Immune checkpoint therapy has revolutionized cancer treatment by blocking inhibitory pathways in T cells that limits the an effective anti-tumor immune response. Therapeutics targeting CTLA-4 and PD1/PDL1 have progressed to first line therapy in multiple tumor types with some patients exhibiting tumor regression or remission. However, the majority of patients do not benefit from checkpoint therapy emphasizing the need for alternative therapeutic options. Lymphocyte Activation Gene 3 (LAG3) or CD223 is expressed on multiple cell types including CD4 + and CD8 + T cells, and T regs , and is required for optimal T cell regulation and homeostasis. Persistent antigen-stimulation in cancer or chronic infection leads to chronic LAG3 expression, promoting T cell exhaustion. Targeting LAG3 along with PD1 facilitates T cell reinvigoration. A substantial amount of pre-clinical data and mechanistic analysis has led to LAG3 being the third checkpoint to be targeted in the clinic with nearly a dozen therapeutics under investigation. In this review, we will discuss the structure, function and role of LAG3 in murine and human models of disease, including autoimmune and inflammatory diseases, chronic viral and parasitic infections, and cancer, emphasizing new advances in the development of LAG3-targeting immunotherapies for cancer that are currently in clinical trials.

Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop “universal” receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells.

Chimeric antigen receptors (CARs) are artificial T cell receptors that re-target patients’ T cells to specifically recognize and kill tumor cells. Despite remarkable success of anti-CD19 CAR therapy against refractory B cell leukemias, there are several limitations to CAR T cell therapies including toxicities and antigen-loss leading to relapse. To address these issues, we previously developed a “universal” CAR, SNAP-CAR, for which antigen-specificity is directed by co-administered tumor-targeted antibodies. Instead of directly recognizing a tumor antigen, the SNAP-CAR carries out an enzymatic reaction to fuse with antibodies conjugated to a benzylguanine (BG) tag. Activation and effector functions of SNAP CAR T cells can be re-targeted by antibody-BG conjugates to several antigens including: CD20, CD19, HER2, and EGFR. We are now developing SNAP-CAR T cells for potential clinical translation including optimization of the CAR expression construct and functional characterization in a human tumor xenograft mouse model. SNAP-CAR-T2A-LNGFR was cloned into a gamma-retroviral expression system, yielding a 10-fold greater expression level in primary human T cells compared to our previous lentiviral vector. SNAP-CAR T cells were potently activated and lysed tumor cells in a target antigen-specific manner in vitro. Challenging NSG mice with CD20+ Raji leukemia cells, significant tumor regression was observed with SNAP-CAR T cells + Rituximab-BG and anti-CD20-CAR positive control T cells but not SNAP-CAR T cells without antibody, as compared to untransduced T cells + Rituximab-BG. The SNAP-CAR provides a powerful system to program the antigen-targeting capability of human T cells with promise for cancer therapy.

As living drugs, engineered T cell therapies are revolutionizing disease treatment with their unique functional capabilities. However, they suffer from limitations of potentially unpredictable behavior, toxicities, and non-traditional pharmacokinetics. Engineering conditional control mechanisms responsive to tractable stimuli such as small molecules or light is thus highly desirable. We and others previously developed universal chimeric antigen receptors (CARs) that interact with co-administered antibody adaptors to direct target cell killing and T cell activation. Universal CARs are of high therapeutic interest due to their ability to simultaneously target multiple antigens on the same disease or different diseases by combining with adaptors to different antigens. Here, we further enhance the programmability and potential safety of universal CAR T cells by engineering OFF-switch adaptors that can conditionally control CAR activity, including T cell activation, target cell lysis, and transgene expression, in response to a small molecule or light stimulus. Moreover, in adaptor combination assays, OFF-switch adaptors were capable of orthogonal conditional targeting of multiple antigens simultaneously following Boolean logic. OFF-switch adaptors represent a robust new approach for precision targeting of universal CAR T cells with potential for enhanced safety.