Search

Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies

The main barrier to HIV virus eradication is the presence of long-lived HIV-1 that persists in latent reservoirs. Current efforts have focused on HIV cure interventions that “shock and kill” virally infected cells to purge the latent pool. Latency reversing agents (LRAs) induce viral activation leading to immune cell recognition and clearance of latently-infected cells. Though several clinical trials with LRAs have demonstrated that activation of viral gene expression is possible in vivo, limited or no clearance of the reactivated cells has been observed. The identification of molecules that can simultaneously activate latent HIV, increase immune activation and enhance the killing of latently-infected cells represents an alternative promising approach. Here, we developed and evaluated a trispecific antibody that targets three independent proteins: (1) the HIV envelope, (2) the T cell antigen CD3, and (3) the co-stimulatory molecule CD28. For targeting HIV Env, we used the broadly reactive CD4-binding site monoclonal antibody (mAb), N6. We found that an antibody with all three specificities (N6/αCD3-αCD28), significantly increased antigen-specific T-cell activation and cytokine release in both CD4+ and CD8+ T cells. Co-culturing CD4+ T cells with autologous CD8+ T cells isolated from ART suppressed HIV+ donors in the presence of N6/αCD3-αCD28, resulted in activation of latently-infected cells and enhancement of the cytolytic activity of CD8+ T cells to eliminate the cells expressing reactivated HIV. This trispecific antibody mediated CD4+ and CD8+ T-cell activation in non-human primates and was well tolerated in vivo, confirming its activity and safety in a relevant animal model. This HIV-directed antibody is therefore merits further development as a potential intervention for the eradication of latent HIV infection.

Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and was accompanied by intratumoral IFN-γ induction, systemic antigen-specific T cell expansion, increased granzyme B

More than 75% of breast cancers are estrogen receptor alpha (ERα) positive (ER+), and resistance to current hormone therapies occurs in one-third of ER+ patients. Tumor resistance is still ERα-dependent, but mutations usually confer constitutive activation to the hormone receptor, rendering ERα modulator drugs such as tamoxifen and aromatase inhibitors ineffective. Fulvestrant is a potent selective estrogen receptor degrader (SERD), which degrades the ERα receptor in drug-resistant tumors and has been approved for the treatment of hormone-receptor-positive metastatic breast cancer following antiestrogen therapy. However, fulvestrant shows poor pharmacokinetic properties in human, low solubility, weak permeation, and high metabolism, limiting its administration to inconvenient intramuscular injections. This Drug Annotation describes the identification and optimization of a new series of potent orally available SERDs, which led to the discovery of 6-(2,4-dichlorophenyl)-5-[4-[(3S)-1-(3-fluoropropyl)pyrrolidin-3-yl]oxyphenyl]-8,9-dihydro-7H-benzo[7]annulene-2-carboxylic acid (43d), showing promising antitumor activity in breast cancer mice xenograft models and whose properties warranted clinical evaluation.

Primary treatment for estrogen receptor-positive (ER+) breast cancer is endocrine therapy. However, substantial evidence indicates a continued role for ER signaling in tumor progression. Selective estrogen receptor degraders (SERD), such as fulvestrant, induce effective ER signaling inhibition, although clinical studies with fulvestrant report insufficient blockade of ER signaling, possibly due to suboptimal pharmaceutical properties. Furthermore, activating mutations in the ER have emerged as a resistance mechanism to current endocrine therapies. New oral SERDs with improved drug properties are under clinical investigation, but the biological profile that could translate to improved therapeutic benefit remains unclear. Here, we describe the discovery of SAR439859, a novel, orally bioavailable SERD with potent antagonist and degradation activities against both wild-type and mutant Y537S ER. Driven by its fluoropropyl pyrrolidinyl side chain, SAR439859 has demonstrated broader and superior ER antagonist and degrader activities across a large panel of ER+ cells, compared with other SERDs characterized by a cinnamic acid side chain, including improved inhibition of ER signaling and tumor cell growth. Similarly, in vivo treatment with SAR439859 demonstrated significant tumor regression in ER+ breast cancer models, including MCF7-ESR1 wild-type and mutant-Y537S mouse tumors, and HCI013, a patient-derived tamoxifen-resistant xenograft tumor. These findings indicate that SAR439859 may provide therapeutic benefit to patients with ER+ breast cancer, including those who have resistance to endocrine therapy with both wild-type and mutant ER.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide, accounting for more than 700,000 deaths per year. Although transforming growth factor beta (TGF-β) can function as a tumor-suppressor in premalignant cells, patient data suggest that it plays a role in the progression of HCC. Recently, immunotherapy targeting PD-1 has been approved for HCC, however, success is limited to only ~17% objective response rate in the treated HCC patients. Analysis of genomic data from two HCC patient cohorts revealed that TGF-β pathway is highly activated in patients with advanced HCC. Collectively, these findings indicate that targeting TGF-β in combination with PD-1 blockade may be a compelling therapeutic strategy. Therefore, using two syngeneic mouse models of HCC (Hepa1-6 and H22), we examined the effect of a pan-TGF-β neutralizing antibody (1D11) in combination with PD-1 blockade. Results from our study show that co-inhibiting TGF-β with PD-1 resulted in significant tumor growth inhibition in both HCC models. Flow cytometry analysis of the tumor revealed the combination treatment group exhibited a reduction in potentially suppressive tumor-associated-macrophages relative to either single agent groups. In addition, we show that combination therapy was associated with increase in the intra-tumoral levels of proinflammatory cytokines - IFNγ, IL-1β, TNF-α as well as enhanced expression of anti-tumor immune related genes. Based on these data, clinical testing of SAR439459 (TGF-β antibody) and Cemiplimab (anti-PD1) combination has recently been initiated in HCC patients (NCT03192345). Citation Format: Natalia Malkova, Joachim Theilhaber, Mikhail Levit, Tatiana Tolstykh, Andrew Hebert, Kevin Atchison, Adalis Maisonet, Sara Sara Sinicropi-Yao, Alexei Protopopov, Jack Pollard, Sukhvinder Sidhu, Tun Tun Lin, Lily Pao, Dmitri Wiederschain, Sharad K. Sharma. Pan-TGF-β inhibition with PD-1 blockade as a combination treatment strategy to augment anti-tumor immune response in hepatocellular carcinoma [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 1909.

In the recent decade several immune checkpoint inhibitors, such as anti-PD1/PD-L1 antibodies, have been approved by regulatory authorities and are actively used in oncology clinical practice. However, these antibodies exhibit durable anti-tumor benefits in only a small fraction of cancer patients. A major unmet medical need in immune oncology (IO) is extending the benefits of these therapies to patients who never experience tumor regression (primary resistance) and those who derive initial benefit before experiencing tumor progression (acquired resistance). Novel IO combinations have the potential to both sensitize primary resistant tumors and combat the acquisition of resistance to checkpoint inhibition. Synthetic, in vitro transcribed messenger RNA (mRNA) is an emerging therapeutic modality that offers the ability to express a broad range of therapeutic combinations. In this project, we proposed to ignite anti-tumor immunity locally through intratumoral (i.t.) delivery of a mixture of mRNAs encoding cytokines and a TNF receptor superfamily (TNFRSF) agonist. Using in vitro and in vivo screening, we identified a combination therapy consisting of three cytokines IFNα, IL-7, and a fusion protein of IL-15 with its receptor alpha chain as well as one TNFRSF agonist, an Fc fusion hexameric OX40L for enhanced receptor clustering. The mRNA mixture enhanced human antigen-specific T cell responses in vitro and displayed potent anti-tumor efficacy in syngeneic tumor models when injected i.t., demonstrating the induction of protective immunity. Importantly, in a preclinical dual-flank syngeneic tumor model, we demonstrated that this protective immunity extended beyond tumors injected with the lead mRNA mixture to control growth of distant untreated lesions. Mechanistically, the lead candidate induced anti-tumor immunity by recruiting dendritic cells to the tumor, leading to activation and expansion of tumor specific CD8 T cells. In summary, this mRNA mixture, encoding IFNα, IL-7, IL-15 and OX40L, induced potent anti-tumor immunity in mice and will be further investigated as a combination partner to overcome resistance mechanisms to immune checkpoint inhibition. Citation Format: Julie-Ann Gavigan, Chaomei Shi, Michael Lampa, Natalia Malkova, Qunyan Yu, Hui Qu, Mikhail Levit, Timothy Wagenaar, Christian Hotz, Xiangming Li. Induction of anti-tumor immunity by intratumoral delivery of mRNA encoding cytokines and TNFRSF agonists [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 1711.

Novel N-substituted azaindoles have been discovered as PIM1 inhibitors. X-ray structures have played a significant role in orienting the chemistry effort in the initial phase of hit confirmation. Disclosure of an unconventional binding mode for 1 and 2, as demonstrated by X-ray crystallography, is presented and was an important factor in selecting and advancing a lead series.

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/β-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (β-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of β-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on β-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on β-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of β-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant β-catenin signaling in the maintenance of oncogenic phenotype in HCC.

Primary treatment for estrogen receptor-positive (ER+) breast cancer is endocrine therapy. However, substantial evidence indicates a continued role for ER signaling in tumor progression. Selective estrogen receptor degraders (SERDs), such as fulvestrant, induce effective ER signaling inhibition, although clinical studies with fulvestrant report insufficient blockade of ER signaling, possibly due to suboptimal pharmaceutical properties. Furthermore, activating mutations in the ER have emerged as a resistance mechanism to current endocrine therapies. New oral SERDs with improved drug properties are under clinical investigation, but the biological profile that could translate to improved therapeutic benefit remains unclear. Here we describe the discovery of SAR439859, a novel, orally bioavailable SERD with potent antagonist and degradation activities against both wild-type and mutant Y537S ER. Driven by its fluoropropyl pyrrolidinyl side chain, SAR439859 has demonstrated broader and superior ER antagonist and degrader activities across a large panel of ER+ cells, compared with other SERDs characterized by a cinnamic acid side chain, including improved inhibition of ER signaling and tumor cell growth. Similarly, in vivo treatment with SAR439859 demonstrated significant tumor regression in ER+ breast cancer models including MCF7-ESR1 wild type and mutant-Y537S mouse tumors, and HCI013, a patient-derived tamoxifen-resistant xenograft tumor. These findings indicate that SAR439859 may provide therapeutic benefit to patients with ER+ breast cancer, including those have resistance to endocrine therapy with both wild-type and mutant ER. This presentation will highlight the global profile and key features of new SERD and its translation to patient. Citation Format: Maysoun Shomali, Fangxian Sun, Mikhail Levit, Jack Pollard, Joon Sang Lee, Laurent Schio, Youssef El-Ahmad, Chris Soria, Patrick Cohen, Laurent Debussche, Monsif Bouaboula. Sar439859, a novel selective estrogen receptor degrader (serd), demonstrates effective and broad antitumor activity in wild-type and mutant er-positive breast cancer models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-16.

N-substituted azaindoles were discovered as potent pan-PIM inhibitors. Lead optimization, guided by structure and focused on physico-chemical properties allowed us to solve inherent hERG and permeability liabilities, and provided compound 27, which subsequently impacted KG-1 tumor growth in a mouse model.

Class IA PI3K pathway activation resulting from PTEN deficiency has been associated with lack of sensitivity of melanoma to BRAF kinase inhibitors. Although previous studies have shown synergistic activity when pan-PI3K inhibitors were combined with MAPK inhibitors in the treatment of melanoma exhibiting concurrent genetic abnormalities, overlapping adverse events in patients limit optimal dosing and clinical application. With the aim of specifically targeting PTEN-deficient cancers and minimizing the potential for on-target toxicity when inhibiting multiple PI3K isoforms, we developed a program to discover PI3Kβ-selective kinase inhibitors and identified SAR260301 as a potent PI3Kβ-selective, orally available compound, which is now in clinical development. Herein, we provide a detailed biological characterization of SAR260301, and show that this compound has outstanding biochemical and cellular selectivity for the PI3Kβ isoform versus the α, δ, and γ isoforms and a large panel of protein and lipid kinases. We demonstrate that SAR260301 blocks PI3K pathway signaling preferentially in PTEN-deficient human tumor models, and has synergistic antitumor activity when combined with vemurafenib (BRAF inhibitor) or selumetinib (MEK inhibitor) in PTEN-deficient/BRAF-mutated human melanoma tumor models. Combination treatments were very well tolerated, suggesting the potential for a superior safety profile at optimal dosing using selective compounds to inhibit multiple signaling pathways. Together, these experiments provide a preclinical proof-of-concept for safely combining inhibitors of PI3Kβ and BRAF or MEK kinase modulators to improve antitumor activity in PTEN-deficient/BRAF-mutant melanoma, and support the evaluation of SAR260301-based combinations in clinical studies. Mol Cancer Ther; 15(7); 1460–71. ©2016 AACR.

Cancer immunotherapies with anti-PD-1/PD-L1 checkpoint blockade antibodies have revolutionized the treatment of a wide variety of malignancies. However, immunotherapy is ineffective in a significant subset of patients, or eventually results in the development of resistance with relapsed disease. Therefore, the future of immuno-oncology is identification of new agents that can be combined to increase the depth and breadth of anti-PD-1/PD-L1 therapies. Local intratumoral delivery of messenger RNA (mRNA)-based immunotherapies provides an opportunity to stimulate innate and adaptive immune responses against tumors and modulate tumor microenvironment, while limiting the side-effects related to systemic administration of immuno-modulatory therapeutics. We examined the local immunotherapy of synthetic mRNAs encoding immunomodulatory cytokines (single chain interleukin-12 (IL-12), interferon alpha (IFN-α), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-15 sushi(IL-15)) alone and in combination with anti-PD-1 antibodies across 12 syngeneic mouse tumor models representing a diverse range of murine tumor types. Single agent treatment with cytokine mRNAs resulted in a significant anti-tumor response in 8 out of 12 tumor models. Combining cytokine mRNA with systemic anti-PD-1 immunotherapy further improved the anti-tumor response and resulted in tumor growth inhibition in two additional models otherwise unresponsive to single agent treatments. Transcriptome and cytokine analyses have been performed to understand factors which determine sensitivity to single agent and combination treatments with cytokine mRNA and anti-PD-1 antibodies. In summary, the combination of local cytokine mRNA immunotherapy with immune checkpoint blockade demonstrated improvement in the anti-tumor activity in a range of preclinical cancer models representing different murine tumor types. Based on these and other data, clinical testing of cytokine encoding mRNA mixture has recently been initiated (NCT03871348). Citation Format: Natalia V. Malkova, Tatiana Tolstykh, Mikhail Levit, Joachim Theilhaber, Andrew Hebert, Kevin Atchison, Adalis Maisonet, Christian Hotz, Ugur Sahin, Alexei Protopopov, Jack Pollard, Sukhvinder Sidhu, Dmitri Wiederschain, Timothy R. Wagenaar. Combination of local mRNA immunotherapy with systemic immune checkpoint blockade demonstrates anti-tumor activity across a diverse range of preclinical syngeneic tumor models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4451.

Abstract: Nearly 70% or more of newly diagnosed cases of breast cancer (BC) are estrogen receptor positive (ER+) where endocrine therapy is a primary treatment. However, substantial evidence describes a continued role of ER signaling in tumor progression, where approximately 40% of patients on endocrine therapy develop resistance that include mutations in ER that drive a constitutively active receptor. SAR439859 is a novel next generation SERD (selective estrogen receptor degrader) with potent antagonist and degradation activity against ER both wild type and mutant and currently evaluated as a single agent and in combination with palbociclib in a Phase I/II clinical trial (NCT03284957). Here we describe the preclinical development of this molecule in multiple xenografts and demonstrated significant anti-tumor efficacy/regression in ER+ BC models including MCF7-ESR1 mutant-Y537S and endocrine therapy resistant patient-derived xenograft tumor models. The molecule also demonstrated strong synergistic activity with CDK4 inhibitor palbociclib. SAR439859 has a favorable safe profile and no uterotrophic activities. Collectively, these results show that SAR439859 is an oral, selective estrogen receptor antagonist and degrader that could provide therapeutic benefit to ER+ breast cancer patients. In conclusion, SAR439859 demonstrates broad anti-tumor activity in ER+ breast cancer cell line/PDX models. Anti-tumor efficacy correlates with PK exposure/PD modulation in target tissue. SAR439859 also has synergistic activity with CDK4 inhibitor Palbociclib. Citation Format: Fangxian Sun, Jane Cheng, Amy Sullivan, Natalia Malkova, Maysoun Shomali, Jesseca McManus, Mikhail Levit, Dinesh Bangari, Youssef El-Ahmad, Sukhvinder Sidhu, Laurent Debussche, Monsif Bouaboula. Pre-clinical development of next generation selective estrogen receptor degrader - SAR439859 [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 3452.

N-substituted azaindoles were discovered as promising pan-PIM inhibitors. Lead optimization is described en route toward the identification of a clinical candidate. Modulation of physico-chemical properties allowed to solve inherent hERG and permeability liabilities. Compound 17 showed tumor growth inhibition in a KG1 tumor-bearing mouse model.

N-Substituted azaindoles have been discovered as pan-PIM kinase inhibitors. Initial SAR, early ADME and PK/PD data of a series of compounds is described and led to the identification of promising pan-PIM inhibitors which validated our interest in the 7-azaindole scaffold and led us to pursue the identification of a clinical candidate.

Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML.

SAR103168, a tyrosine kinase inhibitor of the pyrido [2,3-d] pyridimidine subclass, inhibited the kinase activities of the entire Src kinase family, Abl kinase, angiogenic receptor kinases (vascular endothelial growth factor receptor [VEGFR] 1 and 2), Tie2, platelet derived growth factor (PDGF), fibroblast growth factor receptor (FGFR) 1 and 3, and epidermal growth factor receptor (EGFR). SAR103168 was a potent Src inhibitor, with 50% inhibitory concentration (IC50) = 0.65 ± 0.02 nM (at 100 μM ATP), targeting the auto-phosphorylation of the kinase domain (Src(260-535)) and activity of the phosphorylated kinase. Phosphorylation of Src, Lyn and Src downstream signaling pathways (PYK2, P-130CAS, FAK, JNK and MAPK) were inhibited in a dose-dependent manner. SAR103168 inhibited the phosphorylation of STAT5 in KG1 cells and fresh cells from patients with acute myeloid leukemia (AML). SAR103168 inhibited proliferation and induced apoptosis in acute and chronic myeloid leukemic cells at nanomolar IC50. SAR103168 induced anti-proliferation of leukemic progenitors (CFU-L) from 29 patients with AML, and85% of AML patient samples were sensitive to SAR103168. These antagonist activities of SAR103168 were independent of FLT3 expression. SAR103168 treatment was effective in 50% of high-risk patient samples carrying chromosome 7 abnormalities or complex rearrangement. SAR103168 administration (intravenous or oral) impaired tumor growth and induced tumor regression in animals bearing human AML leukemic cells, correlating with potent inhibition of Src downstream signaling pathways in AML tumors. SAR103168 showed potent anti-tumor activity in SCID (severe combined immunodeficiency) mice bearing AML (KG1, EOL-1, Kasumi-1, CTV1) and chronic myeloid leukemia (CML) (K562) tumors. The combination of cytarabine and SAR103168 showed synergistic activity in AML and CML tumor models. These results highlight the therapeutic potential of SAR103168 in myeloid leukemias and support the rationale for clinical investigations.

Nearly 70% or more of newly diagnosed cases of breast cancer (BC) are estrogen receptor positive (ER+) where endocrine therapy is a primary treatment. However, substantial evidence describes a continued role of ER signaling in tumor progression, where approximately 40% of patients on endocrine therapy develop resistance that include mutations in the ER that drive a constitutively active receptor. Fulvestrant, an estrogen receptor degrader, is effective at shutting down ER signaling. However, fulvestrant efficacy studies report insufficient blockade of ER signaling in patients that may be a consequence of poor pharmaceutical properties. Here we describe the discovery of SAR439859, a novel, orally bioavailable SERD with potent antagonist and degradative properties against ER both in vitro and in vivo. SAR439859 has robust inhibition of ER signaling activity in multiple ER+ breast cancer cell lines including tamoxifen resistant lines harboring ER mutations. Across a large panel of ER+ cells, SAR439859 demonstrated broad and superior ER degradation activity as compared to other SERDs including improved inhibition of ER signaling and inhibition of cell growth. Similarly, in vivo treatment with SAR439859 demonstrated significant tumor regression in ER+ BC models including MCF7-ESR1 mutant-Y537S model and endocrine therapy resistant patient-derived xenograft tumor transplantation. Collectively, these results showed that SAR439859 is an oral, nonsteroidal, selective estrogen receptor antagonist and degrader that could provide therapeutic benefit to ER+ breast cancer patients. SAR439859 is currently being evaluated in a phase I clinical trial. Citation Format: Maysoun Shomali, Youssef El-Ahmad, Frank Halley, Jane Cheng, Michael Weinstein, Muchun Wang, Fangxian Sun, Natalia Malkova, Mikhail Levit, Malvika Koundinya, Zhuyan Gou, Andrew Hebert, Jessica McManus, Dietmar Hoffman, Hui Cao, Joonil Jung, Jack Pollard, Sylvie Vincent, Timothy Ackerson, Francisco Adrian, Chris Winter, Victoria Richon, Hong Chen, Karl Hsu, Joanne Lager, Albane Courjaud, Rosalia Arrebola, Laurent Besret, Pierre-Yves Abecassis, Laurent Schio, Gary McCort, Michel Tabart, Victor Certal, Fabienne Thompson, Bruno Filoche-Romme, Laurent Debussche, Patrick Cohen, Carlos Garcia-Echeverria, Monsif Bouaboula. Identification of SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that has strong anti-tumor activity in wild-type and mutant ER+ breast cancer models [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 5775.

TGFβ may act as a tumor suppressor early in tumorigenesis; however, TGFβ is also known to support tumor growth via multiple mechanisms, including promotion of epithelial-mesenchymal transition, fibroblast recruitment, ECM deposition, neovascularization, and immunosuppression of both adaptive and innate immune cells. SAR439459 is pan-neutralizing TGFβ antibody that is being developed for the treatment of solid tumors. In this study, we investigated the effect of SAR439459 on the immune system in primary human immune cells in vitro and in murine syngeneic tumor models in vivo. Exogenous TGFβ inhibited proliferation of CD8pos primary human T cells and suppressed the production of inflammatory cytokines such as IFNγ. The addition of SAR439459 blocked these effects. Additionally, TGFβ enhanced the development of inducible Tregs by approximately 4-fold, while the inclusion of SAR439459 abrogated this effect. The effects of exogenous TGFβ and SAR439459 were next examined in the context of T Cell Receptor signaling in a co-culture system of Jurkat cells expressing luciferase reporter under the control of NFAT-responsive promoter and PD-L1pos APCs. The addition of anti-PD1 stimulated TCR signaling in this system, while the addition of TGFβ inhibited anti-PD1-mediated response. SAR439459 restored the ability of anti-PD1 to stimulate the TCR signaling. These data support the notion that TGFβ neutralization could positively impact T effector cells and inhibit Tregs development. Therefore, combining a TGFβ-neutralizing antibody with anti-PD1 could enhance T cell responsiveness. Compared to checkpoint inhibitors, which are suggested to cause the reactivation and de-repression of exhausted T cells, the effects of TGFβ on the immune system are thought to be distinct. To test this, the combination of anti-PD1 and SAR439459 was examined in syngeneic mouse models. Established MC38 tumors were treated with anti-PD1, SAR439459 or the combination of both antibodies. In tumor-bearing mice treated with either anti-PD1 or SAR439459 alone, neither Ab resulted in significant tumor growth inhibition. However, the combination of anti-PD-1 and SAR439459 led to tumor regression in the majority of tumor-bearing mice. These results provide the rationale for combining TGF-β neutralization with checkpoint inhibitors, specifically anti-PD1, for the treatment of human cancers. Citation Format: Richard C. Gregory, Rita Greco, Hongjing Qu, Natalia Malkova, Mikhail Levit, Keli Perron, Waldemar Racki, Fangxian Sun, Gary Shapiro, Christopher Winter, Dmitri Wiederschain, Tun Tun Lin, Jack Pollard. The anti-TGFβ neutralizing antibody, SAR439459, blocks the immunosuppressive effects of TGFβ and inhibits the growth of syngeneic tumors in combination with anti-PD1 [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 2790.

Nearly 70% or more of newly diagnosed cases of breast cancer (BC) are estrogen receptor positive (ER+) where endocrine therapy is a primary treatment. However, substantial evidence describes a continued role of ER signaling in tumor progression, where approximately 40% of patients on endocrine therapy develop resistance that include mutations in the ER that drive a constitutively active receptor. Fulvestrant, an estrogen receptor degrader, is effective at shutting down ER signaling. However, fulvestrant efficacy studies report insufficient blockade of ER signaling in patients that may be a consequence of poor pharmaceutical properties. Here we describe the discovery of SAR439859, a novel, orally bioavailable SERD with potent antagonist and degradative properties against ER both in vitro and in vivo. SAR439859 has robust inhibition of ER signaling activity in multiple ER+ breast cancer cell lines including tamoxifen resistant lines harboring ER mutations. Across a large panel of ER+ cells, SAR439859 demonstrated broad and superior ER degradation activity as compared to other SERDs including improved inhibition of ER signaling and inhibition of cell growth. Similarly, in vivo treatment with SAR439859 demonstrated significant tumor regression in ER+ BC models including MCF7-ESR1 mutant-Y537S model and endocrine therapy resistant patient-derived xenograft tumor transplantation. Collectively, these results showed that SAR439859 is an oral, nonsteroidal, selective estrogen receptor antagonist and degrader that could provide therapeutic benefit to ER+ breast cancer patients. SAR439859 is currently being evaluated in a phase I clinical trial. Citation Format: Maysoun Shomali, Youssef El-Ahmad, Frank Halley, Jane Cheng, Michael Weinstein, Muchun Wang, Fangxian Sun, Natalia Malkova, Mikhail Levit, Malvika Koundinya, Zhuyan Gou, Andrew Hebert, Jessica McManus, Dietmar Hoffman, Hui Cao, Joonil Jung, Jack Pollard, Sylvie Vincent, Timothy Ackerson, Francisco Adrian, Chris Winter, Victoria Richon, Hong Chen, Karl Hsu, Joanne Lager, Albane Courjaud, Rosalia Arrebola, Laurent Besret, Pierre-Yves Abecassis, Laurent Schio, Gary McCort, Michel Tabart, Victor Certal, Fabienne Thompson, Bruno Filoche-Rommé, Laurent Debussche, Patrick Cohen, Carlos Garcia-Echeverria, Monsif Bouaboula. Identification of SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that has strong anti-tumor activity in wild-type and mutant ER+ breast cancer models [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 5775.

Introduction: Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer-related mortality. Currently there are few treatment options available for advanced HCC patients. Multi-kinase inhibitors sorafenib and regorafenib are the only approved systemic HCC therapies and are only marginally effective in extending survival. HCC is largely driven by difficult-to-drug oncogenes, including β-catenin, and none of the current HCC therapeutics on the market or in development addresses these major genetic drivers of disease. β-catenin is an integral part of the WNT signaling pathway and plays a major role in regulation of cell survival, apoptosis and developmental processes. The goal of this study was to systematically interrogate contribution of β-catenin to HCC pathogenesis. Methods: The extent of genomic alterations in β-catenin itself or components of its regulatory network was interrogated in two large HCC data sets, The Cancer Genome Atlas and HCC cohort from Asan Medical Center. β-catenin in vitro credentialing (target knock-down, PD modulation and cell growth inhibition) was carried out in cultured HCC cell lines using multiple siRNAs against β-catenin. Stably expressed doxycycline-inducible shRNAs targeting β-catenin were used to establish in vivo dependency in HCC tumor xenografts. Results: Building on previous observations, we detected high frequency of somatic mutations in CTNNB1/β-catenin itself and in constituent members of its regulatory network in two independent cohorts of HCC patients. Furthermore, significant upregulation in β-catenin protein levels was detected in the overwhelming majority of HCC patient samples, patient-derived xenografts and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we discovered that dependency on β-catenin in human HCC cell lines generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1/β-catenin or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1/β-catenin and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in PD marker AXIN2, and decreased cancer cell proliferation. Conclusions: β-catenin is an important therapeutic target in HCC that could potentially be targeted using therapeutic siRNA suitably formulated for delivery to human liver tumors. Genomic alterations in CTNNB1/β-catenin, as well as increased abundance of non-phosphorylated (active) β-catenin, may serve as predictive biomarkers for patient selection. Citation Format: Zhihu (Jeff) Ding, Chaomei Shi, Lan Jiang, Tatiana Tolstykh, Hui Cao, Dinesh Bangari, Susan Ryan, Taiguang Jin, Mikhail Levit, Karl Mamaat, Qunyan Yu, Hui Qu, Joern Hopke, May Cindhuchao, Dietmar Hoffmann, Fangxian Sun, Mike Helms, Kerstin Jahn-Hofmann, Sabine Scheidler, Douglas Fang, Liang Schweizer, Jack Pollard, Christopher Winter, Dmitri Wiederschain. Sensitivity of liver cancer cell lines to B-catenin knock-down correlates with pathway activation [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 2489.

Cancer immunotherapy localized to the tumor microenvironment holds great potential to promote innate and adaptive immune responses against tumors, while avoiding toxicities related to systemic administration of immuno-modulatory therapeutics. Current strategies for tumor-targeted, gene-based delivery of immune therapies face limitations in the clinic due to suboptimal target expression, anti-vector immunity, potential for unwanted genomic rearrangements and other off target effects. We developed a highly potent synthetic mRNA-based platform for in vivo transfection and sustained intratumoral expression of immuno-modulatory molecules that is capable of inducing immunity to tumor specific antigens while avoiding anti-vector immunity and distal tissue expression often associated with viral gene therapy or oncolytic viruses. Using iterative rounds of in vivo screening in murine syngeneic tumor models, we identified a combination of four synthetic mRNAs encoding potent cytokines that mediate high frequency of complete tumor regressions across different tumor models. Mechanistically, anti-tumor activity of cytokine mRNAs relied on the action of T cells as well as NK cells and was accompanied by robust intratumoral induction of interferon gamma, systemic expansion of antigen-specific T cells and increased granzyme B positive CD8+ T cell infiltration. Immunological memory to both dominant and subdominant antigens was formed that protected long-term survivors from re-challenge with autologous tumors. Importantly, although cytokine mRNAs were administered intratumorally, resulting in local target expression, anti-tumor activity extended beyond the injected lesion to effectively control the growth of distant tumors in both a dual tumor model and in an experimental lung metastasis model. Finally, the combination of four cytokine mRNAs with immunomodulatory antibodies further enhanced the abscopal effect leading to improved overall survival and higher incidence of complete tumor regressions across several preclinical models. In summary, the robust and versatile synthetic mRNA platform reported herein was used to identify multi-modal localized cancer immunotherapy with broad anti-tumor activity against treated and untreated lesions in a range of preclinical cancer models. Citation Format: Timothy R. Wagenaar, Christian Hotz, Friederike Gieseke, Hui Cao, Jan Diekmann, Mustafa Diken, Christian Grunwitz, Andy Hebert, Karl Hsu, Marie Jordan, Katalin Kariko, Sebastian Kreiter, Andreas N. Kuhn, Mikhail Levit, Natalia Malkova, Serena Masciari, Jack Pollard, Hui Qu, Abderaouf Selmi, Julia Schlereth, Fangxian Sun, Bodo Tillmann, Tatiana Tolstykh, Lena Wicke, Sonja Witzel, Qunyan Yu, Yu-An Zhang, Gang Zheng, Gary Nabel, Joanne Lager, Ugur Sahin, Dmitri Wiederschain. Combinatorial treatment with intratumoral cytokine mRNAs results in high frequency of tumor rejection and development of anti-tumor immunity across a range of preclinical cancer models [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 LB-130.

Despite resistance to endocrine therapies, estrogen receptor-positive (ER+) breast cancers (BC) still rely on ER. Eliminating ER by inducing its degradation with selective ER downregulators (SERD) should induce complete ablation of ER pathways. The clinical SERD fulvestrant, although it has demonstrated clinical benefits, is hampered by its poor drug properties and undesirable pharmacokinetics, undermining its optimal clinical activity. Therefore there is an unmet need for an improved therapy targeting ER. Here we describe SAR439859, a novel, nonsteroidal, orally bioavailable SERD currently in clinical trials. SAR439859 has a potent ER antagonist and ER degrading activity that translates in a robust inhibition of ER signaling in multiple ER+ breast cancer cell lines, including tamoxifen-resistant lines as well as cell lines harboring ER mutations. SAR439859 displays a broad and superior ER degradation activity across a large panel of ER+ cells. Importantly, based on its mechanism of action SAR439859 shows limited cross-resistance with other clinical SERD molecules. SAR439859 induces strong in vivo antitumor activity against a variety of BC cell lines and patient-derived xenografts, including models that harbor ERα mutations. The transcriptional profile analysis highlighted a crosstalk of ER mutant signaling and other oncogenic pathways. Interestingly, CDK4/6 inhibition by palbociclib induces partial activation of ER pathways as potential mechanism of tumor escape, which is completely abolished by the combination of SAR439859 with palbociclib. Finally, we demonstrate that SAR439859 in combination with palbociclib can lead to higher in vivo efficacy. This study highlights novel mechanism of ER degradation by SAR439859 that leads to profound inhibition of ER signaling as well as modulation of other oncogenic pathways and provides rationale for the ongoing clinical investigation of SAR439859 in ER+ breast cancer patients, both as a single agent and in combination with approved agents, such as CDK4 inhibitor. Citation Format: Monsif Bouaboula, Maysoun Shomali, Jane Cheng, Natalia Malkova, Fangxian Sun, Malvika Koundinya, Zhuyan Guo, Stephane Poirier, Mikhail Levit, Dietmar Hoffman, Hui Cao, Laurent Bestret, Francisco Adrian, Christoph Winter, Youssef El-Ahmad, Sylvie Vincent, Frank Halley, Gary McCort, Laurent Schio, Vicky Richon, Hong Cheng, Karl Hsu, Chris Soria, Patrick Cohen, Joanne Lager, Carlos Garcia-Echeverria, Laurent Debussche. SAR439859, an orally bioavailable selective estrogen receptor degrader (SERD) that demonstrates robust antitumor efficacy and limited cross-resistance in ER+ breast 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 943.

Deregulation of NF-κB signaling pathway has been identified as one of the key drivers of many hematological malignancies. Activation of the NF-κB pathway in these cells can happen either through activating mutations within the pathway, loss of function mutations of inhibitory molecules or through interaction with the stroma that secrete specific cytokines. The strategies for blocking the NF-κB pathway activation have so far focused on NF-κB pathway kinase inhibitors or the more general proteasome inhibitors. However, the adverse events associated with available NF-κB pathway modulators in pre-clinical and clinical settings have limited their general therapeutic use. To discover new targets and modulators of this key survival pathway, we performed a cellular phenotypic screen that led to the identification of a novel class of small molecules that inhibit the activation of the canonical NF-κB pathway. The compounds showed potent inhibition of NF-κB activation by multiple receptors including the TNF, antigen and BAFF/ APRIL receptors. Structure activity relationship analysis for this novel class of molecules identified pairs of stereoisomers wherein the cis isoform showed greater than 100 fold activity than the trans isoform. Detailed interrogation of the mechanism of action of the active cis isomers using biochemical and cellular pathway analysis revealed that the compounds are potent inhibitors of RHO associated kinases, only weakly inhibit IKK1 and IKK2 and strongly inhibit the induction of phosphorylated IkBα upon stimulation. However, the differential activity of the stereoisomers was not seen for the inhibition of the RHO associated kinases, suggesting the inhibition of NF-κB pathway was due to modulation of other targets. This raises the possibility of identifying novel targets associated with this compound. Using in-lysate affinity chemical proteomics with the active and in-active enantiomer, we have identified PKN1 as a key efficacy target for the inhibitors. Knockdown of PKN1 using genetic tools was sufficient to abrogate the response of cells to TNFα stimulation implicating a critical role for PKN1 in the activation of NF-κB pathway. Our findings therefore present an opportunity to develop novel NF-κB inhibitors for the tumors that depend on this key pathway for survival. Citation Format: Katherine Tang, Michael Lampa, Tieu-Binh Le, Matthieu Barrague, Ronald Tomlinson, Brittain Scott, Tahir Majid, Marion Dorsch, Hong Cheng, Christoph Lengauer, Carlos Garcia-Echeverria, Francisco Adrian, Mikhail Levit, Balin Zhang, Kin Yu, Ivan Cornella-Taracido, Lakshmi Srinivasan. Chemical Proteomics effort identifies PKN1 as a key player in the canonical NF-κB signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1135. doi:10.1158/1538-7445.AM2013-1135