Novel autoregulated car t cells and uses thereof

Abstract

This disclosure relates to novel chimeric inhibitory receptors (CIRs), for delivering targeted inhibitory signaling, and methods of making and using thereof. CAR T-cells expressing CIR designed for inhibiting the inflammatory mediator IFN gamma exhibited a significant reduction in levels of the inflammatory mediator and downstream immune activation while not adversely affecting CIR / CAR T cell killing of tumor cells in vivo.

Description

NOVEL AUTOREGULATED CAR T CELLS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims priority to U.S. Provisional Application 63 / 595,225 filed on November 1, 2023, which is incorporated herein by reference in its entirety.INCORPORATION OF ELECTRONICALLY FILED MATERIALThe Instant Application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 31, 2024 is named “UVA0003PCT” and is 128,045 bytes in size.TECHNICAL FIELD

[0001] This disclosure relates to engineered autoregulated chimeric antigen receptor (CAR) T cells and uses thereof.BACKGROUND

[0002] Chimeric antigen receptor (CAR)-T cell therapies targeting CD 19 or CD22 have been highly successful at salvaging children and adults with multiply relapsed or refractory acute lymphoblastic leukemia (r / rALL) and adults with B-cell lymphomas. B-cell maturation antigen (BCMA) targeted CAR-T cell therapies have been successfully used in adults with multiple myeloma. However, they have significant toxicities for which few solutions exist. Cytokine release syndrome (CRS), immune effector cell associated neurotoxicity syndrome (ICANS) and hemophagocytic lymphohistiocytosis / macrophage activation-like syndrome (HLH / MAS) can be life threatening and difficult to manage even with tocilizumab (anti-IL-6R antibody) and corticosteroids. For r / rALL patients, as many as 77% develop Grade 3-5 CRS and 40% Grade 3-5 ICANS. The underlying cause of these toxicities is the exponential and uncontrollable nature of CAR-T cell expansion and activation leading to an as of yet mechanistically poorly characterized supra-physiologic and generalized immune system activation. None of the FDA-approved CAR-T cell products are susceptible to any controllable regulation. Therefore, the field is in dire need of understanding the mechanistic underpinnings of CAR-T cell toxicities as well as novel mechanisms to regulate CAR-T cells while preserving efficacy.

[0003] Severe CRS (sCRS) occurs when hyperactivated CAR-T cells secrete copious inflammatory cytokines that hyperactivate the rest of the immune system. Left unchecked, sCRS can cause multi-organ failure and even death or morph into carHLH / MAS, which is often refractory to aggressive interventions. Many times, sCRS and carHLH / MAS do not respond to multiple doses of tocilizumab, the only FDA approved drug for sCRS, or high-dose steroids suggesting additional undiscovered mechanisms of toxicity exist. The pathophysiology of ICANS is also poorly understood but is related to uncontrolled CAR-T cell activity. There is presently no proven therapy for mitigating or treating ICANS, though steroids are often used.

[0004] Under physiologic conditions, T cell receptor (TCR) engagement by its cognate peptide-MHC complex in the context of co-stimulatory signals results in productive activation. Balancing this, immune checkpoint receptors such as PD-1 and TIGIT (T cell immunoreceptor with Ig and ITIM domains) are upregulated to control the T cell response and maintain immune homeostasis. In contrast, CAR-T cell engagement, at least in B cell malignancies, results in a supraphysiologic and uncontrolled activation of the T cells and many other components of innate immunity such as macrophages and monocytes. Currently, clinically available CAR constructs lack regulatory mechanisms to counterbalance CAR activation.

[0005] Since uncontrolled CAR-T cell activity and cytokine production is the common fundamental source of these toxicities, patients will benefit from a rheostat-controlled CAR-T cell system that autoregulates its own activity in response to circulating inflammatory cytokines or chemokines.SUMMARY

[0006] This disclosure relates to novel chimeric inhibitory receptors (CIRs) for attenuating and mitigating systemic toxicity in a subject, and methods of making and using thereof.

[0007] In an aspect, a mammalian cell comprising a nucleic acid encoding a CIR domain, the CIR domain comprising a ligand-sensing polypeptide able to sense, recognize, or interact with, an extracellular ligand, operably linked to an inhibitory signaling molecule, such that binding of the ligand to the ligand- sensing polypeptide delivers inhibitory signaling to automatically regulate and titrate ligand expression or production.

[0008] In one aspect, a nucleic acid sequence encoding a CIR domain, the nucleic acid identified in any one of SEQ ID No. 1-6, or a nucleic acid with at least 75% homology to said sequence. In yet another aspect, an amino acid sequence encoding a CIR domain, the amino acid sequence identified in any one of SEQ ID NO: 7-12, or an amino acid with at least 75% identity to said sequence.

[0009] In another aspect, a mammalian cell comprising a nucleic acid encoding a CIR domain of the invention and a chimeric antigen receptor (CAR) domain, a CIR / CAR. In an aspect, the mammalian cell is a T-cell transduced with a nucleic acid encoding a CIR / CAR of the invention, a CIR / CAR T-cell.

[0010] In one aspect, a nucleic acid sequence encoding a CIR / CAR identified in at least one sequence set forth in any one of SEQ ID No. 13 - 24, or a nucleic acid sequence with at least 75% homology to said sequence. In yet another aspect, an amino acid sequence encoding a CIR / CAR, the amino acid identified in any of SEQ ID No. 25-33, or an amino acid with at least 75% identity to said sequence.

[0011] In one aspect, a method for producing a CIR / CAR T-cell, comprising transducing a T cell with a nucleic acid encoding a CIR / CAR (or CAR / CIR) described herein. In another aspect, a method for controlling CAR T cell activation and cytokine production comprising transducing a mammalian cell or an immune effector cell with a nucleic acid encoding CIR / CAR described herein.

[0012] In another aspect, a pharmaceutical composition comprising a mammalian cell described herein and a pharmaceutically acceptable carrier.

[0013] In one aspect, a vector comprising one or more of the nucleic acids as described herein.

[0014] In one aspect, a cell comprising the vector as described herein.

[0015] In one aspect, the cell is an immune effector cell.

[0016] In one aspect, a cell comprising one or more of the nucleic acids as described herein. In another aspect, a cell comprising two of the nucleic acids as described herein.

[0017] In one aspect, a method of treating or reducing cytokine toxicity in a subject receiving CAR T-cell therapy, the method comprising administering a therapeutically effective amount of a composition comprising a mammalian cell comprising a nucleic acid encoding a CIR domain described herein to the subject. In another aspect, the mammalian cell comprises anucleic acid encoding both a CIR domain and a CAR domain, or a CIR / CAR (or CAR / CIR) nucleic acid.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Fig. 1 shows proposed model of chimeric inhibitory receptor (CIR) -mediated attenuation of CD19 CAR activity. Development of CAR / CIR T cells to mitigate toxicity while preserving anti-tumor efficacy in an automated and titratable fashion. Co-expression of a chimeric inhibitory receptor (CIR) that can sense an increase in a specific CRS- associated cytokine / chemokine. Upon binding to a specific pro-inflammatory cytokine or chemokine in the extracellular environment, CIRs deliver an intracellular inhibitory signal to counter activating signals of the CAR and deliver inhibitory signaling to automatically regulate and titrate CAR-T cell activity and the resultant global immune hyperactivation, which together mitigate severe CAR-T cell toxicities in a “hands-free” approach.

[0001] Fig. 2 shows a schematic representative model of a chimeric inhibitory receptor (CIR) composed of an anti-IFN-gamma binding scFv coupled to the intracellular inhibitory signaling domain of the TIGIT receptor. The schematic shows interchangeability of extracellular cytokine-binding domain and intracellular inhibitory signaling domain.

[0020] Fig. 3 shows construct design of CD19.28z CAR-T cell co-expressing a representative chimeric inhibitory receptor (CIR). Schematic design of CD19.28z.CIR (anti- IFN-y / TIGIT) used in the examples below. CIR components can be modified to target other cytokines / chemokines or provide alternative intracellular signaling. scFv = single-chain variable fragment; T2A = “self-cleaving” peptide sequence; EGFRt = truncated epidermal growth factor receptor, inert receptor used for isolation, detection, or depletion of CARs; GM-CSFR = granulocyte-monocyte-colony stimulating factor receptor; |32M = Beta 2 microglobulin; LTR = long terminal repeat.

[0021] Figs. 4A and 4B show normal expansion and proliferation of CD19.28z CAR- T cells co-expressing CIR domain during in vitro manufacture. 4A) CAR-T cells were enumerated at indicated timepoints post-activation during in vitro expansion following retroviral transduction of CAR construct. 4B) Fold expansion of CAR-T cells relative to Day 7 postactivation during in vitro production. Results shown are representative of at least six individual experiments using T cells from 3 different normal human donors.

[0022] Figs. 5A and 5B show CD19.28z.CIR CAR-T cells exhibit attenuated activation following exposure to NALM-6 CD19+tumor cells. 5A) CD19.28z CAR-T cells, CD19.28z.CIR CAR-T cells, or non-transduced (NT) T cells were cultured with (dark-shaded histograms) or without (light- shaded histograms) NALM-6 for 24 hours then CAR-T cell activation was evaluated by staining for CD3, CD4, CD8, CD69, CD25, CD38, PD-1, CD107a and analyzed by flow cytometry. Representative flow plots for expression of T cell activation markers CD69 and CD38 are shown for CD4+and CD8+CAR-T cells. 5B) Bar graphs represent the percentage of CAR-T cells expressing the indicated number of activation markers per cell. Results shown are representative of five individual experiments using T cells from 3 different normal human donors. Statistical significance was calculated by Two-way ANOVA with Sidak multiple comparisons test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, ns=not significant.

[0023] Fig. 6 shows in vitro cytotoxicity of CD19.28z CAR-T cells expressing CIR receptor against CD19+leukemia cell line NALM-6. CD19.28z CAR-T cells, CD19.28z.CIR CAR-T cells, or control Nontransduced T cells were co-cultured overnight with NALM-6 tumor cell line expressing firefly luciferase, at various effector to target (E:T) cell ratios. Anti-tumor cytotoxicity was then evaluated by measuring bioluminescent activity of remaining tumor cells following addition of luciferin. Culture plates were subsequently read in a Lago X instrument (Spectral Instruments Imaging). Cytotoxicity was calculated as follows: percent cytotoxicity = 100 - (((average signal of CAR-T cell-treated wells) I (average signal from untreated tumor cell only wells)) x 100). The graphs above are representative individual experiments performed with CARs generated from multiple donors.

[0024] Figs. 7A - 7C show CD19.28z.CIR / CAR-T cells exhibit attenuated cytotoxicity kinetics which is dependent upon CIR-mediated binding to IFN-gamma. 7A) CD19.28z CAR-T cells co-expressing CIR exhibit attenuated cytotoxicity kinetics following in vitro serial stimulation by tumor cells. Representative experiment (n=5) shows slower target cell killing kinetics by CD19.28z.CIR CAR-T cells. NALM-6 were co-incubated with NT, CD19.28z CAR, or CD19.28z CAR / CIR-T cells. Sequential NALM-6 stimulations were carried out as denoted by adding fresh target cells. 7B) IFN-gamma binding by CIR scFv domain is required for attenuated cytotoxic activity during in vitro stimulation by tumor cells. CD19.28z.CIR CAR-T cells require IFN-y-scFv binding domain and are responsive to IFN-y. NALM-6 cells were co-incubated with CD19.28z CAR, CD19.28z.CIR, or CD19.28z.CIR(scFv-less, cannot bind IFN-y) T cells. CD19.28z.CIR (scFv-less) T cells mimic the anti-tumor killing of control CD19.28z CAR-T cells, demonstrating the necessity for IFN-y recognition to drive TIGIT signaling. 7C) Pre-exposure to IFN-y blunts initial cytotoxic activity of CD19.28z CAR / CIR T cells. NT, CD19.28z, or CD19.28z.CIR CAR-T cells were incubated with rIFN-y (0.05, 0.5, 5, 50 ng / ml) for 24 hours to engage CIR signaling then co-cultured with NALM-6 targets. Cytotoxicity was measured at 6 and 20 hours by BLI (representative of three individual experiments).

[0025] Figs. 8A and 8B show CIR signaling in CD19.28z CAR-T cells results in attenuated production of cytokines and chemokines. 8A) Attenuated pro-inflammatory cytokine production by CD19.28z.CIR CAR-T cells relative to CD19.28z CAR-T cells following stimulation by tumor cells. NT, CD19.28z CAR, or CD19.28z.CIR CAR-T cells were exposed to NALM-6 for 24 hrs and the culture supernatants analyzed by Luminex®. Error bars = standard deviation. Representative of two individual experiments using T cells from 2 different normal human donors. 8B) Luminex® results are summarized and presented as the percent change in analyte concentration from CD19.28z.CIR cells relative to CD19.28z controls. Statistical significance was calculated using Two-tailed unpaired t-test. *p<0.05 and ns=not significant.

[0026] Fig. 9 shows overall polyfunctionality of CD19.28z CAR-T cells co-expressing CIR is not compromised by CIR signaling. CD19.28z CAR-T cells or CD19.28z.CIR CAR-T cells were cultured in the presence or absence of NALM-6 cells for 22 hrs followed by singlecell secretome analysis using the Isolight® platform (Bruker© Cellular Analysis).

[0027] Figs. 10A - 10C show CD19.28z CAR-T cells attenuated by CIR signaling results in diminished pro-inflammatory cytokine / chemokine secretion by bystander autologous macrophages and DCs. 10A) Schematic diagram of an in vitro co-culture assay consisting of NALM-6, CD19.28z, or CD19.28z.CIR CAR-T cells, and either dendritic cells (DC) or macrophages (Mac) for the production of CRS-associated cytokines and chemokines. 10B) CD 19 CAR-T cells attenuated by CIR signaling results in decreased release of innate pro- inflammatory CRS-associated cytokines / chemokines by monocyte-derived dendritic cells (MoDC) and monocyte-derived macrophages (MoMac). All groups shown include NALM-6 tumor cells. Co-cultures were incubated for 24 Hrs. and the supernatants were collected for analysis by Luminex®. Error bars represent standard deviation. The above data are representative of two individual experiments. 10C) Luminex® results are summarized andpresented as the percent change in analyte concentration from MoMac cultured withCD19.28z.CIR cells relative to CD19.28z controls.

[0028] Fig. 11 shows Nanostring® nCounter gene expression profiling demonstrates significant regulation of CAR-T cell pathways by the presence of CIR signaling. CD19.28z CAR-T cells or CD19.28z.CIR CAR-T cells were cultured in the presence or absence of NALM- 6 cells for 24 hrs followed by total cell lysis and RNA extraction. RNA was then subjected to Nanostring® nCounter analysis using the CAR-T Characterization Panel, a 780-plex gene expression panel (Nanostring® Technologies). Standard quality control procedures and data normalization were performed using the ROSALIND online platform. RNA Samples from each condition were analyzed in triplicate.

[0029] Figs. 12A-12E show CIR-regulated CD19.28z CAR-T cells exhibit similar anti-tumor activity and survival in vivo relative to CD19.28z controls. 12A) On day -2, NOD.Cg-Prkdcsc’dI12rgtmlwj| / SzJ (NSG) mice were injected with luciferase-expressing NALM-6 cells (0.5xl06). 2 days later, mice were treated i.v. with CD19.28z or CD19.28z.CIR CAR-T cells (10xl06). Bioluminescent imaging (BLI) for disease burden was performed by injecting mice with luciferin and was measured 2-3 times / week. 12B) BLI images from a representative experiment are shown for indicated timepoints (DPT = Days Post-Treatment) following CAR-T cell treatment. 12C) Bioluminescent tumor growth curves. Statistical significance was calculated using Two-way ANOVA with Tukey’s multiple comparisons test. ****p<0.0001. 12D) Kaplan- Meier survival curve analysis. 12E) Peripheral blood (left) and spleens (right) from individual mice were analyzed on day 28 post- treatment for the presence of CAR-T cells using flow cytometry. Individual symbols represent individual mice. Statistical significance was calculated using Two-tailed unpaired t-test. ns=not significant. Results shown are representative of two individual experiments using T cells from 2 different normal human donors; n=4-5 mice / group.

[0030] Figs. 13A-13E show CIR-mediated attenuation of CD19 CAR-T cell responses is observed in vivo in tumor-bearing mice. 13A) On day -2, NSG mice were injected with 0.5xl06NALM-6. Two days later, mice were treated i.v. with CD19.28z or CD19.28z.CIR CAR-T cells (10xl06). 13B) Bioluminescent tumor growth curves. 13C) Serum was pooled from individual mice for each group on days 1 and 2 and IFN-y measured by ELISA. n=4 mice / group. 13D and 13E) Confirmation of CIR-mediated attenuation of CAR-T cells in vivo was performed by evaluating T cell activation (% CD38+and % CD38+PD-1+CAR cells) inthe spleen and bone marrow of mice treated with CD19.28z or CD19.28z.CIR CAR-T cells on Day 2. Individual symbols represent individual mice. Statistical significance was calculated using Two-tailed unpaired t-test. **p<0.001.

[0031] Fig. 14 shows Chimeric Inhibitory Receptors (CIRs) can be applied to any CAR product, such as CD19.BBz CAR-T cells. Schematic design of CD19.BBz.CIR (anti- IFN-y / TIGIT) used in the following studies. scFv = single-chain variable fragment; T2A = “selfcleaving” peptide sequence; EGFRt = truncated epidermal growth factor receptor, inert receptor used for isolation, detection, or depletion of CARs; GM-CSFR = granulocyte-monocyte-colony stimulating factor receptor; |32M = Beta-2 microglobulin; LTR = long terminal repeat.

[0032] Figs. 15A and 15B show CD19.BBz CAR / CIR-T cells exhibit attenuated activation following exposure to NALM-6 CD19+tumor cells. 15A) CD19.BBz CAR-T cells, CD19.BBz.CIR CAR-T cells, or non-transduced (NT) T cells were cultured with (dotted line histograms) or without (full line histograms) NALM-6 for 24 hours then CAR-T cell activation was evaluated by staining for CD3, CD4, CD8, CD69, PD-1, and CD25 and analyzed by flow cytometry. 15B) Bar graphs represent the percentage of CAR-T cells expressing the indicated activation markers. Results shown are representative of three individual experiments using T cells from 3 different normal human donors. Statistical significance was calculated using multiple unpaired t-tests multiple comparisons test. ***p<0.001 and **p<0.01.

[0033] Fig. 16 shows in vitro cytotoxicity of CD19.4-lBBz CAR T cells expressing CIR receptor against leukemia cell line NALM-6 demonstrates similar leukemia control. CD19.BBz CAR T cells, CD19.BBz.CIR CAR T cells, or control Nontransduced T cells were co-cultured overnight with NALM-6 tumor cell line expressing firefly luciferase, at various effector to target (E:T) cell ratios. Anti-tumor cytotoxicity was then evaluated by measuring bioluminescent activity of remaining tumor cells following addition of luciferin. Culture plates were subsequently read in a Lago X instrument (Spectral Instruments Imaging). Cytotoxicity was calculated as follows: percent cytotoxicity = 100 - (((average signal of CAR T cell-treated wells) I (average signal from untreated tumor cell only wells)) x 100). The graphs above are representative individual experiments performed with CARs generated from multiple donors.

[0034] Figs. 17A and 17B show diminished pro-inflammatory cytokine / chemokine secretion by bystander autologous macrophages and DCs during in vitro CRS-assay. 17A) Schematic diagram of an in vitro co-culture assay consisting of NALM-6, CD19.BBz, orCD19.BBz.CIR CAR-T cells, and macrophages (Mac) for the production of CRS- associated cytokines / chemokines. 17B) CD19.BBz CAR-T cells attenuated by CIR signaling results in decreased release of innate pro-inflammatory CRS-associated cytokines / chemokines by monocyte-derived macrophages (MoMac). All groups shown include NALM-6 tumor cells. Cocultures were incubated for 24 Hrs. and the supernatants were collected for analysis by Luminex®. Error bars represent standard deviation. The above data are representative of two individual experiments.

[0035] Figs. 18A-18D show self-regulating CD19.BBz CAR-T cells expressing anti- IFN-y / TIGIT CIR exhibit similar anti-tumor activity and survival but improved persistence in vivo. 18A) On day -2, NOD.Cg-Prkdcsc‘dH2rgtmlw | / SzJ (NSG) mice were injected with 0.5xl06NALM-6. 2 days later, mice were treated i.v. with CD19.BBz or CD19.BBz.CIR CAR-T cells (10xl06). BLI for disease burden was performed 2-3 times / week. 18B) Bioluminescent tumor growth curves. Statistical significance was calculated using Two- way ANOVA with Tukey’s multiple comparisons test. ****p<0.0001. 18C) Kaplan-Meier survival curve analysis. 18D) Peripheral blood (left) and spleens (right) from individual mice were analyzed on day 28 post-treatment for the presence of CAR-T cells using flow cytometry. Individual symbols represent individual mice. Statistical significance was calculated using Two- tailed unpaired t-test. **p<0.001 and *p<0.05. Results shown are representative of two individual experiments using T cells from 2 different normal human donors; n=5 mice / group.

[0036] Fig. 19 shows generic CIR constructs for “sensing” various inflammatory cytokines / chemokines coupled to various inhibitory signaling domains. CD 19 CAR-T cells could also express CIRs engineered to bind additional extracellular cytokines / chemokines and incorporate different inhibitory signaling domains for attenuating CAR-T activation and mitigating toxicity. Schematic design of alternative CIR constructs co-expressed with CD19 CAR-T cells. scFv = single-chain variable fragment; T2A = “self-cleaving” peptide sequence; EGFRt = truncated epidermal growth factor receptor, inert receptor used for isolation, detection, or depletion of CARs; GM-CSFR = granulocyte-monocyte-colony stimulating factor receptor; |32M = Beta-2 microglobulin; LTR = long terminal repeat.

[0037] Fig. 20 shows alternative CIR constructs for “sensing” various inflammatory cytokines / chemokines coupled to various inhibitory signaling domains. CIR domain is designed to be modular so alternative inhibitory signaling domains can be utilized and combinedwith alternative cytokine -binding domains. Schematic design of alternative CIR constructs coexpressed with CD19 CAR-T cells. scFv = single-chain variable fragment; T2A = “selfcleaving” peptide sequence; EGFRt = truncated epidermal growth factor receptor, inert receptor used for isolation, detection, or depletion of CARs; GM-CSFR = granulocyte-monocyte-colony stimulating factor receptor; | 2M = Beta-2 microglobulin; LTR = long terminal repeat.

[0038] Fig. 21 shows potential incorporation of CIR domains into other CAR-T cell constructs. CIR domain could also be co-expressed with other CAR-T cell domains for attenuating activation and mitigating toxicity. Schematic design of CIR constructs co-expressed with other CAR-T cells, including but not limited to, HER2 and av|E CARs. scFv = single-chain variable fragment; T2A = “self-cleaving” peptide sequence; EGFRt = truncated epidermal growth factor receptor, inert receptor used for isolation, detection, or depletion of CARs; GM- CSFR = granulocyte-monocyte-colony stimulating factor receptor; |32M = Beta-2 microglobulin; LTR = long terminal repeat.DETAILED DESCRIPTION

[0039] Described herein is a novel auto-regulatory element in the form of a chimeric inhibitory receptor, CIR, comprising a ligand-sensing polypeptide, or a sensor, that when engaged with its ligand, will trigger inhibitory signaling within the cell. Without being limited to a theory, upon binding to a specific ligand, e.g. a pro-inflammatory cytokine or chemokine, in the extracellular environment, CIRs deliver inhibitory signaling to counter and automatically titrate cell activation signals, for example from an activated chimeric antigen receptor (CAR) or other immune cell producing the signal, resulting in regulation of the amount of ligand produced. In the case of overexpression due to CAR-T cell activity resulting in global immune hyperactivation, CIR can mitigate severe CAR-T cell toxicities in a “hands-free” approach.

[0040] In one embodiment, a mammalian cell comprising a nucleic acid encoding a CIR domain, the CIR domain comprising a ligand-sensing polypeptide able to recognize an extracellular ligand, operably linked to an inhibitory signaling molecule, such that sensing of the ligand by the ligand- sensing polypeptide delivers inhibitory signaling to automatically regulate and titrate ligand expression or production by the cell. By ‘ligand-sensing’ is meant that the polypeptide is able to detect, bind, internalize or react, or a combination of any of these functions, to the presence of the ligand. By ‘inhibitory signaling molecule’ is meant a moleculeable to inhibit or reduce the amount of ligand at the level of transcription or translation, or reduce the function of the ligand, or modify the ligand to reduce its activity, or divert the ligand’s intended effect into a different effect on the cell as designed, or bind the internalized ligand to prevent its activity, or prevent entry of the ligand to the cell. In one aspect, the ligand-sensing polypeptide interacts with the ligand. In another aspect, the ligand-sensing polypeptide recognizes and binds the ligand. In yet another aspect, the ligand- sensing polypeptide recognizes, binds, and internalizes the ligand.

[0041] As is evident from the description, examples below, and drawings herein, the extracellular ligand-sensing polypeptide and the intracellular inhibitory signaling molecule are modular and exchangeable, i.e. can be selected or chosen as needed.

[0042] In one embodiment, the ligand is a circulating signal, a hormone, a neurotransmitter, a lipid, a peptide, a cytokine or a chemokine. In another embodiment, the ligand is toxic to the cell. In another aspect, the ligand is a soluble mediator of toxicity in a cell. In one embodiment, the ligand causes systemic toxicity in a subject. In one aspect, the ligand is a cytokine or chemokine produced by direct or indirect chimeric antigen receptor (CAR) activation. In yet another embodiment, the ligand is interferon gamma (IFNy), interleukin 6 (IL- 6), interleukin 1 (IL-1), monocyte chemoattractant protein 1 (MCP-1), granulocyte macrophage colony stimulating factor (GM-CSF) or a combination thereof.

[0043] In one embodiment, the ligand- sensing polypeptide is able to sense the ligand. By ‘sensing the ligand’ is meant, sensing the presence of, detecting, interacting with, binding, internalizing, or any combination thereof, of the ligand. In one embodiment, the ligand- sensing polypeptide is a naturally occurring or modified ligand receptor. In another embodiment, the ligand-sensing polypeptide is an antibody or antigen-binding fragment that recognizes the ligand. In an embodiment, the antibody or antigen-binding fragment is a single-chain variable fragment (scFv) of an antibody able to recognize the ligand. In one embodiment, the scFv recognizes a cytokine or chemokine. In another embodiment, the scFv recognizes and interacts with a cytokine or chemokine resulting from direct or indirect CAR activation.

[0044] In one embodiment, the inhibitory signaling molecule is any naturally occurring, or naturally or artificially modified, inhibitory or regulatory signal molecule. In another embodiment, the inhibitory molecule is an inhibitory motif derived from a signaling molecule, the inhibitory motif comprising part or whole of an inhibitory molecule. In one embodiment, theinhibitory signals molecule is from an immune checkpoint receptor. In one embodiment, the signaling molecule is from a TIGIT (a T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine -based inhibitory motif or ITIM), from a PD-1 (programmed cell death protein 1), positive programmed death-ligand 1 (PD-L1), from a cytotoxic T-lymphocyte protein 4 (CTLA-4), from a lymphocyte activation gene 3 (LAG-3), from a T-cell immunoglobulin and mucin domain 3 (TIM-3).

[0045] In one embodiment, the inhibitory signaling molecule is TIGIT, comprising the transmembrane and intracellular signaling domain thereof. In one embodiment, the inhibitory molecule TIGIT has a nucleic acid sequence set forth in SEQ ID NO:37, a polypeptide encoded by SEQ ID NO: 37 set for in SEQ ID NO: 38, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;CTGCTTGGAGCTATGGCCGCTACACTGGTGGTCATCTGTACCGCCGTGATCGTGGTG GTGGCCCTGACCAGAAAGAAGAAGGCCCTGAGAATCCACAGCGTGGAAGGCGACCT GCGGAGAAAGTCTGCCGGACAAGAAGAGTGGTCCCCTAGCGCTCCATCTCCACCTG GATCTTGTGTGCAGGCCGAAGCTGCTCCTGCTGGACTGTGTGGCGAACAGAGAGGC GAAGATTGCGCCGAGCTGCACGACTACTTCAACGTGCTGAGCTACAGAAGCCTGGG CAACTGCAGCTTCTTCACCGAGACAGGA (SEQ ID NO: 37).SEQ ID NO: 38 LLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSC VQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG

[0046] In another embodiment, the inhibitory signaling molecule is PD-1, comprising the transmembrane and intracellular signaling domain of PD-1. In one embodiment, the inhibitory molecule PD-1 has a nucleic acid sequence set forth in SEQ ID NO:39, a polypeptide encoded by SEQ ID NO: 39 set forth in SEQ ID NO: 40, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence having a nucleic acid sequence;GTGGGAGTTGTTGGAGGCCTGCTGGGATCTCTGGTGCTGCTTGTTTGGGTGCTCGCC GTGATCTGTAGCAGAGCCGCCAGAGGAACAATCGGCGCCAGAAGAACAGGCCAGCCTCTGAAAGAGGACCCCTCTGCCGTTCCTGTGTTCAGCGTGGACTATGGCGAGCTGG ATTTCCAGTGGCGGGAAAAGACACCCGAGCCTCCAGTGCCTTGTGTGCCTGAGCAG ACAGAGTACGCCACCATCGTGTTCCCTAGCGGCATGGGCACATCTAGCCCTGCCAGA AGAGGATCTGCCGACGGACCTAGATCTGCCCAGCCTCTCAGACCTGAGGATGGCCA CTGTTCTTGGCCTCTG (SEQ ID NO:39)SEQ ID NO: 40 VGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQ WREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0047] In one embodiment, a nucleic acid encoding a CIR domain, wherein the ligandsensing polypeptide is a single-chain variable fragment (scFv) (a single-chain variable fragment heavy chain, a single-chain variable domain light chain, an IgGl hinge sequence) capable of sensing the ligand. In one embodiment, the scFv is an anti-interferon gamma single-chain variable fragment. In one embodiment, the anti-interferon gamma single-chain variable fragment has a sequence set forth in SEQ ID NO: 41, a polypeptide encoded by SEQ ID NO 41 set forth in SEQ ID NO: 42, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence having a nucleic acid sequence;GAAGTGCAGCTGCTGGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAG ACTGTCTTGTGCCGCCAGCGGCTTCACCTTTAGCAGCTACGCCATGAGCTGGGTCCG ACAGGCTCCTGGCAAAGGCCTTGAATGGGTGTCCGCCATCTCTGGCTCTGGCGGCAG CACATATTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCA AGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTAC TACTGTGCCAAGGATGGCAGCAGCGGATGGTACGTGCCCCATTGGTTTGATCCTTGG GGCCAGGGCACACTGGTCACCGTTTCTTCTGGCGGCGGCGGGAGCGGAGGAGGCGG ATCCGGGGGCGGGGGGAGCGGCGGAGGCGGAAGCAACTTCATGCTGACCCAGCCTC ACAGCGTGTCCGAGTCTCCAGGCAAGACCGTGACCATCAGCTGCACAAGAAGCAGC GGCTCTATCGCCAGCAACTACGTGCAGTGGTATCAGCAGAGGCCTGGCAGCAGCCC TACCACCGTGATCTACGAGGACAACCAGAGGCCTAGCGGCGTGCCCGATAGATTTT CCGGCAGCATCGACAGCAGCAGCAACAGCGCCAGCCTGACAATCAGCGGCCTGAAA ACAGAGGACGAGGCCGACTACTACTGCCAGAGCTACGACGGCTCCAACAGGTGGATGTTTGGCGGCGGAACAAAGCTGACAGTGCTGGCTAGCGAGCCCAAGAGCGCCGACAAGACCCACACCGCCCCC (SEQ ID NO:41)SEQ ID NO: 42 VGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQ WREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0048] In another embodiment, the ligand- sensing polypeptide is an anti-interleukin-6 (IL-6) single chain variable fragment (scFv). In one embodiment, the anti-IL-6 scFv has a nucleic acid sequence set forth in SEQ ID NO: 43, a polypeptide encoded by SEQ ID NO: 43 set forth in SEQ ID NO: 44, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence having a nucleic acid sequence;GAGGTGCAGCTGGTGGAGAGCGGCGGCAAGCTGCTGAAGCCCGGCGGCAGCCTGA AGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTTCGCCATGAGCTGGTTCA GGCAGAGCCCCGAGAAGAGGCTGGAGTGGGTGGCCGAGATCAGCAGCGGCGGCAG CTACACCTACTACCCCGACACCGTGACCGGCAGGTTCACCATCAGCAGGGACAACG CCAAGAACACCCTGTACCTGGAGATGAGCAGCCTGAGGAGCGAGGACACCGCCATG TACTACTGCGCCAGGGGCCTGTGGGGCTACTACGCCCTGGACTACTGGGGCCAGGG CACCAGCGTGACCGTGAGCAGCGGCGGCGGCGGGAGCGGAGGAGGCGGATCCGGG GGCGGGGGGAGCGGCGGAGGCGGAAGCCAGATCGTGCTGATCCAGAGCCCCGCCA TCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGC GTGAGCTACATGTACTGGTACCAGCAGAAGCCCGGCAGCAGCCCCAGGCTGCTGAT CTACGACACCAGCAACCTGGCCAGCGGCGTGCCCGTGAGGTTCTCCGGCAGCGGAA GCGGCACCAGCTACAGCCTGACCATCAGCAGGATGGAGGCCGAGGACGCCGCCACC TACTACTGCCAGCAGTGGAGCGGCTACCCCTACACCTTCGGCGGCGGCACCAAGCT GGAGATCAAGGCTAGCGAGCCCAAGAGCGCCGACAAGACCCACACCGCCCCC (SEQ ID NO:43).SEQ ID NO: 44 MARSVTLVFLVLVSLTGLYAHHHHHHMHASGSGSEVQLVESGGKLLKPGGSLKLSCAA SGFTFSSFAMSWFRQSPEKRLEWVAEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEMS SLRSEDTAMYYCARGLWGYYALDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSQIVLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIK

[0049] In an embodiment, the ligand-sensing polypeptide is an anti-MCP- 1 single chain variable fragment having a nucleic acid sequence. In one embodiment, the anti-MCP- 1 scFv has a nucleic acid sequence set forth in SEQ ID NO: 45, a polypeptide encoded by SEQ ID NO:45 set forth in SEQ ID NO: 46, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence having a nucleic acid sequence;CAGGTGGAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGA AGGTGAGCTGCAAGGCCAGCGGCGGCACCTTCAGCAGCTACGGCATCAGCTGGGTG AGACAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCGGCATCATCCCCATCTTCGG CACCGCCAACTACGCCCAGAAGTTCCAGGGCAGAGTGACCATCACCGCCGACGAGA GCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTG TACTACTGCGCCAGATACGACGGCATCTACGGCGAGCTGGACTTCTGGGGCCAGGG CACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGGAGCGGAGGAGGCGGATCCGGG GGCGGGGGGAGCGGCGGAGGCGGAAGCGAGATCGTGCTGACCCAGAGCCCCGCCA CCCTGAGCCTGAGCCCCGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGC GTGAGCGACGCCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGACT GCTGATCTACGACGCCAGCAGCAGAGCCACCGGCGTGCCCGCCAGATTCTCCGGCA GCGGAAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTC GCCGTGTACTACTGCCACCAGTACATCCAGCTGCACAGCTTCACCTTCGGCCAGGGC ACCAAGGTGGAGATCAAGGCTAGCGAGCCCAAGAGCGCCGACAAGACCCACACCG CCCCC (SEQ ID NO: 45)SEQ ID NO: 46MARSVTLVFLVLVSLTGLYAHHHHHHMHASGSGSQVELVQSGAEVKKPGSSVKVSCK ASGGTFSSYGISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYME LSSLRSEDTAVYYCARYDGIYGELDFWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSE IVLTQSPATLSLSPGERATLSCRASQSVSDAYLAWYQQKPGQAPRLLIYDASSRATGVPA RFSGSGSGTDFTLTISSLEPEDFAVYYCHQYIQLHSFTFGQGTKVEIK

[0050] In another embodiment, a nucleic acid encoding a CIR domain, wherein the ligand-sensing polypeptide is anti-IFN gamma scFv and the inhibitory signaling molecule is from a TIGIT, set forth in SEQ ID NO:1, an amino acid sequence encoded by SEQ ID NO:1 set forth in SEQ ID NO: 7, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;SEQ ID NO: 1ATGGCTCGCTCGGTGACCCTGGTCTTTCTGGTGCTTGTCTCACTGACAGGCTTGTATG CTCACCACCACCACCACCACATGCATGCTAGCGGCAGCGGCAGCGAAGTGCAGCTG CTGGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCC GCCAGCGGCTTCACCTTTAGCAGCTACGCCATGAGCTGGGTCCGACAGGCTCCTGGC AAAGGCCTTGAATGGGTGTCCGCCATCTCTGGCTCTGGCGGCAGCACATATTACGCC GACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTA CCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGCCAAGG ATGGCAGCAGCGGATGGTACGTGCCCCATTGGTTTGATCCTTGGGGCCAGGGCACAC TGGTCACCGTTTCTTCTGGCGGCGGCGGGAGCGGAGGAGGCGGATCCGGGGGCGGG GGGAGCGGCGGAGGCGGAAGCAACTTCATGCTGACCCAGCCTCACAGCGTGTCCGA GTCTCCAGGCAAGACCGTGACCATCAGCTGCACAAGAAGCAGCGGCTCTATCGCCA GCAACTACGTGCAGTGGTATCAGCAGAGGCCTGGCAGCAGCCCTACCACCGTGATC TACGAGGACAACCAGAGGCCTAGCGGCGTGCCCGATAGATTTTCCGGCAGCATCGA CAGCAGCAGCAACAGCGCCAGCCTGACAATCAGCGGCCTGAAAACAGAGGACGAG GCCGACTACTACTGCCAGAGCTACGACGGCTCCAACAGGTGGATGTTTGGCGGCGG AACAAAGCTGACAGTGCTGGCTAGCGAGCCCAAGAGCGCCGACAAGACCCACACCG CCCCCCTGCTTGGAGCTATGGCCGCTACACTGGTGGTCATCTGTACCGCCGTGATCG TGGTGGTGGCCCTGACCAGAAAGAAGAAGGCCCTGAGAATCCACAGCGTGGAAGGC GACCTGCGGAGAAAGTCTGCCGGACAAGAAGAGTGGTCCCCTAGCGCTCCATCTCC ACCTGGATCTTGTGTGCAGGCCGAAGCTGCTCCTGCTGGACTGTGTGGCGAACAGAG AGGCGAAGATTGCGCCGAGCTGCACGACTACTTCAACGTGCTGAGCTACAGAAGCC TGGGCAACTGCAGCTTCTTCACCGAGACAGGAACGCGTTAASEQ ID NO: 7MARSVTLVFLVLVSLTGLYAHHHHHHMHASGSGSEVQLLESGGGLVQPGGSLRLSCAA SGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKDGSSGWYVPHWFDPWGQGTLVTVSSGGGGSGGGGSGGGGS GGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQR PSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDGSNRWMFGGGTKLTVLASEP KSADKTHTAPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEW SPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETGTR

[0051] In yet another embodiment, a nucleic acid encoding a CIR domain, wherein the ligand-sensing polypeptide is anti-IL-6 scFv and the inhibitory signaling molecule is from a TIGIT, the nucleic acid set forth in SEQ ID NO:2 below, an amino acid encoded by SEQ ID NO: 2 set forth in SEQ ID NO: 8, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;SEQ ID NO: 2ATGGCTCGCTCGGTGACCCTGGTCTTTCTGGTGCTTGTCTCACTGACAGGCTTGTATG CTCACCACCACCACCACCACATGCATGCTAGCGGCAGCGGCAGCGAGGTGCAGCTG GTGGAGAGCGGCGGCAAGCTGCTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGC CGCCAGCGGCTTCACCTTCAGCAGCTTCGCCATGAGCTGGTTCAGGCAGAGCCCCGA GAAGAGGCTGGAGTGGGTGGCCGAGATCAGCAGCGGCGGCAGCTACACCTACTACC CCGACACCGTGACCGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACACCCTG TACCTGGAGATGAGCAGCCTGAGGAGCGAGGACACCGCCATGTACTACTGCGCCAG GGGCCTGTGGGGCTACTACGCCCTGGACTACTGGGGCCAGGGCACCAGCGTGACCG TGAGCAGCGGCGGCGGCGGGAGCGGAGGAGGCGGATCCGGGGGCGGGGGGAGCGG CGGAGGCGGAAGCCAGATCGTGCTGATCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGTAC TGGTACCAGCAGAAGCCCGGCAGCAGCCCCAGGCTGCTGATCTACGACACCAGCAA CCTGGCCAGCGGCGTGCCCGTGAGGTTCTCCGGCAGCGGAAGCGGCACCAGCTACA GCCTGACCATCAGCAGGATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAG TGGAGCGGCTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGCTAG CGAGCCCAAGAGCGCCGACAAGACCCACACCGCCCCCCTGCTTGGAGCTATGGCCG CTACACTGGTGGTCATCTGTACCGCCGTGATCGTGGTGGTGGCCCTGACCAGAAAGAAGAAGGCCCTGAGAATCCACAGCGTGGAAGGCGACCTGCGGAGAAAGTCTGCCGG ACAAGAAGAGTGGTCCCCTAGCGCTCCATCTCCACCTGGATCTTGTGTGCAGGCCGA AGCTGCTCCTGCTGGACTGTGTGGCGAACAGAGAGGCGAAGATTGCGCCGAGCTGC ACGACTACTTCAACGTGCTGAGCTACAGAAGCCTGGGCAACTGCAGCTTCTTCACCG AGACAGGAACGCGTTAASEQ ID NOG MARSVTLVFLVLVSLTGLYAHHHHHHMHASGSGSEVQLVESGGKLLKPGGSLKLSCAA SGFTFSSFAMSWFRQSPEKRLEWVAEISSGGSYTYYPDTVTGRFTISRDNAKNTLYLEMS SLRSEDTAMYYCARGLWGYYALDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSQI VLIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFS GSGSGTSYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIKASEPKSADKTHTAPL LGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCV QAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETGTR

[0052] In yet another embodiment, a nucleic acid encoding a CIR domain, wherein the CIR domain comprises an scFv recognizing MCP-1 and the inhibitory signaling molecule is from a TIGIT, set forth in SEQ ID NOG, an amino acid sequence encoded by SEQ ID NOG set forth in SEQ ID NO: 9, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;SEQ ID NOG atggctcgctcggtgaccctggtctttctggtgcttgtctcactgacAggCttgtatgctCACCACCACCACCACCACATG CATGCTAGCGGCAGCGGCAGCCAGGTGGAGCTGGTGCAGAGCGGCGCCGAGGTGA AGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCGGCACCTTCAGC AGCTACGGCATCAGCTGGGTGAGACAGGCCCCCGGCCAGGGCCTGGAGTGGATGGG CGGCATCATCCCCATCTTCGGCACCGCCAACTACGCCCAGAAGTTCCAGGGCAGAGT GACCATCACCGCCGACGAGAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGA GAAGCGAGGACACCGCCGTGTACTACTGCGCCAGATACGACGGCATCTACGGCGAG CTGGACTTCTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGGGAG CGGAGGAGGCGGATCCGGGGGCGGGGGGAGCGGCGGAGGCGGAAGCGAGATCGTG CTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGAGAGCCACCCTGAG CTGCAGAGCCAGCCAGAGCGTGAGCGACGCCTACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACGACGCCAGCAGCAGAGCCACCGGCGTG CCCGCCAGATTCTCCGGCAGCGGAAGCGGCACCGACTTCACCCTGACCATCAGCAG CCTGGAGCCCGAGGACTTCGCCGTGTACTACTGCCACCAGTACATCCAGCTGCACAG CTTCACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGGCTAGCGAGCCCAAGAGCG CCGACAAGACCCACACCGCCCCCCTGCTTGGAGCTATGGCCGCTACACTGGTGGTCA TCTGTACCGCCGTGATCGTGGTGGTGGCCCTGACCAGAAAGAAGAAGGCCCTGAGA ATCCACAGCGTGGAAGGCGACCTGCGGAGAAAGTCTGCCGGACAAGAAGAGTGGTC CCCTAGCGCTCCATCTCCACCTGGATCTTGTGTGCAGGCCGAAGCTGCTCCTGCTGG ACTGTGTGGCGAACAGAGAGGCGAAGATTGCGCCGAGCTGCACGACTACTTCAACG TGCTGAGCTACAGAAGCCTGGGCAACTGCAGCTTCTTCACCGAGACAGGAACGCGTt aaSEQ ID N0:9 MARSVTLVFLVLVSLTGLYAHHHHHHMHASGSGSQVELVQSGAEVKKPGSSVKVSCK ASGGTFSSYGISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYME LSSLRSEDTAVYYCARYDGIYGELDFWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSE IVLTQSPATLSLSPGERATLSCRASQSVSDAYLAWYQQKPGQAPRLLIYDASSRATGVPA RFSGSGSGTDFTLTISSLEPEDFAVYYCHQYIQLHSFTFGQGTKVEIKASEPKSADKTHTA PLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGS CVQAEAAPAGECGEQRGEDCAEEHDYFNVESYRSEGNCSFFTETGTR

[0053] In yet another embodiment, a nucleic acid encoding a CIR domain, wherein the CIR domain comprises an scFv recognizing IFN gamma and the inhibitory signaling molecule is from a PD-1, set forth in SEQ ID NO:4, an amino acid sequence encoded by SEQ ID NO:4 set forth in SEQ ID NO: 10, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;SEQ ID NO:4 atggctcgctcggtgaccctggtctttctggtgcttgtctcactgacAggCttgtatgctCACCACCACCACCACCACATG CATGCTAGCGGCAGCGGCAGCGAAGTGCAGCTGCTGGAATCTGGCGGAGGACTGGT TCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTTAGCAG CTACGCCATGAGCTGGGTCCGACAGGCTCCTGGCAAAGGCCTTGAATGGGTGTCCG CCATCTCTGGCTCTGGCGGCAGCACATATTACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGA GCCGAGGACACCGCCGTGTACTACTGTGCCAAGGATGGCAGCAGCGGATGGTACGT GCCCCATTGGTTTGATCCTTGGGGCCAGGGCACACTGGTCACCGTTTCTTCTGGCGG CGGCGGGAGCGGAGGAGGCGGATCCGGGGGCGGGGGGAGCGGCGGAGGCGGAAG CAACTTCATGCTGACCCAGCCTCACAGCGTGTCCGAGTCTCCAGGCAAGACCGTGAC CATCAGCTGCACAAGAAGCAGCGGCTCTATCGCCAGCAACTACGTGCAGTGGTATC AGCAGAGGCCTGGCAGCAGCCCTACCACCGTGATCTACGAGGACAACCAGAGGCCT AGCGGCGTGCCCGATAGATTTTCCGGCAGCATCGACAGCAGCAGCAACAGCGCCAG CCTGACAATCAGCGGCCTGAAAACAGAGGACGAGGCCGACTACTACTGCCAGAGCT ACGACGGCTCCAACAGGTGGATGTTTGGCGGCGGAACAAAGCTGACAGTGCTGGCT AGCGAGCCCAAGAGCGCCGACAAGACCCACACCGCCCCCGTGGGAGTTGTTGGAGG CCTGCTGGGATCTCTGGTGCTGCTTGTTTGGGTGCTCGCCGTGATCTGTAGCAGAGC CGCCAGAGGAACAATCGGCGCCAGAAGAACAGGCCAGCCTCTGAAAGAGGACCCC TCTGCCGTTCCTGTGTTCAGCGTGGACTATGGCGAGCTGGATTTCCAGTGGCGGGAA AAGACACCCGAGCCTCCAGTGCCTTGTGTGCCTGAGCAGACAGAGTACGCCACCAT CGTGTTCCCTAGCGGCATGGGCACATCTAGCCCTGCCAGAAGAGGATCTGCCGACG GACCTAGATCTGCCCAGCCTCTCAGACCTGAGGATGGCCACTGTTCTTGGCCTCTGACGCGTtaaSEQ ID NO: 10MARSVTLVFLVLVSLTGLYAHHHHHHMHASGSGSEVQLLESGGGLVQPGGSLRLSCAA SGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKDGSSGWYVPHWFDPWGQGTLVTVSSGGGGSGGGGSGGGGS GGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDGSNRWMFGGGTKLTVLASEP KSADKTHTAPVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVF SVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRP EDGHCSWPLTR

[0054] In still another embodiment, a nucleic acid encoding a CIR domain, wherein the CIR domain comprises an scFv recognizing IL-6 and the inhibitory signaling molecule is from a PD-1, set forth in SEQ ID NO:5, an amino acid sequence encoded by SEQ ID NO:5 set forth in SEQ ID NO: 11, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence;SEQ ID NO: 5ATGGCTCGCTCGGTGACCCTGGTCTTTCTGGTGCTTGTCTCACTGACAGGCTTGTATGCTCACCACCACCACCACCACATGCATGCTAGCGGCAGCGGCAGCGAGGTGCAGCTGGTGGAGAGCGGCGGCAAGCTGCTGAAGCCCGGCGGCAGCCTGAAGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTTCGCCATGAGCTGGTTCAGGCAGAGCCCCGA GAAGAGGCTGGAGTGGGTGGCCGAGATCAGCAGCGGCGGCAGCTACACCTACTACC CCGACACCGTGACCGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACACCCTG TACCTGGAGATGAGCAGCCTGAGGAGCGAGGACACCGCCATGTACTACTGCGCCAG GGGCCTGTGGGGCTACTACGCCCTGGACTACTGGGGCCAGGGCACCAGCGTGACCG TGAGCAGCGGCGGCGGCGGGAGCGGAGGAGGCGGATCCGGGGGCGGGGGGAGCGG CGGAGGCGGAAGCCAGATCGTGCTGATCCAGAGCCCCGCCATCATGAGCGCCAGCC CCGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGTAC TGGTACCAGCAGAAGCCCGGCAGCAGCCCCAGGCTGCTGATCTACGACACCAGCAA CCTGGCCAGCGGCGTGCCCGTGAGGTTCTCCGGCAGCGGAAGCGGCACCAGCTACA GCCTGACCATCAGCAGGATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAG TGGAGCGGCTACCCCTACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGCTAG CGAGCCCAAGAGCGCCGACAAGACCCACACCGCCCCCGTGGGAGTTGTTGGAGGCC TGCTGGGATCTCTGGTGCTGCTTGTTTGGGTGCTCGCCGTGATCTGTAGCAGAGCCG CCAGAGGAACAATCGGCGCCAGAAGAACAGGCCAGCCTCTGAAAGAGGACCCCTCT GCCGTTCCTGTGTTCAGCGTGGACTATGGCGAGCTGGATTTCCAGTGGCGGGAAAAG ACACCCGAGCCTCCAGTGCCTTGTGTGCCTGAGCAGACAGAGTACGCCACCATCGTG TTCCCTAGCGGCATGGGCACATCTAGCCCTGCCAGAAGAGGATCTGCCGACGGACC TAGATCTGCCCAGCCTCTCAGACCTGAGGATGGCCACTGTTCTTGGCCTCTGACGCG TTAASEQ ID NO: 11MARSVTLVFLVLVSLTGLYAHHHHHHMHASGSGSEVQLVESGGKLLKPGGSLKLSCAASGFTFSSFAMSWFRQSPEKREEWVAEISSGGSYTYYPDTVTGRFTISRDNAKNTEYEEMS SERSEDTAMYYCARGEWGYYAEDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGGSQI VEIQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPREEIYDTSNEASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIKASEPKSADKTHTAPV GVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQ WREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLT R

[0055] In yet another embodiment, a nucleic acid encoding a CIR domain, wherein the CIR domain comprises an scFv recognizing MCP-1 and the inhibitory signaling molecule is from a PD-1, set forth in SEQ ID NO:6; an amino acid sequence encoded by SEQ ID NO:6 set forth in SEQ ID NO: 12, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.SEQ ID NO:6 ATGGCTCGCTCGGTGACCCTGGTCTTTCTGGTGCTTGTCTCACTGACAGGCTTGTATG CTCACCACCACCACCACCACATGCATGCTAGCGGCAGCGGCAGCCAGGTGGAGCTG GTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGCA AGGCCAGCGGCGGCACCTTCAGCAGCTACGGCATCAGCTGGGTGAGACAGGCCCCC GGCCAGGGCCTGGAGTGGATGGGCGGCATCATCCCCATCTTCGGCACCGCCAACTA CGCCCAGAAGTTCCAGGGCAGAGTGACCATCACCGCCGACGAGAGCACCAGCACCG CCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGCGCC AGATACGACGGCATCTACGGCGAGCTGGACTTCTGGGGCCAGGGCACCCTGGTGAC CGTGAGCAGCGGCGGCGGCGGGAGCGGAGGAGGCGGATCCGGGGGCGGGGGGAGC GGCGGAGGCGGAAGCGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAG CCCCGGCGAGAGAGCCACCCTGAGCTGCAGAGCCAGCCAGAGCGTGAGCGACGCCT ACCTGGCCTGGTACCAGCAGAAGCCCGGCCAGGCCCCCAGACTGCTGATCTACGAC GCCAGCAGCAGAGCCACCGGCGTGCCCGCCAGATTCTCCGGCAGCGGAAGCGGCAC CGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCGTGTACTACTG CCACCAGTACATCCAGCTGCACAGCTTCACCTTCGGCCAGGGCACCAAGGTGGAGA TCAAGGCTAGCGAGCCCAAGAGCGCCGACAAGACCCACACCGCCCCCGTGGGAGTT GTTGGAGGCCTGCTGGGATCTCTGGTGCTGCTTGTTTGGGTGCTCGCCGTGATCTGTA GCAGAGCCGCCAGAGGAACAATCGGCGCCAGAAGAACAGGCCAGCCTCTGAAAGA GGACCCCTCTGCCGTTCCTGTGTTCAGCGTGGACTATGGCGAGCTGGATTTCCAGTG GCGGGAAAAGACACCCGAGCCTCCAGTGCCTTGTGTGCCTGAGCAGACAGAGTACGCCACCATCGTGTTCCCTAGCGGCATGGGCACATCTAGCCCTGCCAGAAGAGGATCTGCCGACGGACCTAGATCTGCCCAGCCTCTCAGACCTGAGGATGGCCACTGTTCTTGGCCTCTGACGCGTTAASEQ ID N0:12 MARSVTLVFLVLVSLTGLYAHHHHHHMHASGSGSQVELVQSGAEVKKPGSSVKVSCK ASGGTFSSYGISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYME LSSLRSEDTAVYYCARYDGIYGELDFWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSE IVLTQSPATLSLSPGERATLSCRASQSVSDAYLAWYQQKPGQAPRLLIYDASSRATGVPA RFSGSGSGTDFTLTISSLEPEDFAVYYCHQYIQLHSFTFGQGTKVEIKASEPKSADKTHTA PVGVVGGELGSEVEEVWVEAVICSRAARGTIGARRTGQPEKEDPSAVPVFSVDYGELDF QWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPE TR

[0056] Now that multiple CIR domains and their nucleic acid sequences have been described herein, altering the nucleic acid sequence, to remove, add, or substitute sequences for different ligand- sensing polypeptides or inhibitory signaling molecules in order to design a CIR for a selected purpose is known in the art. Sequences required for cloning a nucleic acid, for proper expression of a nucleic acid, for proper expression of an encoded polypeptide, for proper localization of the encoded peptide, for detection of the encoded peptide, or for transformation or transduction of cells, are known in the art and can be substituted with the ones described herein, such as for example, linkers, signal sequences, His Tags, 5’ETR, 3’ETR, transmembrane localization signals antibiotic resistance, etc. and methods for producing a nucleic acid for its desired function, to name a few.

[0057] In an aspect, the cell is a mammalian cell. In another aspect, the cell is an immune effector cell:-a T cell selected from the group consisting of a helper CD4+T cell, a CD4+stem cell memory T cell, a cytotoxic CD8+T cell, a CD8+stem cell memory T cell, a memory T cell, a naive and / or central memory T cell, a regulatory CD4+T cell, an innate-like T cell, a natural killer T cell, a mucosal associated invariant T cell, and a Gamma Delta T cell;-a monocyte selected from the group consisting of a CD14++CD16 monocyte, a CD14+CD16++ monocyte, and a CD14++CD16+ monocyte;-a B cell selected from the group consisting of a plasmablast, a plasma cell, a lymphoplasmacytoid cell, a memory B cell, a B-2 cell, a B-l cell, and a regulatory B cell (Breg);-a NK cell selected from the group consisting of a CD56BRIGHTNK cell and a CD56DMNK cell; or-a mononuclear phagocytic cell selected from the group consisting of an adipose tissue macrophage, a monocyte, a Kupffer cell, a sinus histiocyte, an alveolar macrophage (dust cell), a tissue macrophage (histiocyte), a microglial cell, a Hofbauer cell, an intraglomerular mesangial cell, an osteoclast, a Langerhans cell, an epithelioid cell, a red pulp macrophage (sinusoidal lining cell), a peritoneal macrophage, a lysomac, and a perivascular macrophage.

[0058] In one aspect, a mammalian cell is a T-cell. In one aspect, a mammalian cell is a CAR T-cell. CARs are artificial receptors that redirect antigen specificity, activate T cells, and further enhance T cell function through their costimulatory component. CAR T-cells are genetically modified with targeted CARs, CARs targeted to a cell surface molecule, or antigen, specific to a disease cell. In most cases, autologous T-cells are transduced with a nucleic acid encoding a CAR which targets the modified T-cell against a specified antigen, i.e. not expressed in normal non-disease cells

[0059] The fundamental structure of a CAR is known in the art. Briefly, it involves 3 components: an extracellular antigen recognition domain, a transmembrane domain, and a cytoplasmic signaling domain. The extracellular antigen-binding domain is usually a singlechain variable fragment (scFv) derived from an antigen -reactive antibody. The scFv comprises of the variable regions of heavy (VH) and light (VL) chains of the antibody joined together by a flexible peptide linker. This ectodomain scFv provides the target antigen specificity for CAR T cells. In contrast to the natural T-cell receptor, the scFv of CAR does not need antigen processing and presentation of peptide epitope in the context of MHC molecules.

[0060] CAR T-cell targeting molecules are known in the art and include a native protein, an antibody or an antigen-binding fragment thereof, a T cell receptor (TCR), an integrin, or a cell adhesion molecule; an integrin selected from the group consisting of integrin beta-1 (ITG|31), integrin alpha-5 (ITGa5), lymphocyte function-associated antigen 1 (LFA-1), and integrin subunit alpha V (ITGaV); integrin-alpha v beta 3 (ITGav / ITGb3 or CD51 / CD61); a cell adhesion molecule selected from the group consisting of intercellular adhesion molecule- 1 (ICAM-1), vascular cell adhesion protein-1 (VCAM-1), neural cell adhesion molecule 1 (N-CAM1), and platelet endothelial cell adhesion molecule-1 (PE-CAM1); a patient-derived TCR; an engineered protein comprising an antibody or an antigen-binding fragment thereof; B-cell activating factor (BAFF) receptor, B-cell maturation antigen (BCMA), CD3, CD5, CD19, CD20, CD22, CD28, CD30, CD38, CD79B; AXF, B7 homolog 3 protein (B7-H3), carcinoembryonic antigen (CEA), CD70, claudinl8.2 (CEDN18.2), delta-like ligand 3 (DEE3), disialoganglioside (GD2), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), guanylyl cyclase C (GUCY2C), human epidermal growth factor receptor 2 (HER2), Kita-Kyushu lung cancer antigen-1 (KK-LC-1), Eewis Y (FEY), mesothelin (MSEN), MUCIN 1 (MUC1), NEW YORK esophageal squamous cell carcinoma 1 (NY-ESO-1), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), receptor-tyrosine-kinase like orphan receptor 1 (R0R1), transforming growth factor beta (TGF-0), Kirsten rat sarcoma vims (KRAS) G12D, melanoma antigen recognized by T cells 1 (MART-1), melanoma-associated antigen 3 (MAGE- A3), tumor protein p53 (TP53), FMS-like tyrosine kinase 3 (FLT3), or alkaline phosphatase placental-like 2 (ALPPL2); LI cell adhesion molecule (LICAM), neurexin 3, vesicular glutamate transporter 1 (VGLUT1), vesicular inhibitory amino acid transporter (VIA AT), neuroligin 1, neuroligin 2, neural cell adhesion molecule 1 (NCAM1), vesicular acetylcholine transporter (VAChT), folate receptor- 1 (FOLR1), gamma-aminobutyric acid B receptor 1 (GABA(b)Rl), GABA(b)R2, glutamate ionotropic receptor NMDA type subunit 1 (GRIN1), GRIN2B, or solute carrier family 6 member 4 (SLC6A4); neural / glial antigen 2 (NG2), oligodendrocyte marker 01, oligodendrocyte marker 04, A2B5, or myelin-oligodendrocyte glycoprotein (MOG); P2Y 12, macrophage colony- stimulating factor receptor (M-CSFR), or CX3C motif chemokine receptor 1 (CX3CR1); CD133 or CD49F; stem cell antigen- 1 (Sca-1), CD27, CD34, CD38, CD43, CD117, or CD150, A2B5, connexin 43, or aquaporin-4 (AQP-4); CD71 or CD24

[0061] The first-generation CAR had only one intracellular signaling domain, most commonly a CD3^ chain, which provided signal 1 for activation of CAR T-cells. To improve the function, proliferation, and persistence of CARs, second- and third-generation CARs were developed by adding one or more costimulatory domains, respectively to provide signal 2 for CAR T-cell activation. The costimulatory domains that are most commonly used are CD28 and CD137 (4-1BB). Costimulatory domains with significant therapeutic potential include CD28, CD137 (4-1BB), 0X40, CD27, and ICOS. Any of these CAR T-cells can be used for expressing a CIR as described herein.

[0062] In another aspect, a method of producing a CAR T-cell with a CIR domain, comprising transducing a CAR T-cell with a nucleic acid encoding a CIR domain. In another aspect, the T-cell can be transduced with a nucleic acid encoding a CIR domain and a nucleic acid encoding a CAR domain targeted to a specific antigen. In one aspect, a T-cell cell can be transduced with two nucleic acids, one encoding a CIR domain as described herein, and one encoding a CAR domain. In another aspect, the T cell can be transduced with one nucleic acid encoding both a CIR domain and a CAR domain as is exemplified herein. A nucleic acid encoding both a CIR domain and a CAR domain, or co-expressing a CIR domain and a CAR domain, is a CIR / CAR nucleic acid (or a CAR / CIR nucleic acid). The cell expressing both a CIR domain and a CAR domain is a CIR / CAR cell or a CAR / CIR cell. When the cell is a T-cell, it is referred to as a CIR / CAR T-cell.

[0063] In one embodiment, a nucleic acid comprising a CIR / CAR nucleic acid described herein. In another embodiment, a CAR targeted to CD4, CD8, CD14, CD18, CD19, CD1D, CD20, CD33, BCMA, or MUC1. In another embodiment, the CAR contains at least one signaling domain, in a non-limiting example, the CAR signaling domain is CD28, CD3z or 4- 1BB, OX-40, CD27, CD30, GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein), herpes virus entry mediator (HVEM), ICOS (inducible T cell co stimulator).

[0064] As mentioned above, and as is known in the art, when designing a CIR / CAR nucleic acid for proper expression, the nucleic acid may encode molecules for detection, isolation, cleavage, integration in the genome, transmembrane or surface localization, secretion, endoplasmic retention, and others as found necessary for the proper expression of the CIR / CAR. In an aspect, the nucleic acid encodes a molecule for detection and isolation of cells transformed with the nucleic acid. In a non-limiting example, an inert receptor EGFRt, a truncated form of EGFR is used in the detection / isolate or depletion of CARs. Other detection / isolation molecules will be evident to those with ordinary skill in the art. In another aspect, a nucleic acid encoding a self-cleaving peptide inserted into the nucleic acid so that the CIR domain is cleaved from the CAR domain after translation. In a non-limiting example, the self-cleaving peptide is a 2A peptide which can induce ribosomal skipping during translation of the protein. Examples of 2A peptides include P2A, T2A, F2A. In another aspect, the nucleic acid encodes a signal sequence for surface expression of the peptide. In a non-limiting example, the signal sequence is a CD8a signal sequence and / or B2M (beta-2-microglobulin) signal sequence, a GM-CSFR signalsequence for surface localization. In yet another aspect, the nucleic acid may be inserted into a retroviral vector or a lenti viral vector by including viral 5’ LTR (long terminal repeat) and 3’ LTR as promoter and terminator regions for transcription and integration into the host cell genome. Other sequences and methods for integration and transcription of the recombinant CAR / CIR nucleic acid sequence into the cell genome, such as CRISPR technology, are known in the art. It is evident that the position of the CIR and CAR domains in the nucleic acid can be interchanged, or the CIR can be positioned upstream or downstream of the nucleic acid encoding the CAR domain.

[0065] In one embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-interferon-gamma scFv for recognizing the ligand IFNy, and an inhibitory signal from TIGIT comprising the transmembrane and intracellular signaling domain of TIGIT receptor; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.28z antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid is identified in SEQ ID NO: 13, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:25 or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0066] In yet another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-interferon-gamma scFv, and an inhibitory signal from TIGIT comprising the transmembrane and intracellular signaling domain of TIGIT receptor; and encoding a CAR domain targeted to CD19 with a CD19.BBz stimulatory domain and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO: 14, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:26, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0067] In still another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-interferon-gamma scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.28z antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO: 15, and the encoded an amino acid sequence is set forth in SEQ IDNO:27 or a sequence having at least about: 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0068] In another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-interferon-gamma scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD19 with a stimulatory CD19.BBz antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO: 16, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence is set forth in SEQ IDNO:28 or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0069] In another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-IL-6 scFv, and an inhibitory signal from TIGIT; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.29z antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO: 17, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence is set forth in SEQ IDNO:29, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0070] In yet another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-IL-6 scFv, and an inhibitory signal from TIGIT; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.BBz antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO: 18, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:30, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0071] In another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-IL-6 scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.28z antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO: 19, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:31,or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0072] In yet another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-IL-6 scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD 19 with a stimulatory CD19.BBz antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO:20, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:32, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0073] In yet another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-MCPl scFv, and an inhibitory signal from TIGIT; and encoding a CAR domain targeted to CD19 with a stimulatory CD19.28z antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO:21, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO: 33 or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0074] In another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-MCPl scFv, and an inhibitory signal from TIGIT; and encoding a CAR domain targeted to CD19 with a stimulatory CD19.BBz antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO:22, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:34, or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0075] In another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-MCP-1 scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD19 with a stimulatory CD19.28z antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO:23, or a sequence with at least70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:35 or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0076] In yet another embodiment, a CIR / CAR nucleic acid encoding a CIR domain comprising an anti-MCP-1 scFv, and an inhibitory signal from PD-1; and encoding a CAR domain targeted to CD19 with a stimulatory CD19.41BBz antigen and a CD3z costimulatory domain. In one aspect, a nucleic acid identified in SEQ ID NO:24, or a sequence with at least 70%, 75%, 80%, 85%, 90%, 95% homology to said sequence; an amino acid sequence set forth in SEQ IDNO:36 or a sequence having at least about: 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to said sequence.

[0077] In one embodiment, a polynucleotide encoding a CIR or CIR / CAR, is introduced and expressed in an immune effector cell using routine methods and readily available reagents. In some embodiments, a polynucleotide encoding a CIR or CIR / CAR is synthesized by conventional techniques, including automated DNA synthesizers. Alternatively, PCR amplification of nucleic acid fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive nucleic acid fragments that can subsequently be annealed and re-amplified to generate a chimeric nucleic acid sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992).

[0078] In one embodiment, a vector comprising one or more of the nucleic acids described herein. In some embodiments, a polynucleotide or a vector encoding a CIR or CIR / CAR is introduced into a cell (e.g., an immune effector cell or a mammalian cell) using a physical or chemical method, for example, by transfection, transformation, or transduction. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray E. J. (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)); diethylaminocthyl (DEAE)-dextran; electroporation; cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-77 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-34 (1987)). Phage or viral vectorscan be introduced into cells, after growth of infectious particles in suitable packaging cells, many of which are commercially available.

[0079] In another embodiment, a retrovirus is used to deliver a polynucleotide encoding a CIR or a CIR / CAR into a cell (e.g., an immune effector cell or a mammalian cell). Retroviruses are a common tool for gene delivery (Miller, 2000, Nature 357: 455-60). Non-limiting examples of retroviruses suitable for use in particular embodiments include Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV), Rous Sarcoma Virus (RSV), and lentivirus. Non-limiting examples of lentiviruses include human immunodeficiency virus (e.g., HIV type 1 and HIV type 2), visna-macdi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0080] In one embodiment, a polynucleotide encoding an CIR or a CIR / CAR is integrated into the genome of a cell (e.g., an immune effector cell or a mammalian cell). In some embodiments, a polynucleotide encoding an CIR or a CIR / CAR is inserted into a gene. In some embodiments, a polynucleotide encoding CIR or CIR / CAR is extrachromosomal in a cell (e.g., an immune effector cell or a mammalian cell).

[0081] In another embodiment, a mammalian cell or an immune effector cell expresses a CIR or a CIR / CAR disclosed herein. In some embodiments, a mammalian cell or immune effector cell expressing a CAR domain is transduced with a nucleic acid encoding a CIR resulting in a CIR / CAR cell as described herein. In one embodiment, the immune effector cell is a CIR / CAR T-cell.

[0082] In one embodiment, the level of a CIR or a CIR / CAR in an immune effector cell or a mammalian cell is conditionally (e.g., tunable, reversible, spatial control, and / or temporal control) regulatable (e.g., inducible or suppressible), for example, at the level of transcription, protein synthesis, protein degradation, or a combination thereof. In some embodiments, the level of a CIR or a CIR / CAR in an immune effector cell or a mammalian cell is conditionally inducible. In some embodiments, the level of a CIR or CIR / CAR in an immune effector cell or a mammalian cell is conditionally suppressible. In some embodiments, the level of a CIR orCIR / CAR in an immune effector cell or a mammalian cell is conditionally regulated by controlling gene expression (e.g., reversibly or irreversibly).

[0083] In another embodiment, the level (e.g., transcription level) of a CIR or a CIR / CAR in an immune effector cell or a mammalian cell is conditionally regulatable (e.g., inducible or suppressible) by a small molecule, a light-switchable system, a ligand-switchable system, or a combination thereof.

[0084] In one embodiment, the level (e.g., transcription level) of a CIR or CIR / CAR in an immune effector cell or a mammalian cell is conditionally regulatable (e.g., inducible or suppressible) by a small molecule. Non-limiting examples of small molecules include tetracycline (TET), doxycycline (DOX), caffeine, 4-hydroxytamoxifen, estrogen, ecdysone, abscisic acid, mifepristone, xylose, FKBP12-rapamycin, and HCV NS3 / 4A protease inhibitors. In some embodiments, a small molecule comprises TET or DOX.

[0085] In another embodiments, the level (e.g., transcription level) a CIR or CIR / CAR in an immune effector cell or a mammalian cell is conditionally regulatable (e.g., inducible or suppressible) by a light-switchable system. In some embodiments, a light-switchable system responds to light in the range of about 450-700 nm, for example, in the range of about 450-500 nm (e.g., about 450 nm) or about 620-700 nm (e.g., about 660 nm). In some embodiments, an immune effector cell comprises: (a) a tetracycline operator (tetO) motif operably linked to a nucleic acid encoding a CIR or CAR / CIR, (b) a phytochrome-interacting factor 6 (PIF6) sequence (e.g., amino acids 1-100 of PIF6 from Arabidopsis lhaliana) fused to tetracycline repressor (TetR), and (c) A. thaliana red- and far-red light receptor phytochrome B (PhyB) (e.g., amino acids 1-650 of PhyB) linked to VP16 transactivation domain and a nuclear localization sequence (NLS).

[0086] In another embodiment, an immune effector cell comprises a transcriptional control element responsive to a customizable zinc finger transcription factor operably linked to a nucleic acid encoding a CIR or CIR / CAR. Non-limiting examples of a transcriptional control element include three repeats of a binding site for GI-ZFP1, three to nine repeats (e.g., 3, 6, 7, or 9 repeats) of a binding site for GI-ZFP2, and three repeats of a binding site for GI-ZFP3.

[0087] In some embodiments, the level (e.g., transcription level) of a CIR or CIR / CAR in an immune effector cell or a mammalian cell is conditionally regulatable (e.g., inducible or suppressible) by a ligand- switchable system. In some embodiments, a ligand- switchable systemcomprises a synthetic Notch receptor (synNotch), a modular extracellular sensor, a synthetic intramembrane proteolysis receptor (SNIPR), or a combination thereof. In some embodiments, an immune effector cell comprises: (a) a nucleic acid encoding a binding-triggered transcriptional switch (e.g., synNotch, a modular extracellular sensor, or SNIPR), and (b) a transcriptional control element responsive to the binding-triggered transcriptional switch operably linked to a nucleic acid a CIR or CIR / CAR. See, e.g., published PCT applications WO2016138034, WO2013022739, and W02021061791, the contents of which are incorporated by reference herein in their entirety.

[0088] In some embodiments, an immune effector cell comprises a transcriptional control element responsive to a small molecule described herein operably linked to a nucleic acid encoding a CIR or CIR / CAR.

[0089] Also provided herein are compositions comprising any one or more mammalian cell or immune effector cells disclosed herein.

[0090] In some embodiments, a composition further comprises a pharmaceutically acceptable carrier. In some embodiments, a composition is a pharmaceutical composition.

[0091] In some embodiments, a composition (e.g., a pharmaceutical composition) is formulated in a serum-free (without serum or substantially free of serum) cry opreservation medium. Any suitable cryopreservation media and / or excipients may be used. In some embodiments, a cryopreservation medium comprises 5% DMSO and Dextran 40.

[0092] Also provided herein are kits comprising a container and optionally an instruction for use, wherein the container comprises any one or more compositions of pharmaceutical compositions disclosed herein.

[0093] Also provided herein are methods of treating a disease in a subject in need thereof, said methods comprising administering to the subject an effective dosage of immune effector cells, mammalian cells or pharmaceutical compositions disclosed herein.

[0094] Also provided herein are use of immune effector cells, mammalian cells or pharmaceutical compositions disclosed herein, for the preparation of a medicament for treating cancer in a subject in need thereof.

[0095] Also provided is a method of treating or reducing cytokine toxicity in a subject receiving CAR T-cell therapy, the method comprising administering a therapeutically effective amount of a composition comprising a mammalian cell comprising a nucleic acid encoding aCIR domain described herein to the subject. In another aspect, the mammalian cell is a CIR / CAR T cell comprising a nucleic acid encoding both a CIR domain and a CAR domain.

[0096] In one embodiment, the cytokine toxicity is one of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), immune effector cell- associated hematotoxicity (ICAHT), immune effector cell- associated hemophagocytic lymphohistiocytosis-like syndrome (CAR-HLH), cerebral edema associated with CAR therapy, parkinsonism-like syndrome associated with CAR therapy.

[0097] In one embodiment, the subject is receiving CAR T therapy for treating a disease. In one embodiment, the disease is cancer. Non-limiting examples of cancers include: acute lymphoblastic leukemia (ALL); Acquired immunodeficiency syndrome (AIDS)-related cancer (e.g., Kaposi Sarcoma, AIDS-related lymphoma, primary central nervous system (CNS) lymphoma); acute myeloid leukemia (AML); adrenocortical carcinoma; anal cancer; appendix cancer; basal cell carcinoma of the skin; bile duct cancer; bladder cancer; bone cancer (including Ewing sarcoma, osteosarcoma and malignant fibrous histiocytoma); brain tumors / cancer; breast cancer; Burkitt lymphoma; carcinoid tumor (gastrointestinal); cervical cancer; childhood adrenocortical carcinoma; childhood astrocytoma; childhood atypical CNS teratoid / rhabdoid tumor; childhood bladder cancer; childhood carcinoid tumor; childhood cardiac (heart) tumors; childhood central nervous system embryonal tumors; childhood cervical cancer; childhood chordoma; childhood CNS germ cell tumors (e.g., childhood extracranial germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, testicular cancer); childhood colorectal cancer; childhood craniopharyngioma; childhood embryonal tumors; childhood ependymoma; childhood esophageal cancer; childhood extracranial germ cell tumor; childhood gastric (stomach) cancer; childhood gastrointestinal stromal tumors; childhood germ cell tumor; childhood intraocular melanoma; childhood laryngeal papillomatosis; childhood lung cancer; childhood melanoma; childhood mesothelioma; childhood ovarian cancer; childhood pancreatic cancer; childhood paraganglioma; childhood pheochromocytoma; childhood rhabdomyosarcoma; childhood skin cancer; childhood testicular cancer; childhood vaginal cancer; cholangiocarcinoma; chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); chronic myeloproliferative neoplasms; colorectal cancer; cutaneous T-cell lymphoma (e.g. mycosis fungoides and Sezary syndrome); Ductal carcinoma in situ (DCIS); endometrial cancer (uterine cancer); esophageal cancer; esthesioneuroblastoma; extragonadal germ celltumor; eye (ocular) cancer; fallopian tube cancer; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumors (GIST); germ cell tumors; gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; hepatocellular (liver) cancer; Hodgkin lymphoma; intraocular (eye) melanoma; kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer; leukemia; liver cancer; lung cancer (non-small cell and small cell); lymphoma; male breast cancer; malignant mesothelioma; melanoma; Merkel cell carcinoma; metastatic cancer; metastatic squamous neck cancer with occult primary; midline tract carcinoma with NUT gene changes; mouth cancer; multiple endocrine neoplasia syndromes; multiple myeloma / plasma cell neoplasms; myelodysplastic syndromes (e.g., myelodysplastic / myeloproliferative neoplasms); nasal cavity and paranasal sinus cancer; neuroblastoma; non-Hodgkin lymphoma; oral cancer (e.g., lip and oral cavity cancer, oropharyngeal cancer); osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; pancreatic cancer; pancreatic neuroendocrine tumors (e.g., islet cell tumors); paraganglioma; paranasal sinus and nasal cavity cancer; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pituitary tumor; plasma cell neoplasm / multiple myeloma; pleuropulmonary blastoma; pregnancy and breast cancer; primary peritoneal cancer; prostate cancer; rectal cancer; recurrent cancer; retinoblastoma; salivary gland cancer; sarcoma (e.g. childhood rhabdomyosarcoma, childhood vascular tumors, Ewing sarcoma, Kaposi sarcoma, osteosarcoma (bone cancer), soft tissue sarcoma, uterine sarcoma); skin cancer; small intestine cancer; squamous cell carcinoma of the skin; testicular cancer; throat cancer (e.g. nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer); thymoma and thymic carcinoma; thyroid cancer; transitional cell cancer of the renal pelvis and ureter; urethral cancer; vaginal cancer; vascular tumors; vulvar cancer; and Wilms tumor and other childhood kidney tumors.

[0098] In another embodiment, the subject is receiving CAR T therapy for treating an autoimmune disease. Non-limiting examples of autoimmune diseases include but are not limited to include but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease,cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, crest syndrome, Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia- fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulindependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemacious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis, and other autoimmune diseases.

[0099] In another embodiment, the subject is receiving CAR T therapy to treat inflammatory disorders. Examples of inflammatory disorders include but are not limited to, chronic and acute inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator induced lung injury.

[0100] In some embodiments, a subject is infused with cells disclosed herein (e.g., immune effector cells with CIR or CIR / CAR or mammalian cells with CIR or CIR / CAR disclosed herein). In some embodiments, cells (e.g., immune effector cells or mammalian cells) are autologous. In some embodiments, cells (e.g., immune effector cells or mammalian cells) are syngeneic. In some embodiments, cells (e.g., immune effector cells or mammalian cells) are allogeneic, i.e from a different animal of the same species. In some embodiments, cells (e.g. immune effector or mammalian cells) are xenogeneic, i.e. from an animal or a different species.

[0101] In one embodiment, the administration of the cells described herein may be carried out in any convenient manner known to those of skill in the art. The cells of the presentinvention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like.

[0102] The cells described herein can also be administered using any number of matrices. The present invention utilizes such matrices within the novel context of acting as an artificial lymphoid organ to support, maintain, or modulate the immune system, typically through modulation of T cells. Accordingly, the present invention can utilize those matrix compositions and formulations which have demonstrated utility in tissue engineering. Accordingly, the type of matrix that may be used in the compositions, devices and methods of the invention is virtually limitless and may include both biological and synthetic matrices. In one particular example, the compositions and devices set forth by U.S. Pat. Nos. 5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900; or 5,626,561 are utilized, as such these patents are incorporated herein by reference in their entirety. Matrices comprise features commonly associated with being biocompatible when administered to a mammalian host. Matrices may be formed from natural and / or synthetic materials. The matrices may be non-biodegradable in instances where it is desirable to leave permanent structures or removable structures in the body of an animal, such as an implant; or biodegradable. The matrices may take the form of sponges, implants, tubes, telfa pads, fibers, hollow fibers, lyophilized components, gels, powders, porous compositions, or nanoparticles. In addition, matrices can be designed to allow for sustained release of seeded cells or produced active agent. In certain embodiments, the matrix of the present invention is flexible and elastic, and may be described as a semisolid scaffold that is permeable to substances such as inorganic salts, aqueous fluids and dissolved gaseous agents including oxygen.

[0103] A matrix is used herein as an example of a biocompatible substance. However, the current invention is not limited to matrices and thus, wherever the term matrix or matrices appears these terms should be read to include devices and other substances which allow for cellular retention or cellular traversal, are biocompatible, and are capable of allowing traversal of macromolecules either directly through the substance such that the substance itself is a semi- permeable membrane or used in conjunction with a particular semi-permeable substance.DEFINITIONS

[0104] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. It will be further understood that terms, such as those defined in commonly-used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and / or as otherwise defined herein.

[0105] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0106] Throughout this specification and the claims which follow, unless the context requires otherwise, the term “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of, e.g., a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step. When used herein, the term “comprising” can be substituted with the term “containing” or “including.”

[0107] When introducing elements disclosed herein, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. Further, the one or more elements may be the same or different. For example, unless the context clearly indicates otherwise, “a polypeptide” includes a single polypeptide, and two or more polypeptides.

[0108] As used herein, the term “consisting of’ excludes any element, step, or ingredient not specified in the claim element. When used herein, the term “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0109] Also provided herein are corresponding embodiments for each and every embodiment featuring the term “comprising,” “containing,” “including,” or “having,” wherein those terms are replaced by the term “consisting of’ and / or “consisting essentially of.”

[0110] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where twoelements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and, therefore, satisfy the requirement of the term “and / or.”

[0111] It should be understood that for all numerical bounds describing some parameter in this application, such as “about,” “at least,” “less than,” “fewer than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description “at least 1, 2, 3, 4, or 5” also describes, inter alia, the ranges 1-2, 1- 3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.

[0112] As used herein, the term “about” means within an acceptable error range for a particular value, as determined by one of ordinary skill in the art. Typically, an acceptable error range for a particular value depends, at least in part, on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of +20%, e.g., +10%, +5% or +1% of a given value. It is to be understood that the term “about” can precede any particular value specified herein, except for particular values used in the Exemplification. When “about” precedes a range, as in “90-99.9%,” the term “about” should be read as applying to both given values of the range, such that “about 90-99.9%” means about 90% to about 99.9%.

[0113] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list, and every combination of that list, is a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0114] As used herein, a “polynucleotide” is defined as a plurality of nucleotides and / or nucleotide analogs linked together in a single molecule. In some embodiments, a polynucleotide disclosed herein comprises deoxyribonucleotides. In some embodiments, the polynucleotide comprises ribonucleotides. Non-limiting examples of polynucleotides include single-, double-, or multi-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), DNA-RNA hybrids(e.g., each “T” position may be independently substituted by a “U” or vice versa), or a polymer comprising purine and pyrimidine bases, or other natural, chemically, or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups, modified or substituted sugar or phosphate groups, a polymer of synthetic subunits such as phosphoramidates, or a combination thereof.

[0115] As used herein, the term “encoding” refers to specific sequences of nucleotides in a polynucleotide, such as a DNA (e.g., a cDNA) or an RNA (e.g., an mRNA), to serve as a template for synthesis of a protein having defined sequences of amino acids. Unless otherwise specified, a polynucleotide encoding an amino acid sequence can have any one nucleic acid sequence of all nucleic acid sequences that are degenerate versions of each other and that encode the amino acid sequence.

[0116] A protein or polypeptide disclosed or described herein (e.g., CIR or CIR / CAR) can comprise any suitable L- and / or D-amino acid, for example, common a-amino acids (e.g., alanine, glycine, valine), non-a-amino acids (e.g., B-alanine, 4- aminobutyric acid, 6- aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine). The amino, carboxyl and / or other functional groups on a peptide can be free (e.g., unmodified) or protected with a suitable protecting group. Suitable protecting groups for amino and carboxyl groups, and methods for adding or removing protecting groups are known in the art and are disclosed in, for example, Green and Wuts, “Protecting Groups in Organic Synthesis,” John Wiley and Sons, 1991. The functional groups of a protein, peptide, or polypeptide can also be derivatized (e.g., alkylated) or labeled (e.g., with a detectable label, such as a fluorogen or a hapten) using methods known in the art. A protein or polypeptide disclosed or described herein (e.g., CIR or CIR / CAR) can comprise one or more modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, terminal modifiers (e.g., cyclizing modifications), N-methyl-a-amino group substitution), if desired. In addition, a protein, peptide, or polypeptide can be an analog of a known and / or naturally occurring peptide, for example, a peptide analog having one or more conservative amino acid residue substitutions.

[0117] As used herein, the term “sequence identity” refers to the extent to which two nucleotide sequences have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage. For sequencealignment and comparison, typically one sequence is designated as a reference sequence, to which test sequences are compared. Sequence identity between reference and test sequences is expressed as a percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity. As an example, two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, the test sequence has the same nucleotide residue at 70% of the same positions over the entire length of the reference sequence.

[0118] Alignment of sequences for comparison to achieve maximal levels of identity can be readily performed by a person of ordinary skill in the art using an appropriate alignment method or algorithm. In some instances, alignment can include introduced gaps to provide for the maximal level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. AppL Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology).

[0119] When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. A commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP) available through National Center for Biotechnology Information, National Library of Medicine, of the United States National Institutes of Health (Altschul et al., 1990).

[0120] In some embodiments, an amino acid substitution is a conservative substitution. The term “conservative amino acid substitution(s)” or “conservative substitution(s)” refers to an amino acid substitution having a value of 0 or greater in BLOSUM62.

[0121] As used herein, the term “antigen” refers to any substance that can be recognized by the immune system. “Antigen” broadly encompasses proteins, such as enzymes; peptides,such as polypeptides; carbohydrates, such as polysaccharides; haptens; nucleic acids, such as polynucleotides; and grafts. An antigen can be a self-antigen, an antigen produced by the body under normal conditions or as part of a disorder, a foreign antigen, or a non-self-antigen. Examples of self-antigens include self-antigens associated with cancers.

[0122] As used herein, the term “tumor-associated antigen” or “TAA” refers to a protein or polypeptide antigen that is expressed by a cancer cell (e.g., a tumor cell). For example, a TAA may be one or more surface proteins or polypeptides, nuclear proteins or glycoproteins, or fragments thereof, of a cancer cell (e.g., a tumor cell).

[0123] Cancer cells may be, for example, cells of a solid tumor (e.g., a tumor of the breast, lung, prostate, colon, bladder, ovary, kidney, stomach, colon, rectum, testes, head and / or neck, pancreas, brain, skin) or a hematologic cancer (e.g., leukemia, lymphoma, myeloma). As used herein, the term “cancer” may also refer to non-malignant hyperplasia, neoplasia, or pre- cancerous cells or lesions.

[0124] As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multi- specific antibodies (e.g., bi-specific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi- specific antibodies formed from antibody fragments.

[0125] As used herein, the term “antigen-binding fragment” refers to a portion of a full- length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain). Nonlimiting examples of antibody fragments include, e.g., Fab, Fab’, F(ab’)2, and disulfide- linked Fvs (sdFv, e.g., diabody, triabody or tetrabody), a single-chain variable fragment (scFv, e.g., comprising an antibody heavy chain variable region (Vn region) and an antibody light chainvariable region (VL region) in either orientation), a domain antibody (dAb) consisting of one VH domain or one VL domain, a small modular immunopharmaceutical (SMIP), and rigG.

[0126] In some embodiments, an antigen binding domain is an scFv. In some embodiments, an scFv is derived from a mouse antibody. In some embodiment, the scFv comprises a humanized VH region, a humanized VL region, or both. In some embodiments, an scFv comprises a fully human VH region, a fully human VL region, or both. In some embodiments, a VH region and a VL region of an scFv are linked via a peptide linker. In some embodiments, a peptide linker has stretches of hydrophilic residues, for example, glycine and / or serine to increase flexibility, and glutamate and / or lysine to increase solubility. In some embodiments, an antigen binding domain comprises a heavy-chain antibody (comprising two or more heavy chains but lacking light chain), or an antigen-binding fragment thereof.

[0127] As used herein, the term “human antibody” refers to an antibody that is encoded by an endogenous nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a bovine) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus).

[0128] As used herein, the term “chimeric antibody” refers to an antibody that contains a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species such as a human and a mouse antibody). A non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and the constant domains of a human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.

[0129] As used herein, the term “humanized antibody” refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g.,CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody), e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically, that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Nonlimiting examples of methods for generating humanized antibodies are described herein.

[0130] As used herein, a “vector” is any construct capable of delivering one or more polynucleotide(s) of interest to a host cell when the vector is introduced to the host cell. As used herein, a “vector” may be employed to introduce a nucleic acid sequence or gene into a cell, either in vitro, ex vivo, or in vivo.

[0131] An “expression vector” is capable of delivering and expressing the one or more polynucleotide(s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and / or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.

[0132] A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus), naked DNA orRNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0133] In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are known in the art. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked,” and the uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.

[0134] For expression, the DNA insert comprising an antibody-encoding or polypeptide- encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. The expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0135] As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.

[0136] Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene®; and ptrc99a, pKK223-3, pKK233-3, pDR540,pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

[0137] Non-limiting bacterial promoters suitable for use include the E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.

[0138] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.

[0139] Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals and are known in the art.

[0140] Transcription of DNA encoding an antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0141] For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.

[0142] The polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of thepolypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.

[0143] As used herein, the term “expression vector” refers to a replicable nucleic acid from which one or more proteins can be expressed when the expression vector is transformed into a suitable expression host cell. As used herein, the term “promoter” refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene. As used herein, the term “operably linked” means that the nucleic acid is positioned in the recombinant polynucleotide, e.g., vector, in such a way that enables expression of the nucleic acid under control of the element (e.g., promoter) to which it is linked. As used herein, the term “selectable marker element” is an element that confers a trait suitable for artificial selection. Selectable marker elements can be negative or positive selection markers.

[0144] As used herein, the term “fusion protein” refers to a synthetic, semi-synthetic or recombinant single protein molecule. A fusion protein can comprise all or a portion of two or more different proteins and / or polypeptides that are attached by covalent bonds (e.g., peptide bonds).

[0145] Methods for preparing CAR T cells are known in the art. In certain embodiments, a source of T cells is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, and tumors. In certain embodiments, any number of T cell lines available in the art, may be used. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriatebuffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0146] In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient. Alternatively, T cells can be isolated from umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.

[0147] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19 and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.

[0148] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.

[0149] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrationsof 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0150] T cells can also be frozen after the washing step, which does not require the monocyte-removal step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C. or in liquid nitrogen.

[0151] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0152] In certain embodiments the T cells can be expanded. Through expansion, T cells can be multiplied by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold.

[0153] Following culturing, the T cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or greater before passing the cells to another culture apparatus. More preferably, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The T cell medium may be replaced during the culture of the T cells at any time. Preferably, the T cell medium is replaced about every 2 to 3 days. The T cellsare then harvested from the culture apparatus whereupon the T cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing nucleic acids into the T cell.

[0154] In another embodiment, the method comprises isolating T cells and expanding the T cells. In another embodiment, the invention further comprises cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.

[0155] Another procedure for ex vivo expansion cells is described in U.S. Pat. No. 5,199,942 (incorporated herein by reference). Expansion, such as described in U.S. Pat. No. 5,199,942 can be an alternative or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of T cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding the T cells comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.

[0156] The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0157] The medium used to culture the T cells may include an agent that can co-stimulate the T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. This is because, as demonstrated by the data disclosed herein, a cell isolated by the methods disclosed herein can be expanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold, or more by culturing the electroporated population.

[0158] In one embodiment, the method of expanding the T cells can further comprise isolating the expanded T cells for further applications. In another embodiment, the method ofexpanding can further comprise a subsequent electroporation of the expanded T cells followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid, into the expanded population of T cells, wherein the agent further stimulates the T cell. The agent may stimulate the T cells, such as by stimulating further expansion, effector function, or another T cell function.

[0159] As used herein, the term “ex vivo” refers to methods conducted within or on cells or tissue in an artificial environment outside an organism with minimum alteration of natural conditions. As used herein, the term “in vivo” refers to a method that is conducted within living organisms in their normal, intact state. As used herein, the term “in vitro” method is conducted using components of an organism that have been isolated from its usual biological context.

[0160] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. In some embodiments, a subject is a mammal. In some embodiments, a subject is a non-human mammal. As used herein, the term “subject” includes humans, domestic animals, such as laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e.g., cats, dogs, rabbits, etc.), livestock (e.g., pigs, cattle, sheep, goats, horses, etc.), and non-domestic animals. In some embodiments, a subject is a human. In some embodiments, a subject (e.g., human) is male. In some embodiments a subject (e.g., human) is female. In some embodiments, a subject is an intersex human. In some embodiments, a human subject: has physiological and / or genetic characteristics associated with one or more sexes; has undergone or received medical interventions that affect physiological characteristics associated with one or more sexes; and / or is intersex. In some embodiments, the sex of a subject is undefined, unknown, or unclear. In some embodiments, a subject has two X chromosomes. In some embodiments, a subject has one X chromosome and one Y chromosome. In some embodiments, a subject has two or more X chromosomes. In some embodiments, a subject has one or more X chromosomes. In some embodiments, a subject has one X chromosome. In some embodiments, a subject has one or more Y chromosomes.

[0161] In some embodiments, a subject (e.g., a human) is a child (e.g., birth to 17 years of age). In some embodiments, a subject (e.g., a human) is an adult (18-64 years of age). In some embodiments, a subject (e.g., a human) is an older adult (65 years of age or older). In someembodiments, a subject (e.g., a human) is at least about 1 year of age, for example, at least about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 19, 20, 25, 30, 35, 40, 45, or 50 years of age. In some embodiments, a subject is at least 14 years of age. In some embodiments, a subject is at least 18 years of age.

[0162] The term “a subject in need thereof’ or “a patient in need thereof’ refers to an animal (e.g., a mammal, such as a human), diagnosed with or suspected of having a disease or condition (e.g., a cancer), or one at risk of developing such a condition. Diagnosis may be performed by any method or technique known in the art. One skilled in the art will understand that a subject to be treated according to the present disclosure may have been subjected to standard tests or may have been identified, without examination, as one at risk due to the presence of one or more risk factors associated with the disease or condition.

[0163] As used herein, when referring to an antibody, the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a target- specific antibody. For example, an antibody that specifically binds to a CD 19 molecule may be referred to as a CD 19- specific antibody or an anti-CD19 antibody.

[0164] A “pharmaceutical composition” refers to a formulation of one or more therapeutic agents and a medium generally accepted in the art for delivery of a biologically active agent to subjects, e.g., humans. In some embodiments, a pharmaceutical composition may include one or more pharmaceutically acceptable excipients, diluents, or carriers. In some embodiments, a pharmaceutical composition suitable for use in methods disclosed herein further comprises one or more pharmaceutically acceptable carriers.

[0165] The phrase “pharmaceutically acceptable” means that the substance or composition the phrase modifies is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio.

[0166] “Pharmaceutically acceptable carrier, diluent, or excipient” includes any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer,surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0167] “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some embodiments, the carrier may be a diluent, adjuvant, excipient, or vehicle with which the agent (e.g., polynucleotide) is administered. Such vehicles may be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating, and coloring agents, etc. The concentration of the agent in such pharmaceutical formulation may vary widely, i.e., from less than about 0.5% to at least about 1%, or to as much as 15% or 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight. The concentration will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the mode of administration. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21stEdition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing: 691-1092 (e.g., pages 958-89).

[0168] Non-limiting examples of pharmaceutically acceptable carriers are solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents, and the like that are physiologically compatible, such as salts, buffers, antioxidants, saccharides, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants or emulsifying agents, or combinations thereof.

[0169] Non-limiting examples of buffers are acetic acid, citric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, histidine, boric acid, Tris buffers, HEPPSO, and HEPES.

[0170] “Administering” or “administration,” as used herein, refers to providing a composition (e.g., a pharmaceutical composition) to a subject in need of treatment or prevention. Administering can be performed, for example, once, a plurality of times, and / or over one or more extended periods. Administration includes both direct administration (including selfadministration), and indirect administration (including an act of prescribing a drug or directing a subject to consume an agent). For example, as used herein, one (e.g., a physician) who instructs a subject (e.g., a human patient) to self-administer a pharmaceutical composition, or to have a pharmaceutical composition administered by another and / or who provides a patient with a prescription for a pharmaceutical composition is administering an agent to a subject.

[0171] As used herein, the term “a therapeutically effective amount,” “an effective amount” or “an effective dosage” is an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result (e.g., treatment, healing, inhibition or amelioration of physiological response or condition, etc.). The full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. A therapeutically effective amount may vary according to factors such as disease state, age, sex, and weight of a mammal, mode of administration and the ability of a therapeutic, or combination of therapeutics, to elicit a desired response in an individual.

[0172] An effective amount of a pharmaceutical composition to be administered can be determined by a clinician of ordinary skill using the guidance provided herein and other methods known in the art. Relevant factors include the given agent (e.g., immune effector cells), the pharmaceutical formulation, the route of administration, the type of disease (e.g., cancer), the identity of the subject (e.g., age, sex, weight) or host being treated, and the like. Determining a dosage for a particular agent, subject and disease is well within the abilities of one of skill in the art. Preferably, a dosage does not cause or produces minimal adverse side effects.

[0173] An effective amount can be administered in one or more administrations. By way of example, an effective amount of the compositions disclosed herein is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and / or delay progression of a disease in a patient. As is understood in the art, an effective amount may vary, depending on, inter alia, patient history as well as other factors such as the type (and / or dosage) of antibody used.

[0174] Effective amounts and schedules for administering the compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein, the route of administration, the particular type of compositions disclosed herein used and other drugs being administered to the mammal.

[0175] It can generally be stated that a pharmaceutical composition comprising the modified cells described herein may be administered by cell number per kg of body weight or by body surface area (BSA) expressed most commonly as m2. In an embodiment, a dosage of 104to 109cells / kg body weight, in some instances 105to 106cells / kg body weight, including all integer values within those ranges. Cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including the mammalian cell or immune effector cell with CIR or CIR / CAR thereof, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the mammalian cell or immune effector cell with CIR or CIR / CAR in vivo.

[0176] As used herein, the term “treating,” or its equivalents (e.g., “treatment” or “treat”), refers to the medical management of a subject with the intent to improve, ameliorate, stabilize (i.e., not worsen), prevent or cure a disease, pathological condition, or disorder, such as the particular indications exemplified herein. This term includes active treatment (treatmentdirected to improve the disease, pathological condition, or disorder), causal treatment (treatment directed to the cause of the associated disease, pathological condition, or disorder), palliative treatment (treatment designed for the relief of symptoms), preventative treatment (treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder); and supportive treatment (treatment employed to supplement another therapy). Treatment also includes diminishment of the extent of a disease or condition (e.g., a cancer such as a solid tumor); preventing spread of the disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0177] As used herein, the term “ameliorating” or “palliating” a disease or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.

[0178] As used herein, the term “cancer” refers to cells having the capacity for autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Also included are malignancies of the various organ systems, such as respiratory, cardiovascular, renal, reproductive, hematological, neurological, hepatic, gastrointestinal, and endocrine systems; as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, and cancer of the small intestine. Cancer that is “naturally arising” includes any cancer that is not experimentally induced by implantation of cancer cells into a subject, and includes, for example, spontaneously arising cancer, cancer caused by exposure of a patient to a carcinogen(s), cancer resulting from insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancer caused by infections, e.g., viral infections. The term “carcinoma” isart recognized and refers to malignancies of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation. The term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin. A hematopoietic neoplastic disorder can arise from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.

[0179] In any of the methods described herein, the pharmaceutical composition (e.g., any of the mammalian or immune effector cells comprising CIR or CIR / CAR, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, the mammalian cell or immune effector cell with CIR or CIR / CAR and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, the at least one mammalian cell or immune effector cell with CIR or CIR / CAR and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least the mammalian cell or immune effector cell with CIR or CIR / CAR and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, the at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.

[0180] In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one the mammalian cell or immune effector cell with CIR or CIR / CAR, or pharmaceutical composition (e.g., any of the mammalian cell or immune effector cell with CIR or CIR / CAR, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and the at least one the mammalian cell or immune effector cell with CIR or CIR / CAR, or pharmaceutical composition (e.g., any of the mammalian cell or immune effector cell with CIR or CIR / CAR, or pharmaceutical compositions described herein) are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the atleast one the mammalian cell or immune effector cell with CIR or CIR / CAR (e.g., any of the mammalian cell or immune effector cell with CIR or CIR / CAR described herein) in the subject.

[0181] In some embodiments, the subject can be administered the at least one the mammalian cell or immune effector cell with CIR or CIR / CAR, or pharmaceutical composition (e.g., any of the mammalian cell or immune effector cell with CIR or CIR / CAR, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of a disease, e.g., immuno-dysfunction). As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of the mammalian cell or immune effector cell with CIR or CIR / CAR (and / or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least the mammalian cell or immune effector cell with CIR or CIR / CAR (and / or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art).

[0182] Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the mammalian cell or immune effector cell with CIR or CIR / CAR described herein. Two or more (e.g., two, three, or four) of any of the mammalian cell or immune effector cell with CIR or CIR / CAR described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

[0183] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions can include a sterile diluent (e.g., sterile water or saline), a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates,citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations), proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza® Corporation and Nova Pharmaceutical, Inc.).

[0184] Compositions containing the mammalian cell or immune effector cell with CIR or CIR / CAR described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, intratumoral, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage).

[0185] Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). One can, for example, determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population): the therapeutic index being the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0186] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of the one or more (e.g., one, two, three, or four) the mammalian cell or immune effector cell with CIR or CIR / CAR (e.g., any of the mammalian cell or immune effector cell with CIR or CIR / CAR described herein) will be an amount that treats the disease in a subject in a subject, or a subject identified as being at risk of developing the disease, decreases the severity, frequency, and / or duration of one or more symptoms of a disease in a subject (e.g., a human). The effectiveness and dosing of any of the mammalian cell or immuneeffector cell with CIR or CIR / CAR described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human). Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases).

[0187] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the mammalian cell or immune effector cell with CIR or CIR / CAR thereof for various uses as described herein.EXAMPLES

[0188] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0189] The following Materials and Methods were used in the examples below.CAR / CIR Construct Design, Synthesis, and Cloning.

[0190] An anti-interferon-gamma single-chain variable fragment (scFv) followed by the transmembrane and intracellular domains of TIGIT (T-cell immunoreceptor with Ig and ITIM domains) were arranged in sequence with the truncated epidermal growth factor receptor (EGFRt) receptor followed by the CD 19 FMC63 CAR (containing either CD28 or 4- IBB costimulatory domain) with intervening Thosea asigna virus 2A cleavage domains. The entire construct was produced by commercial gene synthesis and subsequently cloned into a retroviral backbone.Production of CAR-T cells.

[0191] Retrovirus particles were produced by transfecting 293GP packaging cells with the gene construct using Lipofectamine™ 2000. Retrovirus containing supernatants were then collected and used for transducing human T cells. Prior to transduction with retrovirus, isolated CD3+human T cells were activated for 2-3 days using CD3 / CD28 Dynabeads™ at a 3:1 bead:cell ratio. Next, activated T cells were exposed to retronectin-bound retrovirus on twoconsecutive days. Following exposure to retrovirus Dynabeads™ were magnetically removed and transduced T cells were cultured and expanded in recombinant human IL-2. Transduction efficiency of T cells with CAR construct was assessed by staining with biotinylated CD19-Fc protein or biotinylated EGFR followed by secondary staining with fluorochrome-conjugated streptavidin.Immunophenotyping analysis of CAR-T cells by flow cytometry.

[0192] CAR-T cells were co-cultured with CD19+NALM-6 tumor cells at a 1:1 ratio for 18-22 hours. CAR-T cell immunophenotyping analysis was then performed. CAR-T cells were stained with antibodies against CD3, CD4, CD8, and EGFR. CAR-T cell activation state was measured by staining for CD69, CD25, CD38, PD-1, and CD107a. Dead cell exclusion was performed using Zombie Aqua™ Fixable Viability dye. Data was acquired on a NovoCyte® 3005 flow cytometer (Agilent), and subsequent analysis was performed using FlowJo™ vlO.Cytotoxicity assays and serial restimulation.

[0193] Cytotoxicity was evaluated using a luciferase-based assay measuring bioluminescence. NALM-6 tumor cells expressing GFP / firefly luciferase were co-incubated with non-transduced T cells, CD19.28z, CD19.28z.CIR, CD19.BBz, or CD19.BBz.CIR CAR-T cells for 18 hours at effector-to-target (E:T) ratios ranging from 10:1 to 1.25:1. Tumor cell lysis was determined by measuring residual luciferase activity and calculated as follows: percent lysis=100-(((average signal from T cell treated tumor wells) / ( average signal from untreated target tumor wells))xl00). For serial restimulation with tumor cells, CAR-T cells were initially co-cultured with NALM-6 cells at a 1:1 ratio and then restimulated with fresh tumor cells very 2- 3 days.Evaluation of CAR-T cell cytokine and chemokine production.

[0194] CAR-T cells were co-cultured with NALM-6 tumor cells at a 1:1 ratio for 18-22 hours. Cocultures were incubated for 48 hours and supernatants were collected for cytokine and chemokine analyses by Luminex®.Single-cell secretome analysis for polyfunctionality.

[0195] CAR-T cells were co-cultured with NALM-6 tumor cells at a 2:1 ratio for 18-22 hours. Following incubation, CAR-T cells were collected and separated from tumor cells by negative selection using a Pan T cell Isolation Kit (Miltenyi). CAR-T cells were then subjected to Isolight® platform analysis (Bruker©) using the Human Adaptive Immune Panel. CAR-T cells were loaded into an IsoCode Chip and incubated overnight.In vitro Cytokine Release Assay.

[0196] To mimic cell types present during human cytokine release syndrome (CRS), we utilized a coculture assay that consists of CAR-T cells, NALM-6 tumor cells, and either macrophages or dendritic cells (DC) differentiated from autologous CD14+monocytes. Cocultures were incubated for 48 hours and supernatants were collected for cytokine and chemokine analyses by Luminex®.Nanostring® nCounter gene expression analysis of CAR-T cells.

[0197] CAR-T cells were co-cultured with NALM-6 tumor cells at a 2:1 ratio for 18-22 hours. Following incubation, CAR-T cells were collected and separated from tumor cells by positive selection using an anti-EGFR biotin antibody and anti-biotin microbeads (Miltenyi). CAR-T cells were then subjected to nCounter platform analysis (Nanostring®) using the CAR-T Characterization Panel. Gene expression data was normalized and analyzed using the ROSALIND online platform.In vivo animal studies.

[0198] NOD-scid IL2Rynu11(NSG) mice were purchased from The Jackson Laboratory. Mice were housed according to institutional guidelines. Animal experimentation protocols were performed with approval of the University of Virginia Institutional Animal Care and Use Committee. Six- to eight-week-old NSG mice were i.v. injected with 0.5xl06NALM-6 (GFP+ / Luciferase+) tumor cells. 2-3 days later, mice were i.v. injected with 10xl06CD19 CAR or CD19 CAR.CIR (CAR / CIR) T cells. Tumor growth and progression was monitored using weekly bioluminescent imaging. Bioluminescent tumor data was obtained on a LagoX (Spectral Instruments Imaging) instrument following intraperitoneal injection of D-luciferin (Revvity).Data and Statistical Analysis.

[0199] All data and statistical analyses were completed using GraphPad Prism. Tests used for determining statistical significance are indicated in individual figure legends.EXAMPLE 1: Model of chimeric inhibitory receptor (CIR).

[0200] Severe cytokine release syndrome (sCRS) correlates with rapid CAR-T cell expansion accompanied by a robust and multi-dimensional proinflammatory state. To mitigate sCRS we will engineer and deploy a chimeric inhibitory receptor (CIR), co-expressed with a CAR, that will redirect CRS-associated inflammatory cytokines to inhibitory signals that regulate CAR-T and, subsequently, myeloid cell activity. As cytokine production increases after CAR-T cell activation, CIR activity will attenuate the supraphysiologic immune response responsible for sCRS yet allow continued anti-tumor activity (Figure 2 and 3). The CIR should: (a) be specifically activated by increased levels of a proinflammatory cytokine; (b) dampen overall CAR-T cell activation to blunt inflammatory cytokine production and reduce myeloid cell activation; yet (c) retain CAR-T cell anti-tumor functions.EXAMPLE 2: Normal expansion and proliferation of CD19.28z.CIR CAR-T cells coexpressing CIR domain during in vitro manufacture

[0201] To determine that the presence of a CIR domain does not affect the posttransduction proliferation of CAR-T cells, cell counts of transduced (EGFRt+) cells were monitored during the in vitro expansion phase of CAR-T cell production. Following transduction of activated human T cells with CD 19 CAR co-expressing a CIR domain, cells were expanded in vitro in AIM V™ media (Gibco™) containing recombinant human interleukin-2 (rhIL-2) (300 units / ml). Culture media and rhIL-2 was refreshed every 2-3 days up to day 14 post-activation. Proliferation of transduced CD19.28z and CD19.28z.CIR CAR-T cells (EGFRt+) was comparable with both cell types expanding in number at a similar rate (Figure 4A). The fold expansion of transduced T cells for both cell types was approximately 30-fold by day 14 postactivation relative to the number of transduced cells at day 7 post-activation (Figure 4B). These results confirm that CD 19 CAR-T cells containing a CIR domain undergo normal proliferation during the in vitro expansion phase of CAR-T cell production.EXAMPLE 3: CD19.28z.CIR CAR-T cells exhibit attenuated activation following exposure to NALM-6 CD19+tumor cells.

[0202] The presence of a CIR containing an inhibitory receptor signaling domain is expected to lead to overall attenuation in the activation level of CAR-T cells co-expressing the CIR when CAR-T cells are undergoing stimulation by target expressing tumor cells. To evaluate the ability of the CIR to attenuate activation in CAR-T cells, CD19.28z or CD19.28z.CIR CAR- T cells were exposed to CD19+NALM-6 leukemia cells in vitro at a 1:1 effector cell to target cell ratio for 24 hours. CAR-T cells were then stained and labeled with fluorescent antibodies against T cell surface markers to identify CD4 and CD8 subsets as well as multiple known markers of activation including CD69, CD25, CD38, PD-1, and CD107a. Labeled cells were then analyzed on a flow cytometer. Representative flow cytometry plots for CD69 and CD38 on CD4+and CD8+CAR-T cells are shown for non-transduced (NT) T cells, CD19.28z CAR-T cells, and CD19.28z.CIR CAR-T cells either stimulated with NALM-6 cells or left unstimulated (Figure 5A). The overall levels of activation for CAR-T cells were then evaluated by calculating the percentage of CD4+and CD8+CAR-T cells expressing a specified number of activation markers, ranging from 0 to 5 markers, with 5 markers indicating the most highly activated cells. As shown in the representative bar graphs of the percentage of CAR-T cells expressing the indicated number of activation markers, CIR-expressing CAR-T cells exhibited significantly reduced percentages of cells expressing 4 and 5 activation markers and significantly increased percentages of cells expressing only 1 and 2 activation markers (Figure 5B). These data demonstrate a reduction and attenuation in the overall activation state of CD19.28z CAR-T cells expressing the CIR domain. These results are representative of at least 5 individual experiments using CAR-T cells generated from 3 different human donors.EXAMPLE 4: In vitro cytotoxicity of CD19.28z CAR-T cells expressing CIR receptor against CD19+leukemia cell line NALM-6.

[0203] To determine that the presence of a CIR domain does not adversely affect the anti-tumor activity of CAR-T cells in eliminating tumor cells, standard in vitro cytotoxicity assays were performed. To evaluate the capacity of CIR-expressing CAR-T cells to effectively kill tumor cells, CD19.28z or CD19.28z.CIR CAR-T cells were exposed to CD19+NALM-6 leukemia cells in vitro at various effector cell to target cell ratios (ranging from 10:1 to 1.25:1)for 24 hours. Control non-transduced T cells exhibited little to no cytotoxicity against tumor cells, whereas, both CD19.28z and CD19.28z.CIR CAR-T cells exhibited equivalent cytotoxicity (Figure 6). Thus, although activation levels of CD19.28z.CIR CAR-T cells were attenuated relative to standard CD19.28z CAR-T cells, the CAR-T cells co-expressing a CIR domain displayed similar anti-tumor activity.EXAMPLE 5: CD19.28z.CIR CAR-T cells exhibit attenuated cytotoxicity kinetics which is dependent upon CIR-mediated binding to IFN-gamma.

[0204] CD 19 CAR-T cells expressing a CIR domain show equal tumor cell cytotoxicity during short-term exposure to tumor cells. However, prolonged exposure to tumor cells through repetitive tumor cell challenges would lead to the increasing and / or continual presence of interferon-gamma (IFN-y). Thus, it is expected that this would result in an increase in CIR- mediated sensing of IFN-y through the CIR scFv, which in turn may lead to effects on CD 19 CAR-T cell anti-tumor activity. These effects could be beneficial in that CD 19 CAR-T cell antitumor activity is attenuated but not abrogated, resulting in a more tempered CD 19 CAR-T cell response with reduced cytokine release and toxicity. Alternatively, repeated and prolonged signaling through the CIR could result in a substantial loss of anti-tumor activity and control of tumor cell growth.

[0205] To evaluate the effect of repeated exposure to tumor cells, CD19.28z or CD19.28z.CIR CAR-T cells were cultured with NALM-6 tumor cells for three consecutive challenges and the presence of tumor cells was measured at various timepoints over the course of 96 hours. As expected, both CAR-T cell groups exerted equal cytotoxicity after the first challenge. Upon the second challenge, CD19.28z.CIR CAR-T cells exhibited a delay in tumor cell killing relative to standard CD19.28z CARs, however, CD19.28z.CIR CAR-T cells eventually reached the maximum level of cytotoxicity (Figure 7A). Upon the third tumor cell challenge, CD19.28z.CIR CAR-T cells once again showed attenuated cytotoxicity, however the levels of cytotoxicity were equivalent at the end of the time course. Thus, the presence of a CIR domain results in attenuated cytotoxicity kinetics of CD19.28z CAR-T cells.

[0206] To demonstrate that the attenuation of cytotoxic activity from the CIR is dependent upon recognition of IFN-y by the CIR scFv-binding domain, a CIR construct lacking the anti-IFN-y scFv (scFv-less CIR) was utilized in an experimental setup as in Figure 7A.CD19.28z, CD19.28z.CIR, and CD19.28z.CIR (scFv-less) CAR-T cells were subjected to multiple tumor cell challenges. As above, CD19.28z.CIR CAR-T cells showed attenuated cytotoxicity kinetics. However, CD19.28z.CIR (scFv-less) CAR-T cells which lack to ability to recognize IFN-y through the CIR domain, did not exhibit an attenuation of cytotoxicity kinetics and were equivalent to standard CD19.28z CAR-T cells (Figure 7B). This result demonstrates that IFN-y binding by the CIR is required for attenuated cytotoxicity of CD19.28z CAR-T cells co-expressing a CIR domain.

[0207] To confirm the above results through an additional experimental approach, nontransduced T cells, CD19.28z CAR-T cells, and CD19.28z.CIR CAR-T cells were first exposed in vitro to recombinant human IFN-y (0.05ng / ml-50ng / ml) for 24 hours. T cells were then cultured with target tumor cells to evaluate cytotoxicity. After 6 hours, cytotoxicity of CD19.28z.CIR CAR-T cells was substantially blunted relative to standard CD19.28z CAR-T cells in an IFN-y dose-dependent manner (Figure 7C, left). After 20 hours, cytotoxicity was equivalent between the groups of CAR-T cells, highlighting that attenuation by IFN-y though the CIR is not a permanent, irreversible effect (Figure 7C, right). In conclusion, the blunting of cytotoxicity in CD19.28z.CIR CAR-T cells by pre-exposure to IFN-y adds further evidence of the responsiveness and signaling of the CIR domain in CAR-T cells.EXAMPLE 6: CIR signaling in CD19.28z CAR-T cells results in attenuated production of cytokines and chemokines.

[0208] Highly activated CAR-T cells contribute to severe cytokine release syndrome through the secretion of copious amounts of pro-inflammatory cytokines and chemokines leading to systemic immune activation. Attenuation of CAR-T cell activity via the expression of a CIR domain should result in diminished production of pro-inflammatory mediators by the CAR-T cell, thus leading to a mitigation of systemic immune activation. To confirm that the presence of a CIR domain attenuates pro-inflammatory cytokine / chemokine secretion by CAR-T cells, CD19.28z and CD19.28z.CIR CAR-T cells were stimulated in vitro by NALM-6 tumor cells for 24 hours, cell-free culture supernatants were collected, and subsequently analyzed by Luminex® to measure the levels of specific cytokines and chemokines. In general, CD19.28z.CIR CAR-T cells tended to secrete lower levels of several proinflammatory cytokines including IFN-y, IL-2, TNF-CX, IL-17A and IL-4 relative to standard CD19.28z CAR-T cells, while the secretion of anti-inflammatory cytokines like IL- 13 and IL- 10 were unchanged (Figure 8A). A more comprehensive evaluation of additional cytokines and chemokines showed a strong trend towards a decrease in the secretion of effector, stimulatory, and inflammatory mediators by CD19.28z.CIR CAR-T cells relative to standard CD19.28z CARs (Figure 8B). These results provide strong evidence demonstrating a functional attenuation of CD19.28z CAR-T cells when a CIR domain is present.EXAMPLE 7: Overall polyfunctionality of CD19.28z CAR-T cells co-expressing CIR is not compromised by CIR signaling.

[0209] A deeper and more advanced analysis of the functional characteristics of CD19.28z.CIR CAR-T cells was performed to evaluate the overall polyfunctionality of cells responding to target tumor cells. Polyfunctional CAR-T cells are CAR-T cells that secrete two or more cytokines or chemokines. Polyfunctional CAR-T cells are associated with favorable clinical outcomes and successful CAR-T cell expansion following patient infusion. Poly functionality, specifically the polyfunctionality strength index (PSI), in CAR-T cells is a measure of the percent of polyfunctional cells and the mean level of proteins secreted. Thus, CAR-T cells with a higher PSI are associated with improved clinical outcomes. To determine that the presence of a CIR domain does not adversely affect the overall polyfunctionality of CAR-T cells, single-cell secretome analysis for measuring polyfunctional cells was performed. CD19.28z and CD19.28z.CIR CAR-T cells were stimulated in vitro by NALM-6 tumor cells for 22 hours. CAR-T cells were removed from tumor cells and analyzed using the Isolight® platform utilizing the Human Adaptive Immune Cytokine Panel (32+ cytokine analysis) chip. Analysis of the single-cell secretome of CAR-T cells demonstrated that the overall polyfunctionality of CD19.28z.CIR CAR-T cells co-expressing a CIR is not compromised by CIR signaling, as these cells exhibited a similar PSI relative to CD19.28z CAR-T cells. Thus, despite an attenuation in activation state and diminished secretion of certain proinflammatory mediators, CD19.28z.CIR CAR-T cells retain a normal overall polyfunctional phenotype.EXAMPLE 8: CD19.28z CAR-T cells attenuated by CIR signaling results in diminished pro-inflammatory cytokine / chemokine secretion by bystander autologous macrophages and DCs.

[0210] To validate that attenuation of CD19.28z CAR-T cells by CIR-mediated signaling leads to down-stream mitigation of immune activation, an in vitro co-culture system consisting of bystander myeloid immune cells, which are significant contributors to severe cytokine release syndrome, was utilized. Importantly, this system more closely recapitulates cytokine release syndrome, as it contains the pertinent cell types involved in its development and leads to the production of many severe CRS- associated cytokines, including IFN-y. Thus, this provides a more physiological manner of evaluating the attenuation of CD19 CAR-T cells expressing a CIR domain. In this assay a three-party co-culture system consisting of either CD19.28z CAR or CD19.28z.CIR CAR-T cells, NALM-6 tumor cells, and autologous monocytes, either monocyte- derived dendritic cells (MoDC) or macrophages (MoMac) is established (Figure 10A). Cocultures were established, incubated for 48 hours, and cell-free supernatants were collected for Luminex® analysis using a Human 42-plex panel. The secretion of myeloid cell-derived IL-6, IL-12p70, MIP-la, and IL-8, cytokines that are substantially increased in CAR-T cell patients undergoing sCRS, were diminished in cultures containing CIR-regulated CD19.28z CAR-T cells (Figure 10B). A comprehensive evaluation of additional cytokines and chemokines showed a decrease in the secretion of multiple effector, stimulatory, chemoattractive, and inflammatory mediators in cultures where CD 19.28. CIR CAR-T cells were present relative to standard CD19.28z CARs (Figure 10C). These results provide evidence demonstrating that functional attenuation of CD19.28z CAR-T cells via a CIR domain, leads to a reduction in the activation and secretion of proinflammatory mediators by immune myeloid cells.EXAMPLE 9: Nanostring® nCounter gene expression profiling demonstrates significant regulation of CAR-T cell pathways by the presence of CIR signaling.

[0211] Gene expression characterization of CD19.28z CAR-T cells was performed to evaluate and confirm the observed phenotypic and functional changes introduced by the expression of a CIR domain. Profiling of gene expression was performed on resting unstimulated CAR-T cells and CAR-T cells exposed to NALM-6 tumor cells. Following stimulation, CAR-T cells were removed from tumor cells via isolation of EGFRt+(CAR+) T cells and magneticmicrobead separation. Nanostring® nCounter gene expression profiling of isolated RNA was performed using the CAR-T Characterization Panel (780 target genes). Standard quality control and data normalization was completed in the ROSALIND platform followed by individual pathway analysis and differential expression. The overall attenuation of the activation state of CD19.28z CAR-T cells expressing a CIR domain was confirmed as genes in multiple activation pathways were less enriched relative to CD19.28z CARs, including JAK / STAT, MAPK and PI3K Signaling, NFkB, and Costimulatory Molecules pathways following exposure to tumor cells (Figure 11). In particular, the NFAT signaling pathway was markedly less enriched in CD19.28z.CIR CAR-T cells relative to CD19.28z CAR-T cells. Additional pathways that were reduced included NOTCH, TGF-beta, and Wnt Signaling. The presence of the CIR domain in CD19.28z CAR-T cells led to significant metabolic shifts across many gene pathways involved in cellular metabolism. Overall, CD19.28z.CIR CAR-T cells exhibited reductions in these metabolic pathways, potentially a result of the attenuated activation as changes in T cell metabolism are induced naturally following activation. This altered metabolic phenotype may confer enhanced metabolic fitness to CAR-T cells. Lastly, the exhaustion phenotype of CD19.28z.CIR CAR-T cells was unchanged relative to CD19.28z CAR-T cells, however, the presence of the CIR domain reduced the expression of genes related to apoptosis.EXAMPLE 10: CIR-regulated CD19.28z CAR-T cells exhibit similar anti-tumor activity and survival in vivo relative to CD19.28z controls.

[0212] CAR-T cell anti-tumor efficacy and disease control is typically demonstrated using a standard in vivo mouse model where NOD.Cg-Prkdc^1112rgtmlwj1 / SzJ (NSG) mice are injected with NALM-6 leukemia tumor cells followed by administration of CD19 CAR-T cells. In order to confirm that CD19.28z CAR-T cells expressing a CIR domain retain in vivo antitumor efficacy and durability, in vivo experiments were conducted using NSG mice. NALM-6 (GFP+ / Luciferase+) tumor cells were first injected into mice and two days later mice received either non-transduced T cells, CD19.28z CAR-T cells, or CD19.28z.CIR CAR-T cells via intravenous injection (Figure 12A). Weekly live-animal bioluminescent imaging was conducted to monitor tumor burden over the course of the experiment. Tumor burden and disease rapidly increased in mice that were administered control non-transduced T cells and required humane euthanasia by day 13 post-treatment (Figure 12B-D). As expected, tumor burden and diseasewere efficiently controlled and eliminated in mice receiving either CD19.28z or CD19.28z.CIR CAR-T cells, demonstrating equivalent in vivo efficacy of CAR-T cells expressing the CIR domain (Figure 12B-D). Lastly, the ability of CAR-T cells to persist in vivo was evaluated by measuring the frequency of blood-circulating and number of splenic CAR-T cells at the experimental endpoint on day 28 post-treatment (Figure 12E). CD19.28z.CIR CAR-T cells persisted as well or better than CD19.28z CAR-T cells, demonstrating that persistence is not negatively affected by CIR signaling. These results validate the in vivo anti-tumor activity and efficacy of CIR-regulated CD19.28z CAR-T cells.EXAMPLE 11: CIR-mediated attenuation of CD19 CAR-T cell responses is observed in vivo in tumor-bearing mice.

[0213] To formally demonstrate that CIR-mediated attenuation was occurring in vivo in mice treated with CAR-T cells, a separate experiment was performed to evaluate attenuation. Similar to the experimental setup in Figure 12, NALM-6 (GFP+ / Luciferase+) tumor cells were first injected into mice and two days later mice received either non-transduced T cells, CD19.28z CAR-T cells, or CD19.28z.CIR CAR-T cells via intravenous injection (Figure 13A). Anti-tumor activity was confirmed through live-animal bioluminescent imaging conducted on days 0, 1, and 2 post-treatment (Figure 13B). On days 1 and 2 post-treatment, serum was collected from mice through tail-vein blood collection and the levels of IFN-gamma were measured by ELISA. In agreement with previous results in vitro, mice treated with CD19.28z.CIR CAR-T cells had less systemic IFN-gamma compared to mice treated with standard CD19.28z CAR-T cells (Figure 13C). On day 2 post-treatment mice were sacrificed, and spleen and bone marrow were collected to analyze the activation phenotype of CAR-T cells. The spleen and bone marrow of mice treated with CD19.28z.CIR CAR-T cells contained a significantly reduced percentage of activated CD38+CAR-T cells relative to mice treated with CD19.28z CARs (Figure 13D). Further analysis also revealed a lower percentage of activated CAR-T cells double-positive for both CD38 and PD-1 in the bone marrow of mice treated with CD19.28z.CIR CAR-T cells (Figure 13E). These data confinn that CD19.28z CAR-T cell activation can be attenuated in vivo while retaining normal anti-tumor activity.EXAMPLE 12: CD19.BBz CAR / CIR-T cells exhibit attenuated activation following exposure to NALM-6 CD19+tumor cells.

[0214] The presence of a CIR domain leads to overall attenuation in the activation level of CD19.28z CAR-T cells. To evaluate the ability of the CIR to attenuate activation in CAR-T cells with a 4-1BB co-stimulation domain, CD19.BBz or CD19.BBz.CIR CAR-T cells (Schematic shown in Figure 14) were exposed to CD19+NALM-6 leukemia cells in vitro at a 1:1 effector cell to target cell ratio for 24 hours. CAR-T cells were then stained and labeled with fluorescent antibodies against T cell surface markers to identify CD4 and CD8 subsets as well as multiple known markers of activation including CD69, CD25, and PD-1. Labeled cells were then analyzed on a flow cytometer. Representative flow cytometry plots for CD69, PD-1, and CD25 on CD4+and CD8+CAR-T cells are shown for CD19.BBz CAR-T cells, and CD19.BBz.CIR CAR-T cells either stimulated with NALM-6 cells or left unstimulated (Figure 15A). As shown in the representative bar graphs of the percentage of CAR-T cells expressing the indicated activation markers, CIR-expressing CAR-T cells exhibited significantly reduced percentages of cells expressing CD69, PD-1, and CD25 (Figure 15B). These data demonstrate a reduction and attenuation in the overall activation state of CD19.BBz CAR-T cells expressing the CIR domain. These results are representative of at least 3 individual experiments using CAR-T cells generated from 3 different human donors.EXAMPLE 13: In vitro cytotoxicity of CD19.4-lBBz CAR T cells expressing CIR receptor against leukemia cell line NALM-6 demonstrates similar leukemia control.

[0215] To determine that the presence of a CIR domain does not adversely affect the anti-tumor activity of CAR-T cells in eliminating tumor cells, standard in vitro cytotoxicity assays were performed. To evaluate the capacity of CIR-expressing CAR-T cells to effectively kill tumor cells, CD19.BBz or CD19.BBz.CIR CAR-T cells were exposed to CD19+NALM-6 leukemia cells in vitro at various effector cell to target cell ratios (ranging from 10:1 to 1.25:1) for 24 hours. Control non-transduced T cells exhibited little to no cytotoxicity against tumor cells, whereas, both CD19.BBz and CD19.BBz.CIR CAR-T cells exhibited equivalent cytotoxicity (Figure 16). Thus, although activation levels of CD19.BBz.CIR CAR-T cells were attenuated relative to standard CD19.BBz CAR-T cells, the CAR-T cells co-expressing a CIR domain displayed similar anti-tumor activity.EXAMPLE 14: Diminished pro-inflammatory cytokine / chemokine secretion by bystander autologous macrophages and DCs during in vitro CRS-assay.

[0216] To validate that attenuation of CD19.BBz CAR-T cells by CIR-mediated signaling leads to down-stream mitigation of immune activation, an in vitro co-culture system consisting of bystander myeloid immune cells was utilized. Importantly, this system more closely recapitulates cytokine release syndrome, as it contains the pertinent cell types involved in its development and leads to the production of many severe CRS-associated cytokines, including IFN-y. In this assay a three-party co-culture system consisting of either CD19.BBz CAR or CD19.BBz.CIR CAR-T cells, NALM-6 tumor cells, and autologous monocytes, either monocyte-derived dendritic cells (MoDC) or macrophages (MoMac) is established (Figure 17A). Co-cultures were established, incubated for 48 hours, and cell-free supernatants were collected for Luminex® analysis using a Human 42-plex panel. The secretion of myeloid cell- derived IL-6, IL- la, GROa, IL-8, and MIG, cytokines that are increased in CAR-T cell patients undergoing sCRS, were diminished in cultures containing CIR-regulated CD19.BBz CAR-T cells (Figure 17B). These results provide supporting evidence demonstrating that functional attenuation of CD19.BBz CAR-T cells via a CIR domain, leads to a reduction in the activation and secretion of pro-inflammatory mediators by immune myeloid cells.EXAMPLE 15: Self- regulating CD19.BBz CAR-T cells expressing anti-IFN-y / TIGIT CIR exhibit similar anti-tumor activity and survival but improved persistence in vivo

[0217] To confirm that CD19.BBz CAR-T cells expressing a CIR domain retain in vivo anti-tumor efficacy and durability, in vivo experiments were conducted using NSG mice. NALM-6 (GFP+ / Luciferase+) tumor cells were first injected into mice and two days later mice received either non-transduced T cells, CD19.BBz CAR-T cells, or CD19.BBz.CIR CAR-T cells via intravenous injection (Figure 18A). Weekly live-animal bioluminescent imaging was conducted to monitor tumor burden over the course of the experiment. Tumor burden and disease rapidly increased in mice that were administered control non-transduced T cells and required humane euthanasia by day 14 post-treatment (Figure 18B-C). As expected, tumor burden and disease were efficiently controlled in mice receiving either CD19.BBz or CD19.BBz.CIR CAR- T cells, demonstrating equivalent in vivo efficacy of CAR-T cells expressing the CIR domain(Figure 18B-C). Lastly, the ability of CAR-T cells to persist in vivo was evaluated by measuring the frequency of blood-circulating and number of splenic CAR-T cells at the experimental endpoint on day 35 post-treatment (Figure 18D). CD19.BBz.CIR CAR-T cells persisted significantly better than CD19.BBz CAR-T cells, confirming that persistence is not negatively affected by CIR signaling. These results validate the in vivo anti-tumor activity and efficacy of CIR-regulated CD19.BBz CAR-T cells.OTHER EMBODIMENTS

[0218] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A mammalian cell comprising a nucleotide sequence encoding a chimeric inhibitory receptor (CIR) domain, the CIR domain comprising: a ligand- sensing polypeptide able to sense an extracellular ligand, operably linked to an inhibitory signaling molecule, wherein sensing of the ligand by the ligand-sensing polypeptide delivers inhibitory signaling.

2. The mammalian cell of claim 1, wherein the ligand is a soluble mediator of toxicity to the cell.

3. The mammalian cell of any one of claims 1-2, wherein the CIR domain ligandsensing polypeptide is a naturally occurring or modified ligand receptor, an antibody or antigen binding fragment that recognizes the ligand, or a combination thereof.

4. The mammalian cell of claim 4, wherein the CIR domain ligand-sensing polypeptide is an antibody or antigen binding fragment is a single-chain variable fragment (scFv) of an antibody.

5. The mammalian cell of any one of claims 1-4, wherein the ligand is a circulating signal, a hormone, a neurotransmitter, a lipid, a peptide, a cytokine, or a chemokine.

6. The mammalian cell of any one of claims 1-5, wherein the mammalian cell is an immune effector cell.

7. The mammalian cell of claim 6, wherein the immune effector cell is a T cell, a monocyte, a B cell, a natural killer (NK) cell, a mononuclear phagocytic cell, or a dendritic cell.

8. The mammalian cell of claim 7, wherein the immune effector cell is: a) a T cell selected from the group consisting of a helper CD4+T cell, a cytotoxic CD8+T cell, a memory T cell, a regulatory CD4+T cell, an innate-like T cell, a natural killer T cell, a mucosal associated invariant T cell, and a Gamma Delta T cell; b) a monocyte selected from the group consisting of a CD14++CD16 monocyte, a CD14+CD16++ monocyte, and a CD14++CD16+ monocyte; c) a B cell selected from the group consisting of a plasmablast, a plasma cell, a lymphoplasmacytoid cell, a memory B cell, a B-2 cell, a B-l cell, and a regulatory B cell (Breg); d) a NK cell selected from the group consisting of a CD56BRIGHTNK cell and a CD56DMNK cell; or e) a mononuclear phagocytic cell selected from the group consisting of an adipose tissue macrophage, a monocyte, a Kupffer cell, a sinus histiocyte, an alveolar macrophage (dust cell), a tissue macrophage (histiocyte), a microglial cell, a Hofbauer cell, an intraglomerular mesangial cell, an osteoclast, a Langerhans cell, an epithelioid cell, a red pulp macrophage (sinusoidal lining cell), a peritoneal macrophage, a lysomac, and a perivascular macrophage.

9. The mammalian cell of any one of claims 6-8, wherein the immune effector cell is a human immune cell or a humanized immune cell.

10. The mammalian cell of any one of claims 1-9, wherein the ligand is a result of direct or indirect chimeric antigen receptor (CAR) activation.

11. The mammalian cell of any one of claims 1-10, wherein the ligand is any one of IFN-y, IL-6, IL-1 or MCP-1 (monocyte chemoattractant protein 1) or a combination thereof.

12. The mammalian cell of any one of claims 1-11, wherein the CIR inhibitory signaling peptide is from a TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine -based inhibitory motif or ITIM), a PD-1 (programmed cell death protein 1), positive programmed death-ligand 1 (PD-L1), or from a cytotoxic T-lymphocyte associated protein 4 (CTLA-4).

13. The mammalian cell of any one of claims 1-12, wherein the CIR inhibitory signaling peptide is from a TIGIT.

14. The mammalian cell of any one of claims 6-12, wherein the mammalian cell is a T cell that further comprises a nucleic acid encoding a chimeric antigen receptor (CAR) domain, a CIR / CAR T-cell.

15. The mammalian cell of claim 14, wherein the CAR domain comprises a targeting molecule, resulting in a targeted CIR / CAR T-cell, the targeting molecule chosen from the group consisting of a) a native protein; b) an antibody or an antigen -binding fragment thereof, a T cell receptor (TCR), an integrin, or a cell adhesion molecule; c) an integrin selected from the group consisting of integrin beta-1 (ITG01), integrin alpha-5 (ITGa5), lymphocyte function-associated antigen 1 (LFA-1), and integrin subunit alpha V (ITGaV); integrin-alpha v beta 3 (ITGav / ITGb3 or CD51 / CD61); d) a cell adhesion molecule selected from the group consisting of intercellular adhesion molecule- 1 (ICAM-1), vascular cell adhesion protein- 1 (VCAM-1), neural cell adhesion molecule 1 (N-CAM1), and platelet endothelial cell adhesion molecule- 1 (PE-CAM1); e) a patient-derived TCR; f) an engineered protein comprising an antibody or an antigen-binding fragment thereof; g) B-cell activating factor (BAFF) receptor, B-cell maturation antigen (BCMA), CD3, CD5, CD19, CD20, CD22, CD28, CD30, CD38, CD79B; h) AXL, B7 homolog 3 protein (B7-H3), carcinoembryonic antigen (CEA), CD70, claudinl8.2 (CLDN18.2), delta-like ligand 3 (DLL3), disialoganglioside (GD2), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), guanylyl cyclase C (GUCY2C), human epidermal growth factor receptor 2 (HER2), Kita- Kyushu lung cancer antigen- 1 (KK-LC-1), Lewis Y (LEY), mesothelin (MSLN), MUCIN 1 (MUC1), NEW YORK esophageal squamous cell carcinoma 1 (NY-ESO-1), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), receptor-tyrosine-kinase like orphan receptor 1 (R0R1), transforming growthfactor beta (TGF-0), Kirsten rat sarcoma virus (KRAS) G12D, melanoma antigen recognized by T cells 1 (MART-1), melanoma-associated antigen 3 (MAGE-A3), tumor protein p53 (TP53), FMS-like tyrosine kinase 3 (FLT3), or alkaline phosphatase placental-like 2 (ALPPL2); i) LI cell adhesion molecule (LICAM), neurexin 3, vesicular glutamate transporter 1 (VGLUT1), vesicular inhibitory amino acid transporter (VIAAT), neuroligin 1, neuroligin 2, neural cell adhesion molecule 1 (NCAM1), vesicular acetylcholine transporter (VAChT), folate receptor- 1 (FOLR1), gamma-aminobutyric acid B receptor 1 (GABA(b)Rl), GABA(b)R2, glutamate ionotropic receptor NMDA type subunit 1 (GRIN1), GRIN2B, or solute carrier family 6 member 4 (SLC6A4); j) neural / glial antigen 2 (NG2), oligodendrocyte marker 01, oligodendrocyte marker 04, A2B5, or myelin-oligodendrocyte glycoprotein (MOG); k) P2Y 12, macrophage colony-stimulating factor receptor (M-CSFR), or CX3C motif chemokine receptor 1 (CX3CR1); l) CD133 or CD49F; m) stem cell antigen-1 (Sca-1), CD27, CD34, CD38, CD43, CD117, or CD150, A2B5, connexin 43, or aquaporin-4 (AQP-4); or n) CD71 or CD24.

16. The mammalian cell of claim 14 or 15, wherein the targeting molecule of the CIR / CAR T-cell is an anti-CD19 scFv.

17. The mammalian cell of any one of claims 14-16, wherein the CAR domain comprises at least one signaling peptide.

18. The mammalian cell of claim 17, wherein the at least one signaling peptide is CD28, CD3z, or 4- IBB, OX-40, CD27, CD30, GITR, HVEM, ICOS, or a combination thereof.

19. The mammalian cell of claim 17 or 18, comprising two signaling peptides CD28 and CD3z resulting in a CD19-CD28- CD3z CIR / CAR T cell.

20. The mammalian cell of claim 17 or 18, comprising two signaling peptides CD28 and 4-IBB, resulting in a CD19-41BB-CD28 CIR / CAR T cell.

21. The mammalian cell of claim 17-20, wherein the scFv in the CIR domain recognizes IFN-gamma.

22. The mammalian cell of claim 17-20, wherein the scFv in the CIR domain recognizes IL-6.

23. The mammalian cell of claim 17-20, wherein the scFv in the CIR domain recognizes MCP-1 or CCL2.

24. The mammalian cell of any one of claims 19-21, wherein the inhibitory signaling molecule in the CIR domain is PD-1 signaling.

25. The mammalian cell of any one of claims 19-21, wherein the inhibitory signaling molecule in the CIR domain is TIGIT signaling.

26. A composition comprising the mammalian cell of any one of claims 1-26.

27. A pharmaceutical composition comprising the mammalian cell of any one of claims 16-25 and a pharmaceutically acceptable carrier.

28. A method for treating cancer or autoimmune disease in a subject without inducing cytokine toxicity, said method comprising administering an effective amount of the composition of claim 25.

29. A method for reducing cytokine toxicity in a subject receiving CAR T-cell therapy, said method comprising administering an effective amount of the composition of claim 25.

30. The method of claim 26, wherein the cytokine toxicity is one of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), immune effector cell-associated hematotoxicity (ICAHT), immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (CAR-HLH), cerebral edema associated with CAR therapy, parkinsonism-like syndrome associated with CAR therapy.

31. A nucleic acid encoding a CIR domain, wherein the CIR domain comprises a ligand- sensing polypeptide able to recognize an extracellular ligand resulting from direct or indirect chimeric antigen receptor (CAR) T-cell activation, and an inhibitory signaling molecule.

32. The nucleic acid of claim 31, wherein the ligand- sensing polypeptide is a naturally occurring or modified ligand receptor, or an antibody or antigen binding fragment that recognizes the ligand.

33. The nucleic acid of claim 32, wherein the antibody or antigen binding fragment is a single-chain variable fragment (scFv) of an antibody.

34. The nucleic acid of claims 31-33, wherein the ligand is a circulating signal, a hormone, a neurotransmitter, a lipid, a peptide, a cytokine, or a chemokine.

35. The nucleic acid of claims 31-34, wherein the nucleic acid has at least 85% homology to at least one sequence set forth in SEQ ID Nos 1-6.

36. A nucleic acid encoding a CIR domain and further comprising a chimeric antigen receptor domain, wherein the nucleic acid has at least 85% homology to at least one sequence set forth in SEQ ID Nos 13-24.

37. A polypeptide comprising a CIR domain, wherein the CIR domain comprises a ligand- sensing polypeptide able to recognize a ligand resulting from direct or indirect chimeric antigen receptor (CAR) activation, and an inhibitory signaling molecule.

38. The polypeptide of claim 37, wherein the ligand- sen sing polypeptide is a naturally occurring or modified ligand receptor, or an antibody or antigen binding fragment that recognizes the ligand.

39. The polypeptide of claim 38, wherein the antibody or antigen binding fragment is a single-chain variable fragment (scFv) of an antibody.

40. The polypeptide of any one of claims 37-39, wherein the ligand is a circulating signal, a hormone, a neurotransmitter, a lipid, a peptide, a cytokine, or a chemokine.

41. The polypeptide of any one of claims 37-40, wherein the polypeptide has at least 85% sequence identity to at least one sequence set forth in SEQ ID Nos: 7-12.

42. A polypeptide comprising a CIR domain, and further comprising a chimeric antigen receptor domain, wherein the polypeptide has at least 85% sequence identity to at least one sequence set forth in SEQ ID Nos: 25-36.

43. A method of producing a CAR T-cell with a CIR domain, comprising transducing a T cell with a nucleic acid encoding a CIR / CAR, wherein the nucleic acid encodes both a CAR domain targeted to a specific antigen, and a CIR domain.

44. The method of claim 43, wherein the nucleic acid is any one of SEQ ID NO: 1-6.

45. The method of claim 43, wherein the nucleic acid is any one of SEQ ID NO: 13- 24.

46. A vector comprising one or more nucleic acid sequence identified in any one ofSEQ ID NO: 1-6.

47. A vector comprising one or more nucleic acid sequence identified in any one of SEQ ID NO: 13-24.

48. A method for controlling CAR T cell activation and cytokine production, comprising producing a CAR T cell with a CIR domain by transducing a mammalian cell with a nucleic acid encoding CIR / CAR, wherein the nucleic acid encodes both a CAR domain and a CIR domain.

49. The method of claim 47, wherein the mammalian cell is an immune effector cell.

50. The method of claim 48, wherein the immune effector cell is: f) a T cell selected from the group consisting of a helper CD4+T cell, a cytotoxic CD8+T cell, a memory T cell, a regulatory CD4+T cell, an innate-like T cell, a natural killer T cell, a mucosal associated invariant T cell, and a Gamma Delta T cell; g) a monocyte selected from the group consisting of a CD14++CD16 monocyte, a CD14+CD16++ monocyte, and a CD14++CD16+ monocyte; h) a B cell selected from the group consisting of a plasmablast, a plasma cell, a lymphoplasmacytoid cell, a memory B cell, a B-2 cell, a B-l cell, and a regulatory B cell (Breg); i) a NK cell selected from the group consisting of a CD56BRIGHTNK cell and a CD56DIMNK cell; or j) a mononuclear phagocytic cell selected from the group consisting of an adipose tissue macrophage, a monocyte, a Kupffer cell, a sinus histiocyte, an alveolar macrophage (dust cell), a tissue macrophage (histiocyte), a microglial cell, a Hofbauer cell, an intraglomerular mesangial cell, an osteoclast, a Langerhans cell, an epithelioid cell, a red pulp macrophage (sinusoidal lining cell), a peritoneal macrophage, a lysomac, and a perivascular macrophage.

51. The method of claim 48 or 49, wherein the immune effector cell is a human immune cell or a humanized immune cell.

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