MC38 Syngeneic Models Research Articles

Abstract 2665: Fc silencing of commonly used surrogate anti-PD-1 RMP1.14 increases its therapeutic profile in MC38 syngeneic model

Abstract Introduction: Anti-PD1 treatment has become a backbone treatment in several cancers such as melanoma and lung carcinoma. Adding new drugs to PD-1 requires careful evaluation of combination treatments in preclinical models to evaluate efficacy and tolerability. Syngeneic tumor model remains one of the best way of preclinical evaluation of combinations, as such evaluation requires fully immune competent animals to correctly assess both the PD-1 contribution and the added value or synergistic effect of new drugs in the combination. Anti mouse PD-1 such as RMP1.14, a rat IgG2a, is widely used in these models as anti-PD-1 mAb surrogate for such studies. Although RMP1.14 demonstrated efficacy in several models such as MC38, and CT26, it may not fully recapitulate the use of blocking mAb in the human as it may be immunogenic in mice and may retain some agonist activity due to its rat isotype. Here, we demonstrate that engineering this antibody with a mice Fc silent backbone increases its activity in MC38 model. Methods: VH/VL from RMP1.14 rat IgG2a (mPD1) were sequenced and engineered with a mouse Fc silent IgG1 backbone (N297Q) and produced in CHO. Both antibodies (mPD-1: rat IgG2a; and MOS2-mPD-1: Fc silent mouse IgG1) were purified on protein A using standard procedures. MC38 (1million cells) were injected subcutaneously in C57BL/6 mice, and randomized when tumor volume reached 100mm3. Dose effect of mPD-1 and MOS2-mPD-1 and their corresponding isotype controls were tested in this MC38 model at doses of 0.5; 1.25; 2.5 and 12.5 mg/kg at day 0, day 3, and day 7 post randomization (8 mice per group). Treatment route effect was tested in the same model by comparing intraperitoneal and intravenous injections in an additional experiment using both antibodies at 1.25 mg/kg and 12.5 mg/kg doses. Results: Both mPD-1 and MOS2-mPD-1 demonstrated efficacy in survival in MC38 model at all tested doses (p<0,05 Mantel-Cox test) compared to their isotype controls. MOS2-mPD-1 demonstrated increased efficacy in terms of complete responses in the above escalated doses (CR of 12.5, 12.5, 12.5 and 37.5 % and 0, 75, 50 and 62.5 % for mPD-1 and MOS2-mPD-1 respectively). Tumor volumes and survival were also always lower in the MOS2-mPD-1 groups compared to dose corresponding mPD-1 groups and were statistically different in two independent experiments at medium doses (P<0,05 Dunn’s test performed at 1.25mg/kg at day 27, and Mantel-Cox test). IP and IV routes were equivalent in efficacy at the tested doses. Conclusion: Mouse Fc engineered surrogate have significantly different behavior in mice models compared to the commonly used surrogates usually from rat origin. These new formatted antibodies should better mimick the efficacy and tolerability of antibodies used in human, and should be preferred when new combinations of treatments are tested. Citation Format: Ester Morgado, Laurent Pouyet, Olivier Deas, François Romagne. Fc silencing of commonly used surrogate anti-PD-1 RMP1.14 increases its therapeutic profile in MC38 syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2665.

Abstract 6662: Modified oncolytic viruses with immunomodulatory genes enhance anti-tumor efficacy

Abstract While chemotherapy and radiotherapy are considered first-line treatments for most cancers, their significant side effects have prompted ongoing innovation and the discovery of novel cancer treatment strategies. Oncolytic viruses (OV) as one of the most advanced cancer treatments have shown promising results in treating otherwise untreatable types of cancer in last two decades. After selectively targeting tumor cells, OVs could directly kill cancer cells, and induce a powerful anti-cancer immunity. Recently, we evaluated the anti-tumor effect of a modified OV (mOV) in vitro and in vivo. Firstly, the expression of the transgenes were validated on different tumor cells by FCM and ELISA assay. The results showed that a susceptible cell line was identified which has high expression efficiency of transgenes. In order to evaluate the cytotoxicity of mOV, CCK-8 assay was performed on 30+ tumor cell lines. It indicated that mOV has stronger killing effect on susceptible cells through inducing cellular apoptosis. To further evaluate the biological activity of the transgenes on immune cells, mOV-infected tumor cells and supernatant were collected to treat T cell, NK cell or MoDC. The results demonstrated that mOV infection could significantly promote T cell and NK cell activation, and increase the maturation of moDC cells, resulting in the secretion of IFN-γ by CD8+ T cells. In the in vivo efficacy study, MC38 syngeneic model was used to test the anti-tumor effect of mOV. The result showed that mOV could significantly inhibit the growth of the treatment side tumor, and reduce the contralateral tumor to a certain extent. Blood FCM analysis revealed that the percentage of IFN-γ+CD3+, IFN-γ+CD4+ and IFN-γ+CD8+ T cells were markedly increased by mOV treatment compared with vehicle. In addition, IHC analysis of tumor showed that mOV treatment could promote the infiltration of CD4+, CD8+ and CD86+ positive cells. To study the safety or PK of the mOV, viral bio-distribution and shedding assay were performed to detect viral genome at different tissues or excreta. It indicated that mOV was almost undetectable or at very low levels for 14 days after administration. In conclusion, our study revealed that modified OVs with immunomodulatory genes have good anti-tumor efficacy both in vitro and in vivo, which could obviously induce the activation of several immune cells. Moreover, the mOV genome was almost undetectable or at very low levels in mouse tissues and excreta. Keywords: Oncolytic virus, immunotherapy, immunomodulatory genes Citation Format: Zhongbao Song, Guangxu Ding, Anran Xia, Wanqing Ma, Qiuhong Li, Chenhui He, Xu Chen, Zhixiang Zhang, Qingyang Gu. Modified oncolytic viruses with immunomodulatory genes enhance anti-tumor 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 6662.

Abstract 4213: Multiplex digital spatial profiling of proteins and RNA in MC38 mouse colorectal cancer syngeneic model

Abstract Introduction Characterization of the tumor microenvironment (TME) has become an essential step for the understanding of the complexity of cell interactions and the discovery of biomarkers for prediction and prognosis of immunotherapy response1. The NanoString GeoMx® Digital Spatial Profiler (DSP) technology includes the ability to quantitatively assess RNA and protein levels as well as sample multiple user-defined regions of interest, specifically focused on defining heterogeneity and TME interactions2. This technology has been widely used to identify biomarkers in clinical surgical specimens, but its applications in preclinical models are still needed. MC38 colorectal cancer syngeneic model is one of the most utilized models for assessing anti-cancer immunotherapies. In this study, we present the spatial profiling of proteins and RNA on MC38 model treated with anti-PD-1 through GeoMx® DSP technology. Methods GeoMx protein panel was used to detect the target protein expression differential in tumor-rich region and immune-rich region within MC38 subcutaneous tumor sections treated. Mice were treated with vehicle or anti-PD-1 (10mg/kg, BIW x 4 doses, ip). Whole Transcriptome Atlas (WTA) was employed, and then a comprehensive assessment of the data and comparison was performed. Results Two morphology markers, CD45 and PanCK, were used to separate tumor-rich region and immune-rich region. The GeoMx® protein data showed that the abundance of VISTA in tumor-rich region differed significantly (log2(fold-change) = 0.67, p < 0.01) in anti-PD-1 group compared to vehicle group. While in immuno-rich region, GITR was significantly altered (log2(fold-change) = 0.79, p < 0.05) in anti-PD-1 group (TGI value = 50.39%). From WTA data, we observed the upregulation of 839 genes, while 1048 genes were downregulated in immuno-rich region in anti-PD-1 group compared with vehicle group (p < 0.05). For tumor-rich regions, 550 genes were upregulated while 782 genes were downregulated in anti-PD-1 group versus vehicle group (p < 0.05). From the gene set enrichment analysis, we found that interferon alpha and interferon gamma, which are related to tumor immunity and immunotherapy response, were upregulated in anti-PD-1 group. Conclusion Both the differential expressing genes and proteins in TME of MC38 syngeneic models treated with anti-PD-1 could be well detected by GeoMx® DSP, which has the potential to provide insightful data for use in biomarker discovery in preclinical cancer models. References Charmsaz S, Collins DM, Perry AS, Prencipe M. Novel Strategies for Cancer Treatment: Highlights From the 55th IACR Annual Conference. Cancers (Basel) 2019; 11(8). Toki MI, et al. High-plex predictive marker discovery for melanoma immunotherapy-treated patients using digital spatial profiling. Clin. Cancer Res. 2019;25:5503-5512. Citation Format: Meihui Chen, Hengyuan Liu, Xiaolong Tu, Sheng Guo, Xiaobo Chen. Multiplex digital spatial profiling of proteins and RNA in MC38 mouse colorectal cancer syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4213.

Abstract A034: VV-iVP55: A Superior Vaccinia Virus Platform with Enhanced Oncolytic Potency and Immunogenicity

Abstract This research introduces VV-iVP55, a conditionally replicating vaccinia virus platform, as a promising alternative to thymidine kinase (Tk) deleted oncolytic vaccinia viruses. The unique feature of VV-iVP55 is its growth control using doxycycline, a trait not found in conventional Tk-deleted viruses. Laboratory studies revealed that VV-iVP55 achieves higher virus titers and lower cell viability in B16-F10 and MC38 syngeneic models compared to Tk-deleted viruses. Notably, VV-iVP55 demonstrated lower toxicity in immunocompromised mice upon doxycycline withdrawal, unlike Tk-deleted viruses that showed significant toxicity. In vivo efficacy studies further highlighted the superiority of VV-iVP55 over Tk-deleted viruses in CT26 and B16 subcutaneous models, as well as MC38 subcutaneous and intraperitoneal models. An immune microenvironment analysis conducted six days post-intratumoral injection revealed beneficial alterations induced by VV-iVP55, such as increased T and B cell content in the tumor and reduced MDSCs in both the tumor and spleen. Moreover, VV-iVP55 was linked to a significant change in T cell differentiation. Unlike the PBS and Tk viruses, which showed almost complete differentiation towards effector T cells, VV-iVP55 maintained a substantial population of T cells in naive and transition phases, significantly in both CD4 and CD8 T cells. This likely led to lower expression of exhaustion markers PD1, TIM3, TIGIT, and LAG3, suggesting less exhaustion and more potential for immune response. In summary, VV-iVP55 emerges as a powerful alternative to Tk-deleted oncolytic vaccinia viruses, offering enhanced immunogenicity, controlled toxicity, and preserved oncolytic properties. Citation Format: Reza Rezaei, Taha Azad, Julia Petryk, John C. Bell. VV-iVP55: A Superior Vaccinia Virus Platform with Enhanced Oncolytic Potency and Immunogenicity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A034.

BGB-A445, a novel non-ligand-blocking agonistic anti-OX40 antibody, exhibits superior immune activation and antitumor effects in preclinical models.

OX40 is a costimulatory receptor that is expressed primarily on activated CD4+, CD8+, and regulatory T cells. The ligation of OX40 to its sole ligand OX40L potentiates T cell expansion, differentiation, and activation and also promotes dendritic cells to mature to enhance their cytokine production. Therefore, the use of agonistic anti-OX40 antibodies for cancer immunotherapy has gained great interest. However, most of the agonistic anti-OX40 antibodies in the clinic are OX40L-competitive and show limited efficacy. Here, we discovered that BGB-A445, a non-ligand-competitive agonistic anti-OX40 antibody currently under clinical investigation, induced optimal T cell activation without impairing dendritic cell function. In addition, BGB-A445 dose-dependently and significantly depleted regulatory T cells in vitro and in vivo via antibody-dependent cellular cytotoxicity. In the MC38 syngeneic model established in humanized OX40 knock-in mice, BGB-A445 demonstrated robust and dose-dependent antitumor efficacy, whereas the ligand-competitive anti-OX40 antibody showed antitumor efficacy characterized by a hook effect. Furthermore, BGB-A445 demonstrated a strong combination antitumor effect with an anti-PD-1 antibody. Taken together, our findings show that BGB-A445, which does not block OX40-OX40L interaction in contrast to clinical-stage anti-OX40 antibodies, shows superior immune-stimulating effects and antitumor efficacy and thus warrants further clinical investigation.

Abstract 5463: BPI-442096: A potent and selective inhibitor of SHP2 for the treatment of multiple cancers

Abstract Src homology region 2 domain-containing phosphatase-2 (SHP-2) is a key node in the RAS signaling pathway. Allosteric inhibition of SHP2 phosphatase is a potential therapeutic strategy for cancers harboring oncogenic mutations in the KRAS pathway. SHP2 also participates in the signal transduction downstream of regulatory immunoreceptors, and it has been shown in preclinical models that SHP2 inhibition drives anti-tumor immunity through modulation of both innate and adaptive mechanism. BPI-442096 is a potent, selective, and orally bioavailable small molecule SHP2 inhibitor. It exhibited significant anti-proliferation activities against multiple KRAS mutant cancer cell lines, including those from NSCLC, PDAC, CRPC, etc. BPI-442096 dose-dependently inhibited SHP2 phosphatase and downstream ERK phosphorylation in cancer cells, as well as NFAT reporter gene expression downstream of PD-1/PD-L1 signaling in immune cells. In vivo, BPI-442096 demonstrated strong tumor growth inhibition in KRASG12C, KRASG12D, and KRASG12V mutant xenograft mouse models. BPI-442096 also exhibited anti-tumor immunity in the MC38 syngeneic model, as a single agent or in combination with anti-PD1/PD-L1 drugs. Moreover, BPI-442096 combining with KRASG12C inhibitor may reverse intrinsic and acquired resistance to KRASG12C inhibition. Adequate oral exposure across multiple pre-clinical species and good ADME properties ensured the druggability of BPI-442096. In conclusion, BPI-442096 exhibits a robust anti-tumor effect in multiple KRAS mutant models and enhanced anti-cancer immunity in syngeneic mouse models, and it shows multiple combination potentials to overcome drug resistance. Phase 1 clinical trial is planned in early 2022. Citation Format: Ling Li, Bang Fu, Han Han, Zhongxin Sun, Xiangdong Zhao, Xuepeng Jv, Jun Tong, Jiayu Zhao, Zhengyao Zou, Haibo Chen, Xiaoyun Liu, Wei Ren, Yinlong Li, Wenmao Wu, Jing Guo, Dan Yan, Xiangyong Liu, Hong Lan, Hao Wu, Lieming Ding, Jiabing Wang. BPI-442096: A potent and selective inhibitor of SHP2 for the treatment of multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5463.

Abstract P103: The introduction of a single strain of Bacillus into a germ-free environment did not impact the anti-PD-1 efficacy in a MC38 syngeneic model

Abstract Background. Although immunotherapy has led to exceptional and durable clinical response, the majority of patients respond poorly to the current immunotherapies. Growing evidence has linked some of the poor responsiveness to the gut microbiota, and the modulation of gut microbiome composition is becoming a promising new strategy to enhance immune checkpoint inhibitor (ICI) treatment outcome. Mouse tumor modelling under germ-free (GF) conditions combined with introduction of defined bacterial strains could be a useful approach to investigate the impact of microbiota on ICI efficacy, as well as understanding the underlying mechanisms. We previously demonstrated that GF mice exhibited a significantly poor response to anti-PD-1 therapy when compared to the specific pathogen free (SPF) mice in a subcutaneous MC38 colorectal cancer model, which is consistent with other reports. Methods. Introduction of a single strain of Bacillus in the GF-environment was assessed for its impact on the anti-mouse PD-1 monoclonal antibody (mAb) therapy in GF-mice and SPF mice, both for efficacy and pharmacodynamics tumor infiltrating lymphocytes (TILs) profiling. C57BL/6 mice were inoculated subcutaneously with MC38 tumor cells and when the tumors were reached 80-120mm3, the mice were randomized for isotype or anti-PD-1 mAb treatment. Fecal sample 16S rRNA analysis (NGS) was used to confirm the gut bacteria status. Results. The MC38 tumor in GF mice has significantly fast baseline growth kinetics, even with the introduction of Bacillus under GF conditions compared to SPF mice, suggesting tumor immunity was not enhanced by Bacillus. Despite the introduction of Bacillus, GF mice also showed reduced response to anti-PD-1 treatment when compared to the SPF mice as previously reported, further confirming that introduction of Bacillus had minimal effects on the efficacy of anti-PD-1 therapy. Moreover, the SPF mice and GF mice with Bacillus exhibited distinct profiles of TILs, consistent with distinct efficacies as observed. GF free mice showed a lower frequency of CD45+ TILs in comparison to SPF mice. In addition, GF mice exhibited lower frequency of CD8+ TILs and TIL- NKT when compared to the SPF mice, both of which are consistent with the stronger efficacy seen in SPF mice. Meanwhile, GF mice also exhibited higher granulocytic myeloid derived suppressor cells (gMDSC) and lower M1/M2 ratio, both of which imply a more suppressive tumor microenvironment in GF mice. Fecal sample analysis using 16S rRNA analysis confirmed a single strain of Bacillus was indeed introduced into the guts of all the GF mice. Conclusions. The GF conditions provide a useful environment for the investigation of specific microbiota strains on the impact on ICI treatment outcome. In summary, the introduction of Bacillus in GF mice did not impact the efficacy of anti-PD-1, thus suggesting that other strain(s) of gut microbiota in SPF mice may impact this and need to be investigated. Citation Format: Tao Yang, Bonnie Xiaobo Chen, Rongfei Lu, Xiaoyu An, Mingfa Zang, Jingjun Li, Sheng Guo, Wubin Qian, Jian Fei, Tongyang Hao, Edward Xu, Henry Li. The introduction of a single strain of Bacillus into a germ-free environment did not impact the anti-PD-1 efficacy in a MC38 syngeneic model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P103.

Abstract LBA009: Orally available ENPP1 inhibitor, TXN10128, restores STING activation in tumor microenvironment and confers anti-tumor responses in combination with immune checkpoint blockade

Abstract Background Recently, there are growing needs of immune modulators that can convert cold tumors into hot tumors, which can be utilized for combination treatment with existing immune related therapies. An orally available small molecule that is capable of activating innate immune response can be an ideal candidate to meet those needs. Upon binding to 2’3’-cGAMP, STING activates TBK1-IRF3 signaling cascade in cancer cells as well as host cells and promotes innate immune responses against cancer cells, leading to T cell mediated anti-tumor immunity by facilitating T cell priming and infiltration. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), a transmembrane protein highly expressed in subset of cancer cells, has been known to hydrolyze 2’3’-cGAMP and negatively regulates the STING activation. Inhibition of ENPP1 can prevent 2’3’-cGAMP degradation in tumor microenvironment (TME) and restore STING activation, leading to innate immune responses. It has been shown that ENPP1 inhibitor can enhance anti-tumor immunity by restoring 2’3’-cGAMP level in TME and can prevent tumor growth when co-treated with immune checkpoint blockade (ICB) as well as with radiation therapy. Methods The activity of ENPP1 inhibitors were measured in enzymatic assay using 2’3’-cGAMP as substrate and in cellular STING activation assay using IRF3-responsive reporter. Also, the effects on innate immune response were estimated in LIN (Lymphocyte INfiltration) assay measuring lymphocyte infiltration and TMED (TME in Dish) assay measuring tumor spheroid growth and immune cell activation. In vivo efficacy of TXN10128 (PO dosing) was evaluated in MC38 syngeneic model in combination with and anti-PD-L1 (IV dosing). Tumor growth was monitored and immune cells in tumor were analyzed. Results TXN10128 inhibited ENPP1 activity with single digit nanomolar potency in enzyme assay and further induced STING activation in cellular assay. In 3D spheroid co-culture condition, TXN10128 enhances lymphocyte infiltration and inhibits the spheroid growth. TXN10128 has a drug-like properties desirable for oral administration in terms of physicochemical properties and pharmacokinetics parameters. Systemic exposure of TXN10128 by PO dosing resulted in synergistic tumor growth inhibition with anti-PD-L1 antibody and improved tumor-infiltrating lymphocytes (TIL) profile as expected in MC38 syngeneic model. Additional in vivo studies are on-going to expand applicable cancer types to TXN10128 treatment. Conclusions TXN10128 is a potent and selective ENPP1 inhibitor that can exert immune response in 3D co-culture condition, which is consistent with tumor growth inhibition and favorable TIL profile in animal model. Together with promising drug-likeness, these studies demonstrate that TXN10128 is a suitable candidate for clinical investigation as a combination partner with existing immunotherapies. Citation Format: Sungjoon Kim, Imran Ali, Ahran Yu, Sun woo Lee, Sung young Park, Jung hwan Choi, Yong-yea Park, Chan sun Park. Orally available ENPP1 inhibitor, TXN10128, restores STING activation in tumor microenvironment and confers anti-tumor responses in combination with immune checkpoint blockade [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA009.

Abstract 1586: Oncolytic virus HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 promotes systemic anti-tumor responses and cooperates with immuno-modulatory agents in multiple mouse syngeneic tumor models

Abstract Oncolytic virotherapy is an emerging treatment modality that may hold promise as a therapeutic option for cancer patients who do not respond to checkpoint inhibitors. Oncolytic viruses preferentially replicate in tumor cells, offer the ability to express transgenes and have been shown to modify the local and distant tumor microenvironments from immunosuppressive to immunostimulatory. We generated HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12, an oncolytic virus based on a modified herpes simplex virus type 1 (HSV-1) designed to selectively replicate in tumors and produce murine FMS-like tyrosine kinase 3 ligand (mFLT3L) to enhance dendritic cell expansion and T cell priming, and murine interleukin-12 (mIL12) to potentiate type I helper and cytotoxic T cell responses. We confirmed the expression and bioactivity of mFLT3L and mIL12 in vitro and evaluated the efficacy of HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 alone and in combination with anti-PD-1 or anti-4-1BB treatment in multiple syngeneic mouse tumor models. Bilateral tumor models were used and only one tumor was treated via intratumoral injection with HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12. Anti-tumor responses were evaluated in the injected tumor (local response), the uninjected tumor (abscopal) and via splenic immune profiling (systemic). HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 treatment alone led to tumor growth inhibition in both treated and contralateral tumors, and a significant increase in overall survival in the A20, Neuro2a and MC38 models. HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 in combination with anti-PD-1 blocking antibody further improved tumor growth inhibition in both treated and contralateral tumors in the MC38 syngeneic model, and significantly increased median overall survival compared to either agent as a monotherapy. In addition, HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 in combination with an agonistic antibody targeting 4-1BB significantly increased overall survival compared to either agent as a monotherapy in the MC38 model. To understand antigen-specific anti-tumor T cell responses, MC38 tumor antigen epitopes were identified by combining exome sequencing and MHC/HLA-binding prediction algorithms. Antigen-specific T cell responses were measured in an IFN-γ ELISPOT assay using splenocytes from treated mice. HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 induced and/or enhanced systemic T cell responses directed against both tumor neoantigens and tumor-associated antigens. Together, these data reveal that treatment with HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 can cause direct tumor lysis and induce an adaptive, systemic T cell-mediated anti-tumor immune response that can be further enhanced by concurrent PD-1 blockade or 4-1BB agonism in multiple syngeneic mouse tumor models. Citation Format: Keegan Cooke, Walter H. Meisen, Petia Mitchell, Juan Estrada, Jinghui Zhan, Jessica Orf, Peng Li, Christina de Zafra, Jing Qing, Jason DeVoss. Oncolytic virus HSV-1/ICP34.5-/ICP47-/mFLT3L/mIL12 promotes systemic anti-tumor responses and cooperates with immuno-modulatory agents in multiple mouse syngeneic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1586.

Abstract 1649: Mechanistic understanding of HPK1 inhibition on enhanced human T cell activation and tumor immunity in a syngeneic model

Abstract Background: HPK1, a member of the MAP4K family of protein serine/threonine kinases, is involved in negatively regulating signal transduction cascades in cells of hematopoietic origin. Recent data generated using potent and highly selective HPK1 inhibitors underscores the role of HPK1 in negatively regulating T, B, and dendritic cell activation and thus orchestrating a comprehensive anti-tumor immune response. In the present study, we seek to further understand the biological effects of HPK1 inhibition in primary human T cells and in syngeneic models. Methods: RNA sequencing and multiplex cytokine analyses were utilized to profile both primary human T cells and the MC38 syngeneic model. In vivo efficacy, target engagement and pharmacodynamic data were generated using murine syngeneic tumor models. Results: In vitro, HPK1 small molecule inhibition resulted in enhanced cytokine production in primary human T cells. In vivo, HPK1 inhibition abrogated T cell receptor-stimulated phospho-SLP-76, enhanced cytokine production, and mediated robust tumor growth inhibition in a murine syngeneic tumor model. Conclusion: Pharmacological blockade of HPK1 kinase activity represents a novel and potentially valuable immunomodulatory approach for anti-tumor immunity. Citation Format: David Ciccone, Vad Lazari, Ian Linney, Michael Briggs, Samantha Carreiro, Christine Loh, Peter Tummino, Joshua McElwee, Alan Collis, Neelu Kaila. Mechanistic understanding of HPK1 inhibition on enhanced human T cell activation and tumor immunity in a syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1649.

Abstract 1444: TPX-0022, a potent MET/SRC/CSF1R inhibitor that modulates the tumor immune microenvironment in preclinical models

Abstract Within the tumor microenvironment (TME), CSF1R (colony stimulating factor 1 receptor) signaling plays a pivotal role in recruitment and enrichment of M2 tumor-associated macrophages (TAM) that dampen the anti-tumor immune response and promote tumor progression. Although CSF1R inhibitors have shown promise in the clinic, tumor cells and TAMs can bypass CSF1R inhibition and expand M2 TAMs through other growth factor receptors, such as MET (hepatocyte growth factor receptor) to confer resistance to targeted therapies and immune checkpoint inhibitors. Through autocrine signaling pathways, CSF1R and MET activation in tumors contribute to tumor invasiveness and metastasis. Therefore, inhibitors that target both CSF1R and MET, such as TPX-0022 (a compact macrocyclic inhibitor of MET/SRC/CSF1R), may more effectively suppress CSF1R mediated signaling in tumor cells and the stroma. In the current study, the role of TPX-0022 in the anti-tumor immune response is evaluated in the context of both normal immune cells and immunocompetent syngeneic murine models. TPX-0022 blocks both HGF and CSF1 mediated signaling and proliferation of MET and CSF1R dependent cell lines. In the MC-38 syngeneic model, TPX-0022 treatment repolarized TAMs towards M1 anti-tumorigenic macrophages, increased CD8 T cells, and inhibited tumor growth. In a mixed lymphocyte reaction assay, we demonstrated that pretreatment of macrophages with TPX-0022 enhances macrophage function in driving the activation and proliferation of CD8 T cells. Furthermore, TPX-0022 treatment of anti-C3/CD28 and or SIINFEKL peptide stimulated murine T cells had no deleterious effects on T cell activation and proliferation. Additionally, TPX-0022 enhanced T cell cytotoxic function towards cancer cells in a dose-dependent manner. Detailed analysis of the TME in CT-26 animals treated with TPX-0022 showed a significant reduction in TAMs, rebalancing both immunosuppressive Treg and M2 macrophages towards anti-tumorigenic CTL (CD8+ CD69+ GnzB+) and M1 macrophages. The combination of TPX-0022 and a PD-1 antibody resulted in significant downregulation of pro-tumorigenic cytokines IL-10, IP-10, IL-1β, and HGF, without impacting the anti-tumor cytokine IFN-γ. In the MC-38 model, TPX-0022 demonstrated single-agent activity and a trend to enhanced efficacy in combination with a PD-1 antibody. Taken together, the preclinical data demonstrates that the MET/SRC/CSF1R inhibitor TPX-0022 can reprogram tumor associated macrophages as well as support antigen presentation and activation of T cells within the TME. A TPX-0022 combination with a PD-1 therapy yielded more anti-tumor activity than single agent treatments. Together, the in vitro and in vivo preclinical data provide a mechanistic rationale for the potential utility of TPX-0022 in combination with immune checkpoint inhibitors. Citation Format: Vikas K. Goel, Wei Deng, Dayong zhai, Nathan V. Lee, Ana Parra, Brion W. Murray. TPX-0022, a potent MET/SRC/CSF1R inhibitor that modulates the tumor immune microenvironment in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1444.

Macrophage Activation Status Rather than Repolarization Is Associated with Enhanced Checkpoint Activity in Combination with PI3Kγ Inhibition.

Suppressive myeloid cells mediate resistance to immune checkpoint blockade. PI3Kγ inhibition can target suppressive macrophages, and enhance efficacy of immune checkpoint inhibitors. However, how PI3Kγ inhibitors function in different tumor microenvironments (TME) to activate specific immune cells is underexplored. The effect of the novel PI3Kγ inhibitor AZD3458 was assessed in preclinical models. AZD3458 enhanced antitumor activity of immune checkpoint inhibitors in 4T1, CT26, and MC38 syngeneic models, increasing CD8+ T-cell activation status. Immune and TME biomarker analysis of MC38 tumors revealed that AZD3458 monotherapy or combination treatment did not repolarize the phenotype of tumor-associated macrophage cells but induced gene signatures associated with LPS and type II INF activation. The activation biomarkers were present across tumor macrophages that appear phenotypically heterogenous. AZD3458 alone or in combination with PD-1-blocking antibodies promoted an increase in antigen-presenting (MHCII+) and cytotoxic (iNOS+)-activated macrophages, as well as dendritic cell activation. AZD3458 reduced IL-10 secretion and signaling in primary human macrophages and murine tumor-associated macrophages, but did not strongly regulate IL-12 as observed in other studies. Therefore, rather than polarizing tumor macrophages, PI3Kγ inhibition with AZD3458 promotes a cytotoxic switch of macrophages into antigen-presenting activated macrophages, resulting in CD8 T-cell-mediated antitumor activity with immune checkpoint inhibitors associated with tumor and peripheral immune activation.

Enhanced antitumor immunity by a novel small molecule HPK1 inhibitor

BackgroundHematopoietic progenitor kinase 1 (HPK1 or MAP4K1) has been demonstrated as a negative intracellular immune checkpoint in mediating antitumor immunity in studies with HPK1 knockout and kinase dead mice. Pharmacological...

Abstract 2219: OnxoVEXmGM-CSF promotes systemic response to PD-L1 /PD-1 blockade in multiple mouse syngeneic tumor models

Abstract Inhibition of the immune-checkpoint axis that involves PD-L1/PD-1 has improved responses and outcomes for certain cancer patients, but there remains unmet need as only a fraction of patients respond to these therapies. Talimogene laherparepvec (T-VEC) is an FDA-approved, first-in-class oncolytic immunotherapy based on a modified herpes simplex virus type 1 (HSV-1) designed to selectively replicate in tumors and produce granulocyte-macrophage stimulating factor (GM-CSF) to enhance a tumor-antigen specific adaptive immune response. Intratumoral treatment with T-VEC has been shown to promote an inflammatory tumor microenvironment both preclinically and clinically. Combining T-VEC with immune checkpoint inhibitors is of high interest to potentially broaden and deepen the clinical responses in checkpoint-resistant tumors. In the current study, we evaluated the efficacy of OncoVEXmGM-CSF (an HSV modified like T-VEC, except that it produces murine GM-CSF) in combination with anti-PD-1/PD-L1 treatment in multiple syngeneic mouse tumor models. Bilateral tumor models were used, where only one tumor was treated with OncoVEXmGM-CSF (representing the local oncolytic effect), allowing the evaluation of systemic abscopal effect in the uninjected contralateral tumors. OncoVEXmGM-CSF in combination with anti-PD-L1/PD-1 enhanced tumor growth inhibition in both treated and contralateral tumors in the A20 and MC38 syngeneic models and overcame anti-PD-L1 resistance in the CT26 model. The combination treatment also led to a significant increase in median overall survival compared to either agent as a monotherapy. In addition, tumor antigen epitopes were identified by combining exome sequencing and MHC/HLA-binding prediction algorithms. Antigen-specific T cell response was measured in an IFN-γ ELISPOT assay using splenocytes from treated mice. OncoVEXmGM-CSF induced and /or enhanced systemic T cell responses directed against both tumor neoantigens and tumor-associated antigens. Together, these data reveal that OncoVEXmGM-CSF treatment can cause direct tumor lysis along with the potentiation of an adaptive, systemic T cell-mediated anti-tumor immune response that can be enhanced by the addition of PD-L1/PD-1. Our study provides a strong rationale for the ongoing clinical evaluation of the combination of T-VEC and PD1-/PD-L1-targeted therapy in cancer patients. Citation Format: Keegan Cooke, Juan Estrada, Petia Mitchell, Jinghiu Zhan, Pedro J. Beltran, Jing Qing, Jude Canon. OnxoVEXmGM-CSF promotes systemic response to PD-L1 /PD-1 blockade in multiple mouse syngeneic tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2219.

Abstract 2205: CBT-102, an oral small molecule multi-kinase inhibitor, demonstrates favorable CSF-1R activity, offering means of controlling tumor associated macrophages

Abstract Background: The role of the tumor microenvironment (TME) in fostering the development of malignancies is prompting the pursuit of anticancer therapies that target its components as to opposed to the tumor itself. As part of their immune surveillance duties, immune cells form part of this microenvironment, and yet, cancer cells have devised means to downplay their tumoricidal capabilities. Colony stimulating factor 1 receptor (CSF-1R) may offer such a means of controlling tumor associated macrophages in the tumor microenvironment. Methods: The IC50 of CBT-102 against CSF1R was evaluated in both cell free system and cell based assays, where enzyme activity was evaluated with radiometric assay format, and cell growth inhibition was evaluated in the Ba/F3 hCSF1R cell line by the CellTiter-Glo (CTG) method. Further evaluation of CBT-102 IC50 was undertaken in M-NFS-60 syngeneic cell lines and human monocytes. In vivo studies of CBT-102 in combination with anti-PD1 antibody were performed in H22 and MC38 syngeneic models. Results: CBT-102 demonstrated a reproducible activity against CSF1R in a radiometric enzyme activity assay. Using engineered BaF3 hCSF1R cell lines, we demonstrated the IC50 value of CBT-102 was 0.588 µM compared to 1.333 µM of sulfatinib, a similar multi-kinase inhibitor, and 0.279 µM of GW2580, a more specific CSF1R kinase inhibitor. Similar studies conducted in M-NFS-60 cell lines and human monocyte also showed sub-micromolar activities of CBT-102. In vivo study combining CBT-102 with anti-PD1 antibody in syngeneic models demonstrated a favorable combination effect compared to each of the single agents. Conclusions: We demonstrated previously that CBT-102 effectively inhibits VEGFR and angiogenesis. The newly revealed mechanism of CBT-102, inhibiting CSF1R and macrophages, offers a promising dual mechanism of action in addition to targeting VEGFR and angiogenesis. Rational combination with check-point inhibitors (CPIs) may improve the efficacy of CBT-102 and broaden the impact of CPIs. Further studies may be needed to delineate the interplay among CBT-102’s different mechanisms and its impact in combination with CPIs. Citation Format: Elaine Liu, Lan Yang, Gavin Choy, Xiaoling Zhang, Tillman Pearce, Mamatha Reddy, Sanjeev Redkar, Qian Shi. CBT-102, an oral small molecule multi-kinase inhibitor, demonstrates favorable CSF-1R activity, offering means of controlling tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2205.

Abstract 4559: KY-B602, a humanized anti-human OX40 antibody, enhances CD4+T cell activation and inhibits tumor growth in a syngeneic model

Abstract OX40, a member of the tumor necrosis factor (TNF) receptor superfamily, is mainly expressed on activated T cells. As a secondary costimulatory molecule, activated OX40 can increase effector T-cell survival, proliferation, and memory. Anti-OX40 monoclonal antibodies have shown anti-tumor efficacy. Recent preclinical studies further support the potential synergistic effects of agonist OX40 immunotherapy in combination with cancer vaccination or in sequential co-administration with PD1 antibody to revert PD1 resistance. Several anti-OX40 agonistic monoclonal antibodies are currently tested in early phase cancer clinical trials either in monotherapy or in combination with other immune modulators. KY-B602 is a novel humanized IgG1 anti-OX40 antibody under pre-clinical development. It was generated from hybridoma and humanized by CDR grafting and structure simulation. KY-B602 specifically binds to the extracellular domain of human OX40 with high affinity (KD = 5.1 nM, determined by Biacore). FACS study showed KY-B602 also binds to both human and cynomolgus monkey OX40 over-expressed on 293T cells (EC50 =4.6nM, EC50=6.1nM, respectively). Another, in HT1080 cell reporter assay, KY-B602 significantly increased IL-8 secretion through OX40 pathway. In addition, KY-B602 dose-dependently augmented IL-2 and IFN-gamma secretion from CD4+T cells, that were pre-stimulated with IL-2 and PHA. However, in FACS competition assay, KY-B602 did not affect OX40L binding to OX40, suggesting that KY-B602 binds to a different epitope with that of OX40L. Moreover, KY-B602 inhibits in vivo tumor growth in MC38 syngeneic model. In summary, the current data of KY-B602 support its potency for cancer therapy. Citation Format: feng hao, feng he, mingxuan ning, zhaoshuai bai, jinying ning. KY-B602, a humanized anti-human OX40 antibody, enhances CD4+T cell activation and inhibits tumor growth in a syngeneic model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4559.

Abstract LB-218: Development of Axl/Mer inhibitor, ONO-9330547: preclinical evidence supporting the combination with immunotherapeutics

Abstract Purpose: Recent clinical studies have demonstrated that immunotherapeutics, such as checkpoint inhibitors are a promising approach to the treatment of cancer; however, strategies to improve its clinical efficacy are still required. Axl and Mer, member of the TAM receptor tyrosine kinase family are known to be over-expressed in various types of hematological and solid tumor cancers and have been reported as poor prognostic factors. ONO-9330547 is a highly potent and selective oral Axl/Mer inhibitor. Previous study with ONO-9330547 demonstrated both increase proportion of CD8+ T cell and alternation of immunosuppressive microenvironment in syngeneic tumor graft model (Tanaka AACR 2015). Thus, we next went on to examine whether combining ONO-9330547 with anti-PD1 antibody has the potential to improve efficacy in a mouse colon cancer MC38 syngeneic model. Methods: MC38 tumor cells, a mouse colon cancer cell line, were implanted into female C57/B6 mice. Randomization of mice occurred when mean tumor volume reached 150 mm3. ONO-9330547 was fed in a diet containing 0.0013 or 0.013% ONO-9330547 (equivalent to 2 or 20 mg/kg/day). Anti-mouse PD-1 Ab (4H2) was administered intraperitoneally at a dose of 10 mg/kg every 6 days for 28 days. Tumor volumes were determined using the formula volume ( = width2 x length)/2. Animals were euthanized when the tumors reached a maximum volume of 2,000 mm3. QPCR was used to determine the TAM ligands, Gas6 and Pros1. Results: Treatment with ONO-9330547 combined with 4H2 resulted in a significantly improved inhibition of tumor growth in the MC38 syngeneic model. In contrast, treatment of 4H2 monotherapy showed inferior anti-tumor activity compared with combination therapy. In particular, ONO-9330547 at 2 or 20 mg/kg/day combined with 4H2 resulted in tumor remission in 3/9 or 6/9 animals at day 28, respectively, whereas each monotherapy resulted in tumor remission in only 1/9 animals. Interestingly, the treatment with 4H2 significantly increased TAM ligands mRNA levels, Gas6 and Pros1 in tumor. Conclusion: Our results demonstrate that ONO-9330547, a highly potent dual Axl/Mer inhibitor, shows synergistic anti-tumor effect when combined with anti-PD-1 antibody which induces the expression of TAM ligands. These data support the concept that the immunomudulatory effect of ONO-9330547 is further enhanced when ONO-9330547 is combined with immunotherapy. Rational combination with immunotherapeutics is worth further investigation in the clinical setting. Citation Format: Toshio Yoshizawa, Kohei Tanaka, Tomoko Yasuhiro, Ryu Fujikawa, Suiho Ri, Kazuhito Kawabata. Development of Axl/Mer inhibitor, ONO-9330547: preclinical evidence supporting the combination with immunotherapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-218.