Murine Tumor Models Research Articles

Abstract 52: CRISPR-mediated knock-out of A20 lowers the activation threshold and induces metabolic rewiring of CAR-T cells

Abstract Background: Common failures of chimeric antigen receptor (CAR)-T cell therapy regimens stem from immune escape of target antigen low tumors. Strategies to improve CAR-T cell therapy efficacy include enhancing intrinsic effector function while preventing T cell exhaustion. The ubiquitin-modifying enzyme A20, is a key negative regulator of the NF-kB pathway downstream of T cell receptor activation in T cells. Dysfunctional CD8 T cells in tumors have high expression of A20, and deletion of A20 enhances antitumor activity of CD8 T cells in murine tumor models. We tested whether CRISPR-mediated knock-out (KO) of tumor necrosis factor α-induced protein 3 (TNFAIP3), the gene for A20, increases the effector function and alters the metabolic state of CAR-T cells. Results: A20 deletion led to upregulation of NF-κB target genes and resulted in higher levels of activation marker expression and effector cytokine secretion compared to unarmored CAR-T upon incubation with target cells. In addition, A20 KO CAR-T cells maintained prolonged expression of activation markers upon antigen clearance in the absence of enhanced differentiation, and were more resistant to dysfunction in a repeat challenge serial kill assay. In vivo, A20 deficient CAR-T exhibited increased anti-tumor efficacy against low, medium, and high target expressing tumor models. Consistent with NF-κB upregulation, A20 KO cells significantly upregulated key glycolytic intermediates suggesting that amino acid anabolism was fueled by glycolysis and the pentose phosphate pathway. Moreover, the preferential increase of glycolysis over oxidative phosphorylation confirmed that A20 KO CAR-T cells were skewed towards an effector-like phenotype. Notably, glutathione and its cysteine-glycine bi-products were upregulated upon activation to a higher level in A20 KO CAR-T suggesting enhanced resistance to oxidative stress, in line with their prolonged persistence. Conclusions: CRISPR-mediated knock-out of A20 lowers the activation threshold of CAR-T cells producing enhanced in vitro and in vivo anti-tumor efficacy particularly against tumors with very low levels of target expression. A20 KO induces metabolic rewiring of CAR-T cells by promoting glycolysis and amino acid anabolism to sustain their superior effector functions. This intrinsic armoring strategy can be applied to any CAR-T in any indication to increase tumor control and reduce immune escape. Citation Format: Nina Jia Chu, Carina Nava-Barbero, Ashley Merlino, Ping-Hsien Lee, David Baker, Ben Taylor, Gordon Moody, Letizia Giardino. CRISPR-mediated knock-out of A20 lowers the activation threshold and induces metabolic rewiring of CAR-T cells [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 52.

Abstract 5322: Discovery of CRD1601, a potent and selective HPK1 inhibitor with robust in vivo anti-cancer activity

Abstract Background: Haematopoietic Progenitor Kinase (HPK-1, MAP4K1) is a member of the STE20 family of serine/threonine kinases, expressed predominantly in hematopoietic cells. HPK-1 functions as a negative regulator of T-cell and B-cell receptor activation by triggering proteasomal degradation and disrupting signalosome complexes downstream of TCR and BCR. HPK1 inhibition is a novel IO strategy that could combine with existing chemotherapies and immunotherapies. Methods: Curadev used a pharmacophore based approach to discover novel small molecule HPK-1 inhibitors using in vitro enzymatic and phenotypic cellular screens using primary cells. CRD1601, the clinical candidate was selected based on its ability to enhance human T-cell proliferation through the induction of IL2 and IFNγ. Syngeneic tumor models were used to assess anti-tumor activity of selected compounds as a monotherapy or combination therapy. Results: CRD1601 is a potent HPK1 inhibitor with a single digit nanomolar IC50 value in enzymatic assay and potent cellular activity in the SLP76 assay. A single dose of CRD1601 in mice treated with an anti-CD3 antibody caused systemic enhancement of pro-inflammatory cytokines such as IL2, IFN gamma and reduced pSLP76 levels in the spleen. Further, CRD1601 reversed the immunosuppressive effect of PGE2 and NECA. CRD1601 demonstrated significant single agent activity in murine tumor models while also synergizing with chemotherapy and immunotherapy. Conclusions: Inhibition of HPK-1 is a promising therapeutic modality that could augment the effects of existing anti-cancer treatments including immunotherapy. CRD1601 is a potent and selective HPK-1 inhibitor with favorable drug like properties that has been selected from a rich portfolio of active compounds and shows promising in vivo activity in tumor models. Citation Format: Monali Banerjee, Ritesh Shrivastava, David Pryde, Sandip Middya, Sabyasachi Debnath, Anindita Middya, Dharmendra Yadav, Nidhi Rawat, Rajib Ghosh, Manisha Padaliya, Sourav Basu, Arjun Surya. Discovery of CRD1601, a potent and selective HPK1 inhibitor with robust in vivo anti-cancer activity [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 5322.

Abstract 6523: TOX upregulation promotes a GPX4-dependent CD8+ T cell ferroptosis in tumor microenvironments

Abstract Frequency and function of CD8+ tumor-infiltrating T cells (TILs) decide on clinical outcome of cancer immunotherapy; and TOX function as a crucial regulator of TIL dysfunction. In the present study, we found that multiplex factors, such as tumor cell, viral antigens and hypoxia, in tumor microenvironments (TME) induced CD8+ TIL ferroptosis by upregulating TOX. Single cell RNA-sequencing revealed that TOX modulated Havcr2, Pdcd1, Hmox1, Gpx4, and Ctsb transcription in human and murine CD8+ TILs. We further generated TOX conditional knockout in CD8+ T cell (Toxflox/floxCd8Cre, CKO) mice and the tumor-bearing CKO mice exhibited increased anti-tumor immunity and survival time. TOX upregulation increased reactive oxygen species (ROS), lipid peroxidation, and ferrous iron, leading to a GPX4-dependent CD8+ T cell ferroptosis. Genetic or antibody ablation of TOX, TIM-3, or HO-1 indicated that these molecules orchestrated CD8+ TIL ferroptosis by inducing iron overload and lipid peroxidation. Targeting TOX, PD-1, TIM-3, or ferroptosis boosted the anti-tumor suppression of tumor-specific T cell-based adoptive cell therapy (ACT) in murine tumor model. TOX, TIM-3 and GPX4 were prognostic predictors in human cervical cancer. This finding identifies a novel mechanism of tumor-induced CD8+ T cell ferroptosis underwent by TOX that provides new insights in improving TIL-based ACT immunotherapy. Key words: TOX, CD8+ T cells, ferroptosis, tumor microenvironments, cancer immunotherapy Citation Format: Jiang Li, Qian Zhu, Xiufeng Liu. TOX upregulation promotes a GPX4-dependent CD8+ T cell ferroptosis in tumor microenvironments [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 6523.

Abstract 7458: Inhibition of brain cancer progression via resolution of inflammation

Abstract While inflammation is a hallmark of cancer, the mechanisms that disrupt its resolution in cancer are not well understood. A paradigm shift is emerging in our understanding that the resolution of inflammation is an active process controlled by specialized pro-resolving mediators (SPMs), including protectins and resolvins, which are endogenous lipid autacoids present in multiple tissues including the brain and cerebrospinal fluid. SPMs counter-regulate pro-inflammatory cytokine production and stimulate phagocytosis of cellular debris (“pro-resolution”) without being immunosuppressive. We hypothesized treatment with pro-resolving agonists such as SPMs presents a novel debris-clearing and resolution-enhancing therapeutic approach to complement cytotoxic brain cancer therapies. Here, we demonstrate the failure of resolution of inflammation via dysregulated SPMs may be a critical risk factor for aggressive brain cancer growth. Moreover, chemotherapy and targeted therapy further disrupt inflammation resolution via a tumor cell death (“debris”)-induced cytokine storm by macrophages. The SPM receptors GPR37 (protectin D1), GPR18/DRV2 (resolvin (Rv) D2), GPR32 (RvD1), and ChemR23/ERV (RvE1) are expressed in tumor-infiltrating macrophages and endothelial cells in the brain tumor microenvironment. Plasma levels of SPMs were significantly reduced in the medulloblastoma-bearing mice, suggesting that brain cancer progression is associated with a failure of endogenous resolution of inflammation. To determine whether failed resolution in brain cancer can be rescued by lipid mediator agonists, we systemically administered SPMs to subcutaneous and orthotopic glioblastoma and medulloblastoma murine brain tumor models. Significantly, RvD1 and RvD2 each inhibited the growth of human glioblastoma tumors by 15-fold at nanogram concentrations without toxicity. Likewise, RvD2 inhibited the growth of human medulloblastoma tumors by 3-fold. Protectins, PDX and PD1n-3, each inhibited growth of human medulloblastoma in mice by approximately 5-fold. Systemic administration of PDX or RvD2 prolonged survival of mice injected with orthotopic human D556 medulloblastoma and also glioblastoma models. Flow cytometry analysis of orthotopic glioblastoma tumors revealed increased immune infiltration into the brain tumor microenvironment in the PCTR1-treatment group when compared to the tumor-bearing non-treated group. At nanogram per day doses, protectins and resolvins suppressed orthotopic debris-stimulated brain tumor growth by activating macrophage and microglial phagocytosis of tumor cell debris and counter-regulating pro-inflammatory cytokines, including TNF-α, CCL2, CXCL1, and CCL4. Thus, stimulating the resolution of inflammation via the clearance of cellular debris with pro-resolving agonists represents a novel therapeutic approach to brain cancer, including glioblastoma and medulloblastoma. Citation Format: Jacqueline Capuano, Kimberly Vasquez, Katherine Quinlivan, Rachel Bayer, Sarina Virani, Michael Gillespie, Mark Kieran, Charles Serhan, Diane Bielenberg, Dipak Panigrahy. Inhibition of brain cancer progression via resolution of inflammation [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 7458.

Abstract 5979: Recombinant N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB) inhibits lung metastases in murine B16F10 syngeneic tumor model

Abstract Activity of the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B: ARSB), which removes the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S) and dermatan sulfate, is reduced in several malignant cell lines and human tissues, including melanomas, mammary, prostate, and colon. Following tail vein injection of B16F10 melanoma cells in 12-week-old female C57BL/6J mice (n=11) and subsequent treatment by tail vein injection of bioactive, recombinant human ARSB (0.2 mg/kg), on days 2 and 7, development of metastatic lung lesions was markedly reduced. The average number of lesions declined from mean value of 114 (range: 10-278; n=5) to mean value of 2.5 (range: 0-9; n=6). These findings are consistent with our previous report of significant increase in survival and decrease in size of B16F10 subcutaneous melanomas in C57BL/6J mice treated with exogenous ARSB. The underlying mechanisms leading to inhibition of melanoma proliferation have profound impact on vital signaling pathways and transcriptional events. These pathways involve more or less binding with C4S when ARSB activity is modified. Notably, galectin-3 binding with C4S increases following exogenous ARSB, leading to decline in free nuclear galectin-3. This leads to reduced expression of the melanoma biomarker chondroitin sulfate proteoglycan CSPG)-4; also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP). Expression of Programmed Death-Ligand 1 (PD-L1) is also reduced, declining from 1.34 ± 0.23 ng/mg protein (n=5) to 0.50 ± 0.05 ng/mg protein (n=6) following ARSB treatment. Inverse to the effect on galectin-3, SHP2, the ubiquitous non-receptor tyrosine phosphatase (PTPN11), binds less to C4S following treatment with exogenous ARSB. The resulting activation of SHP2 impacts on MAPK signaling, reducing phospho-ERK1/2 and phospho(T180/Y182)-p38-MAPK. These effects contribute to decline in matrix metalloproteinases MMP2 and MMP9 and thereby restrain invasiveness and metastasis. Following treatment with exogenous ARSB, the serum MMP2 declined from 267 ± 38 (ng/ml; n=5) to 121 ± 16 ng/ml (n=6), consistent with the observed decline in melanoma lesions. The impact of ARSB on signaling pathways and transcriptional events demonstrates how modification of a critical sulfation and the resulting impact on binding with C4S influences vital cell processes and can suppress tumor development. Recombinant human ARSB is an approved agent for the treatment of Mucopolysaccharidosis VI and is used safely and effectively for this congenital deficiency of ARSB. Exogenous ARSB may become an effective treatment for acquired deficiency of ARSB in melanoma. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Insug O-Sullivan. Recombinant N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB) inhibits lung metastases in murine B16F10 syngeneic tumor model [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 5979.

Abstract 2715: Preclinical characterization of SM2248: A dual-action bispecific antibody enhancing immunotherapy for solid tumors

Abstract Background: Innate immune cells, such as macrophages and myeloid-derived suppressor cells (MDSCs), dominate the immune landscape within "cold" solid tumors, which typically exhibit poor responses to immune checkpoint inhibitors (ICIs). The dearth of tumor-infiltrating T cells and the prevailing immunosuppressive microenvironment contribute to this ineffectiveness. To bolster immunotherapy against such tumors, we developed SM2248, a bispecific VHH antibody. SM2248 simultaneously obstructs the CD47-SIRPα phagocytosis checkpoint pathway and conditionally activates CD40 on tumor-infiltrating innate immune cells. CD47/SIRPa blockade enhances macrophage and dendritic cell (DC) phagocytosis and cross-presentation of antigens to cytotoxic T cells. CD40 activation fosters DC licensing, reprograms macrophages, and guides MDSC differentiation towards DCs and macrophages. The combined action of CD47 blockade and CD40 stimulation remodels the tumor microenvironment, fostering T cells infiltration and activation. Methods: SM2248 was developed by fusing a highly selective CD47-blocking VHH module - minimizing binding to human erythrocytes and platelets, with a conditional CD40 agonistic VHH module - selectively activating immune cells upon CD47 engagement, incorporated into an inactivated IgG1 Fc backbone. The specificity and affinity of SM2248 binding to human CD47 and CD40 were assessed via ELISA and flow cytometry assays. CD47 and SIRPα interaction blockade was confirmed through a competitive ligand binding assay. Target-dependent CD40 activation was evaluated via a CD40-NFκB-Luc reporter assay. In vivo anti-tumor efficacy was determined using a syngeneic transplantation model of MC38-hCD47 cancer cells in B-hCD40 humanized mice. Binding to erythrocytes and platelets, compared with magrolimab, was assessed using flow cytometry. Hemolysis toxicity was evaluated through an in vitro erythrocyte lysis assay. Results: SM2248 effectively inhibits cell surface CD47-SIRPα interaction at nanomolar concentrations and activates CD40-NF-κB-luciferase reporters at picomolar concentrations in a target-dependent manner. In contrast to the CD47 monoclonal antibody magrolimab, SM2248 does not bind to human erythrocytes or platelets and does not induce hemolysis. In murine syngeneic tumor models, SM2248 completely halts the growth of MC38-hCD47 tumors in humanized B-hCD40 mice and does not induce dose-dependent hepatotoxicity at doses up to 10 mg/kg. Conclusions: SM2248, a humanized bispecific tetravalent VHH antibody targeting CD47 and CD40, exhibits promising in vivo efficacy and a favorable safety profile in preclinical studies, offering the potential for enhancing immunotherapy in solid tumors. Citation Format: Xiaodan Liu, Hong Zhou, Shihao Lu, Huiqin Geng, Wenjing Song, Siyao Xie, Yi Wei, Xing Zhang, Simin Yang, Sheila Zhou, Yanbin Liang. Preclinical characterization of SM2248: A dual-action bispecific antibody enhancing immunotherapy for solid tumors [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 2715.

Abstract 6752: Targeting of neoantigen cancer vaccines to antigen presenting cells results in faster and broader T cell responses whether encoded in DNA or mRNA

Abstract Individualized cancer vaccines based on patient-specific neoantigens represent a groundbreaking approach in oncology, offering a highly individualized treatment option for cancer patients. Unlike traditional vaccines, which are designed to prevent diseases by stimulating an immune response against a specific pathogen, personalized cancer vaccines are therapeutic, aiming to treat cancer by training the immune system to recognize and attack the patient’s own cancer cells. The key is to induce a fast, strong, broad, and persistent CD8+ T cell response. Nykode Therapeutics has developed a platform that targets antigens directly to antigen presenting cells (APCs) using a modular dimeric protein format known as a VaccibodyTM. Individualized neoantigen vaccines delivered in a DNA-encoded Vaccibody format have been shown to induce strong and broad T cell responses in pre-clinical and clinical settings. Here, we wanted to determine if the APC-targeted approach leads to superior T cell responses if Vaccibody vaccines are encoded in mRNA and administered in a lipid nanoparticle (LNP) formulation. For this purpose, we designed and evaluated a model cancer vaccine composed of 20 unstructured 27 amino acid long neoantigens derived from the murine MC38 tumor model. A DNA-encoded Vaccibody vaccine elicited faster T cell responses compared to an untargeted mRNA-LNP-encoded vaccine. DNA-encoded Vaccibody responses were also broader, targeting a substantially greater number of neoantigens. Next, we compared the APC-targeting Vaccibody vaccine to an untargeted vaccine when both were formulated in an mRNA-LNP format. The APC-targeting Vaccibody vaccine elicited a greater breath and magnitude of T cell responses compared to the untargeted vaccine. Further studies to evaluate the functional differences associated with the various formats, in terms of T cell phenotype and response to tumor challenge, are ongoing. In summary, the data demonstrate that an APC-targeted Vaccibody vaccine containing MC38 neoantigens can be encoded in both DNA and mRNA-LNP to induce faster, broader, and stronger T cell responses compared to a non-targeted neoantigen vaccine. Citation Format: Brian T. Weinert, Lise Skullerud, Eirik Solbakken, Ana Textor, Mirjam Dürkoop, Inga Patrycja Winge, Gunnstein Norheim, Elisabeth Stubsrud, Stine Granum, Mikkel W. Pedersen, Håkan Norell. Targeting of neoantigen cancer vaccines to antigen presenting cells results in faster and broader T cell responses whether encoded in DNA or mRNA [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 6752.

Abstract 5002: Next-generation mRNA-LNP vaccine prototype achieves tumor clearance in a GBM mouse model

Abstract Glioblastoma (GBM) is the most prevalent and aggressive primary brain tumor type with an abysmal prognosis, featuring an immunologically dampened tumor microenvironment and limited neoantigen production. Novel therapeutic strategies are urgently needed. A well-characterized GBM-associated mutation that can elicit an anti-tumor immune response is EGFRvIII mutation. The cancer-driver mutation is detected in approximately 30% of patients at the time of diagnosis and plays a pivotal role in the emergence of GBM. EGFRvIII is an in-frame epidermal growth factor receptor (EGFR) deletion generating a constitutively active oncogenic protein. Its highly immunogenic nature represents an ideal focal point for cutting-edge mRNA-LNP candidate therapeutics. To evaluate proprietary lipid nanoparticle (LNP) compositions for their capacity to induce anti-GBM immunity, we developed a murine transplantable GBM tumor model by neonatal electroporation. The somatically engineered glioblastomas harbor two targeted CRISPR knockouts and stably overexpress EGFRvIII to yield transplantable cell lines for orthotopic tumor initiation. One week after intracranial tumor cell implantation of 6-8 week-old female mice (day 0; n=10), half the animals were administered either buffer (control cohort) or our novel anti-EGFRvIII mRNA-LNP vaccine (experimental cohort). The regimen involved four intramuscular injections before monitoring tumor progression by MRI on day 30. The imaging revealed a high tumor burden in all control group animals, compared to no detectable tumor cells in vaccine-protected mice. All control group animals were humanely euthanized by day 38 due to symptoms associated with the growing brain tumor mass. The asymptomatic experimental animals received a fifth anti-EGFRvIII vaccination before MRI-based examinations on day 43 and day 78 confirmed that no tumor cells could be detected in vaccine-protected mice. The results highlight the potency and potential of our proprietary anti-EGFRvIII mRNA-LNP vaccine as a novel anticancer therapeutic. In addition, the established tumor model represents a valuable tool to test future mRNA-LNP designs. Outstanding cellular and molecular investigations will reveal vaccine-mediated mechanistic insights, allowing for the co-advancement of the tumor model and next-generation multi-target mRNA-LNP vaccine candidates. Citation Format: Robert Nechanitzky, Shannon Snelling Snelling, Kristofor Ellestad, Xueqing Lun, Kirstin Olsen, Yu Wu, Yury Karpov, Jun Liu, Matthew Gold, Haritha Menon, Rajesh Krishnan, Robert Georgantas, Pamela S. Ohashi, Douglas J. Mahoney, Jennifer A. Chan, Natalia Martin-Orozco. Next-generation mRNA-LNP vaccine prototype achieves tumor clearance in a GBM mouse model [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 5002.

Abstract 2652: NDI-101150 is a potent and highly selective HPK1 inhibitor that both synergizes with and differentiates from anti-PD1 immune checkpoint blockade

Abstract Introduction HPK1 is a member of the MAP4K family of protein serine/threonine kinases that negatively regulates activation signals in multiple immune cells and is an attractive therapeutic target for many cancers. Using structure-based drug design, we developed a highly selective HPK1 inhibitor, NDI-101150, with nanomolar potency and physiochemical properties suitable for once daily oral administration. Methods & Results HPK1 inhibition with NDI-101150 has a differentiated pharmacology profile when compared directly to PD1 blockade. NDI-101150 was able to overcome the immunosuppressive effects of prostaglandin E2, adenosine, and TGF-β on human T cell activation. HPK1 inhibition also directly limited the suppressive capacity of T regulatory cells. In contrast, anti-PD1 treatment was not able to overcome the immunosuppressive activity associated with any of these mechanisms. In vivo, HPK1 inhibition significantly enhanced the production of antigen-specific antibodies in response to immunization with both T-dependent and T-independent antigens, while anti-PD1 treatment had minimal to no effects on antibody production. In the EMT-6 syngeneic model, 7/10 mice receiving NDI-101150 exhibited complete tumor regressions, compared to only 1/10 from an anti-PD1 cohort. Furthermore, NDI-101150 established a robust and durable immune memory response as 100% of treated mice showed complete rejection of tumor growth upon subsequent re-challenge. HPK1 inhibition with NDI-101150 also shows robust synergy with PD1 blockade in vitro and in vivo. Both NDI-101150 and anti-PD1 were able to reinvigorate exhausted human T cells, restoring cytokine production and proliferative effects in mixed lymphocyte reactions. Further, in combination HPK1 inhibition and PD1 blockade synergized to enhance the activity of exhausted human T cells to an extent exceeding that observed with naïve human T cells in similar experiments. In vivo, while NDI-101150 or anti-PD1 treatments alone induced tumor growth inhibition in the murine CT-26 syngeneic tumor model, NDI-101150 and anti-PD1 in combination mediated complete tumor regressions in several mice. Furthermore, NDI-101150 seemed to induce a durable immune memory response as evidenced by the combination-treated animals showing complete rejection of tumor growth upon subsequent re-challenge, without any further dosing of NDI-101150. Conclusions Pharmacological inhibition of HPK1 with NDI-101150 represents a powerful system-wide immunomodulatory approach with the potential to enhance anti-tumor immunity in patients failing to respond to currently approved immune checkpoint therapies or in combination with immune checkpoint blockade therapies. NDI-101150 is currently being tested in a Phase 1/2 trial (NCT05128487) in patients with advanced solid tumors. Citation Format: David Ciccone, Fu-Shan Kuo, Scott Daigle, Neelu Kaila, Gene Yau, Mark Carlson, Denise Levasseur, Bhaskar Srivastava, Frank G. Basile, Christine Loh. NDI-101150 is a potent and highly selective HPK1 inhibitor that both synergizes with and differentiates from anti-PD1 immune checkpoint blockade [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 2652.

Abstract 2663: Determining the role of tumor mutational burden in a pre-clinical model of glioblastoma

Abstract Despite advances in care in many cancer types, patients with glioblastoma (GBM) have a grim prognosis of 15 to 21 months and unchanged standard therapy since 2005. These tumors have a profoundly immunosuppressive tumor immune microenvironment. Immunotherapy, including the checkpoint inhibitors (ICIs) targeting PD-1 and CTLA-4, has transformed the treatment of many cancers but has failed in trials of both newly diagnosed and recurrent GBM. Tumor mutational burden (TMB) has been proposed as a predictor of response to ICI treatment, presumably through an increase in neoantigens that can be recognized by cytotoxic CD8+ T cells leading to tumor rejection. GBM typically has a low TMB, a feature speculated as a contributor to poor response to ICI treatment. However, cases with an extremely high TMB (i.e., patients with the biallelic mismatch repair deficiency syndrome) do show response to immunotherapy. To begin to investigate the impact of mutational burden on immune response in GBM, we created a murine syngeneic tumor model using CRISPR-Cas9 to knockout (KO) the mismatch repair protein Msh2, the lynchpin to mismatch recognition, in the SB28 GBM model that has a low TMB (108 mutations) at baseline. Msh2 KO increased the TMB from 2 to 12-fold depending on single cell sorted clone, resulting in between 150 and 200 predicted strong MHC-I binding neoantigens estimated by the NetMHC algorithm. Preliminary studies have demonstrated that in vitro proliferation is similar amongst low TMB and high TMB SB28 clones. Flank implantation of high TMB SB28 clones revealed slower growth compared to the low TMB SB28 tumors. Furthermore, treatment with ICIs did provide a survival advantage in this flank model. However, no survival advantage was noted when the high TMB clones were implanted intracranially either with or without ICI treatment. These results suggested that either there is poor immune cell trafficking to the intracranial tumors, or a robust immune response was causing increased intracranial pressure that is prematurely causing death. Use of dexamethasone starting at day 11 to control cerebral edema has shown some survival improvement with ICI treatment, supporting that an inflammatory response is at least partially responsible for the lack of improved survival in early studies. We are currently evaluating CD4+ and CD8+ T cell clonality, earlier timepoints to evaluate the immune infiltrate and anti-tumor immune response, and alterations in chemokines/cytokines in the tumor microenvironment. By creating high mutation burden clones from a GBM with a low mutational burden, we will be able to interrogate potential mechanisms of heightened immunogenicity that has been reported in other cancers. Citation Format: Kevin Breen, Tuesday Haynes, Masashi Watanabe, Mark Gilbert. Determining the role of tumor mutational burden in a pre-clinical model of glioblastoma [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 2663.

Abstract 3717: Sudocetaxel Zendusortide (TH1902) triggers the cGAS/STING pathway and potentiates anti-PD-L1 immune-mediated tumor cell killing

Abstract The potential impact of the immune tumor microenvironment (iTME) was assessed with respect to the anticancer efficacy of sudocetaxel zendusortide (TH1902), a peptide-drug conjugate internalized through a sortilin (SORT1)-mediated endocytic pathway. In the triple-negative breast cancer (TNBC)-derived MDA-MB-231 immunocompromised xenograft tumor model, TH1902 induced complete tumor regression for more than 40 days after the last treatment. Surprisingly, immunohistochemistry analysis revealed higher staining of CD45 leukocytes and of STING, a master regulator in the cancer-immunity cycle, in comparison to docetaxel. Additionally, in a murine B16-F10 melanoma syngeneic tumor model, weekly administration of TH1902 as a single agent also demonstrated superior tumor growth inhibition than did docetaxel. Although B16-F10 is considered a non-immunogenic “cold” tumor model, we observed a net increase in CD45 leukocyte infiltration within TH1902-treated tumors, especially for tumor-infiltrating lymphocytes and tumor-associated macrophages. Moreover, in TH1902-treated tumors, increased staining of perforin, granzyme B, and caspase-3 was suggestive of elevated cytotoxic T and natural killer cell activities. Combined TH1902/anti-PD-L1 treatment led to increased tumor growth inhibition and median animal survival. Accordingly, TH1902 inhibited cell proliferation and triggered apoptosis and senescence in B16-F10 cells in vitro, while inducing several downstream effectors of the cGAS/STING pathway and the cell surface expression of MHC-I and PD-L1. This is the first evidence that TH1902 exerts its antitumor activity, in part, through modulation of the iTME and that the combination of TH1902 with checkpoint inhibitors (anti-PD-L1) could lead to improved clinical outcomes. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Zgheib, Isabelle Cousineau, Veronique Lullier, Richard Beliveau, Christian Marsolais, Borhane Annabi. Sudocetaxel Zendusortide (TH1902) triggers the cGAS/STING pathway and potentiates anti-PD-L1 immune-mediated tumor cell killing [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 3717.

Abstract 5562: Altered tumor microenvironment in animal model of concomitant GBM and Alzheimer's pathology

Abstract Glioblastoma (GBM) and Alzheimer’s disease (AD) are two devastating central nervous system diagnoses with no known cure. Both diseases are associated with advanced age. The majority of GBM tumors form in the frontal and temporal lobes, two of the brain regions most impacted by amyloid and tau pathology in AD. Females have a higher risk of AD, while males have a higher risk of GBM. Several large case series have established epidemiological evidence that the two diseases are inversely correlated. Nevertheless, it remains unknown whether presence of subclinical amyloid or tau pathology antagonizes the establishment or progression of GBM. The present study sought to characterize GBM growth, progression, and immune response in the setting of concomitant AD pathology using the syngeneic murine GL261 tumor model. To further understand the tumor cell changes that occur in the presence or absence of AD pathology, we engineered GL261 with a dual-tagged vector consisting of RiboTag (to allow for full-length mRNA profiling) and TurboID (to allow for proteomic profiling). GL261 cells were injected intracranially into APPNL-G-F/NL-G-F - MAPThuman/human, APPNL-G-F/+ - MAPThuman/+, and wildtype C57BL/6J mice. Tumors in both wildtype and AD conditions contained ~50% intratumoral myeloid cells (Iba+, P2RY12-). Differential activation of intratumoral myeloid cells will be assessed with immunohistochemistry. Microgliosis (IBA1, P2RY12) and astrogliosis (GFAP) around the tumor in the setting of Alzheimer’s pathology versus normal conditions will be compared. Invasiveness, proliferation, DNA damage (pH2AX), and apoptosis (cleaved PARP, cleaved Caspase 3) will be compared. Tumor cell-specific changes unique to tumors in the AD microenvironment will be assessed transcriptionally with RiboTag profiling as well as proteomically with TurboID purifications. Our preliminary data suggests that the presence of concomitant AD pathology affects GBM growth and progression as well as the immune response to the tumor, and warrants further investigation. Citation Format: David Nascari, Ryan Eghlimi, Angad Beniwal, Drake Alton, John Fryer, Nhan L. Tran. Altered tumor microenvironment in animal model of concomitant GBM and Alzheimer's pathology [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 5562.

Abstract 6740: Next-generation LNPs induce effective anti-tumor T cell responses

Abstract mRNA immunotherapies have gained interest for the treatment of tumors from various origins, however, their ability to provide long -term durable tumor control has been underwhelming. Here we report that novel mRNA lipid nanoparticle (LNP) vaccinations induce robust expansion of antigen-specific T cells, although this does not always correlate with their ability to infiltrate and kill solid tumors. Using a portfolio of novel ionizable lipids and a robust in vivo model that reliably identifies LNPs with prominent anti-tissue T cell activity we have identified a putative LNP candidate that provides long term tumor control in a murine syngeneic tumor model. We utilized the model antigen glycoprotein (Gp), from the lymphocytic choriomeningitis virus (LCMV), and produced mRNA encoding the immunodominant epitopes gp3333-41, gp6161-80 and gp276276-286 (3GP-mRNA). This mRNA was encapsulated in various novel LNP formulations and administered i.m. into naïve C57Bl/6 mice to track the induction of a robust CD8+ T cell response identified using tetramers to gp33 and gp276. Successful candidate LNPs were then tested in the RIPgp model of autoimmune diabetes. RIPgp transgenic mice express LCMV-Gp in the pancreatic β cells and offer a robust assay for the ability to induce antigen-specific T cells capable of breaking self-tolerance and infiltrating and killing target tissue. Utilizing the RIPgp model to screen LNP candidates we identified a lead candidate, C2-5A, that both induced a robust expansion of antigen-specific T cells and tissue destruction. When utilized in a colon adenocarcinoma model expressing the LCMV-Gp antigen (MC38-Gp), therapeutic dosing of LNP C2-5A as a monotherapy lead to long term tumor clearance in 40% of the treated animals. We believe this screening platform allows the selection of LNP candidates capable of breaking self-tolerance, and one such candidate C2-5A provides a potent vehicle and adjuvant for mRNA based cancer vaccines. Citation Format: Douglas G. Millar, Matthew J. Gold, Kirsten Olsen, Yu Wu, Yury Karpov, Robert Nechanitzky, Haritha Menon, Rajesh Krishnan, Robert Georgantas, Pamela Ohashi, Natalia Martin-Orozco. Next-generation LNPs induce effective anti-tumor T cell responses [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 6740.

Abstract 2357: T cell receptor beta chain-directed Tri-specific antibody molecules retarget V-beta T cell subsets to tumors to promote potent and durable antitumor activity

Abstract Background We have previously shown that novel bifunctional “STAR” antibody-fusion molecules comprising germline β chain TCR-targeting antibodies fused to costimulatory cytokines co-engage the TCR and T cell cytokine receptors, thereby promoting activation and expansion of subsets of T cells expressing distinct germline variable β (Vβ) chain TCRs. Here we describe a new class of T cell engager (TriSTAR) that utilizes a tri-specific format comprising the STAR TCR β chain antibody-IL-2 fusion with an additional Fab arm targeting tumor-associated antigens (TAA). By design, TriSTAR molecules promote the activation and expansion of Vβ T cell subsets that adopt a novel memory phenotype and are subsequently re-targeted to tumors expressing specific TAAs. TriSTAR molecules induce potent and durable anti-tumor activity which in refractory murine solid tumor models appear to be superior to established anti-CD3 T cell engager (TCE) formats. Methods Using the gp75 (Tryp1) melanoma antigen as a model TAA, gp75-targeting TriSTARs were tested in vivo in the B16F10 syngeneic mouse tumor model, compared with a conventional gp75-targeting CD3 bispecific TCE. Cured mice were re-challenged with tumors to assess the potential of TriSTARs to induce immunological memory responses. The pharmacodynamics and pharmacokinetics of gp75-targeting TriSTARs and anti-CD3 TCEs were assessed in blood and tumor using flow cytometry, NanoString and other immunoassays. This work was further extended to testing human TAA-targeting TriSTARs using in vitro human cytotoxicity assays and a transgenic MC38 model. Results In the B16F10 murine melanoma model, gp75-targeting TriSTARs induced potent anti-tumor responses that were superior to control constructs and gp75-targeting CD3 TCEs. The superior anti-tumor activity of the gp75-targeting TriSTARs was attributed to a significant increase and accumulation of CD8+, CD25+ and Granzyme B+ T cells in tumors that was due to the expansion of the target Vβ T cell subset. Further immune profiling by flow cytometry and NanoString in tumors and blood isolated from treated B16F10 mice highlighted significant remodeling of tumor-infiltrating lymphocytes (TILs) and other immunological changes associated with immune activation. These observations with murine gp75-targeting TriSTAR molecules were broadly confirmed with TriSTARs targeting human TAAs in both human in vitro studies and human TAA transgenic mouse tumor models. Conclusions Vβ TCR-targeting TriSTAR molecules represent a new class of T cell engager with the potential to deliver improved anti-tumor activity compared with existing anti-CD3 T cell engagers in immunologically “cold” solid tumors with low levels of T cell infiltration and antigen presentation and may therefore offer a new therapeutic approach for the treatment of patients with refractory solid tumors. Citation Format: Madan Katragadda, Jacques Moisan, Gurkan Guntas, Manuel Sequeria, Roya Servattalab, Jessica Lowry, Robert Ruidera, Kent Nybakken, Erik Gerson, Ke Liu, Kevin Chin, Zhen Su, Andrew Bayliffe. T cell receptor beta chain-directed Tri-specific antibody molecules retarget V-beta T cell subsets to tumors to promote potent and durable antitumor activity [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 2357.

Abstract 3339: TTFields for the management of spinal metastases in in vitro and in vivo models

Abstract Spine metastases often affect cancer patients, leading to significant morbidity and mortality. Their prevalence has increased over time due to the progressive improvements in cancer management which led to a longer survival of these individuals. Radiation therapy is the mainstay treatment for local control of these lesions, with surgery reserved for selected cases (e.g., spinal instability, neurological deficits secondary to neural compression requiring urgent decompression, separation surgery). Chemotherapy addresses the diffusely metastatic spread. However, when spinal metastases progress despite using all the appropriate available treatment, there follows a progressive decline in function and further treatment options are limited, highlighting the need for development of novel therapies. Tumor treating fields (TTFields) has demonstrated to impair tumor cell replication and are influenced by surrounding tissue, such as bone (presents non-conductive properties). Our goal was to investigate the effects of TTFields in in vitro and in vivo spinal metastasis models. We hypothesize that the deposition of TTFields is increased in bone, and this would ultimately result in vertebral metastasis growth suppression. A computational model using a simulated spine resection cavity with metallic hardware was used to investigate the TTFields intensity in various tissues. For in vitro studies, luciferase tagged KRIB osteosarcoma and A549 lung adenocarcinoma cell lines were cultured in demineralized human bone graft. These were treated with TTFields and cell viability was quantified. In vivo assays were performed using a previous validated murine orthotopic tumor model. The effects of TTFields in these animals were monitored with imaging (bioluminescence and MRI) and clinical findings (progressive neurological deficits milestones that corresponds to the degree of spinal cord compression). At the end of the study, tissue was collected for histologic analysis. Sham stimulation was used for control groups. Computational simulation demonstrated an elevated TTFields intensity in the tumor-bone interface of the resected vertebral body. In all experimental models, TTFields significantly suppressed tumor cell growth. In vivo models, this effect on cell proliferation prevented tumor growth into the spinal canal, delaying, or even preventing, neurological deficits. TTFields inhibited tumor growth in three-dimensional cultures and orthotopic murine models of spinal metastases, preventing functional loss. The bony environment surrounding the tumor demonstrated to enhance the intensity of electric fields on a computational simulation, which stands as an advantage with the use of TTFields for vertebral tumors. These results provide solid evidence supporting future clinical trials investigating the effects of this novel and promising technology for the treatment of spine metastases. Citation Format: Romulo Augusto Andrade de Almeida, Daniel Ledbetter, Xizi Wu, Ariel Naveh, Chirag Patel, Queena Gonzalez, Thomas H. Beckham, Robert Y. North, Laurence Rhines, Christopher A. Alvarez-Breckenridge, Claudio E. Tatsui. TTFields for the management of spinal metastases in in vitro and in vivo models [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 3339.

Abstract 6336: Unleashing CAR T cell activity against osteosarcoma using tumor specific RNA-based vaccination

Abstract Background: Metastatic osteosarcoma (OSA) has a dismal prognosis despite novel targeted therapies, including CAR T cells. Unleashing CAR T cell therapy against poorly immunogenic cancers requires new technologies that activate the tumor microenvironment (TME), while inducing CAR T cell migration, proliferation, and persistence into tumor to sustain tumor-specific immunity. We have developed tumor RNA-loaded nanoparticle (CD70-encoding RNA-NP) vaccines to overcome CD70-specific CAR T cell limitations in OSA. Methods: Using two murine solid tumor models (K7M2 osteosarcoma and B16F0 melanoma, in Balb/c and C57Bl/6 background) highly metastatic to lungs, we implant K7M2 and B16F0 intravenously (i.v.). We subsequently generated NPs complexed with RNA encoding for CD70 antigen and control vaccine (GFP RNA). RNA-NPs are administered intravenously beginning on day 5 post-tumor implantation and day 7, 14 & 21. Mouse CD70-specific CAR T cells are manufactured after activation of T cells with CD3 and CD28 antibodies for 2 days. CAR T cells are ready for use in downstream experiments 2 days after pMSGV8-mCAR transduction and cells were injected (i.v.) on day 6. Results: In preliminary data, we show that intravenous administration of CD70-encoding RNA-NPs rapidly recruit CD70-directed CAR T cells into tumor stroma unlocking their activity in two immunocompetent aggressive lung metastatic tumor models. Interestingly, regression of CD70-expressing tumors associates with CAR T cell mobilization out of peripheral blood and into reticuloendothelial (RES) tissues (i.e., lung, and spleen), a phenomenon that correlates with upregulation of vaccine-induced type I interferon (IFN) and activation of CCR2 pathway. In addition to the CAR T-mediated antitumor killing, an antigen spreading phenomenon was observed. The long-term survival benefit (150 days) provided by this approach reached 70-80% in both models compared with 0-10% of control groups. Conclusions: Based on these findings, we hypothesize that tumor trafficking and persistence of CD70 CAR T cells depend on coordinated cytokine/chemokine signaling that can be ignited through intravenous RNA-NP vaccination. RNA-NPs in combination with CAR T cells prolong long-term survival of mice bearing these aggressive tumor models which was not only mediated by CAR T cells but also by activation of endogenous adaptive immunity. Future studies include the evaluation of this approach in large animal models prior to translation into first-in-human clinical trials. Citation Format: Arnav Barpujari, Rui Liu, Elizabeth Ogando-Rivas, Xiaojie Ma, Haipeng Taoa, Hector Gomez, Duane Mitchell, Jianping Huang, Elias Sayour, Paul Castillo. Unleashing CAR T cell activity against osteosarcoma using tumor specific RNA-based vaccination [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 6336.

Abstract 5247: Reprogramming tumor microenvironment with intratumor macrophage adoptive cell therapy in melanoma

Abstract The interplay between tumor cells and immune cells within the tumor microenvironment (TME) dictates protumor or antitumor immune responses. Tumor-associated macrophages (TAMs) respond to signals of the TME and exhibit a spectrum of phenotypes ranging between M1 (antitumor) and M2 (protumor) macrophages. In most tumor types, including melanoma, the balance between M1 and M2 macrophages is critical with a higher M1/M2 ratio favoring antitumor immunity. Therefore, strategies enhancing the M1/M2 ratio can significantly alter the TME towards antitumor immunity. In this study, we administered luciferase and GFP-expressing M1 macrophages as an adoptive cell therapy (ACT) through intravenous (i.v) and intratumor routes respectively, into mice bearing SM1 murine melanoma tumors to determine the effective treatment modality. We reprogrammed antitumor M1 macrophages ex-vivo with HDAC6 inhibitors to lock into the M1 phenotype and administered intratumorally as ACT in the syngeneic SM1 murine melanoma and humanized NSG-SGM3 melanoma models. We performed histological analysis of tumors for macrophage markers, immune phenotyping of infiltrated immune cells, single-cell secretome analysis of tumor macrophages, and single-cell RNA-seq analysis of CD45+ immune cell populations to demonstrate the benefit of macrophage ACT. Tail vein injected, luciferase-expressing M1 macrophages localized in the lungs and spleen, failed to reach the tumor as visualized by IVIS imaging, suggesting that i.v administration is ineffective for macrophage therapy. On the contrary, intratumor M1 macrophage ACT resulted in diminished tumor growth. Single-cell RNA-seq analysis of the CD45+ sorted tumor-associated immune cell population revealed distinct macrophage subsets and a significant M1/M2 macrophage ratio increase. NicheNet cell-cell interaction analysis indicated that M1-like TAMs activated infiltrating T-cells and monocytes through ligand-receptor interactions. Trajectory analysis of infiltrated monocytes indicated differentiation toward inflammatory macrophages in ACT tumors. Flow cytometry analysis corroborated that ACT increased the M1/M2 macrophage ratio and an increase in CD8 effector T-cells. Furthermore, HDAC6i-treated macrophages increased antigen cross-presentation. Single-cell secretome analysis of F4/80+ TAMs by the Isoplexis platform revealed polyfunctionality of HDAC6-treated M1 macrophages secreting inflammatory cytokine Tnfa and T-cell recruiting chemokine Cxcl10. Histological examination of tumor sections for macrophage phenotypic markers suggested that transplanted macrophages retained the M1 phenotype post-ACT in both SM1 murine and NSG-SGM3 melanoma tumor models. We demonstrated the potential of reprogramming macrophages ex vivo with HDAC6 inhibitors as a feasible macrophage cell therapy to treat solid tumors. Citation Format: Satish Kumar Reddy Noonepalle, Nithya Gajendran, Manasa Suresh, Xintang Li, Maria D. Hernandez, Christian Zevallos Delgado, Nima Aghdam, Michael Berrigen, Tessa Knox, Karen Tan, Marie Durr, Eduardo Sotomayor, Katherine B. Chiappinelli, Duncan Wardrop, Anelia Horvath, Brett A. Shook, Norman H. Lee, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Maho Shibata, Alejandro Villagra. Reprogramming tumor microenvironment with intratumor macrophage adoptive cell therapy in melanoma [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 5247.

Abstract 7117: Convection enhanced delivery of a novel ATR inhibitor synergizes with systemic lomustine for improved treatment of glioblastoma

Abstract Developing effective strategies for treating glioblastoma (GBM), the most common and aggressive form of brain cancer, has been a long-standing challenge. Despite rigorous treatment regimens including surgical resection with concomitant radiotherapy and chemotherapy, the prognosis for this disease remains poor, largely due to tumor recurrence, resistance to conventional therapeutics, pharmacokinetic and brain delivery challenges, and dose-limiting toxicities of existing therapies. The DNA damage response and DNA repair mechanisms such as the ataxia telangiectasia and Rad3-related (ATR) pathway are key mediators of therapeutic responses, and recent studies have shown that targeting DNA repair proteins alongside standard-of-care options is a promising anti-tumor strategy. Here, we employ a combination approach using a next-generation macrocyclic ATR inhibitor, ATRIN-333, to sensitize GBM tumors to alkylators. We establish the potency of ATRIN-333 and report a robust synergistic interaction between it and the alkylator lomustine in GBM cell-based assays and in a murine flank tumor model. This combinatorial treatment was then applied to a mouse intracranial tumor model, where we utilized a convection enhanced delivery (CED) system combined with nanoparticle technology for direct intracranial administration of ATRIN-333. ATRIN-333 was successfully encapsulated into a novel polymeric nanoparticle formulation using poly(ethylene glycol)-co-poly(ethylene brassylate-co-dioxanone) (PEG-EB-co-DO). CED of both free and encapsulated ATRIN-333 potently sensitized intracranial GBM tumors to low-dose (5 mg/kg) systemically-delivered lomustine, reducing tumor size and conferring a substantial survival benefit compared to cohorts treated with lomustine or the ATR inhibitor alone. The combination approach was well tolerated by the mice, with no hematological toxicity observed as assessed by bone marrow analysis and blood cell counts. Our results demonstrate that ATR inhibitor/lomustine combination therapy, used in conjunction with a CED platform, is a powerful avenue for GBM treatment and a viable option for potential clinical implementation. Note: AJ and TL contributed equally to this work. Citation Format: Alexander Josowitz, Teresa Lee, Ranjini K. Sundaram, Jinal Pothupitiya, Eric J. Brown, Amrita Sule, Jason Beckta, Yongheng Wang, Joseph Vacca, Oren Gilad, Ranjit S. Bindra, W Mark Saltzman. Convection enhanced delivery of a novel ATR inhibitor synergizes with systemic lomustine for improved treatment of glioblastoma [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 7117.

Abstract 6637: The Haemophilus influenzae vaccine mitigates pancreatic tumor progression and lowers immunoregulatory cell levels in the tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease projected to become the second leading cause of cancer deaths by 2030. First-line chemotherapies gemcitabine and FOLFIRINOX provide little therapeutic benefit and are accompanied by severe adverse toxicities, precluding long-term treatment. In addition, PDAC is notoriously refractory to checkpoint inhibitor immunotherapies, due to poor immunogenicity and a tumor microenvironment dominated by suppressive cell types. Thus, there is an unmet need for innovative, immune-based strategies to combat PDAC. A previous study utilized engineered bacteria to effectively kill PDAC tumor cells by directly delivering immunogenic tetanus toxoid to the tumor. Given the potential toxicity in using live bacteria, our group utilized a less toxic alternative, an FDA-approved Haemophilus influenzae type b vaccine (HibVx) also containing a tetanus toxoid component. To determine the antitumor potential of HibVx, our group used a physiologically relevant murine model, low T cell clone murine PDAC tumor model 641c95 and clinically relevant KPC mice model for PDAC. After tumors were established, animals received three consecutive daily treatments of HibVx via intraperitoneal injection. HibVx-treated animals experienced a dramatic reduction in tumor growth compared to PBS-treated controls (p<0.001). Given that tetanus toxoid is highly immunogenic in nature, changes in intratumoral immune cell populations following HibVx treatments were of great interest. Using flow cytometry analysis, HibVx-treated tumors had a significant increase in natural killer cells compared to control tumors (p<0.05). In addition, HibVx affected myeloid-derived suppressor cell populations (MDSCs), which are heavily implicated in the suppressive nature of PDAC tumors. HibVx also significantly decreased MDSCs within the tumor (p<0.05). In all, these findings position HibVx as an effective immunostimulatory agent that can be used as an alternative to live bacteria-based therapy for the treatment of PDAC. Citation Format: Eileena F. Giurini, Sam G. Pappas, Kajal H. Gupta. The Haemophilus influenzae vaccine mitigates pancreatic tumor progression and lowers immunoregulatory cell levels in the tumor microenvironment [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 6637.

Abstract 289: Temporal and spatial dynamics of glial cells apolipoprotein E expression in astrocytoma microenvironment

Abstract Introduction: Brain tumors have proven challenging to treat due to the complex and sensitive architecture of the brain. Previous research has shown that GBM cells depend highly on cholesterol uptake, and apolipoprotein E (APOE) is required to transport cholesterol among tissues or cells. APOE is primarily synthesized by astrocytes and has been extensively studied in neurodegenerative diseases. While extensively studied in neurodegenerative diseases, limited studies are focusing on their roles in brain tumor development. Here, we analyzed the temporal and spatial expression of APOE during brain tumor progression in a murine astrocytoma tumor model. Material and method: ALTS1C1 astrocytoma cells were intracranially injected into the mouse brain. The animal was sacrificed, and the left hemisphere, right hemisphere, and tumor tissue were collected for flow cytometry analysis. CD45, CD11b, and GFAP were used to recognize macrophages, microglia, and astrocytes. CD68, TMEM119, and GFAP were used for tissue immunostaining to label macrophages, microglia, and astrocytes, respectively. Results and Discussion: Flow cytometry analysis revealed upregulated APOE in tumor-infiltrating microglia, macrophages, and lymphocytes. GFAPhigh astrocytes displayed lower APOE levels than GFAPlow astrocytes. IHC analysis showed that CD68+ macrophages exhibited higher APOE expression at the tumor edge than normal tissue. On the contrary, GFAP+ astrocytes at the tumor edge displayed a lower APOE signal than the normal tissue. Previous studies have shown the participation of APOE in regulating immune response in various cancers. Further studies to analyze the dynamic change of APOE on the gene expression level with in situ hybridization and the quantification of cholesterol levels in the microenvironment may help uncover their roles in the glioma microenvironment. Conclusion: This study demonstrated that APOE was temporal and spatial differentially expressed in macrophages and astrocytes in the brain tumor microenvironment. Tumor-infiltrating cells showed an elevated APOE expression, while GFAP+ astrocytes expressed reduced APOE levels. Although the mechanism of these alterations remains unclear, this study provides a basis for future studies about the roles of APOE in the brain tumor microenvironment. Citation Format: Ting-Yi Chien, Chi-Shiun Chiang. Temporal and spatial dynamics of glial cells apolipoprotein E expression in astrocytoma microenvironment [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 289.