Biology subjects

June, C. H.

Publications and source records attributed to June, C. H..

13 recordsLinked to original sources

bioRxiv2026

A generalizable system for antigenic peptide targeting across HLA-I allotypes

T cell receptors (TCRs) and TCR-mimicking antibodies recognize peptide antigens in the context of specific Human Leucocyte Antigen (HLA-I) allotypes, and the extreme polymorphism of the HLA locus limits the breadth of immunotherapy development. Key barriers include divergent molecular surfaces on HLA proteins and differences in the peptide structure. As a result, existing modalities cannot confer therapeutic coverage across patients of divergent genetic backgrounds. Here, we develop an approach which combines a peptide conformational prediction tool, PepPred, with a cross-HLA binding protein engineering system, TRACeR-I1, to outline a generalized framework for developing binders (xTRACeRs) with compatibility across HLA allotypes while maintaining high levels of specificity towards the peptide antigen. We use our system to develop and validate xTRACeRs against clinically relevant, established peptide antigens presented across common alleles within five HLA-A/B/C supertypes2. Cryo-EM structures of xTRACeR-pHLA complexes for an oncofetal antigen from PRAME and a neuroblastoma-specific peptide from PHOX2B reveal effective mechanisms to navigate polymorphic HLA surface residues, and extensive interactions with the peptide. We implemented these two xTRACeRs as Chimeric Antigen Receptor (CAR) T cells and demonstrated their potent killing efficacy and specificity. Overcoming restriction across HLA supertypes lifts a key barrier in HLA-targeted immunotherapy by expanding patient coverage.

synthetic biology↗

bioRxiv2025

Engineering a Juxtamembrane-Targeting CAR T-Cell Against Mesothelin: A Novel Binder Resilient to Shed Antigen in Ovarian and Pancreatic Cancer

Mesothelin is an attractive target for CAR-T therapy on a number of cancer types; however, the efficacy of this therapy is diminished because the bulk of the cell surface-expressed mesothelin is shed through naturally occurring proteolysis leaving behind a short juxtamembrane peptide "stump". The two problems this creates are (1) the bulk of the target protein is no longer on the tumor cell and (2), the free soluble shed mesothelin remains in the tumor microenvironment and becomes present in blood/other body fluids binding to the mesothelin-targeted CAR-T and interfering with their ability to target the mesothelin that remains on the surface of the tumor. These issues have likely contributed at least in part to the lack of desired efficacy of CAR-T cells that target membrane distal regions of mesothelin (i.e., the shed domain) such as those utilizing anti-mesothelin scFvs SS1 and M5. Here we describe CAR T cells that utilize novel antibodies specific for the mesothelin stump domain thus being unaffected by the natural process of mesothelin shedding. Mesothelin "stump" specific CAR T cells (ST4-22) had cytotoxicity and in vivo activity that was comparable to standard anti-mesothelin CAR T cells. Importantly, CAR T cells expressing ST4-22 were effective against tumor cells that were resistant to standard anti-mesothelin CAR T cells.

immunology↗

bioRxiv2025

DR5 CAR-T cells target solid tumors and suppress MDSCs with minimal toxicity

Chimeric antigen receptor (CAR) T cell therapies have poor efficacy in solid tumors due to limited target specificity and an immunosuppressive tumor microenvironment. We investigated death receptor 5 (DR5) as a CAR target based on its high expression in both solid tumors and myeloid- derived suppressor cells (MDSCs). We engineered agonistic DR5-specific CAR constructs and evaluated their activity in multiple models, demonstrating DR5-expression-dependent tumor killing, confirmed by knockout and overexpression experiments. DR5-targeting single-chain variable fragments retained their pro-apoptotic activity when expressed on non-effector cells or extracellular vesicles. Among multiple CAR designs, we identified a construct with optimized binding affinity that maintained T cell viability while preserving strong tumor and MDSC-killing potency. To assess safety and efficacy in an immunocompetent setting, we also developed a murine DR5-targeted CAR. In multiple xenograft and syngeneic mouse models, DR5 CAR-T cells reduced tumor growth, prolonged survival, and did not cause detectable toxicity. In patient- derived organoids and tissue slices, DR5 CAR-T cells infiltrated tumor tissues, reduced MDSCs, boosted CD8+ T cell activity, and inhibited tumor growth. These findings support DR5-targeted CAR-T therapy as a promising strategy for treating solid tumors, which combines direct tumor cytotoxicity with immune activation and minimizes off-target effects. TeaserDR5 CAR-T cells eliminate tumor cells and MDSCs and activate tumor-resident CD8+ T cells within the TME.

cancer biology↗

bioRxiv2025

Bioinspired Oxidized mRNA Lipid Nanoparticles for Ex Vivo Engineering of Chimeric Antigen Receptor Macrophages Targeting Solid Tumors

Solid tumors remain difficult to treat via conventional and novel therapeutic strategies. Immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy have been remarkably effective in treating hematological cancers, but their efficacy is limited in solid tumors. Recently, CAR macrophages (CAR-Ms) have emerged as a promising solid tumor immunotherapy, primarily for their intrinsic tumor infiltration and effector functions. However, CAR-Ms are engineered using viral transduction, which is associated with aberrant immunogenicity and toxicity. To overcome these challenges, we developed a bioinspired oxidized lipid nanoparticle (LNP) platform for mRNA-based engineering of human CAR-Ms. A library of 24 ionizable lipids was synthesized, formulated into LNPs, and screened for delivery to human macrophages. The composition of the top LNP was subsequently optimized using an orthogonal design of experiments (DoE) and physicochemical properties, such as size and mRNA encapsulation, were tuned via optimization of microfluidic mixing parameters, yielding a particle that significantly outperformed a gold standard C12-200 LNP. Utilizing small molecule and antibody inhibitors, we demonstrate that uptake of optimized LNPs into macrophages is driven by apolipoprotein E (ApoE) independent macropinocytosis, which is further supported by potent extrahepatic spleen tropism upon intravenous administration to mice. Lastly, we demonstrate the translatability of this LNP platform and utilize it to engineer functional primary human HER2-CAR-Ms ex vivo with potent antigen-specific tumor killing, validated in an ex vivo co-culture with ovarian cancer cells. This bioinspired oxidized LNP platform can potentially be utilized to engineer a range of human CAR-M immunotherapies to treat various types of solid tumors.

bioengineering↗

bioRxiv2025

Solid tumor CAR T cells engineered with fusion proteins targeting PDL1 for localized IL-12 delivery

CAR T cell efficacy in solid tumors is limited due in part to the immunosuppressive TME. To improve anti-tumor responses, we hypothesized that enabling CAR T cells to secrete bifunctional fusion proteins consisting of a cytokine modifier (e.g., TGF{beta}trap, IL15, or IL12) combined with an immune checkpoint inhibitor (e.g., PDL1) will provide tumor localized immunomodulation to improve CAR T cell functionality. To that end, we engineered CAR T cells to secrete TGF{beta}trap, IL15, or IL12 molecules fused to PDL1 scFv, and assessed in vitro functionality and in vivo safety and efficacy in prostate and ovarian cancer models. CAR T cells engineered with PDL1-IL12 were superior in safety and efficacy compared to CAR T cells alone and to those engineered with PDL1 fused with TGF{beta}trap or IL15. Further, PDL1-IL12 engineered CAR T cells improved T cell trafficking and tumor infiltration, localized IFN{gamma} production, TME modulation, and anti-tumor responses, with reduced systemic inflammation-associated toxicities. We believe our PDL1-IL12 engineering strategy presents an opportunity to improve CAR T cell clinical efficacy and safety across multiple solid tumor types.

bioengineering↗

bioRxiv2025

IL-9 signaling redirects CAR T cell fate toward CD8+ memory and CD4+ cycling states, enhancing anti-tumor efficacy

The success of chimeric antigen receptor T cell therapies targeting solid tumors is limited by the immunosuppressive tumor microenvironment. We demonstrate that endowing CAR T cells with ectopic interleukin-9 (IL-9) signaling by co-expressing an IL-9 receptor, rewires CAR T cell fate under antigen stress to enhance anti-tumor efficacy. In preclinical solid tumor models, IL-9-signaling CAR T cells exhibit increased expansion, persistence, and tumor infiltration, resulting in superior tumor control at significantly lower doses than conventional products. Trajectory and RNA velocity analyses of single-cell RNA sequencing data reveal that IL-9 signaling alters CAR T cell differentiation under antigen stress away from dysfunction, favoring a multipotent transition toward CD8+ cell memory and effector states, and promoting a CD4+ cell proliferative state. Interrogation of transcription factor pathways indicates that IL-9-mediated activation of STAT1 and STAT4 drives the superior phenotype of IL-9-signaling CAR T cells, providing a promising therapeutic strategy for targeting solid cancers.

immunology↗

bioRxiv2023

Disruption of desmoplastic stroma overcomes restrictions to T cell extravasation, immune exclusion and immunosuppression in solid tumors

The desmoplastic stroma in solid tumors presents a formidable challenge to immunotherapies that rely on endogenous or adoptively transferred T cells, however, the mechanisms are poorly understood. To define mechanisms involved, we treat established desmoplastic pancreatic tumors with CAR T cells directed to fibroblast activation protein (FAP), an enzyme highly overexpressed on a subset of cancer-associated fibroblasts (CAFs). Depletion of FAP+CAFs results in loss of the structural integrity of desmoplastic matrix. This renders these highly treatment-resistant cancers susceptible to subsequent treatment with a tumor antigen (mesothelin)-targeted CAR and to anti-PD1 antibody therapy. Mechanisms include overcoming stroma-dependent restriction of T cell extravasation and/or perivascular invasion, reversing immune exclusion, relieving T cell suppression, and altering the immune landscape by reducing myeloid cell accumulation and increasing endogenous CD8+ T cell and NK cell infiltration. These data provide strong rationale for combining tumor stroma-and malignant cell-targeted therapies to be tested in clinical trials.

immunology↗

bioRxiv2023

Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells

CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the chromosome, including in pre-clinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells,1 dramatically reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.

cell biology↗

bioRxiv2022

Induction of a CD8 T cell intrinsic DNA damage and repair response is associated with clinical response to PD-1 blockade in uterine cancer.

Despite the success of immune checkpoint blockade (ICB), many patients fail to achieve durable clinical benefit, and the underlying immunological mechanisms remain poorly understood. Here, we investigated immune reinvigoration by ICB in advanced or recurrent hypermutated or microsatellite instability-high, mismatch repair deficient (MSI-H/MMRd) uterine cancer patients treated with anti-PD-1 (nivolumab). CD8 T cells underwent rapid pharmacodynamic proliferation 2-4 weeks after initiating PD-1 blockade. This immunological response, however, did not correlate with clinical response. We hypothesized that the T cell-intrinsic response to proliferative and genotoxic stress might contribute to the disparity between immunological and clinical response. We developed a high-dimensional single cell cytometric platform to simultaneously analyze T cell differentiation with changes in DNA damage and repair (DDR) pathways. This DDR-Immune platform revealed T cell subset-specific patterns of DDR, and distinct DDR pathways induced by different types of DNA damage. Applying this platform to MSI-H/MMRd or hypermutated uterine cancer patients revealed a signature of DDR exemplified by rapid increase in phosphorylated-ATM (pATM) intrinsic to CD8 T cells proliferating in response to PD-1 blockade that distinguished clinical responders and non-responders. ATM regulated transcriptional circuits in T cells were associated with better clinical response to PD-1 blockade. These findings highlight a previously unrecognized role for CD8 T cell-intrinsic DDR as a potential determinant of immune fitness and clinical outcome of PD-1 blockade therapy. STATEMENT OF SIGNIFICANCEUsing high-dimensional immune and DNA damage response (DDR) profiling in T cell subsets we identified distinct signatures in anti-PD-1 treated MSI-H/hypermutated uterine cancer patients associated with clinical response versus non-response. Thus, T cell-intrinsic DDR is a potential determinant of immune responsiveness and clinical outcome to PD-1 blockade therapy in cancer.

immunology↗

bioRxiv2021

Decade-long remissions of leukemia sustained by the persistence of activated CD4+ CAR T-cells

The adoptive transfer of T lymphocytes reprogrammed to target tumor cells has demonstrated significant potential in various malignancies. However, little is known about the long-term potential and the clonal stability of the infused cells. Here, we studied the longest persisting CD19-redirected chimeric antigen receptor (CAR) T cells to date in two chronic lymphocytic leukemia (CLL) patients who achieved a complete remission in 2010. CAR T-cells were still detectable up to 10+ years post-infusion, with sustained remission in both patients. Surprisingly, a prominent, highly activated CD4+ population developed in both patients during the years post-infusion, dominating the CAR T-cell population at the late time points. This transition was reflected in the stabilization of the clonal make-up of CAR T-cells with a repertoire dominated by few clones. Single cell multi-omics profiling via Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) with TCR sequencing of CAR T-cells obtained 9.3 years post-infusion demonstrated that these long-persisting CD4+ CAR T-cells exhibited cytotoxic characteristics along with strong evidence of ongoing functional activation and proliferation. Our data provide novel insight into the CAR T-cell characteristics associated with long-term remission in leukemia.

immunology↗

bioRxiv2021

Single-cell antigen-specific activation landscape of CAR T infusion product identifies determinants of CD19 positive relapse in patients with ALL

Chimeric antigen receptor-modified (CAR) T cells targeting CD19 have mediated dramatic responses in relapsed/refractory acute lymphoblastic leukemia (ALL), yet a notable number of patients have CD19-positive relapse within one year of treatment. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes prior to treatment. Herein we present 101,326 single cell transcriptomes and surface protein landscape from the CAR T infusion products of 12 pediatric ALL patients upon CAR antigen-specific stimulation in comparison with TCR-mediated activation and controls. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of Th2 function was associated with CD19-positive relapsed patients (median remission 9.6 months) compared with very durable responders (remission>54 months). Proteomic profiles also revealed that the frequency of early memory T cell subsets, rather than activation or co-inhibitory signatures could distinguish CD19-positive relapse. Additionally, a deficit of type 1 helper and cytotoxic effector function and an enrichment for terminally differentiated CD8+ T cells exhibiting low cytokine polyfunctionality was associated with initial non-responders. By contrast, the single-cell transcriptomic data of unstimulated or TCR-activated CAR T cells failed to predict clinical responses. In aggregate, our results dissect the landscape of CAR-specific activation states in infusion products that can identify patients who do not develop a durable response to the therapy, and unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long term remission.

immunology↗

bioRxiv2019

Impaired tumor death receptor signaling drives resistance to CAR T cell therapy

Cellular immunotherapy using T cells engineered to express chimeric antigen receptors targeting CD19 (CART19) leads to long-term remission in patients with B-cell malignancies1-5. Unfortunately, a significant fraction of patients demonstrate primary resistance to CART19 or experience relapse after achieving remission. Beyond loss of target antigen, the molecular pathways governing CART19 failure are unknown. Here we demonstrate that death receptors, cell surface signaling molecules that induce target cell apoptosis, are key mediators of leukemic resistance and CART19 failure. Using a functional CRISPR/Cas9-based genome-wide knockout screen6 in B-cell acute lymphoblastic leukemia (ALL), we identified the death receptor signaling pathway as a central regulator of sensitivity to CART19-induced cell death. In the absence of the pro-apoptotic death receptor signaling molecules BID or FADD, ALL cells were resistant to CART19 cytotoxicity, resulting in rapid disease progression in mice. We found that this initial resistance to cytotoxicity led to persistence of tumor cells, which drove the development of T cell dysfunction that further compromised anti-tumor immunity and permitted tumor outgrowth. We validated these findings using clinical samples collected from patients with ALL treated with CD19-targeted CAR T cells and found that expression of pro-apoptotic death receptor pathway genes in pre-treatment tumor samples correlated with CAR T cell expansion and persistence, as well as patient response and overall survival. Our findings indicate that tumor-intrinsic death receptor signaling directly contributes to CAR T cell failure.

immunology↗

bioRxiv2017

CAR T Cells Secreting IL18 Augment Antitumor Immunity and Increase T Cell Proliferation and Costimulation

Interleukin 18 (IL18) is known to induce the expression of interferon-{gamma} (IFNG), but its effects on T cell proliferation and costimulation are not completely understood. In this study, we demonstrate that ectopic expression of IL18 in CART cells caused significant T cell proliferation in vitro and in vivo, and enhanced antitumor effects in xenograft models. Moreover, IL18 mediated T cell expansion required neither tumor antigen nor CAR expression, and produced severe GVHD in NSG mice. Furthermore, recombinant IL18 costimulated IFNG secretion and proliferation of anti-CD3 beads treated T cells. Interestingly, IL18 costimulation could expand purified CD4 T cells, but not CD8 T cells. However, CD8 T cells proliferated greater than CD4 T cells in magnitude within bulk T cells, suggesting CD4 help effect was involved. Using CRISPR/Cas9 gene editing, we confirmed that IL18-driven expansion was both TCR and IL18 receptor (IL18R) dependent. Importantly, we demonstrated that TCR-deficient, IL18-expressing CD19 CART cells exhibited remarkable proliferation and persistent antitumor activity against CD19-expressing tumor cells in vivo, without eliciting any detectable GVHD symptom. Finally, we describe APACHE T cells, a novel strategy for coupling IL18 expression in CART cells to antigen stimulation, thereby limiting potential toxicity associated with persistent IL18 production. In sum, our study supports human IL18 as a T cell costimulatory cytokine for fueling CART therapy.

synthetic biology↗