Syngeneic Mouse Tumor Models Research Articles

Abstract 736: ACTM-838 safely and comprehensively re-activates the immunosuppressive TME by exploiting myeloid biology intrinsic in many cancers

Abstract ACTM-838 is a genetically modified bacterial vehicle encoding a DNA plasmid with two engineered payloads, IL-15plex and constitutively active eSTING. The bacterial vehicle is a highly attenuated S. typhimurium, engineered to facilitate safe IV administration. ACTM-838 is designed to specifically enrich in the TME via auxotrophic dependency on extracellular adenosine and purine metabolites. ACTM-838 is then selectively internalized by tumor-resident myeloid cells achieving tumor-specific payload delivery. Previously, we showed ACTM-838 exhibited significant and durable anti-tumor efficacy across syngeneic tumor mouse models (EMT6 breast, MC38 colon) and MMTV-PyMT GEMM. To elucidate TME changes induced by ACTM-838, EMT6 tumors were analyzed post-treatment by scRNA-seq, which demonstrated that ACTM-838 induced significant transcriptional changes in several cell populations in the TME. Cytolytic pathways were upregulated in T and NK cells, while type I IFN and PDL1 were upregulated in myeloid populations consistent with previous bulk RNAseq analyses. To understand the tolerability and safety of ACTM-838, a GLP toxicology study was performed on naïve cynomolgus monkeys (NHP). No ACTM-838-related clinical or veterinary observations were reported at any dose level. Acute dose-dependent increases in serum cytokines were observed at 4 hours which reverted to baseline by 24 hours. ACTM-838 was cleared from blood by 24 hours post-dose with no significant shedding in urine or feces. ACTM-838 was only detected in the spleen in 2 and 1 animals at 22- and 49-days post treatment, respectively. Pre-existing ADA was observed in some animals with subsequent increases in ADA upon dosing in most animals. However, no correlation of serum ADA and blood PK levels suggested that the presence of ADA does not impact drug stability. The ACTM-838 mechanism of action requires high levels of adenosine or purine metabolites to enrich and colonize the TME as well as phagocytic internalization by tumor-intrinsic myeloid cells to achieve tumor-specific payload delivery. Myeloid cells comprise roughly half of all cells in the TME in human tumors. Bioinformatics analyses of the public patient datasets identified key tumor indications enriched in both adenosine and myeloid pathways. To further understand the spatial heterogeneity in the TME across tumors, we performed multiplex immunofluorescence on patient tumor microarrays to validate tumor types with a high prevalence of myeloid populations coupled with an active adenosine pathway, which will help guide future clinical study design. In summary, we demonstrate that ACTM-838 modulates the TME and is stable and safe in animal models. Additionally, our studies recommend the prioritization of certain tumor indications for treatment with ACTM-838 in the clinic. Citation Format: William Lu, Kyle Cron, Julie Janes, Leigh Berryman, Gurvinder Singh, Christopher Thanos, Akshata Udyavar. ACTM-838 safely and comprehensively re-activates the immunosuppressive TME by exploiting myeloid biology intrinsic in many cancers [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 736.

Abstract 4060: Characterization of MDNA113, a tumor-targeting anti-PD1-IL-2SK immunocytokine with conditional activation to increase tolerability and maximize efficacy

Abstract Background-MDNA113 consists of an IL-13 decoy receptor-binding domain (MDNA213) fused to anti-PD1-IL-2SK (MDNA223) immunocytokine using a matrix metalloprotease (MMP) sensitive linker (PSL). MDNA213 is an IL-13 superkine with high selectivity towards IL-13Rα2, a tumor associated antigen overexpressed in numerous aggressive solid tumors with limited expression in normal tissues. IL-13Rα2 overexpression is associated with MMPs abundancy in tumor microenvironment (TME) and poor clinical outcomes. MDNA223, a fusion of ‘not-alpha, beta enhanced’ IL-2 superkine with anti-PD1 is designed to facilitate cis-binding to PD1 and IL-2Rβγ on CD8+ T cells. MDNA223 is superior to co-administration of anti-PD1 and IL-2 agonist at inhibiting both immunologically ‘hot’ and ‘cold’ tumors in mice. MDNA113 is designed to target and accumulate in tumors via the MDNA213 domain, which also attenuates immune stimulation by hindering IL-2R activation to reduce peripheral activity. The dual targeting/masking MDNA213 domain is released within tumor microenvironment following MMP cleavage of PSL to fully restore IL-2R agonism while maintaining PD1/PDL-1 blockade. Methods-In vitro signaling studies were performed in reporter cell assays and human PBMCs. Tolerability and pharmacodynamic studies were conducted in mice. Tumor growth inhibition was assessed in syngeneic mouse tumor models in monotherapy and combination settings. Results-MDNA113 showed reduced IL-2R agonism (~38-fold) while maintaining PD1/PD-L1 blockade compared to MDNA223 (non-masked) in cell-based reporter assays. Capacity to stimulate human PBMC proliferation was reduced while potency in CD8+ T cell p-STAT5 signaling also decreased. MMP cleavage of MDNA113 to release MDNA213 domain fully restored IL-2R signaling. Systemic delivery of MDNA113 to mice showed reduction, but not complete blockade, of lymphocyte expansion compared to equimolar dose of MDNA223. A 3x increase in MDNA113 restored lymphocyte expansion to a similar level achieved with MDNA223, indicating a ‘partial masking’ effect of MDNA213 domain. Accordingly, MDNA113 was better tolerated than MDNA223 in viability studies in mice. In syngeneic tumor models, equimolar treatment with MDNA113 and MDNA223 (non-mask version) resulted in similar extent of tumor growth inhibition. In contrast, in vivo activity was partially compromised when mice were treated with a non-cleavable version of MDNA113, consistent with a role for MMP-cleavage to restore full anti-tumor effect within TME. Conclusion-MDNA113 is a tumor-targeting and conditional-activatable anti-PD1/IL-2SK fusion characterized with potent PD1/PDL1 blockade and ‘not-alpha, beta enhanced’ IL-2R agonism. MDNA113 is better tolerated than the non-mask version while achieving similar effect on tumor control, therefore offering a much broader therapeutic index. Citation Format: Aanchal Sharma, Minh D. To, Qian Liu, Fahar Merchant. Characterization of MDNA113, a tumor-targeting anti-PD1-IL-2SK immunocytokine with conditional activation to increase tolerability and maximize efficacy [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 4060.

Abstract 5301: Tumor-targeted activation of CD137 using Bicycles: New insights into mechanism of action and discovery of BT7455, a clinical candidate for the treatment of EphA2-expressing cancers

Abstract Introduction: Clinical studies in cancer patients have validated CD137 agonism as an activator of the immune system to enable tumor rejection. We have demonstrated that small, chemically synthetic bicyclic peptides can drive tumor-localized agonism of CD137 and anti-tumor immunity in mouse models. Here, we report the next stage of our work - delving into the mechanism of action of these novel agents and extending our program to serve patients whose tumors express EphA2. Experimental Procedures: MultiOmyx™ hyperplexed immunofluorescence assay was used to evaluate the expression of CD137 and EphA2 in head and neck squamous cell carcinoma samples. Human PBMC/tumor cell co-culture assays were used to assess CD137 Bicycle TICA™ in vitro bioactivity and syngeneic mouse tumor models were used to evaluate CD137 Bicycle TICA anti-tumor activity as mono- or combination therapies. Pharmacodynamic activity was evaluated by transcriptional profiling using NanoString assays or single cell RNA sequencing. Summary of the Data: Studies utilizing a syngeneic mouse model and deep RNA sequencing of tumors from CD137 Bicycle TICA-treated mice implicated intratumoral dendritic cells in addition to T cells as early responders to localized CD137 agonism and potential contributors to the anti-tumor response. Furthermore, a mouse efficacy model demonstrated synergy with checkpoint inhibitor therapy. Multiplex imaging revealed that EphA2 and CD137 are co-expressed in human tumors of high unmet medical need and therefore, using our highly modular Bicycle platform, we discovered BT7455, a Bicycle TICA that engages EphA2 and CD137 with high affinity, resulting in potent EphA2-dependent activity in vitro and robust anti-tumor activity in vivo with intermittent dosing in mouse models. Gene expression profiling of tumors revealed that BT7455 led to increased production of cytokines and chemokines known to drive T cell infiltration, the extent of which differentiated it from both a checkpoint inhibitor and an anti-CD137 monoclonal antibody agonist. BT7455 was well tolerated in preclinical species and liver function tests indicated no evidence of hepatic toxicity. Statement of Conclusions: In summary, we have identified EphA2 as a promising target to pair with a CD137 agonist. In addition to CD8+ T cells, tumor-resident dendritic cells are a likely contributor to anti-tumor immunity following treatment with Bicycle® TICAs, supporting utility for Bicycle® tumor-targeted CD137 agonists in solid tumors beyond those that are highly T cell-infiltrated. We have advanced a clinical development candidate, BT7455, to realize this potential for patients with EphA2-expressing cancers. Citation Format: Johanna Lahdenranta, Kristen Hurov, Heather Cohen, Lia Luus, Cara Bray, Peter Brown, Anna Devlen, Carly Campbell, Matthew Gray, Mike Kelly, Gemma Mudd, Punit Upadhyaya, Sailaja Battula, Kelvin Zhang, Anne-Sophie Dugast, Kevin McDonnell, Phil Brandish, Nicholas Keen. Tumor-targeted activation of CD137 using Bicycles: New insights into mechanism of action and discovery of BT7455, a clinical candidate for the treatment of EphA2-expressing cancers [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 5301.

An IRF2-Expressing Oncolytic Virus Changes the Susceptibility of Tumor Cells to Antitumor T Cells and Promotes Tumor Clearance.

IFN regulatory factor 1 (IRF1) can promote antitumor immunity. However, we have shown previously that in the tumor cell, IRF1 can promote tumor growth, and IRF1-deficient tumor cells exhibit severely restricted tumor growth in several syngeneic mouse tumor models. Here, we investigate the potential of functionally modulating IRF1 to reduce tumor progression and prolong survival. Using inducible IRF1 expression, we established that it is possible to regulate IRF1 expression to modulate tumor progression in established B16-F10 tumors. Expression of IRF2, which is a functional antagonist of IRF1, downregulated IFNγ-induced expression of inhibitory ligands, upregulated MHC-related molecules, and slowed tumor growth and extended survival. We characterized the functional domain(s) of IRF2 needed for this antitumor activity, showing that a full-length IRF2 was required for its antitumor functions. Finally, using an oncolytic vaccinia virus as a delivery platform, we showed that IRF2-expressing vaccinia virus suppressed tumor progression and prolonged survival in multiple tumor models. These results suggest the potency of targeting IRF1 and using IRF2 to modulate immunotherapy.

Abstract 6535: Targeting estrogen receptor signaling in tumor associated myeloid cells to enhance anti-tumor immunity

Abstract Immune checkpoint inhibitors (ICB) have significantly improved outcomes across the spectrum of cancers, especially in patients with metastatic melanoma or non-small cell lung cancer (NSCLC). Despite encouraging efficacy in some patients, the development of primary and acquired resistance and severe toxicity associated with these treatment regimens, together with the overall low response rates, have limited their widespread use. Mining publicly available transcriptomic datasets, we have demonstrated that the response to ICB in patients with melanoma is influenced by the functionality of tumor associated macrophages (TAMs). Thus, it is of significance that we have demonstrated that 17-β estradiol (E2), working through myeloid cell intrinsic estrogen receptor alpha (ERα), promotes tumor growth in preclinical mouse models by polarizing tumor associated macrophages towards an immune-suppressive state in a manner that suppresses CD8+T cell functionality and limits ICB efficacy independent of gender. Transcriptomic analysis of tumor infiltrating myeloid cells isolated from E2-treated syngeneic tumors revealed that the type I interferon (IFN) signaling pathway was suppressed and that this activity could be reversed by the addition of fulvestrant a Selective Estrogen Receptor Downregulator. The importance of type I IFN signaling in E2-regulated tumor growth was confirmed in syngeneic mouse tumor models (B16F10-melanoma and LLC1-NSCLC cells) by showing that inhibiting type I interferon receptor (IFNAR) activity using blocking antibodies reversed the protective effects of E2 deprivation (ovariectomy) on tumor growth. Further, using in vitro cultures of macrophages from mouse bone marrow or peripheral blood mononuclear cells from deidentified human donors, we demonstrated that E2-dependent inhibition of type I IFN signaling could be attributed to increased efferocytosis by myeloid cells. These findings were recapitulated in vivo using an IFN reporter mouse (Mx1-egfp) implanted with labelled tumor cells (mCherry-Spectrin labelled LLC1), where we demonstrated that E2 mediated-suppression of myeloid cell intrinsic interferon signaling within the tumor microenvironment is contingent upon their engulfment of apoptotic cancer cells. Mechanistically, this enhanced efferocytosis was attributed to increased expression of the fractalkine receptor (CX3CR1) in E2-treated myeloid cells- as either genetic or pharmacological depletion of CX3CR1 in mouse reversed E2-mediated tumor growth (B16F10 and LLC1), myeloid cell polarization (from immunosuppressive to proinflammatory), myeloid cell intrinsic type I IFN signaling and reinvigorated CD8+T cell function. Taken together, our results highlight the potential clinical utility of targeting the E2/ER-CX3CR1 pathway as a means to enhance response to ICBs. Citation Format: Binita Chakraborty, Michael Brown, Prabuddha Chakraborty, Aditi Goyal, Ching Yi Chang, Donald McDonnell. Targeting estrogen receptor signaling in tumor associated myeloid cells to enhance anti-tumor immunity [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 6535.

Abstract 5534: Needle biopsy induces pro-metastatic progression and systemic dissemination of estrogen receptor-positive breast cancer cells through the COX-2/PGE2 cascade

Abstract Breast cancer is the most commonly diagnosed malignancy in the U.S. and over two-thirds of invasive breast cancer cases are diagnosed early stage. However, 15-year survival rates for early-stage breast cancer remain in the range of 70-77%. Mounting evidence showed an increased risk of disease progression and mortality among early-stage breast cancer patients whose surgery was delayed over 60 days after the diagnostic biopsy. Yet, the mechanism(s) underlying the rapidly increased mortality due to delay of surgery after diagnosis remains unknown. Our cohort analyses of early-stage breast cancer patients reveal the emergence of a significantly rising mortality risk when the biopsy-to-surgery interval was extended beyond 53 days. Additionally, histology of post-biopsy tumors shows prolonged retention of a metastasis-permissive wound stroma dominated by M2-like macrophages capable of promoting cancer cell epithelial-to-mesenchymal transition and angiogenesis by secretion of transforming growth factor beta 1 (TGF-β1) and vascular endothelial growth factor (VEGF). We show that needle biopsy promotes systemic dissemination of cancer cells through a mechanism of sustained activation of the cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) cascade, which favors M2-polarization and its associated pro-metastatic changes, but are abrogated by oral treatment with COX-2 inhibitors in estrogen receptor-positive (ER+) syngeneic mouse tumor models. Therefore, we conclude that needle biopsy of ER+ breast cancer provokes progressive pro-metastatic changes, which may explain the mortality risk posed by surgery delay after diagnosis. Also, our data may provide a rationale for a pharmacologic strategy to inhibit the COX-2/PGE2 cascade in cases when delay of surgery is unavoidable. Citation Format: Hiroyasu Kameyama, Macall Leslie, Reese Simmons, Yunguang Sun, Misung Yi, Priya Dondapati, John F. Langenheim, Kar-Ming Fung, Inna Chervoneva, Hallgeir Rui, Takemi Tanaka. Needle biopsy induces pro-metastatic progression and systemic dissemination of estrogen receptor-positive breast cancer cells through the COX-2/PGE2 cascade [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 5534.

Abstract 1321: In vivo immune cell programming using mRNA-LNP chimeric antigen receptors

Abstract Introduction: The tumor microenvironment (TME) is a dynamic and multifaceted system that comprises largely myeloid lineage immune cells including monocytes, neutrophils, macrophages, and dendritic cells. Reprogramming the myeloid compartment within the context of cancer therapy presents an attractive strategy to overcome current limitations in solid tumor treatment. We have developed a novel approach that harnesses the natural ability of Myeloid cells to take up mRNA loaded lipid nanoparticles, to reprogram these cells with chimeric antigen receptors (CAR’s) that arm these cells to recognize and kill tumor cells. Methods: Current LNPs technology is sufficient to deliver significant mRNA cargo to numerous cell types, including B cells, T cells and myeloid cells. Leveraging this phenomenon we have developed a new class of chimeric antigen receptors (CARs), using protein biology that imparts targeted immune cell expression and functionality, specifically in Myeloid cells. These CARs harness Immunoreceptor Tyrosine-based Activation Motif (ITAM) signaling adaptors expressed exclusively in immune cells, achieving immune cell-type specific CAR-directed tumor lysis and cytokine responses. With a FcaR I (CD89) based CAR design, we have developed a CAR that is dependent on the common Fc receptor gamma chain, primarily restricted to myeloid cells, for stable expression and function. Results: We have demonstrated specific expression of CD89-based CAR in myeloid cells in mouse and monkey following systemic mRNA/LNP delivery, and in human myeloid cells following in vitro treatment with mRNA/LNPs. Systemic mRNA/LNP delivery of CD89-based CAR has demonstrated robust anti-tumor efficacy against multiple tumor antigens in various xenograft mouse models of human breast cancer, hepatocellular carcinoma, and ovarian cancer. Furthermore, in syngeneic mouse tumor models, robust anti-tumor immune responses, characterized by tumoral infiltration of activated CD8+ T cells, diminished tumor-associated Tregs, systemic enhancement of dendritic cells and anti-tumor IgG production, were observed for treated animals. Conclusions: Taken together, we present CAR designs that achieve expression and function in targeted immune cell populations, capable of eliciting anti-tumor efficacy against multiple target antigens by leveraging innate and adaptive immune responses, and demonstrating a class of novel CAR designs to overcome limitations faced by current in vivo mRNA/LNP delivery strategy. A phase I clinical trial is currently on-going to assess the safety and preliminary efficacy of CD89-based CAR against TROP2+ epithelial malignancies. Citation Format: Shannon Argueta, Franciele K. Melber, Michael Gorgievski, Neha Divangi, Edward Cochran, Thach Chu, Yuxiao Wang, Jian Ding, Daniel Getts. In vivo immune cell programming using mRNA-LNP chimeric antigen receptors [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 1321.

Abstract 6349: 2MW4991, a novel ADCC-enhanced integrin αvβ8 blocker, exhibits high anti-tumor potency and was well tolerated in cynomolgus monkeys

Abstract Background: The three highly structurally related TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3) exert greatly different effects on cancer and immune cells under various physiological conditions. Efforts to target the TGF-β pathway have been hampered by systemic toxicity or limited efficacy due to challenges associated with the global inhibition of TGF-βs. Recently, TGF-β1 was proven to be a critical player in generating an immunosuppressive tumor microenvironment (TME). TGF-β1 is expressed as an inactive latent form (L-TGF-β1) and activated upon binding to integrins, and integrin αvβ8 is a key activator of TGF-β1 in TME. Here, an ADCC-enhanced αvβ8 blocking antibody, 2MW4991, was developed to specifically inhibit TGF-β1-mediated signaling for the treatment of αvβ8-expressing cancers. Methods: Binding ability, specificity, and affinity of antibodies were measured by Flow cytometry and Biacore T200. A blocking assay was performed using U251MG cells and TGF-β signaling reporter cells. Jurkat/h FcγRIIIa reporter system was used to assess the ADCC activity of 2MW4991. Mouse syngeneic tumor models were used to characterize the in vivo anti-tumor efficacy. A repeat dose administration study was performed in cynomolgus monkeys to evaluate the pharmacokinetic and toxicity profiles of 2MW4991. Results: ScRNA-seq analysis of kidney renal clear cell carcinoma (KIRC) and head and neck squamous cell carcinoma (HNSCC) clinical samples revealed that αvβ8 highly expresses in tumor and some myeloid-derived cells, suggesting the development of ADCC-enhanced αvβ8 blocking antibodies for the treatment of αvβ8-expressing cancers. 2MW4991 showed sub-nanomolar binding affinity and high specificity to αvβ8, with no cross-reactivity to other integrins. 2MW4991 could effectively block TGF-β1 release in a GARP/L-TGF-β1/αvβ8 reporter system. In an immune-excluded EMT6 syngeneic model, 2MW4991 promoted substantial tumor regression and reversed immune suppressive TME by significantly increasing tumor infiltration of CD8+ T and innate immune cells. As expected, TGF-β1 blockade significantly increased the sensitivity of anti-PD-1 therapy in a combination study. Furthermore, 2MW4991 displayed potent anti-tumor efficacy in the human αvβ8 knock-in syngeneic model mimicking αvβ8 expression in human cancers. In a repeat dose administration NHP study, 2MW4991 showed a good pharmacokinetic profile and was well tolerated at a high dose of 100 mg/kg, accompanied by significantly declined pharmacodynamic biomarker TGF-β1 in peripheral blood. Conclusion: Integrin αvβ8 is a main activator of TGF-β1 in the TME and overexpressed in some cancer types. 2MW4991 demonstrated potent anti-tumor activity by unleashing the immune cells suppressed by TGF-β1 in TME and Fc-mediated immune functions. These compelling preclinical results support further evaluation of 2MW4991 in clinical trials. Citation Format: Cuicui Guo, Zhen Han, Xiaowei Cen, Hai Wu, Jinchao Zhang, Xun Gui. 2MW4991, a novel ADCC-enhanced integrin αvβ8 blocker, exhibits high anti-tumor potency and was well tolerated in cynomolgus monkeys [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 6349.

Abstract 1183: A first-in-class and SHP1 allosteric inhibitor, SB8091, shows a good efficacy and safety profile in preclinical models

Abstract Introduction: Src homology 2 domain-containing phosphatase 1 (SHP1) is a cytoplasmic protein tyrosine phosphatase (PTP) that is primarily expressed in hematopoietic cells and is commonly recognized as a negative regulator of multiple signaling pathways1. Previously, we showed that inhibition of the phosphatase activity of SHP1 by a novel SHP1 allosteric inhibitor, SB6299, results in the activation of innate and adaptive immune cells and enhanced anti-tumor immune responses2. Here, we report SB8091, an advanced SHP1 allosteric inhibitor with a promising initial safety profile and improved pharmacokinetic properties. Methods: SHP1 inhibitors were developed through a structure-based drug design approach, and optimized for cellular activity on target, in vitro ADME, and in vivo pharmacokinetic properties. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. The lead series was assessed in in-vitro studies with mouse or human primary immune cells. In-vivo efficacy and pharmacodynamic data were generated using mouse syngeneic tumor models. Results: Structurally, SB8091 seemed to stabilize the autoinhibitory form of SHP1 as an allosteric inhibitor, in a nanomolar range and which can explain the outstanding selectivity compared to other PTP enzymes, including SHP2. In several human primary immune cell types, SB8091 significantly increased cytokine responses and demonstrated significantly enhanced activity in NK cells and macrophages against various cancer target cells. As a single agent, this compound exhibited inhibitory effects on tumor growth when administered orally, and the efficacy was further enhanced when co-administered with anti-PD1 antibodies in various syngeneic tumor models. Notably, this effect was associated with a pharmacodynamic response marked by increased levels of phosphorylated substrates regulated by SHP1. SB8091 exhibited favorable pharmacokinetic properties for oral administration, and SafetyScreen data showed a very low potential for off-target activity and drug-drug interactions. Additionally, during a 2-week repeat pilot toxicity study in mice, SB8091 demonstrated excellent tolerability even at high doses, indicating a high therapeutic index (TI > 100). Conclusion: Our advanced compound, SB8091, is a potent, highly selective, and orally bioavailable allosteric inhibitor of SHP1. It has demonstrated excellent efficacy as a monotherapy or in combination with immune checkpoint blockers, with favorable drug properties and safety profiles. Research to advance to the clinical stage is being conducted.

Abstract 4514: Discovery of potent and orally bioavailable degraders of HPK1 based on a novel HPK1 binder

Abstract Background: Proteolysis targeting chimeras (PROTACs) are novel structures designed to target a protein of interest (POI) for ubiquitination and degradation, leading to the selective reduction in the expression levels of the POI. Hematopoietic progenitor kinase 1 (HPK1) is considered a promising target for tumor immunotherapy. PROTAC-involved disruption of HPK1 protein activity enhanced the treatment efficacy of CAR-T-cell-based immunotherapies in different solid tumors and mouse models. Thus, targeting HPK1 by PROTAC to restore T-cell activity is an attractive approach to induce a greater immune responses in cancer. Results: We designed and synthesized a series of HPK1 degraders by tethering our previously discovered novel HPK1 inhibitor with CRBN binder through various linkage. The most potent PROTAC effectively degrades HPK1 proteins in the Ramous cell in a dose-dependent manner and achieves DC50 value <50 nM. The maximum degradation (D max ) values achieved are >90%. Our PROTAC showed EC50 value <100 nM for IL-2 and IFNγ production. PK studies revealed a low clearance, high plasma exposure and oral bioavailability in mice and rats. Furthermore, our PROTAC is shown to induce robust and statistically significant tumor growth inhibition in the MC38 and CT26 syngeneic model with increased T cell signatures observed within the tumor. The PROTAC in combination with anti-PD1 also resulted in robust anti-tumor activity with TGI > 90%. The 14 days DRF experiment shows that the compound has a great safety window(>200 folds). Conclusion: We show here that a potent oral HPK1 degrader demonstrates strong immune cell activation and robust anti-tumor activity in mouse syngeneic tumor models, as a single agent and in combination with anti-PD1. Further evaluation of these potent degraders in additional in vivo studies will continue to build upon our mechanistic understanding of HPK1 degradation as a novel immunomodulatory approach for anti-tumor immunity. Citation Format: Zhimin Zhang, Mengqiang Wu, Ling Wang, Xi Yang, Zhiping Zhang, Liubin Guo, Hao Pan, Mengting Zhao, Linli Wang, Sirui Liu, Zhao Dong, Chunhua Jiang, Haowen Zheng, Dongzhou Liu. Discovery of potent and orally bioavailable degraders of HPK1 based on a novel HPK1 binder [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 4514.

Abstract 4062: A novel method for generating regulated cytokine therapeutics: Safety and activity of a conditionally active cLAG3-IL2 capable of delivering IL2 to LAG3+ cells while remaining inert on LAG3- cells

Abstract IL-2 is a powerful cytokine central to the generation of an effective immune response. However, its use in cancer immunotherapy has been limited by toxicities arising from its broad activity as well as the narrow patient population in which efficacy is seen. To address these limitations, many strategies have been pursued including IL-2 attenuation and targeting of IL-2 activity. These approaches, however, retain unacceptable toxicity and/or are not targeted to the appropriate cell populations. Using a novel approach that takes advantage of the ability of an antibody to bind specifically and competitively to two distinct antigens, we have generated a conditionally active cLAG3-IL2 therapeutic that targets IL-2 to LAG3+ cells while remaining inert on IL-2Rβγ+ cells lacking LAG3 expression, even at high treatment doses. As a marker of antigen-activated and tumor-specific T cells, LAG3 is an ideal target toward which to direct IL-2 activity. In vitro, using both reporter cell lines and receptor binding assays, the regulated cLAG3-IL2 demonstrates >100 fold difference in activity in the presence or absence of LAG3. In primary immune cells, regulated cLAG3-IL2 preferentially induces IL-2 cis-signaling in activated human LAG3+ CD8 T cells compared to LAG3- T cells. In mouse syngeneic tumor models, cLAG3-IL2 inhibits tumor growth while avoiding clinical signs of IL-2-mediated toxicity at doses well above the level where the non-regulated IL2 is toxic. Additionally, cLAG3-IL2 does not induce expansion of circulating LAG3- IL-2Rβγ+ cell populations, including NK cells and LAG3- T cells. In the TME, cLAG3-IL2 drives the expansion and activation of tumor-specific CD8+ T cells. Furthermore, cLAG3-IL2 demonstrates robust combinatorial activity with anti-PD1. These results demonstrate the strength of the Bonum platform, currently applied to multiple target/effector pairs including PD1, PDL1, ATP, LRRC15 and effectors IFNα, IL12 and TGFβ inhibition, and support the clinical development of our conditionally active cLAG3-IL2. Citation Format: Justin Killebrew, Shannon Okada, Lynn Amon, David Bienvenue, Laura Carlucci, David Colby, Wendy Curtis, Kendyl Daniels, Alton Etheridge, Zane Kraft, Jamie Nguyen, Sandra Notonier, Jacqueline Pham, Megan Sprague, Kerri Thomas, Diane Hollenbaugh, John Mulligan. A novel method for generating regulated cytokine therapeutics: Safety and activity of a conditionally active cLAG3-IL2 capable of delivering IL2 to LAG3+ cells while remaining inert on LAG3- 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 4062.

Abstract 4081: rhIL-7-hyFc (efineptakin-alfa, NT-I7) increases tumor-specific CD8+ T cells despite FOLFOX cytotoxicity effect

Abstract rhIL-7-hyFc (efineptakin-alfa; NT-I7) is a potent T cell amplifier, comprised of two molecules of interleukin-7 (IL-7) fused to the hybrid Fc domain of IgD/IgG4 immunoglobulin. NT-I7 safely, significantly, and persistently increases the absolute lymphocyte counts (ALC) in humans in a dose-proportional manner. Importantly, this increase is primarily due to an increase in T cells (1). Previous studies have shown that NT-I7 not only significantly increases the number of CD8+ T cells in peripheral blood but also dramatically boosts the presence of tumor-infiltrating CD8+ T cells, including both tumor-specific (p15E+PD-1+) and tumor non-specific (PD-1-) CD8+ T cells in mice (2). FOLFOX is a chemotherapy regimen composed of 5-fluorouracil, leucovorin, and oxaliplatin that inhibits DNA duplication and eradicates rapidly dividing cells (3). Although the mechanism of FOLFOX results in direct tumor killing, it also has the potential to kill newly dividing lymphocytes during NT-I7 treatment (4-6). Therefore, in this study, we aimed to evaluate the feasibility of combining FOLFOX with NT-I7 in a syngeneic MC38 mouse tumor model. Our results show that NT-I7 improves the efficacy of FOLFOX, irrespective of the timing of NT-I7 administration: simultaneous administration or delayed 3 hours, or 2 days after FOLFOX administration. This improvement persists despite a significant decrease in ALC for the first 6 days, which was significantly lower than the ALC observed with FOLFOX alone. FOLFOX decreases the absolute numbers of tumor non-specific CD8+ T cells. However, adding FOLFOX does not affect the NT-I7-driven increase of tumor-specific CD8+ T cells within the tumor. Taken together, our data demonstrate that FOLFOX does not compromise NT-I7’s ability to increase tumor-specific T cells within the tumor and the combination enhances the anti-tumor response irrespective of the timing of NT-I7 administration. Citation Format: Soung-Min Lee, Miyoung Kim, Sara Ferrando-Martínez, Sun-Kyoung Im, Mankyu Ji, Donghoon Choi. rhIL-7-hyFc (efineptakin-alfa, NT-I7) increases tumor-specific CD8+ T cells despite FOLFOX cytotoxicity effect [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 4081.

Abstract 4059: Vitokine-2: A TIL-targeting tumor activable engineered IL-2 for cancer treatment

Abstract Background: Interleukin-2 is the first approved immune-oncology (IO) drug to treat multiple metastatic cancers with durable and curative antitumor effects. Yet, its application is restricted due to severe toxicity, short half-life, and a small fraction of response rate. To reduce toxicity and improve efficacy, we developed Vitokine-2, a tumor infiltrating lymphocyte (TIL)-targeting, tumor activable, and precision engineered IL-2 that remains inert systemically but activated in the tumor microenvironment (TME). Methods: Through protein and structure-guided precision engineering, the biological activity of IL-2 was optimized, concealed, guided to exhausted TILs, and activated at TME. The biological activity was determined by measuring proliferation (Ki67) of CD8+ T and NK cells using human peripheral blood mononuclear cells (PBMCs). Pharmacokinetics (PK), pharmacodynamics (PD), and anti-tumor efficacy were evaluated in mice, encompassing healthy and multiple syngeneic tumor mouse models. Results: Vitokine-2 was inactive in stimulating CD8+T and NK cell proliferation in vitro. Yet, its biological activity was fully restored following the cleavage and release of the concealing domain. Vitokine-2 displayed a significantly prolonged in vivo pharmacokinetic profile due to half-life extension, concealing and reduction of target sink. Administration of Vitokine-2 in mice resulted in minimal or no detectable activation, proliferation, and expansion of cytotoxic CD8+T and NK cells in the systemic circulation. However, marked CD8+ T cell proliferation and expansion with enhanced expression of granzyme B, a cytotoxic functional biomarker, was observed within the TME. The effect was specific to cytotoxic lymphocytes but not regulatory T cells. Corroborating with the expansion and enhanced functionality of CD8+ T cells in TME, robust antitumor efficacy was observed with Vitokine-2 but not with the non-activable IL-2 counterpart. Furthermore, dose-dependent tumor killing efficacy and complete eradication of tumors were demonstrated in multiple syngeneic tumor models. Vitokine-2 was found to be safe and well tolerated with minimal interferon gamma release and body weight loss even at much higher doses. Conclusion: Vitokine-2 is a TIL-targeting, tumor activable and precision engineered IL-2. It demonstrated robust anti-tumor efficacy with minimal systemic toxicity. It is safe, well tolerated, and permits high dose administration, yielding a widened therapeutic window. Vitokine-2 presents a paradigm-shifting, potentially a first-in-class, highly safe and efficacious IO therapy with an immense potential to address both IO-responsive and unresponsive patient population. Citation Format: Sreerupa V. Challa, Yao-Te Hsieh, Karen Gelinas, Andrea Umana, Wei-chun Weng, Toska Berisha, Cameron Stephens, Yuesheng Li, Jing Xu. Vitokine-2: A TIL-targeting tumor activable engineered IL-2 for cancer treatment [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 4059.

Abstract 86: Deep immuno-profiling of syngeneic tumor mouse models for preclinical studies

Abstract The identification of the major cellular players involved in the progression of a type of cancer is a key step for the success of new immunotherapies for personalized medicine. Immune cells play critical functions in cancer, and mice with intact immune systems are vital to understanding tumor immunology. It is however a daunting challenge as complex relationships interplay between tumor cells and the immune system. Each component of innate immunity or adaptive immunity, such as T lymphocytes, macrophages or neutrophils, may be directed to a pro- or anti-tumor function. In order to cope with the complexity of the tumor microenvironment, it is necessary to use an experimental approach capable of characterizing the heterogeneity of the cell types present in the tumor, the evolution of their relative proportion and their fine-tuned functional specificity. This approach leads to the identification of new cellular biomarkers of different stages of tumor progression in order to identify new targets for therapeutic time-window of and improve cancer diagnosis To decipher the impact of immunotherapy treatments involved in the anti-tumor response, cellular phenotyping of leukocytes infiltrating a tumor but also those present in peripheral organs is necessary. Essentially based on extracellular labeling, this primary screen aims to quantify the different cell populations present in a syngenic tumor models on B6/N or BalbC genetical backgrounds. In order to increase our understanding in the precise mode of action of anti-PD1 treatment at the cellular level in sensitive and unsensitive models, we investigated immunophenotypes and responses to immune checkpoint inhibitor (ICI) of several hallmark syngeneic tumor models (MC38, CT26, B16F10, B16-OVA, RENCA, EMT6) in immunocompetent mouse models by flow and mass cytometry. We compared growth kinetics and profiled the immune cell composition of tumor microenvironment (TME), draining lymph node (dLN) and blood in order to establish immune-phenotypic cell signatures that correlates with treatment efficacy. Supervised, unsupervised and integrative data analysis as well as visualization tools are used to identify significant changes across different experimental settings. Our results indicate that each model possesses a unique tumor-immune infiltrate profile that can be modulated with immunotherapies. Overall, these studies provide an important resource of highly-characterized syngeneic tumor model and highlight the importance of tumor immune landscape variance across models that will drive selecting the most appropriate model to test novel immunotherapeutic agents and enhance our translation of knowledge from syngeneic models to human tumors. Citation Format: Anais Joachim, Emilie Maturin, Marielle Mello, Lilia Hadjem, Magali Grange, Olivier Deas, Ana Zarubica, Bernard Malissen, Hervé Luche, François Romagne, Stéphanie Blanchin. Deep immuno-profiling of syngeneic tumor mouse models for preclinical studies [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 86.

Abstract 2474: Antitumoral efficacy of ASKG812, a Smartkine® based bifunctional PD-1-eIL-2 molecule, through enhancing T effector cis agonism

Abstract Cytokines are potent molecules, yet their broad application as therapeutics has been significantly hampered due to short PK, severe systemic toxicity, and narrow therapeutic window. To improve the developability of cytokines, AskGene has invented a proprietary cytokine platform (Smartkine®) to achieve its overarching objective of modulating immune reactions at a disease site in a selective and controlled manner. Several antibody-cytokine fusion molecules using the SmartKine® platform have been moving into preclinical and clinical development. ASKG812 is a unique fusion molecule composed of anti-PD-1 antibody and engineered IL-2 (eIL-2). The eIL-2 moiety also comprises a specific mask such that the eIL-2 molecule binding biases towards PD-1+ T effector cells in a cis agonism manner for improved efficacy and therapeutic window. In ex vivo human PBMC assays, we demonstrated that ASKG812 significantly reduced activity in PD-1− T and NK cells, and enhanced activity in PD-1+ T cells judged by levels of pSTAT5, Ki67 and Granzyme B. In a cytotoxicity assay, ASKG812 can efficiently induce PBMC-mediated killing of MDA-MB-231 tumor cells in a dose-dependent manner. Moreover, in both anti-PD-1 therapy responsive and non-responsive syngeneic mouse tumor models, ASKG812 showed robust efficacy; without noticeable toxicity compared to a reference anti-PD-1-IL-2v fusion molecule. In conclusion, these findings provide the basis for the development of this new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the clinical treatment of cancer. Citation Format: Chunxiao Yu, Kurt Shanebeck, Shiguang Yu, Jeanine Ruiz, Shichang Li, Stone D.-H Shi, Hong Ma, Yuefeng Lu. Antitumoral efficacy of ASKG812, a Smartkine® based bifunctional PD-1-eIL-2 molecule, through enhancing T effector cis agonism [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 2474.

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 5282: Sulindac modulates the response of triple negative breast cancer to anti-PD-L1 immunotherapy

Abstract Triple-negative breast cancer (TNBC) is a heterogeneous tumor, and there is a lack of effective therapies. Immune checkpoint inhibitors (ICIs) therapy has been widely used to treat a variety of human cancers including TNBC. Despite the FDA's approval of therapies like Atezolizumab (anti-PD-L1) and Pembrolizumab (anti-PD-1) for certain TNBC cases, more than half of all TNBC patients, especially those with low PD-L1 levels and in advanced stages, remain unresponsive. Therefore, there is urgent clinical need to find other innovative combination therapy strategy for these TNBC patients. In this study, we examined the efficacy of sulindac to enhance the response of TNBC to anti-PD-L1 immunotherapy. We utilized a 4T1 syngeneic mouse tumor model to compare the inhibitory effects of PD-L1 antibody, sulindac, and their combination on 4T1 tumor growth. We found that mice treated with combination therapy showed a significant reduction in tumor volume, along with increased infiltration of activated T lymphocytes (Granzyme B+/CD8+ T cells) in the tumor tissues. We also established a PBMC humanized mouse model to further confirmed that combination therapy could significantly reduce the tumor size of TNBC patient-derived xenografts (56S) and the combination group were found to have the most infiltrating activated human T lymphocytes (Granzyme B+/CD8+ T cells) in the tumor tissues. Immunofluorescent staining of organoids also confirmed that organoids from combination group have more CD8+ T cell and the results of organoid cell viability assay in vitro indicated that combination of sulindac with PD-L1 antibody together with activated human PBMC significantly reduce 56S organoid cell viability, which is consistent with in vivo study. When investigating the mechanism of action, first we found sulindac could downregulate exosomal PD-L1 by decreasing expression of nsMase2 which is a major regulator in production of exosomal PD-L1. Second, we demonstrated that sulindac could downregulate PD-L1 by blocking Stat3 signaling and enhancing the expression of miR-570-3p which can potentially target PD-L1, which in turn led to a further decrease in exosomal PD-L1. Previous study showed that PD-L1 antibody could be bound and consumed by exosomal PD-L1 in the blood circulation. Therefore, in combination therapy, sulindac downregulating exosomal PD-L1 leads to increased availability of PD-L1 Ab, which potentially improves the overall efficacy of anti-PD-L1 therapy. In conclusion, our findings provide unique insights into the mechanism of action and efficacy for sulindac as an immunomodulatory agent in combination with anti-PD-L1 therapy for the treatment of TNBC. Citation Format: Bin Yi, Ruixia Ma, Yaguang Xi. Sulindac modulates the response of triple negative breast cancer to anti-PD-L1 immunotherapy [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 5282.

Abstract 4026: The combination of DLL4/VEGF-a blockade and immunomodulation can eliminate MHC class I negative tumors in mice

Abstract CTX-009 is a bispecific antibody targeting VEGF-A and DLL4 which is undergoing clinical investigation in patients with multiple tumor types, including biliary tract and colon cancers. In biliary tract cancers, checkpoint inhibitor therapy has been shown to improve outcomes when combined with chemotherapy. In an effort to increase the breadth and duration of response of CTX-009, we set out to investigate the combined anti-tumor efficacy of this bispecific antibody and immunotherapy in preclinical models. Here we report the evaluation of the combination of the mouse surrogates of CTX-009 and CTX-471, an agonistic anti-mouse CD137 antibody, or monoclonal antibodies that disrupt the PD-1/PD-L1 signaling axis. Coupling VEGF-A/DLL4 targeting with either CD137 agonism or PD-1/PD-L1 blockade markedly increased the anti-tumor efficacy of the individual treatments alone in multiple syngeneic mouse tumor models. Tumor growth inhibition was accompanied by pharmacodynamic evidence consistent with enhancement of anti-tumor immunity. Notably, the combination of CTX-009 and CTX-471 retained potent anti-tumor activity even in a tumor line (MC38) which was made MHC I deficient through the deletion of the beta 2 microglobulin gene. This system may model the clinically relevant unmet need characterized by tumor escape through the loss of antigen presentation machinery such as HLA LOH. Interestingly, the model cell lines CT26 and MC38 failed to thrive in the B2M-deficient setting and depletion of NK cells markedly increased their ability to grow. This implies that NK cells might be involved in controlling tumor growth in this setting, and that the combined action of CTX-471 and CTX-009 in the tumor stroma enhances NK cell activity. The findings presented here suggest that CTX-009 may provide enhanced clinical benefit when combined with immunomodulatory agents, such as agonistic anti-CD137 antibodies or PD-1/PD-L1 blockers in tumors where immune checkpoint inhibitors have failed. Citation Format: Diana I. Albu, Rachael Duffy, Amy Daniel Ulumben, Jason Kong, Jessica Zolotarevsky, Melissa Brundin, Mei Su, Ruturaj Jadhav, Kris F. Sachsenmeier, Bing Gong, Thomas J. Schuetz, Alberto Visintin. The combination of DLL4/VEGF-a blockade and immunomodulation can eliminate MHC class I negative tumors in mice [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 4026.

Abstract 5079: Comparative studies of chemotherapy and immuno-oncology combinations in multiple mouse syngeneic tumor models

Abstract The development of immune checkpoint inhibitors (ICIs) and combinations of immuno-oncology therapies with standard-of-care (SoC) chemotherapeutic agents has shifted therapeutic paradigms for patients with multiple cancer indications. For many patients, these combinations have shown a beneficial effect on clinical outcomes and disease management. However, additional combination therapies are still needed. Preclinically, a comparison of responses from SoC chemotherapies and ICI combinations in multiple disease models may be helpful in identifying optimal treatment combinations in the clinic. Here we report the results of multiple SoC/ICI in vivo combination efficacy studies in a large panel of mouse syngeneic models spanning seven tissue origins: 1) Colon: CT26, MC38; 2) Breast: EMT6, 4T1; 3) Kidney: Renca; 4) Bladder: MB49, MBT2; 5) Skin: B16F10, CM3; 6) Lung: TC1, LL/2, M109; 7) Brain: GL261. The advantage of using syngeneic models is not limited to the rapid and reproducible tumor growth, but importantly that the animals also have an intact immune system and clinically relevant tumor microenvironment, which are required for ICI combination studies. We tested > 10 chemo SoC combinations with a murinized anti-PD-1 antibody (DX400) in 14 syngeneic mouse models. We found that multiple SoC regimens combined well with DX400 treatment, including FOLFIRI, FOLFOX (colon), Carboplatin/Paclitaxel, Carboplatin/Pemetrexed (lung), Capecitabine/Docetaxel, Cisplatin/Gemcitabine (bladder), Cisplatin/5-FU/or capecitabine, and Oxaliplatin/5-FU/or capecitabine (gastric). Specifically, we observed that the SOC/ICI combinations resulted in enhanced antitumor activities compared to single-agent treatments in many of the models. These data, particularly specific SoC combination regimens that combine well with ICIs in each disease, are useful for optimizing promising combination approaches for clinical development and will be presented here. Citation Format: Eunsil Park, Marlene Hinton, Mingmei Cai, Malgorzata A. Gil, Gustave Hebert, Long Cui, Doug Linn, Brian J. Long, Evan R. Barry. Comparative studies of chemotherapy and immuno-oncology combinations in multiple mouse syngeneic tumor 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 5079.

Abstract 5949: The clinical dual KRASG12C inhibitor FMC-376 has demonstrated potential as both a monotherapy and in combination for the treatment of patients with KRASG12C mutation positive NSCLC

Abstract KRAS, a membrane-associated guanosine triphosphatase (GTPase), is the most frequently mutated oncogene in cancer with activating mutations occurring in approximately 25% of NSCLC. Of these, KRASG12C occurs in approximately 14% of adenocarcinomas and in 0.5 to 4% of squamous NSCLCs. This mutation impairs GTPase activity and GTP-hydrolysis leading to an increase in the active, GTP-bound (ON), state. KRAS was long considered undruggable until the recent discovery of inhibitors that bind the GDP-bound, inactive (OFF) state of KRASG12C. The most clinically advanced of these first-generation molecules have demonstrated clinical response rates of 37-43% and 6 to 7-month progression-free survival in patients with KRASG12C positive NSCLC. While significant, a majority of cancers do not respond, and acquired resistance is common. FMC-376 is a next-generation dual inhibitor of both the ON and OFF states of KRASG12Cwith the potential to deliver improved outcomes as both a monotherapy and in combination, for patients with KRASG12C positive NSCLC. FMC-376 was discovered through the FrontierTM platform, which integrates chemoproteomics, machine-learning, and covalent fragment-based drug discovery. The potential for FMC-376 in the treatment of patients with KRASG12C mutant NSCLC has been assessed in a broad panel of NSCLC PDX models representing settings of sotorasib/adagrasib resistance, in a model of NSCLC CNS-metastasis and in combination with an immune checkpoint inhibitor. The dual ON + OFF state mechanism of action of FMC-376 is highly active across a panel of NSCLC CDX and PDX models, including those that carry co-mutations that are associated with both primary and acquired resistance to the OFF-state inhibitors adagrasib and sotorasib in NSCLC. These drivers of resistance include activating mutations of receptor tyrosine kinases (e.g. EGFR, MET, etc.), co-mutations including KEAP1 and adaptive resistance due to release of negative feedback control. Evaluation of FMC-376 in models where EGFR signaling is induced by EGF demonstrated rapid and durable target engagement in contrast to both sotorasib and adagrasib which show decreased effectiveness after EGF stimulation. As a result, FMC-376 is highly effective in NSCLC PDX models that have EGFR and MET upregulation. In addition, 27-42% of patients with KRASG12C positive NSCLC present with CNS metastasis. FMC-376 is highly active in an intracranial NSCLC model of CNS-metastasis, resulting in tumor regression. Furthermore, FMC-376 has demonstrated activity in combination with an anti-PD-1 mAb in a KRASG12C mutant mouse syngeneic tumor model. Together, these data demonstrate the potential of FMC-376, a next-generation clinical stage dual inhibitor of ON and OFF state KRASG12C, for the treatment of patients with KRASG12C mutant NSCLC as well as other KRASG12C positive tumors. Citation Format: Yan Wang, Allison Roberts, Philamer Calses, Richard M. Neve, Jocelyn Staunton, Kevin R. Webster. The clinical dual KRASG12C inhibitor FMC-376 has demonstrated potential as both a monotherapy and in combination for the treatment of patients with KRASG12C mutation positive NSCLC [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 5949.