Jennifer M. Binning

Idecabtagene vicleucel (ide-cel) induces deep responses in relapsed/refractory multiple myeloma (RRMM), yet more than half of patients relapse within one year. The intrinsic features of CAR-T products that distinguish durable from non-durable responders are poorly defined, particularly at single-cell resolution, and defining drivers of durable response is critical to guide patient counseling and to inform strategies for optimizing CAR-T manufacturing and efficacy. To address this need, 40 ide-cel infusion products (184,398 cells) were profiled using single-cell RNA sequencing. These analyses revealed that a transcriptional program in CD4 CAR-T cells that led to durable responses is characterized by NF-κB signaling, pro-survival circuits, tonic/chemokine signaling, and elevated CAR transgene expression. These features were associated with prolonged progression-free and overall survival irrespective of baseline clinical characteristics. Further, analysis of paired apheresis and tumor microenvironment samples showed that elevated NF-κB activity is an intrinsic hallmark of T-cell fitness that is characterized by a central memory phenotype and the lack of checkpoint receptorligand expression, and that these features were manifest in marrow-derived and peripheral blood T cells prior to CAR-T manufacturing. Finally, validating functional relevance, pharmacologic inhibition of NF-κB abrogated CAR-T cytotoxicity and cytokine production in vitro. Our results support that NFKB in the ide-cel product marks a signaling axis impacting CAR-T function and NFKB activity represents a global marker of T cell fitness present prior to CAR-T manufacture.

Protein glycation is a detrimental byproduct of carbohydrate metabolism, and nearly all organisms encode kinases that facilitate the removal of early glycation products. In humans, these repair functions are performed by Fructosamine-3 and Ketosamine-3 kinases (FN3K, KT3K). Recent structural studies identified conserved residues essential for FN3K activity, but the molecular basis for substrate discrimination between FN3K and KT3K remains unknown. Here, we show that substrate-binding residues are highly conserved and do not confer specificity in the FN3K family. Using APR, we resurrected four ancestral fructosamine kinases that recapitulate the distinct substrate preferences of FN3K and KT3K. Through mutational studies and structural analysis, we identify an evolutionarily tuned allosteric network that modulates substrate selection through long-range intramolecular interactions. Our findings define the mechanism of substrate selectivity within the FN3K family and establish a framework for the development of selective FN3K inhibitors.

Protein glycation is a detrimental byproduct of carbohydrate metabolism, and nearly all organisms encode kinases that facilitate the removal of early glycation products. In humans, these repair functions are performed by Fructosamine-3 and Ketosamine-3 kinases (FN3K, KT3K). Recent structural studies identified conserved residues essential for FN3K activity, but the molecular basis for substrate discrimination between FN3K and KT3K remains unknown. Here, we show that substrate-binding residues are highly conserved and do not confer specificity in the FN3K family. Using APR, we resurrected four ancestral fructosamine kinases that recapitulate the distinct substrate preferences of FN3K and KT3K. Through mutational studies and structural analysis, we identify an evolutionarily tuned allosteric network that modulates substrate selection through long-range intramolecular interactions. Our findings define the mechanism of substrate selectivity within the FN3K family and establish a framework for the development of selective FN3K inhibitors.

Protein glycation is a detrimental byproduct of living cells’ reliance on carbohydrate metabolism, and nearly all organisms encode kinases that facilitate the removal of early glycation products. In humans, these repair functions are performed by Fructosamine-3 kinase (FN3K) and Ketosamine-3 kinase (KT3K) enzymes which share conserved catalytic mechanisms but differ in substrate specificity. Recent structural studies defined key active site residues required for FN3K activity on a model substrate, yet the molecular basis for differential substrate recognition by FN3K and KT3K remains unresolved. Here, we integrate phylogenetic analysis, ancestral protein reconstruction (APR), and mutational biochemistry to to elucidate how substrate specificity evolved within the fructosamine-3 kinase family. We show that conserved substrate-binding residues are required for the phosphorylation of both fructosamines and ketosamines, but do not contribute to substrate specificity. Using APR, we resurrected four ancestral fructosamine kinases that recapitulate the distinct substrate preferences of FN3K and KT3K despite differing by only 12 amino acids. Through mutational studies and structural analysis, we show that substrate specificity is modulated by an evolutionarily tuned allosteric network that enables long-range intramolecular communication. These insights provide a new mechanistic framework for understanding FN3K substrate selection and open avenues for rational design of FN3K-selective therapeutics targeting protein glycation in metabolic disease and aging.

Human Papilloma Virus (HPV)-related cancers are a global health burden, yet there are no targeted therapies available for chronically infected patients. The HPV protein E6 is essential for HPV-mediated tumorigenesis and immune evasion, making it an attractive target for antiviral drug development. In this study, we developed an E6-targeting Proteolysis Targeting Chimera (PROTAC) that inhibits the growth of HPV(+) tumors. To develop E6 antagonists, we generated a panel of nanobodies targeting E6 proteins derived from the oncogenic HPV16 subtype. The highest affinity E6 nanobody, A5, was fused to Von Hippel Lindau protein (VHL) to generate a PROTAC that degrades E6 (PROTAC ^E6 ). Mutational rescue experiments validated specific degradation via the CRL2 ^VHL E3 ligase. Intralesional administration of the PROTAC ^E6 using a clinically viable DNA vaccine reduced tumor burden in an immunocompetent mouse model of HPV(+) cancer. The inhibitory effect of the PROTAC ^E6 was abrogated by CD4 ⁺ and CD8 ⁺ T-cell depletion, indicating that the antitumor function of the PROTAC ^E6 relies in part on a host immune response. Overall, these results suggest that the targeted degradation of E6 inhibits its oncogenic function and stimulates a robust immune response against HPV(+) tumors, opening new opportunities for virus-specific therapies in the treatment of HPV-related cancers.

Immunotherapies are a major breakthrough in oncology, yielding unprecedented response rates for some cancers. Especially in combination with conventional treatments or targeted agents, immunotherapeutics offer invaluable tools to improve outcomes for many patients. However, why not all patients have a favorable response remains unclear. There is an increasing appreciation of the contributions of the complex tumor microenvironment, and the tumor-immune ecosystem in particular, to treatment outcome. To date, however, there exists no immune biomarker to explain why two patients with similar clinical stage and molecular profile would have different treatment outcomes. We hypothesize that it is critical to understand both the immune and tumor states to understand how the complex system will respond to treatment. Here, we present how integrated mathematical oncology approaches can help conceptualize the effect of various immunotherapies on a patient’s tumor and local immune environment, and how combinations of immunotherapy and cytotoxic therapy may be used to improve tumor response and control and limit toxicity on a per patient basis.