Targeting solid tumor antigens with chimeric receptors: cancer biology meets synthetic immunology.

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.

Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma.

PURPOSE: 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.

Genetic retargeting of E3 ligases to enhance CAR T cell therapy.

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 TGFß 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 TGFß 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.

Chemical genetic control of cytokine signaling in CAR-T cells using lenalidomide-controlled membrane-bound degradable IL-7.

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.

Proteolysis-targeting chimeras with reduced off-targets.

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.

Clonal hematopoiesis is associated with protection from Alzheimer's disease.

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.

A molecular glue approach to control the half-life of CRISPR-based technologies.

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.

Distinct cellular dynamics associated with response to CAR-T therapy for refractory B cell lymphoma.

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.

Calreticulin mutant myeloproliferative neoplasms induce MHC-I skewing, which can be overcome by an optimized peptide cancer vaccine.

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are underrepresented in patients with CALRMUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUT MPN would not efficiently respond to a CALRMUT fragment cancer vaccine but would when immunized with a modified CALRMUT heteroclitic peptide vaccine approach. We found that heteroclitic CALRMUT peptides specifically designed for the MHC-I alleles of patients with CALRMUT MPN efficiently elicited a CALRMUT cross-reactive CD8+ T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+ T cell response to the CALRMUT fragment upon immunization with a CALRMUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide-based cancer vaccines in patients with CALRMUT MPN.

SEAKER cells coordinate cellular immunotherapy with localized chemotherapy.

Tumor antigen escape and T cell dysfunction limit the effectiveness of chimeric antigen receptor (CAR) T cell therapies. To overcome these challenges, Gardner et al. engineered synthetic enzyme-armed killer (SEAKER) cells to coexpress a CAR and a prodrug-activating enzyme to orchestrate a dual immunologic and pharmacologic attack at the tumor site.

Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia.

Chimeric antigen receptor (CAR) T cell therapy is effective in lymphoid malignancies, but there has been limited data in myeloid cancers. Here, we start with a CD27-based CAR to target CD70 ("native") in acute myeloid leukemia (AML), and we find modest efficacy in vivo, consistent with prior reports. We then use orthogonal approaches to increase binding on both the tumor and CAR-T cell sides of the immune synapse: a pharmacologic approach (azacitidine) to increase antigen density of CD70 in myeloid tumors, and an engineering approach to stabilize binding of the CAR to CD70. To accomplish the latter, we design a panel of hinge-modified regions to mitigate cleavage of the extracellular portion of CD27. Our CD8 hinge and transmembrane-modified CD70 CAR-T cells are less prone to cleavage, have enhanced binding avidity, and increased expansion, leading to more potent in vivo activity. This enhanced CD70-targeted CAR is a promising candidate for further clinical development.

Concentrate and degrade PFOA with a photo-regenerable composite of In-doped TNTs@AC.

"Concentrate-and-degrade" is an effective strategy to promote mass transfer and degradation of pollutants in photocatalytic systems, yet suitable and cost-effective photocatalysts are required to practice the new concept. In this study, we doped a post-transition metal of Indium (In) on a novel composite adsorptive photocatalyst, activated carbon-supported titanate nanotubes (TNTs@AC), to effectively degrade perfluorooctanoic acid (PFOA). In/TNTs@AC exhibited both excellent PFOA adsorption (>99% in 30 min) and photodegradation (>99% in 4 h) under optimal conditions (25 °C, pH 7, 1 atm, 1 g/L catalyst, 0.1 mg/L PFOA, 254 nm). The heterojunction structure of the composite facilitated a cooperative adsorption mode of PFOA, i.e., binding of the carboxylic head group of PFOA to the metal oxide and attachment of the hydrophobic tail to AC. The resulting side-on adsorption mode facilitates the electron (e‒) transfer from the carboxylic head to the photogenerated hole (h+), which was the major oxidant verified by scavenger tests. Furthermore, the presence of In enables direct electron transfer and facilitates the subsequent stepwise defluorination. Finally, In/TNTs@AC was amenable to repeated uses in four consecutive adsorption-photodegradation runs. The findings showed that adsorptive photocatalysts can be prepared by hybridization of carbon and photoactive semiconductors and the enabled "concentrate-and-degrade" strategy is promising for the removal and degradation of trace levels of PFOA from polluted waters.

CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours.

Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies1-6, but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy7,8. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.

Repurposing the Damage Repair Protein Methyl Guanine Methyl Transferase as a Ligand Inducible Fusion Degron.

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.

Clonal hematopoiesis in patients receiving chimeric antigen receptor T-cell therapy.

Chimeric antigen receptor (CAR) T-cells have emerged as an efficacious modality in patients with non-Hodgkin lymphoma (NHL) and multiple myeloma (MM). Clonal hematopoiesis of indeterminate potential (CHIP), a state in which mutations in hematopoietic cells give rise to a clonal population of cells, is more common in patients exposed to cytotoxic therapies, has been shown to influence inflammatory immune programs, and is associated with an adverse prognosis in patients with NHL and MM receiving autologous transplantation. We therefore hypothesized that CHIP could influence clinical outcomes in patients receiving CAR T-cell therapy. In a cohort of 154 patients with NHL or MM receiving CAR T-cells, we found that CHIP was present in 48% of patients and associated with increased rates of complete response and cytokine release syndrome severity, but only in patients younger than age 60 years. Despite these differences, CHIP was not associated with a difference in progression-free or overall survival, regardless of age. Our data suggest that CHIP can influence CAR T-cell biology and clinical outcomes, but, in contrast to autologous transplantation, CHIP was not associated with worse survival and should not be a reason to exclude individuals from receiving this potentially life-prolonging treatment.

Cancer therapies based on targeted protein degradation - lessons learned with lenalidomide.

For decades, anticancer targeted therapies have been designed to inhibit kinases or other enzyme classes and have profoundly benefited many patients. However, novel approaches are required to target transcription factors, scaffolding proteins and other proteins central to cancer biology that typically lack catalytic activity and have remained mostly recalcitrant to drug development. The selective degradation of target proteins is an attractive approach to expand the druggable proteome, and the selective oestrogen receptor degrader fulvestrant served as an early example of this concept. Following a long and tragic history in the clinic, the immunomodulatory imide drug (IMiD) thalidomide was discovered to exert its therapeutic activity via a novel and unexpected mechanism of action: targeting proteins to an E3 ubiquitin ligase for subsequent proteasomal degradation. This discovery has paralleled and directly catalysed myriad breakthroughs in drug development, leading to the rapid maturation of generalizable chemical platforms for the targeted degradation of previously undruggable proteins. Decades of clinical experience have established front-line roles for thalidomide analogues, including lenalidomide and pomalidomide, in the treatment of haematological malignancies. With a new generation of 'degrader' drugs currently in development, this experience provides crucial insights into class-wide features of degraders, including a unique pharmacology, mechanisms of resistance and emerging therapeutic opportunities. Herein, we review these past experiences and discuss their application in the clinical development of novel degrader therapies.

Reversible ON- and OFF-switch chimeric antigen receptors controlled by lenalidomide.

Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.

The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K.

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.

Recurrent genetic HLA loss in AML relapsed after matched unrelated allogeneic hematopoietic cell transplantation.

Immune evasion is a hallmark of cancer and a central mechanism underlying acquired resistance to immune therapy. In allogeneic hematopoietic cell transplantation (alloHCT), late relapses can arise after prolonged alloreactive T-cell control, but the molecular mechanisms of immune escape remain unclear. To identify mechanisms of immune evasion, we performed a genetic analysis of serial samples from 25 patients with myeloid malignancies who relapsed ≥1 year after alloHCT. Using targeted sequencing and microarray analysis to determine HLA allele-specific copy number, we identified copy-neutral loss of heterozygosity events and focal deletions spanning class 1 HLA genes in 2 of 12 recipients of matched unrelated-donor HCT and in 1 of 4 recipients of mismatched unrelated-donor HCT. Relapsed clones, although highly related to their antecedent pretransplantation malignancies, frequently acquired additional mutations in transcription factors and mitogenic signaling genes. Previously, the study of relapse after haploidentical HCT established the paradigm of immune evasion via loss of mismatched HLA. Here, in the context of matched unrelated-donor HCT, HLA loss provides genetic evidence that allogeneic immune recognition may be mediated by minor histocompatibility antigens and suggests opportunities for novel immunologic approaches for relapse prevention.