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SNAP-tag, a mutant of the O 6 -alkylguanine-DNA-alkyltransferase, self-labels by reacting with benzylguanine (BG) substrates, thereby forming a thioether bond. SNAP-tag has been genetically fused to a wide range of proteins of interest in order to covalently modify them. In the context of both diagnostic and therapeutic applications, as well as use as a biological recording device, precise control in a spatial and temporal fashion over the covalent bond-forming reaction is desired to direct inputs, readouts, or therapeutic actions to specific locations, at specific time points, in cells and organisms. Here, we introduce a comprehensive suite of six caged BG molecules: one light-triggered and five others that can be activated through various chemical and biochemical stimuli, such as small molecules, transition metal catalysts, reactive oxygen species, and enzymes. These molecules are unable to react with SNAP-tag until the trigger is present, which leads to near complete SNAP-tag conjugation, as illustrated both in biochemical assays and on human cell surfaces. This approach holds promise for targeted therapeutic assembly at disease sites, offering the potential to reduce off-target effects and toxicity through precise trigger titration.

Background/objectives: Mucin-1 (MUC1) is a transmembrane glycoprotein that is overexpressed and hypoglycosylated in premalignant and malignant epithelial cells compared to normal cells, creating a target antigen for humoral and cellular immunity. Healthy individuals with a history of advanced colonic adenomas and at high risk for colon cancer were enrolled in a clinical trial to evaluate the feasibility of using a MUC1 peptide vaccine to prevent colon cancer. Anti-MUC1 antibodies elicited by this vaccine were cloned using peripheral blood B cells and sera collected two weeks after a one-year booster. Twelve of these fully human monoclonal antibodies (mAb) were tested for binding to MUC1+ target cells, and three with the highest binding were further evaluated for various effector functions important for tumor rejection., Methods: Immune cells were incubated together with target cells expressing variations in the number, distance, and membrane anchoring properties of the MUC1 epitope in the presence of each mAb., Results: All three mAbs mediated antibody-dependent cytokine release (ADCR), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). Two also mediated antibody-dependent trogocytosis/trogoptosis (ADCT). None were capable of complement-dependent cytotoxicity (CDC)., Conclusions: ADCP and ADCT functions were more efficient when antibodies bound epitopes proximal to and anchored to the membrane, providing insight for future therapeutic antibody validation strategies.

The success of chimeric antigen receptor (CAR) T cells in blood cancers has intensified efforts to develop CAR T therapies for solid cancers. In the solid tumor microenvironment, CAR T cell trafficking and suppression of cytotoxic killing represent limiting factors for therapeutic efficacy. Here, we present a microwell platform to study CAR T cell interactions with 3D breast tumor spheroids and determine predictors of anti-tumor CAR T cell function. To precisely control antigen sensing, we utilized a switchable adaptor CAR system that covalently attaches to co-administered antibody adaptors and mediates antigen recognition. Following the addition of an anti-HER2 adaptor antibody, primary human CAR T cells exhibited higher infiltration, clustering, and secretion of effector cytokines. By tracking CAR T cell killing in individual spheroids, we showed the suppressive effects of spheroid size and identified the initial CAR T cell to spheroid area ratio as a predictor of cytotoxicity. We demonstrate that larger spheroids exhibit higher hypoxia levels and are infiltrated by CAR T cells with a suppressed activation state, characterized by reduced expression of IFN-γ, TNF-α, and granzyme B. Spatiotemporal analysis revealed lower CAR T cell numbers and cytotoxicity in the spheroid core compared to the periphery. Finally, increasing CAR T cell seeding density resulted in higher CAR T cell infiltration and cancer cell elimination in the spheroid core. Our findings provide new quantitative insight into CAR T cell function within 3D cancer spheroids. Given its miniaturized nature and live imaging capabilities, our microfabricated system holds promise for screening cellular immunotherapies., Competing Interests: Yes, J.L. is an inventor on a patent application field by the University of Pittsburgh on the universal SNAP-CAR technology used herein (WO2020072764A1). The remaining authors declare no competing interest., (© 2024 Author(s).)

The success of chimeric antigen receptor (CAR) T cells in blood cancers has intensified efforts to develop CAR T therapies for solid cancers. In the solid tumor microenvironment, CAR T cell trafficking and suppression of cytotoxic killing represent limiting factors for therapeutic efficacy. Here, we present a microwell platform to study CAR T cell interactions with 3D tumor spheroids and determine predictors of anti-tumor CAR T cell function. To precisely control antigen sensing by CAR T cells, we utilized a switchable adaptor CAR system, that instead of directly binding to an antigen of interest, covalently attaches to co-administered antibody adaptors that mediate tumor antigen recognition. Following addition of an anti-HER2 adaptor antibody, primary human CAR T cells exhibited higher infiltration and clustering compared to the no adaptor control. By tracking CAR T cell killing at the individual spheroid level, we showed the suppressive effects of spheroid size and identified the initial CAR T cell : spheroid area ratio as a predictor of cytotoxicity. Spatiotemporal analysis revealed lower CAR T cell numbers and cytotoxicity in the spheroid core compared to the periphery. Finally, increasing CAR T cell seeding density, resulted in higher CAR T cell infiltration and cancer cell elimination in the spheroid core. Our findings provide new quantitative insights into CAR T cell-mediated killing of HER2+ breast tumor cells. Given the miniaturized nature and live imaging capabilities, our microfabricated system holds promise for discovering cell-cell interaction mechanisms that orchestrate antitumor CAR T cell functions and screening cellular immunotherapies in 3D tumor models.

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 nontraditional 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 coadministered 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 the precision targeting of universal CAR T cells with potential for enhanced safety.

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., (© 2023. The Author(s).)

Background: Cellular immunotherapies for cancer represent a means by which a patient's immune system can be augmented with high numbers of tumor-specific T cells. Chimeric antigen receptor (CAR) therapy involves genetic engineering to 'redirect' peripheral T cells to tumor targets, showing remarkable potency in blood cancers. However, due to several resistance mechanisms, CAR-T cell therapies remain ineffective in solid tumors. We and others have shown the tumor microenvironment harbors a distinct metabolic landscape that produces a barrier to immune cell function. Further, altered differentiation of T cells within tumors induces defects in mitochondrial biogenesis, resulting in severe cell-intrinsic metabolic deficiencies. While we and others have shown murine T cell receptor (TCR)-transgenic cells can be improved through enhanced mitochondrial biogenesis, we sought to determine whether human CAR-T cells could be enabled through a metabolic reprogramming approach., Materials and Methods: Anti-EGFR CAR-T cells were infused in NSG mice which bore A549 tumors. The tumor infiltrating lymphocytes were analyzed for exhaustion and metabolic deficiencies. Lentiviruses carrying PPAR-gamma coactivator 1α (PGC-1α), PGC-1α S571A and NT-PGC-1α constructs were used to co-transduce T cells with anti-EGFR CAR lentiviruses. We performed metabolic analysis via flow cytometry and Seahorse analysis in vitro as well as RNA sequencing. Finally, we treated therapeutically A549-carrying NSG mice with either PGC-1α or NT-PGC-1α anti-EGFR CAR-T cells. We also analyzed the differences in the tumor-infiltrating CAR-T cells when PGC-1α is co-expressed., Results: Here, in this study, we show that an inhibition resistant, engineered version of PGC-1α, can metabolically reprogram human CAR-T cells. Transcriptomic profiling of PGC-1α-transduced CAR-T cells showed this approach effectively induced mitochondrial biogenesis, but also upregulated programs associated with effector functions. Treatment of immunodeficient animals bearing human solid tumors with these cells resulted in substantially improved in vivo efficacy. In contrast, a truncated version of PGC-1α, NT-PGC-1α, did not improve the in vivo outcomes., Conclusions: Our data further support a role for metabolic reprogramming in immunomodulatory treatments and highlight the utility of genes like PGC-1α as attractive candidates to include in cargo along with chimeric receptors or TCRs for cell therapy of solid tumors., Competing Interests: Competing interests: GMD has filed patent applications around the use of PGC-1α as a metabolic reprogramming agent in cellular therapies. BB was an employee of 2seventy bio during the period of the research and owns stock of the same company. EC was an employee of 2seventy bio during the period of the research., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The modeling of chimeric antigen receptor (CAR) T cell therapies has been mostly focused on immunodeficient models. However, there are many advantages in studying CAR-T cell biology in an immunocompetent setting. We generated a fully murine CAR targeting CD105 (endoglin), a component of the TGFβ receptor expressed on the surface of certain solid tumors and acute leukemias. CD105-targeted CAR-T cells can be grown from various murine backgrounds, tracked in vivo by congenic marks, and be activated by CD105 in isolation or expressed by tumor cells. CD105-targeted CAR-T cells were toxic at higher doses but proved safe in lower doses and modestly effective in treating wild-type B16 melanoma-bearing mice. CAR-T cells infiltrating the tumor expressed high levels of exhaustion markers and exhibited metabolic insufficiencies. We also generated a human CD105 CAR, which was efficacious in treating human melanoma and acute myeloid leukemia in vivo . Our work details a new murine model of CAR-T cell therapy that can be used from immunologists to further our understanding of CAR-T cell biology. We also set the foundation for further exploration of CD105 as a possible human CAR-T cell target., Competing Interests: G.M.D. has filed patent applications around the use of PGC-1α as a metabolic reprogramming agent in cellular therapies., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Expression of the transmembrane protein Tim-3 is increased on dysregulated T cells undergoing chronic activation, including during chronic infection and in solid tumors. Thus, Tim-3 is generally thought of as an inhibitory protein. We and others previously reported that under some circumstances, Tim-3 exerts paradoxical costimulatory activity in T cells (and other cells), including enhancement of the phosphorylation of ribosomal S6 protein. Here, we examined the upstream signaling pathways that control Tim-3-mediated increases in phosphorylated S6 in T cells. We also defined the localization of Tim-3 relative to the T cell immune synapse and its effects on downstream signaling. Recruitment of Tim-3 to the immune synapse was mediated exclusively by the transmembrane domain, replacement of which impaired the ability of Tim-3 to costimulate T cell receptor (TCR)-dependent S6 phosphorylation. Furthermore, enforced localization of the Tim-3 cytoplasmic domain to the immune synapse in a chimeric antigen receptor still enabled T cell activation. Together, our findings are consistent with a model whereby Tim-3 enhances TCR-proximal signaling under acute conditions., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Successes of immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) T cell therapy in curing patients with otherwise lethal cancers have validated immunotherapy as a treatment for cancer and have inspired excitement for its broader potential. Most promising is the ability of each approach to eliminate bulky and advanced-stage cancers and to achieve durable cures. Despite this success, to date only a subset of cancer patients and a limited number of cancer types respond to these therapies. A major goal now is to expand the types of cancer and number of patients who can be successfully treated. To this end a multitude of immunotherapies are being tested clinically in new combinations, and many new immunomodulatory antibodies and CARs are in development. A third major immunotherapeutic approach with renewed interest is cancer vaccines. While over 20years of therapeutic cancer vaccine trials have met with limited success, these studies have laid the groundwork for the use of therapeutic vaccines in combination with other immunotherapies or alone as prophylactic cancer vaccines. Prophylactic vaccines are now poised to revolutionize cancer prevention as they have done for the prevention of infectious diseases. In this review we examine three major cancer immunotherapy modalities: immunomodulatory antibodies, CAR T cell therapy and vaccines. For each we describe the current state of the art and outline major challenges and research directions forward., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An adaptive variant of the human Ectodysplasin receptor, EDARV370A, is one of the strongest candidates of recent positive selection from genome-wide scans. We have modeled EDAR370A in mice and characterized its phenotype and evolutionary origins in humans. Our computational analysis suggests the allele arose in central China approximately 30,000 years ago. Although EDAR370A has been associated with increased scalp hair thickness and changed tooth morphology in humans, its direct biological significance and potential adaptive role remain unclear. We generated a knockin mouse model and find that, as in humans, hair thickness is increased in EDAR370A mice. We identify new biological targets affected by the mutation, including mammary and eccrine glands. Building on these results, we find that EDAR370A is associated with an increased number of active eccrine glands in the Han Chinese. This interdisciplinary approach yields unique insight into the generation of adaptive variation among modern humans., (Copyright © 2013 Elsevier Inc. All rights reserved.)

One of the longstanding challenges in synthetic biology is rational design of complex regulatory circuitry with multiple biological inputs, complex internal processing, and physiologically active outputs. We have previously proposed how to address this challenge in the case of transcription factor inputs. Here we describe the methods used to construct these synthetic circuits, capable of performing logic integration of transcription factor inputs using microRNA expression vectors and RNA interference (RNAi). The circuits operate in mammalian cells and they can serve as starting point for more complex synthetic information processing networks in these cells.

Molecular-level information processing is essential for 'smart' in vivo nanosystems. Natural molecular computing, such as the regulation of messenger RNA (mRNA) synthesis by special proteins called transcription factors, has inspired engineered systems that can control the levels of mRNA with certain combinations of transcription factors. Here, we show an alternative approach to achieving general-purpose control of mRNA and protein levels by logic integration of transcription factor input signals in mammalian cells. The transcription factors regulate synthetic genes coding for small regulatory RNAs (called microRNAs), which, in turn, control the mRNA of interest (the output) via an RNA interference pathway. The simplicity of these modular interactions makes it possible, in theory, to implement any arbitrary logic relation between the transcription factors and the output. We construct, test and optimize increasingly complex circuits with up to three transcription factor inputs, establishing a platform for in vivo molecular computing.

With the advent of dense maps of human genetic variation, it is now possible to detect positive natural selection across the human genome. Here we report an analysis of over 3 million polymorphisms from the International HapMap Project Phase 2 (HapMap2). We used 'long-range haplotype' methods, which were developed to identify alleles segregating in a population that have undergone recent selection, and we also developed new methods that are based on cross-population comparisons to discover alleles that have swept to near-fixation within a population. The analysis reveals more than 300 strong candidate regions. Focusing on the strongest 22 regions, we develop a heuristic for scrutinizing these regions to identify candidate targets of selection. In a complementary analysis, we identify 26 non-synonymous, coding, single nucleotide polymorphisms showing regional evidence of positive selection. Examination of these candidates highlights three cases in which two genes in a common biological process have apparently undergone positive selection in the same population:LARGE and DMD, both related to infection by the Lassa virus, in West Africa;SLC24A5 and SLC45A2, both involved in skin pigmentation, in Europe; and EDAR and EDA2R, both involved in development of hair follicles, in Asia.