embodiments, the cleavable linker is structured for cleavage by a small molecule such as glutathione. In some embodiments, the cleavable linker is structured for cleavage by an intracellular enzyme. In some embodiments, the cleavable linker is structured for cleavage by cathepsin, glycosidase, phosphatase, sulfatase, legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member Al, (ALDH7A1), lipase C, hepatic type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase-A (KHK-A), hexokinases (HK), nucleoside diphosphate kinase (NDPK or NDK), and 6-phosphofructo- 2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), mitochondrial a-ketoglutarate dehydrogenase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short-chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), a-ketoglutarate dehydrogenase (α-KGDH), CD39, CD73, or fumarase. Other suitable cleavable linkers are well known to those of skill in the art.

[0097] In other embodiments, the adaptor molecule comprises a small molecule therapeutic that is linked to an antigen recognition element by a non-cleavable linker. In these embodiments, the small molecule therapeutic is separated from the antigen recognition element through lysosomal proteolytic degradation. Suitable non-cleavable linkers are well known to those of skill in the art.

[0098] Included herein is a CAR system wherein the adaptor molecule comprises an antigen binding element, a small molecule therapeutic and a tag ligand. In some aspects the tag ligand comprises a benzylguanine (BG), benzylcytosine (BC), chloroalkane, fluoresceine (FITC), SpyTag, leucine-zipper, La-SS-B, CD19, anti-folate receptor antibody, Fc domain, peptide neoepitope (PNE), or biotin. In some embodiments, the tag ligand comprises a benzylguanine. In some embodiments, the tag ligand comprises a biotin.

[0099] In the embodiments wherein the adaptor molecule includes a tag ligand, the CAR system comprises a receptor that binds the tag ligand, a hinge domain, and a signaling domain. Non-limiting examples of a hinge domain are a CD8a domain, CD28 hinge, or modified IgG hinges with a deletion or modification of the CH2 and CH3 domain. Nonlimiting examples of a signaling domain are a DAP10, NKG2D, NKG2C, NKp44, CD28, CD27, Meg 10, CD32, CD 16, 2B4, and CD3^ signaling domain.

[0100] As discussed herein, in some embodiments the CAR system includes 1) an adaptor molecule comprising an antigen binding element and a cytotoxic compound and 2) a CAR comprising a receptor that binds the cytotoxic compound, a hinge domain, and a signaling domain. In some embodiments, the CAR receptor that binds the cytotoxic compound comprises SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO:23, or SEQ ID NO:25. In some embodiments, the CAR receptor that binds the cytotoxic compound comprises SEQ ID NOG, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:23, or SEQ ID NO:25. In some embodiments, the CAR receptor that binds the cytotoxic compound comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO:23, or SEQ ID NO:25. In some embodiments, the CAR receptor that binds the cytotoxic compound comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NOG, SEQ ID NO:9, SEQ ID NO: 13, SEQ ID NO:23, or SEQ ID NO:25. In other or further embodiments, the CAR receptor that binds the cytotoxic compound comprises SEQ ID NO:27, or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO:27. In some embodiments, the CAR receptor that binds the cytotoxic compound comprises SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 11, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO:18, or SEQ ID NO:21. In some embodiments, the CAR receptor that binds the cytotoxic compound comprises SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO:17 or SEQ ID NO: 18. In some embodiments, the CAR receptor that binds the cytotoxic compound comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO: 1 1 , SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO:21 . In some embodiments, the CAR receptor that binds the cytotoxic compound comprises a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18.

[0101] In one aspect, it is understood and herein contemplated that for activation of a T cell comprising the universal CAR to occur, additional cellular signaling events need to occur beyond the CAR T cell antigen recognition element binding to its target. In these aspects, co-stimulation is also required. Co-stimulation can occur via native interactions that occur during the activation of any T cell and already present on any CAR T cell such as the stimulation of CD28 and 4- IBB via interactions with their respective ligands B7 and 4-1 BBL on the surface of the target cell. Alternatively, the universal CAR can further comprise one or more co-stimulation domains (such as, for example, signaling domains for CD27, CD28, ICOS, 4-1BB, or 0X40, such that co-stimulation occurs upon the antigen recognition element binding its target without the further need of the target cell providing the necessary co- stimulatory signals. Thus, in one aspect, disclosed herein are universal CAR comprising a tag that binds a tag ligand, a hinge domain (such as, for example, a CD8oc domain, CD28 hinge, or modified IgG hinges with a deletion or modification of the CH2 and/or CH3 domain), and a signaling domain (such, as for example a DAP10, NKG2D, NKG2C, NKp44, CD28, CD27, MeglO, CD32, CD16, 2B4, or CD3^ signaling domain); and wherein the CAR further comprises one or more costimulation domains (such as, for example, signaling domains for CD27, CD28, ICOS, 4- 1BB, or 0X40). Also disclosed herein are universal CAR T cells expressing any of the universal CARs disclosed herein further comprising one or more co-stimulation domains. Thus, in one aspect, disclosed herein are universal CAR systems, further comprising one or more co-stimulation domains (such as, for example, CD27, CD28, ICOS, 4-1BB, or 0X40).

[0102] It is understood and herein contemplated that the disclosed CAR and/or synNotch receptors are made from and/or ultimately expressed on T cells, NK cells, NK T cells, B cells, and/or macrophage for the CAR and any immune cell (e.g., T cell, a B cell, memory T cell, memory B cell, NK T cell, a monocyte, a natural killer cell, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, y8 T cell, or a cytotoxic T cell), a neuron, an epithelial cell, and endothelial cell for the synNotch receptor. The cells used to make and express the CAR and/or synNotch receptors disclosed herein as well as any cell comprising said receptors may be from an autologous, syngeneic or allogeneic source with the selection dependent on the disease to be treated and the means available to do so. Exemplary sub-populations of T cells include, but are not limited to, those expressing CD3+ including CD3+CD8+ T cells, CD3+CD4+ T cells, and NKT cells. In one aspect, the T cells are peripheral blood mononuclear cells (PBMC) of any HLA background from PBMCs and utilized in an autologous, syngeneic, or allogeneic systems. T cells may also be isolated from any source, including but not limited to a tumor explant of the subject being treated or intratumoral T cells of the subject being treated. For the sake of convenience, the effector cells are commonly referred to herein as T cells, but it should be understood that any reference to T cells, unless otherwise indicated, is a reference to all effector cell types as defined herein.

[0103] Accordingly, included herein are engineered immune cells (e.g., T cell, NK cell, NK T cell, B cell, and/or macrophage) comprising a CAR receptor as disclosed herein — a receptor that binds the tag ligand and comprises a signaling domain or a receptor that binds the small molecule therapeutic and comprises a signaling domain. Also included herein are engineered immune cells (e.g., T cell, NK cell, NK T cell, B cell, and/or macrophage) comprising a synNotch receptor — a receptor that binds the tag ligand, a notch core comprising one or more cleavage sites, and one or more transcription factors. Further included are engineered cells comprising both a CAR as described herein and a synNotch receptor as described herein.

[0104] In some aspects, the engineered T cell (or NK cell, NK T cell, B cell, and/or macrophage) expressing the CAR receptor is engineered to express a polypeptide on the surface that binds to the cytotoxic compound (referred to herein as a “binding protein”), such as, for example, a tubulin polypeptide. In some embodiments, the tubulin polypeptide comprises SEQ ID NO:1. In some embodiments, the tubulin polypeptide comprises SEQ ID NO:1, or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO: 1. Such binding reduces damage to the T cell (or NK cell, NK T cell, B cell, and/or macrophage) by the cytotoxic compound. In certain aspects, the tubulin polypeptide is mutated. For example, the tubulin polypeptide can contain one or more of the following groups of mutations: D203N-D224N, M321A-S322A-K324A-Q245A and R251A/D- K252A/E.

[0105] In other or further aspects, the engineered T cell (or NK cell, NK T cell, B cell, and/or macrophage) is engineered to express one or more polypeptides that form a channel at the cell surface for removal of cytotoxic compound from the inside of the cell. In some embodiments, the channel forming polypeptide is an MDR1 polypeptide, also known as P- glycoprotein 1 or ABCB1 . Accordingly, included herein are immune cells comprising a polynucleotide encoding a CAR receptor, and optionally, a polynucleotide encoding a tubulin polypeptide. Also included herein are immune cells comprising a polynucleotide encoding a CAR receptor, and optionally, a polynucleotide encoding an MDR1 polypeptide. In some embodiments, the MDR1 polypeptide comprises SEQ ID NO:2, or a sequence having at least 80%, 85%, 90%, 95%, or 98% identity with SEQ ID NO:2.

[0106] METHODS

[0107] Included herein is a method of treating a disease or disorder in a subject comprising administering to the subject a CAR and/or synNotch system described herein, wherein the disease or disorder comprises a cancer, an autoimmune disease, an autoinflammatory disease, a viral infection, a bacterial infection, or a fungal infection. In some embodiments, the CAR system and/or synNotch system is administered to the subject for the treatment of a cancer.

[0108] It should be understood that the elements of the CAR and/or synNotch systems described herein can be administered simultaneously or sequentially. In some embodiments, the adaptor molecule is administered prior to administration of a CAR and/or synNotch receptor, and more particularly, one or more immune cells expressing a CAR and/or synNotch receptor. In some embodiments, one or more immune cells expressing a CAR and/or synNotch receptor are administered prior to administration of an adaptor molecule. In some embodiments, the one or more immune cells expressing a CAR and/or synNotch receptor and adaptor molecule are administered simultaneously.

[0109] In some embodiments, the cancer is selected from the group consisting of lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers, small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, rectal cancer, prostatic cancer, and pancreatic cancer. In some embodiments, the cancer is an ovarian cancer. In some embodiments the cancer is a triple-negative breast cancer. In some embodiments, the cancer is a lung cancer.

[0110] A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, rectal cancer, prostatic cancer, or pancreatic cancer.

[0111] Compounds disclosed herein may also be used for the treatment of precancer conditions such as cervical and anal dysplasias, other dysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, and neoplasias.

[01 12] It is intended herein that the disclosed methods of treating a cancer can comprise the administration of any anti-cancer agent known in the art including, but not limited to Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amboclorin Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, Atezolizumab, Avastin (Bevacizumab), Avelumab, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine Hydrochloride, BEP, Besponsa (Inotuzumab Ozogamicin) , Bevacizumab, Bexarotene, Bexxar (Tositumomab and Iodine I 131 Tositumomab), Bicalutamide, BiCNU (Carmustine), Bleomycin, Blinatumomab, Blincyto (Blinatumomab), Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab Vedotin, Brigatinib, BuMel, Busulfan, Busulfex (Busulfan), Cabazitaxel, Cabometyx (Cabozantinib-S-Malate), Cabozantinib-S-Malate, CAF, Campath (Alemtuzumab), Camptosar , (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carac (Fluorouracil— Topical), Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, Carmubris (Carmustine), Carmustine, Carmustine Implant, Casodex (Bicalutamide), CEM, Ceritinib, Cerubidine (Daunorubicin Hydrochloride), Cervarix (Recombinant HPV Bivalent Vaccine), Cetuximab, CEV, Chlorambucil, CHLORAMBUCIL-PREDNISONE, CHOP, Cisplatin, Cladribine, Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clof arabine), Clolar (Clofarabine), CMF, Cobimetinib, Cometriq (Cabozantinib-S-Malate), Copanlisib Hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (Dactinomycin), Cotellic (Cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Daratumumab, Darzalex (Daratumumab), Dasatinib, Daunorubicin Hydrochloride, Daunorubicin Hydrochloride and Cytarabine Liposome, Decitabine, Defibrotide Sodium, Defitelio (Defibrotide Sodium), Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (Cytarabine Liposome), Dexamethasone, Dexrazoxane Hydrochloride, Dinutuximab, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Durvalumab, Efudex (Fluorouracil-Topical), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Elotuzumab, Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Empliciti (Elotuzumab), Enasidenib Mesylate, Enzalutamide, Epirubicin Hydrochloride , EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (Asparaginase Erwinia chrysanthemi) , Ethyol (Amifostine), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista , (Raloxifene Hydrochloride), Evomela (Melphalan Hydrochloride), Exemestane, 5-FU (Fluorouracil Injection), 5-FU (Fluorouracil-Topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil-Topical), Fluorouracil Injection, Fluorouracil-Topical, Flutamide, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI- BEVACIZUMAB, FOLFIRI-CETUXIMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gardasil 9 (Recombinant HPV Nonavalent Vaccine), Gazyva (Obinutuzumab), Gefitinib, Gemcitabine Hydrochloride, GEMCITABINE-CISPLATIN, GEMCITABINEOXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (Gemcitabine Hydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (Imatinib Mesylate), Gliadel (Carmustine Implant), Gliadel wafer (Carmustine Implant), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate), Hemangeol (Propranolol Hydrochloride), Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPV Nonavalent Vaccine, Recombinant, HPV Quadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride), Hydrea (Hydroxyurea), Hydroxyurea, Hyper-CVAD, Ibrance (Palbociclib), Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride), Idamycin (Idarubicin Hydrochloride), Idarubicin Hydrochloride, Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene Laherparepvec), Inlyta (Axitinib), Inotuzumab Ozogamicin, Interferon Alfa- 2b, Recombinant, Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alfa-2b), Iodine I 131 Tositumomab and Tositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride, Irinotecan Hydrochloride Liposome, Istodax (Romidepsin), Ixabepilone, Ixazomib Citrate, Ixempra (Ixabepilone), Jakafi (Ruxolitinib Phosphate), JEB, Jevtana (Cabazitaxel), Kadcyla (Ado-Trastuzumab Emtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance (Palifermin), Keytruda (Pembrolizumab), Kisqali (Ribociclib), Kymriah (Tisagenlecleucel), Kyprolis (Carfilzomib), Lanreotide Acetate, Lapatinib Ditosylate, Lartruvo (Olaratumab), Lenalidomide, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Leustatin (Cladribine), Levulan (Aminolevulinic Acid), Linfolizin (Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), Lomustine, Lonsurf (Trifluridine and Tipiracil Hydrochloride), Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride) , Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride, Neulasta (Pegfilgrastim), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilandron (Nilutamide), Nilotinib, Nilutamide, Ninlaro (Ixazomib Citrate), Niraparib Tosylate Monohydrate, Nivolumab, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Obinutuzumab, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Olaratumab, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ondansetron Hydrochloride, Onivyde (Irinotecan Hydrochloride Liposome), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Osimertinib, Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, PAD, Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, PCV, PEB, Pegaspargase, Pegfilgrastim, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa- 2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride, Portrazza (Necitumumab), Pralatrexate, Prednisone, Procarbazine Hydrochloride , Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Propranolol Hydrochloride, Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium 223 Dichloride, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, Relistor (Methylnaltrexone Bromide), R-EPOCH, Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Ribociclib, R-ICE, Rituxan (Rituximab), Rituxan Hycela (Rituximab and Hyaluronidase Human), Rituximab, Rituximab and , Hyaluronidase Human, ,Rolapitant Hydrochloride, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), Rubraca (Rucaparib Camsylate), Rucaparib Camsylate, Ruxolitinib Phosphate, Rydapt (Midostaurin), Sclerosol Intrapleural Aerosol (Talc), Siltuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sonidegib, Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq , (Atezolizumab), Temodar (Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil-Topical), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine 1 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride), Vectibix (Panitumumab), VelP, Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (Vinblastine Sulfate), Vemurafenib, Venclexta (Venetoclax), Venetoclax, Verzenio (Abemaciclib), Viadur (Leuprolide Acetate), Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, Vinorelbine Tartrate, VIP, Vismodegib, Vistogard (Uridine Triacetate), Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Vyxeos (Daunorubicin Hydrochloride and Cytarabine Liposome), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda (Capecitabine), XELIRI, XELOX, Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Yondelis (Trabectedin), Zaltrap (Ziv-Aflibercept), Zarxio (Filgrastim), Zejula (Niraparib Tosylate Monohydrate), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zofran (Ondansetron Hydrochloride), Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zydelig (Idelalisib), Zykadia (Ceritinib), and/or Zytiga (Abiraterone Acetate). Also contemplated herein are chemotherapeutics that are PD1/PDL1 blockade inhibitors (such as, for example, lambrolizumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559, Atezolizumab, Durvalumab, or Avelumab).

[0113] Examples of autoimmune diseases include, but are not limited to Achalasia, Acute disseminated encephalomyelitis, Acute motor axonal neuropathy, Addison’s disease, Adiposis dolorosa , Adult Still’s disease, Agammaglobulinemia, Alopecia areata, Alzheimer’s disease, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome, Aplastic anemia , Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune enteropathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome , Autoimmune retinopathy, Autoimmune urticaria, Axonal & neuronal neuropathy (AMAN), Balo disease, Behcet’s disease, Benign mucosal emphigoid, Bickerstaffs encephalitis , Bullous pemphigoid, Castleman disease (CD), Celiac disease, Chagas disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS), Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan’s syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn’s disease, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Diabetes mellitus type 1, Discoid lupus, Dressier’s syndrome, Endometriosis, Enthesitis, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Felty syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s encephalopathy, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), Hypogammalglobulinemia, IgA Nephropathy, IgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis (IBM), Interstitial cystitis (IC), Inflammatory Bowel Disease (IBD), Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus nephritis, Lupus vasculitis, Lyme disease chronic, Meniere’s disease, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis optica, Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Ord's thyroiditis, Palindromic rheumatism (PR), PANDAS, Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Pars planitis (peripheral uveitis), Parsonnage-Tumer syndrome, Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), POEMS syndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II, III, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRC A), Pyoderma gangrenosum, Raynaud’s phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Rheumatoid vasculitis, Sarcoidosis, Schmidt syndrome, Schnitzler syndrome, Scleritis, Scleroderma, Sjogren’s syndrome, Sperm & testicular autoimmunity, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac’s syndrome, Sydenham chorea, Sympathetic ophthalmia (SO), Systemic Lupus Erythematosus, Systemic scleroderma, Takayasu’s arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiated connective tissue disease (UCTD), Urticaria, Urticarial vasculitis, Uveitis, Vasculitis, Vitiligo, Vogt-Koyanagi-Harada Disease, and Wegener’s granulomatosis (or Granulomatosis with Poly angiitis (GPA)).

[01 14] Also disclosed herein are methods of treating an autoinflammatory diseases (i.e., disorders where the innate immune response attacks host cells) comprising administering to a subject with an autoinflammatory disease the CAR system and/or synNotch system disclosed herein. Examples of autoinflammatory disorders include asthma, graft versus host disease, allergy, transplant rejection, Familial Cold Autoinflammatory Syndrome (FCAS), Muckle-Wells Syndrome (MWS), Neonatal-Onset Multisystem Inflammatory Disease (NOMID) (also known as Chronic Infantile Neurological Cutaneous Articular Syndrome (CINCA)), Familial Mediterranean Fever (FMF), Tumor Necrosis Factor (TNF) - Associated Periodic Syndrome (TRAPS), TNFRSFHA-associated hereditary fever disease (TRAPS 11), Hyperimmunoglobulinemia D with Periodic Fever Syndrome (HIDS), Mevalonate Aciduria (MA), Mevalonate Kinase Deficiencies (MKD), Deficiency of Interleukin- IB (IL-1B) Receptor Antagonist (DIRA) (also known as Osteomyelitis, Sterile Multifocal with Periostitis Pustulosis), Majeed Syndrome, Chronic Nonbacterial Osteomyelitis (CNO), Early-Onset Inflammatory Bowel Disease, Diverticulitis, Deficiency of Interleukin-36-Receptor Antagonist (DfTRA), Familial Psoriasis (PSORS2), Pustular Psoriasis (15), Pyogenic Sterile Arthritis, Pyoderma Gangrenosum, and Acne Syndrome (PAPA), Congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD), Pediatric Granulomatous Arthritis (PGA), Familial Behcets- like Autoinflammatory Syndrome, NLRP 12- Associated Periodic Fever Syndrome, Proteasome-associated Autoinflammatory Syndromes (PRAAS), Spondyloenchondrodysplasia with immune dysregulation (SPENCDI), STING-associated vasculopathy with onset in infancy (SAVT), Aicardi-Goutieres syndrome, Acute Febrile Neutrophilic Dermatosis, X-linked familial hemophagocytic lymphohistiocytosis, and Lyn kinase-associated Autoinflammatory Disease (LAID).

[0115] Further, the disclosed CAR and synNotch systems can be used to treat disease resulting from an infection with a bacterium, virus, fungi, and/or parasite. In one aspect, the infectious disease being treated can be the result of an infection with a virus selected from Herpes Simplex virus- 1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein- Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (such as, for example, avian coronavirus (IBV), porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus (PRCV), transmissible gastroenteritis virus (TGEV), feline coronavirus (FCoV), feline infectious peritonitis virus (FIPV), feline enteric coronavirus (FECV), canine coronavirus (CCoV), rabbit coronavirus (RaCoV), mouse hepatitis virus (MHV), rat coronavirus (RCoV), sialodacryadenitis virus of rats (SDAV), bovine coronavirus (BCoV), bovine enterovirus (BEV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea virus (PEDV), porcine hemagglutinating encephalomyelitis virus (HEV), turkey bluecomb coronavirus (TCoV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKUl, HCoV-NL63, Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV) (SARS-CoV), Severe Acute Respiratory Syndrome (SARS)-Coronavirus (CoV)-2 (SARS-CoV-2) (including, but not limited to the B1.351 variant, B.1.1.7 variant, USA-WA1/2020, or P.l variant), or middle east respiratory syndrome (MERS) coronavirus (CoV) (MERS-CoV)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papillomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2.

[0116] In one aspect, the infectious disease being treated can be the result of an infection with a bacteria selected from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species. In one aspect the bacteria is not Bacillus anthracis.

[0117] In another aspect, the infectious disease being treated can be the result of an infection with a fungi selected from Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis camii, Penicillium mameffi, and Altemaria altemata.

[0118] In another aspect, the infectious disease being treated can be the result of an infection with a parasite selected from Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica.

[0119] The therapeutically effective amount of the CAR and/or synNotch systems, or components thereof, described herein can be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.5 to about 200 mg/kg of body weight of active composition per day, which can be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 0.5 to about 150 mg/kg of body weight of active composition per day, about 0.5 to 100 mg/kg of body weight of active compound per day, about 0.5 to about 75 mg/kg of body weight of active compound per day, about 0.5 to about 50 mg/kg of body weight of active composition per day, about 0.5 to about 25 mg/kg of body weight of active composition per day, about 1 to about 20 mg/kg of body weight of active composition per day, about 1 to about 10 mg/kg of body weight of active composition per day, about 20 mg/kg of body weight of active composition per day, about 10 mg/kg of body weight of active composition per day, or about 5 mg/kg of body weight of active composition per day.

[0120] Those of skill in the art will understand that the specific dose level and frequency of dosage for any particular subject can be varied and will depend upon a variety of factors, including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.

[0121 ] The disclosed methods can be performed any time after the onset of a cancer, an autoimmune disease, an autoinflammatory disease, a viral infection, a bacterial infection, or a fungal infection. In one aspect, the disclosed methods can be employed 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 months; 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,

14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 days; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 minutes after onset of a cancer, an autoimmune disease, an autoinflammatory disease, a viral infection, a bacterial infection, or a fungal infection; or 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, or 3 days; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 minutes after onset of a cancer, an autoimmune disease, an autoinflammatory disease, a viral infection, a bacterial infection, or a fungal infection.; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,

20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 105, 120 minutes; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

15, 18, 24, 30, 36, 48, 60 hours; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, 60, 90 or more days; 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months; 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41,

40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,

16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 years after the onset of a cancer, an autoimmune disease, an autoinflammatory disease, a viral infection, a bacterial infection, or a fungal infection.

[0122] Dosing frequency for the CAR and/or synNotch systems, or components thereof, of any preceding aspects, includes, but is not limited to, at least once every year, once every two years, once every three years, once every four years, once every five years, once every six years, once every seven years, once every eight years, once every nine years, once every ten year, at least once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, at least once every month, once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, daily, two times per day, three times per day, four times per day, five times per day, six times per day, eight times per day, nine times per day, ten times per day, eleven times per day, twelve times per day, once every 12 hours, once every 10 hours, once every 8 hours, once every 6 hours, once every 5 hours, once every 4 hours, once every 3 hours, once every 2 hours, once every hour, once every 40 min, once every 30 min, once every 20 min, or once every 10 min. Administration can also be continuous and adjusted to maintaining a level of the compound within any desired and specified range.

[0123] It should be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims. All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety for all purposes.

[0124] EXAMPLES

[0125] Example 1

[0126] Antibody-drug conjugate (ADC)-adaptor and SNAP-CAR T cell coincubation - Antigen positive or negative target cells are stained with CellTrace Yellow following the manufacturer’s recommended protocol (ThermoFisher), and 10,000 or 50,000 (E:T = 5: 1 or 1 :1) target cells per well are co-cultured with 50,000 SNAP-CAR T cells or MOCK untransduced T cells in a 96 well V-bottom plate with a BG-conjugated antibody drug conjugate (ADC) or control antibody. Plates undergo a quick spin to collect cells at the bottom of the wells and are then incubated at 37 °C for 24-72 hours. To identify lysed cells, co-incubated cells are stained with Ghost Dye Red Viability Dye (Ton bo Biosciences) and analyzed by flow cytometry. Target cells are identified by CellTrace Yellow and lysed target cells are identified by positive Ghost Dye staining. Percent specific cytotoxicity of target cells is calculated by the equation: 100*(% experimental lysis - % target-only lysis) / (100 - % target-only lysis). T cell viability is evaluated by analyzing the Cell trace Yellow negative population (T cells) for Ghost Dye staining, with the Ghost Dye negative representing the live cells. Furthermore, following co-incubation cells are stained with anti-CD271-BV421 (BD Biosciences) and anti-CD69- BV71 1 (BD Biosciences) to identify LNGFR+ (CAR+ cells) and to evaluate T cell activation by CD69 expression in the LNGFR+ population by flow cytometry.

[0127] Pre-loaded SNAP-CAR with ADC co-incubation - Anti-payload antibody (ex: anti-DM4) is labeled with benzylguanine tag via NHS-ester conjugation. SNAP-CAR T cells are pre-labeled with anti-payload antibody by incubating them for 30 minutes at 37 °C with l-10μg/mL of anti-payload antibody-BG conjugate, and excess antibody is washed away. Antigen positive or negative target cells are then stained with CellTrace Yellow following the manufacturer’s recommended protocol (ThermoFisher), and 10,000 or 50,000 (E:T = 5: 1 or 1 : 1 ) target, cells per well and are co-cultured with 50,000 prelabeled SNAP-CAR T cells or MOCK untransduced T cells in a 96 well V-bottom plate with an antibody drug conjugate (ADC) recognized by the anti-payload antibody or control antibody. Plates undergo a quick spin to collect cells at the bottom of the wells and are then incubated at 37 °C for 24-72 hours. To identify lysed cells, co-incubated ceils are stained with Ghost Dye Red Viability Dye (Tonbo Biosciences) and analyzed by flow cytometry. Target cells are identified by CellTrace Yellow and lysed target cells are identified by positive Ghost Dye staining. Percent specific cytotoxicity of target cells is calculated by the equation: 100*(% experimental lysis - % target-only lysis) / (100 - % target-only lysis). T cell viability is evaluated by analyzing the Cell trace Yellow negative population (T cells) for Ghost Dye staining, with the Ghost Dye negative representing the live cells. Furthermore, following co-incubation cells are stained with anti-CD271 -BV421 (BD Biosciences) and anti-CD69-B V711(BD Biosciences) to identify LNGFR+ (CAR+ cells) and to evaluate T cell activation by CD69 expression in the LNGFR+ population by flow cytometry.

[0128] Anti-payload CAR co-incubation - Antigen positive or negative target cells are stained with CellTrace Yellow following the manufacturer’s recommended protocol (ThermoFisher), and 10,000 or 50,000 (E:T = 5: 1 or 1: 1) target cells per well are cocultured with 50,000 anti-payload Universal CAR T cells or MOCK untransduced T cells in a 96 well V-bottom plate with an antibody drug conjugate (ADC) recognized by the anti-payload CAR T cells or control antibody. Plates undergo a quick spin to collect cells at the bottom of the wells and are then incubated at 37 °C for 24-72 h. To identify lysed cells, co-incubated cells are stained with Ghost Dye Red Viability Dye (Tonbo Biosciences) and analyzed by flow cytometry. Target cells are identified by CellTrace Yellow and lysed target cells are identified by positive Ghost Dye staining. Percent specific cytotoxicity of target cells is calculated by the equation: 100*(% experimental lysis - % target-only lysis) / (100 - % target-only lysis). T cell viability is evaluated by analyzing the Cell trace Yellow negative population (T cells) for Ghost Dye staining, with the Ghost Dye negative representing the live cells. Furthermore, following co-incubation cells are stained with anti-CD271-BV421 (BD Biosciences) and anti-CD69-BV711(BD Biosciences) to identify LNGFR+ (CAR+ cells) and to evaluate T cell activation by CD69 expression in the LNGFR+ population by flow cytometry. [0129] Anti-payload CAR co-incubation with MDR1 transgene protection - Antigen positive or negative target cells are stained with CellTrace Yellow following the manufacturers recommended protocol (ThermoFisher), and 10,000 or 50,000 (E:T = 5:1 or 1:1) target cells per well are co-cultured with 50,000 anti-payload Universal CAR T cells ---/-expression of the MDR1 gene or MOCK untransduced T cells in a 96 well V- bottom plate with an antibody drug conjugate (ADC) recognized by the anti-payload CAR T cells or control antibody. Plates undergo a quick spin to collect cells at the bottom of the wells and are then incubated at 37 °C for 24-72 hours. To identify lysed cells, co-incubated cells are stained with Ghost Dye Red Viability Dye (Tonbo Biosciences) and analyzed by flow cytometry. Target cells are identified by CellTrace Yellow and lysed target cells are identified by positive Ghost Dye staining. Percent specific cytotoxicity of target cells is calculated by the equation: 100 *(% experimental lysis - % target-only lysis) / (100 - % target-only lysis). T cell viability is evaluated by analyzing the Cell trace Yellow negative population (T cells) for Ghost Dye staining, with the Ghost Dye negative representing the live cells. Furthermore, following co-incubation cells are stained with anti-CD271 -BV421 (BD Biosciences) and anti-CD69-BV711(BD Biosciences) to identify LNGFR+ (CAR+ cells) and to evaluate T cell activation by CD69 expression in the LNGFR+ population by flow cytometry .

[0130] As noted above, Figure 5(A-B) shows plots of percent (%) live (A) K562 HER2 or (B) K562+FOLR1 (K562 FR A) target cells after a 72-hour incubation with the indicated concentrations of adaptor antibodies and controls. The MIRV-BG is MIRV conjugated to benzylguanine (BG) (line with closed triangular markers) that was made as follows: Trastuzumab emtansine (MCE, Cat. No.: HY-P9921), Trastuzumab Deruxtecan (MCE, Cat. No.: HY-138298A), Mirvetuximab (MCE, Cat. No.: HY-P99225) and Mirvetuximab soravtansine (MCE, Cat. No.: HY-132258A) were produced by first buffer exchanged the Abs in PBS by using 7 K MWCO Zeba Spin Desalting Columns (ThermoFisher Scientific). Then the Abs were incubated with 20ME (Molar equivalent) of BG-GLA-NHS (NEB) (New England Biolabs, Cat. No.: S9151S) for 30 minutes at room temperature, subsequently buffer exchanged by using 7 K MWCO Zeba Spin Desalting Columns and the concentration was measured by Nanodrop One (Thermofisher Scientific).

[0131] Figure 6(A-C) shows data from co-incubations of K562+FOLR1 target cells with SNAP-CAR T cells (Fig. 6A) or MOCK (untransduced) T cells (Fig. 6C) and the indicated antibody adaptors and controls for 72 hours and assayed for target cell lysis and CD69 activation marker expression (Fig. 6B) on CAR T cells. SNAP CAR and DM1/DM4 CAR T cells were generated by CD3+ T cells isolation with Pan T cell isolation kit (Miltenyi Biotec), then stimulated with TransAct Human T cell activation reagent (Miltenyi Biotec), 100 U/ml human IL-2 IS (Miltenyi Biotec) and 1 ng/ml IL- 15 (Miltenyi Biotec) for 48h. Then the SNAP CAR or DM1 /DM4 virus was spun down in retronectin coated plate at 2000 x g for 2 h at 32 °C, and after removing 2ml of media, 1 x 10⁶ of T cells were added in 4ml of media and spun down for 10 mins 1000 x g at 32 °C. Cells were then expanded every 2-3 days, and fresh IL-2 and IL- 15 were added in each expansion time. Transduction efficiency was tested at day 8 post-transduction.

[0132] Figure 7(A-C) shows data from co-incubations of K562 (FOLR1 antigen negative) target cells and SNAP-CAR T cells (Fig. 7A) or MOCK (untransduced) T cells (Fig. 7C) with the indicated antibody adaptors and controls for 72 hours and assayed for target cell lysis and CD69 activation marker expression (Fig. 7B) on CAR T cells. The ADC-BG is BG conjugated to an antibody drug conjugate (line with closed circular markers) that was made as follows: Trastuzumab emtansine (MCE, Cat. No.: HY-P9921), Trastuzumab Deruxtecan (MCE, Cat. No.: HY-138298A), Mirvetuximab (MCE, Cat. No.: HY- P99225) and Mirvetuximab soravtansine (MCE, Cat. No.: HY-132258A) were produced by first buffer exchanged the Abs in PBS by using 7 K MWCO Zeba Spin Desalting Columns (ThermoFisher Scientific). Then the Abs were incubated with 20ME (Molar equivalent) of BG-GLA-NHS (NEB) (New England Biolabs, Cat. No.: S9151S) for 30 minutes at room temperature, subsequently buffer exchanged by using 7 K MWCO Zeba Spin Desalting Columns and the concentration was measured by Nanodrop One (Thermofisher Scientific).

[0133] Figure 13(A-B) shows that BG-modified trastuzumab ADCs can function as an adaptor for universal SNAP-CAR T cells. The BG-modified trastuzumab ADCs were made as follows: Trastuzumab emtansine (MCE, Cat. No.: HY-P9921), Trastuzumab Deruxtecan (MCE, Cat. No.: HY-138298A), Mirvetuximab (MCE, Cat. No.: HY- P99225) and Mirvetuximab soravtansine (MCE, Cat. No.: HY-132258A) were produced by first buffer exchanged the Abs in PBS by using 7 K MWCO Zeba Spin Desalting Columns (ThermoFisher Scientific). Then the Abs were incubated with 20ME (Molar equivalent) of BG-GLA-NHS (NEB) (New England Biolabs, Cat. No.: S9151S) for 30 minutes at room temperature, subsequently buffer exchanged by using 7 K MWCO Zeba Spin Desalting Columns and the concentration was measured by Nanodrop One (Thermofisher Scientific). [0134] SNAP-CAR T cells function with additional ADC-BGs as adaptors - Figure 14 shows that ADC-based adaptors show potent cell killing that exceeds the ADC alone, and the killing ability is not affected by the hook effect observed for SNAP-CAR with the T- BG adaptor at high adaptor concentrations, likely due to the drug activity from the ADC.

[0135] Anti-DM1/DM4 CAR Design and expression - Figure 15 shows high expression of the anti-DMl/DM4 CAR on primary human T cells using How cytometry, which correlates with LNGFR marker expression. Using the ADC as the primary staining molecule further confirms binding ability to the ADC drug molecule.

[0136] Anti-DM1/DM4 CAR shows potent activity - Figure 16 shows potent T cell activation and target cell killing mediated by the anti-DMl/DM4 CAR T cells and 2 FDA- approved adaptor elements A) mirvetuximab soravtansine and B) trastuzumab emtansine in vitro.

[0137] Anti-DM-1/DM4 CAR in vivo - Figure 17 shows the anti-DMl/DM4-CAR combined with trastuzumab emtansine (T-DM1) provides potent anti-tumor activity compared to the CAR T cells alone or when combined to irrelevant (SNAP-CAR) T cells.

|0138| SEQ ID NO : 1 (Tubulin sequence)

[0139] MREIVHIQAGQCGNQIGAKFWEVISDEHGIDPTGSYHGDSDLQLERINVYY NEATGNKYVPRAILVDLEPGTMDSVRSGPFGQIFRPDNFVFGQSGAGNNWAKGH YTEGAELVDSVLDVVRKESESCDCLQGFQLTHSLGGGTGSGMGTLLISKIREEYPD RIMNTFSVMPSPKVSDTVVEPYNATLSVHQLVENTDETYC1DNEALYDICFRTLKL TTPTYGDLNHLVSATMSGVTTCLRFPGQLNADLRKLAVNMVPFPRLHFFMPGFAP LTSRGSQQYRALTVPELTQQMFDSKNMMAACDPRHGRYLTVAAIFRGRMSMKE VDEQMLNVQNKNSSYFVEWIPNNVKTAVCDIPPRGLKMSATFIGNSTAIQELFKRI SEQFTAMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATADEQG EFEEEEGEDEA

[0140] SEQ ID NO:2 (MDR1)

[0141] MDLEGDRNGGAKKKNFFKLNNKSEKDKKEKKPTVSVFSMFRYSNWLDK LYMVVGTLAAIIHGAGLPLMMLVFGEMTDIFANAGNLEDLMSNITNRSDINDTGF FMNLEEDMTRYAYYYSGIGAGVLVAAYIQVSFWCLAAGRQIHKIRKQFFHAIMR QEIGWFDVHDVGELNTRLTDDVSKINEGIGDKIGMFFQSMATFFTGFIVGFTRGW KLTLVILAISPVLGLSAAVWAKILSSFTDKELLAYAKAGAVAEEVLAAIRTVIAFG GQKKELERYNKNLEEAKRIGIKKAITANISIGAAFLLIYASYALAFWYGTTLVLSG EYSIGQVLTVFFSVLIGAFSVGQASPSIEAFANARGAAYEIFKIIDNKPSIDSYSKSG HKPDNIKGNLEFRNVHFSYPSRKEVKILKGLNLKVQSGQTVALVGNSGCGKSTTV QLMQRLYDPTEGMVSVDGQDIRTINVRFLREIIGVVSQEPVLFATTIAENIRYGREN VTMDEIEKAVKEANAYDFIMKLPHKFDTLVGERGAQLSGGQKQRIAIARALVRNP KILLLDEATSALDTESEAVVQVALDKARKGRTTIVIAHRLSTVRNADVIAGFDDG VIVEKGNHDELMKEKGIYFKLVTMQTAGNEVELENAADESKSEIDALEMSSNDSR SSLIRKRSTRRSVRGSQAQDRKLSTKEALDESIPPVSFWRIMKLNLTEWPYFVVGV FCAIINGGLQPAFAIIFSKIIGVFTRIDDPETKRQNSNLFSLLFLALGIISFITFFLQGFT FGKAGEILTKRLRYMVFRSMLRQDVSWFDDPKNTTGALTTRLANDAAQVKGAIG SRLAVTTQNTANLGTGIIISFIYGWQLTLLLLArVPITAIAGVVEMKMLSGQALKDKK ELEGSGKIATEAIENFRTVVSLTQEQKFEHMYAQSLQVPYRNSLRKAHIFGITFSFT QAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSFAPDY AKAKISAAHIIMIIEKTPLIDSYSTEGLMPNTLEGNVTFGEVVFNYPTRPDIPVLQGL SLEVKKGQTLALVGSSGCGKSTVVQLLERFYDPLAGKVLLDGKEIKRLNVQWLR AHLGIVSQEPILFDCSIAENIAYGDNSRVVSQEEIVRAAKEANIHAFIESLPNKYSTK VGDKGTQLSGGQKQRIAIARALVRQPHILLLDEATSALDTESEKVVQEALDKARE GRTCIVIAHRLSTIQNADLIVVFQNGRVKEHGTHQQLLAQKGIYFSMVSVQAGTK RQ

[0142] SEQ ID NOG Anti-DM4 aa

[0143] QVQLQESGGGLVQPGGSLRLSCAASGSIFGISYMGWYRQAPGKQREPVAM ITSGGSTYYADSVKGRVTISRDNAKKTVYLQMNSLRPEDASTYYCAAGCYRSDSP NRYDYWGQGTQVTVSS

[0144] SEQ ID NO:4 Anti-DM4 DNA

[0145] CAAGTGCAGCTCCAAGAGAGTGGCGGGGGTCTGGTTCAGCCGGGTGGG TCTTTGCGGCTCAGCTGCGCTGCGTCAGGGTCCATCTTCGGAATCTCTTACATG GGTTGGTACAGGCAAGCCCCCGGAAAGCAGAGGGAACCTGTGGCGATGATAA CGTCTGGAGGTTCCACTTACTATGCCGACTCTGTAAAGGGTCGGGTTACTATA AGCCGGGACAACGCTAAGAAAACGGTGTATCTTCAGATGAACTCACTCAGGC CCGAAGATGCAAGCACTTATTATTGTGCTGCAGGCTGCTATCGGTCTGACAGT CCCAATCGCTACGATTACTGGGGGCAAGGCACGCAGGTGACAGTATCTTCT

[0146] SEQ ID NO : 5 Anti-DM4-C AR aa

[0147] METDTLLLWVLLLWVPGSTGQVQLQESGGGLVQPGGSLRLSCAASGS1FG1 SYMGWYRQAPGKQREPVAMITSGGSTYYADSVKGRVTISRDNAKKTVYLQMNS LRPEDASTYYCAAGCYRSDSPNRYDYWGQGTQVTVSSPRTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA

YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE

[0148] SEQ ID NO:6 Anti-DM4 CAR DNA

[0149] ATGGAGACAGACACACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAG

GTTCCACAGGTCAAGTGCAGCTCCAAGAGAGTGGCGGGGGTCTGGTTCAGCC GGGTGGGTCTTTGCGGCTCAGCTGCGCTGCGTCAGGGTCCATCTTCGGAATCT CTTACATGGGTTGGTACAGGCAAGCCCCCGGAAAGCAGAGGGAACCTGTGGC GATGATAACGTCTGGAGGTTCCACTTACTATGCCGACTCTGTAAAGGGTCGGG TTACTATAAGCCGGGACAACGCTAAGAAAACGGTGTATCTTCAGATGAACTCA CTCAGGCCCGAAGATGCAAGCACTTATTATTGTGCTGCAGGCTGCTATCGGTC TGACAGTCCCAATCGCTACGATTACTGGGGGCAAGGCACGCAGGTGACAGTA

TCTTCTCCTAGGACCACTACTCCGGCACCGCGCCCCCCAACTCCTGCACCGAC GATAGCTTCACAACCGCTTTCATTGCGGCCCGAAGCATGTCGGCCAGCCGCCG GAGGCGCTGTGCATACAAGAGGGCTGGATTTTGCATGTGATATATATATTTGG GCGCCCCTTGCTGGCACTTGCGGCGTTCTTCTTCTTAGCCTCGTTATTACGCTC TACTGTAAGCGAGGTAGGAAAAAATTGCTGTATATCTTTAAACAGCCTTTTAT GAGACCCGTGCAAACGACTCAAGAGGAAGACGGGTGTAGCTGTAGATTTCCT GAAGAGGAAGAGGGGGGGTGCGAACTGCGGGTGAAGTTCAGCAGAAGCGCC

GACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACC TGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCC TGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAAC GAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGG GCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCAC CGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGC TCGAG

[0150] SEQ ID NO:7 Anti-DM4-CAR-T2A-MDR1 aa

[0151] METDTLLLWVLLLWVPGSTGQVQLQESGGGLVQPGGSLRLSCAASGSIFGI

SYMGWYRQAPGKQREPVAMITSGGSTYYADSVKGRVTISRDNAKKTVYLQMNS LRPED ASTYYC AAGC YRSDS PNRYD YWGQGTQ VTVS SPRTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE LEGGGEGRG SLLTCGDVEENPGPRMDLEGDRNGGAKKKNFFKLNNKSEKDKKEKKPTVSVFSM FRYSNWLDKLYMVVGTLAAIIHGAGLPLMMLVFGEMTDIFANAGNLEDLMSNIT NRSDINDTGFFMNLEEDMTRYAYYYSGIGAGVLVAAYIQVSFWCLAAGRQIHKIR KQFFHAIMRQEIGWFDVHDVGELNTRLTDDVSKINEGIGDKIGMFFQSMATFFTG FIVGFTRGWKLTLVILAISPVLGLSAAVWAKILSSFTDKELLAYAKAGAVAEEVLA AIRTVIAFGGQKKELERYNKNLEEAKRIGIKKAITANISIGAAFLLIYASYALAFWY GTTLVLSGEYSIGQVLTVFFSVLIGAFSVGQASPSIEAFANARGAAYEIFKIIDNKPSI DSYSKSGHKPDNIKGNLEFRNVHFSYPSRKEVKILKGLNLKVQSGQTVALVGNSG CGKSTTVQLMQRLYDPTEGMVSVDGQDIRTINVRFLRETIGVVSQEPVLFATTIAE NIRYGRENVTMDEIEKAVKEANAYDFIMKLPHKFDTLVGERGAQLSGGQKQRIAI ARALVRNPKILLLDEATSALDTESEAVVQVALDKARKGRTTIVIAHRLSTVRNAD VIAGFDDGVIVEKGNHDELMKEKGIYFKLVTMQTAGNEVELENAADESKSEIDAL EMSSNDSRSSLIRKRSTRRSVRGSQAQDRKLSTKEALDESIPPVSFWRIMKLNLTE WPYFVVGVFCAIINGGLQPAFAIIFSKIIGVFTRIDDPETKRQNSNLFSLLFLALGIISF ITFFLQGFTFGKAGEILTKRLRYMVFRSMLRQDVSWFDDPKNTTGALTTRLANDA AQVKGAIGSRLAVITQNIANLGTGIIISFIYGWQLTLLLLAIVPIIAIAGVVEMKMLS GQALKDKKELEGSGK1ATEAIENFRTVVSLTQEQKFEHMYAQSLQVPYRNSLRKA HIFGITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVG QVSSFAPDYAKAKISAAHIIMIIEKTPLIDSYSTEGLMPNTLEGNVTFGEVVFNYPT RPDIPVLQGLSLEVKKGQTLALVGSSGCGKSTVVQLLERFYDPLAGKVLLDGKEI KRLNVQWLRAHLGIVSQEPILFDCSIAENIAYGDNSRVVSQEEIVRAAKEANIHAFI ESLPNKYSTKVGDKGTQLSGGQKQRIAIARALVRQPHILLLDEATSALDTESEKVV QEALDKAREGRTCIVIAHRLSTIQNADLIVVFQNGRVKEHGTHQQLLAQKGIYFS MVSVQAGTKRQ

[0152] SEQ ID NO:8 Anti-DM4-CAR-T2A-MDR1 DNA

[0153] ATGGAGACGGACACACTTCTCCTGTGGGTGTTGCTGCTGTGGGTGCCAG GAAGTACCGGTCAGGTACAATTGCAGGAGTCAGGAGGGGGACTGGTGCAACC AGGAGGCTCTCTGAGATTGTCCTGTGCAGCTAGCGGTAGCATCTTTGGCATCT CATATATGGGGTGGTATAGGCAAGCTCCCGGCAAGCAGAGAGAACCCGTTGC AATGATCACATCAGGGGGGTCTACCTACTACGCTGATTCCGTAAAAGGCCGGG TCACGATATCTCGCGACAACGCGAAGAAGACAGTTTACCTCCAAATGAATTCT CTTCGACCGGAGGACGCCTCAACATATTACTGTGCGGCCGGTTGTTATAGGAG CGATAGCCCTAACAGATATGACTACTGGGGGCAAGGTACGCAGGTAACAGTT TCCTCTCCTCGGACTACGACACCCGCACCTCGCCCCCCTACTCCGGCCCCTACG ATTGCCAGTCAACCCCTTAGCCTGCGACCTGAGGCATGCCGACCGGCCGCTGG CGGTGCAGTGCACACCCGAGGATTGGACTTCGCATGTGACATTTACATTTGGG

CCCCGTTGGCAGGAACATGTGGCGTACTTCTGCTGTCACTGGTCATCACGCTCT

ATTGTAAGAGAGGTAGGAAGAAACTGCTCTATATTTTCAAGCAACCATTTATG

CGACCTGTTCAGACAACCCAGGAAGAGGATGGTTGTTCCTGTAGGTTCCCAGA

AGAAGAGGAAGGCGGCTGTGAGCTCAGAGTTAAATTTTCTCGATCTGCCGACG

CTCCAGCTTACCAGCAAGGCCAGAATCAACTGTACAACGAGCTGAATCTGGGT

CGGAGGGAAGAGTATGACGTGCTGGACAAGCGGAGAGGTCGGGACCCTGAAA

TGGGCGGCAAGCCTCGAAGAAAGAACCCCCAGGAAGGGCTCTACAACGAACT

GCAAAAGGATAAAATGGCTGAGGCTTATTCAGAAATAGGGATGAAAGGCGAG

CGCCGACGGGGCAAGGGGCACGATGGTCTTTACCAAGGCCTCTCAACGGCTA

CAAAGGACACGTATGATGCCCTTCACATGCAAGCTTTGCCTCCCCGGCTTGAG

CTCGAAGGTGGAGGTGAAGGCAGGGGGAGCCTCTTGACCTGTGGTGACGTTG

AAGAGAATCCGGGCCCCAGGATGGACTTGGAGGGAGACAGAAATGGCGGCGC

TAAGAAAAAAAACTTCTTTAAGCTGAACAACAAATCCGAAAAGGACAAAAAG

GAGAAGAAGCCTACAGTCTCCGTTTTCTCAATGTTCCGATATTCTAACTGGTTG

GATAAACTCTATATGGTCGTGGGCACCCTCGCGGCCATCATCCATGGTGCCGG

TTTGCCGCTGATGATGTTGGTGTTCGGGGAAATGACAGACATCTTTGCGAATG

CCGGTAACCTCGAGGACCTCATGAGTAATATCACTAATCGCAGCGATATAAAC

GATACAGGATTTTTTATGAATCTTGAGGAAGATATGACTAGGTACGCTTACTA

TTATTCCGGGATAGGTGCGGGTGTACTTGTCGCGGCCTACATACAAGTAAGCT

TCTGGTGCCTGGCAGCAGGCAGACAGATACATAAGATACGGAAGCAGTTCTTC

CACGCAATCATGAGGCAGGAAATAGGATGGTTCGATGTACATGATGTGGGGG

AACTCAATACCCGCCTCACTGACGACGTGAGTAAAATCAATGAAGGTATAGG

CGACAAGATAGGCATGTTCTTTCAGTCTATGGCGACGTTCTTCACTGGGTTCAT

CGTCGGTTTTACACGGGGATGGAAGCTTACGCTCGTGATTTTGGCCATTTCACC

CGTACTTGGATTGTCCGCTGCGGTATGGGCCAAGATACTGTCCTCCTTTACAG

ATAAGGAATTGCTCGCGTACGCCAAAGCCGGAGCTGTGGCAGAAGAGGTTCT

TGCAGCGATACGCACCGTCATTGCGTTCGGTGGCCAAAAAAAAGAACTCGAG

AGATATAATAAAAATCTCGAAGAAGCTAAAAGGATCGGAATTAAGAAAGCAA

TAACGGCAAACATCTCTATAGGAGCGGCCTTTCTGCTGATTTATGCGTCCTATG

CTCTGGCCTTTTGGTACGGCACGACTCTCGTTTTGTCCGGTGAGTACAGTATAG

GGCAGGTTTTGACTGTGTTCTTCTCAGTTCTCATCGGGGCTTTCAGTGTAGGGC

AGGCTTCCCCAAGCATTGAAGCATTTGCTAATGCCCGAGGGGCAGCATACGAA

ATATTTAAGATCATTGACAATAAACCATCCATTGACTCCTACAGTAAGAGCGG GCATAAGCCTGACAATATAAAGGGGAACTTGGAATTTCGCAACGTACACTTCA

GCTACCCTTCACGAAAGGAGGTAAAGATCCTCAAAGGACTGAATTTGAAAGT

GCAGTCTGGACAGACAGTTGCGCTGGTAGGCAATTCAGGATGCGGCAAGAGT

ACCACGGTACAACTTATGCAGAGGTTGTACGATCCCACTGAAGGTATGGTATC

AGTAGATGGCCAAGATATACGAACCATTAATGTACGCTTTCTTAGGGAGATTA

TAGGTGTGGTCTCACAGGAACCAGTTCTGTTCGCTACCACCATAGCTGAAAAC

ATTCGATACGGCAGAGAAAATGTCACTATGGACGAGATAGAAAAGGCAGTAA

AAGAAGCCAACGCATACGACTTCATAATGAAACTCCCGCATAAGTTTGATACG

CTCGTAGGGGAAAGGGGGGCCCAGCTGTCAGGCGGACAGAAACAAAGAATA

GCAATTGCCCGCGCACTCGTGAGGAACCCGAAGATATTGCTTCTCGATGAAGC

CACTTCCGCCCTCGATACGGAGAGCGAGGCGGTCGTTCAAGTAGCACTTGACA

AGGCGAGAAAAGGGCGGACAACCATTGTGATTGCTCATCGACTCTCCACGGT

ACGAAATGCTGATGTGATTGCTGGTTTCGACGATGGTGTCATCGTAGAGAAGG

GCAATCACGATGAATTGATGAAGGAAAAAGGGATTTATTTTAAGCTGGTAACT

ATGCAGACAGCGGGGAACGAAGTGGAGTTGGAAAATGCTGCAGACGAATCCA

AGTCAGAGATCGACGCGCTTGAGATGAGTTCCAACGACAGCCGATCATCTCTT

ATCCGAAAGCGGAGTACACGCAGGTCAGTCAGGGGAAGCCAGGCTCAAGACA

GGAAACTCTCAACCAAGGAAGCACTCGACGAATCCATTCCACCCGTAAGTTTC

TGGCGAATAATGAAATTGAATCTCACTGAATGGCCGTACTTCGTCGTGGGGGT

ATTCTGCGCGATTATAAATGGAGGCCTTCAGCCCGCATTCGCTATCATATTCA

GTAAGATAATCGGTGTATTTACTCGAATCGATGACCCTGAAACAAAGAGACA

AAATTCCAACCTGTTCTCCTTGTTGTTTCTCGCCCTCGGCATTATCTCATTCATA

ACGTTTTTCCTGCAGGGTTTTACATTTGGCAAAGCCGGGGAGATCCTTACAAA

AAGATTGAGATACATGGTCTTTCGGTCAATGCTGAGACAAGATGTCTCATGGT

TCGACGATCCCAAGAACACTACAGGGGCCCTTACTACTAGGCTGGCCAACGAT

GCAGCACAGGTCAAAGGCGCAATCGGATCCCGCCTGGCGGTTATAACCCAAA

ACATAGCAAATCTCGGGACGGGTATCATCATAAGTTTCATATATGGATGGCAA

CTGACGCTTTTGCTCTTGGCCATCGTCCCGATTATCGCCATAGCAGGGGTAGTG

GAAATGAAGATGCTCTCAGGGCAGGCACTGAAGGACAAAAAAGAACTTGAAG

GCAGTGGTAAAATCGCTACCGAGGCCATTGAGAATTTCCGGACTGTGGTTTCT

CTGACACAAGAGCAGAAATTCGAACACATGTACGCCCAATCTCTCCAAGTCCC

ATACCGCAATTCTCTCAGAAAGGCTCATATCTTCGGTATTACCTTTTCCTTTAC

TCAAGCGATGATGTACTTCTCCTACGCGGGCTGTTTTCGGTTCGGCGCCTATCT

GGTAGCACACAAACTGATGAGCTTCGAGGACGTTCTTCTGGTCTTTTCAGCGG TTGTATTCGGAGCTATGGCCGTGGGCCAAGTGTCAAGCTTTGCTCCTGATTAC

GCCAAAGCGAAAATATCAGCTGCGCACATTATCATGATTATAGAAAAGACTCC

TTTGATAGACTCTTATAGTACCGAGGGCCTCATGCCTAACACACTCGAAGGTA

ACGTAACCTTTGGGGAGGTTGTATTTAATTACCCGACAAGACCAGACATCCCG

GTGCTTCAAGGGCTGTCTTTGGAAGTGAAGAAAGGACAAACTTTGGCCCTCGT

TGGGTCTAGCGGGTGTGGAAAGAGTACGGTCGTACAGTTGCTCGAGCGGTTCT

ATGACCCACTTGCAGGAAAAGTGCTGCTGGACGGAAAAGAGATCAAAAGATT

GAATGTACAATGGCTGAGGGCCCATCTGGGTATAGTATCTCAGGAACCAATAC

TTTTTGATTGTAGCATCGCAGAAAACATCGCTTACGGCGATAATTCACGGGTT

GTTTCTCAAGAGGAGATTGTAAGAGCCGCCAAGGAAGCGAACATACATGCTTT

TATCGAATCCCTTCCGAACAAATATTCAACCAAGGTGGGCGACAAAGGGACA

CAGCTTAGTGGAGGCCAAAAGCAGAGGATTGCTATTGCCAGGGCTCTGGTAC

GGCAACCCCATATTTTGCTGCTCGATGAAGCGACGAGCGCTCTTGACACCGAA

TCAGAGAAGGTGGTACAGGAAGCACTGGACAAAGCACGAGAAGGTAGAACTT

GCATTGTCATTGCTCATAGACTGTCCACAATCCAGAATGCTGACCTTATTGTTG

TTTTTCAGAACGGTAGAGTAAAAGAACATGGCACGCACCAACAACTTCTTGCA

CAAAAGGGAATATACTTTTCTATGGTTTCCGTGCAGGCCGGTACAAAGAGGCA A

[0154] SEQ ID NO:9 Anti-DMl aa

[0155] QVQLQESGGGLVQPGGSLRLSCAASGGIFGISYMGWYRQAPGKQREPVAL

ITSGGDTAYGDSVKGRFAISRDNAKNTVYLQMNSLKPDDTSAYYCAAGFYRSDSP NRYDYWGQGTQVTVSS

[0156] SEQ ID NO:10 Anti-DMl DNA

[0157] CAAGTCCAACTTCAAGAGTCCGGGGGTGGATTGGTTCAACCAGGCGGG

TCCCTGAGGCTTTCATGTGCGGCGAGCGGTGGGATCTTCGGAATTAGTTATAT

GGGATGGTACCGGCAGGCGCCGGGGAAGCAACGCGAGCCTGTCGCGTTGATA

ACTTCCGGGGGGGATACAGCCTATGGTGACTCCGTTAAGGGCCGCTTTGCGAT

CAGCAGGGATAACGCTAAGAACACTGTCTATCTTCAAATGAACTCTTTGAAAC

CAGACGACACTTCTGCCTATTACTGTGCAGCGGGTTTCTACCGGTCCGACTCA

CCCAACCGGTATGATTATTGGGGCCAGGGTACACAGGTAACCGTATCCAGT

[0158] SEQ ID NO:11 Anti-DMl CAR aa

[0159] METDTLLLWVLLLWVPGSTGQVQLQESGGGLVQPGGSLRLSCAASGGIFG

ISYMGWYRQAPGKQREPVALITSGGDTAYGDSVKGRFAISRDNAKNTVYLQMNS

LKPDDTSAYYCAAGFYRSDSPNRYDYWGQGTQVTVSSPRTTTPAPRPPTPAPTIAS QPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR

KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQ

NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA

YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE

[0160] SEQ ID NO:12 Anti-DMl CAR DNA (Codon optimized for human expression)

[0161] ATGGAGAC AGAC AC ACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCC AG

GTTCCACAGGTCAAGTCCAACTTCAAGAGTCCGGGGGTGGATTGGTTCAACCA

GGCGGGTCCCTGAGGCTTTCATGTGCGGCGAGCGGTGGGATCTTCGGAATTAG

TTATATGGGATGGTACCGGCAGGCGCCGGGGAAGCAACGCGAGCCTGTCGCG

TTGATAACTTCCGGGGGGGATACAGCCTATGGTGACTCCGTTAAGGGCCGCTT

TGCGATCAGCAGGGATAACGCTAAGAACACTGTCTATCTTCAAATGAACTCTT

TGAAACCAGACGACACTTCTGCCTATTACTGTGCAGCGGGTTTCTACCGGTCC

GACTCACCCAACCGGTATGATTATTGGGGCCAGGGTACACAGGTAACCGTATC

CAGTCCTAGGACCACTACTCCGGCACCGCGCCCCCCAACTCCTGCACCGACGA

TAGCTTCACAACCGCTTTCATTGCGGCCCGAAGCATGTCGGCCAGCCGCCGGA

GGCGCTGTGCATACAAGAGGGCTGGATTTTGCATGTGATATATATATTTGGGC

GCCCCTTGCTGGCACTTGCGGCGTTCTTCTTCTTAGCCTCGTTATTACGCTCTA

CTGTAAGCGAGGTAGGAAAAAATTGCTGTATATCTTTAAACAGCCTTTTATGA

GACCCGTGCAAACGACTCAAGAGGAAGACGGGTGTAGCTGTAGATTTCCTGA

AGAGGAAGAGGGGGGGTGCGAACTGCGGGTGAAGTTCAGCAGAAGCGCCGA

CGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAACCTG

GGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTG

AGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGA

ACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGC

GAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTGTCCACCG

CCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGGCTC GAG

[0162] SEQ ID NO : 13 Anti-MMAE/MM AF aa

[0163] QVQLVQSGGGLVQAGGSLRLSCVASGRTFSMYRMGWFRQAPGKEREFVA

SIRWSGGSTYYTDSVKDRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPVWR LNSWYRGAVGYWGQGTQVTVSS

[0164] SEQ ID NO:14 Anti-MMAE/MMAF DNA (Codon optimized for human expression

[0165] CAAGTGCAGCTTGTTCAGTCCGGAGGAGGTCTCGTGCAGGCTGGTGGA AGCCTTCGACTGTCTTGTGTGGCCAGTGGTCGGACATTCTCTATGTATCGCATG GGATGGTTCAGGCAAGCTCCAGGTAAAGAGAGGGAGTTCGTCGCGAGTATAA GATGGTCCGGAGGAAGCACCTATTATACTGATTCCGTAAAAGATCGGTTTACC ATTAGCCGCGACAATGCTAAAAATACCGTTTATCTTCAGATGAACTCACTGAA GCCTGAAGATACAGCCGTTTATTATTGTGCAGCGGACCCTGTATGGAGGTTGA ATAGCTGGTATCGAGGTGCTGTAGGGTACTGGGGGCAGGGCACCCAAGTTAC CGTGAGCAGC

[0166] SEQ ID NO: 15 Anti-MMAE/MMAF CAR aa

[0167] METDTLLLWVLLLWVPGSTGQVQLVQSGGGLVQAGGSLRLSCVASGRTF SMYRMGWFRQAPGKEREFVASIRWSGGSTYYTDSVKDRFTISRDNAKNTVYLQM NSLKPEDTAVYYCAADPVWRLNSWYRGAVGYWGQGTQVTVSSPRTTTPAPRPP TPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT LYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP AYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE

|0168| SEQ ID NO:16 Anti-MMAE/MMAF CAR DNA (Codon optimized for human expression)

[0169] ATGGAGAC AG AC AC ACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCC AG

GTTCCACAGGTCAAGTGCAGCTTGTTCAGTCCGGAGGAGGTCTCGTGCAGGCT GGTGGAAGCCTTCGACTGTCTTGTGTGGCCAGTGGTCGGACATTCTCTATGTAT CGCATGGGATGGTTCAGGCAAGCTCCAGGTAAAGAGAGGGAGTTCGTCGCGA GTATAAGATGGTCCGGAGGAAGCACCTATTATACTGATTCCGTAAAAGATCGG TTTACCATTAGCCGCGACAATGCTAAAAATACCGTTTATCTTCAGATGAACTC ACTGAAGCCTGAAGATACAGCCGTTTATTATTGTGCAGCGGACCCTGTATGGA GGTTGAATAGCTGGTATCGAGGTGCTGTAGGGTACTGGGGGCAGGGCACCCA AGTTACCGTGAGCAGCCCTAGGACCACTACTCCGGCACCGCGCCCCCCAACTC CTGCACCGACGATAGCTTCACAACCGCTTTCATTGCGGCCCGAAGCATGTCGG CCAGCCGCCGGAGGCGCTGTGCATACAAGAGGGCTGGATTTTGCATGTGATAT ATATATTTGGGCGCCCCTTGCTGGCACTTGCGGCGTTCTTCTTCTTAGCCTCGT TATTACGCTCTACTGTAAGCGAGGTAGGAAAAAATTGCTGTATATCTTTAAAC AGCCTTTTATGAGACCCGTGCAAACGACTCAAGAGGAAGACGGGTGTAGCTG TAGATTTCCTGAAGAGGAAGAGGGGGGGTGCGAACTGCGGGTGAAGTTCAGC AGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACG AGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGG CCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGC

CTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCG

GCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGG

GCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTG

CCCCCAAGGCTCGAG

[0170] SEQ ID NO: 17 TUBB2B-CAR aa

[0171 ] METDTLLLWVLLLWVPGSTGMREIVHIQAGQCGNQIGAKFWEVISDEHGI

DPTGSYHGDSDLQLERINVYYNEATGNKYVPRAILVDLEPGTMDSVRSGPFGQIF

RPDNFVFGQSGAGNNWAKGHYTEGAELVDSVLDVVRKESESCDCLQGFQLTHSL

GGGTGSGMGTLLISKIREEYPDRIMNTFSVMPSPKVSDTVVEPYNATLSVHQLVEN

TDETYCIDNEALYDICFRTLKLTTPTYGDLNHLVSATMSGVTTCLRFPGQLNADLR

KLAVNMVPFPRLHFFMPGFAPLTSRGSQQYRALTVPELTQQMFDSKNMMAACDP

RHGRYLTVAAIFRGRMSMKEVDEQMLNVQNKNSSYFVEWIPNNVKTAVCDIPPR

GLKMSATFIGNSTAIQELFKRISEQFTAMFRRKAFLHWYTGEGMDEMEFTEAESN

MNDLVSEYQQYQDATADEQGEFEEEEGEDEAPRTTTPAPRPPTPAPTIASQPLSLR

PEACRPAAGGAVHTRGLDFACD1Y1WAPLAGTCGVLLLSLVITLYCKRGRKKLLY1

FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNE

LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE

[0172] SEQ ID NO: 18 TUBB2B-CAR DNA (Codon optimized for human expression)

[0173] ATGGAGAC AGAC AC ACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCC AG

GTTCCACAGGTATGCGCGAGATCGTCCACATCCAAGCTGGTCAGTGTGGGAAT

CAAATTGGCGCTAAATTCTGGGAAGTCATCAGTGATGAACATGGAATCGACCC

CACTGGGAGCTACCACGGGGACTCTGACCTCCAGTTGGAGCGGATCAATGTAT

ACTACAACGAAGCCACAGGGAACAAGTACGTTCCACGGGCTATCCTCGTAGA

CCTGGAACCAGGTACAATGGACTCCGTTCGCTCAGGTCCTTTCGGTCAAATCT

TCCGACCTGATAACTTCGTTTTCGGACAAAGTGGGGCGGGGAACAACTGGGCC

AAAGGACACTATACTGAAGGTGCGGAATTGGTGGATTCCGTTCTTGACGTGGT

CCGCAAGGAATCTGAATCTTGCGACTGCTTGCAAGGCTTCCAGCTTACACATT

CCCTTGGGGGAGGGACGGGCTCTGGCATGGGAACACTGTTGATTTCTAAAATA

AGAGAGGAGTACCCGGACCGGATCATGAACACGTTCTCAGTGATGCCATCTCC

TAAAGTGAGTGATACTGTGGTAGAGCCATATAACGCGACGTTGTCAGTTCATC

AGTTGGTAGAGAACACGGATGAAACGTATTGTATTGACAACGAGGCCCTGTAT

GATATATGCTTTAGGACATTGAAACTTACGACACCTACATATGGCGACCTGAA CCATCTCGTCAGCGCTACCATGTCTGGCGTTACCACCTGTTTGCGCTTTCCGGG

TCAGCTCAACGCAGACCTCCGAAAACTGGCAGTGAACATGGTCCCTTTCCCAC

GCTTGCATTTTTTCATGCCGGGATTTGCACCACTTACTTCACGCGGGTCCCAAC

AATACAGAGCGCTGACCGTCCCGGAGCTCACGCAGCAAATGTTTGACAGTAA

GAACATGATGGCAGCTTGCGATCCAAGGCATGGGCGATACTTGACAGTTGCA

GCCATTTTTCGGGGAAGAATGTCAATGAAAGAAGTAGATGAGCAGATGTTGA

ACGTGCAAAACAAAAATTCCTCCTATTTCGTGGAGTGGATCCCAAACAACGTC

AAAACCGCAGTATGCGATATACCGCCACGCGGTCTCAAAATGTCCGCAACATT

CATAGGGAACTCTACTGCCATTCAAGAGCTCTTCAAGAGAATTTCAGAACAAT

TTACCGCTATGTTTAGGCGAAAGGCATTTTTGCATTGGTACACAGGTGAGGGA

ATGGATGAGATGGAATTTACAGAAGCTGAGTCCAACATGAATGATTTGGTTTC

AGAATACCAGCAGTATCAGGATGCAACGGCCGACGAGCAGGGGGAATTCGAG

GAAGAGGAAGGGGAAGATGAGGCGCCTAGGACCACTACTCCGGCACCGCGCC

CCCCAACTCCTGCACCGACGATAGCTTCACAACCGCTTTCATTGCGGCCCGAA

GCATGTCGGCCAGCCGCCGGAGGCGCTGTGCATACAAGAGGGCTGGATTTTGC

ATGTGATATATATATTTGGGCGCCCCTTGCTGGCACTTGCGGCGTTCTTCTTCT

TAGCCTCGTTATTACGCTCTACTGTAAGCGAGGTAGGAAAAAATTGCTGTATA

TCTTTAAACAGCCTTTTATGAGACCCGTGCAAACGACTCAAGAGGAAGACGGG

TGTAGCTGTAGATTTCCTGAAGAGGAAGAGGGGGGGTGCGAACTGCGGGTGA

AGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCT

GTACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAG

CGGAGAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCC

AGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAG

CGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCT

GTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGC AGGCCCTGCCCCCAAGGCTCGAG

[0174] SEQ ID NO : 19 Anti-DOTA aa

[0175] EVMLVESGGGLVKPGGSLTLSCAASGFTFTAHAMSWVRQTPEKRLEWVA

TISGGGTYTYFPDSFQGRFTISDNAKNTLYLQMSSLRSEDTSMYFCTRHGDYRYAF GYWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSVVTQESALTTSPGETVTLTCR SSSGAVTTNNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSBSLIGDKAALTIT GAQTEDEAIYFCALWFSDHWVFGGGTELTVLG

[0176] SEQ ID NO:20 Anti-DOTA DNA (Codon optimized)

[0177] GAGGTGATGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGC AGCCTGACCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCACCGCCCACGCCAT

GAGCTGGGTGAGGCAGACCCCCGAGAAGAGGCTGGAGTGGGTGGCCACCATC AGCGGCGGCGGCACCTACACCTACTTCCCCGACAGCTTCCAGGGCAGGTTCAC CATCAGCGACAACGCCAAGAACACCCTGTACCTGCAGATGAGCAGCCTGAGG AGCGAGGACACCAGCATGTACTTCTGCACCAGGCACGGCGACTACAGGTACG

CCTTCGGCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCGGGGGTGG AGGTTCAGGGGGGGGTGGGAGCGGCGGGGGCGGTTCTGGAGGCGGCGGAAG CGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGACCGTGACC CTGACCTGCAGGAGCAGCAGCGGCGCCGTGACCACCAACAACTACGCCAACT

GGGTGCAGGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCGGCACCAA

CAACAGGGCCCCCGGCGTGCCCGCCAGGTTCAGCNNNAGCCTGATCGGCGAC AAGGCCGCCCTGACCATCACCGGCGCCCAGACCGAGGACGAGGCCATCTACT TCTGCGCCCTGTGGTTCAGCGACCACTGGGTGTTCGGCGGCGGCACCGAGCTG ACCGTGCTGGGC

[0178] SEQ ID NO:21 Anti-DOTA-CAR aa

|0179| METDTLLLWVLLLWVPGSTGEVMLVESGGGLVKPGGSLTLSCAASGFTFT AHAMSWVRQTPEKRLEWVATISGGGTYTYFPDSFQGRFTISDNAKNTLYLQMSSL RSEDTSMYFCTRHGDYRYAFGYWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGS VVTQESALTTSPGETVTLTCRSSSGAVTTNNYANWVQEKPDHLFTGLIGGTNNRA

PGVPARFSBSLIGDKAALTITGAQTEDEAIYFCALWFSDHWVFGGGTELTVLGPRT TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ

EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPRLE

[0180] SEQ ID NO:22 Anti-DOTA-CAR DNA (Codon optimized)

[0181] ATGGAGAC AG AC AC ACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCC AG

GTTCCACAGGTGAGGTGATGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCC CGGCGGCAGCCTGACCCTGAGCTGCGCCGCCAGCGGCTTCACCTTCACCGCCC ACGCCATGAGCTGGGTGAGGCAGACCCCCGAGAAGAGGCTGGAGTGGGTGGC

CACCATCAGCGGCGGCGGCACCTACACCTACTTCCCCGACAGCTTCCAGGGCA GGTTCACCATCAGCGACAACGCCAAGAACACCCTGTACCTGCAGATGAGCAG CCTGAGGAGCGAGGACACCAGCATGTACTTCTGCACCAGGCACGGCGACTAC AGGTACGCCTTCGGCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCCG GGGGTGGAGGTTCAGGGGGGGGTGGGAGCGGCGGGGGCGGTTCTGGAGGCG

GCGGAAGCGTGGTGACCCAGGAGAGCGCCCTGACCACCAGCCCCGGCGAGAC

CGTGACCCTGACCTGCAGGAGCAGCAGCGGCGCCGTGACCACCAACAACTAC

GCCAACTGGGTGCAGGAGAAGCCCGACCACCTGTTCACCGGCCTGATCGGCG

GCACCAACAACAGGGCCCCCGGCGTGCCCGCCAGGTTCAGCNNNAGCCTGAT

CGGCGACAAGGCCGCCCTGACCATCACCGGCGCCCAGACCGAGGACGAGGCC

ATCTACTTCTGCGCCCTGTGGTTCAGCGACCACTGGGTGTTCGGCGGCGGCAC

CGAGCTGACCGTGCTGGGCCCTAGGACCACTACTCCGGCACCGCGCCCCCCAA

CTCCTGCACCGACGATAGCTTCACAACCGCTTTCATTGCGGCCCGAAGCATGT

CGGCCAGCCGCCGGAGGCGCTGTGCATACAAGAGGGCTGGATTTTGCATGTG

ATATATATATTTGGGCGCCCCTTGCTGGCACTTGCGGCGTTCTTCTTCTTAGCC

TCGTTATTACGCTCTACTGTAAGCGAGGTAGGAAAAAATTGCTGTATATCTTT

AAACAGCCTTTTATGAGACCCGTGCAAACGACTCAAGAGGAAGACGGGTGTA

GCTGTAGATTTCCTGAAGAGGAAGAGGGGGGGTGCGAACTGCGGGTGAAGTT

CAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTAC

AACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGA

GAGGCCGGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGA

AGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAG

ATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATC

AGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCC

CTGCCCCCAAGGCTCGAG

[0182] SEQ ID NO:23 Anti-DXd aa

[0183] QCQSVEESGGRLVTPGTPLTLTCTASGFSLSSYAMNWVRQAPGKGLEYIGI

IYDSGTTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARAALDIWGPGTL

VTVSSGGGGSGGGGSGGGGSGGGGSMDTRAPTQLLGLLLLWLPGATFAAVLTQT

PSPVSAAVGGTVSISCQASKSVYNNNWLSWVQQTPGQPPKLLIYGASTLASGVPS

RFKGSGSGTQFTLTISDVQCDDAATYYCAGGYGSSSDNAFGGGTEVVVE

[0184] SEQ ID NO:24 Anti-DXd DNA (Codon optimized)

[0185] C AATGCC AG AGTGTGGAGG AGAGTGGGGGCCGGTTGGTAAC ACC AGGC

ACGCCGCTCACACTCACTTGTACGGCGTCAGGATTCAGCCTGAGCTCCTACGC

AATGAACTGGGTCCGCCAAGCCCCTGGTAAAGGCTTGGAGTACATCGGTATCA

TTTATGATTCAGGTACAACTTATTACGCGAGCTGGGCTAAAGGCCGCTTCACT

ATTTCAAAAACATCAACCACGGTTGACTTGAAGATTACGAGCCCTACGACGGA

GGACACCGCGACGTATTTCTGCGCGAGAGCAGCACTTGATATATGGGGTCCAG GCACTCTGGTGACGGTAAGCAGCGGGGGTGGAGGTTCAGGGGGGGGTGGGAG

CGGCGGGGGCGGTTCTGGAGGCGGCGGAAGCATGGATACGCGGGCTCCAACG

CAGTTGCTCGGCCTCCTCCTCCTCTGGCTCCCCGGAGCTACTTTCGCTGCCGTG

TTGACGCAAACCCCCTCTCCGGTATCCGCTGCTGTAGGTGGGACCGTGAGCAT

TAGTTGCCAAGCGTCTAAAAGTGTTTACAATAATAATTGGCTCTCATGGGTTC

AACAGACCCCAGGTCAGCCACCCAAGCTGCTGATTTATGGAGCCTCTACGCTC

GCGAGCGGGGTCCCCTCCCGGTTCAAGGGCTCCGGCAGTGGAACACAATTCAC

CCTTACGATATCCGACGTACAGTGTGATGACGCGGCTACTTACTACTGTGCAG

GAGGTTACGGTAGTAGCAGTGATAACGCATTTGGCGGTGGCACTGAAGTGGTC

GTGGAG

[0186] SEQ ID NO:25 Anti-DXd aa

[0187] METDTLLLWVLLLWVPGSTGQCQSVEESGGRLVTPGTPLTLTCTASGFSLS

SYAMNWVRQAPGKGLEYIGIIYDSGTTYYASWAKGRFTISKTSTTVDLKITSPTTE

DTATYFCARAALDIWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSMDTRAPTQL

LGLLLLWLPGATFAAVLTQTPSPVSAAVGGTVSISCQASKSVYNNNWLSWVQQT

PGQPPKLL1YGASTLASGVPSRFKGSGSGTQFTLT1SDVQCDDAATYYCAGGYGSS

SDNAFGGGTEVVVEPRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL

DFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDG

CSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR

GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG

LSTATKDTYDALHMQALPPRLE

[0188] SEQ ID NO:26 Anti-DXd DNA (Codon optimized)

[0189] ATGGAGAC AG AC AC ACTCCTGCTATGGGTGCTGCTGCTCTGGGTTCC AG

GTTCCACAGGTCAATGCCAGAGTGTGGAGGAGAGTGGGGGCCGGTTGGTAAC

ACCAGGCACGCCGCTCACACTCACTTGTACGGCGTCAGGATTCAGCCTGAGCT

CCTACGCAATGAACTGGGTCCGCCAAGCCCCTGGTAAAGGCTTGGAGTACATC

GGTATCATTTATGATTCAGGTACAACTTATTACGCGAGCTGGGCTAAAGGCCG

CTTCACTATTTCAAAAACATCAACCACGGTTGACTTGAAGATTACGAGCCCTA

CGACGGAGGACACCGCGACGTATTTCTGCGCGAGAGCAGCACTTGATATATG

GGGTCCAGGCACTCTGGTGACGGTAAGCAGCGGGGGTGGAGGTTCAGGGGGG

GGTGGGAGCGGCGGGGGCGGTTCTGGAGGCGGCGGAAGCATGGATACGCGGG

CTCCAACGCAGTTGCTCGGCCTCCTCCTCCTCTGGCTCCCCGGAGCTACTTTCG

CTGCCGTGTTGACGCAAACCCCCTCTCCGGTATCCGCTGCTGTAGGTGGGACC

GTGAGCATTAGTTGCCAAGCGTCTAAAAGTGTTTACAATAATAATTGGCTCTC ATGGGTTCAACAGACCCCAGGTCAGCCACCCAAGCTGCTGATTTATGGAGCCT

CTACGCTCGCGAGCGGGGTCCCCTCCCGGTTCAAGGGCTCCGGCAGTGGAACA

CAATTCACCCTTACGATATCCGACGTACAGTGTGATGACGCGGCTACTTACTA

CTGTGCAGGAGGTTACGGTAGTAGCAGTGATAACGCATTTGGCGGTGGCACTG

AAGTGGTCGTGGAGCCTAGGACCACTACTCCGGCACCGCGCCCCCCAACTCCT

GCACCGACGATAGCTTCACAACCGCTTTCATTGCGGCCCGAAGCATGTCGGCC

AGCCGCCGGAGGCGCTGTGCATACAAGAGGGCTGGATTTTGCATGTGATATAT

ATATTTGGGCGCCCCTTGCTGGCACTTGCGGCGTTCTTCTTCTTAGCCTCGTTA

TTACGCTCTACTGTAAGCGAGGTAGGAAAAAATTGCTGTATATCTTTAAACAG

CCTTTTATGAGACCCGTGCAAACGACTCAAGAGGAAGACGGGTGTAGCTGTA

GATTTCCTGAAGAGGAAGAGGGGGGGTGCGAACTGCGGGTGAAGTTCAGCAG

AAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAG

CTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCC

GGGACCCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCT

GTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGC

ATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCC

TGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCC CCAAGGCTCGAG

[0190] SEQ ID NO:27 CAR aa

PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL

VITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA

DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNE

LQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRLE

Claims

1. A chimeric antigen receptor (CAR) system comprising, a. an adaptor comprising an antigen binding element, a small molecule therapeutic and a tag ligand, and b. a CAR comprising signaling domain and a receptor that binds the tag ligand.

2. A chimeric antigen receptor (CAR) system comprising, a. an adaptor comprising an antigen binding element and a small molecule therapeutic, and b. a CAR comprising a receptor that binds the small molecule therapeutic and a signaling domain.

3. A synthetic notch (synNotch) receptor system comprising, a. an adaptor molecule comprising an antigen binding element, a small molecule therapeutic and a tag ligand, and b. a synNotch receptor comprising a receptor that binds the tag ligand, a notch core comprising one or more cleavage sites, and one or more transcription factors.

4. The CAR system or synNotch receptor system of any one of claims 1-3, wherein the antigen binding element comprises an antibody or antigen-binding fragment thereof.

5. The CAR system or synNotch receptor system of any one of claims 1-4, wherein the adaptor molecule comprises gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotzuzumab ozogamicin, polatuzumab vedotin, enfortumab vedotin, trastuzumab deruxtecan, sacituzumab govitecan, belantamab mafodotin, tisotumab vedotin, loncastuximab tesirine, disitamab vedotin, mirvetuximab soravtansine, or trastuzumab duocarmazine.

6. The CAR system or synNotch receptor system of any one of claims 1 -5, wherein the adaptor molecule comprises mirvetuximab soravtansine.

7. The CAR system or synNotch receptor system of any one of claims 1-5, wherein the adaptor molecule comprises trastuzumab emtansine.

8. The CAR system or synNotch receptor system of any one of claims 1 -5, wherein the adaptor molecule comprises trastuzumab deruxtecan.

9. The CAR system or synNotch receptor system of any one of claims 1 -4, wherein the antigen binding element comprises brentuximab, inotuzumab, polatuzumab, trastuzumab, enfortumab, Sacituzumab, belantamab, tisotumab, loncastuximab, disitamab, rituximnab, FMC63, herceptin, cetuximab, nimotuzumab, panitumumab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacuzimab or an antigen-binding fragment of any one thereof.

10. The CAR system or synNotch receptor system of any one of claims 1-9, wherein the small molecule therapeutic is a cytotoxic compound.

11. The CAR system or synNotch receptor system of claim 10, wherein the cytotoxic compound comprises a maytansinoid, an auristatin, a tubulysin, a calicheamicin, a duocarmycin, an exatecan, a pyrrolobenzodiazepine. a TLR agonist, or a STING agonist.

12. The CAR system or synNotch receptor system of any one of claims 10-11, wherein the cytotoxic compound comprises a maytansinoid.

13. The CAR system or synNotch receptor system of any one of claims 10-11, wherein the cytotoxic compound comprises a DM1 or a DM4.

14. The CAR system or synNotch receptor system of any one of claims 1-13, wherein the small molecule therapeutic is linked to the antigen binding element by a cleavable linker.

15. The CAR system or synNotch receptor system of claim 14, wherein the cleavable linker is cleavable by exposure to glutathione, an enzyme, hypoxia, or acidic pH.

16. The CAR system or synNotch receptor system of any one of claims 14-15, wherein the cleavable linker comprises a disulfide bond cleavable by glutathione.

17. The CAR or synNotch receptor system of any one of claims 14-15, wherein the cleavable linker comprises an acid-sensitive moiety.

18. The CAR system or synNotch receptor system of claim 15, wherein the enzyme comprises cathepsin, glycosidase, phosphatase, sulfatase, legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member Al, (ALDH7A1), lipase C, hepatic type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase-A (KHK-A), hexokinases (HK), nucleoside diphosphate kinase (NDPK or NDK), and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), mitochondrial a-ketoglutarate dehydrogenase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short-chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), a-ketoglutarate dehydrogenase (α-KGDH), CD39, CD73, or fumarase.

19. The CAR system or synNotch receptor system of any one of claim 1 and claims 3-18, wherein the tag ligand comprises a benzylguanine (BG), benzylcytosine (BC), chloroalkane (CA), fluorescein (FITC), SpyTag, leucine-zipper, La-SS-B, CD19, antifolate receptor antibody, Fc domain, peptide neoepitope (PNE), or biotin.

20. The CAR system or synNotch receptor system of any one of claim 1 and claims 3-18, wherein the tag ligand comprises a benzylguanine.

21. The CAR system or synNotch receptor system of any one of claim 1 and claims 3-18, wherein the tag ligand comprises a biotin.

22. The CAR system or synNotch receptor system of any of claims 1-21, wherein the CAR or synNotch receptor is comprised on a CAR T cell, a CAR NK cell, a CAR NK T cell, a CAR B cell, or a CAR macrophage.

23. A method of treating disease or disorder in a subject comprising administering to the subject a therapeutically effective amount of the CAR system or synNotch receptor system of any one of claims 1-22, wherein the disease or disorder comprises a cancer, an autoimmune disease, an autoinflammatory disease, a viral infection, a bacterial infection, or a fungal infection.

24. The method of claim 23, wherein the disease is a cancer.

25. The method of claim 24, wherein the cancer is selected from the group consisting of lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx, or lung, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, large bowel cancer, hematopoietic cancer, testicular cancer, colon cancer, rectal cancer, prostatic cancer, and pancreatic cancer.

26. The method of claim 23, wherein the disease is a cancer selected from the group consisting of ovarian cancer, breast cancer, and lung cancer.

27. The method of claim 23, wherein the disease is an ovarian cancer.

28. The method of claim 23, wherein the disease is a triple-negative breast cancer.

29. An engineered cell comprising a channel-forming protein or a binding protein and a receptor comprising a signaling domain and a domain that binds a cytotoxic compound or a tag ligand.

30. The engineered cell of claim 29, wherein the binding protein comprises a tubulin protein.

31. The engineered cell of claim 29, wherein the channel-forming protein comprises an MDR1 protein.

32. The engineered cell of any one of claims 29-31 , wherein the receptor is a CAR or a synNotch receptor.