CT26 Syngeneic Model Research Articles

Abstract 1716: WTX-330, a conditionally activated IL-12 INDUKINE, releases IL-12 selectively in the tumor microenvironment to activate anti-tumor immune responses and induce regressions in mouse tumor models

Abstract Systemic therapy with proinflammatory immune modulators is a promising approach to treat cancer by activating innate and adaptive anti-tumor immunity. However, dose-limiting toxicities such as inflammation, cytokine release syndrome, and tissue damage rendered the strategy impractical for cytokines such as the cytotoxic T and NK cell activating interleukin 12 (IL-12). WTX-330 is a novel systemically delivered therapy with targeted intra-tumoral activation to deliver wild-type IL-12 in the tumor microenvironment identified using the Predator™ discovery platform. WTX-330 is a member of a class of inducible polypeptides (INDUKINE™ therapies) designed to be an inactive IL-12 pro-drug with a half-life extension (HLE) domain to support infrequent systemic administration. The INDUKINE™ polypeptide is kept inactive in the periphery via high affinity antibody blockade tethered to IL-12 via a tumor protease-sensitive linker. Cleavage of the same linker also removes the HLE domain producing a PK profile similar to wild-type IL-12. WTX-330 was selected as a lead molecule due to its beneficial in vitro profile. The mouse MC38 syngeneic tumor model was used to demonstrate strong anti-tumor activity of lead mouse INDUKINE™ polypeptides. Intraperitoneal (i.p.) administration of mouse WTX-330 led to complete regression of all MC38 tumors at dose levels which only moderately induced systemic IFNγ and were well tolerated by the mice. Similar efficacy was observed in CT26, B16F10, and EMT-6 syngeneic models. Importantly, wild-type IL-12, while active in mouse tumor models, was not tolerated by the mice after i.p. injection at equimolar amounts compared to the IL-12 INDUKINE™ polypeptide. Mouse WTX-330 strongly activated NK and CD8+ cell responses and induced APC and effector cell markers in MC38 tumors supporting a mechanism of action as described for wild-type IL-12. In non-human primates, WTX-330 was well tolerated at exposure levels which vastly exceeded the levels needed for efficacy in mice. In addition, plasma levels of free IL-12 after dosing with WTX-330 were very low compared to tolerated levels after treatment with wild-type IL-12. Ex vivo incubation of WTX-330 with numerous primary human tumors led to the rapid release of IL-12, while WTX-330 proved to be stable in human serum and normal tissues. Pharmacological and translational data obtained so far clearly support continued preclinical development with the goal of moving this innovative and differentiated engineered IL-12 therapy into human clinical testing. Citation Format: Philipp Steiner, Heather Brodkin, Dan Hicklin, Nesreen Ismail, Kristin Morris, Christopher Nirschl, Andres Salmeron, Cindy Seidel-Dugan, Zoe Steuert, Jenna Sullivan, William Winston. WTX-330, a conditionally activated IL-12 INDUKINE, releases IL-12 selectively in the tumor microenvironment to activate anti-tumor immune responses and induce regressions in mouse 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 1716.

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.

Efficacy of Combination Chemotherapy Using a Novel Oral Chemotherapeutic Agent, FTD/TPI, with Ramucirumab Murine Version DC101 in a Mouse Syngeneic Cancer Transplantation Model.

Trifluridine/tipiracil (FTD/TPI) (a.k.a. TAS-102) is a combination drug for metastatic colorectal cancer (CRC) and severely pretreated metastatic gastric/gastroesophageal junction (GEJ) cancers, comprising FTD, a thymidine-based antineoplastic nucleoside analog, and TPI, which enhances FTD bioavailability. Herein, in KRAS mutant murine colorectal cancer CT26 syngeneic models, we investigate whether combination therapy with DC101 (a surrogate ramucirumab antibody, rat antimouse vascular endothelial growth factor receptor (VEGFR)-2 monoclonal antibody (mAb)) improves FTD/TPI efficacy. Tumor growth inhibition (TGI) on day 15 was 38.0% and 30.6% upon DC101 monotherapy and FTD/TPI monotherapy respectively, and 60.3% upon combination therapy. Tumor volume was significantly lower (p < 0.001) upon combination treatment than upon FTD/TPI or DC101 monotherapy, indicating the additive effects of FTD/TPI and DC101. DNA-incorporated FTD levels on Day 8 were significantly higher in combination therapy with FTD/TPI (for 5 consecutive days) and DC101 (on alternate days for 7days) than in FTD/TPI monotherapy. Furthermore, vascular endothelial cell-specific marker CD31 was downregulated in DC101-treated tumors on day 8. These results indicate that combination therapy with FTD/TPI and DC101 is a promising treatment alternative regardless of KRAS mutations.

Combination of Camidanlumab Tesirine, a CD25-Targeted ADC, with Gemcitabine Elicits Synergistic Anti-Tumor Activity in Preclinical Tumor Models

Camidanlumab tesirine (ADCT-301) is an antibody-drug conjugate (ADC) comprised of HuMax®-TAC, a monoclonal antibody directed against human CD25, conjugated to the pyrrolobenzodiazepine dimer payload tesirine[1]. Currently, camidanlumab tesirine is being evaluated in a pivotal Phase 2 clinical trial in patients with relapsed or refractory Hodgkin lymphoma (HL) (NCT04052997) and in a Phase 1b clinical trial in patients with advanced solid tumors (NCT03621982). In pre-clinical studies, camidanlumab tesirine demonstrated strong and durable single agent activity in CD25-expressing lymphoma xenograft models[1] and in vitro it synergised with selected targeted agents[2]. Moreover, CD25-ADC, a mouse CD25 cross-reactive surrogate of camidanlumab tesirine, induced potent anti-tumor immunity against established syngeneic solid tumor models by depleting CD25-positive tumor-infiltrating T regulatory cells (Tregs) and it showed synergistic activity when combined with PD-1 blockade[3]. Here, we investigated the in vitro and in vivo anti-tumor activity of camidanlumab tesirine combined with gemcitabine, a common standard-of-care chemotherapeutic agent used both in a hematological and solid tumor clinical setting. In vitro, the combination of camidanlumab tesirine and gemcitabine was evaluated in three human-derived cancer cell lines (two HL and one anaplastic large cell lymphoma, ALCL) and resulted in synergistic activity as determined by the Chou-Talalay method. In vivo, camidanlumab tesirine was tested either alone (0.05 or 0.1 mg/kg, single dose) or in combination with gemcitabine (80 mg/kg, q3dx4) in the CD25-expressing ALCL Karpas299 xenograft model. At both ADC dose levels, combination with gemcitabine resulted in synergistic anti-tumor activity (coefficient of drug interaction (CDI) 0.51 and 0.17, respectively), better response rates and increased survival compared to monotherapy with camidanlumab tesirine. In order to extend the investigation to solid tumor models, CD25-ADC was tested in the CT26 syngeneic model, a colorectal cancer model with CD25-expressing tumor-infiltrating Tregs. CD25-ADC was administered either alone (0.1, 0.5 or 1 mg/kg, single dose) or in combination with gemcitabine (80 mg/kg, q3dx4). At the 0.1 mg/kg dose of CD25-ADC, combination with gemcitabine resulted in synergistic anti-tumor activity (CDI 0.68). Moreover, at 0.5 and 1 mg/kg, the combination of CD25-ADC and gemcitabine resulted in more durable anti-tumor activity and better response rates compared to both monotherapy treatments. In conclusion, the combination of camidanlumab tesirine and gemcitabine was synergistic both in vitro and in vivo in lymphoma preclinical models. Synergistic anti-tumor activity was also demonstrated in a colorectal cancer model using CD25-ADC, a mouse-cross-reactive version of camidanlumab tesirine, in combination with gemcitabine. Altogether, these novel pre-clinical data warrant translation of the combination between camidanlumab tesirine and gemcitabine into the clinic. 1.Flynn, M.J., et al., ADCT-301, a Pyrrolobenzodiazepine (PBD) Dimer-Containing Antibody-Drug Conjugate (ADC) Targeting CD25-Expressing Hematological Malignancies. Mol Cancer Ther, 2016. 15(11): p. 2709-2721. 2.Spriano, F., et al., The anti-CD25 antibody-drug conjugate camidanlumab tesirine (ADCT-301) presents a strong preclinical activity both as single agent and in combination in lymphoma cell lines. Hematological Oncology, 2019. 37(S2): p. 323-324. 3.Zammarchi, F., et al., A CD25-targeted antibody-drug conjugate depletes regulatory T cells and eliminates established syngeneic tumors via antitumor immunity. Journal for ImmunoTherapy of Cancer, 2020. In press. Disclosures Jabeen: ADC Therapeutics: Current Employment. Hartley:ADC Therapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding. Van Berkel:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zammarchi:ADC-Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Abstract 6529: GI101, A novel CD80-IgG4-IL2 variant bispecific protein, inhibits tumor growth and induces anti-tumor immune response in multiple preclinical models

Abstract Introduction: Immune checkpoint blockades (ICBs) have revolutionized cancer treatment and broadened clinical applicability. However, the majority of patients still fail to respond to standard ICBs. To overcome such unmet needs in a clinical study, we designed GI-101, combining the extracellular domain of CD80 serve as a CTLA-4 blockade and an IL-2 variant that preferentially binds the IL-2 receptor β subunit (IL-2Rβ) together. The harmonizing mechanisms of action are projected to translate into improved clinical benefits for this first-in-class immune checkpoint inhibitor fusion protein, even in non-inflamed “cold” tumors. Methods: Binding affinity of GI101 to IL2Rs, CTLA4, and CD28 was determined by SPR. Immune cell proliferation was analyzed by CFSE assay. In vivo anti-tumor efficacy was tested by single or combination treatment on CT26, MC38 and B16F10 syngeneic tumor models. To elucidate the involvement of GI101 on tumor microenvironment (TME), immune cell population was analyzed by flow cytometry from tumor. Tumor specific T cells (surrogate marker, gp70) were measured by splenocyte proliferation assay and IFN-γ ELISPOT assay. RNA sequencing was performed to elucidate immune mechanism of GI-101. Results: GI101 highly binds to CTLA-4 (Kd, 2.9 nM) which leads to the reinforcement of endogenous CD80 and CD28 interaction resulting in the activation of T cells. Bivalent IL-2 variant of GI101 triggers both CD8+ T and NK cells proliferation in vitro and in vivo without Tregs proliferation. GI101 has no evidence for toxicity associated with IL-2 activity including vascular leakage syndrome and cytokine storm in non-GLP monkey studies whereas isolated mortality was observed in the anti-PD-1 and anti-CTLA4 combination treatment group. GI101 elicits restoration of immune functions in vitro settings using mouse splenocytes co-cultured with different PDL-1 and CTLA-4 expression level tumor cells. A dose-dependent (3 to 12 mg/kg) inhibition of tumor growth was observed in CT26 syngeneic models without toxicity. Immune profiling of tumor samples also revealed that a robust increment of M1 macrophages, CD8+ central memory T cells (Tcm) and Ki-67+ proliferating T cells but not Tregs in TME (p &amp;lt; 0.05). Tumor specific T cells were strongly proliferated when stimulated with CT26 neoantigens (gp70, RSPWFTTLI and MGPLIVLLL) in splenocyte. IFN-γ+ cells were significantly increased in draining lymph nodes from GI101 treated mice. Furthermore, drastic tumor regression was observed in MC38 tumor-bearing mice treated with GI101 and anti-PD-1 combination. Conclusion: GI101 facilitates the dual function of checkpoint blockade and IL2 activity that enhances the proliferation and activation of T and NK cells. This novel target drug is expected to be interpreted as superior clinical efficacy and safety as indicated even in ‘cold tumor' models. GI101 is the promising immune-oncology drug to replace the first-generation ICBs by single or combining with other immunotherapies. Our findings provide a rationale for further clinical investigations. Keywords: CD80, IL-2 variant, GI101, Bispecific fusion protein, immunotherapy Citation Format: Kyoung-Ho Pyo, Young Jun Koh, Chun-Bong Synn, Jae Chan Park, Jae-Hwan Kim, Yeongseon Byeon, Sung Eun Kim, Ji Min Lee, Ha Ni Jo, Wongeun Lee, Do Hee Kim, Sungwon Park, Yoo Jeong Song, Won Jae Lee, Ji Young Kim, Hyung Nam Ji, Sang Su Park, Kyung Wha Lee, Young Gyu Cho, Young Min Oh, Bo Gie Yang, Su Youn Nam, Myoung Ho Jang, Byoung Chul Cho. GI101, A novel CD80-IgG4-IL2 variant bispecific protein, inhibits tumor growth and induces anti-tumor immune response in multiple preclinical 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 6529.

Abstract 4439: BR101801 triggers anti-tumor immunity and enhances efficacy of immune checkpoint antibodies in syngeneic model

Abstract Introduction: BR101801 received U.S. FDA approval of Phase Ia/Ib clinical trial as an anti-cancer drug candidate. PI3K γ / δ and DNA-PK inhibitory potency of BR101801 exerts anti-cancer efficacy as a target anticancer agent in hematologic malignancies. Recent studies have reported anti-cancer immunity by use of PI3K-δ inhibitor (Idelalisib), PI3K-γ inhibitor (IPI-549) and DNA-PK inhibitor (NU7441). Considering triple inhibitory activity of the BR101801. First, we investigated the anti-cancer efficacy of monotherapy by BR101801 in syngeneic model. Second, synergistic efficacy was confirmed through the combination of BR101801 with anti-PD1 or anti-PDL1, which are known as anti-tumor immunotherapeutics. Finally, immune cell response by BR101801 in human PBMC was tested. Overall, This study aims to demonstrate efficacy of the BR101801 as a potent immunocancer drug. Methods: The enzymatic potency of the PI3K isotype and DNA-PK was analyzed by Eurofin. Proliferation of 4T1(breast cancer) and CT26(colon cancer) cells following a 4 h treatment with the indicated BR101801, WST-8 staining after 72 h culture. Mice bearing syngeneic 4T1 or CT-26 cell lines were treated with vehicle, BR101801, anti-PD1, anti-PD1 + BR101801, anti-PDL1, anti-PDL1 + BR101801. Tumor volumes were measured by caliper. Tregs and CD8+ T cells were quantified by flow cytometry from mice bearing spleen. In addition, CD3 + cells were isolated from human PBMCs and stimulated with CD3/CD28 and analyzed for immunofluorescence by FACS for expression of PD-1, TIM3, LAG3 and TIGIT in Tregs and CD8. Results: In vitro selectivity and target potency of BR101801 on different PI3K subtypes including DNA-PK were studied in cell-free system. The biochemical IC50 values of BR101801 for PI3K-α, -β, -γ, -δ and DNA-PK were 106 nM, 171 nM, 15 nM, 2 nM and 6 nM. 50% growth inhibition in 4T1 and CT26 cell lines was measured above 10 uM. BR101801 showed tumor inhibitor efficacy in 4T1 and CT26 syngeneic models. In 4T1 or CT-26 bearing mouse spleens, Tregs decreased and CD8 + T cells increased. Strong anti-tumor efficacy was observed with about 70% reduction in tumor growth when using BR101801 with anti-PD1 or anti-PDL1 in 4T1 and CT26 syngeneic models. Finally, Tregs decreased and anticancer effects were increased by decreasing the expression of PD1, LAG3, TIM3 and TIGIT of CD8+ T cells. Conclusions: The immunosuppressive effect of BR101801 changing the tumor microenvironment was verified without cancer cells. It is demonstrated that anticancer immunity by decreasing Tregs and increasing CD8. Anti-tumor immunity was enhanced by decreasing the expression of PD1, TIM3, LAG3 and TIGIT in CD8 + cells. In this study, In addition to monotherapy of BR101801, It's synergistic effects with PD-1 or PDL1 have been proposed. This data supports the evidence that it is applicable not only to blood cancer but also to various cancer patients. Citation Format: Seungho Wang, Mi kwon Son, Bo Ram Lee, Byeongwook Jeon, Soo Jung Kim, Eunhui Yang, Min Park, Joo Han Lee, Jayhyuk Myung. BR101801 triggers anti-tumor immunity and enhances efficacy of immune checkpoint antibodies in syngeneic 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 4439.

Abstract 3766: Development of new FLT3/AXL dual inhibitor JRF104 with potent FLT3-TKD inhibition

Abstract Background: More than 30% of de novo AML patients were identified with FLT3 mutations. Two kinds of mutations, internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations, account for 23% and 7% of all AML cases, respectively. FLT3 inhibitors, which have been recommended by both NCCN and ELN guidelines, are often used as single or combo agent to treat FLT3 m+ AML. However, primary and secondary acquired resistance to FLT3 inhibitors is a remarkable challenge. It has been revealed that AXL can activate FLT3, resulting in reduction of response to FLT3 inhibitors. Furthermore, TKD mutations can also lead to development of drug resistance. Therefore, FLT3/AXL dual inhibitor with potent FLT3-TKD inhibition may provide better efficacy as novel AML treatment option. Results: JRF104 is a potent FLT3/AXL dual inhibitor with a kinase inhibition IC50 value of 0.42 nM and 1.22 nM, respectively. In vitro studies suggested that JRF104 could potently inhibit the proliferation of MV4-11 and Molm-13 cells, which endogenously express FLT3-ITD mutation. JRF104 shows wide spectrum in vitro antiproliferation activity against all FLT3 mutation types, including the “gate-keeper” F691L mutation. JRF104 can effectively reduce MDA-MB-231 cell migration in vitro, accomplished by blocking tumor migration pathway regulated by AXL. In MV4-11 xenograft model, JRF104 inhibited tumor growth at the dose of 1 mg/kg, QD (TGI: 65%); significantly reduce tumor volume at 5 mg/kg, QD (TGI: 118%). In Molm-13 xenograft model, tumor volume was well controlled at 15 mg/kg, QD (TGI: 94%), and all tumor volume reduced to zero at 50 mg/kg, QD (TGI: 104%). In CT-26 syngeneic model, JRF104 enhanced anti-tumor growth effect when combined with anti-PD-1 antibody and suppressed pERK and pAKT in tumor tissues. Additionally, JRF104 demonstrated high oral bioavailability in mouse, rat, and dog. In 28 days pre-clinical toxicology studies, JRF104 was well tolerated in all tested rats. HNSTD and NOEAL were determined to be 20 mg/kg QD and 3 mg/kg QD, respectively. All results support that JRF104 with high efficacy and safety. Conclusion: JRF104 demonstrated consistently potent inhibition of FLT3 and strong antitumor efficacy against FLT3-driven tumors in preclinical studies. Based on these datas, JRF104 may overcome resistance caused by AXL and FLT3-TKD mutation, representing a novel promising clinical antitumor agent for treating AML patients with FLT3 mutations. Citation Format: yuquan wei, Xiawei Wei, Yanzhong Zhang, Wanru Qin, Xiangyu Fu, Lihong Hu, Yuanfeng Xia, Jian Zuo, Zhong Xiong, Shanshan Yin, Shaohua Qin, Ge Dong, Ying Chen, Charles Ding, Chichung Chan, Shuhui Chen. Development of new FLT3/AXL dual inhibitor JRF104 with potent FLT3-TKD inhibition [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 3766.

Abstract LB-088: A STAT3 selective targeted protein degrader decreases the immunesuppressive tumor microenvironment and drives antitumor activity in preclinical models

Abstract Signal transducer and activator of transcription 3 (STAT3) has been implicated in multiple aspects of tumorigenesis. In addition to increasing cancer cell proliferation and survival, constitutively activated STAT3 is proposed to regulate cross-talk between tumor, stroma and immune cells to promote immune-evasion. STAT3 activity in tumors promotes the production of immune-suppressive factors that activate STAT3 in diverse immune-cell subsets. Mechanistically, genetic studies support a direct role of activated STAT3 in regulating myeloid cell differentiation to contribute to an immune-suppressed tumor microenvironment (TME) (Kortylewski et al.; Nat. Med. 2005 and Curr. Opin. Immunol. 2008) Therefore, STAT3 is a highly attractive target for immune-oncology. Here, we demonstrate that the degradation of STAT3 with a potent and selective STAT3 degrader reverses immune suppression in preclinical models. KYM-003 is a heterobifunctional molecule that hijacks the ubiquitin-proteasome system (UPS) for rapid STAT3 degradation. KYM-003 robustly degraded STAT3 in both human peripheral blood mononuclear cells (hPBMC)-derived monocytes and lymphocytes with DC50 &amp;lt; 100 nM. Degradation in hPBMCs was highly selective for STAT3 vs &amp;gt;10,000 other detected proteins (including all other STAT family members) as evaluated by deep tandem mass tag proteomics. IL-6 treatment of hPBMCs upregulates STAT3 phosphorylation, resulting in transcription of genes involved in myeloid cell-mediated immune suppression, such as IL-10 and CD163, a marker of M2 macrophage differentiation. Treatment of cells with KYM-003 at DC90 for 6 hours abrogated the IL-6 induced up-regulation of immune-suppressive gene signatures consistent with a role of STAT3 in mediating an immune suppressive environment by regulating macrophages and other myeloid cells. In several tumor cell lines, degradation of STAT3 by KYM-003 led to strong downregulation of PD-L1. Importantly, in the BioMap StroNSCLC (DiscoverRx), a co-culture system composed of NCI-H1299 lung cancer cells, hPBMCs and primary human fibroblasts that models immune-suppressed TME biology, KYM-003 treatment decreased angiogenic and immune-suppressive cytokines, including VEGF and IL-10, and promoted the pro-inflammatory anti-tumor cytokines IL-2, IFNg and TNFa. Lastly, administration of KYM-003 to mice bearing syngeneic tumors exhibited anti-tumor activity as monotherapy in the CT26 syngeneic model. We have shown that targeting STAT3 for degradation may have a role in restoring an immune-permissive environment in tumors by both rescuing the suppressed immunologic microenvironment and directly downregulating immune checkpoint signals in tumor cells. These data support STAT3 degraders as a promising new therapeutic modality as immune-oncology agents.Citations: Kortylewski et al (2005) Nat. Med, 11(12):1314-21. Kortylewski et al (2008) Curr. Opin. Immunol, 20(2):228-33 Citation Format: Alfredo Csibi, Bin Yang, Yogesh Chutake, Karen Yuan, Michele Mayo, Veronica Campbell, Alice McDonald, Scott Rusin, Kirti Sharma, Hari Kamadurai, Henry Li, Mike Sintchak, Sean Zhu, Sharon Townson, Anthony Slavin, Haojing Rong, Phillip Liu, Chris De Savi, Jared Gollob, Duncan Walker, Nan Ji, Nello Mainolfi. A STAT3 selective targeted protein degrader decreases the immunesuppressive tumor microenvironment and drives antitumor activity in preclinical 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 LB-088.

Ned-170: A combination anti-cancer therapy targeting late stage, heavily pre-treated solid tumor malignancies.

141 Background: NED-170 is a 7-component regimen of both marketed drugs and nutraceuticals with established safety and tolerability profiles and antineoplastic activity. These components, including metronomically-dosed cyclophosphamide (CTX), metformin, naltrexone, alpha lipoic acid, genistein, curcumin, and melatonin, target four key pathways driving tumor growth and metastasis. Methods: NED-170 was evaluated pre-clinically for tolerability and efficacy using a murine CT-26 syngeneic xenograft model. After tumor volume reached 80-120 mm³, mice were randomized into three groups of 10. Experimental arms included: NED-170 allometrically scaled to provide comparable exposure to human clinical trial doses, vehicle, CTX alone, or an anti-PD-1 antibody. In parallel to these studies, a compassionate expanded access program (EAP) was established in 2013 as a precursor to formal clinical trials. To date, 21 pts. with stage IIIc/IV advanced solid tumor malignancies (5 ovarian, 6 sarcoma, 4 Br. Ca, 2 NSCLC, &amp; 4 other cancers) who either sought alternatives to standard-of-care (SOC) chemotherapy or whose tumors progressed through available SOC options have been enrolled. Results: In pre-clinical studies, NED-170 demonstrated 78% tumor growth inhibition (TBI) which exceeded vehicle in a statistically significant fashion (p &lt; 0.001). In contrast, anti-PD-1 treatment failed to achieve anti-tumor activity and CTX alone achieved 50% TBI. In parallel, 21 EAP pts. have been treated with a combined 1,217 months (mos.) of NED-170 therapy (median = 11.8 mos; range = 1.0-60.2). For all pts., the regimen has been safe and well-tolerated with no drug-related grade-3-4 adverse events or dose reductions/discontinuations reported. Observational data reported by pts. or physicians demonstrated that 81% of pts. derived benefit and improved quality of life in this late stage setting. Conclusions: Based on these encouraging data, NED-170 may provide late stage cancer pts. with a safe, effective, and lower cost alternative to standard chemotherapy. A multinational phase 1-2b clinical trial commencing in late 2019 is planned.

1223P - GI101, a novel triple-targeting bispecific CD80-IgG4-IL2variant fusion protein, elicits synergistic anti-tumour effects in preclinical models

BackgroundDespite mounting interest in combination immunotherapy including immune checkpoint inhibitors (ICIs) to improve anti-tumor responses, clinical translation has been disappointing. To address the significant unmet needs remaining, we designed GI101 comprising the extracellular domain of CD80 acting as a dual checkpoint (CTLA4/PD-L1) inhibitor, together with a long acting IL2v that preferentially binds to the IL2Rβ. MethodsAffinity of GI101 for IL2Rs, CTLA4, and PD-L1 was determined by SPR. Immune cell proliferation was analyzed by CFSE assay and in vivo immune profiling. In vivo efficacy of GI101 were performed in a tumor-bearing mouse model. ResultsGI101 exhibits high affinity to both CTLA4 (Kd, 2.9nM), PD-L1 (Kd, 8.5nM), and preferential binding to IL2Rβ (Kd, 28.4nM). GI101 induces the robust stimulation of in vitro and in vivo CD8+ T and NK cell proliferation without a significant increase in Treg cells. GI101 has high binding affinity to CTLA4 acting as a decoy ligand, thereby enhancing the interaction between endogenous CD80 and CD28, leading to the activation of T cells. GI101 elicits improved restoration of immune functions compared to a CD80-Fc in in vitro settings using human PBMCs co-cultured with PD-L1hi tumor cells. In the CT26 tumor-bearing mice, GI101 was superior at inhibiting tumor growth when compared to a combination of aPD-1/aCLTA4 in association with a profound increase in CD8+ T and NK cells without causing an increase in Tregs in the TME. In addition, a dose-dependent anti-tumor effect was observed in CT26 syngeneic models. Furthermore, isolated mortality was observed in the aPD-1/aCTLA4 combo-treated group whereas GI101-treated group has no evidence for toxicity associated with IL2 activity including vascular leakage syndrome and cytokine storm. ConclusionsThe complementary modes of action of GI101 via dual checkpoint blockade with IL2 activity to enhance the proliferation and activation of Teff and NK cells is projected to translate into superior clinical efficacy and safety as indicated even in ‘cold tumor’ models. GI101 has promising potential to replace first-generation ICIs as a monotherapy or in combination with chemo/radiotherapies and other immunotherapies. Legal entity responsible for the studyGI innovation. FundingGI innovation. DisclosureJ.C. Park: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. K.H. Pyo: Research grant / Funding (institution): Yonsei Medical Center. Y.J. Song: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. W.J. Lee: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. J. Kim: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. H.N. Ji: Full / Part-time employment: GI innovation. S.S. Park: Full / Part-time employment: GI innovation. Y.J. Koh: Full / Part-time employment: GI innovation. K. Lee: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. B. Cho: Research grant / Funding (institution): Yonsei Medical Center. B. Chun: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. Y.M. Oh: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. B. Yang: Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. S.Y. Nam: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation. M.H. Jang: Leadership role, Shareholder / Stockholder / Stock options, Full / Part-time employment: GI innovation.

Abstract 3271: A systemically administered, conditionally active TLR8 agonist for the treatment of HER2-expressing tumors

Abstract Clinical development of systemically administered myeloid cell agonists has been hindered by acute toxicities due to peripheral activation of the targeted cell types. Intratumoral administration, the route of delivery typically used for innate immune/myeloid cell agonists, is limited by tumor accessibility and a dependence on abscopal responses. Here, we describe an example of a new composition class, termed ImmunoTAC, that allows for systemic delivery of an immune modulator with activity localized to tumor sites and secondary lymphoid structures. Agonism of TLR8 in human myeloid cells drives anti-tumor immunity by inducing direct macrophage killing of tumor cells, repolarizing suppressive myeloid cell populations to a pro-immunogenic phenotype, and inducing dendritic cells to drive tumor-specific CTL responses. SBT6050 is an ImmunoTAC comprised of a novel, potent TLR8 agonist conjugated to a HER2-directed monoclonal antibody. SBT6050 is designed to activate human monocytes and macrophages only in the presence of HER2pos tumor cells with moderate (2+ by IHC) or high (3+ by IHC) expression levels. This activity is dependent upon the ability of the Fc domain of the antibody to engage Fcγ receptors on the surface of the myeloid cells. Additionally, SBT6050 potently activates conventional dendritic cells that, in turn, drive a Th1/CTL program in T cells. Systemic delivery of a SBT6050 surrogate in mice shows robust single agent efficacy in tumor models intrinsically resistant to checkpoint blockade, such as the previously characterized HER2+ CT26 syngeneic model. The intratumoral immune response is characterized by robust activation of tumor-associated myeloid cells, infiltration of neutrophils, persistent increases in local cytokine and chemokine production, decreases in Treg infiltrate, and the generation of a potent, neo-Ag specific anti-tumor CTL response. Mice cured with single-agent treatment are resistant to tumor rechallenge, demonstrating induction of long-lived immunological memory. In contrast to observations with small molecule TLR8 agonists, ImmunoTAC does not induce peripheral cytokine production or associated CRS-like toxicity in mice, consistent with the localized activity of the molecule. Silverback’s lead candidate, SBT6050, is currently in preclinical development for patients with moderate or high HER2-expressing tumors. More broadly, the data presented here describe a novel therapeutic modality that allows for systemic administration of immune modulators with tissue-localized activity. Citation Format: Kara Moyes, Ty Brender, Sean W. Smith, Hengyu Xu, Ben Setter, Li-Qun Fan, Rebecca Brunette, Justin Killebrew, Phil Tan, Craig Coburn, Peter Baum, Valerie Odegard. A systemically administered, conditionally active TLR8 agonist for the treatment of HER2-expressing tumors [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 3271.

Abstract 100: Novel selective PI3Kγ inhibitor AZD3458 promotes anti-tumor immune responses and reverts resistance to immunotherapy in checkpoint blockade refractory preclinical models

Abstract The PI3Kγ isoform is a key regulator of immune cell proliferation, survival, migration and activation. PI3Kγ inhibitors have the potential to treat a variety of diseases including respiratory, inflammatory, metabolic disorders and cancer. In the context of cancer PI3Kγ inhibition re-polarizes macrophages to an immuno-stimulatory phenotype activating a T-cell mediated tumor immune response. Selective PI3Kγ inhibitors have potential as monotherapy and in combination with checkpoint inhibition to overcome resistance and to enhance efficacy of checkpoint inhibitors. AZD3458 is a highly selective PI3Kγ clinical candidate kinase inhibitor with a cellular IC50 of 8nM with a 100-fold selectivity over PI3Kδ. In vitro, AZD3458 inhibits pAKTS308/S473 in human macrophages and mouse CD11b activation at 32nM and 30nM free IC50, respectively. At these concentrations AZD3458 reverses macrophage polarization increasing IL12/IL-10 ratio and does not impact T cell proliferation and function analysed by gene expression profiling, cytokine quantification and flow cytometry. Oral administration of AZD3458 (20mg/Kg BID) remodeled the tumor microenvironment in 4T1 orthotopic breast tumor model. AZD3458 decreased tumor associated macrophages by 20% compared to vehicle and reduced overall protein expression of immunosuppressive markers CD206 and PD-L1 measured by flow cytometry. In addition, AZD3458 reduced MDSC/neutrophil activation and promoted cytotoxic T-cell activation in vivo, measured by GzmB and Perforin mRNA and protein expression. Consistent to the observed tumor remodeling, combination with checkpoint inhibitors α-PD-1 or α-PD-L1 antibodies (10mg/kg 3x week) had greater anti-tumor effects than checkpoint inhibitor alone in 4T1, LLC, CT-26 and MC-38 mouse syngeneic models. These data demonstrate that AZD3458 can reverse myeloid suppressive tumor microenvironment and revert tumor resistance to immunotherapy in myeloid-enriched immunosuppressive tumor types. Citation Format: Larissa S. Carnevalli, Danielle Carrol, Michele Moschetta, Pablo Morentin Gutierrez, Cristina Gardelli, Molly A. Taylor, Antonio Montoya, Adina Hughes, Matthew King, Teresa Klinowska, Simon T. Barry. Novel selective PI3Kγ inhibitor AZD3458 promotes anti-tumor immune responses and reverts resistance to immunotherapy in checkpoint blockade refractory preclinical models [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 100.

Abstract 558: MERTK-specific antibodies that have therapeutic antitumor activity in mice disrupt the integrity of the retinal pigmented epithelium in cynomolgus monkeys

Abstract MERTK, a member of the TAM (TYRO3, AXL, MERTK) family of receptor tyrosine kinases, is a pleiotropic immune modulator that controls efferocytosis. Engagement of MERTK with its ligand GAS6, found anchored to phosphatidylserine exposed on the outer membrane of apoptotic cells, triggers MERTK phosphorylation and signaling events that culminate in the removal of apoptotic debris. Recent studies have highlighted the expression of MERTK on tumor-associated macrophages, and Mertk-deficient mice show reduced tumor cell growth accompanied by inflammatory cytokine production and alterations in macrophage activation. Thus, MERTK has emerged as a promising therapeutic target for augmenting innate antitumor immune responses. MERTK is also expressed in retinal pigmented epithelium (RPE) cells of the eye where it mediates phagocytosis of photoreceptor outer segment fragments. Mutations in MERTK that disrupt its expression or kinase activity lead to marked retinal degeneration and blindness in mice, rats, and humans. Due to known differences in blood-retinal permeability, we explored whether therapeutic antibodies targeting MERTK could inhibit macrophage-mediated efferocytosis and promote antitumor activity while sparing RPE toxicity. A diverse panel of high-affinity antibodies was developed to explore MERTK blockade in vitro and in vivo. Multiple antibodies disrupted MERTK-GAS6 binding and blocked human and murine macrophage-mediated efferocytosis. Two antibodies targeting distinct GAS6 binding epitopes were selected for further characterization. Both antibodies demonstrated antitumor activity in murine CT26 and MC38 syngeneic colorectal cancer models and led to alterations in immune cell-related gene expression. To investigate potential effects on RPE biology with MERTK antibodies, a multi-dose, 4-week cynomolgus monkey study with several in-life and post-mortem ophthalmologic endpoints was designed. While no abnormal ophthalmic or electroretinography (ERG) findings were detected, all animals treated with either MERTK antibody at all doses showed histological abnormalities of the retina, including vacuolation of the outer segments of photoreceptors, displacement of RPE cells, and single cell necrosis of the outer nuclear layer. These data suggest that inhibition of efferocytosis by antibody-mediated blockade of MERTK can promote immune activation and inhibit tumor growth in vivo; however, retinal toxicity consistent with histological observations made in Mertk mutant animals is an on-target effect. As several therapeutics that block MERTK function are currently in preclinical development, a thorough evaluation of retinal toxicity is warranted. Citation Format: Kerry F. White, Matthew Rausch, Jing Hua, Katherine H. Walsh, Christine E. Miller, Christopher C. Wells, Devapregasan Moodley, Benjamin H. Lee, Scott C. Chappel, Pamela M. Holland, Jonathan A. Hill. MERTK-specific antibodies that have therapeutic antitumor activity in mice disrupt the integrity of the retinal pigmented epithelium in cynomolgus monkeys [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 558.

Abstract 1214: Enhancement of anti-PD-1 antitumor efficacy in syngeneic preclinical models by the angiogenesis inhibitor lucitanib

Abstract Background: Lucitanib is a small molecule inhibitor of multiple tyrosine kinases, including some that promote angiogenesis. Lucitanib monotherapy has demonstrated potent tumor growth inhibition in xenograft models of various carcinomas, mediated predominately through the inhibition of angiogenesis. In addition to its role in angiogenesis, vascular endothelial growth factor (VEGF) and other angiogenic factors are thought to dampen antitumor immune responses by promoting inhibitory immune subsets, suppressing dendritic cell maturation, and altering lymphocyte development and trafficking. In these studies, we further characterized lucitanib’s kinase selectivity and investigated the antitumor efficacy of lucitanib in combination with anti-PD-1 therapy. Methods &amp; Results: The inhibitory profile of lucitanib was evaluated against 376 wild-type kinases in functional enzymatic assays. At 500 nM, lucitanib demonstrated &amp;gt;35% inhibition of 5.1% (19/376) of the kinases evaluated. The IC50 values of lucitanib against VEGF receptors 1-3 (VEGFR1-3), platelet-derived growth factor receptors alpha/beta (PDGFRα/β), and fibroblast growth factor receptors 1-3 (FGFR1-3) ranged from 8.96 to 95.7 nM. In addition, inhibition was observed against DDR1, CSF1R, RET, and KIT. Cell-based kinase assays confirmed that lucitanib is a potent and selective VEGFR1-3, PDGFRα/β, and FGFR1-3 inhibitor. Lucitanib antitumor activity was evaluated in combination with an anti-PD-1 antibody in a panel of syngeneic murine models. The combination treatment was generally well tolerated. In the H22 hepatocellular carcinoma model, the tumor growth inhibition in animals treated with lucitanib (10 mg/kg once daily), anti-PD-1 (5 mg/kg biweekly), or the combination was 58.6%, 50.4%, and 81.4%, respectively, following 16 days of treatment. The median survival time (MST) for lucitanib and anti-PD-1 treated animals was 35.0 and 46.5 days, respectively, whereas the MST had not been reached for the combination group when the study was terminated on day 63. Importantly, the combination treatment resulted in 2/10 mice that were tumor free at the end of the study; however, there were no tumor-free animals in the groups treated with the single agents. Antitumor efficacy was also greater with the combination of lucitanib and anti-PD-1 therapy than with treatment with the single agents in the MC38 and CT26 syngeneic murine models of colon cancer. Conclusions: Lucitanib demonstrated potent and selective targeting of VEGFR1-3, PDGFRα/β, and FGFR1-3 and enhanced antitumor activity when combined with PD-1 inhibition in multiple syngeneic models. Results from these studies support the clinical development of combined lucitanib and anti-PD-1 treatments to further evaluate the safety and efficacy of the combination in multiple tumor types. Citation Format: Rachel L. Dusek, Liliane Robillard, Thomas C. Harding, Andrew D. Simmons, Minh Nguyen. Enhancement of anti-PD-1 antitumor efficacy in syngeneic preclinical models by the angiogenesis inhibitor lucitanib [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 1214.

Abstract 1859: Quxie capsule inhibits the colon tumor growth through modulating the gut microbiome and tumor myosin 11 expression

Abstract Traditional Chinese Herbal Medicine formula, such as the Quxie Capsule (QXC), has been used for advanced colorectal cancer treatment with favorable clinical outcome at Xiyuan Hospital in Beijing, China. However, the mechanism(s) underlying the anticancer effect of QXC in colon cancer still remain unclear, which hampers its optimal use for the treatment of colon cancer. The purpose of this study is to examine whether, and if yes, how QXC exerts anti-tumor activity via alteration of gut microbiota and related pathways. The antitumor efficacy of QXC was tested in CT-26 syngeneic mouse colon cancer models. Gut microbiome was measured by 16sRNA sequencing in fecal samples. Signaling protein expression profile in tumor tissues was assessed by RPPA and validated by Western blot. QXC gavaged to mice carrying CT26 tumors for 2 weeks significantly reduced the average tumor weight (0.92 ± 0.15g) compared to that of the vehicle control (1.57 ± 0.1689, p &amp;lt; 0.05). In contrast, when mice bearing CT26 tumor were pretreated antibiotics, the tumor weight (0.51±0.11g) in QXC treated mice was not statistically significantly smaller than that of vehicle control (0.76±0.13g, p&amp;gt;0.05). Mice pretreated with vehicle for 12 days then transplanted with fecal from QXC treated mice had smaller tumor compared with vehicle only group (p&amp;lt;0.05). Similarly, the tumor in mice pretreated with QXC and followed by transplantation of fecal from vehicle control mice grew faster than that of QXC treated alone, but not statistically significant (p&amp;gt;0.05). The result of 16sRNA sequencing showed that the ratio of Firmicutes over Bacteriodetes (F/B) was decreased by 2.03 folds in tumor-bearing mice compared to non-tumor-bearing mice (p&amp;lt;0.05). Intriguingly, QXC led to an increase of Firmicutes and reduction of Bacteriodetes and resulted in a 2.0-fold increase in the ratio of F/B compared to that prior to QXC treatment (p&amp;lt;0.05). The OTUs were significantly and markedly decreased (p&amp;lt;0.05) after antibiotics treatment. Both alpha and beta diversity in antibiotic and QXC co-treated mice were similar to that of the vehicle control and antibiotic co-treated group. The result of RPPA showed that protein Myosin 11, which was downregulated in fiber-deprived colon mucosa, was significantly elevated in QXC treated tumor tissues compared to that of control (p&amp;lt;0.05). In contrast, Myosin 11 expression was lower in the tumor tissues of antibiotics and QXC co-treated mice compared to that of antibiotics and vehicle control co-treatment group. QXC elicited upregulation of Myosin 11 protein expression was further validated in QXC treated CT-26 cells in vitro. In conclusion, QXC inhibited the growth of colon tumors, which could be due to its ability to rebalance the intestinal microbiota and upregulate the myosin 11 level in the syngeneic colon cancer model. Note: This study was partially founded by National Natural Science Foundation of China (No. 81373824). Citation Format: Dongmei Chen, Daoyuan Wei, Yufei Yang, Peiying Yang. Quxie capsule inhibits the colon tumor growth through modulating the gut microbiome and tumor myosin 11 expression [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 1859.

Abstract 5019: Allosteric inhibition of SHP2 suppresses CSF1R signaling and selectively reduces viability of M2 tumor associated macrophages contributing to anti-tumor immunity

Abstract The protein-tyrosine phosphatase SHP2 promotes oncogenic RAS/MAPK pathway activation in different tumor types. In immune cells, SHP2 binds to phosphorylated ITIM and ITAM domains on regulatory receptors, including PD-1. We have shown that RMC-4550, a SHP2 allosteric inhibitor, attenuates tumor growth in syngeneic mouse tumor models with effects equivalent to, or greater than those of checkpoint inhibitors. Consistent with anti-tumor immunity as the mechanism, tumor growth inhibition (TGI) was not observed in immunocompromised mice. TGI was associated with changes in the tumor immune microenvironment, in both the adaptive and innate arms. Similar to checkpoint blockade, the frequency of CD8+T cell infiltrates was increased upon treatment with RMC-4550. Polarized macrophage populations were significantly shifted in favor of antitumor immunity, with marked increases in M1 and decreases in M2 cells, effects not seen with checkpoint blockade. The objective of the present study was to determine if the effect on tumor-associated macrophage (TAM) polarization is secondary to a release of the biochemical brakes on adaptive immunity and/or a result of a direct effect of SHP2 on TAMs. In vivo administration of RMC-4550 in the CT26 syngeneic model significantly decreased M2, and increased M1, TAM after CD8+T cell depletion or IFNγ blockade, similar to the response in control animals. In vitro, SHP2 inhibition attenuated CSF1R signaling in murine bone marrow-derived macrophages and selectively induced apoptotic cell death in M2, but not M1, polarized macrophages. In addition, RMC-4550 treatment in vitro reduced the suppressive potential of human M-MDSCs. Collectively these observations suggest that SHP2 inhibition directly impacts the survival and function of suppressive monocytic immune cells. Given the effect of SHP2 inhibition on the CSF1R signaling pathway, we assessed whether the anti-tumor activity of RMC-4550 was similar to that of CSF1R blockade. In contrast to RMC-4550, anti-CSF1R antibody did not induce a significant delay in CT26 tumor growth. These results confirm that RMC-4550 has pleiotropic effects on the immune system including modulation of both adaptive and innate mechanisms. In summary, we propose that SHP2 plays a central role in inducing immune suppression in the tumor microenvironment by both inhibiting T cells and supporting the viability of pro-tumorigenic macrophages. Thus, SHP2 inhibition represents a novel investigational strategy with dual activity: direct inhibition of cancer cell growth in certain tumors as well as promotion of an anti-tumor immune response by direct transformation of the tumor immune microenvironment. Tumors that are intrinsically dependent upon SHP2 and exhibit a myeloid-rich microenvironment could be particularly susceptible to this dual-mechanism therapeutic strategy. Citation Format: Elsa Quintana, Chris J. Schulze, Tiffany J. Choy, Darienne R. Myers, Kasia Mordec, Dylan Daniel, Mark A. Goldsmith, Jan A. Smith. Allosteric inhibition of SHP2 suppresses CSF1R signaling and selectively reduces viability of M2 tumor associated macrophages contributing to anti-tumor immunity [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 5019.

Design, synthesis and antitumor study of a series of N-Cyclic sulfamoylaminoethyl substituted 1,2,5-oxadiazol-3-amines as new indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors

Indoleamine 2, 3-dioxygenase 1 (IDO1) plays a key role in tryptophan catabolism which is an important mechanism in immune tolerance. The small molecule epacadostat is the most advanced IDO1 inhibitor, but its phase III trials as a single agent or in combinations with PD-1 antibody failed to show appreciable objective responses. To gain more insight on the antitumor efficacy of IDO1 inhibitors, we have designed a series of analogues of epacadostat by incorporating a cyclic aminosulfonamide moiety as the sidechain capping functionality. Compound 5a was found to display significant potency against recombinant hIDO1 and hIDO1 expressed HEK293 cancer cells. This compound has improved physico-chemical properties, acceptable PK parameters as well as optimal cardiac safety. Similar to epacadostat, 5a is ineffective as single agent in the CT-26 syngeneic xenograft model, however, the combination of 5a with PD-1 antibody showed both elevated tumor growth inhibition and prolonged median life span.

Abstract A37: Immuno-oncological efficacy of RXDX-106, a novel TAM (TYRO3, AXL, MER) family small-molecule kinase inhibitor

Abstract Background: The TAM family of receptor tyrosine kinases (RTKs), TYRO3, AXL, and MER, have emerged as attractive targets for cancer therapy. In cancer cells, overexpression of TAM RTKs is associated with resistance and a mesenchymal phenotype. In immune cells, TAM RTKs play a key homeostatic role as negative regulators of immune responses, contributing to the evasion of cancer cells from immune surveillance. RXDX-106 is a potent and selective TAM family inhibitor in preclinical development. Previously, in several syngeneic tumor models, we have demonstrated that RXDX-106 alone inhibited tumor growth, whereas such benefit was reduced when the tumors grew in immunocompromised athymic nude mice. In addition, RXDX-106 induced favorable polarization of the tumor infiltrating immune cells towards an anti-cancer phenotype. Here we sought (1) to evaluate the efficacy of RXDX-106 as a single agent and in combination with immune checkpoint inhibitors; (2) to identify the immuno-modulatory mechanisms of action; (3) to explore the reciprocal regulation of TAM expression on cancer and immune cells in the tumor microenvironment; and (4) to decipher how pharmacological inhibition of TAM signaling pathways on both tumor and immune cells would be beneficial, given their complex regulation and intimate relationship in the tumor microenvironment. Methods and Results: In the present study, we demonstrated that tumor growth inhibition in an MC38 model was associated with a significant increase in tumor associated M1 macrophages, an increase in CD169hi antigen presenting macrophages, and upregulation of PD-L1. In addition, we observed a higher ratio of CD8+/CD4+ T cells and increased expression of CD69 and PD-1 on CD8+ T cells, all indicative of activation of cytotoxic T cells. Finally, we observed an increase in Granzyme B and IFNγ with a concomitant decrease in VEGF in tumor cell lysates, evidence supporting T cell activation and M1 polarization of macrophages. Furthermore, in a CT26 syngeneic model, we demonstrated that RXDX-106 inhibited tumor growth as a single agent, and the effect was further potentiated by combination therapy with immune checkpoint inhibitors, as evidenced by upregulation of anti-tumor gene expression patterns, upregulation of anti-tumor cytokines in the tumor cell lysates, and an increase in T cell function. Finally, in an AXL-driven tumor model, we demonstrated that AXL expressing tumors induced a pro-tumorigenic immune environment, and treatment with RXDX-106 resulted in complete tumor regression and re-polarization of macrophages towards an M1, anti-tumor phenotype. Conclusion: RXDX-106 has the potential to restore and enhance immune function in macrophages and T cells, resulting in repolarization of the immune response towards an anti-tumor microenvironment. The unique mechanism of activating both innate and adaptive immunity, plus regulating cross-talk between immune cells and tumor cells by RXDX-106 supports clinical development of RXDX-106 to potentially treat a wide variety of cancers. Citation Format: Yumi Yokoyama, Erin D. Lew, Colin Walsh, Jack Lee, Joanne Oh, Elizabeth A. Tindall, Robin Nevarez, Amy Diliberto, Heather Ely, Ruth Seelige, Amanda Albert, Jack Bui, Gary Li. Immuno-oncological efficacy of RXDX-106, a novel TAM (TYRO3, AXL, MER) family small-molecule kinase inhibitor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A37.

Abstract 3917: Potentiating effect of reovirus in anti-PD1 therapy in colorectal cancer

Abstract Background: Microsatellite instability (MSI) high colorectal cancers (CRCs) have a deficiency in mismatch repair (MMR) and increased levels of PD-L1, LAG-3, and IDO, and respond positively to anti-programmed death (PD) therapy. MSI low or microsatellite stable (MSS) CRCs that make up majority of tumors in clinical practice have not seen any benefit with PD inhibition. MSS CRC have higher proportion of KRAS oncogenic mutations as compared to MSI CRC. Reovirus, a naturally occurring oncolytic double-stranded RNA virus, has intrinsic preference for replication in KRAS mutant cells causing apoptosis in CRC. Current study was designed to investigate if reovirus could potentiate a beneficial effect of anti-PD therapy in MSS CRC. Methods: An array of CRC cell lines were screened for sensitivity to reovirus by MTT assay and expression of stem cell markers by RNA-seq and FACS. Based on MSI and KRAS status, four cell lines were explored further. Cells were treated with 5MOI reovirus for 48hr and expression of PD-L1 and PD-L2 with and without the potentiating effect of IFN-γ was assayed using FACS and qPCR. Combinatorial effect of reovirus with anti-PD-1 agent was studied in syngeneic models of BALB/c (CT26; KRASmut, MSS) and C57BL/6 (MC38; KRASwt, MSI) mice. The mice were grouped as control (PBS/IgG2A isotype control), reovirus, anti-mouse PD-1 antibody, and combination. Reovirus was used at a dose of 10 million/100 uL daily and anti PD-1 antibody was given i.p 200 ug/100 uL twice a week. Survival data and tumor volumes were recorded. HCT116 cells were xenografted in nude mice and treated with equivalent dose of reovirus. At the end point IHC was performed on excised paraffin-fixed tumor tissue with CD8 and granzyme antibodies. Results: Administration of reovirus was found to be most effective in cell lines tested. HCT116 (MSI &amp; KRASmut), SW620 (MSS, KRASmut), LIM2405 (MSI, KRASwt) and HT29 (MSS, KRASwt) were chosen based on expression of CD133, CD44 and CD24. HCT116, LIM2405 and SW837 revealed increased and HT29 reduced expression of PD-L1 upon treating with reovirus. Survival data and tumor volume measurements showed better potentiating effect of reovirus on anti-PD-1 therapy in CT26 syngeneic model when compared with MC38. While single-agent therapy did not increase survival, the combination did improve survival with significance vs. control in both BALB/c (median 42 vs. 16 days, p=0.003) and C57BL/6 (median 24 vs. 17 days, p=0.02). The reovirus-treated xenografted tumor tissue showed a higher infiltration of T lymphocytes as confirmed by CD8-positive and intensified granzyme staining. Conclusion: Reovirus as single agent is more potent in KRASmut CRC. Syngeneic mice models proved synergistic anticancer effect of reovirus and anti-PD1 agent combination. Reovirus administration increased PD-L1 expression in CRC cells; possible mechanistic rationale for synergistic efficacy. Citation Format: Titto A. Augustine, Radhashree Maitra, Peter John, Sanjay Goel. Potentiating effect of reovirus in anti-PD1 therapy in colorectal cancer [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 3917.

Abstract 192: Preclinical evaluation of TQBWX220, a small-molecule inhibitor of IDO1

Abstract Objective: Indoleamine-2, 3-dioxygenase-1 (IDO1) catalyzes conversion of tryptophan to kynurenine (Kyn), which triggers signaling through GCN2, mTOR and AHR and affects differentiation and proliferation of T cells. Expression of IDO1 by tumor cells or host APCs can inhibit tumor-specific effector CD8+ T cells and enhance the immunosuppressing activity of regulatory T cells. Targeting the IDO pathway via inhibition of IDO1 or blocking its downstream signaling pathways is recognized as one of the prime immunomodulation approaches to unleash antitumor immunity in the tumor microenvironment. As reported, inhibition of IDO1 pathway may also synergize with inhibitory Abs of immune checkpoint proteins, PD-1 and CTLA-4, etc. We report herein TQBWX220, a potent small-molecule inhibitor of IDO. Method: Potency of TQBWX220 was determined by corresponding IDO1 biochemical and cell assays. Antitumor efficacy of TQBWX220 was evaluated in the syngeneic CT26 xenograft colon cancer model. CT26 cells were engrafted onto the back of BALB/c mice. BID oral administration of vehicle, positive control (INCB024360, 100 mg/kg) and TQBWX220 at different doses were performed. Tumor volumes and mouse body weight were measured at time points after the start of the treatments. Other assays were carried out by using standard methods. Result: TQBWX220 displayed good IDO1 enzymatic (IDO1 IC50=22 nM) and cellular (Hela EC50=117 nM) potency. It has excellent solubility (&amp;gt;500uM pH7.4) and low inhibition on CYPs and hERG. TQBWX220 significantly inhibited tumor growth at tested doses (60% TGI at 50 mpk/PO BID and 73% TGI at 100 mpk /PO BID) (P&amp;lt;0.05) in the CT-26 syngeneic model. As a result of IDO1 inhibition, reduction of Kyn level in the tumor was also observed. Conclusion: The promising preclinical antitumor results and its favorable DMPK properties indicate that TQBWX220 has high potential in the clinic as a cancer immunotherapy in combination with checkpoint inhibitors such as anti-PD1 antibodies. Citation Format: Shilan Liu, Guibai Liang, Dahai Wang, Shuhui Chen, wenyuan Qian, Chi-Chung Chan, Xiquan Zhang, Lin Yang, Xin Tian, Yong Gao, Tiantian Dong, Mei Liu, Lihua He. Preclinical evaluation of TQBWX220, a small-molecule inhibitor of IDO1 [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 192.