Biology subjects

Maus, M. V.

Publications and source records attributed to Maus, M. V..

10 recordsLinked to original sources

bioRxiv2026

A Lenalidomide-Inducible Suicide Switch for Gene- and Cell-Based Therapy

Genetically modified immune effector cells, including chimeric antigen receptor (CAR) T cells, have emerged as transformative anti-cancer agents. However, these autonomous, living therapies are limited by difficult-to-control hyperactivation toxicities. To achieve their full potential as therapies for a broader range of diseases, CAR T cells must target novel antigens and integrate further potency enhancements that raise the possibility of novel and intensified toxicities. High performance safety systems are needed to restrain these highly active next-generation gene- and cell-based therapies. Cell therapy suicide switches remain infrequently used, and are currently limited by inaccessible controller drugs, incomplete cell depletion, fitness cost, and immunogenicity. To address these issues, we engineered a fully human sequence-based cell therapy suicide switch based on the overexpression of Caspase-Activated DNase (CAD) and its stoichiometric chaperone/inhibitor ICAD fused to a lenalidomide-inducible degron. Acute destabilization of ICAD with the broadly available, off-patent molecular glue degrader drug lenalidomide uncaged CAD to induce DNA damage and cell death. By this degradable cage mechanism, the ICAD-CAD suicide switch achieved long-term expression in primary human T cells without detectable leakiness, and deep drug-induced cell depletion. The ICAD-CAD switch was generalizable to alternative acute destabilization systems. Genome-wide CRISPR screening established the necessity of CAD and the lenalidomide-interacting E3 ubiquitin ligase substrate adaptor CRBN for switch function. When co-delivered with a CAR, ICAD-CAD CAR T cells retained effector functions and could be rapidly depleted with lenalidomide.

synthetic biology↗

bioRxiv2026

Engineering CAR-T cells to remodel the mucin-rich cancer cell glycocalyx

The dense glycocalyx of cancer cells can restrict immune-cell access to surface antigens and limit CAR-T cell activity. Here, we show that mucin density and epitope position determine how glycocalyx remodeling affects CAR-T cell recognition and killing. We identify KLK5 as a human protease that cleaves tumor-associated mucins, increases access to membrane-proximal antigens, and enhances CAR-T cell function. We then engineer CAR-T cells to display or secrete KLK5, enabling remodeling of the tumor glycocalyx during antigen recognition. KLK5-engineered CAR-T cells improved tumor control across multiple xenograft models, and KLK5-secreting MUC17 CAR-T cells produced the strongest in vivo benefit, prolonging survival compared with conventional MUC17 CAR-T cells. These findings show that CAR-T cells can be engineered to breach the mucin-rich glycocalyx while preserving accessible target epitopes.

bioengineering↗

bioRxiv2026

Microfluidic T-Chip enables one-step clinical-scale T-cell purification from blood products for CAR T-cell manufacturing

Treatment with chimeric antigen receptor (CAR) T cells has emerged as a promising immune therapy for relapsed and refractory hematologic malignancies. The CAR T cells are manufactured in a series of steps that involve isolating T cells from the patients leukapheresis product, genetically modifying them to express the CAR against the target antigen, and reinfusing them into the patient. Efficient T-cell enrichment from leukapheresis products is critical to the success of these therapies. Current methods for T-cell sorting on a clinical scale involve several washing steps to remove red blood cells and platelets, followed by T-cell selection and activation. These multi-step processes result in cell loss during processing and involve several handling steps. Here, we utilize fluidically assembled micromagnetic lenses to develop a high-throughput, continuous-flow microfluidic T-cell sorter, designated as the T-Chip, for sorting magnetic bead-labeled CD3+ T cells in a single step. Our approach allows direct sorting of T cells in expansion media from leukopaks without any washing steps, effectively removing 99.999% of RBCs and platelets from the leukapheresis product. A single 1-inch x 3-inch T-Chip can process leukapheresis product at a throughput of 60 mL/hr and 2.56 {+/-} 0.12 billion cells/hr. Using this optimized workflow, we demonstrate clinical-scale enrichment of highly pure CD3+ T cells (97.7 {+/-} 1.3%) with high viability (97.0 {+/-} 1.1%) and recovery (87.3 {+/-} 14.8%) in a functionally closed manner. Downstream processing of T cells isolated using the T-Chip yielded potent anti-mesothelin CAR T cells with demonstrated anti-tumor efficacy. Overall, by exploiting precisely engineered magnetic forces and laminar flow, the microfluidic T-Chip overcomes bottlenecks caused by low throughput and enables single-step large-scale T-cell purification for the rapid development of CAR T cells.

bioengineering↗

bioRxiv2025

Affinity-matured CD72-targeting Nanobody CAR T-cells Enhance Elimination of Antigen-Low B-cell Malignancies

BackgroundChimeric antigen receptor (CAR) T-cell therapies are highly efficacious for several different hematologic cancers. However, for most CAR T targets it is observed that low surface antigen density on tumors can significantly reduce therapeutic efficacy. Here, we explore this dynamic in the context of CD72, a surface antigen we recently found as a promising target for refractory B-cell cancers, but for which CD72 low antigen density can lead to therapeutic resistance in preclinical models. MethodsPrimary samples were accessed via institutional review board-approved protocols. Affinity-matured and humanized nanobody clones were previously described in Temple et al.1 CAR T-cells were generated via lentiviral transduction. In vitro cytotoxicity assays were performed using luciferase-labeled cell lines. In vivo studies were performed using cell line- or patient-derived xenografts implanted in NOD scid gamma (NSG) mice. ResultsWe first confirmed ubiquitous CD72 expression across a range of primary B-cell non-Hodgkin lymphomas. We further found that after resistance to CD19-directed therapies, across both B-cell acute lymphoblastic leukemia (B-ALL) models and primary tumor samples, surface CD72 expression was largely preserved while CD22 expression was significantly diminished. Affinity maturation of a nanobody targeting CD72, when incorporated into chimeric antigen receptor (CAR) T-cells, led to more effective elimination in vitro of isogenic models of CD72 low-expressing tumors. These results suggested that nanobody-based CAR T-cells (nanoCARs) may exhibit a similar relationship between binder affinity, antigen expression, and efficacy as previously demonstrated only for scFv-based CAR T-cells. Surprisingly, however, this significantly improved in vitro efficacy only translated to modest in vivo survival benefit. As a parallel strategy to enhance CAR T function, we found that the small molecule bryostatin could also significantly increase CD72 surface antigen density on B-cell malignancy models. Structural modeling and biochemical analysis identified critical residues improving CD72 antigen recognition of our lead affinity-matured nanobody. ConclusionsTogether, these findings support affinity-matured CD72 nanoCARs as a potential immunotherapy product for CD19-refractory B-cell cancers. Our results also suggest that for B-ALL in particular, CD72 may be a preferable second-line immunotherapy target over CD22. What is already known on this topicPrevious work using single chain variable fragment (scFv) based CAR Ts has suggested that improving affinity for target antigen could potentially help mitigate tumor resistance mediated by antigen downregulation, or baseline low antigen density. However, it is unknown whether this same dynamic holds for CAR T-cells that utilize different antigen recognition elements, such as nanobodies. What this study addsHere we show that affinity maturation of nanobody-based CAR T-cells (nanoCARs) targeting CD72 can improve their in vitro efficacy versus CD72-low tumors; however, in vivo efficacy differences are more modest. Furthermore, we show that for refractory B-cell malignancies, surface CD72 appears preserved after CD19 resistance even in situations where CD22 is strongly downregulated. How this study might affect research, practice or policyCD72 warrants further investigation as a preferred immunotherapy target in the context of CD19-refractory B-cell cancers, though nanobody affinity maturation is not a universal solution to the challenge of low tumor surface antigen density.

immunology↗

bioRxiv2025

Sialylated CD43 is a glyco-immune checkpoint for macrophage phagocytosis

Macrophages in the tumor microenvironment exert potent anti-tumorigenic activity through phagocytosis. Yet therapeutics that enhance macrophage phagocytosis have not improved outcomes in clinical trials for patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). To systematically identify regulators of phagocytosis, we performed genome-scale CRISPR knockout screens in human leukemia cells co-cultured with human monocyte-derived macrophages. Surprisingly, we found that whereas the classic "dont eat me" signal CD47 inhibited mouse macrophages, it did not inhibit phagocytosis by human macrophages. In contrast, the O-linked glycosylation and sialylation pathways were strong negative regulators of phagocytosis. In AML, the cell surface O-linked glycoprotein CD43 was the major effector of the O-linked glycosylation and sialylation pathways. Genetic deletion or antibody blockade of CD43 enhanced macrophage phagocytosis. This work highlights the importance of using human platforms to identify immune checkpoints, and nominates CD43 as a glyco-immune regulator of human macrophage phagocytosis.

cancer biology↗

bioRxiv2025

Drug tolerant persisters and immunotherapy persister cells exhibit cross-resistance and share common survival mechanisms

Persisters are a rare sub-population of tumor cells that survive anti-cancer therapy and are thought to be a major cause of recurrence. These cells have been identified following both drug- and immune-therapy but are generally considered to be distinct entities. Since both pharmacological agents and immune cells often kill via apoptosis, we tested a hypothesis that both types of cells survive based on reduced mitochondrial apoptotic sensitivity, which in turn would yield a similar and reciprocal multi-agent resistant phenotype. Supporting this hypothesis, we indeed observed that IPCs acquired a reduced sensitivity to multiple drug classes and radiotherapy, suggesting non-immune mechanisms are important in the survival of cancer cells after immunotherapy. Likewise, DTPs developed not only a reduced sensitivity to multiple drug classes and radiotherapy, but also acquired a reduced sensitivity to T cell killing. Both IPCs and DTPs developed a decreased sensitivity to mitochondrial apoptosis. A sub-population of IPCs downregulated antigen and upregulated PD-L1. Intriguingly, in the IPCs that didnt employ these mechanisms of resistance, a greater decrease in sensitivity to mitochondrial apoptosis was observed, suggesting that the presence or absence of a resistance mechanism can exert selective pressures over the emergence of others. Targeting anti-apoptotic dependencies in persisters increased sensitivity to chemotherapy or CAR T therapy. These results suggest that common biological mechanisms underly survival of persisters, whether derived from immune or drug therapy, and offer an explanation for the acquired cross-resistance to these two types of therapies often observed in the clinic. HighlightsO_LIImmunotherapy persister cells (IPCs) are less sensitive to drugs and radiation. C_LIO_LIDrug tolerant persisters (DTPs) are less sensitive to radiation and CAR T cell attack. C_LIO_LIIPCs and DTPs are less sensitive to mitochondrial apoptosis. C_LIO_LITargeting anti-apoptotic dependencies helps eliminate IPCs/DTPs. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/641492v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@fe98b4org.highwire.dtl.DTLVardef@db7bfdorg.highwire.dtl.DTLVardef@1a94469org.highwire.dtl.DTLVardef@1ca6b2c_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

In vivo CRISPR screens identify key modifiers of CAR T cell function in myeloma

Chimeric antigen receptor (CAR) T cells are highly effective in hematologic malignancies. However, loss of CAR T cells can contribute to relapse in a significant number of patients. These limitations could potentially be overcome by targeted gene editing to increase CAR T cell persistence. Here, we performed in vivo loss-of-function CRISPR screens in BCMA-targeting CAR T cells to investigate genes that influence CAR T cell persistence, function and efficacy in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR-library edited T cells in vitro and then at early and late timepoints in vivo to track the performance of gene modified CAR T cells from manufacturing to survival in tumors. The screens revealed several context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, while loss of PTPN2, ZC3H12A, and RC3H1 conferred early selective growth advantages to CAR T cells in vivo. Strikingly, we identified cyclin-dependent kinase inhibitor 1B (CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late timepoints in vivo. CDKN1B ablation increased BCMA CAR T cell proliferation and effector function in response to antigen, significantly enhancing tumor clearance and overall survival. Thus, our findings reveal differing effects of gene-perturbation on CAR T cells over time and in different selective environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma, and underscore the importance of in vivo screening as a tool for identifying genes to enhance CAR T cell function and efficacy.

immunology↗

bioRxiv2024

Library-based single-cell analysis of CAR signaling reveals drivers of in vivo persistence

The anti-tumor function of engineered T cells expressing chimeric antigen receptors (CARs) is dependent on signals transduced through intracellular signaling domains (ICDs). Different ICDs are known to drive distinct phenotypes, but systematic investigations into how ICD architectures direct T cell function--particularly at the molecular level--are lacking. Here, we use single-cell sequencing to map diverse signaling inputs to transcriptional outputs, focusing on a defined library of clinically relevant ICD architectures. Informed by these observations, we functionally characterize transcriptionally distinct ICD variants across various contexts to build comprehensive maps from ICD composition to phenotypic output. We identify a unique tonic signaling signature associated with a subset of ICD architectures that drives durable in vivo persistence and efficacy in liquid, but not solid, tumors. Our findings work toward decoding CAR signaling design principles, with implications for the rational design of next-generation ICD architectures optimized for in vivo function.

immunology↗

bioRxiv2022

Leukemia-intrinsic determinants of CAR-T response revealed by in vivo genome-wide CRISPR screening.

CAR-T therapy is a promising new treatment modality for B-cell malignancies. However, the majority of patients inevitably go on to experience disease relapse through largely unknown means. To investigate leukemia-intrinsic CAR-T resistance mechanisms, we performed genome-wide CRISPR-Cas9 loss-of-function screens in an immunocompetent murine model of B-cell acute lymphoblastic leukemia (B-ALL) utilizing a novel, modular guide RNA library. We identified IFN{gamma}/JAK/STAT signaling and components of antigen processing and presentation pathway as key mediators of resistance to CAR-T therapy in vivo, but not in vitro. Transcriptional characterization of this model demonstrated an upregulation of these pathways in CAR-T treated relapsed tumors, and examination of data from CAR-T treated patients with B-ALL revealed an association between poor outcomes and increased expression of JAK/STAT/MHC-I in leukemia cells. Overall, our data identify an unexpected mechanism of resistance to CAR-T therapy in which tumor cell interaction with CAR-T cells in vivo induces expression of an adaptive T-cell resistance program in tumor cells.

cancer biology↗

bioRxiv2021

SARS -CoV-2 T-cell immunity to variants of concern following vaccination

Recently, two mRNA vaccines to severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) have become available, but there is also an emergence of SARS-CoV-2 variants with increased transmissibility and virulence1-6. A major concern is whether the available vaccines will be equally effective against these variants. The vaccines are designed to induce an immune response against the SARS-CoV-2 spike protein7, 8, which is required for viral entry to host cells9. Immunity to SARS-CoV-2 is often evaluated by antibody production, while less is known about the T-cell response. Here we developed, characterized, and implemented two standardized, functional assays to measure T-cell immunity to SARS-CoV-2 in uninfected, convalescent, and vaccinated individuals. We found that vaccinated individuals had robust T-cell responses to the wild type spike and nucleocapsid proteins, even more so than convalescent patients. We also found detectable but diminished T-cell responses to spike variants (B.1.1.7, B.1.351, and B.1.1.248) among vaccinated but otherwise healthy donors. Since decreases in antibody neutralization have also been observed with some variants10-12, investigation into the T-cell response to these variants as an alternative means of viral control is imperative. Standardized measurements of T-cell responses to SARS-CoV-2 are feasible and can be easily adjusted to determine changes in response to variants.

immunology↗