WO2024238769A2 - Inhibitory chimeric antigen receptors that reduce car-t cell "on-target, off-tumor" toxicity - Google Patents

Abstract

CAR T cell therapy has shown clinical success in treating hematological tumors but its treatment of solid tumors has been limited. One of the major challenges is on-target, off-tumor toxicity, where CAR T cells also damage normal tissues that express the targeted antigen. To reduce this detrimental side-effect, Boolean-logic gates like AND-NOT gates have utilized an inhibitory CAR (iCAR) to specifically curb CAR T cell activity at selected non-malignant tissue sites. We determined iCAR generated with a single PD1 inhibitory domain inhibited cytotoxicity with delayed kinetics that were avidity dependent. We further discovered that domains from BTLA, LAIR-1, and SIGLEC-9 can replace the PD-1 domain in conventional iCARs and reduce such delayed kinetics.

Description

INHIBITORY CHIMERIC ANTIGEN RECEPTORS THAT REDUCE CAR-T CELL “ON-TARGET, OFF-TUMOR” TOXICITY CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. Section 119(e) of co- pending and commonly-assigned U.S. Provisional Patent Application No.63/502,548, filed May 16, 2023, entitled “ INHIBITORY CHIMERIC ANTIGEN RECEPTORS THAT REDUCE CAR-T CELL “ON-TARGET, OFF-TUMOR” TOXICITY”, which application is incorporated by reference herein. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under CA092131, awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD Embodiments of the disclosure concern at least the fields of medicine and immunology. BACKGROUND OF THE INVENTION Chimeric antigen receptors (CARs) enable targeted in vivo activation of immunomodulatory cells, such as T cells. These recombinant membrane receptors have an antigen-binding domain and one or more signaling domains (e.g., T cell activation domains). These special receptors allow the T cells to recognize a specific protein antigen on tumor cells and induce T cell activation and signaling pathways. Recent results of clinical trials with chimeric receptor-expressing T cells have provided compelling support of their utility as agents for cancer immunotherapy. However, despite these promising results, a number of side effects associated the CAR T-cell therapeutics were identified, raising significant safety concerns. One side effect is “on-target but off-tissue” adverse events from TCR and CAR engineered T cells, in which a CAR T cell binds to its ligand outside of the target tumor tissue and induces an immune response. Therefore, the ability to identify appropriate CAR targets is important for effectively targeting and treating the tumor without damaging normal cells that express the same target antigen. The ability to regulate an appropriate response to targets and reduce off-target side effects is important in other immune receptor systems as well, such as TCRs, engineered TCRs, and chimeric TCRs. One way to regulate appropriate CAR T cell responses to targets and reduce off-target side effects involves the utilization of inhibitory chimeric antigen receptors. Inhibitory chimeric antigen receptors (also known as iCARs) are protein constructs that inhibit or reduce immunomodulatory cell activity after binding their cognate ligands on a target cell. Conventional iCAR designs leverage PD-1 intracellular domains for inhibition, but have proven problematical. Thus, new iCARs and methods of using them to regulate appropriate CAR T cell responses to targets are needed. SUMMARY OF THE INVENTION Chimeric antigen receptor T cell (CAR-T) therapy has shown great success in the treatment of various cancers. However, due to the concurrent expression of CAR targets on normal tissues, it has had major setbacks in the treatment of solid tumors due to on-target, off-tumor toxicity, where CAR-T cells not only kill cancer cells that express the target antigen but also normal tissues. This complication has led to the termination of many clinical trials due to dose-limiting toxicities and even death. Strategies that utilize Boolean logic gates have been developed to mitigate this toxicity by increasing the specificity of CAR-T cell recognition. One type of logic gate, known as an “AND”-gate, requires the CAR-T cell to recognize two tumor- associated antigens before activating. This gating strategy has utilized alternative receptors such as the Synthetic Notch or Chimeric Costimulatory Receptor. An alternative logic gate developed by Fedorov utilized an inhibitory CAR construct that combined a scFv chain of an antibody with the T cell inhibitory signaling domain PD- 1 to inhibit CAR-T cell activity in the presence of normal tissues (Federov et al., Sci Transl Med.2013 Dec 11;5(215)). Briefly, to generate this “AND-NOT”-logic gate, a primary T cell is transduced with a CAR that recognizes a cell surface Antigen A and an iCAR that recognizes a cell surface Antigen B. When the CAR-T cell interacts with a tumor cell expressing Antigen A, it will engage the CAR and activate the T cell, leading to cytotoxicity. However, when the CAR-T cell interacts with a normal cell that expresses Antigen B, the iCAR will engage and send an inhibitory signal, preventing killing of the normal tissue. While conventional “AND-NOT”-gate CAR-T strategies function in concept, they have major limitations. First, we discovered that when combined with an activating CAR, the inhibitory CAR’s ability to specifically inhibit CAR-T cell cytotoxicity of a “normal” cell was delayed by 48-72 hours. This delay leads to the death of cells that expressed the iCAR target antigen, limiting the protection of the iCAR. Second, we found that the expression of the iCAR target antigen in the “normal” cell or the expression of the iCAR in the CAR-T cells was required to be high in order for inhibition to occur. To improve upon the inhibitory effects of CAR constructs and reduce the delay in inhibition, we developed a series of new inhibitory CAR constructs. Briefly, we combined a scFv chain that recognizes a cell surface antigen (Antigen B, i.e. Trop2) with a IgG4 hinge/spacer, a CD28 transmembrane domain, and then tested twelve different immune cell inhibitory signaling domains which include those found in proteins CTLA4, BTLA, TIM3, LAG3, LAIR1, SIGLEC7, SIGLEC9, Protocadherin 18, IL-10Ra, TIGIT, VISTA, and CD5. We then screened these constructs for their ability to inhibit CAR-T cell activation through a T cell activation reporter assay. In these studies, we discovered that a subset of inhibitory domains (i.e., those from LAIR1, SIGLEC9, and BTLA as well as PD-1) from among the group of proteins noted above that can be combined together in a single polypeptide in order to specifically inhibit off target cytotoxicity in primary T cells. Moreover, none of these domains showed increased specificity or significant reduction in the delay compared to the Trop2 iCAR that contained a PD-1 domain. A schematic depicting the constructs developed are shown in Figure 3A and were composed of the Trop2 scFv chain, the IgG4 Hinge, IgG4 CH2 and CH3 constant domains, a CD28 transmembrane domain, and the intracellular signaling domains of LAIR1, SIGLEC9, and BTLA. These iCARs comprising additional inhibitory domains expand the range of constructs useful for an “AND-NOT”-gate CAR-T strategies that utilize an inhibitory CAR. The additional constructs were generated in the same manner except for the replacement of the intracellular signaling domains with TIM3, LAG3, SIGLEC7, Protocadherin 18, IL-10Ra, TIGIT, VISTA, and CD5 amino acid sequences. Embodiments of the invention include a series of new iCAR constructs which exhibit improved inhibitory kinetics as compared to conventional iCARs. For example, using the inhibitory domains that function as we discovered, we constructed an iCAR that contains two inhibitory domains rather than one, which we term a dual- signaling iCAR. Illustrative working embodiments of these iCAR constructs contain an scFv chain that recognizes cell surface antigen B (i.e. Trop2), an IgG4 hinge domain, IgG4 CH2 and CH3 constant domains, a CD28 transmembrane domain, an intracellular signaling domain from PD1 and either an additional PD1, LAIR1, SIGLEC9, or BTLA intracellular signaling domain (Figure 3B). These constructs were cloned into a lentiviral vector and transduced into primary T cells in combination with an activating CAR that recognizes tumor-associated Antigen A (i.e. CEACAM5). Cells were enriched by magnetic beads for CAR+ and iCAR+ T cells and co-cultured with target cells that expressed the desired antigens to measure cytotoxicity by Incucyte live-cell image analysis. We found that two of these dual- signaling iCAR constructs unexpectedly showed a reduced delay in inhibition of cytotoxicity compared to a single PD1 containing iCAR construct. These two constructs are the ones in which the PD1 domain was combined with either the LAIR1 or SIGLEC9 domains. The dual-signaling iCAR embodiments of the invention can be used to further increase the specificity of CAR-T cells and prevent/reduce “on-target, off-tumor” toxicity. Trop2 was chosen as a “proof-of-concept” target, because it is widely expressed in normal epithelial cells in the esophagus, liver, lung, skin, kidney, and pancreas. Such dual-signaling iCARs can be combined with a range of CARs that have shown on-target, off-tumor toxicity related to those tissues. Additionally, the use of these combinations of PD1 and alternative inhibitory signaling domain can be generalized to other inhibitory CAR targets. The invention disclosed herein has a number of embodiments. Embodiments of the invention include, for example, compositions of matter including an inhibitory chimeric antigen receptor polypeptide. In such embodiments, the inhibitory chimeric antigen receptor polypeptide comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a first segment of amino acids comprising a first protein inhibitory domain and a second segment of amino acids comprising a second protein inhibitory domain, and the first segment of amino acids or the second segment of amino acids comprise amino acids from a PD1 protein inhibitory domain, a BTLA protein inhibitory domain, a SIGLEC9 protein inhibitory domain or a LAIR1 protein inhibitory domain. In illustrative embodiments of the invention, the inhibitory chimeric antigen receptor polypeptide has an at least a 90% or 95% amino acid sequence identity to an inhibitory chimeric antigen receptor polypeptide shown in Table A. Typically in the polypeptide embodiments of the invention, the antigen binding domain comprises amino acids derived from an antibody or a T cell receptor. In certain embodiments of the invention, the inhibitory chimeric antigen receptor polypeptide further comprises a hinge domain and/or a spacer domain. In illustrative embodiments of the invention, the PD1 protein inhibitory domain has an at least 90% or 95% amino acid sequence identity to: CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPE QTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 1); the BTLA protein inhibitory domain has an at least a 90% or 95% amino acid sequence identity to: RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDP DLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYA SICVRS (SEQ ID NO: 2); the SIGLEC9 protein inhibitory domain has an at least a 90% sequence identity to: VRSCRKKSARPAAGVGDTGIEDANAVRGSASQGPLTEPWAEDSPPDQPPPAS ARSSVGEGELQYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 3); and the LAIR1 protein inhibitory domain has an at least a 90% or 95% amino acid sequence identity to: HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDT SALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO: 4). Embodiments of the invention also include compositions of matter comprising a polynucleotide encoding an inhibitory chimeric antigen receptor polypeptide disclosed herein. Typically the polynucleotide is disposed in an expression vector. Such embodiments of the invention include mammalian cells (e.g., human CD 8⁺ T cells) transduced with a vector comprising a polynucleotide encoding an inhibitory chimeric antigen receptor polypeptide disclosed herein. In certain embodiments of the invention, the mammalian cell further comprises an exogenous nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments of the invention, the inhibitory chimeric antigen receptor polypeptide and the chimeric antigen receptor target the same antigen. In other embodiments of the invention, the inhibitory chimeric antigen receptor polypeptide and the chimeric antigen receptor target different antigens. Embodiments of the invention further include methods of modulating the ability of a human CD 8⁺ T cell to kill target cells, wherein the human CD 8⁺ T cell expresses a chimeric antigen receptor, the method comprising introducing an exogenous nucleic acid into the cell, wherein the exogenous nucleic acid expresses an inhibitory chimeric antigen receptor polypeptide disclosed herein so that the ability of a human CD 8⁺ T cell to kill target cells is modulated. Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It is to be understood, however, that the detailed description and specific examples, while indicating some embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Inhibition of CAR T cell cytotoxicity by the TROP2-PD1 iCAR is delayed. A) The model illustrates the “AND-NOT”-gate CAR T strategy for specifically targeting CEA+ tumor cells. The CAR and iCAR target CEA and TROP2 respectively. The CEACAR consists of a FLAG tag, a scFv chain that recognizes CEA, a IgG 42NQ hinge, a IgG4 CH3 constant domain, a CD28 transmembrane domain (TM), a 41BB costimulatory (CS) domain, and a CD3ȗ activation domain. The TROP2-PD1 iCAR consists of a HA tag, a scFv chain that recognizes TROP2, the IgG4 hinge, CH2, and CH3 constant domains, a CD28 TM, and a PD1 signaling domain. B) CAR+/iCAR+ T cells can inhibit CAR T cell IFN-Ȗ production as measured by ELISA 48 hours after co-culture of T cells with DU145 target cells that express CEA and/or TROP2. C) CAR+/iCAR+ T cells that can specifically inhibit CAR T cell cytotoxicity after 48 hours in co-culture with DU145 target cells that express CEA and TROP2. Target cell presence was measured by Total Green Object Area (μm2/well) of DU145 target cells that express CEA and/or TROP2. D) Inhibition of cytotoxicity is delayed in CAR+/iCAR+ T cells when co-cultured with DU145 target cells. Measurements of Total Green Object Area of GFP+ target cells were measured over ~140 hours by Incucyte live cell image analysis at intervals of 2 hours. Statistics are calculated based on the Total green object area (μm2/well) at the last time point compared to the CAR+ only control. The data is reported as a mean + standard error (n = 3 donors). Statistics performed using 1 way ANOVA analysis with Tukey multiple comparison correction. *p-value < 0.05, **p-value < 0.01, ***p- value < 0.001. Figure 2. Increasing the avidity of iCAR interactions reduces the delay in inhibitory signaling kinetics. A) Engineered target cell lines have different surface level expression of CEA and TROP2 measured by flow cytometry. Histograms are representative images from one of three experiments comparing CEA and TROP2 expression of each target cell line. B) Increasing the target antigen density reduces the delay in iCAR inhibition as measured by cytotoxicity over time. The cytotoxicity curves are representative images from one experiment measuring the total green object area of the target cells over time (^m2/well). C) CAR+/iCAR+ T cells co- cultured with target cells that express high levels of TROP2 have reduced delays in inhibition. The delay in inhibition was measured by calculating the Area under each cytotoxicity curve. The AUC was normalized against the AUC calculated for Untransduced T cells co-cultured with target cells. The normalized AUC quantified is the mean + s.d. (n=3). D) Representative histograms measuring the Mean Fluorescence Intensity (MFI) indicate the difference in CAR and iCAR surface expression between CAR T cell groups being tested. Groups have been transduced with CAR lentivirus at a MOI of 1 and iCAR lentivirus at a MOI of 1, 3, and 10 respectively. E) Increasing the surface level expression of the iCAR in primary T cells reduces the delay in iCAR inhibition as measured by cytotoxicity over time. Representative cytotoxicity curves (Total Green Object Area - ^m2/well) from one experiment are displayed comparing the killing ability of CAR T cells with different surface level expression of the iCAR when co-cultured with DU145 target cells that express CEA or CEA and TROP2. F) CAR+/iCAR+ T cells with higher iCAR surface expression have reduced delays in inhibition when co-cultured with CEALO/TROP2HI target cells. The delay in inhibition was measured by calculating the Area under each cytotoxicity curve. The AUC was normalized against the AUC calculated for Untransduced T cells co-cultured with target cells. The normalized AUC quantified is the mean + s.d. (n=2). Statistics performed using 1 way ANOVA analysis with Tukey multiple comparison correction. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. Figure 3. Schematic illustrating the structure of each inhibitory CAR construct. A) The scFv chain that recognizes Antigen B (Trop2) was linked to a spacer (Short IgG4 Hinge; l.ong- lgG4 Hinge + CH2 + CH3 constant domains) to the CD28 transmembrane domain {TM) and to the inhibitory signaling domains of an immune cell inhibitory receptor. B) Schematic describing the dual signaling iCAR construct design. An scFv chain that recognized Antigen B (Trop2) was linked by a long spacer to the PD-l inhibitory signaling domain and followed by an additional immune cell inhibitory receptor domain such as a BTLA protein inhibitory domain, a SIGLEC9 protein inhibitory domain or a LAIR1 protein inhibitory domain. Figure 4. Inhibitory CARs containing signaling domains with ITIM motifs can reduce CAR T cell cytotoxicity. A) The five different populations (Untransduced, CAR, CAR + TROP2-PD1 iCAR, CAR + TROP2-Short iCAR, CAR + TROP2-Long iCAR) tested for each inhibitory signaling domain are plotted in a flow cytometry plot that corresponds to each color in the legend. The flow cytometry plot is a representative from one experiment. B) CAR+/iCAR+ T cells that were engineered with inhibitory signaling domains with an ITIM motif can inhibit CAR T cell cytotoxicity as measured by Total Green Object Area (μm/well) of DU145 target cells that express CEA and/or TROP2 over 150 hours. Each curve represents a co- culture with the CAR T cell population represented by the color in the legend. These cytotoxicity curves are representative images from one experiment. C) CAR+/iCAR+ T cells that were engineered with inhibitory signaling domains with an ITIM motif can inhibit CAR T cell cytotoxicity with a similar efficiency as the TROP2-PD1 iCAR as measured by Area under the Cytotoxicity curve. AUC was normalized to the untransduced population co-cultured with the target cells. The normalized AUC quantified is the mean + s.d. of at least two independent experiments (BTLA – n=2; LAIR1 – n=2, SIGLEC9 – n=3, VISTA – n=2). The significance values shown are comparisons between a control group. For the CEA-/TROP2- cell line, values are compared to the Untransduced control. For the CEA+/TROP2- cell line, values are compared to the CAR control. For the CEALO/TROP2HI or CEAHI/TROP2LO cell lines, values are compared to the CAR + TROP2-PD1 iCAR group. Statistics performed using 1 way ANOVA analysis with Tukey multiple comparison correction. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. Figure 5. Dual-signaling inhibitory domain CARs increases the efficiency of inhibition in the AND-NOT-gate CAR T strategy. A) The models represent the structure of each dual-signaling iCAR tested. The DiCARs are composed of a TROP2 scFv, the IgG4 Hinge, CH2, and CH3 constant domains, a CD28 TM, the PD- 1 inhibitory signaling domain, and the additional inhibitory signaling domains PD-1, BTLA, SIGLEC-9, or LAIR-1. B) The representative histogram indicates that iCAR surface expression level is similar between the groups of DiCARs being compared. The DiCAR surface expression was determined by flow cytometry for a HA-tag located on the N-terminus of the iCAR/DiCAR. C) Representative cytotoxicity curves of each CAR T cell population demonstrate that CAR T cell populations with a dual-signaling iCAR have a reduced delay in inhibition compared to the TROP2-PD1 iCAR. CAR T cells were co-cultured with DU145 target cells that express GFP and CEACAM5 and/or TROP2. Presence of target cells was measured by Total Green Object Area (μm2/well) over time as measured by Incucyte live cell image analysis over 150 hours. D) The delay in inhibition of the iCAR was measured by Area under the cytotoxicity curve analysis of each cytotoxicity curve and normalized to the co- culture with the Untransduced T cell group. This AUC is a representative of one experiment in which triplicate wells were analyzed. Three biological replicates were performed and reported in Supplemental Figure 6 and 7 in Bangayan. The significance values shown are comparisons between a control group. For the CEA- /TROP2- cell line, values are compared to the Untransduced control. For the CEA+/TROP2- cell line, values are compared to the CAR control. For the CEALO/TROP2HI or CEAHI/TROP2LO cell lines, values are compared to the CAR + TROP2-PD1 iCAR group. Statistics performed using 1 way ANOVA analysis with Tukey multiple comparison correction. *p-value < 0.05, **p-value < 0.01, ***p- value < 0.001. DETAILED DESCRIPTION OF THE INVENTION In the description of embodiments, reference may be made to the accompanying figures which form a part hereof, and in which is shown by way of illustration a specific embodiment in which the invention may be practiced. It is to be understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention. Many of the techniques and procedures described or referenced herein are well understood and commonly employed by those skilled in the art. 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 invention 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. As noted above, on-target, off-tumor toxicity is a major barrier to the application of CAR T therapy to solid tumors. Boolean logic gates like the AND- NOT gate have utilized an inhibitory CAR (iCAR) to reduce this toxicity. We have investigated the role of avidity, affinity, and internal signaling domain composition on the kinetics of iCAR inhibition. With this knowledge, we designed a new inhibitory chimeric antigen receptor (DiCAR) that combines two T cell inhibitory signaling domains to specifically regulate CAR T cell cytotoxicity and improve inhibition efficiency compared to an iCAR with a single PD1 domain. Embodiments of the invention include compositions of matter comprising one or more vectors comprising polynucleotides encoding the inhibitory chimeric antigen receptor polypeptides disclosed herein. A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like. Typically, the vector is an expression vector. The term "expression" as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter. In this context, the term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (Į) and beta (ȕ) chain, although in some cells the TCR consists of gamma and delta chains. TCRs may exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. Embodiments of the invention include a number of different TCR alpha/beta nucleic acids and their encoded polypeptides. The invention disclosed herein has a number of embodiments. Embodiments of the invention include, for example, compositions of matter including an inhibitory chimeric antigen receptor polypeptide as well as polynucleotides encoding such polypeptides. In illustrative embodiments of the invention, the inhibitory chimeric antigen receptor polypeptide comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a first segment of amino acids comprising a first protein inhibitory domain and a second segment of amino acids comprising a second protein inhibitory domain, and the first segment of amino acids or the second segment of amino acids comprise amino acids from a PD1 protein inhibitory domain, a BTLA protein inhibitory domain, a SIGLEC9 protein inhibitory domain or a LAIR1 protein inhibitory domain. In illustrative embodiments of the invention, the inhibitory chimeric antigen receptor polypeptide has an at least 90% or 95% identity to an inhibitory chimeric antigen receptor polypeptide shown in Table A. Typically in the polypeptide embodiments of the invention, the antigen binding domain comprises amino acids derived from an antibody or a T cell receptor. In certain embodiments of the invention, the inhibitory chimeric antigen receptor polypeptide further comprises a hinge domain and/or a spacer domain. In illustrative embodiments of the invention, the PD1 protein inhibitory domain has an at least 90%

RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDP DLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYA SICVRS (SEQ ID NO: 2); the SIGLEC9 protein inhibitory domain has an at least a 90% or 95% sequence identity to: VRSCRKKSARPAAGVGDTGIEDANAVRGSASQGPLTEPWAEDSPPDQPPPAS ARSSVGEGELQYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 3); and the LAIR1 protein inhibitory domain has an at least a 90% or 95% sequence identity to: HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDT SALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO: 4). Embodiments of the invention also include compositions of matter comprising a polynucleotide encoding an inhibitory chimeric antigen receptor polypeptide disclosed herein. Typically the polynucleotide is disposed in a expression vector. Such embodiments of the invention include mammalian cells (e.g. human CD 8⁺ T cells) transduced with a vector comprising a polynucleotide encoding an inhibitory chimeric antigen receptor polypeptide disclosed herein. In certain embodiments of the invention, the mammalian cell further comprises an exogenous nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments of the invention, the inhibitory chimeric antigen receptor polypeptide and the chimeric antigen receptor target the same antigen. In other embodiments of the invention, the inhibitory chimeric antigen receptor polypeptide and the chimeric antigen receptor target different antigens. Embodiments of the invention include methods of making an inhibitory chimeric antigen receptor polypeptide comprising forming a polynucleotide encoding: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein the intracellular signaling domain comprises a first segment of amino acids comprising a first protein inhibitory domain and a second segment of amino acids comprising a second protein inhibitory domain, wherein the first segment of amino acids or the second segment of amino acids comprise amino acids from a PD1 protein inhibitory domain, a BTLA protein inhibitory domain, a SIGLEC9 protein inhibitory domain or a LAIR1 protein inhibitory domain; the PD1 protein inhibitory domain has an at least 90% sequence identity to: CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPE QTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 1); the BTLA protein inhibitory domain has an at least 90% sequence identity to: RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDP DLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYA SICVRS (SEQ ID NO: 2); the SIGLEC9 protein inhibitory domain has an at least 90% sequence identity to: VRSCRKKSARPAAGVGDTGIEDANAVRGSASQGPLTEPWAEDSPPDQPPPAS ARSSVGEGELQYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 3); and the LAIR1 protein inhibitory domain has an at least 90% sequence identity to: HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDT SALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO: 4); and then expressing the inhibitory chimeric antigen receptor polypeptide encoded by the polynucleotide such that a inhibitory chimeric antigen receptor polypeptide is made. Typically these methods further comprise disposing the polynucleotide in a mammalian expression vector. Embodiments of the invention further comprise disposing the mammalian expression vector in a mammalian cell (e.g., a human CD 8⁺ T cell obtained from an individual diagnosed with a cancer). Embodiments of the invention further include methods of modulating the ability of a human CD 8⁺ T cell to kill target cells, wherein the human CD 8⁺ T cell expresses a chimeric antigen receptor, the method comprising introducing an exogenous nucleic acid into the cell, wherein the exogenous nucleic acid expresses an inhibitory chimeric antigen receptor polypeptide disclosed herein so that the ability of a human CD 8⁺ T cell to kill target cells is modulated. Typical CD 8⁺ T cell embodiments of the invention comprise an inhibitory CAR (e.g. iCAR) polypeptide which includes intracellular components that dampen or suppress an immune response, such as an ITAM-and/or co stimulatory-promoted response in the cell. Exemplary of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2/4 Adenosine receptors including A2AR. Embodiments of the invention further include engineered cells manipulated to express a inhibitory CAR polypeptide including two distinct signaling domains of or derived from such an inhibitory molecule, such that it serves to dampen the response of the cell, for example, that induced by an activating and/or costimulatory CAR. (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing a different antigen, whereby an activating signal delivered through a CAR recognizing a first antigen is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects. In some embodiments, the cells manipulated to express engineered antigen receptors include CARs, including activating or stimulatory CARs, costimulatory CARs (see WO2014/055668), and/or inhibitory CARs (iCARs, see Fedorov et al., 2013). The CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, the CARs engineered in mammalian cells include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see, e.g., WO2014/055668, the contents of which are incorporated by reference). In some aspects, the cells include one or more stimulatory or activating CAR and/or a costimulatory CAR. In typical embodiments, the cells further include inhibitory CARs disclosed herein such as a CAR recognizing an antigen other than one associated with and/or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects. In some embodiments, the two receptors induce, respectively, an activating and an inhibitory signal to the cell, such that binding by one of the receptor to its antigen activates the cell or induces a response, but binding by the second inhibitory receptor to its antigen induces a signal that suppresses or dampens that response. Examples are combinations of activating CARs and inhibitory CARs or iCARs. Such a strategy may be used, for example, in which the activating CAR binds an antigen expressed in a disease or condition but which is also expressed on normal cells, and the inhibitory receptor binds to a separate antigen which is expressed on the normal cells but not cells of the disease or condition. In another aspect, the invention includes a method for generating a modified T cell comprising introducing one or more nucleic acids (e.g., nucleic acids disposed within a lentiviral vector) encoding a inhibitory chimeric antigen receptor polypeptide disclosed herein into a T cell (e.g. a CD8⁺ T cell obtained from an individual diagnosed with a cancer that expresses a target peptide antigen). The present invention also includes modified T cells with downregulated or knocked out gene expression (e.g., a modified T cell having a knocked out endogenous T cell receptor and an exogenous/introduced T cell receptor that recognizes peptide antigen associated with a HLA). The term "knockdown" as used herein refers to a decrease in gene expression of one or more genes. The term "knockout" as used herein refers to the ablation of gene expression of one or more genes. The modified T cells described herein may be included in a composition for use in a therapeutic regimen. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified T cells may be administered. Pharmaceutical compositions of the present invention may comprise the modified T cell as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for intravenous administration. Adoptive immunotherapy with T cells harboring antigen-specific TCRs have therapeutic potential in the treatment of cancers. Gene-engineering of CD 8⁺ T cells with a specific TCR has the advantage of redirecting the T cell to a selected antigen. In this context, in one aspect, the invention includes methods for stimulating a T cell- mediated immune response to a target cell or tissue in a subject comprising administering to a subject an effective amount of a modified CD 8⁺ T cell. In this embodiment, the CD8⁺ T cell is modified as described elsewhere herein. Embodiments of the invention also include administering multiple modified CD 8⁺ T cells. Embodiments of the invention encompass methods of treating a disease or condition. The treatment methodology comprises comprising administering an effective amount of a pharmaceutical composition comprising a modified T cell described herein to a subject in need thereof. The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A "subject" or "patient”, as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human. In typical embodiments of the invention, the human has a cancer expressing a polypeptide that functions as an antigen. In some embodiments of the invention, the cells of the cancer form solid tumors. A related embodiment of the invention includes a method for prophylaxis and/or therapy of an individual diagnosed with, suspected of having or at risk for developing or recurrence of a cancer, wherein the cancer comprises cancer cells which express a cancer antigen. This approach comprises administering to the individual modified human T cells comprising a recombinant polynucleotide encoding a inhibitory chimeric antigen receptor polypeptide, wherein the T cells are capable of direct recognition of the cancer cells expressing the cancer antigen, and wherein the direct recognition of the cancer cells comprises HLA class II-restricted binding of the TCR to the peptide antigen expressed by the cancer cells. With respect to use of the engineered CD8⁺ T cells of the present invention, the method generally comprises administering an effective amount (e.g. by intravenous or intraperitoneal injections) of a composition comprising the CD8⁺ T cells to an individual in need thereof. An appropriate pharmaceutical composition may be adapted for administration by any appropriate route, such as parenteral (including subcutaneous, intramuscular, or intravenous), enteral (including oral or rectal), inhalation or intranasal routes. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions. In another aspect, the invention includes use of a polynucleotide or a modified CD8⁺ T cell described herein in the manufacture of a medicament for the treatment of a disease or condition characterized by the expression of a cancer antigen, in a subject in need thereof. In illustrative embodiments of the invention, the disease is a cancer. The technology in this area is fairly developed and a number of methods and materials know in this art can be adapted for use with the invention disclosed herein. Such methods and materials are disclosed, for example in U.S. Patent Publication Nos. 20190247432, 20190119350, 20190002523, 20190002522, 20180371050, 20180057560, 20170029483, 20160024174, and 20150141347, the contents of which are incorporated by reference. See also U.S. Patent Nos. 54695797, 58766450, 11433100 and 11602543, U.S. Patent Publication Nos. 20200261499, 20200385438, 20220289842, 20220249637, 20220127373 PCT Application No. US2014/016527; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127- 33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Further aspects and embodiments of the invention are provided in the examples below. EXAMPLES EXAMPLE 1: DUAL INHIBITORY DOMAIN ICARS IMPROVE EFFICIENCY OF THE AND-NOT GATE CAR T STRATEGY Certain disclosure discussed in this Example (e.g. supplemental materials) is found in Bangayan et al., Proc Natl Acad Sci U S A. 2023 Nov 21;120(47):e2312374120, ( hereinafter “Bangayan”) which is incorporated herein by reference. Genetically engineered adoptive cell therapies that target tumor-associated antigens have recently shown success in the clinic. One such therapy is chimeric antigen receptor (CAR) T cell therapy which introduces an engineered receptor that combines an antibody scFv chain and T cell signaling domains into T cells to specifically kill tumor cells. CAR T cells targeting CD19 and BCMA have successfully treated hematological malignancies such as relapsed or refractory acute lymphoblastic leukemia, large B-cell lymphoma, and multiple myeloma (1, 2). Although application of CAR T cell treatment for solid tumors has rapidly grown in number of clinical trials (3), its success has still been limited due to two major obstacles: the immune restrictive tumor microenvironment (4) and on-target, off- tumor toxicity (5–7). To overcome this inhibitory environment, CAR T cells have been generated to be more potent, but these improvements are still accompanied with neurotoxicity, cytokine release syndrome, and/or on-target, off-tumor toxicity (8). Improvements must be made to balance the strength and efficacy of CAR T therapy and the potential toxicities associated with it. On-target, off-tumor toxicity occurs when CAR T cells recognize normal tissues that express the targeted antigen. These toxicities have ranged from manageable with the CD19 CAR and B cell aplasia (8) to lethal with the ERBB2 CAR and respiratory distress (9). Additional CARs targeting CEACAM5 in the lung and CAIX in the liver have also shown toxicities that were considered too debilitating to advance clinically (5–7). Various strategies have been developed to reduce CAR T cell toxicity. Elimination of CAR T cells through drug-induced suicide genes and secondary markers (10–13), affinity tuning of the antigen binding domain (14, 15), and control of CAR T cell recognition through small molecules and targeting modules (16–19) have all been tested. Each of these strategies have been capable of reducing toxicity but at the cost of efficacy due to the loss of persistence or increased tumor escape. Rather than compromising the efficacy of CAR T treatment, Boolean logic gates have been applied to CAR T cells as safety switches. By integrating signals from multiple receptors at once, CAR T cells can regulate their activity based on their environment. For example, AND-gate strategies utilize two receptors that recognize different tumor antigens to trigger CAR T cell activation. Variations of this strategy have combined a masked CAR and proteases (20), a chimeric co-stimulatory receptor and a first-generation CAR (21), and a Synthetic Notch (SynNotch) receptor and a CAR (22). An alternative logic gate is the AND-NOT gate, which utilizes two receptors – an activating chimeric antigen receptor (CAR) that contains T cell co-stimulatory and activation domains and an inhibitory chimeric antigen receptor (iCAR) that contains a T cell inhibitory signaling domain. The CAR recognizes a tumor antigen and activates a T cell, while the iCAR recognizes a normal tissue antigen and inhibits T cell activity. In this manner, the CAR T cell can distinguish a tumor cell and normal cell that express the same CAR target. Over a decade ago, Fedorov et al published the proof-of-concept of this strategy by linking a scFv chain that recognized PSMA to the PD-1 or CTLA-4 inhibitory signaling domains. This iCAR was capable of inhibiting T cell proliferation, cytokine production, and cytotoxicity when combined with a TCR or CD19-targeting CAR. However, its ability to efficiently inhibit T cell activity was limited to when the iCAR specific antigen was highly expressed (23). Improvements to iCAR design have focused on targeting relevant normal tissue antigens and increasing the potency of iCAR signaling. HLA-C1, HLA-A2, and HLA-A3 have all been described as iCAR targets that limit killing to tumor cells with loss of HLA alleles (24–27), but this subjects CAR T therapy to HLA-restriction. LIR-1 and TIGIT have been reported as replacements to PD-1 (25, 28, 29), but how they enhance iCAR inhibition is unknown. The AND-NOT gate strategy is compelling, but a deeper understanding of the mechanisms and key drivers of specific inhibition are necessary to achieve a tighter regulation of CAR T cell activity. Unlike CARs, the role that affinity and avidity play in iCAR function and kinetics has not been well-studied. To better understand how to enhance specific iCAR inhibition of CAR T cell activity, we studied the role of dosage and internal signaling components in iCAR inhibitory kinetics. This knowledge led us to develop a new class of iCAR which combines two different inhibitory signaling domains into a single construct termed the dual-inhibitory domain iCAR (DiCAR). DiCARs more efficiently inhibits CAR T cell activity than an iCAR with a single PD1 domain. Results The TROP2 PD1 iCAR displays a kinetic delay in inhibition of cytotoxicity Selection of target antigens for the AND-NOT gating strategy To develop a model for the AND-NOT gating strategy, we selected two epithelial cell targets as antigens for the CAR and iCAR. CEACAM5 (CEA) was chosen as a CAR target because of its high expression in neuroendocrine prostate cancer (30), colorectal cancer (31), gastric cancers (32), and small cell cancers of the lung (33). Due to its normal tissue expression in the colon, bladder, kidney, and lung, some adoptive cell therapies targeting this antigen have displayed dose-limiting on- target, off-tumor toxicities that could be reduced with an AND-NOT Boolean logic gate (5, 32, 34). As an inhibitory CAR target, we selected TROP2 (TACSTD2 or TROP2), which is widely expressed in epithelial cells of the lungs, skin, esophagus, kidney, liver, and pancreas (35). Antibodies targeting TROP2 have been used as a target for triple-negative breast cancer, making it an amenable target for immunotherapies (36). Our previous work with TROP2 made it a useful surrogate epithelial cell marker for studying the AND-NOT gating strategy (37, 38). Development of a CEACAM5 CAR and TROP2 iCAR gating strategy As an activating module, we enhanced a previously published CEACAM5- Long-CD28-3z CAR by replacing its extracellular spacer and co-stimulatory domain (Supplemental Figure 1A in Bangayan) to increase in vivo functionality (30, 39–41). This new CEACAM5-42NQ-41BB-3z targeting CAR (CEACAR) elicited the same levels of IFN-Ȗ production and cytotoxicity against a CEACAM5⁺ engineered cell line as our previously published CAR (Supplemental Figure 1B and C in Bangayan) (30). This CEACAR is also able to eliminate CEACAM5⁺ tumors in vivo compared to an untransduced T cell control (Supplemental Figure 1D in Bangayan). To develop the iCAR, antibodies were discovered that bound TROP2 by panning a single fold scFv phage display library as further described in methods (42). Using one of the antibodies (H11) with the highest binding affinity, we generated an inhibitory CAR construct as described by Fedorov et al. (23) The TROP2 scFv chain was linked to an extracellular spacer (Long Spacer - IgG4 hinge, CH2, and CH3 domain), a CD28 transmembrane domain (TM), and a PD1 intracellular signaling domain to form the TROP2-Long-PD1 iCAR (TROP2-PD1 iCAR) (Figure 1A). The TROP-PD1 iCAR can inhibit CEACAR T cell cytotoxicity and cytokine production To establish whether the TROP2-PD1 iCAR could inhibit CEACAR T cell activity, T cells transduced with both the CAR and iCAR were co-cultured with engineered DU145 prostate cancer cell lines. A DU145 cell line in which the TROP2 gene was deleted using CRISPR/Cas9n (43) (CEA-/TROP2-) was engineered to express either CEACAM5 alone (CEA⁺/TROP2-) or both CEACAM5 and TROP2 (CEA⁺/TROP2⁺) by lentiviral transduction. T cell activity was expected when CEACAM5 was expressed; but inhibition was expected when TROP2 was present (Figure 1A). Because high levels of iCAR were reported to be necessary to inhibit allogenic T cell cytotoxicity by Fedorov et al. (23), the multiplicity of infection (MOI) of the iCAR was 10-fold higher than the CEACAR. T cells enriched to be at least 80% CAR⁺/iCAR⁺ were then co-cultured with these engineered DU145 cell lines. Two hallmarks of T cell activation that were observed to be inhibited by CAR⁺/iCAR⁺ T cells after co-culture with CEACAM5⁺/TROP2⁺ target cells were cytokine production and cytotoxicity. Approximately 90% less IFN-Ȗ was produced by the CAR⁺/iCAR⁺ T cells compared to the CAR⁺ only control (Figure 1B). This difference was not seen when the target cells expressed CEACAM5 alone. There was also a 30% reduction in the death of CEACAM5⁺/TROP2⁺ target cells by the CAR⁺/iCAR⁺ T cells compared to the CAR⁺ only control forty-eight hours after co- culture (Figure 1C). However, 50% of the population was still killed compared to the untransduced negative control, suggesting that the TROP2-PD1 iCAR could inhibit CEACAR cytotoxicity but not completely. T cells rapidly integrate both positive and negative signals to determine how they will interact with a target cell. Since CAR T signaling and activity is dynamic (44), we hypothesized that this incomplete inhibition might be due to a delay in the TROP2-PD1 iCAR’s inhibitory function. To test this hypothesis, CAR⁺/iCAR+ T cells were co-cultured with CEACAM5⁺/TROP2⁺ target cells and observed by Incucyte live cell image analysis over 150 hours (Figure 1D and Supplemental Figure 2A in Bangayan). As seen previously, 48 hours after co-culture, killing could still be seen by the CAR⁺/iCAR⁺ T cells. However, at 72 hours, the adherent target cells were stretched out and appeared to be replicating compared to those cultured with the CAR⁺ only control. By 150 hours, the target cells were confluent (Supplemental Figure 2A in Bangayan). Flow cytometry analysis of target cells recovered after co- culture confirmed the continued expression of CEA and TROP2, removing the possibility that the target cells survived due to CAR target antigen loss (Supplemental Figure 2B in Bangayan). Regardless of the three independent donors used, the TROP2-PD1 iCAR was able to inhibit CAR T cell activity, but this inhibition was delayed (Figure 1D). Increasing the avidity of iCAR interactions reduces the kinetic delay in inhibition Can avidity affect the delay in inhibition of cytotoxicity? Both affinity and avidity contribute to the efficacy of antibody-based tumor targeting therapies (45). Avidity was recently shown to contribute to an iCAR’s ability to inhibit CAR NK cell activity (27). This prompted us to ask whether the delay in iCAR inhibition of T cells was also affected by its avidity. Since avidity is based on the number of receptors and antigens interacting, we regulated it by changing the surface level expression of the antigens on target cells and the receptors on T cells. Increasing the avidity by higher surface expression of the iCAR target antigen TROP2 reduces the delay in inhibition To control the level of CAR and iCAR target antigen, the CEA-/TROP2- target cell line was transduced with lentiviruses that contained CEA (CAR antigen) and TROP2 (iCAR antigen). These cells were single cell cloned and screened for target cell lines that had high CEA and low TROP2 expression (CEA^HI/TROP2^LO) and low CEA and high TROP2 expression (CEA^LO/TROP2^HI) (Figure 2A, Supplemental Figure 3A in Bangayan). CAR⁺/iCAR⁺ T cells were then co-cultured with both these cell lines, and the delay in inhibition was compared over time. To compare the delays between groups and experiments, the area under each cytotoxicity curve (AUC) was calculated and normalized to an untransduced T cell control. The closer the normalized AUC was to 1 the more the cytotoxicity curve matched the untransduced control, suggesting a shorter delay. When CAR⁺/iCAR⁺ T cells were co-cultured with target cells that expressed higher levels of the iCAR antigen TROP2 and lower levels of the CAR antigen CEA, the delay in inhibition was reduced (CEA^LO/TROP2^HI vs CEA^HI/TROP2^LO, Figure 2B and 2C). The data showed that as target cells became more sensitive to the iCAR and less sensitive to the CAR, inhibition efficiency improved. Increasing the avidity through higher iCAR surface expression reduces the delay in inhibition Another way to adjust the avidity was to alter the levels of CAR and iCAR on the surface of T cells. To increase the surface expression of the iCAR, primary T cells were transduced with increasing MOIs for the TROP2-PD1 iCAR lentivirus (MOI: 1, 3, 10), while holding the MOI of the CEACAR lentivirus constant (MOI: 1). Flow cytometry confirmed that as the MOI increased, the surface expression as measured by mean fluorescence intensity of the iCAR increased. Concurrently, the mean fluorescence intensity of the CAR decreased (Figure 2D, Supplemental Figure 3B in Bangayan). Overall, the iCAR:CAR ratio gradually increased as we raised the MOI of the iCAR, leading to a higher potential avidity for the iCAR. These CAR⁺/iCAR⁺ T cells were then co-cultured with the CEA^LO/TROP2^HI target cell line and observed for approximately 170 hours. As seen in Figures 2E and 2F, as the MOI of the iCAR increased, the efficiency of inhibition increased as measured by the cytotoxicity curves and AUCs. The CAR T cells with an iCAR at a MOI of 10 had a curve that was similar to the untransduced control (Figure 2E). Although we could not determine the exact iCAR:CAR ratio necessary for complete inhibition, the data suggests that efficiency of inhibition can be controlled through avidity. iCARs with immunoreceptor tyrosine-based inhibition or switch motifs (ITIM/ITSM) can inhibit CAR T cell activation and cytotoxicity CARs have been enhanced by replacing their co-stimulatory and activation domains with alternative domains (i.e. 41BB, ICOS, JAK/STAT, OX-40) that improve proliferation, cytokine production, and in vivo persistence (40, 47–49). It was recently shown that alternative domains, such as CTLA-4, LIR-1, and TIGIT, could also replace the function of PD1 in an iCAR in T cells (23, 25, 28). Domains including KIR2DL1, LIR-1, CD300A, NKG2a, and LAIR-1 were also tested in an iCAR construct in NK cells (50). Selection of Inhibitory Domains for iCAR Construction A series of inhibitory receptor signaling domains were selected as potential modules that could inhibit CAR T cell activity. Some domains were derived from receptors that have been targeted as checkpoint inhibitors like TIM-3, CTLA-4, and LAG-3 (51–53). Other domains like CD5, PCDH18, and VISTA were selected due to their previous roles in T cell inhibition in mouse knock-out models (54–58). Domains from BTLA, LAIR-1, TIGIT, SIGLEC-7, and SIGLEC-9 were all chosen for their inclusion of ITIM/ITSMs, which both inhibit signaling through the recruitment of phosphatases (59). Generation of iCAR Constructs and Confirmation of Their Ability to Traverse to the Cell Surface To test these domains for inhibitory function, twenty-two iCARs were constructed by linking the H11 TROP2 scFv chain, an extracellular spacer of variable length (Short or Long as described in Methods), a CD28 TM, and the intracellular domain of the inhibitory receptor as designated by Uniprot (Figure 4B; Supplementary Table 1 in Bangayan). All constructs were confirmed to be expressed on the cell surface by flow cytometry against a HA-tag on its N-terminus (Supplemental Figure 5C in Bangayan). Short spacer iCARs trafficked less effectively to the surface of the cell compared to those that contained long spacers regardless of the inhibitory signaling domain used (Supplemental Figure 5C in Bangayan). ITIM/ITSM-containing iCARs can inhibit T cell activation in a Jurkat reporter assay To rapidly screen through these iCARs, a Jurkat-NFAT-ZsGreen reporter cell line was co-transduced with both an iCAR and a CEACAM5-Long-CD28-3z CAR (iCAR MOI: 25, CAR MOI: 1) and tested for activation after co-culture. When activated, these Jurkat cells increase the expression of ZsGreen and can be detected by flow cytometry, but if inhibited, they cannot (Supplemental Figure 5A in Bangayan). Sorted CAR⁺/iCAR⁺ Jurkat cells were co-cultured with target cells that expressed CEA and/or TROP2 for 24 hours. Specific inhibition of the iCAR was calculated by comparing the percentage of ZsGreen+ Jurkat cells when co-cultured with CEA⁺/TROP2- target cells compared to CEA⁺/TROP2⁺ cells. Approximately 75% of CAR⁺ Jurkat cells were activated when co-cultured with target cells that expressed CEACAM5 regardless of TROP2 expression. However, the TROP2-PD1 iCAR decreased the percentage of activated cells to ~40% when TROP2 was present (Supplemental Figure 5D in Bangayan). In total, eleven additional inhibitory signaling domains were screened for their ability to inhibit CAR T cell activity. Of the twenty-two constructs tested, eight of them specifically inhibited CAR T cell activation when co-cultured with the CEA⁺/TROP2⁺ line compared to the CEACAM5⁺ line. These eight constructs were all found in iCARs that contained an ITIM/ITSM motif (Supplemental Figure 5D in Bangayan). The TROP2-Long-SIGLEC9 iCAR showed the greatest specific inhibition with a difference of ~40%. iCARs containing BTLA, LAIR-1, and TIGIT inhibited CAR T cell activation even when TROP2 was not expressed. This ligand-independent inhibition may be due to tonic signaling of the iCAR at this avidity. Some non-ITIM-containing iCARs such as LAG-3 and CD5 also showed ligand-independent inhibition, but because no specific inhibition was observed, they were not further pursued. ITIM/ITSM-containing iCARs can inhibit CAR T cell cytotoxicity A selection of iCARs (BTLA, LAIR-1, SIGLEC-9) that functioned best in the reporter assay were then tested for their ability to inhibit cytotoxicity in primary T cells equipped with the CEACAR (Figure 4A). As a negative control, the VISTA iCARs were included. To lower the contribution of avidity and potential tonic signaling seen in the Jurkat reporter assay, the MOI of the iCAR was reduced to a MOI of 10. All iCARs were confirmed to be expressed on the surface of primary T cells (Figure 4B). Observing the kinetics of cytotoxicity, we found that the TROP2-Long-BTLA, LAIR-1, and SIGLEC-9 iCARs all inhibited CAR T cell cytotoxicity at a similar rate as the TROP2-PD1 iCAR when co-cultured with target cells that expressed high levels of TROP2 (Figure 4B, C). When the TROP2 level was reduced in target cells (CEA^HI/TROP2^LO), the TROP2-Long-SIGLEC9 iCAR showed a reduced delay in inhibition compared to the TROP2-PD1 iCAR, suggesting that it might be more efficient. To test whether changing the extracellular spacer length might improve the efficiency of the iCAR, we tested these same constructs with a shorter spacer length. We found that these iCARs had approximately 30-70% less surface expression and were less efficient at inhibiting cytotoxicity compared to their long spacer counterparts (Figure 4B, C). Dual-inhibitory domain iCARs improve iCAR inhibitory kinetics and efficiency Third-generation CARs, which combine multiple co-stimulatory domains into one construct, have been reported to increase CAR T cell survival and antitumor efficacy (60–62). This led us to ask whether combining multiple inhibitory signaling domains into a single construct could further enhance inhibition efficiency. A series of dual-inhibitory domain iCARs (DiCARs) were designed by linking the TROP2-PD1 iCAR with an additional domain from PD-1, BTLA, SIGLEC-9, or LAIR-1 on its C-terminus (Figure 5A). These domains were chosen since they functioned as a single domain iCAR. Only the long extracellular spacer was incorporated into the DiCARs, because short spacer constructs were consistently shown to be less efficient as single domain iCARs (Figure 4B, C). Primary T cells were transduced with the CEACAR at a MOI of 1 and the DiCAR at a MOI of 1 to further reduce the contribution of avidity to inhibition. All DiCARs were confirmed to traverse to the cell surface as detected by flow cytometry (Figure 5B). DiCAR surface expression was similar between all constructs except for the PD1-BTLA DiCAR, which always had the lowest expression and transduction efficiency (Supplemental Figure 6 in Bangayan). Enriched CAR⁺/iCAR⁺ T cells (>94%) were co-cultured with target cells that expressed CEACAM5 and/or TROP2 and monitored for cytotoxicity over a week to observe the delay in inhibition. As represented in Figure 5C, three DiCARs (PD1-PD1, PD1-SIGLEC9, PD1-LAIR1) inhibited CAR T cell cytotoxicity more efficiently than the TROP2-PD1 iCAR as indicated by a faster recovery of target cells. The delay in inhibition was significantly decreased as calculated by AUC (Figure 5C) regardless of high or low TROP2 expression. This trend of improved inhibition by DiCARs was found to be reproducible in three independent experiments although the quantitative effect varied (Supplemental Figure 7 in Bangayan). To match the spacer in the CEACAR, the extracellular spacer of three inhibitory constructs (PD1, PD1-LAIR1, and PD1-SIGLEC9) were replaced with the 4/2NQ spacer (39) to reduce FcȖR binding for future studies (Supplemental Figure 8A in Bangayan). These iCAR and DiCAR constructs, hereby only referred to by their internal signaling domains (i.e. TROP2-42NQ-PD1-LAIR1 DiCAR à PD1-LAIR1 DiCAR), were confirmed to show similar or better inhibition efficiencies as compared to their original counterparts (Supplemental Figure 8B in Bangayan). Interestingly, in two additional experiments where the MOI of the CAR was 1 and the MOI of the DiCAR was 3, the PD1-LAIR1 DiCAR inhibited more efficiently than the PD1- SIGLEC9 DiCAR, especially at a higher effector:target ratio (5:1 E:T) (Supplemental Figure 9A, B in Bangayan). Epithelial cell markers like TROP2 can be used as iCAR targets for AND-NOT gating strategies By combining a CD-19 targeting CAR and a PSMA targeting iCAR, Federov et al. Sci Transl Med. 2013 Dec 11;5(215) showed that an AND-NOT gating strategy could potentially solve the on-target, off-tumor toxicity problem of CAR T cells (23). To make it clinically applicable, many groups began to target HLA molecules with the iCAR. Because HLA is expressed on most normal tissues but downregulated by tumor cells, this target could provide broad protection (24–27, 29). However, by using HLA-directed iCARs, CAR T therapy becomes subject to HLA-restriction, circumventing a key benefit it provided over TCR-based immunotherapies. Here we report that a CAR can also be combined with an iCAR targeting a normal epithelial cell marker like TROP2. Because TROP2 is widely expressed on the kidneys, lung, and skin, this iCAR can provide protection with different CAR modules without HLA-restriction (35). Future work targeting other broadly expressed epithelial cell markers like EpCAM (63), E-Cadherin (64), and Claudin-4 (65) could be promising. Balancing the levels of CAR and iCAR signaling is critical to obtaining specific inhibition While testing the TROP2-PD1 iCAR for specific inhibition against the CEACAR, we observed a delay in its ability to inhibit cytotoxicity. This delay was found to be avidity dependent and correlated to the iCAR:CAR ratio. This result may explain why in previous studies with both T cells and NK cells, iCAR inhibition was enhanced with its overexpression (23, 27). Interestingly, as the amount of iCAR increased, the level of ligand independent inhibition also increased (Figure 2E). Our data suggests that a balance between the number of CARs and iCARs signaling is critical to obtain specific inhibition. Accurate quantification of CAR and iCAR is necessary to determine a therapeutic window for this strategy. Why do the non-ITIM/ITSM inhibitory domains and higher affinity iCARs not improve iCAR inhibition? As an alternative to balancing the ratio of CAR and iCAR, we sought to build a more efficient iCAR. A modification we made was to replace the PD-1 domain with a non- ITIM/ITSM containing domain like LAG-3, TIM-3, or CTLA-4. None of the seven domains, including CTLA-4 which was reported by Fedorov et al. (23) to function, were capable of specifically inhibiting activity in our Jurkat activation screen (Supplemental Figure 5 in Bangayan). Although thirteen constructs were evaluated, the potential combinations of spacer/hinge, transmembrane domain, and signaling domain were not exhausted. Because spacers and transmembranes are known to affect CAR function (67, 68), we cannot exclude the possibility that inhibition could have been seen if another construct was used. It is unclear as to why intracellular signaling domains from known checkpoint inhibitors like LAG-3 and TIM-3 could not inhibit CAR T cell activation in this assay. Alternative inhibitory mechanisms utilized by these non-ITIM containing inhibitory receptors may be unable to inhibit CAR T cell activation. LAG-3 functions through its KIEELE and FxxL motif, but its mode of inhibition is unknown (69, 70). TIM-3 is thought to function by either destabilizing the immunological synapse through the recruitment of phosphatases or recruiting FYN and CSK to the membrane to inactivate Lck (71, 72). It may be that SHP-1 and/or SHP-2 phosphatases that are recruited via the ITIM motif are necessary for CAR inhibition. ITIM/ITSM domains are important motifs for iCAR inhibition and function This concept is further strengthened by the fact that the domains that have been shown capable of replacing PD-1 in an iCAR by other groups and ours all contain ITIM/ITSM motifs. The LIR-1 domain described by Hamburger et al contains four ITIM motifs (25), while all the NK receptor domains tested by Li et al. (KIR2DL1, LIR-1, CD300a, NKG2A, and LAIR-1) all contain varying numbers of ITIM or ITIM-like motifs (50). Because these motifs are important for the recruitment of the phosphatases SHP-1 and/or SHP-2, which dephosphorylate T cell activation proteins like Zap-70 and LAT (59), the number of ITIMs may correlate to iCAR inhibition efficiency. This may explain why the PD1-LAIR1 DiCAR outperforms all others although its surface expression is lower than the PD1-PD1 and PD1-SIGLEC9 DiCARs (Supplemental Figure 6, 9B in Bangayan). In total, the PD1- LAIR1 DiCAR would have three ITIMs and one ITSM, while the PD1-BTLA, PD1- PD1, and PD1-SIGLEC9 DiCARs would all have two ITIMs with varying numbers of ITSM or ITIM-like domains (59, 73, 74). By having one additional ITIM, the PD1- LAIR1 DiCAR may recruit more phosphatases to the membrane, increase dephosphorylation, and more rapidly inhibit CAR T cell activation. Furthermore, the LAIR-1 domain has been found to be constitutively associated with the phosphatase SHP-1 (75) and could be the reason why at higher avidities it shows increased ligand independent inhibition. Alternatively, since these phosphatases bind ITIM domains via a SH2 domain that can affect their activation (76, 77), as well as proximity to CARs, the geometry of ITIMs in these DiCARs may contribute to its inhibition efficiency. Future work should be focused on two major aspects of enhancing this AND- NOT gate design. First, efforts must be concentrated on determining which combinations of spacers, transmembrane domains, and inhibitory domains can be combined to generate DiCARs with enhanced specificity and reduced ligand- independent inhibition. The combination of domains assessed in DiCARs here were not exhaustive, and additional constructs may further enhance the dynamic range of this strategy. Second, additional experiments should be performed to determine the in vivo specificity of CAR⁺, DiCAR⁺ T cells. For these in vivo studies, a replacement pair of CAR and DiCAR antigens that are clinically relevant should be investigated. We suggest that these antigens match the following criteria: 1) the CAR antigen should have low expression in normal tissues, 2) the DiCAR antigen should have high expression in normal tissues that express the CAR antigen, and 3) the DiCAR antigen should be stably expressed on the surface of the cell and not be prone to cleavage. The TROP2 antigen selected in this study is suspected to be cleaved in vivo by proteases like ADAM17 (38), matriptase (78), and/or ADAM10 (79), which may explain why in preliminary studies we have found reduced expression of this antigen. Optimization of CAR dosage, DiCAR dosage, and T cells injected will need to be determined to achieve tumor elimination with reduced toxicity. Just as second-generation CARs combined a costimulatory domain with the activation domain to enhance CAR T cell function, the DiCARs presented here combine two inhibitory domains to become a second-generation iCAR. The AND- NOT gating strategy can be applied to reduce on-target, off-tumor toxicity by balancing the enhanced strength of CARs with the better regulation of DiCARs. Materials and Methods Cell Line Generation The DU145 prostate cancer target cell line was previously modified to knock- out TROP2 expression (CEA-/TROP2- )using a CRISPR-Cas9 strategy (43). To generate target lines that express CEA and/or TROP2, CEA and TROP2 were cloned into separate lentiviral constructs and transduced into the CEA-/TROP2- cell line. Each cell line was also engineered to express GFP for cytotoxicity assays. Following transduction, cells were single cell sorted for CEA, TROP2, and/or GFP expression. Clones were selected that had the desired surface expression of CEA and/or TROP2. Surface expression of CEA and TROP2 were confirmed by flow cytometry using the antibodies listed in Supplementary Table 2 in Bangayan. The Jurkat-NFAT-ZsGreen reporter cell line was a gift generated and given by Dr. David Baltimore’s lab. Lentivirus Production Lentivirus for the various CARs and iCARs were generated using a previously published protocol (80). Briefly, 293T cells were grown in DMEM + 10% FBS. 293T cells were transfected with Mirus TransIT 293 (Mirus, MIR2705). One day after transfection, cells were treated with 10mM Sodium Butyrate for 6-8 hours. Media was replaced with Collection Media (Ultraculture/Pro293-AM + Glutamax + 20mM HEPES). Two days later, viral supernatant was collected, filtered through a 0.45uM filter, and concentrated using Amicon Ultra-15 (100,000 NMWL) filters (Millipore, UFC910024). Virus was frozen and titered on 293T cells. Discovery of TROP2 binding antibodies using phage display A human scFv phage display library previously published by Li et al was used to discover antibodies binding TROP2 (42). The phage library was panned with recombinant TROP2 extracellular domain-Fc chimera (R&D Systems, 650-T2-10). Clones that bound TROP2 were found using an anti-M13 antibody that recognizes the phage by ELISA. Complete antibody molecules (scFv-Fc) were generated by linking the scFv to human IgG1 Fc on the C-terminus and cloned into an expression vector. Stable transfectants for antibody production were generated using Zeocin selection. Supernatant from these transfections were collected, filtered, purified, and concentrated to yield a concentration of 0.1-1mg/mL. These antibodies were confirmed to specifically bind TROP2 by flow cytometry against an engineered TROP2⁺ cell line. Sequences of the desired scFv’s were then utilized as the antigen- binding domain of iCARs. CAR and iCAR Vector Construction The CEACAM5 CAR was previously designed and produced by combining the CEACAM5-targeting scFv (Labetuzumab), an IgG4 hinge, the IgG4 CH2 and CH3 constant domains, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ȗ activation domain.(30) Modifications to the CEACAM5 CAR were made to replace the spacer region (IgG4 Hinge + CH2 + CH3) with a spacer developed by Hudecek et al, which we termed the 42N/Q spacer (39). Additional changes were made to replace the CD28 co-stimulatory domain with the 41BB costimulatory domain to generate the CAR used throughout this paper (CEACAR). Inhibitory CARs were generated using a similar structure to that previously published (23). Antibodies that react to TROP2 as discovered by screening a phage display library were converted into scFv chains. The scFv chain was linked to various extracellular spacers (Short – IgG4 Hinge; Long – IgG4 Hinge + CH2 + CH3; 42NQ – Modified IgG4 Hinge), the CD28 transmembrane domain, and a series of intracellular signaling domains from immune cell inhibitory receptors. The 42NQ hinge is described in Hudecek et al., Cancer Immunol Res. 2015 Feb;3(2):125-35. Hinge elements can typically be a combination of an IgG4 spacer with IgG2. For example, one using a hinge and CH3 constant domains of IgG4 but further incorporating an IgG2 CH2 constant domain in the middle. In addition, in such elements, artisans can make mutations in the sequence to remove N-linked glycosylation sites. The exact amino acids that were used for the intracellular signaling domains are listed in Supplementary Table 1 in Bangayan. DiCARs are generated by linking an anti-TROP2 scFv chain to an extracellular spacer, a CD28 transmembrane domain, a PD-1 signaling domain as listed in Supplementary Table 1 in Bangayan, and an additional signaling domain (i.e. PD-1, BTLA, SIGLEC-9, LAIR-1) as listed in Supplementary Table 1 in Bangayan. Both the CAR and iCAR were cloned into a third-generation lentiviral vector pCCL-c-MNDU3 generously given by Dr. Gay Crooks and Dr. Donald Kohn. Primary CAR T cell Generation, Enrichment, and Characterization Peripheral blood mononuclear cells (PBMCs) were purchased from All Cells, LLC from various donors. Unless stated otherwise, in each experiment, a single donor was used for all groups being compared to remove donor variability within the experiment. T cells and PBMCs were grown in TCM Base supplemented with the listed cytokines (TCM Base = AIM-V Media (Thermo Fisher, 12055) supplemented with 5% human heat-inactivated AB serum (Omega Scientific, HS-25), Glutamax (Thermo Fisher, 35050-061), and 55uM of Beta-mercaptoethanol). PBMCs were initially thawed and cultured in TCM Base + 50U/mL IL-2 (Peprotech, 200-02). PBMCs were activated with Human T-Activator CD3/CD28 Dynabeads (Thermo Fisher, 11132D) at a 1:1 Cell:Bead ratio and plated overnight at 37°C at a concentration of 1x10^6 cells/mL. The following day activated cells with beads were collected and resuspended in fresh TCM + 50U/mL IL-2 and diluted to a concentration of 0.5x10^6 cells/mL and plated into a 24-well plate. Cells were transduced with lentivirus containing the iCAR at the appropriate MOI of 1, 3, or 10. Infections were supplemented with Protamine Sulfate at a concentration of 100ug/mL. Six hours after incubation with the iCAR lentivirus, supernatant was removed, and CAR lentivirus was added with fresh Protamine Sulfate. The next day an additional 1mL of media was added to each well. Seven days after activation, Dynabeads were removed, and T cells were transferred to TCM Base + 50U/mL IL-2 + 0.5ng/mL IL- 15 (Peprotech, 200-15) media at a concentration of 1x10^6 cells/mL. On day 9, T cells were enriched for CAR+, iCAR+ T cells using magnetic bead enrichment. Briefly, CAR⁺ T cells were selected after staining with an Anti-FLAG-PE antibody and enriched using the EasySep Release Human PE Positive Selection Kit (Stemcell, 17654) since CARs were linked to a FLAG-tag on their N-terminal end. These cells were then selected for iCAR⁺ T cells by staining with an Anti-HA-APC antibody and enriched using the EasySep APC Positive Selection Kit (Stemcell, 17681) since iCARs were linked to a HA-tag on their N-terminal end. On day 11, magnetic beads used for enrichment were removed. On day 12, T cells were characterized by flow cytometry and used for various cytotoxicity assays. For ELISAs, forty-eight hours after co-culture began, supernatant was harvested from each well. Supernatant was used to measure IFN-Ȗ using the BD OptEIA Human IFN-Ȗ Set (BD, 555142). Jurkat Activation Reporter Assay To rapidly screen an iCAR’s potential to inhibit CAR T cell activity, a Jurkat reporter assay was utilized. The Jurkat-NFAT-ZsGreen reporter cell line was generously provided by Dr. David Baltimore. These cells were transduced with a lentivirus containing the CEACAM5-Long-CD28-3z CAR previously published by our lab at a MOI of 1 (30). CAR⁺ Jurkat cells were also transduced with a lentivirus containing the selected iCAR at a MOI of 25. CAR⁺/iCAR⁺ Jurkat cells were sorted and used in a co-culture assay. Jurkat cells were incubated with DU145 target cells for 24-hours at an Effector:Target ratio of 1:1 in RPMI + 10% FBS + Glutamine (RPMI10+). Jurkat cells were then collected from the culture and the percentage of ZsGreen+ cells were measured by flow cytometry. Gating was performed on CD3+ cells to ensure that ZsGreen+ target cells were not contributing to the measurement. T Cell Kinetic Cytotoxicity Assay Plates are coated with 0.001% Poly-L-Lysine for at least 30 minutes at 37°C. DU145 target cells that are GFP+ are collected from culture and plated in RPMI10+ (RPMI + 10% FBS + 40mM Glutamine) at the desired concentration into the coated plate. Effector CAR T cells are collected from culture and washed with 1X PBS. CAR T cells are counted and plated at the desired concentration in RPMI10+ into wells that contain target cells. Co-cultures are performed at the Effector:Target ratio described in the figures. Co-cultures are imaged using an Incucyte Zoom Live Cell Analysis System (Sartorius) over a week at approximately 2 hour intervals. Masking is performed to calculate the area covered by GFP+ target cells. Area under the curve analysis is performed using GraphPad Prism over time. Flow Cytometry Analysis Cells are collected from culture and washed with 1X PBS. Cells are stained with the selected antibodies in FACS Buffer (1X PBS + 3% Fetal Bovine Serum + 0.09% Sodium Azide). Antibodies that were used are listed in Supplementary Table 2 in Bangayan. After staining, cells are washed with 1X PBS and resuspended in FACS Buffer. Cells are run on the BD FACS Canto, the BD FACSAria, or the HT LSR II. Quantification of the amount of CAR and iCAR surface expression was performed using Quantum Simply Cellular anti-Mouse IgG (Bangs Laboratories, 815A) and anti- Rat IgG beads (Bangs Laboratories, 817A) using geometric mean fluorescence intensity. Xenograft model for CEACAR tumor killing Animal experiments were conducted according to a protocol approved by the Division of Laboratory Medicine at the University of California, Los Angeles. NSG mice were obtained from The Jackson Laboratory at six-eight weeks of age. Engineered DU145 lines that express CEACAM5 and/or TROP2, GFP, and YFP- Luciferase were mixed with Matrigel Matrix Basement Membrane (Corning 354234) and engrafted into mice subcutaneously on the right flank. T cells were prepared as described in Primary CAR T cell Generation, Enrichment, and Characterization. Approximately, three weeks after engraftment, when tumors were measurable (10-100 mm³), 2 X 10⁶ or 4 X 10⁶ T cells were injected into mice via teil-vein. Weekly caliper measurements were obtained of the tumors starting the second week after T cell injection. References 1. P. Zachery Halford, A Review of CAR T-Cell Therapies Approved for the Treatment of Relapsed and Refractory B-Cell Lymphomas (2022) (August 17, 2022). 2. A. Mullard, FDA approves second BCMA-targeted CAR-T cell therapy. Nature Reviews Drug Discovery 21, 249–249 (2022). 3. K. M. Maalej, et al., CAR-cell therapy in the era of solid tumor treatment: current challenges and emerging therapeutic advances. Molecular Cancer 22, 20 (2023). 4. A. Rodriguez-Garcia, A. Palazon, E. Noguera-Ortega, D. J. Powell, S. Guedan, CAR-T Cells Hit the Tumor Microenvironment: Strategies to Overcome Tumor Escape. Front Immunol 11, 1109 (2020). 5. F. C. Thistlethwaite, et al., The clinical efficacy of first-generation carcinoembryonic antigen (CEACAM5)-specific CAR T cells is limited by poor persistence and transient pre-conditioning-dependent respiratory toxicity. Cancer Immunol Immunother 66, 1425–1436 (2017). 6. C. H. J. Lamers, et al., Treatment of metastatic renal cell carcinoma with autologous T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin Oncol 24, e20-22 (2006). 7. C. H. Lamers, et al., Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity. Mol Ther 21, 904–912 (2013). 8. J. N. Brudno, J. N. Kochenderfer, Toxicities of chimeric antigen receptor T cells: recognition and management. Blood 127, 3321–3330 (2016). 9. R. A. Morgan, et al., Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 18, 843–851 (2010). 10. L. E. Budde, et al., Combining a CD20 Chimeric Antigen Receptor and an Inducible Caspase 9 Suicide Switch to Improve the Efficacy and Safety of T Cell Adoptive Immunotherapy for Lymphoma. PLOS ONE 8, e82742 (2013). 11. F. Ciceri, et al., Infusion of suicide-gene-engineered donor lymphocytes after family haploidentical haemopoietic stem-cell transplantation for leukaemia (the TK007 trial): a non-randomised phase I–II study. The Lancet Oncology 10, 489–500 (2009). 12. M. Griffioen, et al., Retroviral transfer of human CD20 as a suicide gene for adoptive T-cell therapy. Haematologica 94, 1316–1320 (2009). 13. X. Wang, et al., A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood 118, 1255–1263 (2011). 14. H. G. Caruso, et al., Tuning Sensitivity of CAR to EGFR Density Limits Recognition of Normal Tissue While Maintaining Potent Antitumor Activity. Cancer Res 75, 3505–3518 (2015). 15. X. Liu, et al., Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice. Cancer Res 75, 3596–3607 (2015). 16. R. Sakemura, et al., A Tet-On Inducible System for Controlling CD19- Chimeric Antigen Receptor Expression upon Drug Administration. Cancer Immunol Res 4, 658–668 (2016). 17. M. Cartellieri, et al., Switching CAR T cells on and off: a novel modular platform for retargeting of T cells to AML blasts. Blood Cancer Journal 6, e458–e458 (2016). 18. J. Qi, et al., Chemically Programmable and Switchable CAR-T Therapy. Angew Chem Int Ed Engl 59, 12178–12185 (2020). 19. M. S. Kim, et al., Redirection of Genetically Engineered CAR-T Cells Using Bifunctional Small Molecules. J. Am. Chem. Soc.137, 2832–2835 (2015). 20. X. Han, et al., Masked Chimeric Antigen Receptor for Tumor-Specific Activation. Mol Ther 25, 274–284 (2017). 21. C. C. Kloss, M. Condomines, M. Cartellieri, M. Bachmann, M. Sadelain, Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells. Nat Biotechnol 31, 71–75 (2013). 22. K. T. Roybal, et al., Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell 167, 419-432.e16 (2016). 23. V. D. Fedorov, M. Themeli, M. Sadelain, PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. Sci Transl Med 5, 215ra172 (2013). 24. L. Tao, et al., CD19-CAR-T Cells Bearing a KIR/PD-1-Based Inhibitory CAR Eradicate CD19+HLA-C1í Malignant B Cells While Sparing CD19+HLA-C1+ Healthy B Cells. Cancers (Basel) 12, 2612 (2020). 25. A. E. Hamburger, et al., Engineered T cells directed at tumors with defined allelic loss. Molecular Immunology 128, 298–310 (2020). 26. M. S. Hwang, et al., Targeting loss of heterozygosity for cancer-specific immunotherapy. Proceedings of the National Academy of Sciences 118, e2022410118 (2021). 27. F. Fei, L. Rong, N. Jiang, A. S. Wayne, J. Xie, Targeting HLA-DR loss in hematologic malignancies with an inhibitory chimeric antigen receptor. Molecular Therapy 30, 1215–1226 (2022). 28. R. M. Richards, et al., NOT-Gated CD93 CAR T Cells Effectively Target AML with Minimized Endothelial Cross-Reactivity. Blood Cancer Discov 2, 648–665 (2021). 29. M. L. Sandberg, et al., A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo. Science Translational Medicine 14, eabm0306 (2022). 30. J. K. Lee, et al., Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer. Proceedings of the

32. J. Zhou, et al., Identification of CEACAM5 as a Biomarker for Prewarning and Prognosis in Gastric Cancer. J Histochem Cytochem 63, 922–930 (2015). 33. R. H. Goslin, M. J. O’Brien, A. T. Skarin, N. Zamcheck, Immunocytochemical staining for CEA in small cell carcinoma of lung predicts clinical usefulness of the plasma assay. Cancer 52, 301–306 (1983). 34. M. R. Parkhurst, et al., T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 19, 620–626 (2011). 35. L. P. Stepan, et al., Expression of Trop2 cell surface glycoprotein in normal and tumor tissues: potential implications as a cancer therapeutic target. J Histochem Cytochem 59, 701–710 (2011). 36. M. Marhold, Current state of clinical development of TROP2-directed antibody–drug conjugates for triple-negative breast cancer. memo 15, 129–132 (2022). 37. A. S. Goldstein, et al., Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics. Proceedings of the National Academy of Sciences 105, 20882–20887 (2008). 38. T. Stoyanova, et al., Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via ȕ-catenin signaling. Genes Dev. 26, 2271– 2285 (2012). 39. M. Hudecek, et al., The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res 3, 125–135 (2015). 40. A. H. Long, et al., 4-1BB costimulation ameliorates T cell exhaustion induced by tonic signaling of chimeric antigen receptors. Nat Med 21, 581–590 (2015). 41. G. Li, et al., 4-1BB enhancement of CAR T function requires NF-^B and TRAFs. JCI Insight 3 (2018). 42. K. Li, et al., A fully human scFv phage display library for rapid antibody fragment reformatting. Protein Eng Des Sel 28, 307–316 (2015). 43. E.-C. Hsu, et al., Trop2 is a driver of metastatic prostate cancer with neuroendocrine phenotype via PARP1. Proceedings of the National Academy of Sciences 117, 2032–2042 (2020). 44. A. I. Salter, et al., Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function. Science Signaling 11, eaat6753 (2018). 45. S. I. Rudnick, G. P. Adams, Affinity and avidity in antibody-based tumor targeting. Cancer Biother Radiopharm 24, 155–161 (2009). 46. M. Chmielewski, A. Hombach, C. Heuser, G. P. Adams, H. Abken, T cell activation by antibody-like immunoreceptors: increase in affinity of the single-chain fragment domain above threshold does not increase T cell activation against antigen- positive target cells but decreases selectivity. J Immunol 173, 7647–7653 (2004). 47. S. Guedan, et al., Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. JCI Insight 3, 96976 (2018). 48. Y. Kagoya, et al., A novel chimeric antigen receptor containing a JAK–STAT signaling domain mediates superior antitumor effects. Nat Med 24, 352–359 (2018). 49. A. A. Hombach, J. Heiders, M. Foppe, M. Chmielewski, H. Abken, OX40 costimulation by a chimeric antigen receptor abrogates CD28 and IL-2 induced IL-10 secretion by redirected CD4+ T cells. Oncoimmunology 1, 458–466 (2012). 50. Y. Li, et al., KIR-based inhibitory CARs overcome CAR-NK cell trogocytosis-mediated fratricide and tumor escape. Nat Med, 1–12 (2022). 51. N. Acharya, C. Sabatos-Peyton, A. C. Anderson, Tim-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer 8, e000911 (2020). 52. M. Z. Wojtukiewicz, et al., Inhibitors of immune checkpoints—PD-1, PD-L1, CTLA-4—new opportunities for cancer patients and a new challenge for internists and general practitioners. Cancer Metastasis Rev 40, 949–982 (2021). 53. L. Long, et al., The promising immune checkpoint LAG-3: from tumor microenvironment to cancer immunotherapy. Genes Cancer 9, 176–189 (2018). 54. G. Voisinne, A. Gonzalez de Peredo, R. Roncagalli, CD5, an Undercover Regulator of TCR Signaling. Frontiers in Immunology 9 (2018). 55. E. J. Vazquez-Cintron, et al., Protocadherin-18 is a novel differentiation marker and an inhibitory signaling receptor for CD8+ effector memory T cells. PLoS One 7, e36101 (2012). 56. A. B. Frey, The Inhibitory Signaling Receptor Protocadherin-18 Regulates Tumor-Infiltrating CD8+ T-cell Function. Cancer Immunology Research 5, 920–928 (2017). 57. J. L. Lines, L. F. Sempere, T. Broughton, L. Wang, R. Noelle, VISTA Is a Novel Broad-Spectrum Negative Checkpoint Regulator for Cancer Immunotherapy. Cancer Immunology Research 2, 510–517 (2014). 58. W. Xu, T. HiӃu, S. Malarkannan, L. Wang, The structure, expression, and multifaceted role of immune-checkpoint protein VISTA as a critical regulator of anti- tumor immunity, autoimmunity, and inflammation. Cell Mol Immunol 15, 438–446 (2018). 59. T. Thaventhiran, et al., T Cell Co-inhibitory Receptors-Functions and Signalling Mechanisms. J Clin Cell Immunol S12 (2012). 60. D. Abate-Daga, M. L. Davila, CAR models: next-generation CAR modifications for enhanced T-cell function. Mol Ther Oncolytics 3, 16014 (2016). 61. A. A. Hombach, M. Chmielewski, G. Rappl, H. Abken, Adoptive immunotherapy with redirected T cells produces CCR7- cells that are trapped in the periphery and benefit from combined CD28-OX40 costimulation. Hum Gene Ther 24, 259–269 (2013). 62. S. Tammana, et al., 4-1BB and CD28 Signaling Plays a Synergistic Role in Redirecting Umbilical Cord Blood T Cells Against B-Cell Malignancies. Hum Gene Ther 21, 75–86 (2010). 63. U. Schnell, V. Cirulli, B. N. G. Giepmans, EpCAM: Structure and function in health and disease. Biochimica et Biophysica Acta (BBA) - Biomembranes 1828, 1989–2001 (2013). 64. E. Burandt, et al., E-Cadherin expression in human tumors: a tissue microarray study on 10,851 tumors. Biomarker Research 9, 44 (2021). 65. F. Facchetti, et al., Claudin 4 identifies a wide spectrum of epithelial neoplasms and represents a very useful marker for carcinoma versus mesothelioma diagnosis in pleural and peritoneal biopsies and effusions. Virchows Arch 451, 669– 680 (2007). 66. P. Sharma, et al., Structure-guided engineering of the affinity and specificity of CARs against Tn-glycopeptides. Proceedings of the National Academy of Sciences 117, 15148–15159 (2020). 67. S. Guedan, H. Calderon, A. D. Posey, M. V. Maus, Engineering and Design of Chimeric Antigen Receptors. Molecular Therapy - Methods & Clinical Development 12, 145–156 (2019). 68. L. Alabanza, et al., Function of Novel Anti-CD19 Chimeric Antigen Receptors with Human Variable Regions Is Affected by Hinge and Transmembrane Domains. Molecular Therapy 25, 2452–2465 (2017). 69. C. J. Workman, K. J. Dugger, D. A. A. Vignali, Cutting Edge: Molecular Analysis of the Negative Regulatory Function of Lymphocyte Activation Gene-31. The Journal of Immunology 169, 5392–5395 (2002). 70. T. K. Maeda, D. Sugiura, I. Okazaki, T. Maruhashi, T. Okazaki, Atypical motifs in the cytoplasmic region of the inhibitory immune co-receptor LAG-3 inhibit T cell activation. Journal of Biological Chemistry 294, 6017–6026 (2019). 71. K. L. Clayton, et al., T Cell Ig and Mucin Domain–Containing Protein 3 Is Recruited to the Immune Synapse, Disrupts Stable Synapse Formation, and Associates with Receptor Phosphatases. The Journal of Immunology 192, 782–791

73. J. Q. Zhang, G. Nicoll, C. Jones, P. R. Crocker, Siglec-9, a Novel Sialic Acid Binding Member of the Immunoglobulin Superfamily Expressed Broadly on Human Blood Leukocytes*. Journal of Biological Chemistry 275, 22121–22126 (2000). 74. F. Van Laethem, et al., LAIR1, an ITIM-Containing Receptor Involved in Immune Disorders and in Hematological Neoplasms. International Journal of Molecular Sciences 23, 16136 (2022). 75. J. G. Sathish, et al., Constitutive Association of SHP-1 with Leukocyte- Associated Ig-Like Receptor-1 in Human T Cells1. The Journal of Immunology 166, 1763–1770 (2001). 76. D. N. Burshtyn, W. Yang, T. Yi, E. O. Long, A Novel Phosphotyrosine Motif with a Critical Amino Acid at Position í2 for the SH2 Domain-mediated Activation of the Tyrosine Phosphatase SHP-1*. Journal of Biological Chemistry 272, 13066– 13072 (1997). 77. M. Marasco, et al., Molecular mechanism of SHP2 activation by PD-1 stimulation. Science Advances 6, eaay4458 (2020). 78. P. R. Kamble, et al., Proteolytic cleavage of Trop2 at Arg87 is mediated by matriptase and regulated by Val194. FEBS Letters 594, 3156–3169 (2020). 79. M. Trerotola, et al., Trop^2 cleavage by ADAM10 is an activator switch for cancer growth and metastasis. Neoplasia 23, 415–428 (2021). 80. C. S. Seet, et al., Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids. Nat Methods 14, 521–530 (2017).

IgG4 Hinge ESKYGPPCPPCP (SEQ ID NO: 5) IgG4 CH2 Domain APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAK (SEQ ID NO: 6) IgG4 CH3 Domain GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK (SEQ ID NO: 7) *As an alternative spacer that we will be using in the in vivo studies, we used a variant of the IgG4 Hinge/CH2/CH3 spacer with reduced binding in the lung. This spacer was designed by Hudecek et al (Hudecek, Cancer Immunol Res, 2015). IgG4 Alternative 42NQ Spacer ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 8) For Intracellular Signaling Domains Uniprot Code for PD1: Q15116 (Based on the Fedorov, Sci Trans Med, 2013 construct) PD1 Domain CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPV PCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 1)

PSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKL TVLGTRESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWV CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPE QTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 9) H11Trop2scFv-IgG4Hinge-CH2-CH3-CD28TM-PD1PD1 AEVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLE WVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR FGLGYCSSTSCRGGYYGMDVWGQGTTVTVSSGGSTGGGSGGGGSTGAPQSV LTQPPSVSGAPGQRVTISCAGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNR PSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKL TVLGTRESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWV CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPE QTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLCSRAARGTI GARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFP SGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 10) H11Trop2scFv-42NQHinge-CD28TM-PD1 AEVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLE WVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR FGLGYCSSTSCRGGYYGMDVWGQGTTVTVSSGGSTGGGSGGGGSTGAPQSV LTQPPSVSGAPGQRVTISCAGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNR PSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKL TVLGTRESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

AEVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLE WVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR FGLGYCSSTSCRGGYYGMDVWGQGTTVTVSSGGSTGGGSGGGGSTGAPQSV LTQPPSVSGAPGQRVTISCAGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNR

RPAAGVGDTGIEDANAVRGSASQGPLTEPWAEDSPPDQPPPASARSSVGEGEL QYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 14) H11Trop2scFv-IgG4Hinge-CH2-CH3-CD28TM-PD1-LAIR1 AEVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLE WVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR FGLGYCSSTSCRGGYYGMDVWGQGTTVTVSSGGSTGGGSGGGGSTGAPQSV LTQPPSVSGAPGQRVTISCAGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNR PSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKL TVLGTRESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWV CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPE QTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLHRQNQIKQG PPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQ EVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO: 15) H11Trop2scFv-42NQHinge-CH2-CH3-CD28TM-PD1-SIGLEC9 AEVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLE WVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR FGLGYCSSTSCRGGYYGMDVWGQGTTVTVSSGGSTGGGSGGGGSTGAPQSV LTQPPSVSGAPGQRVTISCAGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNR PSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKL TVLGTRESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWV CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPE QTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLVRSCRKKSA RPAAGVGDTGIEDANAVRGSASQGPLTEPWAEDSPPDQPPPASARSSVGEGEL QYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 16) H11Trop2scFv-42NQHinge-CH2-CH3-CD28TM-PD1-LAIR1 AEVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLE WVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR FGLGYCSSTSCRGGYYGMDVWGQGTTVTVSSGGSTGGGSGGGGSTGAPQSV LTQPPSVSGAPGQRVTISCAGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNR PSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKL TVLGTRESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGK EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGKMFWVLVVVGGVLACYSLLVTVAFIIFWV CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPE QTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLHRQNQIKQG PPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQ EVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO: 17) CONCLUSION This concludes the description of the illustrative embodiments of the present invention. The foregoing description of one or more embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of the above teaching.

Claims

CLAIMS: 1. A composition including an inhibitory chimeric antigen receptor polypeptide, wherein the polypeptide comprises: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein the intracellular signaling domain comprises a first segment of amino acids comprising a first protein inhibitory domain and a second segment of amino acids comprising a second protein inhibitory domain, wherein the first segment of amino acids or the second segment of amino acids comprise amino acids from a PD1 protein inhibitory domain, a BTLA protein inhibitory domain, a SIGLEC9 protein inhibitory domain or a LAIR1 protein inhibitory domain.

2. The composition of claim 1, wherein: the PD1 protein inhibitory domain has an at least 90% sequence identity to: CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQT EYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 1); the BTLA protein inhibitory domain has an at least 90% sequence identity to: RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLC FRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVR S (SEQ ID NO: 2); the SIGLEC9 protein inhibitory domain has an at least 90% sequence identity to: VRSCRKKSARPAAGVGDTGIEDANAVRGSASQGPLTEPWAEDSPPDQPPPASAR SSVGEGELQYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 3); and the LAIR1 protein inhibitory domain has an at least 90% sequence identity to: HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSA LAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO: 4).

3. The composition of claim 1, wherein the inhibitory chimeric antigen receptor polypeptide comprises a hinge domain and/or a spacer domain.

4. The composition of claim 1, wherein the antigen binding domain comprises amino acids derived from an antibody polypeptide or a T cell receptor polypeptide.

5. A composition of matter comprising a polynucleotide encoding an inhibitory chimeric antigen receptor polypeptide of claim 1.

6. The composition of claim 5, wherein the polynucleotide is disposed in a mammalian cell expression vector.

7. A composition of matter comprising a mammalian cell transduced with a vector comprising a polynucleotide of claim 5.

8. The composition of claim 7, wherein the mammalian cell is a human CD 8⁺ T cell.

9. The composition of claim 8, wherein the mammalian cell further comprises an exogenous nucleic acid encoding a chimeric antigen receptor (CAR).

10. The composition of claim 9, wherein the inhibitory chimeric antigen receptor polypeptide and the chimeric antigen receptor target the same antigen.

11. The composition of claim 9, wherein the inhibitory chimeric antigen receptor polypeptide and the chimeric antigen receptor target different antigens.

12. The composition of claim 1, wherein the inhibitory chimeric antigen receptor polypeptide has an at least 90% identity to an inhibitory chimeric antigen receptor polypeptide shown in Table A.

13. A method of modulating the ability of a human CD 8⁺ T cell to target an antigen, wherein the human CD 8⁺ T cell expresses a chimeric antigen receptor, the method comprising introducing an exogenous nucleic acid into the cell, wherein the exogenous nucleic acid expresses an inhibitory chimeric antigen receptor polypeptide of claims 1-4 so that the ability of a human CD 8⁺ T cell to target an antigen is modulated.

14. The method of claim 13, wherein the human CD 8⁺ T cell is obtained from an individual diagnosed with a cancer.

15. The method of claim 14, wherein the chimeric antigen receptor targets a peptide expressed by cancer cells.

16. A method of making an inhibitory chimeric antigen receptor polypeptide comprising: forming a polynucleotide encoding: an antigen binding domain; a transmembrane domain; and an intracellular signaling domain, wherein: the intracellular signaling domain comprises a first segment of amino acids comprising a first protein inhibitory domain and a second segment of amino acids comprising a second protein inhibitory domain, wherein the first segment of amino acids or the second segment of amino acids comprise amino acids from a PD1 protein inhibitory domain, a BTLA protein inhibitory domain, a SIGLEC9 protein inhibitory domain or a LAIR1 protein inhibitory domain, the PD1 protein inhibitory domain has an at least 90% sequence identity to: CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQT EYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 1); the BTLA protein inhibitory domain has an at least 90% sequence identity to: RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLC FRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVR S (SEQ ID NO: 2); the SIGLEC9 protein inhibitory domain has an at least 90% sequence identity to: VRSCRKKSARPAAGVGDTGIEDANAVRGSASQGPLTEPWAEDSPPDQPPPASAR SSVGEGELQYASLSFQMVKPWDSRGQEATDTEYSEIKIHR (SEQ ID NO: 3); and the LAIR1 protein inhibitory domain has an at least 90% sequence identity to: HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSA LAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH (SEQ ID NO: 4); and expressing the inhibitory chimeric antigen receptor polypeptide encoded by the polynucleotide such that a inhibitory chimeric antigen receptor polypeptide is made.

17. The method of claim 16, further comprising disposing the polynucleotide in a mammalian expression vector.

18. The method of claim 17, further comprising disposing the mammalian expression vector in a mammalian cell.

19. The method of claim 18, wherein the mammalian cell is a human CD 8⁺ T cell. 20 The method of claim 19, wherein the human CD 8⁺ T cell is obtained from an individual diagnosed with a cancer.