Biology subjects

Majzner, R. G.

Publications and source records attributed to Majzner, R. G..

10 recordsLinked to original sources

bioRxiv2026

A framework for reparative CAR T engineering in the CNS

Chimeric antigen receptor (CAR) T cells have shown remarkable therapeutic promise in hematological malignancies and, more recently, in autoimmunity. They also hold considerable potential for neurodegenerative diseases and CNS injury, where the therapeutic objective shifts from cell depletion to immune modulation and tissue repair. Using ischemic stroke as proof-of-concept, we engineered MOG-targeting CAR T cells to dissect how distinct CAR designs shape the CNS microenvironment. CD4/CD8 CAR T cells (a mixture of CD4 and CD8 subsets) proliferated robustly and efficiently infiltrated the ischemic hemisphere, but induced broad immune recruitment and exacerbated neuroinflammation. In contrast, CD4 restricted CAR T cells markedly reduced immune infiltration, reprogrammed microglia, and minimized inflammatory activation. We engineered CD4 CAR T cells to secrete brain-derived neurotrophic factor (BDNF) to determine whether they could be redirected toward a reparative, non-cytotoxic phenotype. CD4 BDNF-CAR T cells further attenuated inflammation, reduced immune infiltration, and promoted the expansion of regulatory T cells. CD4 BDNF-CAR T treated mice showed significantly improved gait performance following stroke. Together, these findings establish a cellular framework and outline principles for engineering reparative CAR T platforms for neurological diseases.

immunology↗

bioRxiv2025

Synthetic signaling platform uncovers and rewires cellular responses to PD-1 perturbation

Tyrosine phosphorylation motifs are central regulators of cell signaling, yet methods to selectively detect and reprogram these events have been lacking. Here we introduce Sphyder (Selective PHosphotYrosine DEtection and Rewiring), which enables precise detection of signaling at the resolution of individual phosphorylation motifs. Using Sphyder biosensors, we resolved phosphorylation dynamics and uncovered regulatory mechanisms of the checkpoint receptor PD-1 in living cells. Sphyder also provided a framework for reconstructing phosphosignaling pathways. With this approach, we redirected PD-1 signaling from immunosuppressive to immunoactivating outputs and engineered synthetic receptors that linked extracellular sensing to customized transcriptional programs. In addition, Sphyder biosensors revealed previously unrecognized mechanisms of the PD-1/VEGF bispecific antibody Ivonescimab, showing that it induces VEGF-dependent clustering, phosphorylation, and degradation of PD-1. These findings may underlie its promising clinical activity relative to conventional PD-1 blockade. Together, our study establishes a broadly applicable strategy for sensing and reprogramming cell signaling, while also providing mechanistic insights into a new class of immune checkpoint inhibitors of major clinical interest.

immunology↗

bioRxiv2024

IKAROS facilitates antigen escape in the face of CD19- and CD22-targeted therapies for B-cell acute lymphoblastic leukemia

Relapse due to antigen escape is a major cause of treatment failure for patients with B-cell malignancies following targeted immunotherapies, including CD19- and CD22-directed chimeric antigen receptor T (CAR T) cells. To identify tumor intrinsic factors associated with antigen loss, we performed single-cell analyses on 61 primary patient samples or patient-derived xenografts from patients with B-cell acute lymphoblastic leukemia (B-ALL) treated with CAR T cells. We identified that low levels of the transcription factor IKAROS in pro-B-like B-ALL cells before CAR T treatment are associated with antigen escape. We demonstrate that IKAROSlow B-ALL cells lose features of B cell identity and resemble progenitor cells based on their epigenetic and transcriptional state, resulting in the downregulation of B-cell immunotherapy antigens, including surface expression of CD19 and CD22. We find that modulation of CD19 and CD22 protein expression is IKAROS dose-dependent and reversible. Further, we demonstrate that IKAROSlow cells are resistant to CD19- and CD22-targeted therapies. Together, we describe a novel role for IKAROS in the regulation of B-cell immunotherapy targets and the risk of antigen escape relapse, identifying it as a potential prognostic target. HighlightsO_LIIKAROSlow pro-B-like B-ALL cells are associated with CD19neg relapse C_LIO_LIIKAROSlow B-ALL cells resemble progenitor cells and have lower B-cell commitment C_LIO_LIIKAROS modulates CD19 and CD22 surface expression in a dose-dependent and reversible manner C_LIO_LIIKAROSlow B-ALL cells are more resistant to CD19- and CD22-targeted therapies C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/621347v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@109f75eorg.highwire.dtl.DTLVardef@14f41fborg.highwire.dtl.DTLVardef@74e428org.highwire.dtl.DTLVardef@2f275c_HPS_FORMAT_FIGEXP M_FIG C_FIG Before immunotherapy, IKAROSlow pro-B-like B-ALL cells possess chromatin and gene expression states poised for loss of B-cell identity while maintaining expression of CD19 and CD22. Under immune pressure, IKAROShigh cells maintain their antigen expression, making them more susceptible to T cell-mediated killing. Conversely, IKAROSlow cells are more likely to downregulate their antigen expression, giving them a relative advantage to escape immunotherapies, resulting in antigen escape relapse.

cancer biology↗

bioRxiv2024

Immunotherapy-related cognitive impairment after CAR T cell therapy in mice

Persistent central nervous system (CNS) immune dysregulation and consequent dysfunction of multiple neural cell types is central to the neurobiological underpinnings of a cognitive impairment syndrome that can occur following traditional cancer therapies or certain infections. Immunotherapies have revolutionized cancer care for many tumor types, but the potential long-term cognitive sequelae are incompletely understood. Here, we demonstrate in mouse models that chimeric antigen receptor (CAR) T cell therapy for both CNS and non-CNS cancers can impair cognitive function and induce a persistent CNS immune response characterized by white matter microglial reactivity and elevated cerebrospinal fluid (CSF) cytokines and chemokines. Consequently, oligodendroglial homeostasis and hippocampal neurogenesis are disrupted. Microglial depletion rescues oligodendroglial deficits and cognitive performance in a behavioral test of attention and short-term memory function. Taken together, these findings illustrate similar mechanisms underlying immunotherapy-related cognitive impairment (IRCI) and cognitive impairment following traditional cancer therapies and other immune challenges.

neuroscience↗

bioRxiv2023

Engineered CD47 protects T cells for enhanced antitumor immunity

Adoptively transferred T cells and agents designed to block the CD47/SIRP axis are promising antitumor therapeutics, which activate distinct arms of the immune system. We administered anti-CD47 (CD47) with adoptively transferred T cells with the goal of enhancing antitumor efficacy but observed rapid macrophage-mediated clearance of T cells expressing chimeric antigen receptors (CARs) or engineered T cell receptors, which blunted therapeutic benefit. CD47 mediated CAR T clearance was potent and rapid enough to serve as an effective safety switch. To overcome this challenge, we engineered a CD47 variant (47E) that engaged SIRP and provided a "dont-eat-me" signal that was not blocked by CD47 antibodies. TCR or CAR T cells expressing 47E were resistant to clearance by macrophages following CD47, and mediated significant, sustained macrophage recruitment into the TME. Although many of the recruited macrophages manifested an M2-like profile, the combined therapy resulted in synergistic enhancement in antitumor efficacy. This work identifies macrophages as major regulators of T cell persistence and illustrates the fundamental challenge of combining T cell directed therapeutics with those designed to activate macrophages. It further delivers a therapeutic approach capable of simultaneously harnessing the antitumor effects of T cells and macrophages that manifests markedly enhanced potency against solid tumors.

bioengineering↗

bioRxiv2022

Enhanced Effector Activity of Mediator Kinase Module Deficient CAR-T Cells

Adoptive T cell immune therapies mediate impressive clinical benefit in a fraction of patients, but anti-tumor effects are often limited by inadequate T cell potency. To identify genes limiting T cell effector function, we conducted genome-wide CRISPR knock-out screens in human primary CAR-T cells. The top hits were MED12 and CCNC, components of the cyclin-dependent kinase (CDK) module of the Mediator complex, an evolutionarily conserved regulator of gene transcription. MED12 or CCNC deficient CAR-T cells manifest increased expansion, cytokine production, metabolic fitness, effector function, anti-tumor activity and reduced terminal effector differentiation. Chemical inhibition of CDK8/19 kinase activity recapitulated some features of genetic loss of MED12, including increased T cell expansion. MED12 deficient CAR-T cells showed widespread but selective increases in chromatin accessibility, MED1 chromatin occupancy, and H3K27 acetylation at enhancers used by transcription factors playing a critical role in T cell fate, including several STAT and AP1 family members. The most pronounced enhancement was observed for STAT5 which manifested as increased sensitivity to IL-2 in MED12 deficient T cells. These results link Mediator induced transcriptional coactivation with T cell effector programming and identify the CDK module as a target for enhancing the potency of anti-tumor T cell responses. One Sentence SummaryThe Mediator kinase module is a primary regulator of T cell differentiation, and genetic or small molecule-based inhibition of this module enhances effector T cell potency.

immunology↗

bioRxiv2022

Latent human herpesvirus 6 is reactivated in chimeric antigen receptor T cells

Cell therapies have yielded durable clinical benefits for patients with cancer, but the risks associated with the development of therapies from manipulated human cells are still being understood. For example, we currently lack a comprehensive understanding of the mechanisms of neurotoxicity observed in patients receiving T cell therapies, including recent reports of encephalitis caused by human herpesvirus 6 (HHV-6) reactivation1. Here, via petabase-scale viral RNA data mining, we examine the landscape of human latent viral reactivation and demonstrate that HHV-6B can become reactivated in human CD4+ T cells in standard in vitro cultures. Using single-cell sequencing, we identify a rare population of HHV-6 super-expressors (~1 in 300-10,000 cells) that possess high viral transcriptional activity in chimeric antigen receptor (CAR) T cell culture before spreading to infect other cells in vitro. Through the analysis of single-cell sequencing data from patients receiving cell therapy products that are FDA-approved2 or used in clinical studies3,4, we identify the presence of CAR+, HHV-6 super-expressor T cells in vivo. Together, our study implicates cell therapy products as a source of lytic HHV-6 reported in clinical trials1,5-7 and has broad implications for the design, production, and monitoring of cell therapies.

immunology↗

bioRxiv2022

Coopting T cell proximal signaling molecules enables Boolean logic-gated CAR T cell control

Introductory paragraphWhile CAR T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumor toxicity has hampered their development for solid tumors because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean logic gating to CAR T cells to prevent on-target, off-tumor toxicity3-7; however, a truly safe and effective logic-gated CAR has remained elusive8. Here, we describe a novel approach to CAR engineering in which we replace traditional ITAM-containing CD3{zeta} domains with intracellular proximal T cell signaling molecules. We demonstrate that certain proximal signaling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumors in vivo while bypassing upstream signaling proteins such as CD3{zeta}. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for the propagation of T cell signaling. We leveraged the cooperative role of LAT and SLP-76 to engineer Logic-gated Intracellular NetworK (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and the prevention of on-target, off-tumor toxicity. LINK CAR will dramatically expand the number and types of molecules that can be targeted with CAR T cells, enabling the deployment of these powerful therapeutics for solid tumors and diverse diseases such as autoimmunity9 and fibrosis10. In addition, this work demonstrates that the internal signaling machinery of cells can be repurposed into surface receptors, a finding that could have broad implications for new avenues of cellular engineering.

synthetic biology↗

bioRxiv2021

Anti-GD2 antibody disrupts GD2:Siglec-7 interactions and synergizes with CD47 blockade to mediate tumor eradication

The disialoganglioside GD2 is consistently overexpressed in neuroblastoma and osteosarcoma, and is variably expressed in other sarcomas, gliomas, neuroendocrine tumors, and epithelial cancers. Anti-GD2 antibodies have improved the survival rates of patients with neuroblastoma only when administered as part of intense chemotherapy-based cytotoxic regimens, which are associated with debilitating late effects including hearing loss, growth retardation, and secondary leukemias. Despite broad expression of GD2 on osteosarcoma, anti-GD2 antibody has not mediated significant antitumor activity in that disease or any other GD2+ cancers. CD47 is a checkpoint molecule overexpressed on tumor cells that inhibits macrophage activity, and CD47 blockade has demonstrated promising clinical activity in early human trials. We investigated whether anti-CD47 antibody could enhance the efficacy of anti-GD2 antibody in neuroblastoma and other GD2+ malignancies. We demonstrate substantial synergy of these two agents, resulting in the recruitment of tumor associated macrophages (TAMs) to mediate robust and durable anti-tumor responses. The responses are driven by GD2-specific factors that reorient the balance of macrophage activity towards phagocytosis of tumor cells, including disruption of a newly described GD2:Siglec-7 axis. These results demonstrate the unique synergy of combining anti-GD2 with anti-CD47, which has the potential to significantly enhance outcomes for children with neuroblastoma and osteosarcoma and will soon be investigated in a first-in-human clinical trial.

immunology↗

bioRxiv2020

Transient "rest" induces functional reinvigoration and epigenetic remodeling in exhausted CAR-T cells

T cell exhaustion limits immune responses against cancer and is a major cause of resistance to CAR-T cell therapeutics. Using a model wherein tonic CAR signaling induces hallmark features of exhaustion, we employed a drug-regulatable CAR to test the impact of transient cessation of receptor signaling (i.e. "rest") on the development and maintenance of exhaustion. Induction of rest in exhausting or already-exhausted CAR-T cells resulted in acquisition of a memory-like phenotype, improved anti-tumor functionality, and wholescale transcriptional and epigenetic reprogramming. Similar results were achieved with the Src kinase inhibitor dasatinib, which reversibly suppresses CAR signaling. The degree of functional reinvigoration was proportional to the duration of rest and was associated with expression of transcription factors TCF1 and LEF1. This work demonstrates that transient cessation of CAR-T cell signaling can enhance anti-tumor potency by preventing or reversing exhaustion and challenges the paradigm that exhaustion is an epigenetically fixed state.

immunology↗