MC38 Tumor-bearing Mice Research Articles

Abstract 6013: TST005, a bifunctional fusion protein of PD-L1/TGF-βRII, demonstrates potent anti-tumor activities with good safety profiles

Abstract Antibodies targeting PD1/PD-L1 are emerging as effective cancer immunotherapies, however, not all patients with PD-L1 expression respond to the treatment. A growing body of data demonstrates the pivotal roles of the master regulators from the tumor microenvironment (TME), such as transforming growth factor β (TGF-β), in the development of resistance to Immune checkpoint inhibitors. Therefore, co-inhibition of TGF-β signaling is expected to enhance the antitumor responses of PD1/PD-L1-based immunotherapies. TST005 is a bifunctional fusion protein composed of the truncated extracellular domain of the TGF-βRII receptor (a TGF-β trap) fused to a humanized anti-PD-L1 IgG1 antibody with ablated Fc immune effector function. Previously we showed the potent antitumor activity of TST005 in MC38/hPD-L1 and EMT6/hPD-L1 xenograft models. Here we report TST005 anti-tumor activities in MC38 colorectal cancer and EMT-6 breast cancer models compared to M7824 (Merck’s PD-L1/TGF-βRII) analog and its safety profiles following single or repeated doses in rats and non-human primates (NHP). TGF-β1depletion from the plasma of MC38 tumor-bearing mice was observed even at the lowest dose (1mg/kg). At higher dose (10mg/kg), TGF-β1 depletion persisted longer. The same trend was also found in MC38 tumor tissue. In EMT-6 tumor model upon TST005 treatment (30mg/kg), TST005 showed better tumor inhibition compared to M7824 analog at high dose (30mg/kg), which correlates well with the extent of TGF-β1depletion. For TST005, TGF-β1depletion was persistent throughout all time points tested (up to Day 14) in plasma and tumor tissue compared to M7824 analog at the same dose/dosing frequency. TST005 treatment (3mg/kg,10mg/kg) significantly lowered phosphorylated SMAD2 (pSMAD2) in MC38 tumors. TST005 was well tolerated in both rats and monkeys, no significant safety issues were observed following single or repeated doses in general toxicity studies and safety pharmacology assessment, the exposure of TST005 in both species was comparable between two genders and increased in an approximately dose-proportional manner. No cytokine release was observed in monkeys. Based on the exposure of TST005 in monkeys and predicted exposure at First in Human (FIH) dose, the safety margin of TST005 will be higher than 200 and 500 folds calculated on Cmax and AUC respectively. In conclusion, we have demonstrated the antitumor activity of TST005 in PD/PD-L1 sensitive and resistant tumor models as well as the safety profile in NHP, TST005 has been granted for phase 1 clinical trials in USA (NCT04958434). Citation Format: Huanhuan Guo, Xinlai Yao, Chunming Wang, Linlin Mao, Hongjun Li, Shuang Lu, Di Sun, Chengwei Jiang, Shenjie Zhang, Lisa Zheng, Fan Zhang, Fei Teng, Steven Yu, Yi Gu, Xueming Qian. TST005, a bifunctional fusion protein of PD-L1/TGF-βRII, demonstrates potent anti-tumor activities with good safety profiles [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 6013.

M1-derived extracellular vesicles enhance photodynamic therapy and promote immunological memory in preclinical models of colon cancer

Extracellular vesicles (EVs) are promising drug carriers of photosensitizers for photodynamic therapy (PDT) in cancer treatment, due to their ability to circulate in blood and enter cells efficiently. The therapeutic potential of EVs has been suggested to depend on the type and physiological state of their cell of origin. However, the effects of deriving EVs from various cells in different physiological states on their antitumor capacity are rarely evaluated. In the present study, we compared the antitumor efficacy of EV-mediated PDT by incorporating the photosensitizer Zinc Phthalocyanine (ZnPc) into EVs from multiple cells sources. ZnPc was incorporated by a direct incubation strategy into EVs derived from immune cells (M1-like macrophages and M2-like macrophages), cancer cells (B16F10 melanoma cancer cells) and external sources (milk). Our data show that all EVs are suitable carriers for ZnPc and enable efficient PDT in vitro in co-culture models and in vivo. We observed that EV-mediated PDT initiates immunogenic cell death through the release and exposure of damage associated molecular patterns (DAMPs) on cancer cells, which subsequently induced dendritic cell (DC) maturation. Importantly, of all ZnPc-EVs tested, in absence of light only M1-ZnPc displayed toxicity to MC38, but not to DC, in monoculture and in co-culture, indicating specificity for cancer over immune cells. In MC38 tumor-bearing mice, only M1-ZnPc induced a tumor growth delay compared to control in absence of light. Interestingly, M1- but not M2-mediated PDT, induced complete responses against MC38 tumors in murine models (100% versus 38% of cases, respectively), with survival of all animals up to at least 60 days post inoculation. Finally, we show that all cured animals are protected from a rechallenge with MC38 cells, suggesting the induction of immunological memory after EV-mediated PDT. Together, our data show the importance of the cell type from which the EVs are obtained and highlight the impact of the immunological state of these cells on the antitumor efficacy of EV-mediated PDT.Graphical

Photodynamic Therapy in Combination with the Hepatitis B Core Virus-like Particles (HBc VLPs) to Prime Anticancer Immunity for Colorectal Cancer Treatment.

Simple SummaryPhotodynamic therapy (PDT) by means of a photosensitizer is a clinically used therapeutic treatment in a variety of cancers. To further improve the anti-cancer efficiency of PDT, combination therapy with immune agents is a promising option. In this study, we used a viral vaccine as the immune therapeutic partner for PDT. We studied the biological properties of single and combined modalities. Our research suggests that combination therapy enhances innate and humoral immunity, improved survival, and generated a long-term memory capacity in the MC-38 murine colorectal tumor model to prevent a recurrence.Photodynamic therapy (PDT), which combines light and oxygen with a photosensitizer to induce reactive oxygen species (ROS)-mediated killing of primary tumor cells, benefits from non-invasive properties and its negligible toxicity to surrounding healthy tissues. In this study, we have shown that the second-generation photosensitizer FOSCAN can be internalized by tumor cells and effectively induce tumor cell death when exposed to laser irradiation in vitro. In addition, these dying tumor cells can be phagocytosed by dendritic cells and lead to their activation and maturation as assessed by in vitro co-culture models. While PDT induces immunogenic tumor cell apoptosis, its application for the treatment of tumors located in deep tissues and advanced malignancies has been limited. In this study, we demonstrate that hepatitis B core virus-like particles (HBc VLPs) can serve as a vaccine to enhance PDT-induced anti-cancer immunity by priming humoral immune responses and inducing CD8+ T cell responses. The combination of PDT and HBc VLPs increased the survival rate of MC-38 tumor-bearing mice to 55%, compared to 33% in PDT alone and no tumor-free mice in vaccine alone. Moreover, the combination effectively prevented tumor recurrence in vivo through enhanced immune memory T cells after therapy. Therefore, as both are clinically approved techniques, this combination provides a promising strategy for cancer therapy.

AHCC®, a Standardized Extract of Cultured Lentinula Edodes Mycelia, Promotes the Anti-Tumor Effect of Dual Immune Checkpoint Blockade Effect in Murine Colon Cancer.

Treatment strategies combining immune checkpoint blockade (ICB) with other agents have emerged as a promising approach in the treatment of cancers. AHCC®, a standardized extract of cultured Lentinula edodes mycelia, has been reported to inhibit tumor growth and enhance immune cell function. Here we investigated whether AHCC® promotes the therapeutic effect of immunotherapy in cancers. A combination of oral AHCC® and dual immune checkpoint blockade (DICB), including PD-1/CTLA-4 blockade, had reduced tumor growth and increased granzyme B and Ki-67 expression by tumor-infiltrating CD8+ T cells in MC38 colon cancer bearing mice compared to a combination of water and DICB. In the same tumor bearing mice, AHCC® and DICB treatment also altered the composition of the gut microbiome with the increased abundance of the species of Ruminococcaceae family which is associated with increased therapeutic efficacy of immunotherapy. The anti-tumor effect of AHCC® and DICB was not found in MC38 tumor-bearing mice treated with antibiotics. These data suggest that AHCC® increases the anti-tumor effect of DICB by enhancing T cell function and affecting the gut microbiome.

Neoantigen cancer vaccine augments anti-CTLA-4 efficacy

Immune checkpoint inhibitors (ICI) based on anti-CTLA-4 (αCTLA-4) and anti-PD1 (αPD1) are being tested in combination with different therapeutic approaches including other immunotherapies such as neoantigen cancer vaccines (NCV). Here we explored, in two cancer murine models, different therapeutic combinations of ICI with personalized DNA vaccines expressing neoantigens and delivered by electroporation (EP). Anti-cancer efficacy was evaluated using vaccines with or without CD4 epitopes. Therapeutic DNA vaccines showed synergistic effects in different therapeutic protocols including established large tumors. Flow cytometry (FC) was utilized to measure CD8, CD4, Treg, and switched B cells as well as neoantigen-specific immune responses, which were also measured by IFN-γ ELIspot. Immune responses were augmented in combination with αCTLA4 but not with αPD1 in the MC38 tumor-bearing mice, significantly impacting tumor growth. Similarly, neoantigen-specific T cell immune responses were enhanced in combined treatment with αCTLA-4 in the CT26 tumor model where large tumors regressed in all mice, while monotherapy with αCTLA-4 was less efficacious. In line with previous evidence, we observed an increased switched B cells in the spleen of mice treated with αCTLA-4 alone or in combination with NCV. These results support the use of NCV delivered by DNA-EP with αCTLA-4 and suggest a new combined therapy for clinical testing.

Abstract P248: The tyrosine kinase inhibitor XL092 promotes an immune-permissive tumor microenvironment and enhances the anti-tumor activity of immune checkpoint inhibitors in preclinical models

Abstract Background: Cabozantinib, an inhibitor of MET, VEGFR2, AXL, and MER, has shown significant clinical activity in multiple solid tumors, both alone and in combination with immune checkpoint inhibitors (ICIs). XL092 has a similar target profile to cabozantinib but with a significantly shorter clinical half-life and pharmacokinetic properties suitable for once daily oral dosing (Hsu, Eur J Cancer, 2020). In preclinical studies, the combination of XL092 and anti–PD-1 exhibited greater anti-tumor activity than either agent alone. Here, we describe further preclinical characterization of XL092 in combination with ICIs, including modulation of immune cells in vivo and in vitro. Methods: Pharmacodynamic activity was evaluated in the MC38 syngeneic tumor model following treatment with XL092 alone or in combination with anti–PD-1 or anti–PD-L1. Tumor CD31+ microvessel density and CD8+ T cell infiltration were assessed by immunohistochemistry, and circulating immune cells were measured by FACS. Macrophage repolarization and efferocytosis were evaluated in vitro using primary human macrophages. Results: Treatment of MC38 tumor-bearing mice with XL092 inhibited tumor angiogenesis in a dose-dependent manner, and combination treatment with XL092 and anti–PD-1 increased CD8+ lymphocyte tumor cell infiltration compared with either agent alone. XL092 and ICIs also promoted significant changes in peripheral immune cell subsets in the MC38 model. Treatment with XL092 alone or in combination with ICIs increased circulating levels of CD4+ and CD8+ T cells and B cells, and combination treatment with anti–PD-1 decreased levels of proliferating and IFNg-producing effector T cells in circulation. While the level of peripheral NK cells was unaffected by these treatments, XL092 alone or in combination with ICIs decreased levels of proliferating and GrzB+ effector cells in circulation. Treatment with XL092 alone or in combination with ICIs reduced the levels of circulating myeloid cells, including myeloid-derived suppressor cells, dendritic cells, and macrophages. In vitro treatment of human macrophages with XL092 inhibited macrophage efferocytosis with IC50 ~81 nM and promoted repolarization from an M2 anti-inflammatory to an M1 pro-inflammatory phenotype. Conclusions: Immune cell profiling showed that treatment with XL092 and ICIs promotes CD8+ T cell tumor infiltration, increases in peripheral lymphocytes, decreases in peripheral myeloid cells, and macrophage repolarization, suggesting that XL092 promotes an immune-permissive tumor microenvironment. Consistent with these immunomodulatory effects, the combination of XL092 and ICIs has shown greater anti-tumor activity in syngeneic tumor models than either agent alone. XL092 is currently being evaluated in multiple solid tumors as a single agent and in combination with ICIs in the phase 1/1b STELLAR trials (NCT03845166). Citation Format: Jeff Hsu, Colin Chong, Jeffrey Serrill, Sharon Wu, Kevin Leong, Ted Yun, Lynne Bannen, Peter Lamb, Wei Xu, Peiwen Yu. The tyrosine kinase inhibitor XL092 promotes an immune-permissive tumor microenvironment and enhances the anti-tumor activity of immune checkpoint inhibitors in preclinical models [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 P248.

Coadministration of a Clinically Relevant Dexamethasone Dosage With Ablative Radiation Therapy Reduces Peripheral Lymphocytes But Does Not Alter In Vivo Intratumoral Lymphocyte Phenotype or Inhibit Efficacy of Radiation Therapy in a Murine Colorectal Tumor Model.

Dexamethasone is commonly given during radiation therapy (RT) to manage toxicities. Our study examines if dexamethasone coadministration with RT inhibits the RT-induced antitumor T cell response in mouse. Intramuscularly implanted MC38 tumors were irradiated with 15 Gy after establishing for 7 days. Tumor bearing mice were administered dexamethasone using multiple schedules and doses. Peripheral lymphocyte reduction was monitored by complete blood count and intratumoral and tumor draining lymph node (tdLN) populations by flow cytometry. Effector phenotype and function of ex vivo stimulated tumor-infiltrating lymphocytes (TILs) and naïve splenocytes as well as in vivo TILs with or without dexamethasone were monitored by flow cytometry and ELISA. Long course high dose, short course high dose, and short course human equivalent dose dexamethasone reduced peripheral lymphocytes yet did not inhibit survival after irradiation. Short course high dose administration decreased TIL and tdLN lymphocyte activation as well as tdLN mass but did not affect TIL frequencies or change tdLN cell population composition. Dexamethasone inhibited effector function of ex vivo stimulated naïve splenocytes and TILs, but magnitude of IFN-γ secretion was consistently higher in TILs regardless of dexamethasone dose. In vivo analysis of TILs after irradiation and HE dexamethasone treatment showed that TILs had a similar effector phenotype compared with vehicle controls. Dexamethasone reduces blood and tdLN lymphocytes. Dexamethasone also suppresses TIL activation/effector function yet does not affect survival in irradiated MC38 tumor bearing mice, which depend on RT-induced immune responses for therapy efficacy. Additional study in human subjects is warranted.

Non-invasive Detection of Immunotherapy-Induced Adverse Events.

Cancer immunotherapy has markedly improved the prognosis of patients with a broad variety of malignancies. However, benefits are weighed against unique toxicities, with immune-related adverse events (irAE) that are frequent and potentially life-threatening. The diagnosis and management of these events are challenging due to heterogeneity of timing onset, multiplicity of affected organs, and lack of non-invasive monitoring techniques. We demonstrate the use of a granzyme B-targeted PET imaging agent (GZP) for irAE identification in a murine model. We generated a model of immunotherapy-induced adverse events in Foxp3-DTR-GFP mice bearing MC38 tumors. GZP PET imaging was performed to evaluate organs non-invasively. We validated imaging with ex vivo analysis, correlating the establishment of these events with the presence of immune infiltrates and granzyme B upregulation in tissue. To demonstrate the clinical relevance of our findings, the presence of granzyme B was identified through immunofluorescence staining in tissue samples of patients with confirmed checkpoint inhibitor-associated adverse events. GZP PET imaging revealed differential uptake in organs affected by irAEs, such as colon, spleen, and kidney, which significantly diminished after administration of the immunosuppressor dexamethasone. The presence of granzyme B and immune infiltrates were confirmed histologically and correlated with significantly higher uptake in PET imaging. The presence of granzyme B was also confirmed in samples from patients that presented with clinical irAEs. We demonstrate an interconnection between the establishment of irAEs and granzyme B presence and, for the first time, the visualization of those events through PET imaging.

Abstract 2954: Humanized CCR8 mouse model provides a translational tool for anti-human CCR8 antibody drug development

Abstract CCR8 is reported as an important chemokine receptor expressed on intra-tumoral Tregs and plays a critical role in CCR8+Treg-mediated immunosuppression via inducing STAT3-dependent overexpression of FOXP3, CD39, IL-10, and granzyme B on Tregs with interaction of CCL1 (CCR8 ligand). CCR8 becomes a promising target in the anticancer drug development. Generation of humanized CCR8 mouse model can greatly improve translation and accelerate CCR8-related drug development. To evaluate the anti-tumor efficacy of human CCR8 blockade in tumor mouse models, Biocytogen has successfully generated a novel CCR8 humanized knock-in mouse strain to support anti-human CCR8 antibody drug development. In this mouse model, the full length of encoding sequences of mouse CCR8 gene were replaced by the full length of encoding sequences of human CCR8 (hCCR8) gene. CCR8 gene expression in WT and B-hCCR8 mice were tested by RT-PCR. Human CCR8 mRNA was detected in hCCR8 mice model but not in wild-type mice. Protein expression of human CCR8 in hCCR8 mice was evaluated by flow cytometer in a colon cancer cell line MC38 tumor bearing mouse model. The human CCR8 protein were successfully detected in tumor-infiltrating Tregs in B-hCCR8 mice bearing MC38 tumor, but not in Treg cells from spleen. An anti-tumor efficacy study showed significant tumor growth inhibition of hCCR8 antibodies in B-hCCR8 mice bearing MC38 tumors, suggesting that the B-hCCR8 mouse model is an effective tool for in vivo efficacy evaluation of therapeutic hCCR8 antibodies to support anti-human CCR8 antibody clinical development. Citation Format: Linlin Wang, Chengzhang Shang, Jing Zhang, Yichao Chen, Leila Kokabee, Qingcong Lin. Humanized CCR8 mouse model provides a translational tool for anti-human CCR8 antibody drug development [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 2954.

Abstract 1811: A novel OX40 humanized mouse model for efficacy assessment of OX40-targeting therapies

Abstract Immunotherapies through targeting of activating and inhibitory immune checkpoints (ICP) has revolutionized the treatment of cancers. To facilitate the translation of basic research into clinical application of these immunotherapies, we developed a pipeline of immunocompetent mouse models expressing humanized ICP instead of their mouse counterparts, thus compounds can be tested in the absence of endogenous cross-reacting mouse targets. The humanization strategy is target-dependent but ensures that the biology of the target, its physiological regulation and interacting partners are preserved. Herein, we describe a novel OX40 humanized mouse model (hOX40 model), designed to assess the efficacy and mechanism of action of compounds targeting human OX40 (hOX40). The expression of the fully human OX40 protein (human extracellular, transmembrane and intracellular domains) did not alter the distribution of immune cells in spleen, blood and bone marrow. hOX40 was expressed on CD4 and CD8 T cells upon activation and the kinetic of expression mirrored the expression of mouse OX40 in WT cells activated under the same conditions. Treg cells isolated from hOX40 mice expressed hOX40 and this expression was upregulated upon activation, also recapitulating the expression of mouse OX40 in WT cells, and suggesting that the regulation of OX40 expression was kept in the hOX40 mice. hOX40 was also found to be functional on CD4 T cells, as evidenced by the increased proliferation and cytokine production upon stimulation with human OX40 ligand. Importantly, hOX40 mice bearing MC38 tumors showed a tumor growth inhibition upon treatment with an anti-human OX40 therapy with agonist activity. This model should also enable the investigation of mechanism of action as well as tumor immunological memory, as a similar design of the hOX40 model was used for the assessment of a bispecific therapy targeting OX40 and CTLA-4 (Kvarnhammar et.al, 2019). The hOX40 mouse model is currently being crossed with other models expressing humanized immune checkpoint to enable the assessment of bispecifics and combo therapies. Citation Format: Fabiane Sônego, Gaëlle Martin, Audrey Beringer, Chloé Beuraud, Yacine Cherifi, Alexandre Fraichard, Patricia Isnard Petit, Kader Thiam. A novel OX40 humanized mouse model for efficacy assessment of OX40-targeting therapies [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 1811.

Abstract 1883: Mechanism of action and biomarker strategy for HFB200301, an anti-TNFR2 agonist antibody for the treatment of cancer

Abstract Despite the success of immune check point inhibition, identification of other pathways capable of modulating the immune response against the tumor remains challenging. T-cell co-stimulation has been investigated with limited clinical success so far due in part to the fine tuning required for agonist antibodies against those co-stimulatory receptors and to the lack of biomarkers to facilitate the selection of patients likely to benefit from T-cell co-stimulation. TNFR2 belongs to the TNFR family of costimulatory molecules, and its expression on tumor infiltrating lymphocytes across a wide range of tumors make it an attractive target for T-cell co-stimulation. Recently, we identified HFB200301, an anti-TNFR2 antibody with Fc-independent agonist activity that does not block TNFR2 interaction with TNFα. HFB200301 activates CD4+, CD8+ T cells, and NK cells in vitro. In vivo, HFB200301 demonstrated potent single agent anti-tumor activity in syngeneic tumor models and can further increase the antitumor activity in combination with PD-1 blockade. To understand the immunological basis for the anti-tumor efficacy of HFB200301, we investigated the pharmacodynamic effects of HFB200301 in syngeneic mouse tumor models, including immuno-phenotyping and receptor occupancy of tumor infiltrating cells. In hTNFR2 knock-in mice bearing MC38 tumors, HFB200301 induces expansion of CD4+ and CD8+ T cells, and NK cells in the tumor micro-environment without affecting regulatory T cell numbers. We also demonstrate that the anti-tumor efficacy of HFB200301 is correlated with receptor occupancy and circulating soluble TNFR2 in a dose-dependent manner in this model. To discover predictive biomarkers of response to HFB200301, we used primary tumor samples and our proprietary Drug Intelligent Science (DIS™) single-cell platform to establish an immune-related signature. Single-cell RNA sequencing and clonotype barcoding of ex-vivo tumor cultures treated with HFB200301 were used to identify unique T cell profiles with a T cell centric gene panel. These unique T cell profiles may help identifying patients more likely to respond to HFB200301 treatment. In summary, HFB200301 exhibits a unique mechanism of action mainly relying on its agonistic activity on several effector cell types in tumor micro-environment that we expect will benefit a patient population selected with a unique biomarker signature. HFB200301 is currently in preclinical development and a biomarker-driven Phase 1 clinical study is projected for 2021. Citation Format: Shuo Wei, Ross Fulton, Yun-Yueh Lu, Qian Zhang, He Zhou, Andreas Raue, Mingjie Chen, Wenhua Xu, Xing Cai, Juliana Crivello, Zachary Duda, Zhiyuan Wang, Rebecca Silver, Alexandra Staskus, Charina Ortega, Sami Ellouze, Carine George, Sophie Foulon, Dean Lee, Monika Manne, Nicola Beltraminelli, Jinping Gan, Francisco Adrian, Liang Schweizer, Jennifer Watkins-Yoon. Mechanism of action and biomarker strategy for HFB200301, an anti-TNFR2 agonist antibody for the treatment of cancer [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 1883.

Novel immunotherapeutic Albumin-Flt3L enhances antigen-specific immunity and tumor control following chemoradiation: An analysis of underlying mechanism

Abstract CD8+ cells are essential to anti-cancer immunity following chemoradiation and immunotherapy. The induction of tumor specific T cells depends on the presentation of tumor associated antigens/neoantigens. This generates a bottleneck to effective antitumor immunity, where only antigens efficiently presented on MHCI can serve as CD8 T cell targets. To bypass this, we have developed a novel immunotherapeutic by fusing albumin to Flt3L (Alb-Flt3L), a cytokine known to enhance cross-presenting cDC1s, and described its function. Previously, we observed that MC38-tumor bearing mice treated with Alb-Flt3L + radiotherapy engenders neoantigen-specific T cells by tetramer staining. Now we have expanded this analysis to query the breadth and depth of CD8+ TILs by TCRb sequencing. Sorted and sequenced ADPGKneo-specific CD8 T cells encompassed a multitude of unique rearrangements, suggesting polyclonal neoantigen coverage, and bulk CD8+ cells showed a greater Diversity Index in Alb-Flt3L + radiotherapy treated mice compared to controls. We also sought to investigate the mechanism of tumor control following treatment with Alb-Flt3L + chemoradiation. The role of Batf3, a cDC1 transcription factor, type-I-interferons, cytokines essential to immune activation following chemoradiation, and pDCs, an Alb-Flt3L-tropic cell type also capable of cross-presentation, were investigated using knockout mice or antibody depletion in tumor bearing mice treated with cisplatin. This study confirmed the role of these factors in Alb-Flt3L-mediated responses, as worse tumor control was observed in the absence of Batf3, pDCs, or type-I-interferon signaling. Altogether, we were able to characterize mechanisms mediating Alb-Flt3L’s antitumor effects.

Abstract PO-088: Bintrafusp alfa enhances anti-tumor efficacy in combination with radiation therapy plus the ataxia telangiectasia mutated (ATM) inhibitor, M4076

Abstract Ionizing radiation therapy (RT) is a standard of care for many cancer indications. RT promotes the activation of transforming growth factor (TGF)-β, a pleotropic cytokine that mediates RT-induced fibrosis and suppresses anti-tumor immunity. Additionally, RT induces the expression of the immunosuppressive checkpoint PD-L1 on tumor cells. Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor, functioning as a TGF-β “trap”, fused to a human IgG1 antibody blocking PD-L1. Bintrafusp alfa enhances anti-tumor immunity elicited by RT and reverses RT-induced fibrosis in preclinical models. Inhibition of ataxia telangiectasia mutated (ATM) increases the cancer cell's sensitivity to radiotherapy by blocking cellular pathways which facilitate the response to, and repair of, RT induced DNA double strand breaks. We hypothesize that a combination therapy consisting of bintrafusp alfa, RT and the ATM kinase inhibitor M4076 will show greater anti-tumor efficacy than the respective mono- or dual-combination therapies in immune competent preclinical solid tumor models. The anti-tumor efficacy of bintrafusp alfa, RT, and M4076 triple combination therapy was explored in the immune therapy resistant 4T1 murine syngeneic breast carcinoma model. This triple combination therapy significantly increased anti-tumor efficacy and prolonged overall survival relative to the bintrafusp alfa + RT or M4076 + RT dual combination therapies. The triple combination was further evaluated in the more immunogenic MC38 colon carcinoma model. MC38 tumor bearing mice treated with the triple combination therapy showed a significantly greater tumor growth inhibition and long term tumor free survival when compared with the bintrafusp alfa + RT or M4076 + RT treatment groups. Finally, the bintrafusp alfa + RT + M4076 combination therapy also showed significantly greater tumor growth inhibition and long term tumor free survival when compared to the combination of anti-PD-L1 + RT + M4076. The bintrafusp alfa triple combination resulted in 8 of 10 mice showing long term tumor free survival, whereas the anti-PD-L1 triple combination failed to elicit tumor free survival (0 of 10 mice tumor free). These studies demonstrate the superior anti-tumor efficacy of the Bintrafusp alfa + RT + M4076 triple combination therapy over that of the dual combination therapies of RT + bintrafusp alfa or RT + M4076. These data in the MC38 model also suggest that bintrafusp alfa may promote a superior anti-tumor immune response relative to that of anti-PD-L1 when in combination with RT and M4076. Together, these data support the rational to develop a bintrafusp alfa + RT + M4076 combination therapy for the treatment of solid tumors. Citation Format: Adam Lazorchak, Huakui Yu, Guozhong Qin, Jin Qi, Bo Marelli, Yan Lan. Bintrafusp alfa enhances anti-tumor efficacy in combination with radiation therapy plus the ataxia telangiectasia mutated (ATM) inhibitor, M4076 [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-088.

Amino acids and RagD potentiate mTORC1 activation in CD8+ T cells to confer antitumor immunity

BackgroundIn the tumor microenvironment, tumor cells are able to suppress antitumor immunity by competing for essential nutrients, including amino acids. However, whether amino acid depletion modulates the activity of CD8+...

61892 Systemic TLR3-targeting Combinatorial Chemokine Modulation Sensitizes Murine Tumors to PD-1 Blockade

ABSTRACT IMPACT: This work will lead to improved efficacy of immunotherapy directly impacting the survival of patients with hard to treat cancers. OBJECTIVES/GOALS: Immune checkpoint inhibitors (ICI) are most effective against ‘hot’ tumors highly infiltrated with cytotoxic T lymphocytes (CTLs) but have not worked well in poorly infiltrated ‘cold’ tumors. Thus, we are working to achieve a pretreatment regimen that will create a favorable immune profile allowing more effective ?PD-1 therapy. METHODS/STUDY POPULATION: BALB/c or C57BL/6 mice were inoculated with CRC murine cells CT26 or MC38, respectively. Mice were inoculated by two injection types: subcutaneous (SC), for systemic therapy, or intraperitoneal (IP), for local therapy. Tumor-bearing mice were given a two dose course of CKM consisting of IFN-? and rintatolimod via IP injection. Following CKM administration, mice were treated with three doses of ?PD-1 via IP injection. Mice were monitored for the kinetics of tumor growth and survival following treatment. The tumor microenvironment of treated mice was analyzed for production of chemokines, inflammatory cytokines and immune cell infiltration. RESULTS/ANTICIPATED RESULTS: CKM consisting of combination IFN-? and rintatolimod, but neither monotherapy alone, sensitized murine CRC tumors to subsequent ?PD-1 treatment. In both CT26 and MC38 tumor-bearing mice, tumor growth was hindered by CKM plus ?PD-1 treatment, independently on the route of treatment (local or systemic). Mice which experienced complete tumor regression were protected from re-challenge with a dose of tumor cells double that of the initial inoculation. Sensitizing tumors to ?PD-1 did not require intratumoral CKM administration and was observed with systemic application at distant sites. In accordance with these observations we expect that systemic CKM will induce strong increases of total and tumor-specific CTL counts in the tumor tissues as measured by both PCR and flow cytometry. DISCUSSION/SIGNIFICANCE OF FINDINGS: CKM sensitizing cold tumors to ?PD-1 indicates that intratumoral CTLs are an important factor dictating therapeutic effectiveness, independent of other factors such as tumor mutational load. The benefit of the sequential short-term CKM followed by routine ?PD-1 make this strategy feasible for rapid inclusion of into routine immunotherapy plans.

Tumor-targeted superfluorinated micellar probe for sensitive in vivo19F-MRI.

We describe herein the assembly and in vivo evaluation of a tailor-made micellar carrier system designed for the optimized encapsulation of a superfluorinated MRI probe and further targeting of solid tumors. The in vivo validation was carried out on MC38 tumor-bearing mice which allowed the confirmation of the efficient targeting properties of the nano-carrier, as monitored by 19F-MRI.

Targeting the myeloid checkpoint receptor SIRP\u03b1 potentiates innate and adaptive immune responses to promote anti-tumor activity

BackgroundSignal regulatory protein α (SIRPα) is a myeloid-lineage inhibitory receptor that restricts innate immunity through engagement of its cell surface ligand CD47. Blockade of the CD47–SIRPα interaction synergizes with tumor-specific antibodies and T-cell checkpoint inhibitors by promoting myeloid-mediated antitumor functions leading to the induction of adaptive immunity. Inhibition of the CD47–SIRPα interaction has focused predominantly on targeting CD47, which is expressed ubiquitously and contributes to the accelerated blood clearance of anti-CD47 therapeutics. Targeting SIRPα, which is myeloid-restricted, may provide a differential pharmacokinetic, safety, and efficacy profile; however, SIRPα polymorphisms and lack of pan-allelic and species cross-reactive agents have limited the clinical translation of antibodies against SIRPα. Here, we report the development of humanized AB21 (hAB21), a pan-allelic anti-SIRPα antibody that binds human, cynomolgus monkey, and mouse SIRPα alleles with high affinity and blocks the interaction with CD47.MethodsHuman macrophages derived from donors with various SIRPα v1 and v2 allelic status were used to assess the ability of hAB21 to enhance phagocytosis. HAB21_IgG subclasses were evaluated for targeted depletion of peripheral blood mononuclear cells, phagocytosis and in vivo efficacy in xenograft models. Combination therapy with anti-PD1/anti-PD-L1 in several syngeneic models was performed. Immunophenotyping of tissues from MC38 tumor-bearing mice treated with AB21 and anti-PD-1 was evaluated. PK, PD and tolerability of hAB21 were evaluated in cynomolgus monkeys.ResultsSIRPα blockade with hAB21 promoted macrophage-mediated antibody-dependent phagocytosis of tumor cells in vitro and improved responses to rituximab in the Raji human tumor xenograft mouse model. Combined with PD-1/PD-L1 blockade, AB21 improved response rates by facilitating monocyte activation, dendritic cell activation, and T cell effector functions resulting in long term, durable antitumor immunity. In cynomolgus monkeys, hAB21 has a half-life of 5.3 days at 10 mg/kg and complete target occupancy with no hematological toxicity or adverse findings at doses up to 30 mg/kg.ConclusionsThe in vitro and in vivo antitumor activity of hAB21 broadly recapitulates that of CD47 targeted therapies despite differences in ligand expression, binding partners, and function, validating the CD47–SIRPα axis as a fundamental myeloid checkpoint pathway and its blockade as promising therapeutic intervention for treatment of human malignancies.

Repositioning liothyronine for cancer immunotherapy by blocking the interaction of immune checkpoint TIGIT/PVR

BackgroundInhibitors targeting immune checkpoint were proved effective in cancer immunotherapy, such as PD-1/PD-L1 blockade. The novel immune checkpoint TIGIT/PVR plays critical roles in suppressing the anti-tumor effects of CD8+ T and NK cells, and dual blockade of TIGIT/PVR and PD-1/PD-L1 by antibody can elicit synergistic effects in tumor models and clinical trials. However, small molecules for TIGIT/PVR blockade have not been investigated.MethodsThe expression of PVR in tumors were analyzed by using TCGA, Oncomine and GEO database, and in cancer cell lines examined by flow cytometry. Natural product compounds were docked to PVR for virtual screening by using the software Molecular Operating Environment (MOE). Candidate compounds were further tested by biolayer interferometry-based binding assay, microscale thermophoresis assay and cell based blocking assay. The in vitro activity of the candidate compound was determined by MTT, peripheral blood mononuclear cells (PBMCs) activation assay and coculture assay. The anti-tumor effects and mechanism were also investigated by using MC38 tumor-bearing mice model and immune cell depletion tumor model.ResultsPVR was over-expressed in many tumor tissues and cancer cell lines, making it a promising therapeutic target. Through virtual screening, binding, and blocking assay, liothyronine was discovered to bind PVR and block the interaction of TIGIT/PVR. Liothyronine could enhance the function of CD4+ and CD8+ T cells in PBMCs. Besides, in the Jurkat-hTIGIT and CHOK1-hPVR coculture assay, liothyronine could reverse the IL-2 secretion inhibition resulted by TIGIT/PVR ligation. Although had no influence on the proliferation of tumor cells in vitro, liothyronine could significantly inhibit tumor growth when administrated in vivo, by enhancing CD8+ T cell infiltration and immune responses in the tumor bearing mice. The immune cell depletion model showed that the anti-tumor effects of liothyronine depends on CD4+ T cells, CD8+ T cells and NK cells.ConclusionsA small molecule liothyronine was discovered to serve as a potential candidate for cancer immunotherapy by blocking the immune checkpoint TIGIT/PVR.-Ev9Py5y6QZb5r8dVDwR9cVideo abstractGraphical abstract

Abstract 3368: A novel anti-CTLA-4 antibody with potent anti-tumor activity emerged using humanized CTLA-4 mouse model

Abstract CTLA-4 (Cytotoxic T-lymphocyte antigen 4), also known as CD152, belongs to immunoglobulin superfamily. It is an intracellular protein expressed in resting T cells; CTLA-4 translocates to the cell surface and transmits an inhibitory signal to T cells. In addition, CTLA-4 competes with its homologous T-cell co-stimulatory protein CD28 for ligands expressed on APC, namely CD80 (B7-1) and CD86 (B7-2), thereby antagonizing CD28 and inhibiting CD28 signaling, resulting in inhibition of IL-2, IFN-γ, IL-4 cytokines production, IL-2 receptor expression and cell cycle progression. Because Tregs constitutively express CTLA-4 at high levels, anti-CTLA-4 therapy would be expected to have the greatest effect on these cells. Ipilimumab, a fully human IgG1 monoclonal antibody, is the only commercially available CTLA-4 antagonist. It has been approved by FDA for treatment of metastatic melanoma, adjuvant melanoma and advanced renal cell carcinoma, based on its significant effect of tumor suppression demonstrated in clinical trials. We have interests in searching for potent CTLA-4 antagonists. We selected an antibody with the best efficacy, namely YH001, from the candidate molecules through Biocytoge's in vivo drug screening platform. YH001 is a recombinant humanized anti-CTLA-4 IgG1 monoclonal antibody and it can bind with human CTLA-4 potently and specifically, without reactivity with other CD28 family receptors. YH001 was able to block the CTLA-4/CD80 and CTLA-4/CD86 interactions and potentiate CD28 signaling in the presence of cognate TCR stimulation. Moreover, YH001 also induced stronger bioluminescence than ipilimumab in the reporter assay. YH001 showed more potent ADCC activities than ipilimumab. YH001 showed dose-dependent in vivo anti-cancer efficacy, as well as Treg depletion and increased CD8+/Treg ratio in human CTLA-4 knock-in mice bearing MC38 tumors. Exposure to YH001 was dose proportional with a half-life of approximately 5 days in cynomolgus monkey. Our preclinical investigation of YH001 demonstrates Biocytogen's capability in discovering clinical candidates using its robust antibody discovery platform. Citation Format: Chaoshe Guo, Yi Yang, Zhihong Li, Yanan Li, Yunyun Chen. A novel anti-CTLA-4 antibody with potent anti-tumor activity emerged using humanized CTLA-4 mouse model [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 3368.

Abstract 5635: Enhanced anti-tumor lymphocyte function and frequencies measured by multichromatic flow cytometry in human CTLA-4 knock-in mice in a colorectal carcinoma model after treatment with ipilimumab

Abstract Targeting CTLA-4 has shown remarkable long-term benefits and thus remains a valuable approach for combating cancers of many types. A number of preclinical models have been developed over the years to evaluate the efficacy of immune checkpoint blockade in promoting antitumor immunity. In particular, knock-in (KI) humanized mouse models offer the possibility to study clinical grade immune checkpoint inhibitors (ICI) in the context of a fully functional immune system. Here we show the response to ipilimumab in a newly developed hCTLA-4 KI humanized mouse model. Our results demonstrate significant tumor growth inhibition as well as complete tumor regressions in the MC38 colorectal cancer model following treatment with ipilimumab. We have extended these studies by re-challenging the tumor-free surviving animals with tumors cells implanted opposite to the original tumor site. We established that all re-challenged hCTLA-4 KI mice remained tumor free suggesting potent T cell memory was maintained. To identify the immune cells and cytokines that could be responsible for the durable responses, we used multiparameter flow cytometry to characterize and compare blood, spleen and tumors tissues from the control, ipilimumab and mCTLA-4 treated mice. Comprehensive multichromatic phenotyping and functional intracellular cytokine staining (ICS) using validated 18-color flow cytometry panels showed a significant increase in CD8+ T cell frequencies when mice were treated with anti-hCTLA-4 but not with the mouse counterpart or the isotype control. Importantly, hCTLA-4 blockade reduced the regulatory T cell (FoxP3+ Treg) frequency and increased leukocyte infiltration into the tumor in the hCTLA-4 treated mice but not in the other two treated groups. Although equivalent numbers of CD4+ and CD8+ total T lymphocytes where found in the blood of all groups after each treatment, the quality of the T cell responses was found to be significantly different. CD45+ lymphocytes from the blood analyzed from the hCTLA-4 treated mice showed significant increases in both central memory (CM) CD8+ and CD4+ T cells consistent with increased anti-tumor efficacy seen in these mice. Furthermore, treatment of MC38 tumor-bearing mice with ipilimumab enhanced significantly the secretion of IFNγ from CD8+ and CD4+ T cells and the proliferation of both T cell populations as shown by the upregulation of Ki67 staining. These results indicate that immune cells were actively proliferating and had the appropriate effector functions to impact tumor growth. Altogether, the data presented here demonstrates that hCTLA-4 KI humanized mice are a robust model for evaluating the immune-modulatory effects and the activity of clinical grade ICI against tumors. Citation Format: Elizabeth Reap, Fabiane SÔNEGO, Emily O'Koren, Martin Gaëlle, Anya Avrutskaya, Jacob Hauser, Robin Ball, Thi Bui, Ian Belle, Chassidy Hall, Paula L. Miliani De Marval, Kader Thiam. Enhanced anti-tumor lymphocyte function and frequencies measured by multichromatic flow cytometry in human CTLA-4 knock-in mice in a colorectal carcinoma model after treatment with ipilimumab [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 5635.