Responses In Preclinical Models Research Articles

Abstract LB119: The immune-modulation of HM16390, firing up the poor tumor microenvironment to induce a potent anti-tumor efficacy

Abstract Immunotherapy, encompassing immune checkpoint blockades (ICBs) and immune stimulators, has become a widespread approach in cancer treatment. However, the effectiveness of these strategies relies significantly on the characteristics of the current tumor microenvironment (TME). This reliance underscores the urgent need for a potent immune modulator capable of inducing a favorable TME, particularly in non-immunogenic cold tumors. Here, we demonstrate that HM16390, a long acting IL-2 analog, has the potential to modify the immunogenicity of the TME by activating, expanding, and recruiting cytotoxic effector cells in cold tumor. In the murine B16F10 melanoma model, following the early upregulated proliferative potential, the proportions of CD8+ T as well as NK cells increased in both blood and tumor. While NK cell frequency initially increased and then recovered, CD8 T cells exhibited a dose-dependent upregulation in the late phase in both blood and tumor. Given the IL-2Rα (CD25) affinity of HM16390, there was an increase in Treg proportions in blood; however, intriguingly, Treg frequency decreased in the tumor. This pattern emphasizes the crucial role of HM16390 in balancing the hyper-activation of peripheral effector cells while simultaneously inducing a profoundly immune-favorable TME. Effector functions were also upregulated in both blood and tumor. Early upregulation of IFN-γ and TNF occurred in both blood and tumor, but the upregulation of granzyme B displayed a sustained expression pattern in the tumor. These immune-modulated features correlated with the exposures of HM16390, particularly noting a negative correlation between Treg down-regulation and Its exposure in blood. The immune modulations of HM16390 were validated in terms of efficacy in the Pancreatic Ductal Adenocarcinoma (PDAC) model. HM16390 was administered subcutaneously to panc02 tumor syngeneic mice via once-a-week administration for 6 weeks, with or without PD-1 combination treatment. HM16390 treatment group was well-tolerated and resulted in a dose-dependent anti-tumor effect. Notably, all mice treated with the highest dose exhibited a complete response. Furthermore, in panc02 syngeneic mice, known for a poor response to ICBs, the combination treatment of HM16390 with mAnti-PD-1 demonstrated synergistic anti-tumor effects. In conclusion, HM16390 demonstrates improved expansion and functions of effector tumor-infiltrating lymphocytes (TILs), correlating with exposure, and exhibits a safe Treg modulation pattern. These modulations culminate in a significant anti-tumor effect and synergies with PD-1 blockade in the B16F10 and PDAC model. Given that both B16F10 melanoma and Panc02 PDAC are recognized as poor immunogenic murine models with low TIL frequency, HM16390 shows promise in modifying the immunogenicity of the TME, thereby activating proper immune responses in preclinical models. Citation Format: Seongju Jeong, Yunjae Kim, Jooyun Byun, Jinyoung Kim, Jaehyuk Choi, Sungmin Bae, Daejin Kim, In Young Choi. The immune-modulation of HM16390, firing up the poor tumor microenvironment to induce a potent anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB119.

Abstract 720: Combining zelasudil, a small molecule ROCK2 inhibitor, with chemotherapy or immunotherapy improves response in preclinical models of pancreatic cancer

Abstract Zelasudil (RXC007) is an orally available, highly selective small molecule inhibitor of Rho-associated coiled-coil containing protein kinase 2 (ROCK2), which has demonstrated pre-clinical anti-fibrotic efficacy and is currently being tested in a Phase 2a clinical trial for the treatment of idiopathic pulmonary fibrosis (IPF) (NCT05570058). Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and therapeutically challenging disease with a dense and highly fibrotic tumor microenvironment which may limit efficacy of conventional therapies. Here, we investigate the benefit of combining an anti-fibrotic, RXC007, with either standard of care chemotherapy or immunotherapy in mouse models of PDAC.In a fibrotic patient-derived metastatic PDAC orthotopic model, RXC007 (10mg/kg, 50mg/kg and 100mg/kg BID 5d on/2d off PO) in combination with SoC chemotherapy Gemcitabine (100mg/kg IP QW)/Abraxane (30 mg/kg IP QW) improved survival in a dose-dependent manner (all P<0.0001) compared to Gemcitabine/Abraxane alone.In the immunocompetent orthotopic LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) model of metastatic PDAC, survival was significantly increased (P<0.0001) upon treatment with a combination of RXC007 (50 mg/kg BID 5d on/2d off PO) and anti-PD1 (200 ug BIW) compared to anti-PD1 alone. ROCK2 inhibition elicited positive immunomodulatory effects in KPC pancreatic tumors, with increase of CD8+ (P=0.0031) and CD4+ (P=0.008) T cell infiltrate into the tumor cortex and reduction in immunosuppressive FOXP3+ regulatory T cells at the tumor border. Furthermore, RXC007 monotherapy exhibited anti-fibrotic effects, via a decrease in collagen content and organization, effects associated with CAF reprogramming and reduced intra-tumoral aSMA+ PDPN+ myofibroblast-like CAFs (myCAFs; P=0.0406).This data highlights the potential for treating highly fibrotic tumours with RXC007 in combination with SoC chemotherapy or immunotherapy in order to improve patient outcomes in this aggressive and treatment-refractory cancer. Citation Format: Marina Pajic, Benjamin McLean, Diego Chacon Fajardo, Sean Porazinski, Dannielle H. Upton, Diana Schuhmacher, Aji Istadi, Thomas Cox, Paul Timpson, Daniel J. Wilcock, Katie J. Anderson, Helen Mason, Nicolas E. Guisot, Clifford D. Jones, Caroline Phillips, Richard Armer. Combining zelasudil, a small molecule ROCK2 inhibitor, with chemotherapy or immunotherapy improves response in preclinical models of pancreatic cancer [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 720.

Abstract 1899: Novel combinations of aplitabart, a DR5 agonist IgM antibody, with ADCs or chemotherapeutic agents lead to robust anti-tumor responses in solid tumor models

Abstract Agonism of death receptor 5 (DR5), a tumor necrosis factor receptor superfamily member, induces the killing of tumor cells via the extrinsic apoptotic pathway upon receptor multimerization. We have engineered a multivalent IgM DR5 agonist antibody, aplitabart (IGM-8444), that induces robust anti-tumor activity in both in vitro and in vivo preclinical models. We also demonstrated potent synergistic in vitro activity of aplitabart with different classes of chemotherapeutic agents including microtubule inhibitors, nucleoside analogs and topoisomerase inhibitors in a range of solid cancer cell lines. Preliminary results from an ongoing phase 1a/ab clinical study show that aplitabart has an encouraging safety profile and promising activity in combination with FOLFIRI ± bevacizumab in heavily pretreated metastatic colorectal cancer patients. To further explore the combination therapies for aplitabart in lung and breast cancers, we evaluated standard of care chemotherapies, such as paclitaxel, carboplatin, and antibody drug conjugates (ADCs) in vitro on a range of human lung and breast cancer cell lines. We observed dose-dependent synergistic cytotoxic effects of aplitabart on tumor cells when combined with these agents. Additionally, these agents increased the expression of DR5 on tumor cells and could explain their synergistic combinatorial activity with aplitabart. Representative cancer cell lines were selected to evaluate these combinations in vivo using xenograft mouse models where robust anti-tumor responses were demonstrated in the xenograft lung and breast cancer models, resulting in enhanced tumor growth inhibition and extended survival. Our findings demonstrate potent in vitro synergistic cytotoxicity and enhanced anti-tumor response in preclinical models in vivo by combining aplitabart with ADCs, or carboplatin and paclitaxel. These results support the combination of DR5 agonist IgM antibodies with agents known to upregulate DR5 expression on solid tumors. Aplitabart is currently under evaluation in a randomized clinical study in combination with FOLFIRI + bevacizumab in comparison with FOLFIRI + bevacizumab in patients with colorectal cancer (NCT04553692). Citation Format: Mélanie Desbois, Susan E. Calhoun, Kevin C. Hart, Carolyn R. Denson, Poonam Yakkundi, Thomas J. Matthew, Maya K. Leabman, Sanela Bilic, Genevive Hernandez, Eric W. Humke, Albert F. Candia, Bruce Keyt, Angus M. Sinclair, Maya F. Kotturi, Beatrice T. Wang. Novel combinations of aplitabart, a DR5 agonist IgM antibody, with ADCs or chemotherapeutic agents lead to robust anti-tumor responses in solid 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 1899.

Abstract 4074: Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse tumor models

Abstract Systemic administration of proinflammatory cytokines is a promising approach to treat cancer. IL-18 has been shown to promote activation of both innate and adaptive anti-tumor responses in pre-clinical models. Specifically, IL-18 has been shown to impact macrophage suppressive function as well as drive CD4+ T cells towards a TH1 phenotype. However, IL-18 therapies have been hampered by a lack of efficacy due to the suppressive activity of IL-18 binding protein (IL-18BP), which binds to IL-18 and prevents its interaction with its receptor. Several groups have taken the approach of modifying IL-18 to render it resistant to IL-18BP as a potential strategy to enhance anti-tumor activity. To explore the therapeutic benefit of delivering a conditionally-activated IL-18 to the tumor microenvironment, we have developed inducible polypeptides (INDUKINE™ molecules) consisting of mouse IL-18 or IL-18BP resistant IL-18 (BPR IL-18) tethered via protease-sensitive linkers to a high affinity antibody blockade domain, and a half-life extension (HLE) domain which improves exposure in the tumor. IL-18 INDUKINE™ molecules are inactive until reaching the tumor microenvironment, where the linkers are cleaved by intra-tumoral proteases, releasing active IL-18. Intraperitoneal (i.p.) administration of an IL-18BP resistant IL-18 (BPR IL-18) INDUKINE™ molecule led to complete tumor regression in the MC38 tumor model. In contrast, equimolar doses of wild-type IL-18 INDUKINE™ protein were not as efficacious. Moreover, BPR IL-18 INDUKINE treatment led to increased activation and frequencies of NK cells and tumor specific CD8 T cells in MC38 tumors. WTX-518 is a novel INDUKINE™ molecule that is designed to selectively deliver active human BPR IL-18 to the tumor microenvironment. WTX-518 is inducible in vitro, either when tested using a reporter assay or primary immune cells. Importantly, the active BPR IL-18 payload is resistant to IL-18BP in reporter or primary immune cell assays. WTX-518 is also selectively inducible, as incubation ex vivo with various primary human tumors led to the release of active BPR IL-18, while WTX-518 was stable in human serum and after exposure to primary cells from healthy tissues. Pharmacological and efficacy data support continued preclinical development of this innovative and differentiated engineered IL-18 therapy. Citation Format: Kristin R. Morris, Heather Brodkin, Kyriakos Economides, Daniel J. Hicklin, Julie LePrevost, Celesztina Nagy-Domonkos, Christopher Nirschl, Andres Salmeron, Cynthia Seidel-Dugan, Cierra Spencer, Zoe Steuert, William M. Winston. Discovery of WTX-518, an IL-18 pro-drug that is conditionally activated within the tumor microenvironment and induces regressions in mouse 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 4074.

Abstract 170: Targeting of macrophage PI3Kγin prostate cancer using Eganelisib (IPI-549) reprograms immune-suppressive infiltrating macrophages to enhance anti-tumor immune responses and promote immunologically mediated tumor growth

Abstract Background: Prostate cancer (PCa) is traditionally considered an immunologically 'cold’ indication, as evidenced in part by significant infiltration of immunosuppressive tumor associated macrophages (TAMs) and few activated effector CD8+ T cells. Attempts to (re)invigorate an anti-prostate cancer immune response with immune checkpoint blockade (ICB) such as anti-CTLA-4 and anti-PD-1 have been largely ineffective clinically, despite promising results in other cancers. This is likely due to a multitude of immunosuppressive cell types and secreted factors within the tumor microenvironment (TME) that diminish T cell activation and trafficking. Reducing the immunosuppressive burden intratumorally by targeting TAMs is a clinically attractive approach and offers opportunity to sensitize PCa to immune checkpoint blockade. While PI3K signaling is ubiquitous in most cell types, specific catalytic isoforms and regulatory elements are differentially expressed depending on cell type, with PI3Kγ being significantly enriched in myeloid cells and therefore offering a unique therapeutic opportunity for specific targeting. Methods: Utilizing novel syngeneic prostate cancer tumors, PI3Kγ deletion and antagonism using Eganelisib (a small molecule inhibitor for PI3Kγ), we tested our hypothesis that targeting PI3Kγ signaling in PCa TAMs would lead to tumor growth inhibition and anti-tumor immune responses. Results: We show here that PCa tumor cells do not express the PI3Kγ isoform and, therapeutic inhibition of PI3Kγ with Eganelisib reduces the TAM suppressive phenotype. We also show PI3Kγ signaling is essential to monocyte anti-apoptotic signaling, and inhibition of PI3Kγ can enhance anti-tumor immune responses in preclinical models of PCa. Specifically, inhibition of PI3Kγ alone is sufficient to enhance anti-tumor immune responses in the less aggressive B6CaP PCa model, but insufficient in the more aggressive RM-1 PCa model. However, treatment of RM-1 tumors with Eganelisib produces improved tumor control when combined with anti-PD-1 therapy, relative to Eganelisib or anti-PD1 as a monotherapy. Conclusions: Altogether, these data highlight the integral role of PI3Kγ signaling in TAMs and the potential for therapeutic targeting with Eganelisib to sensitize PCa to ICB. Citation Format: Kenneth M. Adusei, Thomas R. Nirschl, Alex J. Lee, Fan Shen, Xiaoxu Wang, Monali Praharaj, Jade Alvarez, Iyana Gross, Kara A. Lombardo, Tamara L. Lotan, Judith A. Varner, Jelani C. Zarif. Targeting of macrophage PI3Kγin prostate cancer using Eganelisib (IPI-549) reprograms immune-suppressive infiltrating macrophages to enhance anti-tumor immune responses and promote immunologically mediated tumor growth [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 170.

Abstract 6650: Macrophage pro-inflammatory phenotype skewing by the application of tumor treating fields (TTFields)

Abstract Introduction: Tumor Treating Fields (TTFields) are low intensity electric fields that disrupt cellular processes critical for cancer cell viability. TTFields have previously been shown to induce immunogenic cell death, promote dendritic cell maturation, and elicit a systemic anti-cancer immune response in preclinical models. The benefit of applying TTFields together with immune checkpoint inhibitors (ICI) was demonstrated in a phase 3 study in non-small cell lung carcinoma. In the current research, we investigated possible effects of TTFields on regulation of macrophage phenotype. Methods: Bone marrow-derived macrophages (BMDMs) were generated from the femurs and tibias of 5-8-week-old Balb\C mice. LPS+IFN-γ or IL-4 were applied to the BMDMs to induce polarization (M1 and M2, respectively). The BMDMs were then treated with TTFields (150 kHz, 24 h), and then examined by flow cytometry for surface expression of the macrophage biomarker F4/80 and the activation markers CD80, major histocompatibility complex class II (MHC II), inducible nitric oxide synthase (iNOS), CD206, and ARG-1. Cell supernatants were collected and analyzed by multiplexed secretion assay for secretion levels of CXCL1 (KC), IL-18, IL-23, IL-12p70, IL6, TNF-α, IL-12p40, TGF-β1, CCL22 (MDC), IL-10, IL-6, G-CSF, CCL17 (TARC), and IL-1β. Cell lysates were examined using a RhoA activation kit and Western blot to determine GEF-H1, c-Jun, and p65 phosphorylation levels. Results: BMDMs exposed to TTFields demonstrated elevated expression of the pro-inflammatory M1 markers CD80+ and MHC IIhigh, and decreased expression of the M2 markers CD206 and ARG-1. A pro-inflammatory secretion pattern was detected, with increased levels of CXCL1, IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, CCL22, G-CSF, CCL17 and IL-1β. The treated cells further demonstrated RhoA activation and GEF-H1 phosphorylation, with subsequent activation of the transcription factors c-Jun and p65. Conclusions: This study reveals a novel immunomodulatory role for TTFields, promoting in vitro pro-inflammatory polarization of macrophage. Citation Format: Tal Kan, Yiftah Barsheshet, Boris Brant, Tharwat Haj Khalil, Tali Voloshin, Alexandra Volodin, Lilach Koren, Bella Koltun, Anat Klein-Goldberg, Efrat Zemer-Tov, Rom Paz, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Macrophage pro-inflammatory phenotype skewing by the application of tumor treating fields (TTFields) [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 6650.

Inhibition of MALT1 and BCL2 induces synergistic anti-tumor activity in models of B cell lymphoma.

The activated B cell (ABC) subset of diffuse large B cell lymphoma (DLBCL) is characterized by chronic B cell receptor signaling and associated with poor outcomes when treated with standard therapy. In ABC-DLBCL, MALT1 is a core enzyme that is constitutively activated by stimulation of the B cell receptor or gain-of-function mutations in upstream components of the signaling pathway, making it an attractive therapeutic target. We discovered a novel small molecule inhibitor, ABBV-MALT1, that potently shuts down B cell signaling selectively in ABC-DLBCL preclinical models leading to potent cell growth and xenograft inhibition. We also identified a rational combination partner for ABBV-MALT1 in the BCL2 inhibitor, venetoclax, which when combined significantly synergizes to elicit deep and durable responses in preclinical models. This work highlights the potential of ABBV-MALT1 monotherapy and combination with venetoclax as effective treatment options for patients with ABC-DLBCL.

Abstract IA20: Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer

Abstract Oncogenic KRAS (KRAS*) is a signature mutation in pancreatic ductal adenocarcinoma (PDAC). While genetic extinction or pharmacological inhibition of KRAS* elicits anti-tumor responses in preclinical models and patients, we and others have identified cancer cell intrinsic (YAP, RTKs) and paracrine immune-mediated (TGFb) mechanisms enabling KRAS* bypass and tumor recurrence. Here, we explored the potential role of cancer associated fibroblasts (CAFs) as contributors to mechanisms driving KRAS* bypass and identified CAF-derived NRG1 activation of cancer cell ERBB2 and ERBB3 receptor tyrosine kinases as a mechanism by which KRAS* independent tumor regrowth is supported. We showed that genetic extinction or pharmacological inhibition of KRAS* results in upregulation of ERBB2 and ERBB3 expression in human and murine cancer cell models. We demonstrated that CAF-derived NRG1 supports KRAS*-dependent tumor growth. These insights prompted genetic depletion or pharmacological inhibitions studies targeting ERBB2/3 or NRG1, which showed abolition of KRAS* bypass and synergy with KRASG12D inhibitor in mouse and human PDAC models. Thus, CAFs via the NRG-ERBB2/3 paracrine axis provide yet another mechanism enabling KRAS* inhibitor therapy resistance, providing a highly actionable combination therapeutic strategy to improve the effectiveness of KRAS* inhibitors in PDAC patients. Citation Format: Jincheng Han, Jiaqian Xu, Yonghong Liu, Shaoheng Liang, Kyle A. LaBella, Deepavali Chakravarti, Denise J. Spring, Yan Xia, Ronald A. DePinho. Stromal-derived NRG1 enables oncogenic KRAS bypass in pancreas cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr IA20.

Donor-derived regulatory dendritic cell infusion modulates effector CD8+ T cell and NK cell responses after liver transplantation.

Immune cell-based therapies are promising strategies to facilitate immunosuppression withdrawal after organ transplantation. Regulatory dendritic cells (DCreg) are innate immune cells that down-regulate alloimmune responses in preclinical models. Here, we performed clinical monitoring and comprehensive assessment of peripheral and allograft tissue immune cell populations in DCreg-infused live-donor liver transplant (LDLT) recipients up to 12 months (M) after transplant. Thirteen patients were given a single infusion of donor-derived DCreg 1 week before transplant (STUDY) and were compared with 40 propensity-matched standard-of-care (SOC) patients. Donor-derived DCreg infusion was well tolerated in all STUDY patients. There were no differences in postoperative complications or biopsy-confirmed acute rejection compared with SOC patients up to 12M. DCreg administration was associated with lower frequencies of effector T-bet+Eomes+CD8+ T cells and CD16bright natural killer (NK) cells and an increase in putative tolerogenic CD141+CD163+ DCs compared with SOC at 12M. Antidonor proliferative capacity of interferon-γ+ (IFN-γ+) CD4+ and CD8+ T cells was lower compared with antithird party responses in STUDY participants, but not in SOC patients, at 12M. In addition, lower circulating concentrations of interleukin-12p40 (IL-12p40), IFN-γ, and CXCL10 were detected in STUDY participants compared with SOC patients at 12M. Analysis of 12M allograft biopsies revealed lower frequencies of graft-infiltrating CD8+ T cells, as well as attenuation of cytolytic TH1 effector genes and pathways among intragraft CD8+ T cells and NK cells, in DCreg-infused patients. These reductions may be conducive to reduced dependence on immunosuppressive drug therapy or immunosuppression withdrawal.

Combination of BET Inhibition with Radiation Induces Anti-Tumor Immune Response in Pre-Clinical Models of ER-Positive Breast Cancer

Despite advances in multidisciplinary care, the risk of late recurrence in Estrogen Receptor-positive (ER+) breast cancer (BC) patients following standard-of-care treatment ranges from 20-52% at 20 years. While immune checkpoint inhibitors (CPI), have shown enormous potential in inducing long term remission in some cases, patients with ER+ BC show poor response to these agents. The objective of this study is to develop effective radiation therapy (RT)-based combination regimens that induce anti-tumor immunity against immunologically cold ER+ breast tumors. We have previously shown that pharmacological inhibition of bromodomain and extraterminal domain (BET) family of epigenetic regulator proteins overcomes both endocrine therapy resistance and radiation resistance in pre-clinical models of ER+ BC by reversing pathological reprogramming of transcriptional and DNA repair pathways, respectively. In this study, we explore the potential of RT+BET inhibition combination to elicit anti-tumor immunity against ER+ BC. A unique ER+ BC model, MXT+, was used to generate tumors in female, BDF1 mice. PD-L1 expression following RT or RT+OTX015 (a BET inhibitor) was assessed by Western blot analysis. Comet assay was used to assess the degree of unrepaired DNA damage following treatment with RT alone, OTX015 alone and RT+OTX015. Multiplex IHC was used to assess changes in tumor microenvironment after various treatments. Tumor growth characteristics were established following treatment with vehicle, RT alone (8 Gy x 3), OTX015 alone (100 mg/kg x 3 days) and RT+OTX015. Using murine ER+ BC model, MXT+, we show that RT is a potent inducer of PD-L1 expression. Combination of RT+OTX015 completely ablated RT-mediated PD-L1 induction at the transcription level. OTX015 also accentuated RT-induced DNA damage through inhibition of NHEJ pathway, resulting in increased micronuclei formation (which activate cGAS/STING pathway, a critical bridge between innate and adaptive immunity). Combination of OTX015 with RT also caused a statistically significant (on unpaired t test) increase tumor infiltrating lymphocytes. In in vivo therapeutic studies, combination of OTX015 with RT (8 Gy x 3) resulted in extraordinary synergy and caused complete inhibition of long-term tumor growth. Treatment with RT alone (8 Gy x 3) or OTX015 alone (on 3 consecutive days) only caused modest inhibition of tumor growth. ANOVA with Dunnett's test was used to adjust for multiple comparisons and showed statistically significant difference in tumor growth between OTX015+RT arm and OTX alone as well as OTX015+RT arm and RT alone. Patients with ER+ BC face high risk of late recurrence after definitive treatment and show poor response to immune CPIs. We show that combination of BET inhibition with RT induces anti-tumor immunity and completely prevents tumor growth. Clinical validation of this regimen is warranted to prevent late recurrences in ER+ BC patients.

Turning up the heat: Oncolytic virus MEM-288 induces anti-tumoral immune response in pre-clinical models of high grade serous ovarian cancer (190)

Turning up the heat: Oncolytic virus MEM-288 induces anti-tumoral immune response in pre-clinical models of high grade serous ovarian cancer (190)

OA-09 Combination of forimtamig with standard of care improves depth and duration of response in preclinical models of multiple myeloma

OA-09 Combination of forimtamig with standard of care improves depth and duration of response in preclinical models of multiple myeloma

CLDN18.2 BiTE Engages Effector and Regulatory T Cells for Antitumor Immune Response in Preclinical Models of Pancreatic Cancer

BiTE (bispecific T-cell engager) immune therapy has demonstrated clinical activity in multiple tumor indications, but its influence in the tumor microenvironment remains unclear. CLDN18.2 is overexpressed in solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC), both of which are characterized by the presence of immunosuppressive cells, including regulatory T cells (Tregs) and few effector T cells (Teffs). We evaluated the activity of AMG 910, a CLDN18.2-targeted half-life extended (HLE) BiTE molecule, in GC and PDAC preclinical models and cocultured Tregs and Teffs in the presence of CLDN18.2-HLE-BiTE. AMG 910 induced potent, specific cytotoxicity in GC and PDAC cell lines. In GSU and SNU-620 GC xenograft models, AMG 910 engaged human CD3+ T cells with tumor cells, resulting in significant antitumor activity. AMG 910 monotherapy, in combination with a programmed death-1 (PD-1) inhibitor, suppressed tumor growth and enhanced survival in an orthotopic Panc4.14 PDAC model. Moreover, Treg infusion enhanced the antitumor efficacy of AMG 910 in the Panc4.14 model. In syngeneic KPC models of PDAC, treatment with a mouse surrogate CLDN18.2-HLE-BiTE (muCLDN18.2-HLE-BiTE) or the combination with an anti-PD-1 antibody significantly inhibited tumor growth. Tregs isolated from mice bearing KPC tumors that were treated with muCLDN18.2-HLE-BiTE showed decreased T cell suppressive activity and enhanced Teff cytotoxic activity, associated with increased production of type I cytokines and expression of Teff gene signatures. Our data suggest that BiTE molecule treatment converts Treg function from immunosuppressive to immune enhancing, leading to antitumor activity in immunologically "cold" tumors.

Targeting the Siglec-sialic acid axis promotes antitumor immune responses in preclinical models of glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain tumor, for which effective therapies are urgently needed. Cancer cells are capable of evading clearance by phagocytes such as microglia- and monocyte-derived cells through engaging tolerogenic programs. Here, we found that high expression of sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) correlates with reduced survival in patients with GBM. Using microglia- and monocyte-derived cell-specific knockouts of Siglec-E, the murine functional homolog of Siglec-9, together with single-cell RNA sequencing, we demonstrated that Siglec-E inhibits phagocytosis by these cells, thereby promoting immune evasion. Loss of Siglec-E on monocyte-derived cells further enhanced antigen cross-presentation and production of pro-inflammatory cytokines, which resulted in more efficient T cell priming. This bridging of innate and adaptive responses delayed tumor growth and resulted in prolonged survival in murine models of GBM. Furthermore, we showed the combinatorial activity of Siglec-E blockade and other immunotherapies demonstrating the potential for targeting Siglec-9 as a treatment for patients with GBM.

A Chemically Defined TLR3 Agonist with Anticancer Activity

ABSTRACT Toll-like receptor 3 (TLR3) agonists such as polyinosinic:polycytidylic acid (poly(I:C)) have immunostimulatory effects that can be taken advantage of to induce anticancer immune responses in preclinical models. In addition, poly(I:C) has been introduced into clinical trials to demonstrate its efficacy as an adjuvant and to enhance the immunogenicity of locally injected tumors, thus reverting resistance to PD-L1 blockade in melanoma patients. Here, we report the pharmacokinetic, pharmacodynamic, mechanistic and toxicological profile of a novel TLR3 agonist, TL-532, a chemically synthesized double-stranded RNA that is composed by blocks of poly(I:C) and poly(A:U) (polyadenylic – polyuridylic acid). In preclinical models, we show that TL-532 is bioavailable after parenteral injection, has an acceptable toxicological profile, and stimulates the production of multiple chemokines and interleukins that constitute pharmacodynamic markers of its immunostimulatory action. When given at a high dose, TL-532 monotherapy reduced the growth of bladder cancers growing on mice. In addition, in immunodeficient mice lacking formylpeptide receptor-1 (FPR1), TL-532 was able to restore the response of orthotopic subcutaneous fibrosarcoma to immunogenic chemotherapy. Altogether, these findings may encourage further development of TL-532 as an immunotherapeutic anticancer agent.

Prolonged cell cycle arrest by the CDK4/6 antagonist narazaciclib restores ibrutinib response in preclinical models of BTKi‐resistant mantle cell lymphoma

Introduction: Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of mantle cell lymphoma (MCL); however, primary and acquired resistance to these agents remains a challenge. Previous studies have suggested that narazaciclib (ON123300), a second-generation, orally bioavailable and clinical-stage CDK4/6 inhibitor (CDKi), may trigger cell cycle arrest and significant tumor growth inhibition (TGI) in BTKi-resistant MCL models. Methods: We compared the efficacy and safety of narazaciclib with the health authority approved CDKi in association with different BTKi, in a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib. We used the CellTiter-Glo proliferation assay, FACS-mediated quantification of cell cycle and apoptosis, and RNA sequencing followed by gene set enrichment analysis (GSEA), RT-PCR and western blot validations. In addition, we evaluated the safety and efficacy of narazaciclib/ibrutinib combo in vivo in an immune-competent chicken embryo chorioallantoic membrane (CAM) MCL xenograft. Results: Narazaciclib exhibited the highest antitumor activity among MCL cell lines (mean IC50: 3.61 ± 2.1 µM), regardless of their sensitivity to ibrutinib. Although there was no correlation between CDKi sensitivity and activation of the CDK4/CDK6-pRb pathway in MCL, transcriptomic and phenotypic analyses revealed a predominant downregulation of E2F target genes and G2/M checkpoint response (NES > 2.5) upon narazaciclib treatment. This feature was associated to intracellular accumulation of p21, p16, and phospho-p27, decreased mitotic index, G1 cell cycle blockade, and apoptosis onset. When combined with ibrutinib, but not with the second generation therapeutic acalabrutinib, narazaciclib achieved significant synergistic antitumor activity in both BTK-sensitive and BTK-resistant cells. The combination was not associated with improved apoptosis, but rather with a slight but constant (+10%–15%) augmentation in G1 phase blockade and the down-modulation of cell cycle-associated transcriptome. Both the downregulation of phospho-histone H3 and the upregulation of p-p27/p27 and p16, also underwent a 10%–15% improvement in combination-treated cells. In vivo, while narazaciclib single agent achieved a 28% TGI in the CAM model, the narazaciclib-ibrutinib combination reduced tumor spreading by 65% and allowed a 50% reduction in malignant B cell infiltration into the bone marrow, with no detectable toxicity. Conclusions: Narazaciclib, due to its completely distinct MoA from BTKi involving the direct modulation of the cell cycle, can achieve significant synergistic activity with ibrutinib in vitro and in vivo, especially in BTKi-resistant models of MCL. Ongoing phospho-proteomics and genetic edition assays will help deciphering the molecular bases of this unique drug cooperation at the cell cycle level. Encore Abstract - previously submitted to AACR 2023 The research was funded by: This study was financially supported by Onconova Therapeutics, Spanish Ministry of Science and Innovation (grant PID2021-123039OB-C21) (to GR). MFS holds a predoctoral fellowship (FI19/00338) from Instituto de Salud Carlos III. This work was carried out under the CERCA Program (Generalitat de Catalunya). Keywords: Aggressive B-cell non-Hodgkin lymphoma, Combination Therapies, Molecular Targeted Therapies Conflicts of interests pertinent to the abstract. A. Makovski-Silverstein Employment or leadership position: Onconova Therapeutics S. Cosenza Employment or leadership position: Onconova Therapeutics G. Roué Research funding: Onconova Therapeutics

Lefitolimod (TLR agonist) and ipilimumab in patients with advanced solid tumors: A phase I trial.

2564 Background: The TLR-9 agonist lefitolimod (MGN1703) significantly increases the Th1 response in pre-clinical models and has demonstrated antitumor activity in early phase clinical trials. This trial assessed the safety and preliminary efficacy of the rational combination of lefitolimod with ipilimumab in patients (pts) with advanced solid tumors. Methods: This was a single-center, open-label, investigator-initiated, dose-finding, and expansion Phase I trial (ClinicalTrials.gov no. NCT0266877). Key inclusion criteria were histologically confirmed advanced solid tumor, ECOG performance status ≤ 2; age ≥ 18 years. Patients (pts) were enrolled at escalating doses of lefitolimod (15-120 mg/kg), subcutaneously (SC) administered weekly and a fixed dose of ipilimumab 3 mg/kg in a 3-week cycle. For the expansion cohort, lefitolimod was administered intratumorally (IT) at 15 mg weekly. The primary endpoint was safety and tolerability according to CTCAE v4.0, dose-limiting toxicity (DLT) and maximum tolerated dose (MTD)/recommended Phase II dose (RP2D) for the lefitolimod in this combination. The secondary endpoint was efficacy, evaluated according to Immune-Related Response Criteria (irRC). Exploratory endpoints included changes in the intra-tumoral T-cell microenvironment in pre- and post- treatment tumor biopsies and the correlation of immune biomarkers with response. Results: From March 2017 to February 2020, 28 pts were enrolled. Median age was 56 years (range: 19-92). Median prior lines of therapy was 4 (range: 0-12). Pts 1-4 received lefitolimod 15 mg SC, pts 5-8 received lefitolimod 30 mg SC, pts 9-12 received lefitolimod 60 mg SC, pts 13-19 received lefitolimod 120 mg SC, and pts 20-28 received lefitolimod 15 mg IT. Eleven pts had at least one treatment-related AE (TRAE). The most common TRAE was skin rash (n=4, 14.2%), followed by fatigue (n=3, 10.7%) and pruritis (n=2, 7.1%). There were no grade 4 or 5 AEs, none of the pts required dose reduction, and there was no discontinuation of treatment due to AEs. DLT and MTD were not reached and the RP2D was established as lefitolimod SC 120 mg weekly with ipilimumab 3mg/Kg every 3 weeks. Of the 28 pts, 8 pts had stable disease as best response. 7 pts could not be evaluated by irRC due to clinical progression before restaging, non-measurable lesions and withdrawal of consent. Paired biopsy samples were analyzed by flow cytometry. Pro-inflammatory immune conditioning of the tumor microenvironment by the combination of the TLR9 agonist MGN1703 in combination with ipilimumab showed increases in intra-tumoral CD8 T cell frequency, memory CD8 phenotype (CD45RO+) and proliferation (Ki67+). Conclusions: The combination of high subcutaneous doses or intra-tumoral administration of lefitolimod in combination with ipilimumab is safe and well tolerated in patients with advanced cancers, with preliminary antitumor activity observed. Clinical trial information: NCT0266877 .

OncoVEXmGM-CSFexpands tumor antigen-specific CD8+ T-cell response in preclinical models

BackgroundCheckpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) have demonstrated clinical efficacy in advanced melanoma, but only a...

PAR2 activation in the dura causes acute behavioral responses and priming to glyceryl trinitrate in a mouse migraine model

BackgroundMigraine is a severely debilitating disorder that affects millions of people worldwide. Studies have indicated that activation of protease-activated receptor-2 (PAR2) in the dura mater causes headache responses in preclinical models. It is also well known that vasodilators such as nitric oxide (NO) donors can trigger migraine attacks in migraine patients but not controls. In the current study we examined whether activation of PAR2 in the dura causes priming to the NO donor glyceryl trinitrate (GTN).MethodsA preclinical behavioral model of migraine was used where stimuli (PAR2 agonists: 2at-LIGRL-NH2 (2AT) or neutrophil elastase (NE); and IL-6) were applied to the mouse dura through an injection made at the intersection of the lamdoidal and sagittal sutures on the skull. Following dural injection, periorbital von Frey thresholds and facial grimace responses were measured until their return to baseline. GTN was then given by intraperitoneal injection and periorbital hypersensitivity and facial grimace responses observed until they returned to baseline.ResultsWe found that application of the selective PAR2 agonist 2at-LIGRL-NH2 (2AT) onto the dura causes headache-related behavioral responses in WT but not PAR2−/− mice with no differences between sexes. Additionally, dural PAR2 activation with 2AT caused priming to GTN (1 mg/kg) at 14 days after primary dural stimulation. PAR2−/− mice showed no priming to GTN. We also tested behavioral responses to the endogenous protease neutrophil elastase, which can cleave and activate PAR2. Dural neutrophil elastase caused both acute responses and priming to GTN in WT but not PAR2−/− mice. Finally, we show that dural IL-6 causes acute responses and priming to GTN that is identical in WT and PAR2−/− mice, indicating that IL-6 does not act through PAR2 in this model.ConclusionsThese results indicate that PAR2 activation in the meninges can cause acute headache behavioral responses and priming to an NO donor, and support further exploration of PAR2 as a novel therapeutic target for migraine.

Abstract 1644: Dual inhibition of MAT2A and PRMT5 delivers synergistic anti-tumor responses in preclinical models of MTAP-deleted cancer

Abstract Approximately 15% of all tumors harbor deletions in the 9p21 locus that encompass CDKN2A and MTAP. MTAP fuels the adenine and methionine salvage pathways by converting MTA into precursors of building blocks for protein and DNA production. In MTAP-deleted (MTAPdel) tumors, cellular MTA accumulates and partially inhibits the essential protein methyl transferase, PRMT5, through direct displacement of the cofactor S-adenosyl methionine (SAM) from the PRMT5 active site. This context provides therapeutic opportunities, currently under clinical evaluation, to selectively extinguish PRMT5 activity in tumor cells with MAT2A inhibitors that limit SAM synthesis or with direct PRMT5 inhibitors that are MTA-cooperative. Preclinical modeling of these synthetic lethal relationships, and elucidation of their cellular mechanisms of action, suggests efficacy can vary depending upon the extent of MTA accumulation and/or adaptive compensation to PRMT5 inhibition. To potentially maximize therapeutic activity among mechanistically heterogenous MTAPdel tumors, we evaluated combinatorial pathway suppression with MAT2A and PRMT5 inhibitors in MTAPWT and MTAPdel tumor models in vitro and in vivo. We noted synergistic antiproliferative effects when the potent and selective MAT2A inhibitor IDE397 was combined with any of multiple MTA-cooperative PRMT5 inhibitors in MTAPdel cell lines that exhibit sensitivity to each single agent. In vivo, the combination of IDE397 and MTA-cooperative PRMT5 inhibitors were well tolerated, and induced durable tumor regressions, including complete responses, at dose levels well below the maximally efficacious preclinical dose of each individual agent in MTAPdel lung adenocarcinoma and pancreas cancer models H838 and BXPC3. Pharmacodynamic target engagement in the tumor was evaluated by quantitative assessment of symmetric dimethyl arginine (SDMA), as detected by IHC, and indicated earlier onset and greater extent of PRMT5 inhibition by combination treatment as compared to either agent alone. The potentiation of the antitumor response with the combination did not occur in the MTAPWT setting. Thus, combined inhibition of MAT2A and PRMT5 potentially offers a compelling dual synthetic lethal opportunity to address unmet need for the many patients afflicted with MTAPdel cancers. Citation Format: Marcus M. Fischer, Kimberline Gerrick, Brian Belmontes, Katherine Slemmons, Yevgeniy Freyman, Jay Jain, Steve Federowicz, Isaac Bishof, Arjun A. Rao, Melissa Fleury, Zineb Mounir, Mark R. Lackner, Paul E. Hughes, Mike White, Claire L. Neilan. Dual inhibition of MAT2A and PRMT5 delivers synergistic anti-tumor responses in preclinical models of MTAP-deleted cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1644.