Epidermal Growth Factor Receptor Variant III Research Articles

Overexpressing Bcl-2 enhances murine chimeric antigen receptor T cell therapy against solid tumor.

Chimeric antigen receptor T (CART) cell therapy has demonstrated promising potential in the treatment of hematologic malignancies. However, its application to solid tumors is limited due to the restrictive nature of the tumor microenvironment, resulting in functional failure and poor persistence of CART cells. Overexpression of Bcl-2 in human CART cells (hCART) has been found to significantly enhance their anti-apoptotic effects both in vitro and in vivo. Nevertheless, the evaluation of hCART cells in preclinical studies has predominantly relied on immunodeficient mice xenograft tumor models, making it challenging to assess the impact of hCART cells on normal tissues and the immune system. We established a murine CART (mCART) that overexpresses Bcl-2 and targets the epidermal growth factor receptor variant III (EGFRvIII), named EGFRvIII·mCART-Bcl2. It demonstrated superior proliferation, cytotoxicity, and anti-apoptotic capabilities in vitro. In an immunocompetent mouse model of abdominal metastasis of colorectal cancer, EGFRvIII·mCART-Bcl2 exhibited improved survival of CART in the abdomen, increased tumor clearance, and significantly prolonged overall mouse survival. In summary, our study provides evidence that the introduction of Bcl-2 into mCART cells can enhance their therapeutic efficacy against solid tumors while ensuring safety.

Preparation and evaluation of the PD0721‑DOX antibody‑drug conjugate targeting EGFRvIII to inhibit glioblastoma

Preparation and evaluation of the PD0721‑DOX antibody‑drug conjugate targeting EGFRvIII to inhibit glioblastoma

Abstract 5496: Multi-omic spatial analysis of the tumor microenvironment in gliomas

Abstract Introduction: The diverse tumor environment of high-grade glioma, which remains refractory to treatment, demands new innovative, multi-omic approaches to characterize tumor heterogeneity and expression profiles associated with progression and therapeutic response. Approach: We have applied an integrated, multi-omic approach using paired spatial in situ sequencing (Xenium) and protein profiling (PhenoCycler® Fusion) of glioma tissue to identify distinct glioma cellular phenotypes, activation states and metabolic pathways at true single cell resolution. Our multi-omic approach used hundreds of phenotypic RNA target probes and >50 antibodies in an unbiased single-cell analysis of primary, recurrent, and IDH1 mutant/wild type tumors with a range of heterogeneity. Summary: We were able to distinguish differentially regulated genes and pathways between aggregated primary and recurrent GBM, including cell cycle pathways being upregulated in primary vs recurrent GBM glial cell clusters and a down regulation of ERBB4 signaling in vascular cell clusters in primary GBMs. Our data also suggests that SOX11, known to be involved in tumorigenesis, is >2-fold upregulated in annotated tumor cells in primary compared to recurrent tumors. We were also able to quantitatively localize the expression of Epidermal Growth Factor Receptor, variant III (EGFRvIII) to specific tumor-cell sub-types, which is important given its association with therapeutic resistance. Further, we have been able to establish the cellular neighborhoods and cell-cell and receptor-ligand relationships within the tumors. Our spatial protein analysis identified distinct tumor and immune phenotypes with varying abundance of myeloid and lymphoid populations in IDH1 wt and mt tumors. Moreover, spatial proximity analyses and cellular neighborhood analyses revealed differences in higher order organizational landscapes that may contribute to the differential outcomes across wt and mt tumor subtypes. Conclusion: Multiomic spatial analysis enables deeper characterization of the glioma cellular and functional landscape to broaden our understanding of the key TME features that contribute to disease pathogenesis and prognoses. Our study provides an analytical framework to combine RNA and protein-based spatial data for a holistic investigation into a variety of glioma subtypes and aid in the identification of novel biomarkers, spatial neighborhoods, and functional states that drive glioma progression. Citation Format: Dmytro Klymyshyn, Vaibhav Jain, Lauren Whaley, Emily Hocke, Bassem Ben Cheikh, Alan Smith, Karen Abramson, Nadine Nelson, Diane Satterfield, Elizabeth Thomas, Giselle Lopez, Seetha Hariharan, Michael Brown, Niyati Jhaveri, Roger McLendon, David Ashley, Matthew Waitkus, Simon Gregory. Multi-omic spatial analysis of the tumor microenvironment in gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5496.

Abstract A040: Characterization of a humanized monoclonal antibody targeting cancer-expressed EGFR

Abstract The epidermal growth factor receptor (EGFR), an established oncology target, is a transmembrane protein highly expressed on the surface of malignant tumors such as glioblastoma, lung, liver, bladder and breast cancers. In addition to overexpression, EGFR mutants are frequently associated with malignancy. For example, EGFR variant III (EGFRvIII), deletion of exons 2-7, is only expressed on cancer cells and is associated with poor prognosis and chemotherapy resistance. To target cancer-expressed EGFR, including EGFRvIII, we generated the 40H3 monoclonal antibody which is reactive with the 287-302 loop that is fully exposed on the extracellular domain of EGFRvIII. The 40H3 antibody also showed strong reactivity for cells expressing gene-amplified EGFR but little or no reactivity for cells expressing normal levels of wild type EGFR. When conjugated with toxic payloads (either tesirine or deruxtecan), the resulting antibody-drug conjugates (ADCs) exhibited antitumor activity in several xenograft models. To improve its utility as a clinical candidate, we humanized the variable portions of the heavy and light chains of 40H3, without changing the original CDRs. The variable regions of 40H3, heavy and light chains, were compared to the closest human immunoglobulin families and then framework residues were altered to produce candidate humanized antibodies. The humanized variable chains were then fused with a human IgG1 heavy chain and a kappa light chain to generate full-sized humanized antibodies. A total of 9 humanized antibodies were generated by pairing 3 variable humanized heavy chains (VH1, VH2, VH3) with 3 variable humanized light chains (VL1, VL2, VL3). To evaluate the binding activity of humanized antibodies, we used either immobilized EGFRvIII (by ELISA or Octet) or various cancer cells expressing either EGFR or EGFRvIII (by flow cytometry). All humanized antibodies retained binding activity to immobilized EGFRvIII but bound with different apparent affinities to cells overexpressing EGFR or cells transfected with EGFRvIII. The ‘A10’ antibody was the best candidate. Mutations introduced in A10 preserved the binding affinity of the 40H3 antibody but exhibited an increased affinity compared to the corresponding chimeric antibody constructed with the original mouse VH and VL. The other eight antibodies retained EGFRvIII binding activity, albeit with a lower cell binding affinity. Specifically, A10 showed improved binding to cells with EGFR gene amplification including two TNBC lines, MDA-MB-468 and BT-20, epithelial cancer cells, A431, head and neck squamous carcinoma, UMSCC17B, as well to cells expressing EGFRvIII, F98npEGFRvIII. No binding of A10 was detected to cell lines expressing wild type EGFR, WI-38 or U87MG. In summary, humanized candidates of the 40H3 monoclonal antibody retained EGFR binding. Of 9 antibodies, A10 exhibited the best cell binding activity elevating it to our top clinical candidate for the production of ADCs or similar antibody-based therapeutics. Citation Format: Tamara G Fernandes Costa, Robert Sarnovsky, Maria Teresa Bilotta, Antonella Antignani, David FitzGerald. Characterization of a humanized monoclonal antibody targeting cancer-expressed EGFR [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A040.

CTIM-24. REPEATED PERIPHERAL INFUSIONS OF ANTI-EGFRVIII CAR T CELLS IN COMBINATION WITH PEMBROLIZUMAB REMODELS THE TUMOR MICROENVIRONMENT IN DE NOVO GLIOBLASTOMA

Abstract Treatment efficacy with chimeric antigen receptor (CAR) T cell therapy in glioblastoma (GBM) is undermined by an immunosuppressive tumor microenvironment (TME). We previously showed that CAR T cell therapy targeting epidermal growth factor receptor variant III (EGFRvIII) produces anti-tumor activity against recurrent GBM and causes upregulation of programmed death-ligand 1 (PD-L1) in the TME. Here, we conducted a phase I trial to study the safety and tolerability of CART-EGFRvIII cells administered concomitantly with the PD-1 inhibitor pembrolizumab in patients with newly diagnosed, EGFRvIII+ GBM (n = 7). Treatment was well tolerated without incidence of dose-limiting toxicity. However, no signal of efficacy was detected with a median progression-free survival of 5.2 months (90% CI, 2.9 – 6.0 months) and overall survival of 11.8 months (90 % CI, 9.2 – 14.2 months). We aimed to elucidate reasons for limited efficacy through correlative analyses. Using BBZ qPCR, we found circulating CAR T cells in 5 out of 7 patients at the time of repeat resection, but only in one patient in the tumor. However, shared TCRs were found between the infusion product and the relapsed tumors, which could indicate an infiltration but lack of persistence of the CART. We further compared the tumor microenvironment of the tumors harvested before and after CAR+aPD1 administration using single cell RNAseq, and observed comparable proportions of the major immune cell subsets. However, the myeloid and T cells infiltrating the tumors significantly evolved, with more exhausted, regulatory and IFN-stimulated T cells at the relapse. At that time, the amount of IFN-stimulated T cells positively correlated with time from relapse to death. Together, these findings suggest that the combination of CAR T cells and PD-1 inhibition in GBM is safe and biologically active but, given the lack of efficacy, also indicate a need to consider alternative immunotherapeutic strategies. ClinicalTrials.gov registration: NCT03726515.

STEM-06. OSMR INTERACTOME MAPPING REVEALS THE SIGNIFICANCE OF A CHLORIDE CHANNEL IN CYTOKINE REGULATION AND BRAIN TUMOUR STEM CELL SELF-RENEWAL

Abstract Oncostatin M Receptor (OSMR) is a key regulator of brain tumour stem cell (BTSC) self-renewal and glioblastoma (GB) tumourigenesis. This receptor is found in a complex with Epidermal Growth Factor Receptor variant III (EGFRvIII) and orchestrates a positive feedback loop with STAT3. OSMR also regulates oxidative phosphorylation and participates in promoting the transition of cells towards mesenchymal-like glioma states. How OSMR drives GB tumorigenesis via multiple mechanisms remains largely unclear. Here, we map OSMR interactome in different GB subtypes using the Mammalian Membrane Two Hybrid High Throughput Screen (MaMTH-HTS). Analysis of these data followed by functional validation has uncovered distinct OSMR interactome in a context dependent manner. Follow up investigations, however, have led to the discovery of the Chloride Intracellular Channel 1 (CLIC1), essential in the regulation of OSMR cytokine signaling and GB progression independently of the genetic profile of the GB tumour. CLIC1 forms a complex with OSMR to promote BTSC self-renewal and knockout of CLIC1 or pharmacological inhibitors to CLIC1 impairs OSM mediated STAT3 phosphorylation. Our work suggests that CLIC1 and OSMR form a complex in BTSCs and functionally interact to activate STAT3.

CTIM-09. PHASE 1/1B TRIAL OF FC-ENGINEERED ANTI-CD40 AGONIST MONOCLONAL ANTIBODY (2141-V11) INFUSED WITH D2C7-IT IN ENHANCING DISEASE BY CONVECTION-ENHANCED DELIVERY (CED) FOR RECURRENT MALIGNANT GLIOMA (RMG)

Abstract BACKGROUND D2C7-IT is a dual-specific immunotoxin. Recombinant antibody fragments bind the wild-type epidermal growth factor receptor (EGFRwt) and its mutant EGFR variant III (EGFRvIII), triggering internalization and intracellular delivery of the Pseudomonas exotoxin, D2C7-IT cell-killing component. Single intratumoral delivery of D2C7-IT in animals generates an antitumor response by directly killing tumor cells and indirectly activating an adaptive T-cell response. However, tumor-associated macrophages (TAMs)-mediated immunosuppression limits its efficacy. Eliminating MG immunosuppression via CD40 co-stimulation enhances D2C7-IT-induced antitumor responses by activating a TAMs proinflammatory phenotype and promoting long-term tumor-specific CD8+ T-cell immunity. We initiated a phase 1 trial of D2C7-IT+2141-V11 (Fc-engineered anti-CD40 agonist) administered via CED in rMG patients. METHODS Eligibility includes adult patients with solitary supratentorial rMG (WHO grade 3/4); ≥ 4weeks after chemotherapy, bevacizumab, or investigational agent; adequate organ function; and KPS ≥70%. Cohorts of 3 patients were treated with increasing doses of 2141-V11 to determine the recommended phase 2 dose (RP2D) when administered sequentially following D2C7-IT (166,075ng) via CED. Five dose levels (DLs) were evaluated (2141-V11 at: DL1: 0.70mg; DL2: 2.0mg; DL2**: 3.0mg; DL2*: 4.0mg; DL3: 7.0mg). RESULTS As of June 5, 2023, 27 patients had been treated (3 patients on DL1 and DL2; 2 on DL3 and DL2*; 17 on DL2**). No dose-limiting toxicities were observed; however, lower DLs were added due to higher frequency of adverse events (AEs) expected with D2C7-IT+2141-V11 within DL3 and DL2* (fever, neurologic symptoms). 13/27 patients remain alive (range 4-22months after therapy). No grade 4 or 5 AEs related to D2C7-IT+2141-V11 were observed, while grade 3 related AEs include one each of: dysphasia, encephalopathy, headache, hydrocephalus, paresthesia, and pyramidal tract disorder. CONCLUSIONS The RP2D for intratumoral infusion of D2C7-IT+2141-V11 via CED is identified. The protocol was amended to evaluate the addition of cervical perilymphatic injections of 2141-V11 post CED of D2C7-IT+2141-V11.

Ceramide Is Involved in Temozolomide Resistance in Human Glioblastoma U87MG Overexpressing EGFR.

Glioblastoma multiforme (GBM) is the most frequent and deadly brain tumor. Many sphingolipids are crucial players in the regulation of glioma cell growth as well as in the response to different chemotherapeutic drugs. In particular, ceramide (Cer) is a tumor suppressor lipid, able to induce antiproliferative and apoptotic responses in different types of tumors including GBM, most of which overexpress the epidermal growth factor receptor variant III (EGFRvIII). In this paper, we investigated whether Cer metabolism is altered in the U87MG human glioma cell line overexpressing EGFRvIII (EGFR+ cells) to elucidate their possible interplay in the mechanisms regulating GBM survival properties and the response to the alkylating agent temozolomide (TMZ). Notably, we demonstrated that a low dose of TMZ significantly increases Cer levels in U87MG cells but slightly in EGFR+ cells (sensitive and resistant to TMZ, respectively). Moreover, the inhibition of the synthesis of complex sphingolipids made EGFR+ cells sensitive to TMZ, thus involving Cer accumulation/removal in TMZ resistance of GBM cells. This suggests that the enhanced resistance of EGFR+ cells to TMZ is dependent on Cer metabolism. Altogether, our results indicate that EGFRvIII expression confers a TMZ-resistance phenotype to U87MG glioma cells by counteracting Cer increase.

CHCHD2 mediates glioblastoma cell proliferation, mitochondrial metabolism, hypoxia‑induced invasion and therapeutic resistance.

Glioblastoma (GBM) is the most common and malignant primary brain tumor affecting adults and remains incurable. The mitochondrial coiled‑coil‑helix‑coiled‑coil‑helix domain‑containing protein 2 (CHCHD2) has been demonstrated to mediate mitochondrial respiration, nuclear gene expression and cell migration; however, evidence of this in GBM is lacking. In the present study, it was hypothesized that CHCHD2 may play a functional role in U87 GBM cells expressing the constitutively active epidermal growth factor receptor variant III (EGFRvIII). The amplification of the CHCHD2 gene was found to be associated with a decreased patient overall and progression‑free survival. The CHCHD2 mRNA levels were increased in high‑vs. low‑grade glioma, IDH‑wt GBMs, and in tumor vs. non‑tumor tissue. Additionally, CHCHD2 protein expression was greatest in invasive, EGFRvIII‑expressing patient‑derived samples. The CRISPR‑Cas9‑mediated knockout of CHCHD2 in EGFRvIII‑expressing U87 cells resulted in an altered mitochondrial respiration and glutathione status, in decreased cell growth and invasion under both normoxic and hypoxic conditions, and in an enhanced sensitivity to cytotoxic agents. CHCHD2 was distributed in both the mitochondria and nuclei of U87 and U87vIII cells, and the U87vIII cells exhibited a greater nuclear expression of CHCHD2 compared to isogenic U87 cells. Incubation under hypoxic conditions, serum starvation and the reductive unfolding of CHCHD2 induced the nuclear accumulation of CHCHD2 in both cell lines. Collectively, the findings of the present study indicate that CHCHD2 mediates a variety of GBM characteristics, and highlights mitonuclear retrograde signaling as a pathway of interest in GBM cell biology.

OSMR INTERACTOME MAPPING REVEALS THE SIGNIFICANCE OF A CHLORIDE CHANNEL IN CYTOKINE REGULATION AND BRAIN TUMOUR STEM CELL SELF-RENEWAL

Abstract Oncostatin M Receptor (OSMR) is a key regulator of brain tumour stem cell (BTSC) self-renewal and glioblastoma (GB) tumourigenesis. This receptor is found in a complex with Epidermal Growth Factor Receptor variant III (EGFRvIII) and orchestrates a positive feedback loop with STAT3. OSMR also regulates oxidative phosphorylation and participates in promoting the transition of cells towards mesenchymal-like glioma states. How OSMR drives GB tumorigenesis via multiple mechanisms remains largely unclear. Here, we map OSMR interactome in different GB subtypes using the Mammalian Membrane Two Hybrid High Throughput Screen (MaMTH-HTS). Analysis of these data followed by functional validation has uncovered distinct OSMR interactome in a context dependent manner. Follow up investigations, however, have led to the discovery of the Chloride Intracellular Channel 1 (CLIC1), essential in the regulation of OSMR cytokine signaling and GB progression independently of the genetic profile of the GB tumour. CLIC1 forms a complex with OSMR to promote BTSC self-renewal and knockout of CLIC1 or pharmacological inhibitors to CLIC1 impairs OSM mediated STAT3 phosphorylation. Our work suggests that CLIC1 and OSMR forms a complex in BTSCs and functionally interact to activate STAT3.

Chimeric Antigen Receptor T Cells in Glioblastoma-Current Concepts and Promising Future.

Glioblastoma (GBM) is a highly aggressive primary brain tumor that is largely refractory to treatment and, therefore, invariably relapses. GBM patients have a median overall survival of 15 months and, given this devastating prognosis, there is a high need for therapy improvement. One of the therapeutic approaches currently tested in GBM is chimeric antigen receptor (CAR)-T cell therapy. CAR-T cells are genetically altered T cells that are redirected to eliminate tumor cells in a highly specific manner. There are several challenges to CAR-T cell therapy in solid tumors such as GBM, including restricted trafficking and penetration of tumor tissue, a highly immunosuppressive tumor microenvironment (TME), as well as heterogeneous antigen expression and antigen loss. In addition, CAR-T cells have limitations concerning safety, toxicity, and the manufacturing process. To date, CAR-T cells directed against several target antigens in GBM including interleukin-13 receptor alpha 2 (IL-13Rα2), epidermal growth factor receptor variant III (EGFRvIII), human epidermal growth factor receptor 2 (HER2), and ephrin type-A receptor 2 (EphA2) have been tested in preclinical and clinical studies. These studies demonstrated that CAR-T cell therapy is a feasible option in GBM with at least transient responses and acceptable adverse effects. Further improvements in CAR-T cells regarding their efficacy, flexibility, and safety could render them a promising therapy option in GBM.

Identification of the E2F1-RAD51AP1 axis as a key factor in MGMT-methylated GBM TMZ resistance.

Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme (GBM). Temozolomide (TMZ) is a standard chemotherapeutic for GBM, but TMZ treatment benefits are compromised by chemoresistance. This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance. CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM. Western blot, real-time PCR, flow cytometry, and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1 (RAD51AP1). Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells. Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment. Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells. Knockdown of E2F1 increased sensitivity to TMZ. Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1, mediates TMZ resistance, and has a potential E2F1 binding site on the promoter. Knockdown of RAD51AP1 enhanced the sensitivity of TMZ; however, overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells. Furthermore, RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase (MGMT) expression. The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated, but not MGMT-unmethylated TMZ-treated GBM patients. Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment. RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair. Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.

Abstract LB097: Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma

Abstract Introduction: We have completed two CAR T cell clinical trials for glioblastoma (GBM) and have identified several key challenges to therapeutic efficacy, including the inherently heterogenous genomic landscape and the immunosuppressive tumor microenvironment (TME) found in GBM. Our previous study showed that EGFR variant III (EGFRvIII)-targeting monovalent CAR T cells reduced target-positive tumor cell populations, but tumor recurrence resulted from target-negative tumor cells, highlighting the limitation of single-target approaches in heterogenous tumors. With regards to the highly immunosuppressive TME in GBM, we found that transforming growth factor-β (TGFβ) was present in the GBM TME as a major driver of suppression of the anti-GBM response in clinical samples. TGFb is consistently highly expressed in both GBM tumor cell lines and patient tumor tissues. Methods: We used two parallel scFv constructs, independently targeting both IL13Rα2 and EGFRvIII, in combination with a truncated dominant negative (dn) TGFβ receptor II. This trivalent construct was designed to explore possible additive effects in both in vitro and in vivo GBM model systems to limit tumor escape and overcome the immunosuppressive GBM TME. The CART-EGFR-IL13Rα2-dnTGFb construct broadened the targeted tumor cell repertoire, blocked TGFβ signaling, and served as a sink for free TGFβ in the GBM TME to overcome the suppressive function of TGFβ. Results: The tri-modular CAR T construct had an enhanced proliferative response when compared with the CART-EGFR-IL13Rα2 construct, in vitro. In co-culture assays, this construct led to reduced PD-1 expression and increased central memory phenotype, when compared to the bicistronic CAR T construct, which suggested a lower fraction of exhausted T cells. Tri-modular CAR T cells blocked the suppressive pSmad2/3 signaling pathway, leading to the increased tumor killing activity in co-culture experiments with both adherent and suspension GBM cell lines. In an immunodeficient mouse model, tri-modular CAR T cells eradicated tumor cells efficiently and mice had a longer median survival when compared those treated with the bicistronic CART-EGFR-IL13Rα2 cells, lacking the dnTGFb receptor II. Conclusion: Overcoming the adaptive changes in the local TME and addressing antigen heterogeneity will be required to improve the clinical efficacy of CAR T-directed strategies. Our combination work showed that bicistronic CART constructs cooperate with truncated TGFβ receptor II efficiently. In summary, the dominant-negative TGFβ RII CART-EGFR-IL13Rα2 structure is a promising strategy to address the clinical challenges of antigenic heterogeneity and the immunosuppressive TME in GBM we have observed in our two GBM CART cell trials at UPenn. Citation Format: Nannan Li, Jesse Rodriguez, Zev Binder, Donald O’Rourke. Armored bicistronic CAR T cells with dominant-negative TGF-β receptor II to alleviate antigenic heterogeneity and suppressive immune microenvironment in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB097.

IGFBP3 induced by the TGF-β/EGFRvIII transactivation contributes to the malignant phenotype of glioblastoma

Dual or multi-targets therapy targeting epidermal growth factor receptor variant III (EGFRvIII) and other molecular may relax the constraint for glioblastoma (GBM), putting forward the urgent requirement of finding candidate molecules. Here, the insulin-like growth factor binding protein-3 (IGFBP3) was considered a candidate, whereas the mechanisms of IGFBP3 production remain unclear. We treated GBM cells with exogenous transforming growth factor β (TGF-β) to simulate the microenvironment. We found that TGF-β and EGFRvIII transactivation induced the activation of transcription factor c-Jun, which specifically bound to the promoter region of IGFBP3 through Smad2/3 and ERK1/2 pathways and promoted the production and secretion of IGFBP3. IGFBP3 knockdown inhibited the activation of TGF-β and EGFRvIII signals and the malignant behaviors triggered by them invitro and invivo. Collectively, our results indicated a positive feedback loop of p-EGFRvIII/IGFBP3 under administration of TGF-β, blocking IGFBP3 may be an additional target in EGFRvIII-expressing GBM-selective therapeutic strategy.

Immunotoxin-αCD40 therapy activates innate and adaptive immunity and generates a durable antitumor response in glioblastoma models.

D2C7-immunotoxin (IT), a dual-specific IT targeting wild-type epidermal growth factor receptor (EGFR) and mutant EGFR variant III (EGFRvIII) proteins, demonstrates encouraging survival outcomes in a subset of patients with glioblastoma. We hypothesized that immunosuppression in glioblastoma limits D2C7-IT efficacy. To improve the response rate and reverse immunosuppression, we combined D2C7-IT tumor cell killing with αCD40 costimulation of antigen-presenting cells. In murine glioma models, a single intratumoral injection of D2C7-IT+αCD40 treatment activated a proinflammatory phenotype in microglia and macrophages, promoted long-term tumor-specific CD8+ T cell immunity, and generated cures. D2C7-IT+αCD40 treatment increased intratumoral Slamf6+CD8+ T cells with a progenitor phenotype and decreased terminally exhausted CD8+ T cells. D2C7-IT+αCD40 treatment stimulated intratumoral CD8+ T cell proliferation and generated cures in glioma-bearing mice despite FTY720-induced peripheral T cell sequestration. Tumor transcriptome profiling established CD40 up-regulation, pattern recognition receptor, cell senescence, and immune response pathway activation as the drivers of D2C7-IT+αCD40 antitumor responses. To determine potential translation, immunohistochemistry staining confirmed CD40 expression in human GBM tissue sections. These promising preclinical data allowed us to initiate a phase 1 study with D2C7-IT+αhCD40 in patients with malignant glioma (NCT04547777) to further evaluate this treatment in humans.

A Case Study of Chimeric Antigen Receptor T Cell Function: Donor Therapeutic Differences in Activity and Modulation with Verteporfin.

Chimeric antigen receptor (CAR) T cells have recently been demonstrated to extract and express cognate tumor antigens through trogocytosis. This process may contribute to tumor antigen escape, T cell exhaustion, and fratricide, which plays a central role in CAR dysfunction. We sought to evaluate the importance of this effect in epidermal growth factor receptor variant III (EGFRvIII) specific CAR T cells targeting glioma. EGFRvIII-specific CAR T cells were generated from various donors and analyzed for cytotoxicity, trogocytosis, and in vivo therapeutic activity against intracranial glioma. Tumor autophagy resulting from CAR T cell activity was evaluated in combination with an autophagy inducer (verteporfin) or inhibitor (bafilomycin A1). CAR T cell products derived from different donors induced markedly divergent levels of trogocytosis of tumor antigen as well as PD-L1 upon engaging target tumor cells correlating with variability in efficacy in mice. Pharmacological facilitation of CAR induced-autophagy with verteporfin inhibits trogocytic expression of tumor antigen on CARs and increases CAR persistence and efficacy in mice. These data propose CAR-induced autophagy as a mechanism counteracting CAR-induced trogocytosis and provide a new strategy to innovate high-performance CARs through pharmacological facilitation of T cell-induced tumor death.

Abstract 2077: The Epidermal Growth Factor Receptor variant III (EGFRvIII) induces the assembly of a unique intracellular compartment with mixed early endosomal and endoplasmic reticular nature

Abstract 2077: The Epidermal Growth Factor Receptor variant III (EGFRvIII) induces the assembly of a unique intracellular compartment with mixed early endosomal and endoplasmic reticular nature

MUC1 promotes glioblastoma progression and TMZ resistance by stabilizing EGFRvIII

Epidermal growth factor receptor variant III (EGFRvIII) is a mutant isoform of EGFR with a deletion of exons 2–7 making it insensitive to EGF stimulation and downstream signal constitutive activation. However, the mechanism underlying the stability of EGFRvIII remains unclear. Based on CRISPR-Cas9 library screening, we found that mucin1 (MUC1) is essential for EGFRvIII glioma cell survival and temozolomide (TMZ) resistance. We revealed that MUC1-C was upregulated in EGFRvIII-positive cells, where it enhanced the stability of EGFRvIII. Knockdown of MUC1-C increased the colocalization of EGFRvIII and lysosomes. Upregulation of MUC1 occurred in an NF-κB dependent manner, and inhibition of the NF-κB pathway could interrupt the EGFRvIII-MUC1 feedback loop by inhibiting MUC1-C. In a previous report, we identified AC1Q3QWB (AQB), a small molecule that could inhibit the phosphorylation of NF-κB. By screening the structural analogs of AQB, we obtained EPIC-1027, which could inhibit the NF-κB pathway more effectively. EPIC-1027 disrupted the EGFRvIII-MUC1-C positive feedback loop in vitro and in vivo, inhibited glioma progression, and promoted sensitization to TMZ. In conclusion, we revealed the pivotal role of MUC1-C in stabilizing EGFRvIII in glioblastoma (GBM) and identified a small molecule, EPIC-1027, with great potential in GBM treatment.

Selected Articles from This Issue

Glioblastoma (GBM) is one of the most aggressive cancers and it is usually refractory to immunotherapies. EGFR variant III (EGFRvIII) is an EGFR mutation commonly present in GBM that generates a unique cell surface neoantigen in tumor cells. Here, Iurlaro and colleagues have designed and developed a novel T cell bispecific antibody (TCB) with specificity for EGFRvIII and CD3. The EGFRvIII-TCB promoted T cell activation and tumor cell killing in EGFRvIII-expressing patient-derived models, and induced tumor regression in GBM patient-derived xenografts (PDX). These results led to the clinical testing of the EGFRvIII-TCB for the treatment of EGFRvIII-expressing GBMs.Comparative oncology clinical trials, carried out in pet dogs with spontaneously occurring cancers, are increasingly recognized a valuable component of the anti-cancer drug discovery and development pathway. In this report, LeBlanc and colleagues conducted a clinical study of a novel orally bioavailable inhibitor of valosin-containing protein (VCP), CB-5339, in pet dogs with a variety of malignancies. This canine trial generated key pharmacokinetic and pharmacodynamic data, defined a tolerable dose and schedule, and discovered an efficacy signal in multiple myeloma. The data generated herein provided a foundation for future studies of this drug in humans with multiple myeloma as well as other tumor types.Glutamine metabolism has been considered an important mechanism for control of tumor growth and survival. Here, Yokoyama and colleagues describe the preclinical pharmacology and mechanism of action of the novel clinical stage glutamine antagonist, DRP-104 (sirpiglenastat). Broadly targeting all 10 glutamine metabolizing enzymes, DRP-104 leads to profound anti-tumor activity related to both direct effects on tumor metabolism as well as immune mediated activity via remodeling of the tumor microenvironment and induction of both innate and adaptive anti-tumor immunity. This unique activity shows promise for the treatment of various tumors including those resistant to immune checkpoint inhibitors.Prostate cancer is the second leading cause of cancer related deaths and most commonly diagnosed cancer in US males. Although androgen receptor-targeted therapies such as enzalutamide are initially effective for the treatment of advanced prostate cancer, resistance occurs frequently. Here, Ning and colleagues have demonstrated that Wnt5A/FZD2 signaling plays vital roles in conferring resistance to enzalutamide and the levels of their expression correlates with aggressive prostate cancer with shortened disease-free survival. Furthermore, a novel bioengineered BERA-Wnt5A siRNA construct significantly suppressed tumor growth and enhanced enzalutamide treatment, suggesting targeting Wnt5A could be developed as a potential therapy for advanced prostate cancer.

240 (PB020) - Antitumor activity of Antibody-Drug Conjugates targeting cancer-expressed EGFR in Preclinical Models

Background: The epidermal growth factor receptor (EGFR) plays a key role in the growth and survival of many human tumors of epithelial origin. The monoclonal antibody, 40H3, targeting overexpressed EGFR and the truncated form EGFR variant III (EGFRvIII) on tumor cells, was conjugated to small molecules with potent cytotoxic activity, generating five antibody-drug conjugates (ADCs). Their lethality for tumor cells was evaluated in vitro and in vivo. Materials and methods: ADC construction: Purified 40H3 antibody was conjugated to five different payloads: two tubulin inhibitors, monomethyl auristatin E (MMAE) and DM1, two topoisomerase inhibitors, SN38 and deruxtecan (DXd1) and a PBD dimer (SG-3199). Binding assay: The binding affinity constants of the various 40H3-based ADCs were determined against the His-tagged EGFR peptide loop (aa 287– 302) immobilized on Ni-NTA biosensors with the Octet Red96 analyzer. Bystander assay: Cocultured F98npEGFRvIII and F98 cells were treated with media, free payload (SG-3199), 40H3 antibody, 40H3-tesirine or IgG-tesirine at the indicated concentrations. After 48 hours, the cells were labeled with cetuximab-PE and SYTOXTM Red viability dye and then analyzed for bystander killing by flow cytometry. In vivo studies: MDA-MB-468 or BT-20 tumor xenografts were treated with unmodified 40H3 antibody or 40H3-tesirine. Tumor volumes and mouse weights were measured at least three times weekly. Results: The five ADCs retained antigen binding activity of the unmodified 40H3 antibody. They showed a potent cytotoxicity on a panel of EGFR-expressing cells including three triple negative breast cancer lines. Cell killing was correlated to the number of binding sites for the 40H3 antibody. The 40H3 conjugate with the PBD dimer (40H3-tesirine) was the most active killing agent and it also exhibited bystander killing of cells not expressing human EGFR. Moreover, in vivo, on two different models of tumor xenografts, treatment with 40H3-tesirine achieved complete remissions. Conclusions: The 40H3 antibody is a valid delivery agent for toxic payloads. Among the five ADCs, 40H3-tesirine showed the highest cytotoxic effect toward EGFR-expressing tumor cells in vitro and in vivo. No conflict of interest.