Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (> 230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors, CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.

Supplementary Figure S5. Intratumoral multiplex RNA-ISH IF imaging and analysis of CAR-TEAM treated mice.

Chimeric antigen receptor (CAR) T cell therapy is a medical breakthrough in the treatment of B cell malignancies. There is intensive focus on developing solid tumor-targeted CAR-T cell therapies. Although clinically approved CAR-T cell therapies target B cell lineage antigens, solid tumor targets include neoantigens and tumor-associated antigens (TAAs) with diverse roles in tumor biology. Multiple early-stage clinical trials now report encouraging signs of efficacy for CAR-T cell therapies that target solid tumors. We review the landscape of solid tumor target antigens from the perspective of cancer biology and gene regulation, together with emerging clinical data for CAR-T cells targeting these antigens. We then discuss emerging synthetic biology strategies and their application in the clinical development of novel cellular immunotherapies.

AbstractPurpose: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell–engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). Experimental Design: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. Results: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. Conclusions: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

Supplementary Figure S7. Superior anti-tumor effect of meso^FAP compared to the combination of meso-CAR and FAP-CAR or meso^CD19.

Supplementary Figure S8. Target expression and individual analysis of patient-derived organoids (PDO) with matching CAFs.

Abstract While CAR T cell therapy is a medical breakthrough against various hematological malignancies, yet treatment failures often occur at least in part due to excessive engineered T cell activity leading to severe toxicities. To combat this, strategies pairing CAR T cells with safety systems, for example, suicide switches engineered to induce cell death are being actively pursued. However, existing designs have limited performance characteristics and generally use unapproved small molecule controllers or non-human sequences. To overcome these issues, we have developed a novel suicide switch leveraging the well-tolerated, FDA-approved anti-cancer drug lenalidomide, as a controller of the stability of fusion proteins tagged with a “degron” sequence. Specifically, we engineered a stoichiometric pair of transgenes using the apoptotic DNase pair, caspase activating DNase (CAD) and its degron tagged inhibitor (ICAD-degron). We hypothesized that the overexpressed ICAD-degron could restrain CAD, until lenalidomide treatment degrades ICAD to unleash CAD for apoptosis. We found that our system was stably overexpressed and rapidly induced cell death even at sub-nanomolar concentrations of lenalidomide. A comparison of our design with the frontline safety switch, inducible Caspase 9 in primary human T cells revealed that the lenalidomide-induced suicide switch enabled more complete depletion of engineered cells. We tested the effects of our suicide switch on CAR T cell function and found comparable proliferation and tumor cell cytolysis with fast depletion upon lenalidomide addition. In vivo, lenalidomide suicide switch CAR T cells exhibited similar anti-tumor function as control CAR T cells and were rapidly depleted after drug treatment. In summary, utilizing all human sequences and a clinically approved drug controller, we developed a suicide switch that is well tolerated and can rapidly induce cell death even at sub-therapeutic concentrations of lenalidomide. More broadly, chemogenetic regulation of stoichiometric protein pairs is a generalizable strategy for post-translational control of highly active transgenic elements. Citation Format: Ditsa Sarkar, William Lin, Joanna Y. Kim, Nelson H. Knudsen, Isabel C. Lane, Tamina Kienka, Michael C. Kann, Amanda A. Bouffard, Andy Cheng, Marcela V. Maus, Robert T. Manguso, Max Jan. Developing a lenalidomide-inducible safety switch for CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6321.

Supplementary Figure S2. In vitro setup of CARTEAM to CAF:AsPC-1 with different ratios showing superiority of mesoFAP compared to CARs targeting mesothelin only.

Supplementary Figure S6. Human CAF-1 cells do not expand in vivo in NSG mice in the presence or absence of AsPC-1 or PDX1294 cells.

Supplementary Figure S4. Still frames from live cell microscopy videos demonstrating reduced edge dynamics of meso^FAP CAR T-cells compared to controls and target cell layer on flow chamber of acoustic microscopy.

Supplementary Video S9. UTD T cells on AsPC1 + CAF

Proteolysis-targeting chimeras (PROTACs) are molecules that induce proximity between target proteins and E3 ligases triggering target protein degradation. Pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other proteins, including zinc-finger (ZF) proteins, with vital roles in health and disease. This off-target degradation hampers the therapeutic applicability of pomalidomide-based PROTACs, requiring development of PROTAC design rules that minimize off-target degradation. Here we developed a high-throughput platform that interrogates off-target degradation and found that reported pomalidomide-based PROTACs induce degradation of several ZF proteins. We generated a library of pomalidomide analogues to understand how functionalizing different positions of the phthalimide ring, hydrogen bonding, and steric and hydrophobic effects impact ZF protein degradation. Modifications of appropriate size on the C5 position reduced off-target ZF degradation, which we validated through target engagement and proteomics studies. By applying these design principles, we developed anaplastic lymphoma kinase oncoprotein-targeting PROTACs with enhanced potency and minimal off-target degradation.

Supplementary Table Legends 1-3, Methods, References from Therapeutic Antibody Targeting of CD47 Eliminates Human Acute Lymphoblastic Leukemia

CAR-T cell therapy has emerged as a breakthrough therapy for the treatment of relapsed and refractory hematologic malignancies. However, insufficient CAR-T cell expansion and persistence is a leading cause of treatment failure. Exogenous or transgenic cytokines have great potential to enhance CAR-T cell potency but pose the risk of exacerbating toxicities. Here we present a chemical-genetic system for spatiotemporal control of cytokine function gated by the off-patent anti-cancer molecular glue degrader drug lenalidomide and its analogs. When co-delivered with a CAR, a membrane-bound, lenalidomide-degradable IL-7 fusion protein enforced a clinically favorable T cell phenotype, enhanced antigen-dependent proliferative capacity, and enhanced in vivo tumor control. Furthermore, cyclical pharmacologic combined control of CAR and cytokine abundance enabled the deployment of highly active, IL-7-augmented CAR-T cells in a dual model of antitumor potency and T cell hyperproliferation.

Chimeric antigen receptor (CAR) T cell therapies are medical breakthroughs in cancer treatment. However, treatment failure is often caused by CAR T cell dysfunction. Additional approaches are needed to overcome inhibitory signals that limit anti-tumor potency. Here, we developed bifunctional fusion "degrader" proteins that bridge one or more target proteins and an E3 ligase complex to enforce target ubiquitination and degradation. Conditional degradation strategies were developed using inducible degrader transgene expression or small molecule-dependent E3 recruitment. We further engineered degraders to block SMAD-dependent TGFβ signaling using a domain from the SARA protein to target both SMAD2 and SMAD3. SMAD degrader CAR T cells were less susceptible to suppression by TGFβ and demonstrated enhanced anti-tumor potency in vivo. These results demonstrate a clinically suitable synthetic biology platform to reprogram E3 ligase target specificity for conditional, multi-specific endogenous protein degradation, with promising applications including enhancing the potency of CAR T cell therapy.

Cellular immunotherapies, including chimeric antigen receptor (CAR) T cells, have demonstrated promising efficacy against hematologic malignancies and liquid tumors but run the risk of severe life-threatening toxicities. Moreover, further modifications to enhance cellular immunotherapy potency are being tested to overcome treatment failures due to T cell dysfunction or suppression in the tumor microenvironment. Consequently, for novel and/or highly active cellular immunotherapies the inclusion of “suicide switches” may be prudent to suppress treatment-related adverse events by inducing specific depletion of the engineered cells. The clinical deployment of existing cell therapy suicide switches has been limited in part by imperfect performance characteristics, reliance on non-FDA-approved controller drugs, and the potential immunogenicity of non-human sequences. Herein, we report the engineering of a clinically suitable cell therapy suicide switch activated by targeted protein degradation, and the credentialing of this system to rapidly and irreversibly deplete CAR T cells in vitro and in vivo. We and others have previously demonstrated that lenalidomide-inducible degron tags can be linked to proteins of interest to allow for their selective degradation (Jan et al, Science Translational Medicine, 2021; Carbonneau et al, Cell Chemical Biology, 2021; Koduri et al, PNAS, 2019). Lenalidomide acts as a “molecular glue” that recruits neosubstrate proteins to the CRL4CRBN E3 ubiquitin ligase to be ubiquitinated and subsequently degraded by the proteasome. Caspase-activated DNase (CAD) and its inhibitor (ICAD) are broadly expressed pro- and anti-apoptotic proteins, respectively. ICAD serves as a chaperone for CAD folding and sequesters CAD activity by forming an inactive heterodimer with CAD. Upon apoptosis signaling, activated Caspase 3 cleaves ICAD, liberating CAD to form an active homodimer that acts as a pair of “molecular scissors” to create double strand breaks in the genome. We hypothesized that the overexpression of CAD and an ICAD-degron fusion protein at a stoichiometric 1:1 ratio could be well-tolerated, and furthermore, that lenalidomide treatment would deplete the ICAD-degron protein, thereby freeing CAD to cause cell death. After iterative optimization of promoter strength, transgene order, and degron placement, we evaluated an optimized ICAD-degron-CAD lenalidomide suicide switch versus inducible Caspase 9 in primary human T cells. The lenalidomide suicide switch induced more complete cell depletion versus iCasp9, and maximal cell depletion was seen with subtherapeutic nanomolar concentrations of lenalidomide. In multi-day co-culture live cell imaging assays with co-transduced and sorted CAR suicide switch T cells, tumor cell cytolysis and CAR T cell proliferation were comparable with or without expression of the suicide switch. Lenalidomide addition rapidly inhibited tumor cell cytolysis and depleted the suicide switch CAR T cells without subsequent re-expansion. In an in vivo NSG murine xenograft model with JeKo-1 tumor cell engraftment, suicide switch and control CAR T cells demonstrated comparable anti-tumor activity. For the suicide switch CAR T cells, tumor expansion accelerated after pomalidomide treatment, consistent with engineered cell suppression after suicide switch induction. We have also co-delivered the CAR and 2.2 kilobase suicide switch as a single multi-cistronic lentivector. In summary, a lenalidomide-inducible suicide switch composed of the stoichiometric pair of CAD and ICAD-degron proteins enabled rapid, near-complete engineered cell depletion without interfering with CAR T cell growth or effector functions in the models tested. Composed of all-human sequences and a controller available as a generic drug, the lenalidomide-inducible suicide switch is a clinically suitable system that may have broad applications to safeguard the development of highly potent investigational cellular immunotherapies.

Clonal hematopoiesis of indeterminate potential (CHIP) is a premalignant expansion of mutated hematopoietic stem cells. As CHIP-associated mutations are known to alter the development and function of myeloid cells, we hypothesized that CHIP may also be associated with the risk of Alzheimer’s disease (AD), a disease in which brain-resident myeloid cells are thought to have a major role. To perform association tests between CHIP and AD dementia, we analyzed blood DNA sequencing data from 1,362 individuals with AD and 4,368 individuals without AD. Individuals with CHIP had a lower risk of AD dementia (meta-analysis odds ratio (OR) = 0.64, P = 3.8 × 10 −5 ), and Mendelian randomization analyses supported a potential causal association. We observed that the same mutations found in blood were also detected in microglia-enriched fraction of the brain in seven of eight CHIP carriers. Single-nucleus chromatin accessibility profiling of brain-derived nuclei in six CHIP carriers revealed that the mutated cells comprised a large proportion of the microglial pool in the samples examined. While additional studies are required to validate the mechanistic findings, these results suggest that CHIP may have a role in attenuating the risk of AD.

We successfully repurpose the DNA repair protein methylguanine methyltransferase (MGMT) as an inducible degron for protein fusions. MGMT is a suicide protein that removes alkyl groups from the O6 position of guanine (O6G) and is thereafter quickly degraded by the ubiquitin proteasome pathway (UPP). Starting with MGMT pseudosubstrates (benzylguanine and lomeguatrib), we first demonstrate that these lead to potent MGMT depletion while affecting little else in the proteome. We then show that fusion proteins of MGMT undergo rapid UPP-dependent degradation in response to pseudosubstrates. Mechanistic studies confirm the involvement of the UPP, while revealing that at least two E3 ligase classes can degrade MGMT depending on cell-line and expression type (native or ectopic). We also demonstrate the technique's versatility with two clinically relevant examples: degradation of KRASG12C and a chimeric antigen receptor.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of hematologic malignancies. Approximately half of patients with refractory large B cell lymphomas achieve durable responses from CD19-targeting CAR-T treatment; however, failure mechanisms are identified in only a fraction of cases. To gain new insights into the basis of clinical response, we performed single-cell transcriptome sequencing of 105 pretreatment and post-treatment peripheral blood mononuclear cell samples, and infusion products collected from 32 individuals with large B cell lymphoma treated with either of two CD19 CAR-T products: axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel). Expansion of proliferative memory-like CD8 clones was a hallmark of tisa-cel response, whereas axi-cel responders displayed more heterogeneous populations. Elevations in CAR-T regulatory cells among nonresponders to axi-cel were detected, and these populations were capable of suppressing conventional CAR-T cell expansion and driving late relapses in an in vivo model. Our analyses reveal the temporal dynamics of effective responses to CAR-T therapy, the distinct molecular phenotypes of CAR-T cells with differing designs, and the capacity for even small increases in CAR-T regulatory cells to drive relapse.