Biology subjects

Largaespada, D. A.

Publications and source records attributed to Largaespada, D. A..

17 recordsLinked to original sources

bioRxiv2026

Targeting NF-κB prevents Schwann cell reprogramming and plexiform neurofibroma initiation in mouse model of Nf1

Plexiform neurofibromas arise in approximately half of all individuals with Neurofibromatosis Type I (NF1). Genetic lesions apart from NF1 pathogenic variants are not present in NF1 mutant Schwann cells, suggesting that tumorigenesis may instead require cellular rewiring. To identify putatively re-wired tumor cells, we used multiplex imaging and flow cytometry to delineate markers that distinguish tumor Schwann cell from non-tumor Schwann cells. We identified tumor Schwann cells as CD44+;CD49f+ cells that show increased transcription of epithelial to mesenchymal plasticity (EMP)- associated genes. CD44+;CD49f+ expression was induced by treating Nf1-/- Schwann cells with stressors or by direct NF-{kappa}B pathway activation via IKK2; treated Schwann cells increased secretion of NF-{kappa}B targets and immune cell chemoattractants CCL2 and CCL3. In established tumors, MEK inhibition plus NF-{kappa}B blockade diminished tumor cell proliferation, tumor Schwann cells, immune cell populations, tumor cytokines and tumor number. Notably, transient single agent NF-{kappa}B pathway blockade was sufficient to significantly delay tumor formation in mice. Thus, Nf1 loss in Schwann cells is followed by NF-{kappa}B activation and cellular reprogramming that drives tumor initiation and targeting NF-{kappa}B activation can reduce NF1-deficient tumor formation.

cancer biology↗

bioRxiv2026

Biomarker-driven and gene-enhanced viral therapy for malignant peripheral nerve sheath tumor

Background: High-grade malignant peripheral nerve sheath tumors (MPNST) frequently lose Polycomb Repressive Complex 2 (PRC2), reducing the repressive histone mark, H3K27me3. We hypothesized that H3K27me3 loss sensitizes MPNST to oncolytic HSV due to impaired transcriptional resistance. HSV replication is also restricted by the integrated stress response, normally countered by HSV ICP34.5, representing an additional resistance pathway to ICP34.5-attenuated HSV. Methods: PRC2 loss-of-function was evaluated as a biomarker of HSV G47{Delta} ({Delta}ICP34.5) susceptibility in MPNST using isogenic, pharmacologic inhibition, and genetic rescue models. CUT&RUN and RNA-sequencing defined PRC2-dependent viral and host responses. We engineered a G47{Delta}-derived vector (G47{Delta}2) with tumor-regulated ICP34.5 expression [G47{Delta}2,ins(BIRC5>ICP34.5)] and evaluated efficacy in MPNST xenografts. Results: H3K27me3 deficiency increased G47{Delta} sensitivity and viral spread in vitro. CUT&RUN demonstrated PRC2-dependent H3K27me3 accumulation on the viral genome, explaining G47{Delta} activity in H3K27me3-deficient MPNST. RNA sequencing of G47{Delta} infected MPNST cells revealed heightened integrated stress response activity and blunted induction of translation-associated programs in the PRC2-deficient state (both p<0.001), consistent with compensatory resistance at the level of viral protein translation. Accordingly, tumor-regulated ICP34.5 HSV limited daily tumor-volume growth to 0.6% (95% CI, -2.9-4.2%), improving upon both G47{Delta}2 (8.3%; 4.5-12.2%) and controls (22.5%; 17.4-27.8%). A murine gastrointestinal toxicity phenotype reduced all-cause event-free survival, indicating vector and dosing optimizations are needed to widen the therapeutic window. Conclusions: H3K27me3 loss creates a paradoxical antiviral state that tumor-selective ICP34.5 overcomes to rescue HSV anti-tumor activity, supporting biomarker-driven viral engineering to exploit the permissive transcriptional state of H3K27me3-deficient MPNSTs.

cancer biology↗

bioRxiv2026

Integrated ex vivo screening and transcriptomic profiling to prioritize drug combinations for rare cancers

Discovering effective drug combinations requires testing many dose combinations across a diverse panel of tumor models. This approach is limited in rare cancers by a scarcity of cell lines and representative high-throughput models that would make exhaustive screening feasible and predictive. Patient-derived xenograft (PDX) models are genomically representative but too low-throughput for this purpose. Culturing PDX cells ex vivo in three-dimensional (3D) matrices offers a genomically representative and clinically relevant platform for preclinical drug testing, capturing the microenvironmental cues that shape in vivo drug response while requiring only limited tissue per assay. Toward this end, we designed and validated an experimental-computational framework, "ex vivo assessment of combination therapies" (EXACT), to enable drug combination discovery in rare tumors. Using PDX models of malignant peripheral nerve sheath tumors (MPNST), we built a platform to culture PDX cells ex vivo over multiple days, monitoring drug sensitivity and measuring transcriptomic responses to treatment. Computational analysis of these transcriptomic responses then identifies which compensatory pathway creates a unique vulnerability to a second drug. EXACT thus offers a biologically informed, scalable approach for prioritizing drug combinations in rare tumors, nominating drugs alongside biological rationale. Using this methodology, we identified a MEK inhibitor plus HDAC inhibitor combination with enhanced activity in vitro and in vivo, forming the basis of an active clinical trial. This platform could be adapted for real-time use with primary patient specimens, enabling personalized therapeutic discovery. SignificanceEXACT integrates PDX-derived 3D drug screening with biologically informed computational analysis to identify and explain effective combinations, providing a scalable strategy for therapeutic discovery in rare cancers such as MPNST.

cancer biology↗

bioRxiv2026

The Cell Surface Proteome of Malignant Peripheral Nerve Sheath Tumors Reveals Therapeutic Targets

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas and the most common cause of disease-associated death for Neurofibromatosis Type 1 (NF1) patients. In the context of NF1, MPSNTs develop from benign premalignant precursors. The transition to malignancy is usually accompanied by loss of the polycomb repressive complex 2 (PRC2), leading to aberrant upregulation of many genes. The specific mechanisms disrupted by PRC2 loss remain incompletely understood. There is a significant gap in our knowledge of which cell-surface targets become derepressed and therapeutically actionable following PRC2 loss, contributing to the current lack of effective targeted therapies for MPNSTs. This study aims to address this gap by using cell-surface capture technology with mass spectrometry to profile MPNST models. In doing so, we define PRC2-dependent effects on the cell surface proteome, including specific biological pathways that are enhanced or suppressed at the cell surface protein level. We also create an MPNST cell-surface protein compendium comprised of proteins that are highly expressed across a variety of well-defined MPNST models. We prioritized proteins that are preferentially expressed in MPNST or other cancers and for which FDA-approved therapies already exist. Specific proteins from this compendium were molecularly targeted with antibody-drug conjugates in these models to surmise their therapeutic efficacy. Results reveal PTK7 as a novel and promising target for MPNST. In total, these efforts represent a step toward addressing the knowledge gap in MPNST genesis and identifying new therapeutic targets for further testing. Additionally, this data serves as a resource for other researchers wishing to characterize specific molecular targets. KEY POINTSPRC2 modulates key MPNST signaling pathways through the cell surface proteome Cell surface proteomics identifies a plethora of therapeutic targets for MPNST targeted therapy Antibody-drug conjugates targeting PTK7 show enhanced efficacy in reducing MPNST viability IMPORTANCE OF THE STUDYThis study utilizes advances in biochemistry to profile the surface proteome of malignant peripheral nerve sheath tumors. In doing so, it identifies many proteins whose presence is abundant on the cell surface of MPNST cells. Pre-clinical drug testing shows that use of antibody-drug conjugates may be effective in killing MPNST cells when targeted to epitopes identified in our MPNST cell surface proteome compendium. This study is a departure from more commonly used transcriptomic methods to identify cell surface proteins by using direct surface capture and mass spectrometry, providing a more direct measurement of cell surface protein abundance. Additionally, it identifies a handful of proteins which can be directly targeted pharmaceutically and one in particular, PTK7, whose targeting is highly effective in killing MPNST cells.

cancer biology↗

bioRxiv2025

Sleeping Beauty mutagenesis identifies BACH2 and other regulators of CD8+T cell exhaustion, persistence in vivo, and CAR-T function under tumor-associated chronic antigen stimulation

Genes that enhance T cell function represent promising targets for improving engineered T cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T cell persistence, employing Sleeping Beauty (SB) insertional mutagenesis, which induces both gain-(GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. We developed transgenic mice carrying Doxycycline (Dox)-inducible SB mutagenesis system (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T cell persistence under chronic antigen exposure. Specifically, CD8 T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8 T cells using enhanced-specificity tagmentation sequencing (esTag-seq) and RNA-seq, respectively. Under chronic stimulation, SB-mutagenized CD8 T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation stress. Among these, T2/Onc2 insertions into Bach2 and Elmo1 were repeatedly found at the genomic level and were associated with altered nascent transcript expression. Bach2, known as a key regulator of T cell memory formation and resistance to chronic viral infection but less characterized in engineered T cells for cancer therapy, was found to enhance in vivo tumor persistence in the B16-Ova tumor model. We showed that ectopic Bach2 expression levels influence engineered T cell differentiation lineage. A Bach2low signature allowed differentiation into both KLRG1 and CD62L phenotypes, whereas Bach2high restricted differentiation predominantly to the CD62L subset. Finally, in human CART19-28{zeta} cells, BACH2 overexpression enhanced cytotoxicity and improved tumor control following chronic cancer stimulation. Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T cell therapeutic phenotypes. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.

immunology↗

bioRxiv2025

A modular, Galaxy-based immunopeptidogenomic (iPepGen) analysis pipeline for discovery, verification, and prioritization of candidate cancer neoantigen peptides

Characterizing tumor-specific neoantigen peptides, derived from genomic or transcriptomic aberrations and presented to the immune system, is critical for immuno-oncology studies. To this end, the modular iPepGen immunopeptidogenomics pipeline provides these functions: (1) Neoantigen prediction and protein database generation from genomic or transcriptomic sequencing data; (2) Peptide identification (3) Verification from immunopeptidomic mass spectral data; (4) Neoantigen classification and visualization; (5) Candidate prioritization for further study. Easy access via a publicly available, scalable cloud-based gateway coupled with online, interactive training materials streamlines the adoption by cancer researchers who require immunopeptidogenomic analysis tools but lack advanced computational expertise and resources.

bioinformatics↗

bioRxiv2024

The assessment of therapeutic autophagy inhibition in NF1 mutated tumor cells

MAPK pathway activation is found across central nervous system tumors. Neurofibromin 1 (NF1) protein is a GTPase-activating protein and tumor suppressor that negatively regulates RAS protein activity. A NF1 loss of function mutation results in MAPK pathway upregulation and is associated with pediatric gliomas. We previously demonstrated autophagy inhibition improves tumor response to BRAF inhibition. Here we investigate the role of autophagy in NF1 cells in the presence or absence of MEK inhibition (MEKi). NF1 wild type (WT) and NF1 knockout (KO) (HSC1{lambda}, immortalized human Schwann) cells were evaluated. NF1 loss was confirmed, and cell growth was monitored by Incucyte. MAPK/ERK pathway upregulation and autophagy were evaluated via Western blot analysis and RAS pull-down. Effects of MEKi, autophagy inhibition (pharmacologic and genetic), and combination therapy were evaluated via short- and long-term growth assays. NF1 KO cells exhibited MAPK pathway upregulation and increased growth. Additionally, NF KO cells demonstrated increased autophagic activity. Loss of NF1 resulted in increased sensitivity to MEKi and autophagy inhibition alone and in combination. We also demonstrate that the autophagy inhibitor chloroquine (CQ) can improve MEKi sensitivity in a patient harboring a somatic mutation of NF1. This combination is currently being evaluated in clinical trial (NCT04201457).

cancer biology↗

bioRxiv2024

Cell type specific suppression of hyper-recombination by human RAD18 is linked to PCNA K164 ubiquitination

Homologous recombination (HR) and translesion synthesis (TLS) promote gap-filling DNA synthesis to complete genome replication. One factor involved in both pathways is RAD18, an E3 ubiquitin ligase. Although RAD18s role in promoting TLS through the ubiquitination of PCNA at lysine 164 (K164) is well established, its requirement for HR-based mechanisms is currently less clear. To assess this, we inactivated RAD18 in three human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e. HCT116 mutants were hyper-recombinogenic (hyper-rec). Loss of RAD18 also impaired TLS activity in HCT116 cells, but unexpectedly, did not reduce clonogenic survival. Interestingly, these phenotypes appear linked to PCNA K164 ubiquitination, as HCT116 PCNAK164R/+ mutants were also hyper-rec and showed reduced TLS activity, consistent with previous studies in rad18-/-or pcnaK164R avian DT40 mutant cells. Importantly, knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. Taken together, these data suggest that the roles of human RAD18 in directing distinct gap-filling DNA synthesis pathways varies depending on cell type and that these functions are linked to PCNA ubiquitination.

molecular biology↗

bioRxiv2024

Pharmacogenomic synthetic lethal screens reveal hidden vulnerabilities and new therapeutic approaches for treatment of NF1-associated tumors

Neurofibromatosis Type 1 (NF1) is a common cancer predisposition syndrome, caused by heterozygous loss of function mutations in the tumor suppressor gene NF1. Individuals with NF1 develop benign tumors of the peripheral nervous system (neurofibromas), originating from the Schwann cell linage after somatic loss of the wild type NF1 allele, some of which progress further to malignant peripheral nerve sheath tumors (MPNST). There is only one FDA approved targeted therapy for symptomatic plexiform neurofibromas and none approved for MPNST. The genetic basis of NF1 syndrome makes associated tumors ideal for using synthetic drug sensitivity approaches to uncover therapeutic vulnerabilities. We developed a drug discovery pipeline to identify therapeutics for NF1-related tumors using isogeneic pairs of NF1-proficient and deficient immortalized human Schwann cells. We utilized these in a large-scale high throughput screen (HTS) for drugs that preferentially kill NF1-deficient cells, through which we identified 23 compounds capable of killing NF1-deficient Schwann cells with selectivity. Multiple hits from this screen clustered into classes defined by method of action. Four clinically interesting drugs from these classes were tested in vivo using both a genetically engineered mouse model of high-grade peripheral nerve sheath tumors and human MPNST xenografts. All drugs tested showed single agent efficacy in these models as well as significant synergy when used in combination with the MEK inhibitor Selumetinib. This HTS platform yielded novel therapeutically relevant compounds for the treatment of NF1-associated tumors and can serve as a tool to rapidly evaluate new compounds and combinations in the future.

cancer biology↗

bioRxiv2023

CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon restoration of RB function in malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft tissue sarcomas with limited treatment options, and novel effective therapeutic strategies are desperately needed. We observe anti-proliferative efficacy of genetic depletion or pharmacological inhibition using the clinically available SHP2 inhibitor (SHP2i) TNO155. Our studies into the signaling response to SHP2i reveal that resistance to TNO155 is partially mediated by reduced RB function, and we therefore test the addition of a CDK4/6 inhibitor (CDK4/6i) to enhance RB activity and improve TNO155 efficacy. In combination, TNO155 attenuates the adaptive response to CDK4/6i, potentiates its anti-proliferative effects, and converges on enhancement of RB activity, with greater suppression of cell cycle and inhibitor-of-apoptosis proteins, leading to deeper and more durable anti-tumor activity in in vitro and in vivo patient-derived models of MPNST, relative to either single agent. Overall, our study provides timely evidence to support the clinical advancement of this combination strategy in patients with MPNST and other tumors driven by loss of NF1.

cancer biology↗

bioRxiv2023

Perinatal folate levels do not influence tumor latency or multiplicity in a model of NF1 associated plexiform-like neurofibromas

ObjectiveIn epidemiological and experimental research, high folic acid intake has been demonstrated to accelerate tumor development among populations with genetic and/or molecular susceptibility to cancer. Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder predisposing affected individuals to tumorigenesis, including benign plexiform neurofibromas; however, understanding of factors associated with tumor risk in NF1 patients is limited. Therefore, we investigated whether pregestational folic acid intake modified plexiform-like peripheral nerve sheath tumor risk in a transgenic NF1 murine model. ResultsWe observed no significant differences in overall survival according to folate group. Relative to controls (180 days), median survival did not statistically differ in deficient (174 days, P=0.56) or supplemented (177 days, P=0.13) folate groups. Dietary folate intake was positively associated with RBC folate levels at weaning, (P=0.023, 0.0096, and 0.0006 for deficient vs. control, control vs. supplemented, and deficient vs. supplemented groups, respectively). Dorsal root ganglia (DRG), brachial plexi, and sciatic nerves were assessed according to folate group. Mice in the folate deficient group had significantly more enlarged DRG relative to controls (P=0.044), but no other groups statistically differed. No significant differences for brachial plexi or sciatic nerve enlargement were observed according to folate status.

cancer biology↗

bioRxiv2022

RAD-TGTs: Measurement of cellular tensions via flow cytometry and DNA sequencing enabled by force-dependent rupture and delivery of DNA tension probes

Mechanical force is a key driver of cellular processes and is dysregulated in many diseases. Measuring cellular tensions to elucidate mechanotransduction pathways typically involves high-resolution but low throughput imaging of surfaces and arduous experimental preparation of materials. We present here Rupture and Deliver DNA-duplex based molecular tension sensors-RAD-TGTs. RAD-TGTs consist of immobilized DNA duplexes conjugated to a ligand and indicator (fluorophore, barcode etc) which rupture in a force-dependent manner when cells are bound. Readout of rupture is performed in cells of interest using high throughput methods such as flow cytometry and leveraging covalent DNA-protein linking HUH-tags simplifies the preparation of the tension sensor to allow use of "off-the-shelf" oligos. We demonstrate that rupture and delivery is decreased by inhibitors of cytoskeletal dynamics and knockout of mechanosensing proteins. We also show that rupture and delivery correlates with ligand affinity. Excitingly, we demonstrate that rupture and delivery of barcoded DNA-duplexes can be quantified using DNA sequencing, propelling cellular force measurements into the -omics era.

biophysics↗

bioRxiv2022

In vivo functional genomics identifies essentiality of potassium homeostasis in medulloblastoma

The identification of cancer maintenance genes--driver genes essential to tumor survival--is fundamental for developing effective cancer therapy. Transposon-based insertional mutagenesis screens can identify cancer driver genes broadly but not discriminate maintenance from progression or initiation drivers, which contribute to cancer phenotypes and tumorigenesis, respectively. We engineered a nested, double-jumping transposon system to first dysregulate gene expression during tumorigenesis and then restore gene expression following tumor induction, allowing for genome-wide screening of maintenance essentiality in vivo. In a mouse model of medulloblastoma, the most common pediatric malignancy, insertion and remobilization of this nested transposon uncovers potassium channel genes as recurrent maintenance drivers. In human medulloblastoma, KCNB2 is the most overexpressed potassium channel across Group 3, Group 4, and SHH subgroups, and Kcnb2 knockout in mice diminishes the replicative potential of medulloblastoma-propagating cells to mitigate tumor growth. Kcnb2 governs potassium homeostasis to regulate plasma membrane tension-gated EGFR signaling, which drives proliferative expansion of medulloblastoma-propagating cells. Thus, our novel transposon system reveals potassium homeostasis as essential to tumor maintenance through biomechanical modulation of membrane signaling.

cancer biology↗

bioRxiv2022

Differential migration mechanics and immune responses of glioblastoma subtypes

Glioblastoma remains a deadly cancer driven in part by invasion of tumor cells into the brain. Transcriptomic analyses have identified distinct molecular subtypes, but mechanistic differences that account for clinical differences are not clear. Here, we show that, as predicted by the motor-clutch model of cell migration, mesenchymal glioma cells are more spread, generate larger traction forces, and migrate faster in brain tissue compared to proneural cells. Despite their rapid migration and comparable proliferation rates in vitro, mice with mesenchymal tumors survive longer than those with proneural tumors. This improved survival correlated with an immune response in the mesenchymal tumors, including T cell-mediated. Consistently, inducing mesenchymal tumors in immunodeficient mice resulted in shorter survival supporting a protective immune role in mesenchymal tumors. Thus, mesenchymal tumors have aggressive migration, but are immunologically hot which suppresses net proliferation. These two features counteract each other and may explain the lack of a strong survival difference between subtypes clinically, while also opening up new opportunities for subtype-specific therapies. Significant StatementThis study highlights new mechanical and immunological insights into glioblastoma molecular subtypes using an integrated modeling-genome engineering strategy, which can potentially facilitate glioblastoma subtype-specific therapeutic strategies.

cancer biology↗

bioRxiv2022

Ex vivo to in vivo model of malignant peripheral nerve sheath tumors for precision oncology

Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that often develop in patients with neurofibromatosis type 1 (NF1-MPNST), but can occur sporadically. Through a multi-institution collaboration, we have developed 13 NF1-associated MPNST patient-derived xenografts (PDX). Genomic analysis of the PDX-tumor pairs identified somatic mutations in NF1 (61%), SUZ12 (61%), EED (15%), and TP53 (15%), and chromosome 8 (Chr8) gain (77%), consistent with published data. Pre-clinical models that capture this molecular heterogeneity are needed to identify and prioritize effective drug candidates for clinical translation. Here, we describe the successful development of a medium-throughput ex vivo 3D microtissue model with several advantages over 2D cell line growth, which can be utilized to predict drug response in vivo. Herein, we present proof-of-principle of this PDX-to-microtissue system, using four genomically representative MPNST and three drugs. This work highlights the development of a novel ex vivo to in vivo preclinical platform in MPNST that successfully captures the genomic diversity observed in patients and represents a resource to identify future therapeutic strategies.

cancer biology↗

bioRxiv2022

Multivalent, Bispecific ????B7-H3-????CD3 Chemically Self-Assembled Nanorings Direct Potent T-cell Responses Against Medulloblastoma

Few therapeutic options have been made available for treating central nervous system tumors, especially upon recurrence. Recurrent medulloblastoma is uniformly lethal with no approved therapies. Recent preclinical studies have shown promising results for eradicating various solid tumors by targeting the overexpressed immune checkpoint molecule, B7-H3. However, due to several therapy-related toxicities and reports of tumor escape, the full potential of targeting this pan-cancer antigen has yet to be realized. Here, we designed and characterized bispecific chemically self-assembling nanorings (CSANs) that target the T cell receptor, CD3{varepsilon}, and tumor associated antigen, B7-H3, derived from the humanized 8H9 single chain variable fragment (scFv). We show that the B7-H3-CD3 CSANs increase T cell infiltration and facilitate selective cytotoxicity of B7-H3+ medulloblastoma spheroids and that activity is independent of target cell MHC class I expression. Importantly, non-specific T cell activation against the ONS 2303 medulloblastoma cell line can be reduced by tuning the valency of the CD3 targeted monomer in the oligomerized CSAN. Intraperitoneal injections of B7-H3-CD3 bispecific CSANs were found to effectively cross the blood-tumor barrier into the brain and elicit significant anti-tumor T cell activity intracranially as well as systemically in an orthotopic medulloblastoma model. Moreover, following treatment with B7-H3-CD3 CSANs, intratumoral CD4+ and CD8+ T cells were found to primarily have a central memory phenotype that displayed significant levels of characteristic activation markers. Collectively, these results demonstrate the ability of our multi-valent, bispecific CSANs to direct potent anti-tumor T cell responses and indicate its potential utility as an alternative or complementary therapy for immune cell targeting of B7-H3+ brain tumors. SignificanceThis study presents B7-H3-CD3 bispecific CSANs as a promising, new immunotherapeutic option for patients with established B7-H3+ medulloblastomas.

cancer biology↗

bioRxiv2019

CD200 immune checkpoint reversal at the site of tumor vaccine inoculation: A novel approach to glioblastoma immunotherapy

PurposeAdvances in immunotherapy have revolutionized care for some cancer patients. However, current checkpoint inhibitors are associated with significant toxicity and yield poor responses for patients with central nervous system tumors, calling into question whether cancer immunotherapy can be applied to glioblastoma multiforme. We determined that targeting the CD200 activation receptors (CD200AR) of the CD200 checkpoint with a peptide inhibitor overcomes tumor-induced immunosuppression. We have shown the clinical efficacy of the peptide inhibitor in a trial in companion dogs with spontaneous high-grade glioma; adding the peptide to autologous tumor lysate vaccines significantly increased overall survival relative to tumor lysate alone (median survival of 12.7 and 6.36 months, respectively).\n\nExperimental design: This study was developed to elucidate the mechanism of the CD200ARs and develop a humanized peptide inhibitorWe developed macrophage cell lines with each of four CD200ARs knocked out to determine their binding specificity and functional responses. Using proteomics, we developed humanized peptide inhibitors to explore their effects on cytokine/chemokine response, dendritic cell maturation and CMV pp65 antigen response in CD14+ cells. GMP-grade peptide was further validated for activity.\n\nResultsWe demonstrated that the peptide specifically targets the CD200AR complex to induce an immune response. Moreover, we developed and validated our humanized peptides for inducing chemokine response, stimulating immature dendritic cell differentiation and significantly enhancing an antigen-specific response. We also determined that the use of the peptide downregulated the expression of CD200 inhibitory and PD-1 receptors.\n\nConclusionThese results support consideration of a CD200 peptide ligand as a novel platform for immunotherapy against multiple cancers including glioblastoma multiforme.\n\nTranslational relevanceThis report evaluates the ability to modulate the CD200 immune checkpoint by employing synthetic peptides directed as ligands to its paired immune activation receptor. We previously reported the presence of CD200 in the sera and tumor vasculature of patients with glioblastoma multiforme (GBM). We have also shown that a canine CD200 activation receptor ligand extends the lives of companion dogs with high grade glioma. The data we present here show that the human peptide ligand (hCD200ARL) directed to the CD200 activation receptor on CD14+ cells activates immune upregulation through induction of a cytokine response and dendritic cell differentiation. In addition, hCD200ARL downregulates the inhibitory CD200 and PD-1 receptors. These findings provide a basis to evaluate hCD200ARL as a novel immune therapy for patients with GBM. Downregulation of PD-1 suggests that hCD200ARL may also obviate the need for PD1 and PD-L1 directed therapies for GBM and other malignancies.

cancer biology↗