BackgroundProstaglandin E2 (PGE2) is one of the most abundant prostaglandins, with crucial roles in normal and pathologic physiology. Especially, PGE2 levels are abnormally elevated in many cancers, and high levels of PGE2 are known to be pro-tumorigenic, likely due to the immune suppressive effect in the tumor microenvironment.1–4 There are four types of PGE2 receptors; EP1, EP2, EP3 and EP4. Among them, EP2 and EP4 activate adenylate cyclase and increase cAMP levels, which induce the cAMP-dependent protein kinase (PKA) signaling pathway. EP2 and EP4 are expressed in various immune cells (e.g. macrophages, dendritic cells, NK cells and CTLs), and genetic and pharmacological inhibition of EP2 and EP4 increases immune activity and suppresses tumor growth.MethodsTo evaluate the binding affinity against EP2 and EP4, a radioligand binding assay was conducted using EP2 or EP4 transfected HEK293 cells. Cell membrane homogenates were incubated with [3H]PGE2 in the absence or presence of the test compounds. Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters and rinsed several times with cold Tris-HCl. The filters were dried then counted for radioactivity in a scintillation counter using a scintillation cocktail. The results were expressed as a percent inhibition of the control radioligand specific binding.The antagonistic activity against EP2 and EP4 was assessed via LANCE Ultra cAMP assay (PerkinElmer). HEK293 cells overexpressing EP2 or EP4 were seeded into the plate and treated by PGE2 and compounds. After 30 minutes of incubation, cAMP levels were measured by FRET signal using Varioskan plate reader, following the manufacturer’s protocol.Anti-tumor activity of KT-00113 was evaluated using LLC1 syngeneic model. When tumor volume reached approximatively 100 mm3, mice were treated PO, QD. Tumor size was measured twice every week.ResultsSystematic structure-activity relationship (SAR) investigation identified novel EP2 and EP4 dual antagonists. The most promising compound KT-00113 possesses high potency against both EP2 and EP4, while maintaining high selectivity over other prostanoid receptors. In vitro and in vivo ADMET studies show that KT-00113 has a favorable profile, apt for further examination in in vivo cancer models and immune cell function in tumors.ConclusionsKT-00113, a highly potent and selective EP2/4 dual antagonist has strong potential to become the best-in-class immune suppression lifting cancer immunotherapy and may be suitable for further development in a clinical setting.ReferencesCordes T, Hoellen F, Dittmer C, Salehin D, Kummel S, Friedrich M, Koster F, Becker S, Diedrich K, Thill M. Correlation of prostaglandin metabolizing enzymes and serum PGE2 levels with vitamin D receptor and serum 25(OH)2D3 levels in breast and ovarian cancer. Anticancer Res;32 ( 2012):351–357.Diakowska D, Markocka-Maczka K, Nienartowicz M, Lewandowski A, Grabowski K. Increased level of serum prostaglandin-2 in early stage of esophageal squamous cell carcinoma, Arch Med Sci ( 2014);10:956–961.Akbari N, Ghorbani M, Salimi V, Alimohammadi A, Khamseh ME, Akbari H, Nourbakhsh M, Sheikhi A, Taghavi SF, Tavakoli-Yaraki M. Cyclooxygenase enzyme and PGE2 expression in patients with functional and non-functional pituitary adenomas. BMC Endocr Disord 2020;20:39.Gomes RN, Felipe da Costa S, Colquhoun A. Eicosanoids and cancer, Clinics (Sao Paulo), 73 ( 2018) e530s.
Background Cytokines are well-known immunomodulators. Thanks to recent success of immune checkpoint inhibitors there is a renewed interest in cytokines as a promising cancer immunotherapy option. Several inflammatory cytokines including IL-12 showed potent anti-tumor activities but severe immune adverse events when administered systemically greatly hindered using them as anti-tumor agents. Methods Previously (SITC 2020) we showed that IL-12 activity was reduced by our introduced mutation (termed as mut1) if measured by pSTAT4 AlphaLISA assay. But when it was treated in human immune cells IFNg production was not reduced as expected. Thus, we further attenuated IL-12 activities by protein engineering and created our candidate molecule KNP-101. ResultsWe showed that KNP-101 maintained potent antitumor activities in vivo but gained greatly improved toxicity profiles. When we measured pSTAT4 signals, KNP-101 showed about 30-fold attenuation in IL-12 activities compared with rIL-12. IFNg production from human PBMC was also reduced. Although the IL-12 activity was weakened in order to reduce its systemic toxicity, our KNP-101 mouse surrogate still maintained good anti-tumor potency in various mouse syngeneic models with a single intravenous injection as low as 2 ug/head. In combination with anti-PD-L1, KNP-101 surrogate showed a synergistic anti-tumor effect and further FACS analysis of tumor infiltrated lymphocytes demonstrated that the effects were mediated by immune cell infiltration. Importantly, in CD1 naïve mouse toxicity test, KNP-101 surrogate was tolerable up to 50 ug/head and no survival issue was observed. However, that was not the case with the control group, non-tumor-targeting null/IL-12 showing survival issues with all tested dose levels. Compared with the control, KNP-101 surrogate also showed much safer profiles in terms of organ weight and serum chemistry such as ALT level. We also performed similar toxicity study in tumor-bearing mice. KNP-101 surrogate again showed a very safe profiles being tolerable up to 500 ug/head and no survival issue. Noticeably, when serum IFNg was measured in tumor-bearing mice, our KNP-101 surrogate induced far less IFNg in serum compared to null/IL-12 suggesting that systemic toxicity was greatly reduced. Conclusions Together, we demonstrated that systemic toxicity of IL-12 cytokine therapy can be overcome by tumor-targeting and IL-12 attenuation. Our KNP-101 has a widen therapeutic window by maintaining potent anti-tumor activities and showing much improved safety profiles. We hope that KNP-101 can benefit patients in the future who suffer from primary and acquired resistance of the current anti-PD-1/PD-L1 treatments.
Prostaglandin E2 (PGE2) is widely recognized as one of the major bioactive lipids that, with the striking regenerative potential, promote drug-resistance in cancer cells as well as immune evasion in the tumor microenvironment (TME). Primarily driven by apoptotic cell death, PGE2 is thought to elicit wound-healing responses to help provide an immunosuppressive and proliferative niche that supports cancer stem cell repopulation and thereby therapy-resistance. While COX1/2 inhibitors that attenuate PGE2 production have shown promising anti-cancer effects in various (pre-)clinical settings, the gastrointestinal- and cardiotoxicities precluded their development as anti-cancer agents. It is anticipated that specific targeting of PGE2 signaling via its cognate receptors constitutes a safer and potentially more effective approach. Of the receptor subtypes EP1-4, Gα,s-coupled EP2 and EP4 are believed to be directly involved in immunosuppressive effects of PGE2.OCT-598 is a novel, highly potent and selective EP2/EP4 dual antagonist with Ki values of 23 nM and 0.2 nM vs EP2 and EP4, respectively. PGE2 inhibited normal differentiation of human monocytes into CD1a+CD16- dendritic cells under the presence of GM-CSF and IL-4 and promoted differentiation towards CD1a-CD16+ macrophages in vitro. However, EP2/EP4 dual inhibition by OCT-598 reversed this phenomenon to a greater extent than either EP2- or EP4-specific inhibitor alone. In vivo, OCT-598 effected tumor growth inhibition in multiple syngeneic mouse models as a single agent as well as in combination with an immune checkpoint blocker (ICB). Furthermore, the addition of OCT-598 to the lung cancer standard-of-care regimen (anti-PD-1 plus chemotherapy) in TC-1 mouse lung adenocarcinoma model gave rise to complete tumor regression. In conclusion, dual blockade of EP2 and EP4 by OCT-598 is shown to be a compelling strategy to reinforce antitumor effects by thwarting PGE2-mediated therapy resistance and immune evasion.Findings from this study provide a rationale for clinical development of OCT-598 as a therapeutic option for human malignant cancers. Citation Format: Youngrae Lee, Sujeong Baek, Dong Kwon Kim, Yeri Lee, Donggeon Kim, Seongin Jo, Sang Kyun Lim, Young Sook Shin, Soonsang Kwon, Seung Min Yang, Young Taek Kim, Seong-San Kang, Chun-Bong Synn, Kwangmin Na, Mi Hyun Kim, Heekyung Han, Yu Jin Han, Sungwoo Lee, Jae Hwan Kim, Mi Ran Yun, Youngseon Byeon, Young Seob Kim, Ji Yun Lee, Jii Bum Lee, Chang Gon Kim, Min Hee Hong, Sun Min Lim, Kyoung-Ho Pyo, Byoung Chul Cho, Taeyoung Yoon. OCT-598, a novel EP2/EP4 dual antagonist, promotes anti-tumor immune responses in syngeneic mouse tumor models in combination with standard-of-care chemo- and immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3234.
Abnormal activating mutation of KRAS is frequently found in many human cancers, including pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and non-small cell lung cancer (NSCLC). KRAS activating mutations lead to hyperactivation of the MAPK/ERK signaling pathway, resulting in promotion of cell proliferation and growth. SOS1 is one of the major guanine nucleotide exchange factors (GEFs) that regulates RAS proteins including KRAS. Since SOS1 plays a critical role in converting the GDP-bound inactive KRAS “off” state to the GTP-bound active KRAS “on” state, disruption of SOS1 and KRAS protein-protein interaction would be effective to block KRAS-driven oncogenic signaling regardless of its mutation status. Importantly, SOS1 activity is crucial during the reactivation of the KRAS/MAPK signaling upon the treatment of RAS/MEK/ERK inhibitors, thus a SOS1 inhibitor would be an effective therapeutic option to treat KRAS-driven tumors in combination with RAS pathway inhibitors. We developed potent, selective, and orally available small molecules that effectively disrupt the interaction between SOS1 and KRAS. Current lead compounds originated from a virtual screening displayed excellent ADME and PK profiles. In cellular assays, a robust reduction of phospho-ERK level and cancer cell growth were shown. We also observed excellent in vivo antitumor activity in the mouse xenograft models. Moreover, combination with Sotorasib synergistically inhibited tumor cell growth both in vitro and in vivo. Overall, our SOS1 inhibitors demonstrate great therapeutic potential for cancer patients with KRAS mutations. Citation Format: Ha Na Yu, Dong Hyuk Ki, Joonwoo Nam, Eun-Jung Kim, Sungeun Kim, Hunmi Choi, Jieun Kim, Jihyun Yu, Donggeon Kim, Dohyun Park, Kyeong Jin Yoon, Seongin Jo, So-Hyeon Hwang, Sang Kyun Lim, Young Sook Shin, Wooseok Han. Discovery of potent and orally available small molecule inhibitors of the SOS1-KRAS interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 515.
BackgroundAlthough cancer immunotherapy showed promising results in hematological malignancies, it has come up with relatively low tumor response for many solid tumors partly due to immune-suppressive tumor microenvironment (TME). Because of the immune-suppressive nature of TME, TME has been an active area of research and therapeutic target for restoring immune system and subsequent tumor growth inhibition. Among the many components in TME, cancer-associated fibroblasts (CAFs) are one of the key cell components of TME where one of the promising solid-tumor TME marker, fibroblast-activating protein (FAP) is highly expressed. Here we have developed an antibody-cytokine fusion protein from our TMEkine™ platform containing anti-FAP and IL-12. Our TMEkine™ (anti-FAP-IL-12) molecule induced strong anti-cancer effects in preclinical solid tumor models by immune-modulation.MethodsIL-12 cytokine was mutated in TMEkine™ (anti-FAP-IL-12) to reduce systemic toxicity and its binding affinity was tested to FAP and IL-12 receptor. The anti-tumor activity of anti-FAP-IL-12 was investigated on CT26 (murine colorectal cancer) syngeneic mouse models with/without NIH-3T3 (murine fibroblast). Additionally, mice showing complete response after anti-FAP-IL-12 administration were re-injected CT26 with/without 4T1 cells for re-challenge study to monitor long-term durable response generated from the initial immune activation.ResultsWe showed that TMEkine™ (anti-FAP-IL-12) interacts with FAP and IL-12 receptor. IL-12 activity was attenuated by our IL-12 mutants. We also showed that TMEkine™ (anti-FAP-IL-12) induced IFN-γ from primary human T cells and NK cells. TMEkine™ (anti-FAP-IL-12) administration resulted in significant reduction of the tumor burden in both CT26+NIH-3T3/FAP+ and CT26/FAP+ models. In the re-challenge experiments, CT26 tumor growth was inhibited significantly compared to 4T1 tumor suggesting memory immune response was generated in TMEkine™ (anti-FAP-IL-12) treated mice.ConclusionsThese findings provide evidences that the treatment of anti-FAP/IL-12 TMEkine™ induced anti-cancer effects without serious adverse effects. Anti-FAP/IL-12 has a strong potential to provide a therapeutic option for cancer-specific immunomodulator and cancer cell eradication.
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